
Proceedings of the AACR

Volume 60 | April 2019

Part A: Abstracts 1-2748

TABLE OF CONTENTS

BIOINFORMATICS AND SYSTEMS BIOLOGY

  * Convergence Science for Therapeutics and Precision Medicine
  * Tools for Cancer Genome Analysis
  * Convergence Science for Biomarkers, Early Detection, and Diagnosis
  * Laboratory and Computational Methods for Cancer Analysis
  * Convergence Science for Tumor Biology, Molecular Biology, and Microenvironment
  * Databases and Computational Tools for Cancer Discovery

CANCER CHEMISTRY

  * Novel Small Molecules for Cancer Therapy
  * Innovative Concepts for Drug Discovery
  * Anticancer Agents from Natural Sources

CLINICAL RESEARCH

  * Circulating and Cell-free Biomarkers for Diagnosis and Monitoring of Cancer 1
  * Current Developments in Non-invasive Biomarkers for Assessment of Cancer 1
  * Precision Medicine
  * Biomarkers for Early Detection and Biologic Assessment of Cancer
  * Circulating and Cell-free Biomarkers for Diagnosis and Monitoring of Cancer 2
  * Current Developments in Non-invasive Biomarkers for Assessment of Cancer 2
  * Deep Learning
  * Circulating and Cell-free Biomarkers for Diagnosis and Monitoring of Cancer 3
  * Combination Immunotherapies 1
  * Current Developments in Non-invasive Biomarkers for Assessment of Cancer 3
  * Present and Future of Biomarkers for Anticancer Immunotherapy

ENDOCRINOLOGY

  * Endocrine-Related Cancer Research
  * Endocrine-related Cancers

EPIDEMIOLOGY

  * Biomarkers in Risk and Outcomes
  * Lifestyle Factors and Cancer Risk
  * Genetics and Genomics 1: Outcomes
  * Genetics and Genomics 2: Risk
  * Early Detection and Risk Prediction
  * Exposures and Genetics in Cancer Risk

EXPERIMENTAL AND MOLECULAR THERAPEUTICS

  * Antibody-Drug Conjugates: New Agents and Technologies
  * Brain Cancers
  * Cell Death and DNA Repair Pathways
  * Combination Approaches to Novel Therapies
  * Drug Resistance 1
  * New Anticancer Agents
  * New Molecular Targets
  * Drug Resistance
  * Cancer Immunotherapy
  * Cellular Responses to Anticancer Agents 1: The Microenvironment and Metastasis
  * Credentialing of Molecular Targets
  * Drug Resistance 2
  * Novel Targets and Pathways
  * Tyrosine Kinase and Phosphatase Inhibitors 1
  * Apoptosis, Necrosis, and Cancer Cell Survival
  * Cellular Responses to Anticancer Agents 2 / Overcoming Resistance
  * Drug Resistance 3
  * Molecular Classification of Tumors
  * Novel Screening and Delivery Technologies
  * Novel Therapeutics and Pathways
  * Tyrosine Kinase and Phosphatase Inhibitors 2
  * Novel Therapeutic Agents and Screening Approaches
  * Preclinical Drug Development

IMMUNOLOGY

  * Imaging the Tumor Microenvironment
  * Metabolic Mechanisms That Modulate the Immune Response to Cancer
  * Radiation-induced Immunomodulation
  * Therapeutic Antibodies 1
  * Tumor Antigens and Neoantigens
  * Adoptive Cell Therapy
  * Cancer Vaccines and Intratumoral Immunomodulation
  * Adoptive Cell Therapy 1
  * Cancer Vaccines and Intratumoral Immunomodulation
  * Cellular Interactions in the Tumor Microenvironment
  * Suppressive Myeloid Cells
  * Therapeutic Antibodies 2
  * Adoptive Cell Therapy 2
  * Cancer and Inflammation
  * Molecular Mechanisms in the Immune Response to Cancer
  * Therapeutic Antibodies 3
  * Immune Cell Subsets in the Suppressive Tumor Microenvironment
  * Rational Combinations of Immunotherapy

MOLECULAR AND CELLULAR BIOLOGY / GENETICS

  * Apoptosis
  * Cancer Genomics 1
  * miRNA Regulation of Cancer Biology
  * Mitochondrial Function and Metabolism in Cancer
  * New Trends in DNA Methylation
  * Oncogenic Gene Dysregulation and Carcinogenesis
  * Targeting Oxidative Stress and Senescence
  * Emerging Targets in Cancer Epigenetics
  * Receptor Tyrosine Kinase and GTPase Signaling in Cancer
  * Cancer Genomics 2
  * Deregulated Cell Processes and Carcinogenesis
  * DNA Damage and Repair 1
  * Kinases, GTPases, and Phosphatases
  * miRNA Profiling in Cancer
  * Noncoding RNAs 1
  * Signaling Pathways in Cancer Metabolism
  * BCL-2 Family Proteins
  * Cancer Genomics 3
  * Cell Cycle Biology and Signaling
  * DNA Damage and Repair 2
  * miRNA-based Diagnostics and Therapeutics
  * Oncogenic Transcription Factor Function
  * Receptors and Growth Factors
  * Targeting Hypoxia and Endoplasmic Reticulum Stress
  * Exploiting Metabolic Vulnerabilities of Cancer
  * Structural and Functional Cancer Genomics

PREVENTION RESEARCH

  * Experimental Models and Biomarkers in Cancer Prevention Research
  * Nutrition, Physical Activity, and Cancer Prevention: Current Status and Emerging Strategies
  * Nutrition, Screening, and Early Detection for Cancer Prevention
  * Behavioral Science and Cancer Control
  * Molecular and Cellular Mechanisms in Cancer Prevention Research

TUMOR BIOLOGY

  * 3D Models
  * Cell-cell and Cell Matrix Interactions
  * Developmental Phenotypes and Molecular Imaging
  * Drug Targets in the Microenvironment
  * Gene Expression in the Tumor Microenvironment
  * Genes That Regulate Migration and Invasion
  * Targeting Tumor-Host Interactions
  * Novel Approaches to Understand Cancer Stem Cells
  * The Immune Microenvironment in Cancer
  * Epithelial/Mesenchymal Transition (EMT and MET) 1 / Actin Cytoskeleton
  * Human in Mouse
  * Immune Cells in the Tumor Microenvironment 1
  * Metastasis Promoter and Suppressor Genes
  * Novel Imaging Targets
  * Regulation of Cancer Stem Cell Stemness, Epithelial-Mesenchymal Transition, and the Microenvironment
  * Tumor-Immune Interactions
  * Epithelial/Mesenchymal Transition (EMT and MET) 2 / Premetastatic Niche
  * Extracellular Matrix and Mechanotransduction
  * Models and Drug Discovery
  * Novel Imaging Techniques
  * Secreted Changes in the Tumor Microenvironment: Exosomes and Chemokines
  * Therapeutic Metastasis Prevention
  * Tumor-Stroma Interactions
  * Invasion and Metastasis 1: Cell-Intrinsic
  * Signaling in the Tumor Microenvironment

# Sunday, March 31, 2019

## CANCER CHEMISTRY

### Novel Small Molecules for Cancer Therapy

#1

A small molecule inhibits Nrf2 expression and activity in cancer cells.

Di Zhang, Corbin Livingston, Thomas Dexheimer, Edmund Ellsworth, Aaron Odom, Karen Liby. _Michigan State Univ., East Lansing, MI_.

Activation of the Nrf2-Keap1-ARE pathway is a master defense mechanism protecting against oxidative and electrophilic stress. Nrf2 was traditionally considered cancer-preventing, as Nrf2 deficiency enhances susceptibility to carcinogens in a variety of mouse models. However, increasing evidence suggests a tumor-promoting role for Nrf2 in established tumors: gain of function mutations in NFE2L2, which activates the Nrf2 pathway, or loss of function mutations in its negative regulator, Keap1, have been found most frequently in lung cancer. Constitutive activation of the Nrf2 pathway is associated with chemoresistance and poor survival. Nrf2 inhibitors, therefore, become potential novel drugs for cancer treatment and needed tool compounds to study the Nrf2 pathway in cancer. To identify Nrf2 inhibitors, we conducted a high throughput screen with a diverse set of small molecules (~6500 compounds). 890 compounds were identified as hits, and a funnel strategy was applied to filter through the hits and select lead compounds. Although other known Nrf2 inhibitors were identified, a potentially novel compound, MSU225, was chosen as a lead for further evaluation. MSU225 downregulated tBHQ (tert-butyl hydroquinone)-induced Nrf2 transcriptional activity in a luciferase assay without inducing cell death. When A549 human lung cancer cells, in which Keap1 is mutated inducing constitutive activation of Nrf2, were treated with 5 µM MSU225, the mRNA expression of HMOX1, NQO1, GST1A1, GCLC, GCLM, and UGT1A6, all downstream targets of Nrf2, were decreased. Moreover, MSU225 significantly (p<0.05) decreased the protein level of Nrf2 in A549 cells. The effect of MSU225 on Nrf2 protein levels was blocked by the proteasome inhibitor MG132, suggesting MSU225 enhances Nrf2 degradation through the proteasome system. In addition, MSU225 inhibited the growth of human lung cancer cells (A549, H460, A427) in soft agar. Cells addicted to Nrf2 were more susceptible to MSU225 for suppression of cell proliferation than cells not addicted to Nrf2. MSU225 also sensitized A549 cells to carboplatin treatment. Our results suggest that MSU225 is a novel Nrf2 inhibitor, and additional in vivo studies will be done to determine if it can be used to treat experimental lung cancer.

#2

Discovery of BAY 2402234 by phenotypic screening: A human Dihydroorotate Dehydrogenase (DHODH) inhibitor in clinical trials for the treatment of myeloid malignancies.

Stefan N. Gradl,1 Thomas Mueller,1 Steven Ferrara,2 Sherif El Sheikh,3 Andreas Janzer,1 Han-Jie Zhou,4 Anders Friberg,1 Judith Guenther,1 Martina Schaefer,1 Timo Stellfeld,1 Knut Eis,1 Michael Kroeber,1 Duy Nguyen,1 Claudia Merz,1 Michael Niehues,1 Detlef Stoeckigt,1 Sven Christian,1 Katja Zimmermann,1 Pascal Lejeune,1 Michael Bruening,1 Hanna Meyer,1 Vera Puetter,1 David T. Scadden,5 David B. Sykes,5 Henrik Seidel,1 Ashley Eheim,1 Martin Michels,1 Andrea Haegebarth,1 Marcus Bauser1. 1 _Bayer AG, Pharmaceuticals Division, Berlin, Germany;_ 2 _Broad Institute of MIT and Harvard, Boston, MA;_ 3 _Technical University Cologne, Cologne, Germany;_ 4 _Essa Pharmaceuticals, San Francisco, CA;_ 5 _Massachusetts General Hospital, Boston, MA_.

DHODH is a key enzyme in the biosynthesis of pyrimidines and recent studies have renewed interest in this old anti-cancer target. Here, we disclose the discovery of 4-triazolosalicylamides as inhibitors of DHODH and their structure activity relationship (SAR). The hit cluster was discovered during a phenotypic high throughput screen (HTS) of 2.5 million compounds where proliferation of H460 lung cancer cells was used as read-out. DHODH was successfully identified as the molecular target by comparing the activity profile of the hits in a panel of cell lines to a set of inhibitors with known pharmacological activity. The hit compounds showed good cellular potency but had undesirable DMPK properties. Interestingly, the compounds are non-ionizable in contrast to many other DHODH inhibitors and show no potency shift from biochemical to cellular assays. Structural modifications lead to compounds with sub-nanomolar potency in cellular assays and increased metabolic stability enabling the proof of concept in vivo xenograft experiments. Further optimization guided by lipophilicity efficiency and identification of metabolic hot spots resulted in molecules with low clearance and improved solubility. BAY 2402234 was selected as the clinical candidate after side by side comparison of a number of promising compounds. It shows great oral bioavailability, target engagement in all preclinical species tested, induces differentiation in AML models, and has excellent activity in a variety of leukemia models. A clinical phase I study has been initiated in patients with myeloid malignancies. (NCT03404726)

#3

Discovery and optimization of small molecule Bax activators for cancer therapy.

Gang Liu,1 Hyejin Kim,2 Chunyong Ding,1 Zhuo Yu,2 Hong Wang,2 Haiying Chen,1 Qiang Shen,2 Jia Zhou1. 1 _UT Medical Branch, Galveston, TX;_ 2 _UT M. D. Anderson Cancer Center, TX_.

Bax, an indispensable executioner protein of B-cell lymphoma 2 (Bcl-2) family, plays a pivotal role in controlling the mitochondrial dysfunction and apoptosis in normal and cancer cells. High throughput screening to combat the nicotine-induced Bax phosphorylation at serine 184 (S184) to activate the proapoptotic function of Bax has revealed compound SMBA1 as a promising Bax direct activator as we previously reported. Initial medicinal chemistry campaign by choosing SMBA1 as a lead compound for breast cancer treatment resulted in the identification of compound CYD-2-11 with low micromolar IC50 values against MDA-MB-231 and MCF-7 as well as in vivo inhibitory effects against MDA-MB-231 xenograft tumor growth. Herein, we report the comprehensive structural optimization based on CYD-2-11 as the lead by introducing various alkylamino side chains to have a deeper access to S184 pocket and replacing carbon atoms with nitrogen as well as reducing the nitro group of 9H-fluorene scaffold to construct diverse derivatives for enhanced drug properties. CYD-4-61 and GL0385 have been discovered as the most potent anti-breast cancer agents with submicromolar IC50 values of 0.34 and 0.33 µM against MDA-MB-231 as well as 0.65 and 0.26 µM against MCF-7, respectively. More importantly, these drug candidates exhibited significant in vivo efficacy suppressing xenograft tumor growth by activating Bax to induce cancer cell apoptosis with the potential to overcome resistance against Adriamycin (ADR) in breast cancer. This work was supported by Breast Cancer Research Program (BCRP) Breakthrough Award BC160038 (to J.Z.) and BC160038P1 (to Q.S.) from the Department of Defense (DoD).

#4

Discovery of novel and highly potent small molecule inhibitors of chemokine receptor CXCR4.

Xiong Fang,1 Qian Meng,1 Xiao Fang,1 Siyu Zhu,1 Yazi Huang,1 Jing An,2 Yan Xu,1 Ziwei Huang1. 1 _Tsinghua University, Beijing, China;_ 2 _University of California at San Diego, San Diego, CA_.

Introduction: CXCR4 is a member of chemokine receptor and G protein coupled receptor (GPCR) families. Its interaction with the chemotactic ligand stromal cell-derived factor 1 (SDF-1 or CXCL12) plays important roles in physiological and pathological processes. Recent studies have showed that the blockade of CXCL12-CXCR4 interaction by small molecules can be used in several clinical applications, including sensitizing cancer cells to chemotherapy, mobilizing hematopoietic stem cells (HSCs) to the blood for HSC collection, and inhibiting the tumor metastasis. To date, AMD3100 (Plerixafor) is the only clinically approved drug used in combination with G-CSF for mobilizing HSCs. Here, we report the discovery and development of a new class of highly potent small molecule CXCR4 inhibitors which can strongly antagonize CXCL12-CXCR4 interaction and mobilizing HSCs. Methods: Based on the crystal structures of CXCR4 showing a major and minor subpockets, we applied a fragment-based combinatorial design (FBCD) strategy to design a new class of hybrid molecules to recognize these two subpockets. A series of designed compounds were chemically synthesized and tested in a panel of biological assays including competitive CXCR4 binding, chemotaxis, calcium mobilization and CXCR4 internalization to characterize their biological activities. Furthermore, site-directed mutagenesis coupled with molecular docking simulation was carried out to determine the binding modes of the most potent compounds. Results: More than 30 compounds were synthesized and characterized. Among them, compound HFX51116 showed the most potent CXCR4 binding activity (IC50 = 12.2 nM), which was more potent than AMD3100 tested in parallel (IC50 = 325.3 nM). Furthermore, HFX51116 displayed remarkable inhibition of calcium mobilization (IC50 = 1.478 nM) and chemotaxis activity (IC50 = 252.7 nM), both of which are better than those of AMD3100. In addition, mutational studies and molecular docking simulation suggested that HFX51116 recognizes the minor subpocket mainly and major subpocket partially. Conclusions: Our findings have demonstrated that HFX51116 is a promising novel lead compound for developing a new class of small molecule therapeutics targeting CXCR4.

#5

Pre-clinical development of CDK9 inhibitor - LS007 for the treatment of acute myeloid leukemia.

Wang Hui,1 Wang Shudong2. 1 _Changzhou Lesun Pharmaceuticals Ltd, ChangZhou, China;_ 2 _University of South Australia, Adelaide, Australia_.

Acute myeloid leukemia (AML) is the most common form of acute leukaemia with an increasing incidence and dismal long-term prognosis with age. The most aggressive subtype, MLL-AML, is characterized by translocations of the mixed-lineage leukaemia gene (MLL) in chromosome band 11q23 and resistance to conventional chemotherapy with a short overall survival of only 9 months. Cyclin-dependent 9 (CDK9) activity is required for MLL-driven oncogenic transcription. Inhibition of CDK9 directly targets the transcriptional machinery required for MLL-fusion proteins (e.g. MLL-AF4, MLL-AF9 and MLL-ENL), thereby leading to apoptosis.

We have developed a highly effective drug candidate - LS007 that inhibited CDK9 with a Ki = 4 nM. It suppressed proliferation and induced apoptosis in a range of AML cell lines and patient samples. LS007 was highly efficacious against several AML cell line xenografts and patient-derived xenografts, and well tolerated in mice after oral administration. In a Molm-13 xenograft model, after 21 days of orally daily dosing at 25 mg/kg and 50 mg/kg LS007

inhibited tumor growth (p < 0.001) with T/C = 44 % and 18 %, respectively. Similarly, in a HL-60 xenograft model, LS007 exhibited a significant tumor growth inhibition at 45 mg/kg and 60 mg/kg daily by oral administration with T/C = 40 % and 20 %, respectively.

The pharmacokinetic profiles of LS007 was further assessed in the cynomolgus monkeys. After oral doses at the dose of 2, 4 and 8 mg/kg, LS007 was detected in the plasma with the maximum concentration (Cmax) from 55.9 to 279.5 ng/mL and the area under the plasma drug concentration-time graph (AUC) up to 1862.7 h·ng/mL. The mean terminal half-life (T 1/2) was around 3 h. LS007 possessed the favorable pharmacokinetic profiles with oral bioavailability F = 56% in the cynomolgus monkey (at dose 4 mg/kg), suggesting its therapeutic potential as an orally bioavailable anti-AML agent. The GLP- toxicological studies of LS007 in the cynomolgus monkey were completed, and the maximum tolerated doses were identified, which were used to estimate the safe starting dose for the Phase I clinical trials.

With its excellent anti-cancer efficacy along with appreciable absorption and safety profiles, LS007 offers a very exciting prospect as a clinical development candidate. The application of Investigational New Drug (IND) has been approved allowing the commencement of clinical trials of LS007 in patients with AML.

#6

Computationally assisted target screening of STING agonist for immunologic therapy.

Grace A. Binder, Christine S. Gambino, Anna Kharitonova, Rainer S. Metcalf, Kenyon G. Daniel, Wayne C. Guida. _University of South Florida, Tampa, FL_.

Stimulator of interferon genes (STING) is a receptor protein involved in the propagation of innate immune sensing of cytosolic DNA through the production of IFN-β. Mechanistic studies have shown IFN-β production within the tumor microenvironment can result in activation of tumor antigen-specific CD8+ T-cell immunity that can lead to tumor regression [1, 2]. STING activation by STING agonists should result in innate T-cell mediated anti-tumor immunity in the tumor microenvironment and have significant potential as a cancer therapeutic. Conversely, inhibition of STING would lead to a decreased production of IFN-β which could have implications in the treatment of autoimmune disease such as lupus erythematosus.

MD equilibrated crystal structures for human HAQ, REF, and WT alleles were clustered to find optimal conformations for diverse chemical library screening. Novel consensus docking protocols utilizing rigid receptor, induced fit, and quantum polarized ligand docking were applied for quantifying and refining proposed binding mechanisms of STING isoforms. Models for STING agonists and antagonists were developed.

From directed virtual screening, a novel low-molecular-weight organic molecule (GF3-002) that is not based on a cyclic dinucleotide was found as a potential STING activator and is currently under investigation.

GF3-002 and analogs were synthesized and structures were confirmed with LCMS and proton NMR. Both the HAQ and WT alleles, representing 78.3% of the human population were tested against compounds and controls. 2,3-cGAMP and DMSO were used as positive and negative controls, respectively. Microscale thermophoresis and SPR confirmed binding of GF3-002 to WT STING CTD with a kD of 3.2 ± 1.7 μM. IFN-dependent luciferase expression was measured by luminescence in THP-1 monocytic leukemia cells and found an EC50 of 29 ± 1.6 μM, compared to the native ligand 2,3-cGAMP EC50 of 42 ± 4.1 μM. Finally, qPCR was used quantify production of IFN-β via treatment of dendritic cells with GF3-002.

1. Sali, Characterization of a Novel Human-Specific STING Agonist that Elicits Antiviral Activity Against Emerging Alphaviruses. 2017.

2. Corrales, L., et al., Direct activation of STING in the tumor microenvironment leads to potent and systemic tumor regression and immunity. Cell reports, 2015. 11(7): p. 1018-1030.

#7

Discovery of OICR-10268: A potent and selective BCL6 inhibitor.

Iain D. Watson,1 Methvin Isaac,1 Brian Wilson,1 Anh Chau,1 Justin Morin,1 Pandiaraju Subramanian,1 Ahmed Mamai,1 Babu Joseph,1 Michael Prakesch,1 David Uehling,1 Ayome Abibi,1 Richard Marcellus,1 Craig Strathdee,1 Ratheesh Subramaniam,1 Brigitte Theriault,1 Jeffrey Winston,1 Manuel Chan,1 Carly Griffin,1 Herman Cheung,1 Taira Kiyota,1 Elijus Undzys,1 Ahmed Aman,1 Gennady Poda,1 Doug Kuntz,2 Neil C. Pomroy,2 Gil G. Privé,2 Rima Al-awar1. 1 _Ontario Inst. for Cancer Research, Toronto, Ontario, Canada;_ 2 _University of Toronto, Toronto, Ontario, Canada_.

The transcription factor B cell lymphoma 6 (BCL6) is required for the generation of an effective humoral immune response through the development and maintenance of germinal centers (GCs). The inhibition of the protein−protein interaction between BCL6 and its corepressors has been implicated as a therapeutic target for diffuse large B-cell lymphoma (DLBCL), a type of non-Hodgkin's lymphoma (NHL). Using structure-based drug design, we initiated a program to identify novel BCL6 inhibitors. We identified a high micromolar virtual screening hit which was then optimized for potent biophysical binding and anti-proliferative cellular activity resulting in the identification of OICR-10268, a potent and selective Bcl6 inhibitor.

#8

Targeting DNA-damage response pathway with a novel nano-liposomal ATR inhibitor in solid tumors.

Alexander Koshkaryev, Ozan Alkan, Bolin Geng, Lia Luus, Andreas Raue, Walid Kamoun, Suresh Tipparaju, Dmitri Kirpotin, Daryl Drummond. _Merrimack Pharmaceuticals, Cambridge, MA_.

Introduction.

Ataxia telangiectasia and Rad3-related (ATR) protein kinases have been identified as a key part of the DNA damage repair processes (DDRP) and cell cycle signaling. The DDRP is known to stimulate DNA repair, promote cell survival and, consequently, can diminish the therapeutic effect of existing DNA-damaging chemotherapy agents and ionizing radiation (IR) therapy. Therefore, there is a need for the development of potent and selective therapies to deliver ATR inhibitors for the treatment of cancer, as part of rational combination regimens with DNA damaging therapies. In this work, we develop and characterize a novel inhibitor of ATR kinase (BG129) and its nano-liposomal formulation (nLs-BG129).

Methods. In vitro activity of novel ATR inhibitors (BG129) was tested in a broad panel of cancer cell lines as a monotherapy or in combination with DNA-damaging chemotherapeutics (carboplatin, gemcitabine, and SN-38). On-target effects of BG129 in vitro were evaluated by Western-Blot to demonstrate dose-dependent inhibition of pChk1 and pRPA2, and a corresponding increase in ɣH2AX. The ATR inhibitor was encapsulated into unilamellar liposomal vesicles formed from hydrogenated soy phosphatidylcholine (HSPC), cholesterol, and methoxy-poly(ethylene glycol)-1,2-distearoyl-sn-glyceryl (PEG2000-DSG) at a 3:2:0.15 molar ratio and loaded stably into liposomes employing a triethylammonium sucroseoctasulfategradient. Liposomes were characterized by size and drug loading efficiency. Pharmacokinetic (PK) properties of nLs-BG129 were evaluated in mice. The antitumor activity of nLs- BG129 in combination with nano-liposomal irinotecan (nal-IRI) was tested in vivo in ovarian and various lung cancer xenograft models.

Results.

BG129 synergized with SN-38, gemcitabine, and carboplatin in vitro in cell-based assays. On-target dose-dependent inhibition of pChk1 and pRPA2 and a corresponding increase in ɣH2AX were observed in cells, following treatment with BG129. Nano-liposomal BG129 showed dramatically improved pharmacokinetics (AUC = 2835 μg/ml*h), compared to either free BG129 or VE-822/M6620/Berzosertib (AUC = 18.18 μg/ml*h). nLs-BG129 also enhanced antitumor activity of nal-IRI in multiple ovarian and SCLC and NSCLC xenograft models, when given in combination and was well tolerated.

Conclusions. Here we introduced a novel nano-liposomal formulation of an ATRi with good in vivo stability and prolonged clearance, with the ability to enhance the anti-tumor activity of DNA-damaging chemotherapeutics in vivo. The nano-liposomal formulation of ATRi can potentially sensitize tumor responses to DNA-damaging agents and improve their activity and tolerability in cancer patients when compared to unencapsulated and fast clearing ATR inhibitors, such as those currently in the clinic.

#9

Effect of C8-side chain on the cytotoxicity and NF-kB inhibitory capacity of pyrrolobenzodiazepines.

David B. Corcoran,1 Thomas Lewis,2 Kazi S. Nahar,1 Christopher Fegan,2 Chris Pepper,3 David E. Thurston,1 Khondaker Miraz Rahman1. 1 _King's College London, London, United Kingdom;_ 2 _University of Cardiff, Cardiff, United Kingdom;_ 3 _University of Sussex, Brighton, United Kingdom_.

Pyrrolobenzodiazepines (PBDs) have long been of interest in chemotherapeutic research as potential clinical agents. Recently, the conjugation of non-covalently interactive moieties to the C8-position of the PBD structure via a linker of variable length has emerged as a strategy to enhance the DNA sequence specificity of PBDs. This has led to the development of highly cytotoxic agents, which also act as inhibitors of transcription factors such as NF-κB. For example, GWL-78, a C8-linked PBD-Py-Py (Py = pyrrole) conjugate, has been shown to block interaction of the transcription factor NF-Y, and KMR-28-39, a C8-linked PBD-MPB conjugate with an affinity for GC-rich sequences of DNA inhibits activity of the transcription factor NF-κB. As up-regulation of NF-κB has been implicated in the development and progression of several cancer types, this may contribute to the high potency observed for PBD monomers such as KMR-28-39 (i.e., femtomolar IC50 values in some cell lines). In this study, the relationship between the cytotoxicity and transcription factor inhibition of a series of C8-linked PBD hybrid structures was explored. The systematic shortening of the non-covalent element of a C8-linked PBD conjugate, DC-1-92 led to the synthesis of a 19-member library of novel C8-PBD monomers. The critical elements of DC-1-92, which were required to render the molecule cytotoxic, were elucidated by an annexin V assay. The effects of shortening the non-covalent element of the molecule on transcription factor inhibitory capacity were also explored through an ELISA-based measurement of nuclear NF-κB subunit DNA binding following exposure of JJN3 cells to the synthesised molecules. While shortening the non-covalently interactive component of DC-1-92 had less of an effect than expected upon cytotoxicity, the shortening notably reduced the NF-κB inhibitory activity of the shortened analogues. DC-1-92, containing the longest non-covalently interactive C8-element, displayed the most effective inhibition of NF-κB, and effects upon nuclear p65, and RelB levels appeared to diminish in tandem with the shortening of the length of the C8 side chain. These data suggest that the length of the C8 side chain of PBD hybrid structures impacts upon the DNA binding of both canonical and non-canonical NF-κB subunits but this is independent of their cytotoxic effects.

#10

Novel derivatives of anaplastic lymphoma kinase inhibitors: Synthesis, radiolabeling and preliminary biological studies on crizotinib and alectinib analogues.

Mian M. Alauddin, Bhasker Radaram, Yi Rao, Ping Yang, David Piwnica-Worms. _UT MD Anderson Cancer Center., Houston, TX_.

Anaplastic lymphoma kinase (ALK), an oncogenic receptor tyrosine kinase, has emerged as a therapeutic target in various cancers, including lung cancer. Although several ALK inhibitors have gained regulatory approval, some of them do not cross the blood-brain barrier;1 furthermore, non-invasive indicators of target engagement or biomarkers predicting response to these agents in vivo are lacking. To overcome these limitations, fluorinated analogues of the ALK inhibitors crizotinib and alectinib (fluoroethyl crizotinib [FECr] and fluoroethyl alectinib [FEAl]) were synthesised and radiolabeled. We report the radiosynthesis and preliminary biological results on these compounds, fluoroethyl crizotinib (19F/18F-FECr) and fluoroethyl alectinib (19F/18F-FEAl). Briefly, 19F/18F-FECr and 19F/18F-FEAl were synthesized as previously reported.2 19F/18F-fluoroethyl tosylate was prepared, purified and coupled with crizotinib or alectinib. The products were purified by flash column chromatography to yield 19F/18F-FECr and 19F/18F-FEAl. Alternatively, precursor compounds were synthesized, directly fluorinated with 19F/18F-fluoride, and purified to yield 19F/18F-FECr and 19F/18F-FEAl. In vitro cytotoxicity assays were performed in lung cancer ALK-positive H2228 and ALK-negative H441 cells using non-radioactive compounds. In vivo biodistribution and PET/CT imaging studies were performed on tumor-free nude mice using 18F-labeled compounds. Chemically, the first method produced 18F-FECr in 20-24% yield (n=8), molar activity of 37 GBq/μmol, and >99% purity. The second method produced 18F-FECr and 18F-FEAl in 40% (n=6) and 20% yields, respectively. In in vitro cytotoxicity assays with ALK-positive H2228 cells, FECr showed an IC50 = 7.5 μM and FEAl an IC50 = 40 nM, similar to their cognate parental compound, confirming that the modification was not detrimental to drug potency. Positron emission tomographic (PET) imaging in tumor-free mice showed significant brain uptake of the 18F-labeled analogues at 10 min post-injection (6.5 %/ID/g, 18F-FECr; 8.2 %ID/g, 18F-FEAl). In conclusion, novel fluoroethyl-modified crizotinib and alectinib and their 18F-labeled analogues were synthesized with good yields, high purity, and high specific activity. PET and biodistribution results suggest that these fluorinated analogues may penetrate the blood-brain barrier and be potential targeted drugs for treatment of lung cancer that has metastasised to brain. References: 1. Kodama T, et al. Cancer Chemother Pharmacol 2014, 74, 1023-1028. 2. Perera et al. J. Labelled Com Radiopharm. 2016, 59 103-108.

#11

Novel dual inhibitors of LSD1-HDAC6/8 for treatment of cancer.

Dhanalakshmi Sivanandhan, Sridharan Rajagopal, Sreekala Nair, Chandru Gajendran, Dimpy Ghosh, P Nagaraj, Subramanyam J. Tantry, Purushottam Dewang, Mahanandeesha S. Hallur, Kannan Murugan, Srinatha K. C, Damodara Kuntrapaku, M Dilipkumar, R Sharma, S Meghashree, Durga Prasanna Kumar, Suraj P. Ingle, Mohd Zainuddin, A B. Vinod, Sriram Rajagopal. _Jubilant Biosys, Bangalore, India_.

Introduction: Lysine Specific Demethylase 1 (LSD1) is a flavin adenine dinucleotide (FAD)-dependent amine oxidase that has been reported to be over-expressed in many malignant tumors. Down-regulation of LSD1 has been shown to effectively treat cancers by inducing re-expression of aberrantly silenced genes. Studies have shown that LSD1 may contribute to acute myelogenous leukemia pathogenesis by inhibiting the normal pro-differentiative function of ATRA, paving the way for new combinatorial therapies for AML. Similarly, HDAC isoform selective inhibitors are beginning to be explored as less toxic alternatives to panHDAC inhibitors in select cancers. Further, combined inhibition of LSD1 and HDAC has been shown to be more efficacious in inhibiting multiple cancers. Here, we show that JBI-295, a dual inhibitor targeting both LSD1 and HDAC6/8 shows stronger efficacy without enhancing systemic toxicity, in a subset of AML and JAK-dependent myeloproliferative cancer.

Methods: Computational chemistry approaches were used to design LSD1 specific and LSD1-HDAC dual inhibitors. To assess in vitro LSD1 potency, TR-FRET assay was used. For assessing in vitro HDAC activity fluorescence based HDAC6 activity assay was performed. Western blotting was used to assess biomarkers of LSD1 and HDAC inhibition. Alamar blue cytotoxicity assay was used to assess cell proliferation.

Results: Several compounds from this series show strong in vitro potency against LSD1 with more than excellent selectivity against MAOs. JBI-295 one of the lead dual molecules, showed strong LSD1 potency (IC50 of 0.07 μM) and isoform selective HDAC6/8 activity (IC50 of 0.006 and 0.08 µM on HDAC6 and HDAC8, respectively), with about 100 fold selectivity against other HDAC isoforms. JBI-295 showed strong anti-proliferative activity on leukaemia and multiple myeloma cell lines. In cell based and in vivo target engagement studies there was a concomitant increase in CD11b, CD86 and GFI1b and tubulin acetylation levels. JBI-295 was more efficacious in inhibiting the growth of leukemia HEL92.1.7 xenograft by oral administration when compared to IP administration of ACY-1215, a HDAC6 selective inhibitor. Stronger tumor growth inhibition was also observed in melanoma A375 xenograft model as compared to inhibitors with single activity. In addition, JBI-295 showed single agent activity in a syngeneic murine colon cancer model CT26 and also resulted in stronger tumor growth inhibition when combined with anti-PDL1 antibody.

Conclusion: The data obtained demonstrate that dual LSD1-HDAC6/8 inhibitors could serve as novel, effective therapeutic agents for treatment of select subset of cancer. Advanced efficacy and toxicology studies with JBI-295 are in progress.

#12

A novel Hsp90 machine inhibitor combining chemotherapy and immunotherapy.

Ahmed Chadli,1 Nada Eisa,1 Vincent Crowley,2 Sumin Lu,1 Yasmeen Jilani,1 Hasan Korkaya,1 Brian Blagg3. 1 _Augusta Univ. Cancer Ctr., Augusta, GA;_ 2 _The University of Kansas, Lawrence, KS;_ 3 _University of Notre Dame, Notre Dame, IN_.

Molecular chaperones are guardians of the proteome and of cellular homeostasis. Dysregulation in the function of the Hsp90 chaperoning machine in particular has been implicated in metabolic, oncological, neurodegenerative, and cardiovascular diseases. Targeting Hsp90 has been shown to have a combinatorial impact on dysfunctional circuitries that underlie human cancers. Thus, several inhibitors targeting the N-terminal ATP-binding pocket of Hsp90 are undergoing clinical trials as anticancer agents. They inactivate the ATPase activity of the chaperone, causing proteasomal degradation of its "client" oncogenic proteins. Although these inhibitors provided a proof-of-principal that Hsp90 is a valuable therapeutic target, they exhibit modest activity in the clinic, in part because they concomitantly induce a heat shock response (HSR), which turns on pro-survival pathways. Consequently, new mechanisms to inhibit the Hsp90 chaperone machine that do not induce the heat shock response are needed.

To address this, we have developed a unique high throughput screen (HTS) based on the progesterone receptor (PR) and the five purified chaperones required for folding steroid receptors, i.e., Hsp90, Hsp70, Hsp40, Hsp70/Hsp90 organizing protein (Hop), and p23. During our preliminary screening of natural products, we discovered that the compound AD07 kills cancer cells through inhibition of the Hsp90 machine without a significant induction of HSR. In addition, AD07 induces a powerful T cell-mediated immune response, resulting in highly efficient tumor killing in a syngeneic mouse model and long-term memory against the primary tumor. At the molecular level, AD07 reduces the protein levels of two key mediators of tumor-induced immune tolerance: programmed cell death ligand-1 (PDL-1) and indolamine 2,3 dioxygenase (IDO). We therefore propose that AD07 is a promising anti-tumor agent targeting the Hsp90 machine through a novel mechanism of action involving cancer cell toxicity, which increases its immunogenicity, and modulation of the tumor microenvironment to reduce immunotolerance.

#13

Structure elucidation, metabolism, and drug interaction potential of ACP-5862, an active, major, circulating metabolite of acalabrutinib.

Terry Podoll,1 Paul G. Pearson,2 Jerry Evarts,3 Tim Ingallinera,3 Hao Sun,4 Stephen Byard,5 Adrian J. Fretland,6 J. Greg Slatter3. 1 _IV-PO, LLC, Seattle, WA;_ 2 _Pearson Pharma Partners, Westlake Village, WA;_ 3 _Acerta Pharma, a member of the AstraZeneca group, South San Francisco, CA;_ 4 _Covance Laboratories, Madison, WI;_ 5 _Arcinova, Alnwick, United Kingdom;_ 6 _Oncology DMPK, IMED Biotech Unit, AstraZeneca, Boston, MA_.

Acalabrutinib (Calquence®) is a potent, selective, orally administered, covalent inhibitor of Bruton tyrosine kinase (BTK) that received accelerated approval for relapsed/refractory mantle cell lymphoma from the US FDA in October 2017. Profiling of acalabrutinib metabolites in human plasma revealed a late-eluting, +16 Da metabolite circulating at concentrations higher than parent drug. Metabolite regiochemistry could not be determined by mass spectrometry. In vitro metabolism and preparative HPLC was used to generate a pure sample of the metabolite for structural characterization by NMR. Confirmatory chemical synthesis revealed a pyrrolidine ring-opened ketone. The structure of the metabolite, designated ACP-5862, and a smaller -2 Da peak, identified as dehydropyrrolidine, M25, inferred a common carbinolamide intermediate in their genesis. Both metabolites retained the butynamide electrophile responsible for the inactivation of BTK. In vitro studies on the inhibition of BTK and related Tec and Src kinases revealed that ACP-5862 was active against BTK with similar selectivity and potency to acalabrutinib (Kaptein et al, 2019) This work then investigated the in vitro metabolism and drug transport features of acalabrutinib, and the metabolite ACP-5862, to establish the potential for clinical drug-drug interactions (DDI) via CYPs, UGTs and drug transporters. CYP reaction phenotyping indicated CYP3A4 was responsible for both the formation and further metabolism of ACP-5862. Km and Vmax values for the formation of ACP-5862 using rCYP3A4 were 2.78 μM and 4.13 pmol/pmol CYP/min, respectively. The in vitro intrinsic clearance of ACP-5862 was 23.6 μL/min/mg. Acalabrutinib weakly inhibited CYP2C8, CYP2C9 and CYP3A4 in vitro, and ACP-5862 weakly inhibited CYP2C9 and CYP2C19, with no inhibition of CYP1A2, CYP2B6, or CYP2D6. Similarly, UGT1A1, UGT2B7, and aldehyde oxidase were not inhibited. Neither parent or ACP-5862 strongly induced CYP1A2, CYP2B6, or CYP3A4 mRNA. Acalabrutinib and ACP-5862 were substrates of MDR1 and BCRP in vitro, but were not substrates of OATP1B1 or OATP1B3. Acalabrutinib was not a substrate of OAT1, OAT3, and OCT2. Based on static PK model calculations, acalabrutinib may cause a modest increase in exposure to coadministered BCRP substrates by inhibition of intestinal BCRP, but with no inhibition of BCRP at the systemic level. The PK of substrates of MDR1, MATE1, MATE2-K, OATP1B1, OATP1B3, OAT1, OAT3, and OCT2 are not likely to be altered by acalabrutinib or ACP-5862. These data were combined with clinical DDI data (Izumi et al, 2017) to simulate DDI in the presence of CYP3A inhibitors and inducers. PBPK models confirmed that acalabrutinib and ACP-5862 were not likely to perpetrate CYP2C8 or CYP3A4 mediated drug interactions (Zhou et al., 2019). Overall, acalabrutinib and major metabolite, ACP-5862 have a favorable drug interaction profile.

#14

A new orally available drug for the treatment of metastatic cancers.

Kun Zhou,1 Jae Eun Cheong,2 Michaela Zaffagni,1 Yingjie Xu,1 Lijun Sun,2 Bruce Zetter1. 1 _Boston Children's Hospital, Boston, MA;_ 2 _Beth Israel Deaconess Medical Center, Boston, MA_.

Metastatic cancers account for the great majority of cancer deaths. We conducted a screen to indetify known drugs that selectively inhibit the viability of metastatic tumor cells. Results of the screen identified members of the FDA-approved benzimidazole methylcarbamate family (e.g. mebendazole (MBZ) and albendazole (ALB)) as potential therapies for metastatic disease. Earlier work also supports a role for this chemical family in the potential treatment of activity in multiple cancers, but progress has been stalled by their poor water solubility and poor bioavailability for systemic delivery to disseminated tumors. We therefore synthesized a novel compound (OBD9) containing the scaffold of MBZ coupled to an oxetane group to enhance aqueous solubility to 361 μM. OBD9 demonstrates significant cytotoxicity toward a variety of cancer cell types including colon, lung, and prostate cancers (IC50: 0.9-2 μM). In a mouse xenograft model of human A549 lung cancer cell line OBD9 dramatically inhibited the growth of established tumors at 30 or 90 μM without noticeable toxicity. In a mouse xenograft model using highly aggressive PCMLN3 prostate cancer cells, oral administration of OBD9 at 30 mg/kg also significantly repressed growth of established tumors with no visible toxicity.

We have studied the working mechanisms behind OBD9 and found that the Wnt signaling pathway is suppressed by OBD9 treatment. Both qPCR and Western blot data suggest that the beta-catenin/TCF complex in the Wnt signaling pathway are inhibited by OBD9. Among the downstream targets of Wnt signaling pathway, the oncogene cMyc is also significantly decreased in different cancers including colon, lung and prostate cancer. Overall, our in vitro and in vivo data suggest that OBD9 potentially represents a novel therapeutic option for multiple cancers especially metastatic cancer through targeting Wnt signaling pathway and the downstream oncogene cMyc.

#15

Targeting vismodegib-resistant medulloblastoma using novel hedgehog pathway inhibitor and miR-29b mimic.

Virender Kumar, Bharti Sethi, Vinod Kumar, Timothy R. Mcguire, Don W. Coulter, Ram I. Mahato. _University of Nebraska Medical Center, Omaha, NE_.

The purpose of this study to develop targeted liposomes of MDB5 and miR-29b combination for medulloblastoma (MB) treatment. Due to high heterogeneity and diverse genetic make-up, MB treatment is a challenge. Aberrant activation of hedgehog (Hh) pathway regulates cell growth, cancer stem cell (CSC) proliferation, and tumorigenicity in sonic Hh subgroup of MB. Further, Hh signaling regulates yes-associated protein 1 (YAP1) and glutaminolysis for energy need of tumor cells. GDC-0449 a smoothened receptors (SMO) antagonist treats (SHH)-MB initially, but resistance develops after repeated administration. Cholesterol potentiates Hh pathway independent of SHH by binding directly to the SMO. On the other hand, our RNA-seq and miRNA profiling confirmed downregulation of tumor suppressor miR-29b-3p in human MB samples. miR-29b control genes of apoptosis; p53, MDM2, AKT2, and cholesterol biosynthesis including HMGCR and SREBP1. We recently synthesized a potent GDC-0449 analog MDB5 (SMO inhibitors), which binds strongly to various Smo-mutants compared to GDC-0449. We found that a combination of MDB5 and miR-29b can treat MB synergistically. When we modified miR-29b backbone with 2-O'-methoxyethyl phosphorothioate (OMe-PS-miR-29b-3p), it showed enhanced stability in 50% serum. We designed a cationic lipid MDC2 for liposomal delivery of MDB5 and miR-29b1 simultaneously. Liposomes were prepared using film hydration technique and characterized for particle size and zeta potential, drug loading and release profile, miRNA complexation, stability, and transfection efficiency. These liposomes incorporated 88% MDB5 when the drug to phospholipid molar ratio was kept at 5%. Complete complexation was observed at N/P ratio of 3:1. These liposomes when loaded with green fluorescent protein (GFP) siRNA, resulted in 70% of signal silencing into DAOY MB cells (Shh driven). Liposomal MDB5 had higher cytotoxicity in DAOY cells compared to GDC-0449. Further, MDB5 and OMe-PS-miR-29b loaded liposomes resulted in higher, cytotoxicity and % of apoptosis, G0-G1 phase arrest, and decreased target gene expression in DAOY cells. Combination of drugs to simultaneously target multiple pathways related to Hh, glutaminolysis, cholesterol biosynthesis, and apoptosis has the potential to treat MB. Successful completion of this study will provide a nanomedicine for treating SHH-MB and other brain tumors.

#16

3,3'-diindolylmethane induces cytoglobin expression and synergizes with poly ADP ribose polymerase inhibitor PJ34 to inhibit triple negative breast cancer cell growth.

Jonathan V. Wooten,1 Nicole Mavingire,1 Leah Rowland,1 Jason Matthews,2 Eileen Brantley1. 1 _Loma Linda University, Loma Linda, CA;_ 2 _University of Oslo, Oslo, Norway_.

Triple negative breast cancer (TNBC), characterized by tumors that lack expression of the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), carries a poor prognosis. While recently approved poly ADP ribose polymerase (PARP) inhibitor olaparib shows favorable activity in patients with TNBC harboring tumors deficient in DNA repair enzyme BRCA1, patients with TNBC possessing BRCA1-proficient tumors are largely unresponsive to olaparib. Emerging evidence suggests that small molecules that activate aryl hydrocarbon receptor (AhR) signaling have the capacity to confer anticancer actions. 3,3′-diindolylmethane (DIM), an AhR ligand, is the major metabolite of indole-3-carbinol (I3C) found in cruciferous vegetables such as broccoli. Patients often consume natural product-derived AhR ligands while undergoing chemotherapy. It is therefore crucial to enhance our understanding of nutraceutical-pharmaceutical interactions. We previously demonstrated the ability of synthetic AhR ligands to induce the expression of putative tumor suppressor cytoglobin (CYGB) and inhibit Akt signaling in breast cancer cells. Since PARP inhibitor PJ34 has been shown to synergize with Akt inhibitors we hypothesize that DIM induces CYGB expression and synergizes with PJ34 to inhibit TNBC cell growth. Using the Alamar Blue assay, we found that DIM substantially enhanced the sensitivity of TNBC BRCA1 proficient cells to PJ34, though knockdown of CYGB did not significantly influence the responsiveness of cells to DIM. Chromatin immunoprecipitation followed by next-generation sequencing data revealed that DIM promoted the binding of AhR to the CYGB promoter in breast cancer cells. Finally, DIM induced CYGB gene expression in TNBC cells as determined by quantitative PCR. Taken together, our data suggest that DIM upregulates CYGB in TNBC cells and synergizes with PJ34 to confer anticancer actions in TNBC. Our data provide a rationale for incorporating the use of natural product-derived AhR ligands as a strategy to enhance PARP inhibitor efficacy in patients with TNBC that have BRCA1-proficient tumors.

#17

Novel STAT3 inhibitors identified by Structure-Based Virtual Ligand Screening incorporating SH2 domain flexibility.

Ren Kong,1 Uddalak Bharadwaj,2 Thomas Kris Eckols,2 Moses Kasembeli,2 Mikhail Kolosov,2 Anh Tran,2 Oluwatomilona I. Ifelayo,2 Hong Zhao,3 Stephen T. Wong,3 David J. Tweardy2. 1 _Jiangsu University of Technology, Changzhou, China;_ 2 _UT MD Anderson Cancer Ctr., Houston, TX;_ 3 _Houston Methodist Research Institute, Weill Cornell Medicine, Houston, TX_.

Efforts to develop STAT3 inhibitors have focused on its SH2 domain, starting with short phosphotyrosylated peptides with STAT3 binding motifs, e.g. pY905LPQTV within gp130. Despite binding to STAT3 with high affinity, issues regarding stability, bioavailability, and membrane permeability of these peptides, as well as peptidomimetics such as CJ-887, have limited their further clinical development and led to increased interest in small-molecule inhibitors. Some small molecule STAT3 inhibitors have been identified using structure-based virtual ligand screening (SB-VLS). While having favorable drug-like properties, most suffer from weak binding affinities, possibly due to the high flexibility of the target domain, especially within the region involved in pY-peptide binding. We conducted molecular dynamic (MD) simulations of the SH2 domain in a complex with CJ-887, with a focus on ligand-induced protein conformation changes that increase binding affinity. We used an averaged structure from this MD trajectory as "induced-active site" receptor model for SB-VLS of 110,000 compounds in the SPEC database. Screening was followed by re-docking, re-scoring of the top 30% of hits and selection for compounds that directly interact with pY+0 binding pocket residues R609-S613, resulting in 110 initial hits, which were then tested for STAT3 targeting using a panel of cellular and biochemical assays. 24 out of 110 compounds could inhibit G-CSF-stimulated pY-STAT3 by > 50% at 10 µM with 9 compounds inhibiting by > 99%; 8/24 compounds (SPEC-29, 8, 93, 98, 106, 57, 101 and 85) inhibited STAT3-binding to EGFR pY-peptide (SPR) by 29-71% at 10 µM; 5 of these (SPEC-29, 8, 93, 98, and 106) had pY-STAT3 IC50s ranging from 2.7-19.0 µM; 3 compounds (SPEC-29, 8, and 93) potently inhibited growth of pSTAT3-high MDA-MB-468 (IC50 = 2.4-12.1 µM) and MDA-MB-231 (IC50s = 3.0-12.2 µM) cells and the growth-inhibitory abilities correlated to abilities to inhibit pY-STAT3. Thus, we used a dynamic ligand-STAT3, bound-fit docking model to screen libraries that successfully identified two hits with potent activity against STAT3 both of which comply with all of Lipinski's "rules of five" for drug-likeness. Importantly, while most STAT3 inhibitors previously identified have negatively charged moieties, which mediate their binding to the pY+0 binding pocket but limit cell penetration, the two hits identified are uncharged and likely will serve as superior candidates for future hit-to-lead development leading to an effective direct small-molecule STAT3 inhibitor. Furthermore, the ability of the technique employed here for identifying neutral hits may also be useful in designing inhibitors targeting SH2 domains within other oncogenic targets.

#18

**Doubled lifespan in APC** Min/+ **mice by targeting MTAP.**

Ross S. Firestone, Mu Feng, Vern L. Schramm. _Albert Einstein College of Medicine, Bronx, NY_.

Humans with Familial Adenomatous Polyposis (FAP) have increased risk of colon cancer. Mice with the APCMin/+ mutation provide an animal model of FAP. Interruption of epigenetic control by preventing salvage of S-adenosylmethionine from 5′-methylthioadenosine (MTA) was tested as a potential FAP therapy. 5′-Methylthioadenosine phosphorylase (MTAP) catalyzes the phosphorolysis of MTA to adenine and methylthioribose-1-phosphate for methionine recycling. The effect of MTDIA, a transition state analogue of MTAP, was tested as an oral agent in APCMin/+ mice. Survival analysis with 10, 20 and 30 mg/kg/day oral doses of MTDIA showed a 2-fold increase in mouse lifespan at the optimal 20 mg/kg/day dose. Metabolomic analysis (mouse liver) showed significant changes in metabolites relevant to methionine metabolism. The >4-fold increase in MTA, confirmed the physiologic target. Histologic analysis of intestinal tissue revealed slowed tumor progression in treated mice. RNAseq and Western blots provide additional information about the consequences of MTAP inhibition.

#19

Design and synthesis of biaryl indolyl benzamides as HDAC1-selective inhibitors via a fragment-based lead generation approach.

Ahmed T. Negmeldin,1 Mary Kay H. Pflum2. 1 _Gulf Medical University, Ajman, United Arab Emirates;_ 2 _Wayne State Univeristy, Detroit, MI_.

Histone deacetylase (HDAC) proteins are epigenetic regulatory enzymes that deacetylate protein substrates, leading to subsequent changes in cell function. HDAC proteins are implicated in cancers and several HDAC inhibitors have been approved by the FDA as anti-cancer drugs. Unfortunately, most of the FDA-approved drugs non-selectively inhibit most of the HDAC isoforms, which may lead to side effects in the clinic and limit their use on the bench top. Isoform-selective HDAC inhibitors may decrease or eliminate the side effects associated with non-selective inhibitors treatment. In addition, isoform-selective HDAC inhibitors can be used as molecular tools to study HDAC-related cancer biology. In this work, several biaryl indolyl benzamide compounds were designed and synthesized based on a fragment-based lead generation approach as potential HDAC1-selective inhibitors. The design was based on combining two different fragments from known HDAC1/2 and HDAC1/3 selective inhibitors. These fragments can discriminate against both HDAC2 and HDAC3 and possibly impart selectivity towards HDAC1. Docking studies were performed to validate the design of the inhibitors and to understand the binding of different isoform-selective inhibitors to HDAC1, HDAC2, and HDAC3. Potency and isoform selectivity of the new compounds were assessed via in vitro screening with different HDAC isoforms. All analogs showed modest selectivity for HDAC1 over HDAC2. Bnz-3 was identified as the best compound in terms of potency and selectivity. In particular, Bnz-3 displayed roughly 10- to 100-fold higher potency than the other compounds, with an IC50 value of 548 nM with HDAC1. In terms of selectivity, Bnz-3 showed about 10.6-fold selectivity for HDAC1 over HDAC2, with almost no inhibition of either HDAC3 or HDAC6. The new biaryl indolyl benzamide HDAC inhibitors represent potential lead compounds that can help to further develop more selective HDAC1 inhibitors.

#20

Pharmacology and metabolism of GLL398, an oral selective estrogen receptor degrader for endocrine therapy of breast cancer.

Shanchun Guo, Changde Zhang, Madhu Mottamal, Ahamed Hossain, Jiawang Liu, Jiawang Liu, Guangdi Wang. _Xavier University of Louisiana, New Orleans, LA_.

Selective estrogen receptor degrader (SERD) has proven clinically effective in treating advanced or metastatic breast cancer since the approval of fulvestrant by FDA in 2002. Recent expansion of indications as a first line monotherapy and as combination therapy with CDK4/6 inhibitors further demonstrates its clinical utility as an efficacious breast cancer endocrine regimen. However, the poor pharmacokinetic properties of fulvestrant and its injection-only administration route has driven continued efforts to develop orally bioavailability SERD that could potentially improve clinical response to SERD treatment. GLL398, a boron-modified GW5638 analog, showed superior oral bioavailability while retaining both antiestrogenic activity and ER degrading efficacy at a potency level comparable to the more active metabolite of GW5638, GW7604. Here we report further studies on the pharmacology, pharmacokinetics, and metabolism of GLL398. Consistent with GLL398's robust activities in breast cancer cells that are tamoxifen resistant or express constitutively active, mutant ESR1 (Y537S), it was found to bind the mutant ERY537S at a high affinity. Molecular modeling of the binding mode of GLL398 to ER also found its molecular interactions consistent with the experimentally determined high binding affinity towards WT ER and ERY537S. To test if the superior oral bioavailability can be translated to potent efficacy in vivo, mice bearing MCF-7 derived xenograft breast tumors and patient derived xenograft (PDX) tumors harboring ERY537S were treated with GLL398 which potently inhibited tumor growth in mice. Finally the metabolism of GLL398 was also investigated to elucidate the biotransformation of the drug and its potential contribution to the superior oral bioavailability over GW7604.

#21

Generation and characterization of potent analogues of ERX-11.

Suryavathi Viswanadhapalli,1 Mengxing Li,1 Shi-Hong Ma,2 Gangadhara Reddy Sareddy,1 Tae-Kyung Lee,3 Mei Zhou,1 Yiliao Liu,1 Xihui Liu,2 Dede N. Bahun-Wilson,2 Kara Kassees,3 Jung-Mo Ahn,3 Ganesh V. Raj,2 Ratna K. Vadlamudi1. 1 _UT Health Science Ctr., San Antonio, TX;_ 2 _UT Southwestern, Dallas, TX;_ 3 _UT Dallas, TX_.

Breast cancer (BC) is the most common malignancy in women. Approximately 70% BC are Estrogen receptor (ER) positive, However, most patients develop resistance to current therapies and progress to incurable metastases. We had earlier reported a novel therapeutic agent, ERX-11, that modulates estrogen receptor coregulator interactions. For lead optimization, we designed, synthesized, and tested over 500 analogs of ERX-11 in multiple models of BC.

Methods: In vitro activity was tested using cell titer glo, MTT, and apoptosis assays. The utility of the ERX analogs in treating therapy resistant ER-positive BC was evaluated using models with acquired resistance (Tamoxifen, Letrozole), and engineered models that express ER mutations. Xenografts, patient derived xenografts (PDX), and patient derived xenograft explants (PDEx) were used for testing the utility of ERX analogs.

Results: Our screening studies identified several ERX analogs with potent activity against BC cells. Subtle changes in the ERX analogs appear to have significant ramifications on both their potency against ER-positive BC cell lines and against other tumors types. Some analogs like ERX-41 were more potent than ERX-11 in their ability to block the proliferation of multiple ER-positive BC cell lines (IC50 ranging from 50-200 nM). Other analogs like ERX-208 showed similar activity as ERX-11 against ER-positive BC cell lines but had potent activity (IC50 ranging from 50 -100 nM) against ovarian cancer cell lines. Through iterative changes, we have identified lead compounds with significant activity against other cancers, including in gliomas, ovarian and pancreatic cancers. Although all these compounds were designed to better target the ligand binding pocket of ER, CRISPR-Cas9 based KO screening revealed that these active compounds do not all target ER and appear to target other proteins, including other nuclear receptors. Some compounds for example have activity in ER-negative BC. In several xenograft and models, including pancreatic cancer and ER-negative BC, the activity of the compounds have been confirmed by oral administration of the ERX analogs in vivo. We have also validated the activity of these analogs using PDX and PDEX models.

Conclusions: From our studies to develop a more potent ERX-11 lead analog, we have identified multiple analogs with activities against multiple cancers. While the intended target of these analogs was ER, our library of analogs has potent activity against both ER-positive and ER-negative tumors. In collaboration, we are pursuing further leads in multiple cancers to further delineate the mechanism of action of these various analogs.

#22

Design and synthesis of substituted tetrahydroisoquinoline derivatives as anti-angiogenic and anti-breast cancer agents.

Kinfe Ken Redda,1 Madhavi Gangapuram,2 Suresh Eyuinni1. 1 _Florida A &M Univ., Tallahassee, FL; _2 _Florida A &M University, Tallahassee, FL_.

Angiogenesis is the formation of new blood vessels and plays a critical role in the development, invasion and metastasis of breast cancer pathogenesis. Vascular Endothelial Growth Factor (VEGF) is one of the important angiogenic factors secreted by tumor cells. Overexpression of VEGF significantly increases tumor growth and angiogenesis in a murine model of breast cancer. Inhibition of VGEF pathway by limiting new blood vessels formation has proven to be a valuable approach in reducing tumor growth. During the last two decades, diversified small molecules were evaluated and developed as anti-angiogenic agents. Trabectedin (ET-743; Yondelis) is a tetrahydroisoquinoline alkaloid derived from a Caribbean tunicate Ecteinascidia turbinata and has a potent in vitro and in vivo antitumor activity, including prostate and breast cancers. Trabectedin significantly inhibited the mRNA and protein expression levels of VEGF in all breast cancer cells tested. Previously, we developed a series of tetrahydroisoquinoline (THIQs) derivatives and their in-vitro anti-proliferative activities were tested against MCF-7, MDA-MB-231 human breast cancer cell lines and Ishikawa human endometrial adenocarcinoma cell lines. These THIQ derivatives have the same core structure as that of Trabectedin alkaloid and are considered potential anti-angiogenic agents. Novel substituted THIQ derivatives were designed and synthesized in our laboratory. N-amination of substituted isoquinolines by the aminating agent, O-mesytelenesulfonylhydroxylamine and treated with acyl chlorides led to the formation of ylides. The ylides were reduced using sodium borohydride to yield the desired substituted tetrahydroisoquinolines in moderate to good yields. The synthesized compounds were characterized using NMR and elemental analysis. Of the synthesized compounds, fifteen compounds were screened for anti-angiogenesis activity in the Eli Lilly's Open Innovation Drug Discovery Program (OIDD). Five compounds exhibited greater than 50% inhibition of the tube area at 10µM in the primary single point assay. These five compounds were further screened at different concentrations to obtain dose response curves and IC50 Values. Compound 4-ethyl-N-(3,4-dihydroisoquinolin-2(1H)-yl) benzamide showed an IC50 Value of 1.72 µM.

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

#23

Small molecule targeting of gankyrin reduces cell migration and proliferation in certain cancers.

Pamela Farrales, Aaron Muth. _St. John's University, Queens, NY_.

Introduction: Gankyrin is an oncoprotein involved in regulating numerous pathways important to cell growth, signaling, proliferation and death. The overexpression of gankyrin and subsequent increased protein-protein interactions have been identified as key to increased proliferation in a variety of cancers. Consequently, small molecules that bind to and disrupt these protein-protein interactions are promising therapeutic strategies for the treatment of certain cancers. The first small molecule binder of gankyrin, cjoc42, was recently developed and exhibited efficacy in inhibiting the gankyrin-26S proteasome interaction. The aim of this work is to determine the effects of cjoc42 and its derivatives on cell migration and proliferation in gankyrin-overexpressing cancers.

Methods: Wound healing and CyQuant proliferation assays were used to determine the ability of cjoc42 to inhibit migration and proliferation in the gankyrin-overexpressing cell lines A549, MDA-MB231 and Huh6 cells. A protein thermal shift assay was used to screen new cjoc42 derivatives for their ability to bind gankyrin. Western blot experiments will determine if binding of cjoc42 and its derivatives to gankyrin is the mechanism of action in cells.

Results: Cjoc42 demonstrated an ability to inhibit the migratory and proliferative activity of A549 and MDA-MB231 cells. While only a modest inhibition of migration was observed, cjoc42 exhibited a significant antiproliferative effect against these cell lines. Utilization of a protein thermal shift assay yielded cjoc42 derivatives with more potent gankyrin-binding ability. These compounds will be evaluated in-cellulo for their ability to inhibit cell migration and proliferation. Western blot experiments will confirm that gankyrin-binding of cjoc42 attributes to the anti-migration and anti-proliferative effects observed.

Conclusions: These studies show that cjoc42 directly inhibits cell migration and proliferation by inhibiting the interaction between gankyrin and the 26S proteasome. Consequently, this work provides background for the development of small molecule inhibitors as a gankyrin-mediated therapy for the treatment of lung, breast and liver cancers.

#24

Novel STAT3 & HDAC inhibitors that selectively target triple-negative breast cancer cell.

Bocheng Wu. _Georgia Institution of Technology, Atlanta, GA_.

Signal transducer and activator of transcription 3 (STAT3) is an oncogenetic transcriptional factor which can modulate cancer cells apoptosis. Stimulation of cancer cells by cytokines such as IL-6, INF-a, etc., can induce phosphorylation of STAT3, resulting in the translocation of p-STAT3 into the nucleus where it facilitates the expression of anti-apoptosis genes. This p-STAT3 anti-apoptotic activity is one drug resistance mechanism developed by cancer cells against chemotherapies including histone deacetylase inhibitors (HDACi). Pyrimethamine has been reported to inhibit STAT3 phosphorylation, mRNA transcriptional level, and STAT3-dependent cell viabilities. In this presentation, we disclosed novel pyrimethamine-based bifunctional STAT3-HDAC inhibitors as potential therapeutic agents for p-STAT3-dependent cancers. We will describe the design, synthesis and biological activities of STAT3-HDAC inhibitors. We will show that these bifunctional agents inhibit HDAC deacetylase activity and STAT3 phosphorylation. A subset of these compounds is more cytotoxic to MDA-MB-231, a triple-negative breast cancer cell line that is highly dependent on STAT3.

#25

Identifying new inhibitors of ABCG2 by high-throughput screening.

Shoji Kokubo, Shinobu Ohnuma, Hideaki Karasawa, Akihiro Yamamura, Hideyuki Suzuki, Megumi Murakami, Norihiko Sugisawa, Keigo Kanehara, Keisuke Sato, Takanori Ishida, Takashi Kamei, Takeshi Naitoh, Michiaki Unno. _Tohoku University Graduate School of Medicine, Sendai, Japan_.

Background: ABCG2, also known as breast cancer resistance protein (BCRP), is a member of the ATP-binding cassette (ABC)-family of membrane transport proteins associated with multidrug resistance (MDR). No ABCG2 inhibitor has shown clinical significance yet.

Aim: To identify novel inhibitors of ABCG2 by high-throughput screening.

Material & Methods: After higher expression of ABCG2 in HCT-116/BCRP cells was confirmed by flow cytometry (FACSVerse) using 5D3 antibody, high-throughput fluorescent cell-based assay was performed with the original chemical library of Tohoku University containing about 6000 compounds using ABCG2-specific fluorescent substrate, Pheophorbide a (PhA) in HCT-116/BCRP cells overexpressing ABCG2. Ko143, a potent ABCG2 inhibitor, was used as positive control. Fluorescence intensity in the cells was read by a fluorescence plate reader (SpectraMax M2e) in bottom read mode, with 395 nm excitation, 670 nm emission. To validate the results, flow cytometry assay was performed with 10 µM PhA in HCT-116/BCRP cells. The fluorescence intensity was analyzed by flow cytometry (FACSVerse).

Results: Fluorescenct cell-based assay with high-throughput screening could detect 23 candidate compounds, which showed higher fluorescence intensity as positive control Ko143 showed. Then, these 23 compounds were validated with flow cytometry assays. The flow cytometry assays revealed that 16 compounds increased fluorescence intensity of PhA in HCT-116/BCRP cells in dose dependent manner, suggesting that these candidate compounds inhibited transport function of ABCG2. Among 16 compounds, 6 compounds showed further inhibitory effect for transport function of ABCG2 as equivalent to that of Ko143.

Conclusion: These 6 compounds may provide a basis for further investigation of ABCG2 function.

#26

Development of potent lead compounds in multiple tumor types.

Ganesh V. Raj,1 Xihui Liu,1 Dede Ekoue,1 Jung-Mo Ahn,2 Ratna Vadlamudi3. 1 _UT Southwestern Medical Ctr., Dallas, TX;_ 2 _UT Dallas, Dallas, TX;_ 3 _UT Health Sciences Center at San Antonio, San Antonio, TX_.

Background: We had earlier reported a novel therapeutic agent, ERX-11, that modulates estrogen receptor coregulator interactions. For lead optimization, we designed, synthesized and tested over 500 analogs of ERX-11 in multiple models of BC. We also tested this library of compounds for activity against other cancer cell lines both in vitro and in vivo and against primary tumors ex vivo.

Methods: In vitro activity was tested using Cell titer glo, MTT, and apoptosis assays.The utility of the ERX analogs in treating therapy resistant ER-positive BC was evaluated using models with acquired resistance (Tamoxifen, Letrozole), and engineered models that express ER mutations. Xenografts were used for testing the utility of ERX analogs in vivo. Primary patient tumor derived explants were used for ex vivo testing of ERX analogs.

Results: Our screening studies identified several ERX analogs with potent activity against BC cells. Subtle changes in the ERX analogs appear to have significant ramifications on both their potency against ER+ BC cell lines and against other tumors types. Some analogs like ERX-41 were more potent than ERX-11 in their ability to block the proliferation of multiple ER-positive BC cell lines (IC50 ranging from 20-200nM). Other analogs like ERX-208 showed similar activity as ERX-11 against ER-positive BC cell lines (IC50 ranging from 100-500nM) but had potent activity against ovarian cancer cell lines. Through iterative changes, we have identified leads compounds with significant activity against other cancers, including in gliomas, ovarian and pancreatic cancers. Although all these compounds were designed to better target the ligand binding pocket of ER, these active compounds do not all target ER and appear to target other proteins, including other nuclear receptors. Some compounds for example have activity in ER-negative breast cancers. In several xenograft models, including pancreatic cancer and ER-negative, the activity of the compounds have been confirmed by oral administration of the ERX analog in vivo. We have also validated the activity of these analogs in patient-derived explants cultured ex vivoin multiple tumor types.

Conclusions: From our studies to develop a more potent ERX-11 lead analog, we have identified multiple analogs with distinct activities against multiple cancers. While the intended target of these analogs was ER, our library of analogs has potent activity against both ER-positive and ER-negative tumors. In collaboration, we are pursuing further leads in multiple cancers to further delineate the mechanism of action of these various analogs.

#27

Identification and characterization of a novel dual FLT3/microtubule polymerization inhibitor through cell-based screening.

Haleema Sadia Malik,1 Aishah Bilal,1 Rahim Ullah,1 Maheen Iqbal,1 Rahman Shah Saleem,1 Hidayat Hussain,2 Amir Faisal1. 1 _Lahore University of Management Sciences, Lahore, Pakistan;_ 2 _Leibniz Institute of Plant Biochemistry, Halle, Germany_.

Activating mutations in FLT3 receptor tyrosine kinase are found in one-third of acute myeloid leukemia (AML) patients and are associated with disease relapse and bad prognosis. Majority of these mutations are Internal tandem duplications (ITD) in the juxtamembrane domain of FLT3 and have been validated as a therapeutic target. Clinical success of selective inhibitors targeting oncogenic FLT3, however, has been limited due to the acquisition of drug resistance. Here we report the identification of a dual FLT3/microtubule polymerization inhibitor, HH-IA-208, through cell-based screenings for the differential killing of leukemia cell lines expressing FLT3-ITD (MV411) or BCR-ABL (K562) using an MTS proliferation assay and for mitotic inducers using a dot-blot assay for histone H3 phosphorylation in HCT116 cell line. HH-IA-208, a chalcone derivative, inhibits FLT3 in vitro and treatment of FLT3-ITD+ cell lines with HH-IA-208 results in reduced FLT3 signaling. Similarly, HH-IA-208 inhibits tubulin polymerization in a biochemical assay and in cells that results in a mitotic arrest. HH-IA-208 is selectively more potent in leukemia cell lines expressing FLT3-ITD and treatment for 24 and 48 hours with the inhibitor results in apoptotic cell death. Furthermore, HH-IA-208 is able to overcome TKD mutation-mediated acquired resistance to FLT3 inhibitors in a MOLM13 cell line expressing FLT3-ITD with the D835Y mutation. HH-IA-208, therefore, is a promising lead for the discovery of dual target FLT3 inhibitors that can overcome resistance to selective FLT3 inhibitors.

#28

Dihydrolipoyl dehydrogenase is a key molecule inducing ferroptotic cell death.

Jihee Kim, Daiha Shin, Jaewang Lee, Jong-Lyel Roh. _Asan Medical Center, Unv. of Ulsan College of Medicine, Seoul, Republic of Korea_.

Ferroptosis is iron-dependent, non-apoptotic cell death from lipid peroxidation. Cystine-glutamate transporter (xCT) and glutathione peroxidase 4 (GPX4) are recognized as the key regulators of ferroptosis. Glutathione depletion via cystine deficiency or inhibition of xCT or GPX4 accumulates cellular lipid peroxidation and ferrous iron, resulting in ferroptosis. Glutamine is an amino acid essential for growth of cancer cells as well as induction of ferroptotic cell death via the production of alpha-ketoglutarate (αKG) from glutaminolysis. However, it is still unclear whether αKG or its relevant metabolic process is engaged in ferroptotic cell death. Therefore, we examined the role of αKG dehydrogenase (αKGDH) in induction of ferroptosis in head and neck cancer (HNC) cells. Cystine-deficient conditioned medium or xCT inhibition with sulfasalazine induced typical ferroptotic cell death, which was prevented by the inhibition of glutaminolysis or αKG production. Next, we focused the dihydrolipoyl dehydrogenase (DLD) and dihydrolipoyl succinyltransferase (DLST), consisting of the E3 and E2 components of αKGDH respectively. Ferroptosis was significantly prevented by genetic inhibition of the DLD but not the DLST, which was rescued by re-insertion of the DLD gene. Cellular reactive oxygen, lipid peroxidation, and ferrous iron accumulation were induced by the increased function of αKGDH, that is mainly the DLD, when αKG was converted to succinate in the tricarboxylic acid cycle. Sulfasalazine did not significantly suppress the in vivo growth of the shDLD-transfected HNC cells but vector control transplanted in nude mice. Taken together, our data support that the DLD is a key molecule inducing ferroptosis in cancer cells. 

## TUMOR BIOLOGY

### 3D Models

#29

Activity of trastuzumab emtansine (T-DM1) in 3D cell culture.

Jean Z. Boyer,1 Gail Lewis Phillips,2 Hiro Nitta,1 Karl Garsha,1 Eric May,1 Brittany Admire,1 Robert Kraft,1 Megan Peccarelli,1 Andre Zamorano,1 Scott Gill,1 Eslie Dennis,1 Liz Vela,1 Penny Towne1. 1 _Ventana Medical Systems, Inc. Roche Tissue Diagnostics, Oro Valley, AZ;_ 2 _Genentech, Inc, CA_.

Introduction: Cell spheroids/aggregates generated from three dimensional (3D) cell culture methods are similar to real tumors in terms of tissue morphology, biology, and gene expression. We performed a unique 3D cell culture drug efficacy study with Trastuzumab emtansine (T-DM1) across a number of breast cancer cell lines that were previously investigated in 2D cell culture (Lewis Phillips, et al, 2008). We obtained significantly different results for some cell lines grown as 3D spheroids/aggregates when compared to those grown as 2D cultures.

Methodology: We performed 3D cell culture and produced FFPE blocks (using novel methods) from 3D spheroids/aggregates to determine HER2 (IHC) protein expression levels for five cell lines: SK-BR-3; BT-474; MDA-MB-361; MDA-MB-175 and MCF-7. We also performed HER2 gene-protein assay (HER2 GPA) to determine HER2 gene amplification along with IHC protein expression status in four cell lines: SK-BR-3; BT-474; MDA-MB-361 and MDA-MB-175. Drug (T-DM1) activity testing using CellTiterTM-Glo 3D cell viability assay was performed on 3D cell spheroids/aggregates for comparison with 2D cells. Images were obtained of T-DM1 internalization in BT-474 cells and spheroids using pHrodo™ iFL Human IgG Labeling Reagent.

Results: In 3D spheroids/aggregates, HER2 IHC staining and GPA assay showed for SK-BR-3 and BT-474 HER2 3+ expression and HER2/CEP17 of ≥ 2; MDA-MB-361 cells with HER2 2+ expression and HER2/CEP17 of ≥ 2.0; MDA-MB-175 cells with HER2 1+ expression and HER2/CEP17 < 2.0 and MCF-7 cells with HER2 0+ (IHC staining only without HER2 GPA data). Some of the 3D spheroids/aggregates in MDA-MB-361 cells showed heterogeneous expression of HER2 protein. The IC50 values of 3D spheroids/aggregates for some cell lines were significantly higher than were demonstrated for cell lines grown in 2D cell cultures. The fold changes between 3D spheroids and 2D cells (72h T-DM1 treatment time) are: 4.2 for SK-BR-3; ≳ 10 for BT-474 and 22 for MDA-MB-361. Additionally, the fluorescent images showed that a longer incubation time was required for the T-DM1 drug (3 µg/ml) to be internalized effectively into BT-474 3D spheroids; for example, about 120h for 3D spheroids in comparison to about 36h in 2D cells. Interestingly, the 3D spheroids incubated for 120h with T-DM1 (470 µm) are smaller in size than 3D spheroids in the control group (600 µm) incubated for 120h without T-DM1 treatment.

Conclusions: Drug efficacy studies performed on 3D cultured spheroids/aggregates are expected to be very important and biologically relevant for determining drug activity in tumor tissue. Our drug efficacy study using 3D cell culture demonstrated greater concentrations of T-DM1 and longer incubation times were required than for cells grown as 2D in some cell lines, likely due to less efficient internalization.

#30

The development of a 3D cell-culture model of drug-induced oral mucositis.

Maria P. Lambros,1 QinQin Fei,1 Jonathan Moreno2. 1 _Western University of Health Sciences, Upland, CA;_ 2 _Axar Pharmaceuticals, Corona, CA_.

Purpose: Mucositis is a debilitating disease affecting cancer patients. Mucositis is an adverse effect of radiation or chemotherapy. In the past, our lab has developed a model of radiation-caused mucositis using a validated human tissue substitute of oral mucosa and studied the post radiation cellular and molecular stresses and their prevention. In this project, we wished to develop an in-vitro model of chemical-, or drug-induced oral mucositis. For this purpose, we used a three dimensional (3-D) cell culture model of oral mucosa and as the drug to induce mucositis we used everolimus. Everolimus belongs to rapalogs which represent important therapies against cancer but one of its adverse-effects is mucositis. Mucositis caused by everolimus develops early in therapy and progresses quickly to grade 3 making patients to postpone life-saving therapies. Thus, it is very important to understand and prevent the development of everolimus-caused mucositis.

Methods: The 3-D cell cultures of primary human oral keratinocytes grown on top of primary human oral fibroblasts were purchased from MatTek Corporation. These 3-D cell cultures (tissues) are similar to human oral mucosal tissue and have been validated as its substitutes. Different 3-D tissues were treated for 24, 40 and 60 hours with 8, 16, 32, and 64 ng/ml of everolimus. Tissues which received no treatment were used as controls. We then evaluated the tissue histology using H and E staining, and gene expression using RNA-sequencing.

Results: Tissues which were treated with everolimus show signs of distress based on the concentration of everolimus and the duration of the treatment. Low and medium concentration of everolimus (8ng/ml) and (16 or 32 ng/ml), respectively, show no, or minimal tissue distress after 24 hours of treatment duration. Tissues which received a high concentration of everolimus (64 ng/ml) even at a short duration of treatment, show signs of distress, as evidenced by atypical proliferation at the keratinous epithelium, vacuoles, or shredded keratin layers, as well as, higher apoptosis, and expression of pro-inflammatory cytokine genes. In general, the higher the concentration of everolimus and the longer the duration of the treatment, the more the signs of the stress in tissues.

Conclusion: The cell and molecular stress that the 3-D tissue cultures of human oral mucosa experience depends on the drug concentration and the duration of treatment.

Acknowledgment We thank Novartis for their support.

#31

Organoid derived from human operative sample of NET shows NET characteristics.

Yasushi Ichikawa,1 Yukihiko Hiroshima,1 Noritoshi Kobayashi,1 Yusaku Masuda,1 Ayumu Goto,1 Motohiko Tokuhisa,1 Yuma Takeda,1 Takashi Ishikawa,2 Itaru Endo1. 1 _Yokohama City University, Yokohama, Japan;_ 2 _Tokyo Medical University, Tokyo, Japan_.

Background: Neuroendocrine tumor is a rare neoplasm, then, it was difficult to examine using in vitro or animal models. Recently, patient-derived tumor organoid (PDTO) is considered as a promising method to analyze natural characteristics of human tumor, because PDTO is cultured in near-natural circumstances compared with cell lines. We established PDTO of neuroendocrine tumor (NET) utilizing operative samples from a pancreatic NET patient. Aim: Aim of this study is to establish organoid derived from an operative sample of pancreatic NET patient and to compare its characteristics with original tumor using immuno-histochemistry. Material and Method: A pancreatic NET tissue derived from operative sample was cut into small pieces and was treated by collagenase. Prepared tissues were 3-dimensionally cultured on the Matrigel in a serum-free medium (Advanced MEM/F12) with 4 growth factors (Respondin, EGF, Rock inhibitor, Noggin), then PDTO was established. To evaluate growth rate of PDTO, Major axis of the organoid was measured on the day 1, 2, 3, and 6. One week after primary culture of the organoid, subculture was performed. Then major axis of the 2nd, 6th and 7th filial organoid was also measured and compared with each filial. To evaluate characteristics of PDTO as neuroendocrine tumor, immunohistochemistry was performed for the 1st, 2nd and 3rd filial PDTO using anti-chromogranin-A antibody (Dako, mouse IgG), anti-synaptophysin antibody (Abcam, rabbit IgG), anti-NCAM antibody (Abcam mouse IgG) and Neuro-specific enolase (Dako, rabbit IgG). Results: Growth rate of the 2nd and 3rd filial on the day 6 did not show significant difference compared with that of the 1st filial. On the other hand, Growth rate of the 2nd and 3rd filial was significantly lower than that of the 1st filial. Expression of the 4 factors in the 1st, 2nd and 3rd filial PDTO was kept as much as the primary tumor. Conclusion: PDTO derived from neuroendocrine tumor showed the same characteristics as the primary tumor. The 6th and 7th filial PDTO might show cellular aging.

#32

Development of a three-dimensional spheroid model system to more accurately evaluate schedule dependent mechanistic differences between metronomic and conventional dosing of topotecan.

Joshua Davis,1 Taraswi Mitra Ghosh,1 Lani Jasper,1 Matthew Eggert,1 Jeff Warner,1 Brian Cummings,2 Robert Arnold1. 1 _Auburn University, Auburn, AL;_ 2 _Georgia University, GA_.

Alternative treatment schedules, such as low-dose, high-frequency (metronomic; METRO) of some chemotherapeutics enhance efficacy and reduce toxicity in some cancers when compared to chemotherapy given conventionally (CONV). Previously, we demonstrated that low-dose, prolonged exposure to topotecan (30-day infusion 0.1 mg/kg/day via Alzet pump) improved survival and reduced tumor volume ~120%, compared to topotecan given CONV (4 mg/kg, every 4 days) in xenograft model of aggressive prostate cancer in athymic mice. Yet, detailed examination of the mechanisms underlying this enhanced activity and optimization of treatment schedules is limited with existing 2-dimension (2D) cell culture models that do not adequately reflect in vivo reality. Therefore, we developed and applied a 3D spheroid model that allows for the evaluation of longer drug exposures. We used PC-3-Luc-GFP, an aggressive, metastatic castration-resistant prostate cancer that expresses luciferase (Luc) and green fluorescent (GFP) that support bioluminescent and fluorescence imaging. Cell growth and cytotoxicity studies were performed using 2D or 3D models. Briefly, 3D spheroids were formed by seeding cells in a poly-HEMA coated 96-well plate with 2.5% recombinant basement membrane followed by centrifugation (5 min at 1000 g) to encourage spheroid generation. The 2D cell cultures reached confluency in 72-96 hr. While we previously demonstrated enhanced activity in vivo, the 2D model showed no benefit (improved potency) over this time frame, ie, METRO vs CONV dosing (logIC50 2.278 (177nM), 95% CI [1.781-2.776] vs logIC50 2.248 (189 nM) 95% CI [1.723-2.773]. Attempts to reduce seeding density to increase study duration resulted in poor and inconsistent cell growth and greater variability. 3D spheroids yielded reproducible round spheroids of similar size (~800 μm) that grew for up to 28 days, thus permitting evaluation of long treatment exposures. Tumor stromal cells are also known to alter tumor pathology, drug delivery and responsiveness to therapy. Therefore, 3D spheroids with PC-3-Luc-GFP and macrophages and endothelial cells in different cell proportions (1:1:1, 1:2:1, etc.,) were examined. Growth of 3D spheroids was altered based on cell type and cell proportion. Further, differences in rates of growth and cellular morphology were observed for tumor activated vs wild macrophages. Overall, we developed a high-throughput, liquid overlay, scaffold-based 3D co-culture spheroid that can be kept for 28 days. This model will permit evaluation of single agent and concomitant therapy over prolonged periods better mimicking clinical practice. Further, this model will allow for more detailed molecular analysis and ability to discern the effect of tumor microenvironment on alternate treatment schedules.

#33

Ex vivo **drug sensitivity assay with a panel of cancer patient-derived spheroids of small cell neuroendocrine carcinoma of uterine cervix.**

Mie Tanaka,1 Satoshi Kubota,2 Yu Ito,1 Yumiko Kiyohara,3 Hiroko Endo,4 Jumpei Kondo,5 Satoshi Nakagawa,1 Akiko Okazawa,1 Shinya Matsuzaki,1 Toshihiro Kimura,4 Eiji Kobayashi,1 Yutaka Ueda,1 Kiyoshi Yoshino,6 Shoji Kamiura,4 Tadashi Kimura,1 Masahiro Inoue5. 1 _Osaka Univ. Faculty of Medicine, Suita, Japan;_ 2 _Osaka General Medical Center, Japan;_ 3 _Japan Community Health Care Organization Osaka Hospital, Japan;_ 4 _Osaka International Cancer Institute, Japan;_ 5 _Kyoto Univ. Faculty of Medicine, Kyoto, Japan;_ 6 _Univ. of Occupational & Environmental Health Faculty of Medicine, Japan_.

Small cell neuroendocrine carcinoma (SCNEC) of uterine cervix is a rare tumor accounting for approximately 1-2% of uterine cervical tumors. SCNEC of uterine cervix is highly invasive and has an extremely poorer prognosis than other histologic types for which no standard treatment is established. To establish a new management strategy against SCNEC of uterine cervix, we developed a drug sensitivity assay based on a 3D primary culture system. We previously developed the cancer tissue-originated spheroid (CTOS) method, a primary culture method from patients' tumors. To date, we established a panel of 12 SCNEC CTOS lines with the success rate of 100%. For each line, multiple CTOSs were placed in one well of a 96 well plate and dose response analysis was performed for seven anticancer drugs; paclitaxel, carboplatin, irinotecan, SN-38, cisplatin, etoposide and gemcitabine. After 7 days of culture, the CTOSs viability was evaluated by measuring intracellular ATP levels, corrected by the size of CTOS at day0. All drugs had substantial variations in sensitivity among the lines. Cerv54 was far more sensitive to irinotecan than other lines while was similarly sensitive to SN-38. We revealed that cancer cells of cerv54 had high levels of carboxylesterase(CES)1 expression, which converts irinotecan to SN38. Since cerv54 with CES1 knockdown by pGFP-C-shCES1 lentivirus had lower sensitivity than sh-scramble, CES1 expression is related to the irinotecan sensitivity in cerv54. CES1 expression in cancer cells may be useful for predicting the effect of irinotecan based chemotherapy. In addition, this study indicates that sensitivity test using CTOS might be applicable to precision medicine by selecting sensitive drugs for each patient.

#34

A high-content imaging platform for quantitative assessment of immunogenic tumor cell killing and T-cell proliferation in 3D tumoroids of fresh patient tumor samples.

Melanie Mediavilla-Varela, Vijayendra Agrawal, Melba Marie Page, Jenny Kreahling, Soner Altiok. _Nilogen Oncosystems, Tampa, FL_.

Introduction: Challenges in the development of immunotherapeutics still remain due to the lack of clinically relevant drug screening platforms that recapitulate the complexity of the tumor immune microenvironment. T-cell activation and immunogenic tumor cell death have been the most desired assay readouts to fully explore the efficacy of immuo-oncology drugs and to develop most effective rational drug combinations. Conventional cytotoxicity assays such as Cr51 and LDH release assays are limited in providing clinically relevant data about treatment mediated tumor cell killing by immune cells. Here, we describe a novel 3D live tumor explant model of fresh patient tumor tissue (3D-EXplore) to simultaneously and cost effectively evaluate T-cell activation and tumor cell killing by immuno-oncology drugs.

Materials and Methods: All human tumor samples were obtained with patients' consent and relevant IRB approval. Fresh patient tumor samples of various cancer types (lung, bladder, kidney) were processed to generate unpropagated 3D tumoroids of standard size (100-150 microns). 3D tumoroids were treated with pembrolizumab (Keytruda) and atezolizumab (Tecentriq) alone or in combination with conventional chemotherapeutic drugs. Various combinations of viability, vitality, cell proliferation and cell death fluorogenic dyes were utilized to label tumor cells prior to high content confocal imaging. Treatment-mediated changes in tumor cell death and T-cell activation were quantified by Nilogen's QTDI-algorithm. The results were corroborated by multiplex flow cytometry and cytokine relase assays.

Results: 3D-tumoroids were successfully prepared from different tumor types. Tumor cell viability and immune cell composition including CD4 and CD8 T-cells, Tregs, NK cells and M1/M2 macrophages were monitored over 5 days to ensure obtaining reliable results. Using high-content image analysis and flow cytometric analysis, in selected tumors we observed a significant increase in T-cell activation and tumor cell killing upon ex vivo treatment with pembrolizumab or atezolizumab alone, which was enhanced in the presence of carboplatin.

Conclusion: Here we have described a cost-effective and rapid drug testing platform using clinically relevant unpropogated fresh patient tumor tissue for rapid testing of multiple drugs and drug combinations based on tumor cell killing and T-cell activation to accelerate drug discovery, which can be applied in clinical studies to improve personalized immunotherapy for individual patients.

#35

3D in vitro system for immuno-oncology: Real-time imaging of drug delivery, tumoroids, and immune cell activity.

Eric O. McGhee, Alex J. McGhee, Derek L. Hood, Kylie E. Van Meter, Juan M. Urueña, Duane A. Mitchell, Catherine T. Flores, Steven C. Ghivizzani, C Parker Gibbs, Padriac P. Levings, Colin J. Anderson, W Gregory Sawyer. _University of Florida, Gainesville, FL_.

A Liquid Like Solid (LLS) 3D culture system in a convenient microtiter plate format enables long-duration culture of patient derived microtumors (>30 days), in situ confocal imaging, 3D cytotoxic drug studies, inclusion of T cells, and measurements of T cell migration, infiltration, and killing. Introduction: Cancer is a disease in 3-dimensions and there is a desperate need for new tools and infrastructure to study immuno-oncology treatment methods in 3D. Fabrication of microtumors using 3D printing in LLS media comprised of soft granular microgels (3-5 μm crosslinked polyacrylamide (PAA) microgel particles: 7.5% PAA) facilitate precise arrangement of detailed assemblies of extra-cellular matrix components and cells (1), including: epithelial cells (breast ATCC MCF10A), stem cells (bone marrow MSC), cancer cells (breast ATCC MCF7, prostate ATCC-PC3, osteosarcoma ATCC-MG63, melanoma ATCC-A375, primary glioblastoma, and osteosarcoma), fibroblasts (ThermoFisher dermal fibroblasts C0045C), endothelial cells (ThermoFisher HUVECs C0035C), and CD4+/CD8+ T cells (PBMC and Jurkat E6-1). Methods: Long term culture was enabled through the design, development, and validation of a modular perfusion system that uses passive negative pressure within a 96-well microtiter plate format to transport liquid growth medium, drugs (doxorubicin and puromycin), antibodies (aPD1 J43 clone, aCD3, aCD28), growth factors, FBS, and metabolic waste without disturbing the spatial organization and positioning of the experiments (i.e. 3D orientation is preserved and the packed bed of microgel particles remain solid). The perfusion velocities are precisely controlled to between 1-100 nm/s by setting the negative pressure, and complete exchange of liquid media can be tailored from hours to days. Results: Real-time imaging in these 3D assays is performed using in situ confocal microscopy under controlled perfusion. Cell motility, adhesion, and dynamic rearrangement of fibroblasts and endothelial cells within a 3D co-culture of microtumors evolved over the first 72 hours. Immunohistochemistry with Ki-67 and PCNA staining indicated active cell proliferation of the tumoroids after 28 days of continuous culture. Tracking of activated CD8+ T cells revealed super-diffusive motion in the presence of 3D tumors within a range of 250 µm. Activated T cell migration speeds have been measured to be between 1.3 and 2.0 μm/s in the 3D LLS media, and preliminary estimates of T cell migration forces are on the order of 1 nN. Conclusions: This integrated system of 3D bioprinting, perfusion culture plates, and confocal microscopy enables in situ 3D studies of cancer biology, immunotherapy, and drug treatment regimens and provides unique insights and measurements of immune cell invasion dynamics in 3D microtumors. References: 1. Battacharjee et al. Science Advances Sept. 2015 1:8

#36

Application of patients derived organoids as co-clinical model.

Young-Won Cho,1 Hwang-Phill Kim,1 Dong Wook Min,1 Yoojoo Lim,2 Sang-Hyun Song,1 Sae-Won Han,2 Tae-You Kim2. 1 _Cancer Research Institute, Seoul National University, Seoul, Republic of Korea;_ 2 _Seoul National University Hospital, Seoul, Republic of Korea_.

Organoids are known to best in vitro model system which mostly mimic genetic, phenotypic feature of its origin. And it is expected and suitable to variable disease modeling and study. For cancer modeling using organoids as co-clinical model, our group derived organoids from 40 colorectal tumor including lymph node, terminal ileum metastatic tumor and ascites. Whole exome sequencing of matched tumor organoids and tissues was performed and their somatic mutation concordance was analyzed. As a results, Organoids reflect their genetic statues of the original tumor tissue and their phenotypic features are maintained. In order to prove the appliance of these organoids as a co-clinical model, clinical drugs on colorectal cancer such as 5-Fluorouracil, oxaliplatin, SN38 and cetuximab were tested. The drug results reflected the actual clinical outcome of the corresponding patients. To find the precise drug treatment option for conventional chemo-refractory patients, we performed 134 anti-cancer drug library screening. Organoids with specific mutations were sensitive to drugs which target these corresponding mutations. For instance, organoid with RNF43 mutation was sensitive to porcupine inhibitor which suppresses auto- and paracrine WNT- driven growth. In conclusion, we suggest that patient derived organoids can be applied in cancer treatment as a co-clinical model, due to their highly correlated genetic information and drug sensitivity with those of the patients themselves.

#37

**RNA-seq comparisons of** in vitro **and** in vivo **cancer model platforms: Monolayer, spheroids, immunodeficient, and syngeneic mouse model.**

Nicholas Hum, Aimy Sebastian, Wei He, Monica L. Moya, William F. Hynes, Jonathan J. Adorno, Aubree Hinckley, Elizabeth K. Wheeler, Matthew A. Coleman, Gabriela G. Loots. _Lawrence Livermore National Laboratory, Livermore, CA_.

Preclinical cancer models have been vital contributors in minimizing this burden as well as understanding basic cancer cell biology, however conventional and modern cancer models do not accurately or reliably recapitulate the complex in vivo tumor environment. Clinical significance of discoveries made using in vitro models requires an understanding of the limitations imparted from cancer cells in a non-native environment. An ideal pre-clinical cancer platform that mimicks in vivo molecular phenotypes is essential for achieving effective drug screening and personalized treatments. This study aims to elucidate biological processes deficient in conventional in vitro methods from in vivo grown allograft cancer cells via transcriptome analysis.

The effects of culturing conditions on cancer cells were analyzed via whole transcriptome RNA sequencing on a mouse mammary carcinoma (4T1) cell line grown in multiple culture conditions: 2D (monolayer) or 3D (spheroid) constructs under static or dynamic flow in addition to 4T1 cells isolated from subcutaneous or orthotopically grown tumors into the mammary fat pad of immune-competent, BALB/c mice.

Comparative analysis of whole transcriptomic profiles of 4T1 cells in differing culturing conditions reveals distinct biological processes fostered by their environment. Monolayer culture shows enrichment in gene ontologies promoting proliferation, cell cycle progression, and protein synthesis. Compared to monolayer culture all 3-dimensional culturing methods encouraged the expression of proteins known to be critical to tumor progression such as extracellular matrix remodeling, adhesion, and differentiation. Furthermore, spheroid culture introduced heterogeneity as evidenced by upregulation of hypoxic induced genes and regulation of multicellular organism development processes. As expected, 4T1 cells expanded in vivo upregulated genes associated with processes difficult to recapitulate in vitro such as cell migration, inflammatory response, and angiogenesis.

3D culturing methods are able to recapitulate aspects of tumor heterogeneity yet fail to incorporate the complex heterogeneous cell composition and transient fluxes in nutrients and drugs found in vivo. Findings from this study demonstrate the behavioral and transcriptional alterations imparted from environmental factors. Additionally, clinically relevant in vitro testing can be improved by the incorporation of factors found in the native tumor microenvironment to existing 3D culturing approaches.

This study received funding from LLNL LDRD grant 19-SI-003. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344).

#38

NSCLC patient-derived organoids to guide personalized therapy.

Seok-Young Kim,1 Dong Hwi Kim,1 Hyeong-Seok Joo,1 Mi Ran Yun,1 Ji Yeon Lee,2 Sang Min Kim,2 Hyunki Kim,2 Min Hee Hong,2 Hye Ryun Kim,2 Byoung Chul Cho2. 1 _JE-UK Laboratory of Molecular Cancer Therapeutics, Seoul, Republic of Korea;_ 2 _Yonsei University College of Medicine, Seoul, Republic of Korea_.

Purpose: Translational research in non-small cell lung cancer (NSCLC) has been largely hampered by relatively low success rates for establishing patient-derived cell model or patient-derived xenograft model. Recently, NSCLC patient-derived organoid model (PDO) has been introduced, showing a high take rate and stable growth in long-term in vitro cultures. Translational relevance of NSCLC PDOs remains unclear. In this study, we evaluated whether NSCLC PDOs can predict anti-cancer treatment responses and provide therapeutic strategies to overcome drug resistance.

Experimental design: Nineteen malignant effusions and 13 biopsied/surgical specimens were obtained from 31 patients with lung adenocarcinoma. Eleven patients (35%) had no prior therapy, 7 (23%) chemotherapy or radiotherapy, 6 (19%) EGFR-tyrosine kinase inhibitors (TKI), 4 (13%) ALK/ROS1-TKIs, and 3 (10%) immunotherapy. Samples were processed and cultured as reported previously. In brief, specimens were reviewed by pathologists to confirm malignancy. Then, samples were cultured in Advanced DMEM/F12 containing 10% R-spondin 1 conditioned medium, 25 ng/mL FGF7, 100 ng/mL FGF10, 100 ng/mL noggin, 500 nM A83-01, 500 nM SB202190, 1X B27, 1.25 mM N-acetylcysteine, 5 mM nicotinamide, 1X glutamax, 10 mM HEPES, and 1X primocin. For efficient establishment, we stained organoids with H&E at early passages to confirm histological features of NSCLC. Cell viability assay was performed using CellTiter-Glo 3D. PDOs (<P10) were analyzed by whole-exome sequencing and RNA-seq and cryopreserved to establish a biobank.

Results: A total of 22 PDOs derived from NSCLC patients was established. Take rates from malignant effusions and biopsied/surgical specimens were 89% and 38%. Of these established PDOs, 9 were wild-type, 9 EGFR-mutant, 3 ROS1-rearranged, and 1 ALK-rearranged. Genomic and transcriptomic profile of established PDOs were concordant to those of matching parental tumors. Of note, we established clinically important models progressing to targeted therapy or immunotherapy as follows: osimertinib-resistant EGFR mutant PDOs (n=4), repotrectinib-resistant ROS1-rearranged PDO (n=1), and pembrolizumab-resistant PDO (n=1). For example, YUO-2 was established from progressing pleural effusion after 5 months of osimertinib and had lost EGFR T790M but maintained EGFR exon 19 deletion. YUO-2 was resistant to gefitinib, afatinib, and osimertinib with IC50 values of 27300 nM, 9296 nM, and 5691 nM, respectively, implicating clinical relevance of NSCLC PDO models. TKI-resistant PDOs were screened with combinations of TKI targeting the driver mutation of the established models and each drug from the FDA-approved drug library.

Conclusions: NSCLC PDO models could recapitulate the characteristics of corresponding NSCLC tumors and clinical response. PDO-based co-clinical trial will accelerate translational research in NSCLC. Further results will be presented at 2019 AACR meeting.

#39

Microvessel-on-a-chip for investigating glioma-vascular interactions.

Magda Gerigk, Harry J. Bulstrode, Yan Yan Shery Huang. _University of Cambridge, Cambridge, United Kingdom_.

Introduction: Gliomas, which are heterogeneous tumors made up of malignant glial and stromal cells, can often grow and progress without angiogenesis and thus escape anti-angiogenic therapies. One of the alternative mechanisms of tumor blood supply is vessel co-option, where cancer cells migrate along the pre-existing vessel of the host organ, preserving the blood-brain barrier (BBB). However, studying this phenomenon is currently limited mainly to animal models. With the push to reduce in vivo approaches, developing an experimental, organ-on-a-chip model to encompass one or more tractable microenvironmental factors, will enable us to better understand their mechanistic roles in brain tumor progression.

Methods and Results: An extracellular matrix-integrated PDMS-based microfluidic chip with a rounded microvessel, mimicking the BBB, was generated using a human microvascular cell line (hCMEC/D3), in the presence of flow. In the chip, a vessel of ~100µm diameter was interfaced with a 3D brain cancer cell culture (either U87, glioma neural stem or normal neural stem cell, embedded in a collagen-based ECM). The system was coupled with live-cell imaging and image analysis, which enabled tracking of cell-cell and cell-microenvironment interactions. Changes in gene expression and protein distribution in endothelial cells were successfully quantified, thus enabling the characterization of the influence of cancer cells population on the microvessel.

Conclusions: Development and optimization of the novel device has given us the opportunity to study the influence of glioma cells on normal brain endothelium, when agiogenesis does not occur. Crucially, this can be done in controlled, user-defined environment (i.e. choice of ECM components and stiffness, microvessel size and flow rate) unlike in animal models.

#40

Biomaterials and systems biology to guide breast cancer drug screening.

Alyssa Schwartz,1 Lauren Barney,2 Lauren Jansen,1 Christopher Hall,1 Aaron Meyer,3 Shelly R. Peyton2. 1 _University of Massachusetts, Amherst, MA;_ 2 _University of Massachusetts, Amherst, Amherst, MA;_ 3 _UCLA, MA_.

Improved in vitro models are needed to better understand cancer progression and bridge the gap between in vitro proof-of-concept studies, in vivo validation, and clinical application. Many methods exist to create biomaterial platforms, including hydrogels, which we use to study cells in contexts more akin to what they experience in vivo. Our lab has multiple approaches to create such biomaterials, based on combinations of poly(ethylene glycol) (PEG) with peptides and zwitterions. In this presentation, I will discuss our findings in using these cell culture environments to understand the role of the extracellular matrix (ECM) in controlling cancer cell innate drug response via adaptive signaling. Specifically, I will present data comparing the behavior of breast, prostate, and ovarian cancer cells to chemotherapy and targeted drugs when cultured as 3D spheroids, on 2D gels, and as a function of the stiffness of the tumor microenvironment. This approach uncovered that cells on 2D hydrogels and spheroids encapsulated in 3D hydrogels were less responsive to receptor tyrosine kinase (RTK)-targeting drugs sorafenib and lapatinib, but not cytotoxic drugs, compared to single cells in hydrogels and cells on plastic. We found that transcriptomic differences between these in vitro models and tumor xenografts did not reveal mechanisms of ECM-mediated resistance to sorafenib. However, a systems biology analysis of phospho-kinome data uncovered that variation in MEK phosphorylation was associated with RTK-targeted drug resistance. Using sorafenib as a model drug, we found that co-administration with a MEK inhibitor decreased ECM-mediated resistance in vitro and reduced in vivo tumor burden compared to sorafenib alone. In sum, we provide a novel strategy for identifying and overcoming ECM-mediated resistance mechanisms by performing drug screening, phospho-kinome analysis, and systems biology across multiple biomaterial environments. Our work suggests that different model systems are important for evaluating cell response to receptor tyrosine kinase inhibitors, whose efficacy depends on cell-ECM interactions.

#41

A novel high-throughput microfluidic drug screening platform using pancreatic ductal adenocarcinoma derived organoids.

Bart Kramer, Wijnand van Paassen, Luuk de Haan, Henriette Lanz, Jos Joore. _Mimetas B.V., Leiden, Netherlands_.

Pancreatic cancer is one of the deadliest tumors due to the limited treatment options and late diagnosis. Here, we describe a novel high throughput drug screening platform combining a microfluidic based 3D-culture plate, and the recently described pancreatic ductal adenocarcinoma (PDAC) derived organoids. The microfluidic plate is a high throughput microfluidic 3D cell culture platform, supporting physiologically relevant models with a minimal requirement of cell material and enabling a wide range of flow and co-culture options (e.g. with blood vessels). Organoids were derived from human PDAC xenografts and seeded in the microfluidic plate. The low amount of tissue material required (4000 cells per chip) and the high number of replicates on one plate (n=96 on a standard microtiter format plate) renders the microfluidic plate an efficient and cost-effective platform for drug screening and toxicity assays on complex, 3D models. Organoids were exposed to various chemotherapeutic drugs for 72 hours. The viability of the organoids before and after drug treatment is monitored with standard viability assays and subsequently used to generate dose response curves. In conclusion, we showed that the microfluidic plate can be used for high throughput drug screening assays and toxicity screening, and demonstrated its compatibility with human pancreatic PDAC derived organoids.

#42

The importance of intact tumor immune microenvironment in unpropagated 3D fresh patient tumoroids in immuno-oncology drug testing for immunogenic tumor cell death and T-cell activation.

Vijayendra Agrawal, Melanie Mediavilla-Varela, Melba Marie Page, Jenny Kreahling, Soner Altiok. _Nilogen Oncosystems, Tampa, FL_.

Background: The tumor stroma contains important components of the tumor microenvironment including fibroblasts, immune cells, and the extracellular matrix. While none of these cells are malignant, the dynamic interplay of these cells drive alterations in cellular functions. While informative, conventional 2D dissociated tumor models do not maintain the stromal-stoichiometry of the tumor microenvironment to accurately assess ex-vivo tumor cell viability and T-cell activation after drug treatment. In this study we performed a comparative assessment of tumor immune microenvironment, and therapy mediated changes in tumor cell viability and T-cell activation in live 3D tumoroids versus matched 2D dissociated cells of the same patients' fresh tumors side-by-side.

Methods: All human tumor samples were obtained with patients' consent and relevant IRB approval. Un-propagated live 3D tumoroids measuring 100-150 micron in size were prepared from fresh patient tumor samples. In parallel, the same patient tumor sample was enzymatically digested into 2D tumor dissociations to obtain single cell populations for direct comparison to live 3D tumoroids. Cytokine analysis was performed to determine immunomodulatory cytokine profile. To better understand the role of tumor extracellular matrix on immuno-oncology drug response, tumor models were treated with pembrolizumab (Keytruda) and atezolizumab (Tecentriq) (10ug/ml) for 48 hours and multiplex cytokine release assay, flow cytometry and high content confocal image analysis were performed.

Results: Comparative studies revealed that removal of extracellular matrix by digestive enzymes significantly affects tumor immune microenvironment as assessed by multiplex cytokine release assays. Flow cytometry analysis demonstrated differential T-cell and NK cell activation upon immuno-oncology drug treatment between the unpropagated 3D-tumoroid and dissociated cell suspension models. High-content imaging studies combined with flow death analysis indicated that tumor extracellular matrix plays an important role in tumor cell killing by immuno-oncology drugs.

Summary: Our data shows the importance of maintaining the stoichiometry and cell-to-cell interactions of the tumor microenvironment. Using a powerful combinatorial approach including high-content image analysis, flow cytometry, and cytokine analysis we are able to accurately assess therapy-mediated tumor cell death in a quantitative manner. Furthermore, these data demonstrate that intact tumor extracellular matrix within unpropagated 3D tumoroids is critical for a more accurate assessment of therapy-mediated changes in tumor immune microenvironment.

#43

A living biobank of triple-negative breast cancer organoids for screening drug sensitivity.

Malin Hong, Lin Gao, Wenlong Hu, Junying Qiu, Zheng Peng, Wenbin Zhou, Chang Zou. _Shenzhen People's Hospital, Shenzhen, China_.

Introduction: Triple Negative Breast Cancer (TNBC) is the only type of breast cancer for which no specific targeted therapy, it presents drug resistance to targeted therapies of hormomal therapy, HER2-targeting therapy and chemical treatment. At present, due to patient of TNBC suffered with drug resistance, there are no beneficial human cancer models to screening anti-cancer drug in vivo. In this study, we performed TNBC tissues from patients were used for a long-term expansion by using self-proliferating organoids to test drugs in ex vivo.

Methods Triple-negative breast cancer (TNBC) tissues from patients were cut and digested with the human tumor dissociation kit. The digested pellet was resuspended in cold Matrigel and 50μl drops of Matrigel-Cell suspension were allowed to solidify on prewarmed 24-well culture plates at 37℃ for 10 min. Upon completed solidification, cytokines and OAD12 medium was added advanced DMEM/F12 medium, the organoids were cultured at 37℃, 5% CO2 incubators. Medium was replaced every 1-2 weeks and TNBC organoids were passaged every 1-3 weeks. For immunofluorescence assay, TNBC organoids were fixed with 2% formaldehyde for 30 min, and then incubated with antibodies. For counting the percentage of tumoroids, four markers of CD44, CD24, CD133 and CD338 were used to identify tumoroids. IC50 of alazoparib (target PARP), ribociclib (CDK4/6) and SNX-2112 (Hsp90) were used for drug sensitivity screens through CCK-8 assay.

Results For establishing a living TNBCs tissues from patients, tissue samples were operated to isolate cells through mechanical and enzymatic digestion. Digested cells were resuspended with BME drops and seeded into organoid culture medium with various factors. The results showed that TNBC organoids were generated efficiently as well as term expansion over 10 passages. For testing whether TNBC organoids match the cutting TNBC tissues type, we performed a classical combined (CD44+; CD24-; CD133+; CD338-) markers in the tumoroids through immunofluorescence staining, the results showed that CD44 and CD133 were highly expressed in TNBC organoids and in the corresponding tissues. In addition, the flow cytometry assay was used to detect expression of positive, it showed that positive rates of tumoroids expressed up to 80%. In order to test TNBC tumoroids as functional in vitro cultured, talazoparib (target PARP), ribociclib (CDK4/6) and SNX-2112 (Hsp90) were used for drug sensitivity screens. The results indicated an effective response to 3 drugs yielding a single IC50. The tumoroids were sensitive to 3 drugs inhibiting PARP, CDK4/6 and Hsp90 signaling pathway, respectively.

Conclusions In this study, we reveal a novel culture system of living TNBC organoids. Described TNBC organoids are available for clinical treatment of drug sensitivity. It may provide a novel of drugs testing for anti-TNBC therapy in ex vivo.

#44

Assessment of pancreatic cancer 3D organotypic cultures as models for functional preclinical analysis.

Shannon R. Nelson,1 Sandra Roche,1 Fiona O'Neill,1 AnneMarie Larkin,1 John Crown,2 Naomi Walsh1. 1 _Dublin City University, Glasnevin, Ireland;_ 2 _St Vincent's University Hospital, Elm Park, Dublin, Ireland_.

Pancreatic ductal adenocarcinoma (PDAC) is the deadliest of all cancers, with a five-year survival rate of 8%. Decades of research has done little to improve patient outcomes. To identify key therapeutic strategies, appropriate research preclinical models are critical for testing the efficacy of drugs or downstream functional genomic analysis. Current methods of growing PDAC established cell lines in a 2D monolayer do not fully represent how the tumor cells grow in vivo as they fail to recapitulate the physiology of tumors in vivo due to different architecture, adherence structures and biochemical gradients. Patient-derived xenografts can retain the histological and genetic characteristics of their donor tumor and can remain stable across passages. Three-dimensional (3D) organoids grown ex vivo are tractable also retain the architecture, genotype and phenotype of a patients' tumor.

However, a detailed comparative assessment of the utility of organoids, matched 2D cell lines and 3D cell organotypic models to assess the complex biology, interactions, response to therapeutic regimens has not been performed.

Using our unique protocol, we have established PDAC organoids and isogenetically matched 2D cell lines derived from PDAC PDX tumors. To establish if these matched 2D cell lines grown in 3D can recapitulate the organoid model, we cultured the matched cell lines in 3D organoid media in non-adherent, Matrigel containing cultures. The expression of PDAC histological markers between the original tumor, the PDX tumor, organoid and matched cell line in 3D and 2D has identified differential expression of tumor initiating cell (TIC) markers. RNA-seq transcriptomics of the 2D cell line models, matched recapitulated 3D organotypic models, organoids, PDX-tumor and patient tumor will reveal the application of these models for ex vivo downstream functional analysis.

#45

Patient-derived 3D tumor cultures for compound screening and pre-clinical drug development.

Sander Basten,1 Bram Herpers,1 Kuan Yan,1 Julia Schueler,2 Leo S. Price1. 1 _OcellO, Leiden, Netherlands;_ 2 _Charles River Laboratories, Freiburg, Germany_.

Patient-derived xenograft (PDX) models from human cancers allow propagation, characterization and drug testing on a large panel of tumor variants. To expand the potential of these human-relevant PDX models, we sought to develop 3D ex vivo culture methods for PDX-derived tumor cells that show in vivo-like growth characteristics such as polarization and invasion, and allow measurement of response to therapeutics. By embedding dissociated PDX material in extracellular matrix enriched hydrogels we generate ex vivo cultures that form organotypic 3D tissues driven by relevant interaction with the stromal microenvironment in 384 well plates. Our automated high content screening platform with image analysis powered by OMiner software is used to measure morphologic features of the microtissues. This combination creates a unique high throughput ex vivo PDX screening platform that not only allows efficient profiling drug of responses and quantify tumor spheroid volume, apoptosis and tumor invasion, but also enables selection of the optimal PDX tumor models for subsequent validation of candidates in vivo.

Results Hydrogel and growth media composition were optimized to support growth of tumor tissues in vitro from cells derived from various tumor indications, including breast, pancreas, colon, melanoma, and lung cancer PDX tumors. Tumor tissues were cultured in a 384-well format and used to test standard of care chemotherapeutics (e.g. cisplatin, 5-Fu), small molecules (e.g. lapatinib, paclitaxel), and antibodies/ADCs (Trastuzumab, T-DM-1). High content 3D image analysis and data analysis generates a tumor model specific drug profile, reporting therapeutically relevant responses such as cell growth, killing and invasion. These responses serve as a benchmark allowing comparison to novel and experimental drugs, as well as responses across tumors with different mutation profiles. We identified an absolute dependency of a pancreatic PDX model to the WNT-signaling pathway potentiator R-spondin. This allows specific stimulation and targeting of a pathway that is particularly relevant in the development pancreatic cancer.

Conclusions In contrast to organoid cultures, short-term ex vivo culture of tumor captures the heterogeneity of the primary tumor as well as enabling access to large collections of well-characterized tumors. We established several PDX model-derived 3D tumor cultures in which standard-of-care, targeted therapies and novel therapeutic agents against specific pathways can efficiently be screened, based on therapeutically relevant parameters and their changing morphological profile. This method enables both the ex vivo selection of drug candidates in a pre-clinical setting as well as efficient selection of PDX models for in vivo follow-up in the same tumor. This highly translational in vitro-in vivo PDX pipeline is expected to reduce attrition and increase efficiency in early drug-discovery.

#46

3D reconstruction and quantitative analysis of histology for prostate cancer.

Pekka Ruusuvuori,1 Kimmo Kartasalo,1 Mira Valkonen,1 Masi Valkonen,1 Tapio Visakorpi,1 Matti Nykter,1 Leena Latonen2. 1 _University of Tampere, Tampere, Finland;_ 2 _University of Eastern Finland, Kuopio, Finland_.

The integration of serial sectioning of tissue, digital whole slide imaging (WSI) and computational reconstruction algorithms enable the examination of histological samples in 3D at subcellular resolution. This allows visualizing and analyzing the imaged sample in its true three-dimensional context, offering a more comprehensive view on its spatial and morphological characteristics than that obtained via typical 2D examination. The advantages of reconstructing 3D models computationally using WSI over direct 3D imaging include the combination of high resolution, large sample sizes and compatibility with existing biochemical techniques such as in situ hybridization, immunohistochemistry and established histological staining and interpretation protocols.

For this purpose, we developed a software pipeline to perform routine 3D reconstruction tasks for large image sizes and datasets in a fully automatic manner. The steps in our 3D reconstruction process are image acquisition, alignment of images to a shared coordinate space, and visualization of the reconstructed 3D data. We optimized algorithm selection and computationally intensive hyperparameter tuning using a quantitative benchmarking framework and Bayesian optimization. Further, we applied our existing pipeline for feature based analysis of tissue and extended it into 3D, allowing quantification and visualization of hundreds of features characterizing the tissue.

We currently apply this system to characterize prostate cancer tumors. Prostate cancer is a heterogenous, often multifocal disease. In order to understand why and how certain tumor foci develop to a life-threatening disease over others, the tissue growth patterns and evolution of tumors with different genetic backgrounds need to be studied. We use mouse models of prostate cancer to study early tumor development connected to common genetic cancer alterations. By computing hundreds of features from the histology, and studying these in the spatial context, we gain important information of tumor characteristics as well as intra- and intertumor heterogeneity. In the future, we will further scale up the protocol to perform 3D reconstruction for serially sectioned human prostates in the near future.

#47

Analyzing immune cell infiltration of cancer spheroids in a 3D cell culture platform.

Sei Hien Lim,1 Chee Mun Kuan,1 Marco Campisi,2 Valeria Chiono,2 Andrea Pavesi3. 1 _AIM Biotech Pte Ltd, Singapore;_ 2 _Politecnico di Torino, Torino, Italy;_ 3 _Institute of Molecular and Cell Biology :: Agency for Science, Technology and Research (A*STAR), Singapore_.

The adoption of in vitro 3D cell culture models as a bridge between conventional 2D cell culture models and the complex in vivo animal models has been increasing. A carefully designed 3D model can run biological assays with animal model-like complexities but with the simplicity and affordability of traditional cell culture. For example, traditional 2D and transwell assays of immune cell infiltration efficiency are unrealistic, as cell migration is gravity-driven. AIM 3D cell culture chips offer a more realistic immune cell infiltration model by compartmentalizing immune cells and cancer spheroids in parallel channels. The chips consist of a 3D hydrogel channel and two flanking media channels. Cancer spheroids are cultured within the hydrogel channel while immune cells are seeded in one of the media channels. The seeded immune cells are required to actively invade and seek out target cancer cells in 3D hydrogel in AIM chips before they can infiltrate the cancer spheroids. This is a process similar to immune cell infiltration in vivo. By utilizing high content confocal imaging, fluorescent-labeled immune cells that migrate into the 3D hydrogel can be visualized and quantified. This assay, in combination with adoptive T cell therapy or immune checkpoint blockade, can determine the roles of tumor infiltrated lymphocytes in immunotherapy through quantifying the live: dead ratio of cancer spheroids in the chips. This is particularly useful as a quality control tool for cellular adoptive immunotherapy where the infiltration efficiency and tumoricidal activity of engineered immune cells can be accessed in vitro. In summary, AIM 3D cell culture chips create a more physiologically relevant 3D microenvironment for visualizing immune cell infiltration of cancer spheroids.

#48

Cellular dynamics and heterogeneity of active Kras induced-gastric pre-neoplasia in mouse.

Eunyoung Choi,1 Paige N. Vega,1 Jimin Min,1 Amy C. Engevik,1 janice A. Williams,1 Qing Yang,1 Loraine M. Patterson,2 Alan J. Simmons,1 Ken S. Lau,1 Scott T. Magness,2 James R. Goldenring1. 1 _Vanderbilt Univ. Medical Ctr., Nashville, TN;_ 2 _University of North Carolina at Chapel Hill, Chapel Hill, NC_.

Cellular heterogeneity in cancer development is considered as a source of cancer cell generation and drug resistance. However, cellular heterogeneity has not been fully eluciated during metaplasia progression to gastric pre-neoplasia and neoplasia in gastric carcinogenesis. Recent investigations have suggested that Ras activation, rather than Ras mutation, may promote the gastric carcinogenesis. We have recently established several in vitro metaplastic/pre-neoplastic organoid lines; Meta3-metaplasia and Meta4-pre-neoplasia, derived from mouse stomachs induced active Kras expression in chief cells and investigated cellular heterogeneity and plasticity that may lead to the progression of metaplasia to gastric neoplasia. While both Meta3 and Meta4 initially formed spherical or spheroidal structures and generated complicated budding or cyst-like structures between 2 and 4 weeks, Meta4 cells displayed more disorganized structures and aggressive behaviors than Meta3. We also examined altered cellular behaviors, structural changes and survival of Meta4 organoids after MEK inhibition which is a downstream mediator of Kras signaling pathway. The Meta4 treated with Selumetinib for 3 days died or did not show an increase in size at various concentrations. However, some Meta4 organoids showed an ability to survive and retained a spheroidal structure despite the Selumetinib treatment. Single cell-RNA sequencing (scRNA-seq) data analysis demonstrated distinct characteristics of Meta4 cells as pre-neoplastic cells and a signature of presence and heterogeneity of pre-neoplasia stem cell (PNSC) populations. The scRNA-seq data analysis also revealed that cellular behavior and dynamics of pre-neoplastic cells can be controlled by MEK inhibition, but the PNSC populations still maintain the niche for stem-like cells in gastric pre-neoplasia. The isolated two PNSC populations showed functional differences and the stemness of the PNSCs in pre-neoplastic stage through high-throughput functional analysis. Therefore, our study concludes that putative gastric cancer stem cells in gastric pre-neoplasia might be the source for maintenance of heterogeneity and contribute to the progression of gastric pre-neoplasia to cancer.

#49

ET-1 receptor blockade in engineered 3D high-grade serous ovarian cancer tumoroids.

Laura Rosanò,1 Judith Pape,2 Umber Cheema,2 Marilena Loizidou,3 Anna Bagnato1. 1 _Regina Elena National Cancer Inst., Rome, Italy;_ 2 _UCL Institute of Orthopaedics and Musculoskeletal Sciences, UCL Division of Surgery and Interventional Science, Stanmore Campus, London, United Kingdom;_ 3 _UCL Division of Surgery and Interventional Science, Royal Free Campus,, London, United Kingdom_.

High-grade serous ovarian cancer (HG-SOC), accounting for 70-80% of ovarian cancer deaths, is characterized by widespred and rapid metastatic nature, suggesting that unraveling determinants of metastasis may implement new therapeutic strategies on clinical care. In this tumor, autocrine or paracrine activation of the endothelin-1 receptors (ET-1R) are recognized drivers of tumor progression, promoting epithelial-mesenchymal transition (EMT), invasion, and metastasis, as well as tumor angiogenesis and lymphangiogenesis. However, since the metastatic spread and response to therapy are influenced by the extracellular matrix (ECM) density and composition of the surrounding tumour microenvironment (TME), the development of a three-dimensional in vitro model mimicking the in vivo tumors is necessary to better understand the dynamic interactions between tumor cells and TME elements, including fibroblasts, endothelial cells and the ECM, regulated by ET-1R. With this in mind, we have developed HG-SOC tumoroids by engineering a dense central artificial cancer mass (ACM) containing HG-SOC cells, nested within a compressed hydrogel representing the stromal compartment comprising type I collagen, laminin, fibronectin, and stromal cells (fibroblasts and endothelial cells). After 21 days, ET-1-stimulated HG-SOC cells showed an altered migration pattern and formed cellular aggregates, mimicking micrometastases that invaded the stroma. As compared to control cells, ET-1 treated cells showed a higher number of cellular aggregates, which were reduced by treatment with the dual receptor antagonist macitentan. In addition, ET-1 increased the size of the invading aggregates compared to control cells. These findings suggest that HG-SOC tumoroids might be useful to investigate the cross-talk between HG-SOC cells and TME in cancer progression and the response to combinatorial treatment, including ET-1R antagonist.

#50

Anti angiogenesis tyrosine kinase inhibitor vandetanib as a radiosensitizer for hepatocellular carcinoma.

Sami Znati. _University College London, London, United Kingdom_.

Background: The hypervascularity of Hepatocellular Carcinoma (HCC) has let anti-angiogenic therapies to be used clinically. Vandetanib is a selective inhibitor of VEGFR-II, -III and other pathways of angiogenesis. Previous investigators have suggested that vandetanib may be used as a radiosensitizer with radiotherapy to treat HCC; we evaluated the mechanism of this treatment effect.

Methods: A panel of 20 human HCC cell lines was treated with drug plus radiation to measure radiosensitization by 2-D clonogenic survival assays. A gap was created in confluent cells and gap-filling invasion was measured following vandetanib and radiation treatment. Three-dimensional-spheroids of HCC cells were grown alone or in co-culture with fibroblasts and treated with 5 or 10 μM vandetanib alone or in conjunction with 4 Gy radiation. Spheroid growth and invasion was measured daily following treatment and results compared to a control without treatment. A paired t-test was performed for all groups to measure significance between treatments and Coefficient of Drug Interaction (CDI) and two-factor ANOVA was calculated to measure interaction.

Results: In 2-D clonogenic survival assays, HCC cell lines showed inhibition when treated with vandetanib but no significant intrinsic radiosensitization was observed. 2-D migration assays showed a significant, dose-dependent reduction in migration after vandetanib treatment (p<0.05) and additive reduction in cells treated with vandetanib plus radiation (p<0.05). In 3-D spheroid assays, Hepa1-6 cells showed significant reduction in growth after 4 Gy radiation treatment versus controls (p<0.05). When Hepa1-6 cells were grown in co-culture with NIH3T3 fibroblasts, growth compared with controls was significantly reduced after treatment with 4 Gy radiation, 5 and 10 μM vandetanib, and combinations thereof (p<0.01). Similar results were demonstrated for Hep55.1-C grown in spheroids alone (p<0.05) and in co-culture with NIH3T3 (p<0.01). In human cell lines, 3-D spheroids of HLF cells treated with both vandetanib and radiation showed significant growth inhibition when compared to 4 Gy radiation (p<0.005) and 5 µM vandetanib (p<0.0001). In 3-D co-culture of HLF and NIH3T3 receiving combination treatment, invasion was significantly reduced over radiation (p<0.05) and vandetanib (p<0.0001) and in 3-D co-culture of JHH7 and LX2 cells, similar results were observed, with spheroid growth inhibited in combination treatment compared to 5 µM vandetanib (p<0.05) and 4 Gy radiation (p<0.005).

Conclusions: We have shown additive radiosensitization effects of vandetanib in 2-D migration assays and 3-D spheroid culture. Co-culture of HCC cells with fibroblasts showed significant inhibition among groups treated with both vandetanib and radiation therapy. Future studies will examine the role of immune cells in the mechanism of action of this novel combination therapy.

#51

Propagation of human colon, mammary, and lung cancer organoids in growth medium utilizing tissue-specific reagent kits and ready-to-use Wnt-3a and R-Spondin1 conditioned media.

James Clinton, Allison Ruchinskas, Himanshi Desai, Nanda Mahashetty, Dezhong Yin. _ATCC, Gaithersburg, MD_.

Three-dimensional "organoid" growth of tumors may represent a more physiologically relevant in vitro model system than traditional two-dimensional monolayer cultures of cancer cell lines. With the increased availability of cryopreserved human cancer organoids generated by academic laboratories, large-scale biobanking initiatives, and commercial sources, there is an unmet need for simplified, standardized, and cost-effective methods for preparation of the complex growth media required by these models. Human organoid culture media contains a variety of recombinant proteins, small molecules, and other growth factors that are costly to purchase in small-scale, time consuming to reconstitute and aliquot, and have varying stability and shelf life once prepared. Organoid culture media often also utilizes undefined conditioned media (CM) from one or more engineered cell lines that must be cultured separately, requiring additional time and resources to maintain. These lines secrete critical growth factors and the CM generated must be carefully prepared, collected, and stored. CM is subject to variability in activity levels due to batch-to-batch and protocol-to-protocol differences that can affect subsequent organoid culture performance. To address these challenges and to facilitate the wide-spread adoption of human organoid culture we are developing reagent kits containing essential growth medium components in an individually lyophilized format for long-term storage and easy single-use preparation of organoid growth media for a variety of tissue types including human colon, mammary, and lung. We compared growth medium formulated with our components with small-scale "homebrew" preparations and found equivalent or better culture performance (as calculated by doubling times) and similar morphology. Both approaches were able to generate media capable of supporting growth for at least three passages (~30 days) in 15 different organoid models. Additionally, we compared protocols and cell lines for CM generation to support organoid culture and found that an optimized, scaled-up production method could generate Wnt-3a and R-spondin1 CM with higher activity levels (up to 52%) and reduced lot-lot variability in comparison with commonly used small-scale protocols. We found that the optimized protocols produced CM that was stable for at least 2 months at 4°C and at least 6 months at -20°C, and was resilient against freezing and thawing. Previously frozen CM was also able to support organoid growth in culture. These results show that our tissue-specific kits and ready-to-use CM can provide a simplified, cost effective method to support the long-term propagation of human cancer organoids from multiple tissues and cancer types.

#52

**PDX-derived 3D InSight** TM **tumor microtissues as** ex-vivo **human experimental models for evaluating therapeutic responses.**

Francesca Chiovaro,1 Irina Agarkova,1 Nicole Buschmann,1 Chloe' Pichon,1 Teresa Langova,1 Armin Maier,2 Julia Schueler,2 Patrick Guye1. 1 _InSphero AG, Zürich, Switzerland;_ 2 _Charles River DRS Germany GmbH, Freiburg, Germany_.

Background The selection of appropriate preclinical models comes always with the major question on how accurately and robustly they can represent the complexity of human disease. Patient-derived xenograft (PDX) models faithfully preserve the biological features and the genetic expression profile of human tumor specimens. However, one limiting aspect of patient-derived models is the replacement of the human host microenvironment by murine stroma within the tumor. Lack of cross-species compatibility compromises the induction of a broad range of signaling pathways that cannot be entirely recapitulated. With our in vitro 3D InSightTM Tumor Microtissues derived from PDX lines, we provide a relevant physiological environment and a strategy to assess candidate drugs for novel therapeutic approaches.

Aim Development of in vitro 3D InSightTM Tumor Microtissues from PDX lines aimed to retain the cellular heterogeneity found in the original human tumor tissue.

Material & Methods and Results PDX cell suspensions of lung, breast and melanoma origin were successfully used to assess 3D aggregation in 96-well format and characterized over 10 days in culture. After careful removal of mouse cell contaminants in each PDX sample, 3D PDX cell cultures were supplied with exogenous normal human dermal fibroblasts (nHDF). Furthermore, to provide a more physiological cancer niche, PDX cells were also co-cultured with tumor-matched cancer-associated fibroblasts (CAFs). 3D in vitro tumors were analyzed histologically and cancer phenotypic alterations were evaluated through the analysis of epithelial-to-mesenchymal transition (EMT) markers. The morphology, viability and growth rate of PDX-derived microtumors were assessed by size analysis (cell scanner) and ATP assay. To assess the distribution of various cell populations within the tumor, 3D PDX samples were screened for standard stromal vs. epithelial-tumor cells markers (e.g. FAP, pan-CK, E-Cadherin), and diagnostic cancer type-specific biomarkers. 3D PDX samples were also employed to investigate the efficacy of specific targeted therapies based on distinct molecular signatures of PDX tumor models. Immunohistochemistry assessment of 3D microtumors validated the resemblance with their respective PDX tumor models. 3D tumor growth rate and cell behavior observations reflected the diversity of disease progression in vivo.

Conclusion Further efforts will focus on employing this platform to establish more complex co-cultures with integration of additional relevant stromal and immune cells, to enable a reliable preclinical translational research of tumor/immune cell interactions. We suggest that in vitro 3D PDX models offer a more suitable and robust approach to expedite faithful efficacy assessment of immunomodulators and approval of optimal drug candidates.

#53

Ex vivo **models of glioblastoma: a comparison of 3D tissues and patient-derived xenografts to clinical response.**

Ashley M. Smith,1 Lillia Holmes,1 Lacey E. Dobrolecki,2 Charles Kanos,3 Stephen Gardner,3 Philip Hodge,3 Michael Lynn,3 Jeff Edenfield,3 Michael T. Lewis,2 Howland E. Crosswell,1 Teresa M. DesRochers1. 1 _KIYATEC, Inc., Greenville, SC;_ 2 _StemMed, Houston, TX;_ 3 _Greenville Health System, Greenville, SC_.

Standard first-line treatment of newly diagnosed Glioblastoma (GBM) is primarily radiotherapy and Temozolomide (TMZ) with a strong NCCN recommendation of enrollment in clinical trials. The only biomarker that can currently provide a stratification of strong and poor responders to standard of care is methylation of MGMT which indicates a median PFS of 10.3 months for methylated patients and 5.3 months for unmethylated patients. Development of patient-specific in vitro models of GBM for rapid testing of therapeutic options may yield more efficacious therapies and faster, more accurate assignation of therapies to each patient. To that end, we have developed a multi-faceted, patient-based 3D GBM model with modularity that facilitates increasing levels of model complexity such as the inclusion of immune cell components. Stable populations of glioma stem cells (GSC) from 24 of 41 patient samples have been successfully established, verified for stemness through limited dilution in in vitro and in vivo studies, and cultured long-term with minimal molecular changes, as determined from genetic analyses including RT-PCR arrays and MGMT methylation status, flow cytometry, and IHC. These cell populations have been used to establish and validate our 3D model system as well as generate comparable patient-derived xenografts (PDX). KIYATEC's 3D microtumor in our 3DKUBE™ perfusion system provides a moderate throughput, dynamic system that is easily controlled to establish complex microtumors. GSC cell populations were cultured in monoculture only, co-cultured with human brain endothelial cells (HBEC), and tri-cultured with HBEC and CD14+ peripheral blood mononuclear cells (PBMC). Three microtumors were characterized by drug response to TMZ and axitinib, IHC, and molecular profiling including RNA expression and MGMT methylation status. Interestingly, increasing the complexity of the microtumor was capable of reestablishing the primary tumor MGMT methylation status if it were lost during culturing. We also generated PDX models from the same 3 patient tumor tissues as the above 3D models. PDX are a low throughput, time consuming, and expensive model that are still utilized for many systemic drug response studies and therefore a good comparator to the 3D tissue models we established. In vitro drug response in the 3D tissue models has been compared to both the matched PDX in vivo drug response and the patient's clinical response to TMZ and MGMT methylation. Our data supports that KIYATEC's complex patient-derived GBM model can be successfully used to identify, screen, and characterize novel treatments of GBM.

#54

Characterization of patient-derived organoids cultured on a gas-rich, liquid-liquid interface.

James T. Shoemaker,1 Katherine R. Richardson,1 Jamie Arnst,2 Adam Marcus,2 Jelena Vukasinovic1. 1 _Lena Biosciences, Atlanta, GA;_ 2 _Winship Cancer Institute at Emory University, Atlanta, GA_.

Modeling tissues in vitro has long consisted of two-dimensional (2D) monolayer cultures grown in a dish, flask, or multi-well plate. While these methods remain adequate for some applications, the advent of personalized cancer therapies has necessitated the development of more complex models that more accurately represent the in vivo environment. Attempting to characterize tumor cell biology and response to candidate pharmaceuticals in 2D cultures has led to results that fail to translate to clinical therapies. A key source of this failure is the loss of cell diversity due to selective pressure resulting in cell populations that favor adhering to a rigid substrate. Three-dimensional (3D) cell culture methods have produced models that capture many of the dynamics of the in vivo tissue environment. Patient-derived organoids (PDOs) are quickly becoming the most attractive solution for creating predictive, patient-specific tumor models in vitro. Typically grown as spheroids, these models still present limitations in terms of cell survival and recapitulation of tumor development.

Lena Biosciences has previously developed complex, vascularized tissue models using patented 3D scaffolds and a perfusion system. Hepatocytes grown in this system demonstrated increased cell viability and recovery of cytochrome P450 enzyme activity compared to 2D controls. Integral to the perfusion system is a pure, synthetic liquid that is twice as dense as and immiscible with cell culture medium and reagents. It is permissive to atmospheric gases, providing a gas-rich, liquid-liquid interface below 3D cultures maintained in the system. We hypothesized that PDOs could be cultured without a 3D scaffold, directly at the interface between the cell culture medium and this synthetic liquid. A specific preparation of this interface resulted in lung-derived PDOs that spontaneously formed dense, 3D, tissue-like structures that showed increased viability (as measured by a 50% reduction in LDH release) and cellular respiration (three-fold increase as measured by an alamarBlue assay). Ongoing studies are focused on characterizing PDOs from other patients and other tissues as they develop in long-term in vitro studies. Should this PDO model prove to be a faithful representation of in vivo tumor development, this system would be ideal for applications such as testing drug efficacy and studying mechanisms of tumor metastasis.

#55

**Predicting drug sensitivity in patient derived** ex vivo **3D mesothelioma cultures.**

Robin Cornelissen,1 Daphne Dumouline,1 Jan vd Thusen,1 Sander Basten,2 Fanny Grillet,3 Kuan Yan,2 Leo Price,3 Willemijn Vader3. 1 _Erasmus MC, Rotterdam, Netherlands;_ 2 _Ocello, Leiden, Netherlands;_ 3 _Vitroscan, Leiden, Netherlands_.

Background

Malignant pleural mesothelioma (MPM) is almost exclusively linked to asbestos exposure with a latency period that is usually more than 30 years. Once diagnosis is made, the prognosis is poor. Systemic treatment combines an antifolate combined with platinum, resulting in modest responses for 40% of the patients. Recently, the addition of bevacizumab to this combination has shown to be of benefit. To date it is impossible to identify the patients who will respond before start of treatment. Second line treatments (either gemcitabine or vinorelbine) have shown to be of benefit in only a small percentage of patients. Tools to improve stratification for systemic treatment in the clinic would be of great value.

Our technology based on image analysis of 3D tumor cultures accommodates accurate evaluation of drug sensitivity with small amounts of heterogeneous tumor material (tumor, pleural effusion). We have initiated clinical trials to establish the value of the platform for treatment response prediction for cancer patients, including mesothelioma. Based on the results from these trials we plan to develop diagnostics to predict drug responses for cancer patients.

Methods

3D mesothelioma cultures embedded in a protein-rich hydrogel are generated from pleural effusion of chemo naïve patients, and exposed to standard-of-care therapies, targeted therapies and drug combinations. An automated high content screening platform measures cell and tissue morphology, and reports responses such as tumor cell killing, growth arrest, apoptosis, and local invasion. Morphological features are per drug selected as standard read-outs for the response. We correlate clinical response on standard of care drugs with drug response of patients' tumor cultures.

Results

We present first results of drug sensitivity in patient derived ex vivo 3D pleural effusion cultures. Results are generated within three weeks after the pleural fluid is obtained from the patients. Take rates are >80%. Standard-of-care therapies were tested and results are compared with clinical response. Patient specific drug responses are identified.

Conclusion

Ex vivo 3D mesothelioma cultures enable drug sensitivity testing in weeks. Measuring patient-specific treatment responses to standard-of-care drugs, and potential novel therapeutic approaches offer opportunities to personalize treatment for mesothelioma patients. Ongoing trials will reveal the correlation of our in vitro test with treatment responses and relevant diagnostics parameters in the clinic.

Keywords: predictive test, mesothelioma, ex vivo 3D tumor cultures

#56

Establishment and characterization of new tumor xenografts and cancer cell lines from EBV positive nasopharyngeal carcinoma.

Weitao Lin,1 Yim Ling Yip,1 Lin Jia,1 Wen Deng,1 Kwok Wai Lo,2 Chin Man Tsang,1 Maria Li Lung,1 George Sai Tsao1. 1 _Univ. of Hong Kong, Pokfulam, Hong Kong;_ 2 _Chinese Univ. of Hong Kong, Satin, Hong Kong_.

Nasopharyngeal carcinoma (NPC) is rare worldwide but common in southern China, including Hong Kong. The endemic NPC among southern Chinese is typically non-keratinizing carcinoma, which is almost 100% associated with Epstein-Barr virus (EBV) infection. Patient-derived xenografts (PDXs), given their close resemblance with patient tumors, serve as important models in preclinical evaluation for novel therapeutic drugs. For unclear reasons, it has been difficult to establish NPC PDXs in vivo and cell lines in vitro. Currently, there are few NPC PDXs available for research, including X666, X2117, C15, C17 and C18. However, all of them have been passaged in nude mice for over 25 years and may deviate from their original NPC tumors in patients. In vitro, C666-1 is the only EBV-positive (EBV+ve) NPC cell line which has been used extensively in investigations. C666-1 was established from the NPC xenograft (X666), which had been propagated for a long period of time. Most if not all the other previously reported NPC cell lines have lost their EBV episomes upon in vitro propagation. Furthermore, many of these reported NPC cell lines have been shown with genetic contamination of HeLa cells. Hence, their applications in NPC studies are limited. The lack of representative nasopharyngeal carcinoma (NPC) models has also seriously hampered research on EBV carcinogenesis and preclinical studies in NPC.

In this study, we report the successful establishment and comprehensive characterization of four new NPC PDXs (all EBV+ve) and three NPC cell lines (one EBV+ve; two EBV-ve). These newly established EBV+ve NPC PDXs and cell lines significantly recapitulate the mutation profiles of their original NPC tumors, and harbor common genetic alterations reported in NPC, which supports their potential applications in the investigations of NPC pathogenesis. We postulate that lytic EBV reactivation may be an intrinsic barrier to the successful establishment of EBV+ve NPC PDXs and cell lines. The ROCK inhibitor (Y-27632) was shown to be able to suppress epithelial differentiation and lytic reactivation of EBV in infected NPC cells. Establishment of a new EBV-positive NPC cell line, NPC43, was achieved directly from patient NPC tissues by including Y-27632 in culture medium. NPC43 cells exhibited tumorigenicity in immunodeficient mice, and could be induced to undergo EBV lytic reactivation with production of infectious visions. The establishment and characterization of new NPC PDXs and cell lines will provide valuable experimental tools for NPC and EBV research. Funding support: Research Grant Council, Hong Kong: AoE NPC grant (AoE/M-06/08), Theme-based Research Scheme grant (T12-401/13-R) and Collaborative Research Fund (C7027-16G), General Research Fund (106140069, 106160185, 17111516, 17110315) and HMRF grant (04151726, 13142201).

#57

**Development of an** in vitro **3D model system for testing PD-1/ PDL-1 immune checkpoint inhibitors.**

Nicole Buschmann, Silvan Strebel, Francesca Chiovaro, Patrick Guye, Irina Agarkova. _InSphero AG, Zürich, Switzerland_.

Background Immune checkpoint inhibitors provide a new hope for cancer patients not responding to chemotherapy by removing the tumor protection against immune cell attacks. Nevertheless, only a subset of patients responds well to these therapies, and recent studies demonstrated that the mechanisms of action are more complex than expected, often involving multiple cell types of the immune system. Importantly, for a better understanding and assessment of immune cell eliciting cell tumor death, there is a need to have fully human, complex 3D in vitro models. We developed a 3D tumor-immune cell coculture model and show here acquired insights on the modulatory capacity of immune checkpoint inhibitors. Aim In the present study we have developed a 3D tumor - immune cell coculture model and evaluated its application to study the effect of immune checkpoint inhibitors on tumor viability, cytokine secretion, and immune cell infiltration. Material & Methods GFP-labelled A549 tumor cells were aggregated together with human dermal fibroblast to produce 3D tumor microtissues in Akura 96-well and 384-well format. Immune cells were pre-treated using different activation protocols to induce priming and pT-cell exhaustion. 3D tumor-immune cell cocultures were treated for 10 days with Nivolumab, its corresponding isotype and non-treated controls. Tumor viability was measured by GFP-fluorescence over time. Additionally, we have evaluated the effect of nivolumab by measuring the microtissue size by automatic stage fluorescence microscopy. Cytokine levels of IL-2, TNFα, IFNγ and GS-CSM were measured with a MAGPIXTM Luminex system and were monitored over time. The validation of immune cell infiltration and characterization of tumor and immune cell specific markers was based on histological analysis. Results Based on tumor fluorescence we have shown a superior effect of Nivolumab versus its isotype control antibody on tumor viability at the end of the treatment period. The cytokine analysis of supernatants has shown higher levels of IFNγ and GS-CSM in the wells treated with Nivolumab compared to the isotype control. CoCultures with PBMCs have shown stronger activation upon treatment compared to the T-cells alone. The histological analysis has demonstrated a higher number of T-cell infiltration and activation, as well as a stronger induction of apoptosis in the tumor microtissues treated with Nivolumab. Conclusion In summary, our novel in vitro immuno-oncology model system allows high throughput screening of immune check point inhibitors alone and in combination with other anti-cancer drugs for making it efficient for a broad range of tumor diseases.

#58

High throughput 3D tumor model drug screening technology.

Stephanie L. Ham, Emily Mulvany, Hossein Tavana. _OncoSolutions LLC, Akron, OH_.

Two-dimensional (2D) cancer cell cultures, or monolayers, remain the standard for testing cancer drugs prior to animal studies due to their simplicity and compatibility with standard plates and commercially available robotic tools. However, monolayer cell assays often fail to predict the efficacy of cancer drug candidates resulting in 80% of cancer drugs failing in animal drug studies and contributing to the extremely high costs of cancer drug discovery. Unlike solid tumors, the standard 2D cell cultures allow cancer cells unrestricted access to nutrients and oxygen and lack intercellular communications that result in inaccurate drug efficacy data. Three-dimensional (3D) tumor models are biologically relevant cell cultures that reproduce key properties such as diffusion limitations and cellular network of solid tumors that influence cancer drug efficacy. To promote the use of 3D cancer cell cultures in oncology drug discovery, OncoSolutions has engineered a novel technology to generate biologically relevant 3D tumor models that uniquely meets the needs of the pharmaceutical industry. OncoSolutions' technology is highly robust and reliable for drug screening and available in high throughput format (1536 microplate) to accommodate the large drug libraries of the pharmaceutical industry. Importantly, the technology utilizes standard microplates that are compatible with commercially available automated liquid handling and analysis tools to reduce associated costs and increase speed for formation and analyses. OncoSolutions' groundbreaking 3D culture technology is based on the immiscibility of highly aqueous solutions of polyethylene glycol (PEG) and dextran (DEX) to form a stable two-phase system. Cancer cells are mixed with the denser aqueous DEX phase solution and robotically dispensed as a nanodrop into a well of a standard microwell plate containing the immersion aqueous PEG phase. Cells remain entrapped in the nanodrop to autonomously form a single, compact 3D culture per well of the microplate within 24 hours. Unlike existing techniques, our technology quickly and reliably forms highly reproducible 3D cancer cell cultures with a standard deviation of less than 10% of the average 3D culture diameter within microplates. OncoSolutions' technology has been used for biologically relevant standard chemotherapeutic testing and displayed superior robustness through Z-factor measurement (a statistical robustness measurement). In addition to evaluating the viability response of 3D cancer cell cultures generated with OncoSolutions' technology, an assay for determining the apoptotic effects on 3D cancer cell cultures following drug treatment was optimized. Importantly, tumor models grown with OncoSolutions' technology have provided more predictive models of animal drug responses than standard monolayer cultures and can reduce the time and costs associated with the high failure rate of cancer drugs in animal studies.

### Cell-cell and Cell Matrix Interactions

#59

The pioneer transcription factor FOXA2 regulates integrin alpha 1 expression controlling prostate cancer bone colonization.

Zachary M. Connelly,1 Shu Yang,1 Nazih Khater,1 A. Wayne Orr,1 Renjie Jin,2 David Degraff,3 Colm Morrissey,4 Eva Corey,4 Robert Matusik,2 Xiuping Yu1. 1 _LSUHSC Shreveport, Shreveport, LA;_ 2 _Vanderbilt University Medical School, Nashville, TN;_ 3 _Penn State University College of Medicine, Hershey, PA;_ 4 _University of Washington, Seattle, WA_.

Introduction. Approximately 30,000 men will die from prostate cancer (PCa) in 2018. When localized and detected early, it has a low rate of progression and metastasis, with successful treatment. However, some patients may progress to a more lethal castrate-resistant PCa. While this occurs, frequent bone metastases will arise, and patient prognosis typically falls to 3-5 years due to lack of therapy available. The purpose of this study was to unveil the mechanisms of PCa bone colonization, allowing us to identify novel targeted therapies to prevent and treat PCa.

Methods. Gene expression profiling studies revealed a subset of metastatic PCa samples express the pioneer forkhead transcription factor, FOXA2. Consistently, we found FOXA2 abundance in a sample set of human PCa bone metastases, which may suggest a functional role for FOXA2. Protein analysis of common PCa cell lines revealed high levels of FOXA2 in aggressive bone metastatic PCa PC3 cells. To understand the role of FOXA2 in PCa metastasis, FOXA2 was stably knocked down in PC3 cells.

Results. PC3 cells with FOXA2 knocked down (FOXA2-KD) failed to generate in vivo bone destruction following intra-tibial injection contradictory to their parental cell line. To elucidate how FOXA2 is orchestrating these changes, we assessed the expression of all integrins and observed that loss of FOXA2 decreased mRNA and protein expression of the collagen-I binding integrin α1. Furthermore, we found FOXA2-KD decreased PC3 cells' adhesion, spreading, and downstream Integrin α1 signaling cascades on collagen-I (a major component of bone ECM) coated surfaces. Additionally, a neutralizing antibody to integrin α1 was administered to PC3 Control cells, prompting the same adhesion pattern, solidifying that this integrin is critical to collagen-I adherence. Through ChIP analysis, we learned that FOXA2 directly regulates the ITGA1 gene promoter. Lastly, we overexpressed integrin α1 in PC3 FOXA2-KD cells and rescued the adherence properties.

Conclusions. Taken together, the FOXA2-controlled expression of integrin α1 resulted in changes to adherence, spreading, and integrin signaling, providing a mechanism for how PCa cells colonize the bones.

Funding: This research is supported by DOD grant (W81XWH-12-1-0212), NIH grants (R03 CA212567 and R01 CA226285), LSUHSC startups (FWCC and Office of Research) to XY, and Carroll Feist Pre-doctoral Fellowship to ZC.

#60

Heparan sulfate proteoglycans can mediate cancer cell oriented migration through integrin independent Rac 1 activation.

Luisa Bracci, Lorenzo Depau, Jlenia Brunetti, Giulia Riolo, Elisabetta Mandarini, Marta Zanchi, Fabrizia Zevolini, Alessandro Pini, Chiara Falciani. _University of Siena, Siena, Italy_.

Cancer cell membranes and tumor associated extracellular matrix are characterized by a predominant presence of highly sulfated Heparan Sulfate Proteoglycans (HSPGs), which have already been identified as potential tumor markers in different cancers.

We previously reported on stable tetra-branched peptides named NT4, which selectively bind different human cancer tissues and cells, thanks to their high affinity binding to sulfated glycosaminoglycans (GAGs). Using drug-conjugated NT4 peptides as selective tumor targeting agents, we obtained a significant reduction in tumor growth, compared with animals treated with the unconjugated drug under identical conditions. Drug-conjugated NT4 can also by-pass drug resistance mediated by membrane transporters (1-8).

Besides being potential tumor markers, HSPG are also interesting potential drug targets since they appear to take part in many crucial events of cancer progression, such as epithelial mesenchymal transition, migration and invasion.

We identified a putative sulfated oligosaccharide motif allowing high-affinity binding of NT4 to sulfated GAGs. NT4 was then used as a specific tool to analyze the role of sulfated GAGs in signaling events regulating oriented cancer cell migration.

NT4 binding exerts different effects on adhesion and migration in different cancer cells. In PANC-1 human pancreatic adenocarcinoma cells, NT4 binding to sulfated GAGs abolishes cell adhesion and dramatically affects directional migration, inducing disorganization of actin filaments and stress fibers and increasing the number filopodia. This occurs without any effect on activation of beta1 integrins or focal adhesion kinase, but with a clear reduction in Rac1 activity. Otherwise, in TE-671 human rhabdomyosarcoma cells, binding of NT4 to sulfated GAGs has little effect on cell adhesion and cell migration. Untreated TE671 have a slow and scarcely oriented migration and a general morphology which recalls that assumed by PANC-1 upon treatment with NT4, with many filopodia and scarcely organized stress fibers. Interestingly, activated Rac1 is not detectable in untreated migrating TE671 cells. PANC-1 and TE671 have a very different expression of syndecans, particularly syndecan 2 and syndecan 4.

Our results suggest that HSPGs can work as primary players in directional cell migration and actin filaments assembly, by regulating Rac1 activity through an integrin-independent signaling pathway, which seems to require the expression of syndecan 4.

1. Falciani C et al Mol. Cancer Ther. 2007; 6:2441−2448.

2. Falciani C et al ChemMedChem. 2010; 5:567−574.

3. Falciani C et al Curr. Cancer Drug Targets. 2010; 10:695−704.

4. Falciani C et a. ChemMedChem. 2011; 6:678−685.

5. Falciani C et al J Med Chem. 2013; 56:5009-18.

6. Brunetti J et al Sci Rep. 2016; 6:27174.

7. Brunetti J et al Sci Rep. 2015; 5:17736.

8. Depau L et al Oncotarget. 2017; 8:76141-76152.

#61

ECM scaffolds to elucidate the role of Col11a1 in head and neck cancer progression.

Sufi M. Thomas, Jonathan Enders, Greta Isai, Andras Czirok. _Univ. of Kansas Medical Ctr., Kansas City, KS_.

Cancer progression is linked to physical properties, cellular and biochemical composition of the tissue environment including the extracellular matrix (ECM). The ECM not only provides signaling cues by presenting ligands, but also plays a key role in mechano-sensing: modulation of biochemical pathways by physical forces. Collagens are one of the most abundant components of the ECM. We previously reported that head and neck squamous cell carcinoma (HNSCC), which is the 6th most common cause of cancer mortality worldwide with < 40% 5-year survival rates, express higher levels of collagen 11a1 (Col11a1) than normal adjacent mucosa. Col11a1 is a minor fibrillar collagen whose main physiologic role is to regulate the diameter of major collagen fibrils in the cartilage. Although reported that Col11a1 facilitates HNSCC proliferation and invasion, not much is known about its role in the tumor microenvironment. We tested the hypothesis that Col11a1-cancer interdependence facilitates tumor adhesion and metastatic colonization. We developed ECM substrates from cell lines and HNSCC tumor tissue that were characterized by scanning electron microscopy and immunofluorescence microscopy. Electrospun meshes with ECM substrates were developed and used to assess HNSCC adhesion, proliferation and motility. We used a 3D multi-cell type spheroid culture model called the primitive-lung-in-a-dish (PLiD) to demonstrate that Col11a1 siRNA targeting attenuates HNSCC colonization in the lung. Further, using homogenized and lyophilized decellularized ECM from mouse lungs, we demonstrate that Col11a1 regulates motility of HNSCC. Overall, we have used several models to elucidate the role of Col11a1 in HNSCC adhesion and metastatic colonization.

#62

HIC1 inhibits cells metastasis via epithelial-mesenchymal transition in esophageal squamous cell carcinoma.

Beibei Sha, Ping Chen, Xingge Liu, Xuanyu Hu, Miaomiao Li, Pei Li. _Zhengzhou University, Zhengzhou, China_.

Invasion and metastasis are the main causes of death in patients with esophageal squamous cell carcinoma (ESCC). Epithelial-mesenchymal transition (EMT) has been reported to be closely related with tumor metastasis. Although HIC1 (hypermethylated in cancer 1) gene has been observed to be epigenetically modified in many cancers, its intrinsic role and mechanism in ESCC metastasis, especially in EMT, remains unclear. Our previous study showed that hypermethylation of the HIC1 promoter markedly elevated in metastatic ESCC tissues compared with primary and adjacent normal tissues and it was associated with poor patients' survival. In the present study, we used immunohistochemistry (IHC) to detect the expression of HIC1 in ESCC tissues and adjacent normal tissues, and the results showed that HIC1 was down-expressed in ESCC tissues and overexpressed in adjacent normal tissues. Results from trans-well-migration and wound healing experiments showed that overexpression of HIC1 in ESCC cells (KYSE 30) can reduce cell migration and metastasis behavior. Moreover, Western blot showed that overexpression of HIC1 could down-regulate EphA2 expression, simultaneously induced the markers of EMT target changed, in which E-cadherin was observed up-regulated, Snail and Vimentin were down-regulated. In summary, our results suggests that HIC1 may regulate EphA2-SirT1-P53 signaling pathway to mitigate ESCC aggressiveness.

#63

Breast and colon cancer cells express L-selectin ligands that interact with L-selectin on white blood cells under flow conditions.

Nicholas J. Cellars, Ariel L. Lanier, Monica M. Burdick. _Ohio University, Athens, OH_.

Evidence suggests circulating tumor cells (CTCs) can interact with white blood cells (WBCs) and promote metastasis under certain circumstances. However, the interaction between the L-selectin ligands expressed on CTCs and the L-selectin expressed on WBCs is less understood. We hypothesize that the L-selectin of WBCs will bind to the L-selectin ligands of cancer cells under hemodynamic flow conditions. BT-20, Hs-578T, MDA-MB-231 and SK-BR-3 breast cancer cells, and LS174T colon cancer cells were tested for expression of L-selectin ligands using L-selectin hFc (R&D Systems) in flow cytometry. Flow cytometry analysis revealed all five cancer cell lines expressed L-selectin ligands that were detected by L-selectin hFc. The L-selectin ligands on the surface of cancer cells were shown to be protease sensitive, as when the cells were treated with 0.1% bromelain (a broadly active protease), L-selectin hFc binding to all cancer lines was significantly reduced and exhibited binding levels similar to the negative isotype control (hIgG). The parallel plate flow chamber adhesion assay was used to examine the interaction between cancer cell lines (BT-20, Hs-578T, and LS174T) and WBCs isolated from normal healthy human donors in an IRB approved protocol. Two populations of WBCs, PMNs and PBMCs, were separated from whole blood and separately perfused at a wall shear stress of 0.7 dyn/cm2 over a cancer cell monolayer. Interacting cells, defined as PMNs or PBMCs tethered to or rolling on the monolayer, were visualized in real time using video microscopy. Both populations of WBCs interacted with BT-20, Hs-578T, and LS174T cells. Afterward, 5 mM EDTA was perfused across the monolayer, causing the release of WBCs consistent with Ca++ dependent binding. When the WBCs were incubated with an L-selectin antibody (anti-human CD62L, BD Pharmingen) for 30 minutes on ice before perfusion, total interactions were reduced compared to the negative isotype control (mIgG, BD Pharmingen) treated WBCs. These flow chamber results indicate that the L-selectin of PMNs and PBMCs specifically bind to the L-selectin ligands of the breast and colon cancer cell lines. Altogether, the five cell lines that were tested express L-selectin ligands that bind to L-selectin hFc. BT-20, Hs-578T, and LS174T cells interact with WBCs under flow conditions in an L-selectin and divalent cation dependent manner. Ultimately, elucidating the molecular mechanisms of adhesion between WBCs and cancer cells in vasculature may provide a rational basis for developing strategies against metastasis.

#64

Regulation and functional role of the cell-cell junction-associated RNAi machinery in colon cancer .

Amanda C. Daulagala, Catherine Bridges, Lauren Rutledge, Joyce Nair-Menon, Micheal Yost, Antonis Kourtidis. _Medical University of South Carolina, Charleston, SC_.

Colon cancer is the third most common and the second deadliest type of cancer in the United States. A typical characteristic of colon tumors is the compromised integrity of the colonic epithelial monolayer. The Adherens Junction (AJ) is a cell-cell adhesion complex essential for the maintenance of the epithelial integrity, composed of E-cadherin and the catenin family of proteins. We have recently shown that the E-cadherin-p120 catenin partner PLEKHA7 is critical for epithelial monolayer integrity. We have also revealed that PLEKHA7 recruits the core components of the RNA interference (RNAi) machinery, such as Ago2, DROSHA and DGCR8, as well as miRNAs at the mature apical adherens junctions, to suppress expression of pro-tumorigenic markers, through miRNA-mediated silencing. However, we still haven't examined the extend of the contribution of this mechanism to colon tumorigenesis and its modes of regulation. We hypothesize that the adherens junction-RNAi mechanism acts as an epithelial sensor that tethers monolayer integrity to cell behavior. To examine this, we generated 3D cultures of well-differentiated Caco2 colon epithelial cells on Matrigel to examine the distribution of RNAi proteins. Indeed, co-localization of PLEKHA7 with RNAi proteins occurs specifically at the apical surface of these spheroids, whereas PLEKHA7 depletion results in loss of junctional localization of RNAi proteins and in enlarged, multilayered structures. Corroborating these results, PLEKHA7 and RNAi components co-localize specifically at the apical surface of normal colon crypts, whereas extensive disruption or loss of this co-localization was observed in colon tumor patient samples. Fibrosis and extensive deposition of extracellular matrix (ECM) due to chronic inflammation is a precursor to colon cancer. ECM can affect adherens junctions integrity, through integrin-Src signaling. Indeed, we have found that Src activity disrupts localization of PLEKHA7 and RNAi proteins to the junctions. To investigate the potential role of ECM in the regulation of the junctional RNAi mechanism, we are now examining Caco2 cells grown on different ECM substrates, as well as under different conditions of mechanical strain, since fibrosis poses mechanical stress on cells. In summary, our data point towards a novel putative tumor suppressor mechanism, of which we are investigating its modes of regulation and its involvement in colon tumorigenesis.

#65

Gel-forming mucin MUC5AC as the nexus for cell-adhesion molecules governing pancreatic cancer aggressiveness and chemoresistance.

Koelina Ganguly,1 Shiv Ram Krishn,1 Rahat Jahan,1 Pranita Atri,1 Satyanarayana Rachagani,1 Sanchita Rauth,1 Huang Xi,2 Yongfeng Lu,2 Surinder Batra,1 Sukhwinder Kaur1. 1 _University of Nebraska Medical Center, Omaha, NE;_ 2 _University of Nebraska Lincoln, Lincoln, NE_.

Background: Pancreatic cancer (PC) ranks as the third leading cause of cancer-related deaths in the U.S. Poor prognosis of this malignancy stems from propensity for early metastasis and resistance to chemotherapeutic drugs. Tumor-associated extracellular matrix (ECM) plays a key role in impeding drug penetration and regulating tumor aggressiveness by modulating the cell-adhesion molecules and cytoskeletal rearrangement. Heavily glycosylated secreted mucin MUC5AC, one of the most differentially expressed molecule in PC, is associated with poor prognosis of PC patients. We hypothesize, MUC5AC, with the virtue of its gel forming property, promotes tumor-associated ECM deposition that impedes intratumoral drug delivery. Further, abrogation of MUC5AC decreases PC cell aggressiveness and chemoresistance by acting as an interacting nexus for multiple cell-surface mechanotransducers.

Results: RNA sequencing and immunohistochemical analysis revealed significant decrease in profibrotic factors like fibronectin, collagen and α-smooth muscle actin in the pancreatic sections of KCM (KrasG12D; Pdx1-Cre; Muc5ac-/-) mice as compared to KC (KrasG12D; Pdx1-Cre) mice along PC progression. Second harmonic generation imaging revealed less cross-linked collagen organization in KCM as compared to KC. Further, decrease in invasive precursor lesions (p<0.01) and Ki67-positive proliferative cells were observed in KCM mice pancreas. Orthotopic transplantation of human PC cells showed decreased tumor weight (p<0.05) and metastatic incidences upon MUC5AC knock-down (KD). A global mechanistic insight using gene set enrichment analysis from RNA seq. data suggested significant downregulation in genes involved in cell-adhesion, cell-migration and ECM organization in the KCM mice. Among the cell-adhesion molecules, MUC5AC was found to interact with E-cadherin and CD44 on the surface of human PC cells and mouse-derived organoids. Furthermore, MUC5AC expressing cells showed high expression of integrin αvβ5 and pSrc/ pSTAT3 activation. Additionally, MUC5AC-KD cells and organoids from KCM mouse pancreas exhibited greater apoptotic propensity with lesser nuclear localization of β-catenin upon gemcitabine treatment as compared to control. Pharmacologic inhibition of β-catenin nuclear localization sensitized the MUC5AC expressing cells and organoids to gemcitabine.

Conclusion: Our results indicate that gel-forming mucin MUC5AC serves as key node for cell-cell and cell-matrix interactions thereby leading to bidirectional pro-tumorigenic signaling in PC. While, MUC5AC may disrupt E-cadherin-based intercellular junctions to promote transcriptional activation of β-catenin and thereby chemoresistance in PC cells; it also aggravates PC progression and desmoplasia via integrin-mediated mechanosensing.

#66

MMP-7 cleavage of the Perlecan-Sema3A-Plexin(A1)-Neuropilin-1 complex promotes the progression of metastatic prostate cancer.

Tristen V. Tellman,1 Lissette A. Cruz,1 Brian J. Grindel,2 Leland W.K. Chung,3 Mary C. Farach-Carson1. 1 _University of Texas Health Science Center at Houston, School of Dentistry, Houston, TX;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _Cedars-Sinai Medical Center, Los Angeles, CA_.

Introduction: Perlecan/HSPG2 is a major component of the basement membrane and of reactive stroma. PCa relies on matrix metalloproteinase activity to pass through five perlecan-rich boundaries to metastasize to bone. Semaphorin 3A (Sema3A), a secreted factor, complexes with perlecan at the cell surface and binds to a plexin-neuropilin receptor, resulting in a cohesive tumor state. In prostate cancer (PCa) metastasis, cleavage of this complex and disruption of the perlecan-rich stroma promotes migration to secondary sites. Loss of the Perlecan-Sema3A-Plexin(A1)-Neuropilin-1 (PSPN)-complex by MMP-7 cleavage of perlecan disrupts PCa cohesion and enables the formation of circulating tumor cells (CTCs). Downstream FAK, Akt, and FOXM1 are activated by loss of the PSPN-complex in the tumor microenvironment, promoting growth and metastasis. The extent of MMP-7 cleavage of the other components of the PSPN-complex was unknown.

Methods: Knockdowns using shRNA of various PSPN-complex components were used to identify the perlecan-Sema3A binding partners (plexin A1 and neuropilin-1) on the PCa cell surface. Both in silico and in vitro analysis of plexin A1 and neuropilin-1 revealed potential MMP-7 cleavage sites within the PSPN-complex. Silver staining was used to analyze the extent of cleavage of the PSPN-complex components by MMP-7. Western blot analysis was used to probe for any resulting molecular changes in downstream signaling from PSPN-complex components (FAK, Akt, and FOXM1). Changes in cell cohesion were monitored to associate changes in signaling with phenotypic changes in cell behavior.

Results: Knockdowns of the plexin A1 component of the PSPN-complex prevented tumoroid-like formation of PCa cells, implicating this plexin as having a key role in maintaing PCa cohesion mediated through the PSPN-complex. MMP-7 cleavage of various PSPN-complex components resulted in complex destruction, PCa dyscohesion, and the activation of downstream metastasis-promoting factors FAK, Akt, and FOXM1.

Conclusion: The dynamic interactions between the PSPN-complex and MMP-7 at the PCa cell surface governs the cohesion-dyscohesion behavior of PCa cells. Loss of the PSPN-complex by MMP-7 cleavage results in a dyscohesive and metastatic phenotype. Downstream factors FAK, Akt, and FOXM1 can be activated by loss of the PSPN-complex, further implicating the importance of this complex in the metastasis of PCa. These components offer a novel target to perturb the progression of metastatic disease and to prevent CTC formation in PCa patients.

#67

MMP-7 decreases E-cadherin localization at cell-cell contacts resulting in F-actin cytoskeletal remodeling in prostate cancer micro-tumors formed by perlecan/HSPG2.

Lissette A. Cruz,1 Tristen V. Tellman,1 Mary C. Farach-Carson,1 Brian Grindel,2 Daniel D. Carson3. 1 _University of Texas Health Science Center at Houston, Houston, TX;_ 2 _The University of Texas MD Anderson Cancer Center at Houston, Houston, TX;_ 3 _Rice University at Houston, Houston, TX_.

Introduction: Polarized epithelium is maintained by cell-cell interactions via cadherins and cell adhesion molecules (CAMs). These interactions are further stabilized by cell-matrix interactions on the basement membrane. Perlecan/HSPG2 is a major component of the basement membrane, controlling signaling in resting tissues with unperturbed matrices. During epithelial to mesenchymal transition in prostate cancer (PCa), cell-cell adhesions decrease and the cells acquire an invasive phenotype. Proteolytic cleavage of perlecan decreases cell-matrix interactions and dysregulates cell signaling, permitting migration. In vivo, patients with bone metastases were found to have circulating DmIV fragments, with a positive correlation between MMP-7 staining and loss of perlecan in tissue samples from these same patients. Recent studies showed perlecan domain IV-3 (DmIV-3) drives cell cohesion and, when digested with matrix metalloproteinase-7 (MMP-7), drives cell dyscohesion. MMP-7 can cleave cadherins and other CAMs disrupting cell-cell adhesions. Additionally, DmIV-3 fragments generated by MMP-7 cleavage may further induce cell dyscohesion by disrupting interactions between CAMs and/or cadherins.

Methods: To evaluate the impact of MMP-7 and domain IV-3 fragments, uniformly sized PCa cell clusters were pre-formed using a microwell system, enabling control of cluster size and cell number. These pre-formed cell clusters were transferred to DmIV-3 coated wells for 16-24 hours. Clusters then were treated with MMP-7 alone or MMP-7 plus DmIV-3 fragments and stained for E-cadherin and F-actin.

Results: Consistent with previous data, PCa cell clusters maintained strong cell-cell contacts in the presence of DmIV-3. In contrast, we showed that in pre-formed PCa cell clusters cultured in the presence of DmIV-3 cleaved by MMP-7, E-cadherin localization at cell-cell interfaces was reduced. Pre-formed PCa cell clusters treated with MMP-7 had increased F-actin stress fibers present, with a decrease in F-actin organization at cell-cell contacts.

Conclusion: Creating PCa micro-tumors using a microwell system provides a good model to study dynamic changes in cell-cell interactions. The purpose of this study was to identify which cell adhesion complexes are perturbed by MMP-7 in these PCa cell clusters. E-cadherin is known to regulate the cortical actin cytoskeleton; thus, decreased E-cadherin at cell-cell contacts results in actin reorganization and an increase in F-actin stress fiber formation, promoting a migratory cell phenotype. Perlecan fragments may augment this behavior by increasing MMP-7 access to the ectodomains of CAMs. This study will be novel in its identification of a DmIV fragment(s) that may provide a prediction marker for PCa metastasis as well as fragments that are positively associated with tumor dyscohesion.

#68

Comparative transcriptome analysis of primary tumor and matched tumor thrombus reveals that coagulation cascades activation contributes to the venous metastases of osteosarcoma.

Tao Ji,1 Yifei Wang,1 Yi Guo,2 Dasen Li,1 Haijie Liang,1 Tingting Ren,1 Wei Guo1. 1 _Peking University, People's Hospital, Beijing, China;_ 2 _Beaumont Hospital, Royal Oak, MI_.

Comparative transcriptome analysis of primary tumor and matched tumor thrombus reveals that coagulation cascades activation contributes to the venous metastases of osteosarcoma

Osteosarcoma is the most frequent malignant primary tumor of bone in children and adolescents with an approximately 5-year survival about 50-60%. Lung metastasis remains as the most challenging condition during treatment and follow-up, which has been regarded as the main reason for bottle neck of prognosis. Tumor thrombus is occasionally diagnosed in patients with malignant tumor. It usually exists during tumor angiogenesis associated with proliferation and metastasis. The presentation shows aggressive features and advanced stage of the disease. Tumor thrombus is associated with highly metastasis potential. Based on the evolution theory of tumor metastasis, tumor thrombus can be the intermediate phase between local invasion and distal metastasis. In order to investigate the underlying mechanism and identify candidate metastasis driver genes, we collected a set of high quality paired tumor tissue, both the primary site and corresponding tumor thrombus that were treated by thrombectomy. In this study, we present a large-scale transcriptome analysis, by RNA sequencing, of 4 patients diagnosed with large pelvic osteosarcoma with tumor thrombus. The dysregulated genes between primary site and thrombus suggested that the complement and coagulation cascades, focal adhesion and extracellular matrix receptor was correlated with the formation of tumor thrombus. Tissue factor was identified as a major marker in coagulation pathway activation. We also identified NOTCH2NL-NBPF1 fusion genes in all four patients. All of these findings broaden our knowledge of osteosarcoma thrombus and may also contribute to the novel mechanism of metastasis in osteosarcoma.

#69

Integrin α6β4 associates with plectin and vimentin in focal adhesions to enhance breast cancer cell invasion.

Lei Qi, Teresa Knifley, Min Chen, Kathleen L. O'Connor. _University of Kentucky, Lexington, KY_.

Integrin α6β4 binds plectin to associate with intermediate filaments such as vimentin. While vimentin and integrin α6β4 are associated with the aggressiveness of triple negative breast cancer (TNBC), the biological importance of their association is not clear. Here, we utilized naturally occurring Epidermolysis Bullosa mutations in the integrin β4 subunit that are deficient in plectin binding to investigate the association between integrin α6β4 and vimentin. Using integrin β4-deficient BT549 cells, we generated cell lines that expressed integrin β4 wild type (β4-wt), plectin binding mutants (β4-R1225H or β4-R1281W), 1355-1662 deletion mutant (β4-Δ1355-1662; which retains plectin binding but deletes the signaling domain) or an empty control vector (EV). Migration assay and three-dimensional (3D) culture were performed to test whether integrin α6β4 signaling and its association with plectin contribute to the migratory and invasive phenotypes. Compared to EV cells, β4-wt increases cell migration and 3D invasive growth, but the β4-R1225H and β4-R1281W could not. CRISPR knockout of plectin also blocked β4-wt 3D cell growth. Utilizing total internal reflection fluorescence (TIRF) microscopy, we found that integrin β4-wt and β4-Δ1355-1662 were recruited to focal adhesions (FAs, indicated by paxillin co-staining) on laminin-1 plus fibronectin matrices, but not when plated on laminin-1 or fibronectin alone. Integrin β4-R1225H and β4-R1281W did not localize to FAs. Similarly, integrin β4-wt in plectin knockout cells could not aggregate in FAs. Furthermore, we observed that integrin αv and β1 colocalized with integrin β4 in FAs. Proximity ligation assay (PLA) indicated that the close interaction of integrin β1 and plectin. Interestingly, depletion of integrin β1 significantly reduced integrin β4 accumulation in FAs. Further immunostaining showed that integrin β4, plectin, and vimentin aggregated in FAs in the central area of the cell where FAs predominantly formed. Depletion of vimentin did not impact the recruitment of integrin β4 in FAs or cell proliferation, but significantly suppressed cell migration and 3D growth in β4-wt cells. Integrin β4-plectin-vimentin complexes were also found in MDA-MB-231 cells where they co-localized with paxillin and connected with stress fibers. In summary, our study demonstrates the essential roles of plectin binding and vimentin in promoting the migratory and invasive phenotypes by integrin α6β4 signaling in TNBC. Our study further highlights a novel function of plectin binding and integrin β1 in enhancing integrin β4 dynamics.

#70

Oxidative stress and cell death in multidrug-resistant cancer.

Baikuntha P. Aryal,1 Jean-Pierre Gillet,2 V Ashutosh Rao1. 1 _FDA, Silver Spring, MD;_ 2 _University of Namur, Namur, Belgium_.

Reactive oxygen species (ROS) are constantly generated in mitochondria as a consequence of normal cellular metabolic reactions and play vital roles in cell signaling and homeostasis. Prolong production of elevated levels of ROS may lead to oxidative damage, genetic instability, and genetic mutation causing cancer initiation and progression. One proposed therapeutic strategy is to alter oxidative stress from a ROS-induced tumor promoting event to a ROS-induced apoptotic signaling to kill cancer cells. Most ROS inducing therapeutic agents initially induce ROS to kill cancer cells by apoptosis; however, prolonged exposure of such drugs may increase ROS scavenging enzymes resulting in a reduced ROS levels and an adaptive microenvironment that makes cancer cells resistant to the same ROS-inducing therapeutic agents. Chronic exposure of ROS may also upregulate the multidrug resistance associated protein expression in cancer cells. We hypothesize that induction of oxidative stress may sensitize drug resistant cancer cells leading to cell death. We used two pairs of cell lines, a mitoxantrone sensitive and mitoxantrone resistant MCF7 human breast adenocarcinoma (MCF7/MCF7MX) and a H460 non-small lung cancer cell line (H460/H460MX) for our experiments to study the impact of oxidative stress on drug resistance. We employed western blot, ELISA assays, and FACS analysis to determine the total and specific protein oxidative carbonylation, protein aggregation, and cell death by apoptosis or necrosis. At basal level, both mitoxantrone resistant MCF7MX and H460MX cells showed slightly lower level of total protein carbonylation compared to their mitoxantrone sensitive counterparts. Chemical induction of oxidative stress by external sources increased the total protein carbonylation and cell death in both mitoxantrone sensitive and resistance cancer cells. We also observed an increase in protein aggregation with increasing oxidative stress. Our results indicate that oxidative stress-induced protein oxidation by carbonylation and aggregation of oxidized protein which may sensitize drug resistant cancer cells leading to cell death.

#71

**NEDD8-activating enzyme inhibitor MLN4924 reduces cell viability and induces apoptosis in chemoresistant cancer stem-like cells of human urothelial carcinomas** in vitro **and** in vivo **.**

Shih-Ming Liao,1 Fu-Shun Hsu,2 Shao-Ping Yang,1 Yu-Wei Chang,1 Po-Ming Chow,1 Yeong-Shiau Pu,1 Yu-Chieh Tsai,1 Kuan-Lin Kuo,1 Kuo-How Huang1. 1 _National Taiwan University Hospital, Taipei, Taiwan;_ 2 _New Taipei City Hospital / Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, New Taipei City / Taipei, Taiwan_.

Emerging evidence indicates that cancer stem cells (CSCs) may play a crucial role in cancer tumorigenesis, metastasis, and drug resistance. To develop treatment to inhibit CSCs provides promising target for cancer therapy. In this study, we identified a subpopulation of chemoresistant cancer stem-like cells of UCs (T24/R) after chemotherapeutic drug selection from T24, a bladder UC cell line. The T24/R cells displayed stemness markers (SOX2, Nanog, and Kruppel-like factor 4), high tumorigenicity in vivo, and enhanced invasion ability because of their epithelial-mesenchymal transition (EMT) properties. A neddylation inhibitor, MLN4924, efficiently inhibited viability and migration as well as induced apoptosis in the T24/R cells with concomitant suppression of stemness and EMT markers. Our findings suggest that MLN4924 effeiciently suppressed chemoresistant cancer stem-like UC cells and was a promising agent to conquer drug resistance in human bladder UCs.

#72

YAP1 mediates initial survival of alectinib therapy in ALK-rearranged lung cancer via pro-apoptotic protein regulation.

Takahiro Tsuji,1 Hiroaki Ozasa,1 Wataru Aoki,2 Shunsuke Aburaya,2 Tomoko Funazo,1 Koh Furugaki,3 Yasushi Yoshimura,3 Hitomi Ajimizu,1 Yuto Yasuda,1 Takashi Nomizo,1 Yuichi Sakamori,1 Hironori Yoshida,1 Mitsuyoshi Ueda,2 Young Hak Kim,1 Toyohiro Hirai1. 1 _Kyoto Univ. Graduate School of Medicine, Kyoto, Japan;_ 2 _Kyoto Univ. Graduate School of Agriculture, Kyoto, Japan;_ 3 _Chugai Pharmaceutical, Co., Ltd., Kamakura, Japan_.

Anaplastic lymphoma kinase (ALK) inhibitors, such as alectinib (ALC), have dramatic therapeutic effects on ALK-rearranged lung cancer, but cures are usually not achieved. We focused on tumor cells that survive ALK inhibitor administration and hypothesized that targeted therapy for these cells could provide complete remission. To explore survival factors, we established patient-derived cell lines and screened them using proteome analysis. Three ALK-rearranged ALC-sensitive cell lines (KTOR-1, KTOR-2, KTOR-3) were established from 3 patients; the 50% inhibitory concentrations (IC50)s for ALC were 24-65 nM. Comprehensive protein expression profiles of the 3 cells indicated that exposure to ALC significantly enriched proteins related to actin and extracellular matrix (ECM) adhesion. We focused on Yes-associated protein 1 (YAP1), which is activated by ECM adhesion and actin fiber accumulation. Nuclear localization of YAP1 (an activation marker of YAP1) was assessed using immunohistostaining. In KTOR1-3 and H2228 cells from an ALK-rearranged line purchased from ATCC, exposure to ALC in vitro promoted YAP1 accumulation in the nucleus. BALB/nu mice xenograft models of H2228 or KTOR1 were administered ALC (8 mg/kg/day, N=4) or a vehicle (N=4) for 7 days, and tumors were evaluated. In ALC-administered tumors, YAP1 was localized to the nucleus, which was rarely the case in vehicle-administered tumors. The expression of pro-apoptosis factors Mcl-1 and Bcl-xL also increased after exposure to ALC in vitro, but the increment was cancelled by YAP1 inhibition by siRNA or verteporfin (VER), a non-specific YAP1 inhibitor. Exposure to ALC with combinatorial YAP1 inhibition significantly increased Caspase 3/7 activity. To address the treatment effects of YAP1 inhibition, a YAP1-activated H2228 cell line (H2ARY) was established by exposing H2228 cells to 100-300 nM of ALC for 3 months and thorough subsequent cloning. The H2ARY had lower sensitivity to ALC in vitro than parental H2228 (IC50: 1.4 μM vs 315 nM, 96 h) and restored the sensitivity by YAP1 inhibition (208 nM with VER 1 μM, 312 nM with siYAP1). Twenty-four xenograft models (mean volume: 199 mm3) of H2ARY on BALB/nu mice were randomized (Day 0) into 4 treatment groups to receive ALC monotherapy (8 mg/kg daily, N=6), VER monotherapy (12.5 mg/kg twice a week, N=7), combination (N=7), or vehicle (N=5). On day 15, the tumor volume of the vehicle and VER monotherapy groups reached > 800 mm3, with no significant differences among the groups. On day 33, the tumors of the combination group were significantly smaller than those of the ALC monotherapy group (187 vs 761 mm3, P = 0.0125). Exposure to ALC-activated YAP1 may regulate anti-apoptotic activity by controlling the expression of Mcl-1 and Bcl-xL in ALK-rearranged lung cancer cells. This is the first evidence that combinatorial therapy against ALK and YAP1 could enhance ALK-rearranged tumor treatment.

#73

Inhibition of ACK1 delays and overcomes acquired resistance of EGFR mutant NSCLC cells to the third generation EGFR inhibitor, osimertinib.

Jiajia Gu,1 Xia He,2 Nupam P. Mahajan,3 Taofeek K. Owonikoko,1 Suresh S. Ramalingam,1 Shi-Yong Sun1. 1 _Emory University School of Medicine and Winship Cancer Institute, Atlanta, GA;_ 2 _Jiangsu Cancer Hospital & Jiangsu Institute of Cancer Research & The Affiliated Cancer Hospital of Nanjing Medical University, Nanjing, China; _3 _Washington University in St. Louis, St. Louis, MO_.

The approval of osimertinib (AZD9291) as a third generation EGFR inhibitor for treating non-small cell lung cancer (NSCLC) patients with activating EGFR mutations (first-line) or those who have become resistant to 1st generation EGFR inhibitors through the T790M mutation (second-line) have generated tremendous benefits to these NSLCL patients. However, the unavoidable development of acquired resistance to osimertinib limits the long-term benefits of patients. Thus, effective treatment options are urgently needed. Activated Cdc42-associated kinase 1 or ACK1 (also named as TNK2) is a non-receptor tyrosine kinase with oncogenic function. Although ACK1 gene is amplified in lung squamous cell carcinoma, its involvement in lung cancer including regulation of drug resistance has largely not been investigated. We found that inhibition of ACK1 with (R)-9bMS, a specific ACK1 inhibitor, synergized with AZD9291 in inhibiting the growth of EGFR mutant NSCLC cell lines. Similar results were also generated when ACK1 is genetically inhibited through transient shRNA gene knockdown. The combination of AZD9291 and (R)-9bMS enhanced induction of apoptosis as evaluated by detecting annexin V-positive cells with flow cytometry and PARP cleavage using Western blotting. In an in vitro long-term resistance delay assay, the combination of (R)-9bMS and AZD9291 clearly prevented the emergence of AZD9291-resistance as evaluating resistant cell colony formation and growth. Beyond the combination was also effective in inhibiting the growth of EGFR mutant NSCLC cell lines with acquired resistance to AZD9291. In some resistant cell lines, the combinations induced senescence in addition to induction of apoptosis. These preliminary findings suggest that ACK1 inhibition might be a potential and innovative strategy for preventing and overcoming AZD9291 acquired resistance. Hence further in vivo and mechanistic studies are warranted. (This work was supported by the NIH/NCI R01 CA223220 and Winship lung cancer pilot award to SYS; NIH/NCI R01 CA208258, Bankhead Coley Award-6BC08 and Prostate Cancer Foundation (PCF) Challenge award-17CHAL06 to NPM).

#74

Heterogeneity response to afatinib in gastric cancer patient with uncommon EGFR mutations.

Qin Liu,1 Jia Wei,1 Yang Yang,1 Yue Wang,1 Baorui Liu,1 Yang Shao2. 1 _The Comprehensive Cancer Center of Drum-Tower Hospital, Medical School of Nanjing University, Nanjing, China;_ 2 _University of Toronto, Ontario, Canada_.

Introduction: Gastric cancer is the third leading cause of cancer mortality worldwide. Gatric cancer based on driver gene mutation detection may benefit patients by facilitating molecular targeted drugs development and improving survival in gastric cancer patients. On the other hand, the complex and heterogeneous molecular mechanisms of gastric cancer also plays an essential role in the drug resistance.

Methods: Whole exome sequencing (WES) was performed, revealing uncommon EGFR ex21 L861Q and ex18 G719S mutation of formalin-fixed paraffin-embedded sections from primary tumor and different metastatic lesions in a gastric cancer patient. The periodic circulating tumor DNA (ctDNA) was also determined by next generation sequencing (NGS). Stable gastric cancer cell and NIH-3T3 cell lines harboring the mutations were established to investigate the effect and mechanisms.

Results: Tumors with compond EGFR L861Q/G719S mutations and EGFR gene amplfication are sensitive to afatinib, which caused tumor progression in short time. However, the lung metastatic lesion, which lacked EGFR gene amplification demonstrated primary resistance to afatinib. A dramatic increase of MET gene copy number may collectively related to the patient's rapid progression. Periodic mutation profiling of patient's ctDNA by NGS correspondingly revealed compond EGFR L861Q/G719S mutations, and a consitent increase of MET gene amplification. In in vitro studies, afatinib treatment reduced proliferation and inhibited EGFR phosphorylation in L861Q/G719S and L861Q mutant cells.

Conclusions: Afatinib may be a beneficial therapeutic option for a subset of gastric cancer patients with rare EGFR mutations in their tumors. Our results also illustrated the great potential of ctDNA profiling for treatment decision-making to patients with gastric cancer.

#75

Anti-tumor activity of a novel selenonucleoside via targeting Skp2 degradation in paclitaxel-resistant prostate cancer.

Woong Sub Byun, Minkyung Jin, Jinha Yu, Won Kyung Kim, Lak Shin Jeong, Sang Kook Lee. _Seoul National Univ. College of Pharmacy, Seoul, Republic of Korea_.

Prostate cancer (PC) is the most common disease in men over age 50, and its prevalence rate has been gradually increasing since 1980. Taxane-derived anticancer agents are the primary agents used to treat metastatic prostate cancer patients; however, the side effects and acquired drug resistance limit the success of these therapies. Because there is no specific treatment for paclitaxel-resistant prostate cancer, it is necessary to develop new targets and therapeutic strategies to overcome the acquired resistance. In this study, the antitumor activity of a novel selenonucleoside (4′-selenofuranosyl-2,6-dichloropurine, LJ-2618), a third-generation nucleoside, and its plausible mechanisms of action in paclitaxel-resistant prostate cancer (PC-3-Pa) cells were investigated. The established PC-3-Pa cells exhibited over 100-fold resistance against paclitaxel compared to the paclitaxel-sensitive PC-3 cells. LJ-2618, however, effectively inhibited the proliferation of both cell lines with similar IC50 values in vitro. In PC-3-Pa cells, the activated PI3K/Akt signaling pathway was suppressed by LJ-2618 treatment. In addition, Skp2 was found to be over-expressed in paclitaxel-resistant cells, and the transfection of Skp2 siRNA recovered the sensitivity of paclitaxel in PC-3-Pa cells. Furthermore, LJ-2618 significantly down-regulated Skp2 expression in PC-3-Pa cells by promoting degradation and inducing destabilization of Skp2, which triggers G2/M cell cycle arrest. In a xenograft mouse model implanted with PC-3-Pa cells, LJ-2618 (3 or 10 mg/kg) effectively inhibited tumor growth with the enhancement of Skp2 degradation and induction of p27 expression in tumor tissues. These findings suggest that LJ-2618 may have a potential for overcoming paclitaxel resistance via promoting Skp2 degradation and stabilizing p27 expression in PC-3-Pa cells. Therefore, the novel selenonucleoside LJ-2618 may lead to the development of a new treatment strategy for patients with paclitaxel-resistant, castration-resistant prostate cancer. This study was supported by a National Research Foundation of Korea (NRF) Grant funded by the Korean Government (NRF-2016M3A9B6903499).

#76

Exosomes secreted by 5-FU resistant cells promote cell survival in colorectal cancer.

Qian Zhang, Rui-Xian Liu, Jiancong Hu, Xiangling Yang, Huanliang Liu. _Guangdong Institute of Gastroenterology, Guangdong Provincial Key Laboratory of Colorectal and Pelvic Floor Diseases, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China_.

Purpose: Colorectal cancer (CRC) is the third common cancer in the world. 5-Fluorouracil (5-FU) is one of the first-line adjuvant chemotherapy regimens of colorectal cancer. Resistance to 5-FU based chemotherapy is the main course of treatment failure in colorectal cancer. However, decisive evidence that cells resist to chemotherapy intrinsically or acquire resistance during treatment remains uncertain. Recently, evidence indicates that exosomes play a crucial role in chemotherapy resistance during treatment. As a kind of small membrane vesicles secreted by various cells, exosomes contain various cellular constituents including proteins and nucleic acids that are associated with tumor aggressiveness. In this study, we aim to investigate whether exosomes secreted by resistant cells could transfer 5-FU resistance in colorectal cancer.

Methods: 5-FU resistant cell line (RKO/R) was generated from colorectal cancer cell line (RKO/P) with increased concentration of 5-FU in our group. Exosomes secreted from RKO/R and RKO/P were isolated by ultracentrifugation and their characteristics were identified by transmission electron microscope (TEM), high-sensitivity flow cytometer (HSFCM) and western blot. The functions of exosomes were evaluated by real-time cell analyzer (RTCA) and flow cytometry (FCM). The RNA profiles of RKO/P and RKO/R were analyzed by RNA-sequencing. Proteomics analysis of exosomes from RKO/R was performed to identify targets related to chemotherapy resistance.

Results: Exosomes from RKO/R converted apoptosis rate of 5-FU-treated RKO/P. By analyzing the results of RNA-seq, we observed that 2,591 mRNAs were up-regulated in RKO/R compared with RKO/P, which meant those mRNAs could translate into proteins in RKO/R. 964 proteins were identified in exosomes from RKO/R. The overlapped proteins between exosomes and cells were associated with significant cellular pathways including proliferation and cell survival in colorectal cancer.

Conclusion: Our results have strongly suggested that exosomes are associated with conferring chemotherapy resistance. More investigations of these data are underway to find biomarkers to stratify patients and develop new therapeutic strategies to target associated proteins.

#77

Therapeutic potential of ERK inhibitors in overcoming acquired resistance to third generation EGFR tyrosine kinase inhibitors.

Yiting Li, Guoqing Qian, Taofeek K. Owonikoko, Suresh S. Ramalingam, Shi-Yong Sun. _Emory University School of Medicine and Winship Cancer Institute, Decatur, GA_.

Targeting epidermal growth factor receptor (EGFR) activating mutations, 90% of which present as an exon 19 deletion (Del19) or exon 21 point mutation (L858R), with first and second generation EGFR tyrosine kinase inhibitors (EGFR-TKIs; e.g., erlotinib and afatinib) and T790M resistant mutation with third generation EGFR-TKIs (e.g., AZD9291 or osimertinib) has provided significant clinical benefit in patients with non-small cell lung cancer (NSCLC) carrying these mutations. AZD9291 selectively and irreversibly inhibits EGFR activating and T790M mutants while sparing wild-type EGFR. It is now an approved therapeutic option for NSCLC patients with activating EGFR mutations (first-line) or those who have become resistant to the 1st generation EGFR-TKIs through the T790M mutation (second-line). Unfortunately, all patients eventually relapsed and developed resistance to AZD9291 treatment, limiting the long-term benefit of this targeted therapy. Hence the effective strategies that can overcome the resistance are urgently needed in the clinic. We have recently demonstrated that modulation of MEK/ERK-dependent Bim and Mcl-1 degradation critically mediates sensitivity and resistance of EGFR-mutant NSCLC cells to AZD9291 and loss of MEK/ERK-mediated Bim and Mcl-1 modulation is a key mechanism accounting for development of acquired resistance to AZD9291. Accordingly we have suggested that inhibition of the MEK/ERK signaling, which enforces Mcl-1 reduction and Bim elevation, might lower the threshold of AZD9291-resistant cells to re-respond to AZD9291 for undergoing apoptosis and achieve the goal of overcoming acquired resistance. Indeed, the combination of AZD9291 with a MEK inhibitor effectively decreases cell survival with enhanced apoptosis irrespectively of resistant mechanisms and inhibited the growth of AZD9291-resistant xenografts in vivo as we recently reported. In the current study, we further show that the combination of AZD9291 with an ERK inhibitor synergistically decreased the survival of AZD9291-resistance cell lines with enhanced induction of apoptosis. Moreover, the combination of a MEK or ERK inhibitor with a first (e.g., erlotinib) or second (e.g., afatinib) generation EGFR-TKIs also very effectively decreased the survival of AZD9291-resistance cell lines although these cell lines were cross-resistant to first and second generation EGFR-TKIs. Therefore, our results have provided additional evidence supporting the notion that co-targeting the MEK/ERK signaling is an effective strategy for overcoming AZD9291 acquired resistance irrespectively of the underlying resistance mechanisms. Our findings warrant further investigation of this therapeutic strategy to overcome AZD9291 resistance in the clinic. (This study was supported by the NIH/NCI R01 CA223220 and Winship lung cancer pilot award)

#78

IGF-1R inhibition activates p70S6K1 to promote survival via MEK1/2 activation in colon cancer cells.

Qing Wang, Hsin-Sheng Yang. _University of Kentucky, Lexington, KY_.

The insulin-like growth factor 1 receptor (IGF-1R) is a heterotetramer with tyrosine kinase activity, composed of two α subunits containing the ligand-binding site and two β subunits harboring the tyrosine kinase activity. Upon binding with ligands (IGF-1, IGF-2, and insulin), IGF-1R leads to activation of downstream pathways including PI3-K/AKT and Ras/MAPK, two frequently activated pathways for promoting cell survival and proliferation in various cancers including colorectal cancer. Immunohistochemical studies revealed that IGF-1R is over-expressed in colorectal cancer tissues compared to the adjacent normal tissues. Most importantly, a higher expression of IGF-1R is associated with a higher grade and stage in colorectal cancer patients. Since the IGF-1R signaling pathway plays an essential role in development, maintenance, and survival of tumors, many small molecule compounds and antibodies targeting IGF-1R have been developed. Unfortunately, these anti-IGF-1R agents demonstrate very limited efficacy in clinical trials, suggesting the existence of a mechanism to overcome the inhibitory effect of IGF-1R in cancer cells. In this study, we aim to understand the resistance mechanism of IGF-1R inhibition in colon cancer cells. Prolonged treatment of IGF-1R resistant colon cancer cells with IGF-1R inhibitors (OSI-906, BMS-754807, and GSK1838705A) stimulates p70S6K1 activation, a well-known kinase signaling for cell survival. We also found that p70S6K1 activation is independent of mutation of K-RAS and PIK3CA, frequently occurring in colorectal cancer. Genetic knockdown or pharmacologic inhibition of p70S6K1 efficiently suppresses cell viability in response to IGF-1R inhibition, indicating that p70S6K1 activation contributes to the survival of IGF-1R inhibitor-treated cells. In addition to the increased phosphorylation of p70S6K1, phosphorylation of MEK1/2 is also elevated in colon cancer cells with prolonged inhibition of IGF-1R. Knockdown of MEK1/2 results in reduction of p70S6K1 activation in response to IGF-1R inhibition and subsequently increases apoptosis, suggesting that MEK1/2 is involved in p70S6K1 activation for survival. Furthermore, the combination of BMS754807 and U0126, a MEK1/2 inhibitor, effectively decreases the cell viability and increases apoptosis. Our data suggest that inhibition of MEK1/2 enhances the anti-proliferation effects of IGF-1R inhibitors, pointing out a new direction for overcoming the resistance of IGF-1R inhibition in colorectal cancer.

#79

Overexpression of BQ323636.1 modulates tamoxifen resistance in ER positive breast cancer via IL-8-mediated signaling pathway.

Ling Shi, Ho Tsoi, Miao Dong, Ellen Pui Man, Ui Soon Khoo. _The University of Hong Kong, Hong Kong_.

Background

Tamoxifen has been used as first-line adjuvant treatment for estrogen receptor positive (ER+) breast cancer; however, the development of acquired resistance compromises its efficacy. Previously, our group identified BQ323636.1(BQ), a novel splice variant of nuclear receptor repressor 2 (NCOR2), which confers tamoxifen resistance by enhancing ER transcriptional activity1, 2. We hypothesize that BQ may also modulate tamoxifen resistance via androgen receptor (AR) activity. The current study investigates the effect of BQ in modulating the activities of ER and AR in tamoxifen resistant breast cancer.

Methods

In silico search was employed to identify candidate genes that contained both the androgen responsive element (ARE) and estrogen responsive element (ERE) genome-wide. Expression of candidate genes were evaluated by RT-qPCR. Western blot was used to determine the protein expression. Stably transfected BQ overexpressing MCF7 cell line (MCF7-BQ) and control cell line (MCF7-His) were established. ELISA was employed to determine the amount of IL-8. Repertaxin was used to inhibit IL-8 mediated signaling pathway.

Results

There were 22 candidate genes identified by in silico search. qPCR confirmed that IL-8 gene contained both functional ERE and ARE in MCF-7 cells. Overexpression of BQ could enhance the expression of IL-8 at both mRNA and protein levels. ELISA assay confirmed that BQ overexpression could promote the secretion of IL-8 in breast cancer cells. Western blot analysis showed that BQ overexpression could not alter the expression of IL-8 receptor CXCR1. The treatment with repertaxin could recover the response of MCF7-BQ to tamoxifen.

Conclusions

Our results suggest that BQ323636.1 could up-regulate IL-8 to modulate tamoxifen resistance. Targeting IL-8 signaling may provide insights to overcome tamoxifen resistance in ER+ breast cancer.

References

1. Zhang L., Gong C., Lau S.L.Y., Yang N., Wong O.G.W., Cheung A.N.Y., Tsang J.W.H., Chan K.Y.K. and Khoo US. (2013) Cancer Research 73:246-255

2. Gong, C., Man, E. P., Tsoi, H., Lee, T. K., Lee, ⋯.. Khoo, U. (2018). BQ323636.1, a Novel Splice Variant toNCOR2, as a Predictor for Tamoxifen-Resistant Breast Cancer. Clinical Cancer Research,24(15), 3681-3691. doi:10.1158/1078-0432.ccr-17-2259

#80

Electronic cigarette aerosols increase clonogenic survival in oral cancer cells after cisplatin treatment.

Jimmy Manyanga, Toral R. Mehta, Lurdes Queimado. _The University of Oklahoma Health Sciences Center, Oklahoma City, OK_.

Background and Aim: Are electronic cigarettes (ECs) safer than combustible tobacco? No one knows the answer yet. ECs are devices that deliver an aerosol from a heated mixture of propylene glycol, vegetable glycerin, and flavors, with or without nicotine. Short-term EC studies are mostly limited and inconclusive. Despite unknown long-term health effects, the use of ECs has increased alarmingly over the past decade. EC aerosols contain unique chemicals as well as other chemicals similar to those found in tobacco smoke. Tobacco smoking during cancer treatment has been associated with increased drug resistance and reduced overall survival rate. The awareness of the dangers of cigarette smoking and the paucity of studies evaluating the safety of ECs pose a dilemma for cancer patients who might be considering switching to ECs during chemotherapy. Here, we sought to examine the effects of EC aerosol extracts exposure on cell survival in oral cancer cells during cisplatin treatment.

Methods: EC aerosol extracts were collected from two brands of ECs as previously described. Mainstream tobacco smoke (MS) extract was used as a positive control. To evaluate clonogenic survival after cisplatin treatment, oral cancer cells were exposed for 48 h to EC aerosol extracts at nicotine doses comparable to those observed in EC users. Next, cells were treated with both EC aerosol extracts and cisplatin (or vehicle-control) for another 48 h. After treatment, cells were counted and seeded in 6-well plates and colony formation was assessed two weeks later. Colonies were fixed with methanol followed by staining with 0.5% crystal violet in 25% methanol and counted manually. Only colonies with at least 50 cells were counted. Data were analyzed by Student's t-tests and one-way analysis of variance (ANOVA).

Results: After cisplatin treatment, cells exposed to EC aerosol extracts showed a significant increase in clonogenic survival fraction compared to vehicle-control treated cells. Furthermore, cells exposed to EC aerosol extracts formed visibly larger colonies. An increase in clonogenic survival was also observed in MS extract-treated cells.

Conclusions: Collectively, our data suggest that short-term exposure to EC aerosol can induce cisplatin resistance in oral cancer cells, thus allowing the cells to survive and form colonies. These findings are particularly important in paving the way to understanding whether ECs are a safer alternative for oral cancer patients undergoing chemotherapy.

#81

ARL13B interacts with IMPDH2 to modulate purine synthesis and temozolomide resistance in glioblastoma.

Jack Shireman, Eunus Ali, Miranda Saathoff, Cheol Park, Issam Ben-Sahra, Atique U. Ahmed. _Northwestern University, Chicago, IL_.

Glioblastoma, a universally lethal primary brain tumor, harnesses cellular plasticity to drive therapeutic adaptation. Critical factors in developing this plasticity are histone modifiers such as Polycomb Repressor Complex 2 protein EZH2. In order to examine tumor cell plasticity in depth, we conducted multiple ChIP Sequencing runs and demonstrate that EZH2 binds within an enhancer region of ARL13B during temozolomide (TMZ) therapy and induces an H3K4 mono-methylation mark. Concurrently, we observed an increase in H3K27ac at the transcription start site of ARL13B as well as a lack of H3K27 tri-methylation, EZH2's canonical histone mark. Based on this we hypothesize that EZH2 could be non-canonically regulating ARL13B to allow for cellular plasticity and ultimately drive therapeutic adaptation. Delving further into this regulation we demonstrate that knockdown of ARL13B in patient derived xenograft cells significantly increased survival of mice in an orthotopic GBM model when compared to controls (p-value <0.0001). The Cancer Genome Atlas (TCGA) patient dataset demonstrates time to recurrence in patients with downregulated ARL13B is substantially increased as compared to ARL13B upregulated patients (log-rank p-value=0.0012). Searching for a mechanism behind this survival benefit, we preformed mass spectrometry on an ARL13B pulldown in a patient derived xenograft line during TMZ therapy and identified inosine monophosphate dehydrogenase 2 (IMPDH2), the rate-limiting enzyme in de-novo guanine nucleotide biosynthesis, as a significant binding partner of ARL13B during TMZ chemotherapy (p-value <0.0001). Probing this novel interaction further we examined the de-novo and salvage purine biosynthesis pathways using radiolabeled carbon tracing experiments. In ARL13B knockdown cells, purine salvage pathway usage is upregulated 7-fold (p-value <0.0001) while de-novo pathway usage was decreased about 50% (p-value=0.004) in a TMZ specific manner. Examination of IMPDH2 enzymatic activity using a formazin reduction assay demonstrated a decrease in activity over 8 days of TMZ exposure (p<.001). Moreover, ARL13B knockdown GBM cells treated with TMZ show a robust increase in DNA double-strand breaks compared to control cells exposed to TMZ, demonstrated by γH2X staining. Finally, a potent inhibitor of IMPDH2 (Mycophenolate Mofetil) significantly extended median survival in an orthotopic PDX mouse model only when in combination with TMZ (p<.01). Based on these data we hypothesize that EZH2 regulates a novel ARL13B and IMPDH2 interaction which when lost forces cells into salvage synthesis exclusively. This synthesis shift forces cells to uptake and incorporate purines that have been alkylated by TMZ therapy which increases DNA double strand breaks and ultimately impairs therapeutic adaptation.

### Developmental Phenotypes and Molecular Imaging

#82

Transforming of human osteoblasts with the combination of hTERT (T), SV40Tag (S) and c-Myc: Changes in phenotype and tumorigenesis capability.

Zhongting Zhang, Yifei Wang, Wendong Zhang, Michael Roth, Jonathan Benjamin Gill, Zhaohui Xu, Xiangjun Tian, Jing Wang, Richard Gorlick. _MD Anderson Cancer Center, Houston, TX_.

The etiology of osteosarcoma (OS), the most common primary malignant bone cancer in pediatric patients, still remains unknown. It is not clear where tat which point he osteosarcoma is originated from the osteogenic lineagein the osteogenic osteosarcoma arises. , some consider Potential candidates in the literature include the human mesenchymal cells (hMSC) as the cell of origin in OS, whereas others believe theand osteoblast (OB) to be the most likely cell of origin. To understand the origin and molecular pathogenesis of osteosarcoma, we have transformed hMSCs and OBs differentiated from the same MSC with oncogenes hTERT (T), SV40Tag (S) and H-RAS (R) in series to check evaluate for spindle cell tumor formation in mice. Transformed MSC did not form osteoid, whereas OB-TSR tumor showed only scant production of osteoid, suggesting MSC may not be the origin of OS and the hTERT, SV40Tag and h-Ras may not be sufficient for osteoblast to form OS.

C-Myc is one of the genes that play a major role in oncogenic transformation of normal cells. It has been shown that c-Myc is overexpressed in OS cells and more than 10% OS patients carry c-Myc amplification mutations. We further decide to look intoThis served as the basis to examine the role of c-Myc in OS formation in the background of cells transformed by hTERT and SV40. OB transformed with hTERT and SV40Tag cells were further transfected with a retrovirus containing human c-Myc to obtain stable cell lines. Quantitative PCR and western blots assays detected both gene and protein expression patterns of c-Myc, respectively, in the transformed cell lines. Transformed cell lines were analyzed for proliferation, invasion, migration, and spheroid formation to determine the tumorigenic capacity capability. TThe tumorigenesis capability is found to be correlated with the level of c-Myc expression level based onby RT-PCR data. Furthermore, we used RNA-seq to analyze gene expression profiles of the transformed cell lines, compared with those forin comparison to primary OS cell lines and patient derived xenografts (PDXs). In -vivo tumorigenic assays, histological examination for osteoid production and chromosome analysis are still under way. Our study using an osteosarcoma model with transformed human cells may provide new insights on the cell origin of OS.

#83

The critical role of seven-in-absentia (SINA) family E3 ligases in normal development and oncogenic K-RAS-driven human cancer.

Robert E. Van Sciver,1 Yajun Cao,1 Atique U. Ahmed,2 Amy H. Tang1. 1 _Eastern Virginia Medical School, Norfolk, VA;_ 2 _Mayo Clinic, Rochester, MN_.

There will be an estimated 1.8 million new cancer diagnoses in the US this year alone, with >80% of these cases driven by oncogenic K-RAS/SIAH pathway activation, even in the absence of oncogenic RAS mutations. Over three decades of intense research has focused on conquering RAS pathway activation in cancer, yet oncogenic K-RAS has remained essentially undruggable. Seven-IN-Absentia (SINA) is an evolutionarily conserved E3 ubiquitin ligase that is the most downstream signaling module identified in the RAS signaling pathway thus far. Underscoring SINA's importance is its high degree of evolutionary conservation with over 83% amino acid identity shared between Drosophila SINA and its human SINA homologs (SIAHs). As a major signaling "gatekeeper" in the RAS pathway, we have shown that SIAH is required for oncogenic K-RAS-driven tumorigenesis and metastasis in human pancreatic, lung, and breast cancers. Since SIAHs appear to be the ideal drug target to inhibit "undruggable" K-RAS activation, it is important to precisely characterize the activity, regulation, and substrate targeting mechanism(s) of this highly conserved family of SINA/SIAH E3 ligases. By deploying the Drosophila photoreceptor development system, we conducted a genetic modifier screen and identified 28 new sina mutant alleles that exhibit a range of mutant phenotypes. For example, sina complete loss of function (null alleles) is cell lethal, and several newly identified point mutant alleles exhibit stronger mutant phenotypes than those of previously published truncated alleles. Sequencing analysis of these sinamutant revealed critical roles of several immutable amino acid residues indispensable for SINA function. To demonstrate the functional conservation of SINA/SIAH proteins, we generated a collection of transgenic fly lines that carry either wild-type (WT) or dominant negative (DN) DmSINA and human SIAH1/2. Tissue-specific SINA/SIAH expression using GAL4 drivers revealed the biological consequences of gain or loss of function of SINA/SIAH1/2 in transmitting RAS signaling in Drosophila development. Immunofluorescent (IF) staining of developing imaginal discs and electron micrographs of adult tissues show that ectopic expression of SINAWT/DN/SIAHWT/DN in neurons resulted dramatic changes in neuronal cell fate in the developing eye and sensory neuronal cells on the notum. Furthermore, IF staining of larval salivary glands revealed a critical role for SINA/SIAH in regulating cell shape, focal adhesions, and cellular junctions. Results from these transgenic models suggest that Drosophila SINA and human SIAH1/2 are highly evolutionarily conserved and functionally interchangeable. Ultimately, we aim to translate these preclinical findings to demonstrate the high potency and anticancer efficacy of an anti-SIAH-based anti-K-RAS strategy against multidrug-resistant and incurable human cancer.

#84

Inhibition of Hedgehog signalling in ligand dependent tumours.

Vijesh G. Vaghjiani,1 Catherine Cochrane,1 Neil Watkins,2 Stuart Mudge,3 Jason E. Cain1. 1 _Hudson Institute of Medical Research, Clayton, Australia;_ 2 _Garvan Institute of Medical Research, Australia;_ 3 _Mayne Pharma Pty Ltd, Melbourne, Australia_.

Hedgehog (Hh) signalling is evolutionarily conserved pathway that is critical during development and maintenance of stem cells in adults. Activation of the Hedgehog pathway can occur through either ligand-dependent or ligand-independent mechanisms. While mutations in key Hh signalling components (PTCH1, SUFU, SMO, GLI1) result in ligand-dependent signalling and constitutive activation in some cancers, the vast majority of cancers in which Hh signalling is implicated do not have pathway mutations and are therefore driven upstream at the level of Hh ligand. Despite this, there are no biomarkers that can predict which tumours are dependent on Hh ligand and are likely to be responsive to Hh pathway inhibition.

Using a panel of osteosarcoma cell lines derived from either radiation-induced or conditional Trp53 and Rb1 genetic inactivation murine models of osteosarcoma, we observed a dramatic increase in Gli1 mRNA, a downstream target of the Hh pathway, in response to Hh ligand stimulation in Trp53 and Rb1 deficient cells. This was accompanied by a significant increase in primary cilia frequency, a critical organelle for Hh pathway activation. Importantly, Hh response to ligand was abolished using the SMO inhibitor LDE225. Using osteosarcoma allograft models, we showed that LDE225-mediated inhibition of the Hh pathway resulted in reduced tumour growth and increased survival in ciliated Trp53 and Rb1-deficient allografts but no response in a non-ciliated Trp53 and Rb1 wildtype-allograft.

The sustained clinical use of SMO inhibitors ultimately leads to development of resistance. We next evaluated the efficacy of Itraconazole, a SMO inhibitor previously demonstrated to be effective in Hh-therapy resistant ligand-independent tumour models, in our ligand-dependent osteosarcoma models. We show that Itraconazole inhibits Hh pathway activation in response to ligand leading to reduced Gli1 mRNA in vitro, and reduced tumour growth and increased survival in vivo.

Collectively, this data implicates Trp53, Rb1 and primary cilia as a potential biomarker of Hh ligand responsiveness in osteosarcoma and response to Hh pathway inhibition by SMO inhibitors LDE225 and Itraconazole.

#85

Noninvasive imaging of tumor hypoxia during radiation-induced tumor vascular disruption.

Olivia J. Kelada,1 Sijumon Kunjachan,2 Needa A. Virani,2 Alexandre Detappe,3 Jennifer Hayashi,4 Thomas Ireland,5 Douglas E. Biancur,3 Rajiv Kumar,4 Srinivas Sridhar,4 Mike Makrigiorgos,2 Ross I. Berbeco2. 1 _PerkinElmer Inc, Boston, MA;_ 2 _Dana-Farber Cancer Institute, Brigham and Women's Hospital and Harvard Medical School, Boston, MA;_ 3 _Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA;_ 4 _Northeastern University, Boston, MA;_ 5 _Boston University, Boston, MA_.

Purpose Tumor vascular targeted gold nanoparticles induce tumor vascular disruption when combined with external beam radiation therapy. Although effective in suppressing tumor growth and improving progression-free survival, tumor hypoxia may be a potential challenge in this anti-vascular therapy. Here, we investigate, the dynamic changes in tumor hypoxia pre- and post-radiation therapy using a gold nanoparticle-based tumor vascular disrupting agent.

Materials and methods 4-6 weeks old female nude-FOXn1 mice were subcutaneously inoculated with human A549 cells (~3×106) into the left flank. Functionalized gold nanoparticles (AuNP) were used to target the tumor blood vessels and tumor vascular disruption was induced via radiation. 10 Gy radiation treatment was delivered using a clinical radiation beam (6 MV) and HypoxiSense680 based in vivo fluorescence imaging was performed to visualize changes in tumor hypoxia at 24 h, 48 h and an extended period of 10 days. Tumor hypoxia was confirmed via immunohistochemistry. Mice were divided into four treatment groups: control, AuNP only, IR only, and AuNP+IR. Following treatment, tumor progression and overall survival was measured. Further analysis of nanoparticle biodistribution and toxicity were accessed using TEM Imaging, IC-PMS, and immunohistochemistry.

Results Longitudinal changes in tumor hypoxia were observed in all radiation-based treatment conditions. Combining gold nanoparticle and radiation resulted in an increase in tumor hypoxia at 48 h (p < 0.05) and a return to baseline in 10 days. In contrast, the 'radiation only' group showed an increase in tumor hypoxia by a factor of 0.5 at 48 h post-IR compared to baseline while 10 days later the tumor hypoxia remained stable. Quantitative variation in the hypoxia blood factor, CA9 increased 24 h post IR in the AuNP+IR, followed by a decrease in hypoxic by day 10 in accordance with in vivo imaging data. No change was observed with the IR only group. These findings were confirmed with representative pimonidazole staining that showed an increase in tumor hypoxia a few hours after AuNP+IR treatment. The mean relative reduction in tumor size post-treatment was a factor of 5.2 (p < 0.05) in the AuNP+IR group compared to the control and 3.5 compared to the IR only group, nearly 80 days post-treatment. Overall survival showed an average gain of up to 24 days in the AuNP+IR group compared to all other treatments. Almost 100 days post-treatment, 50% survival was observed in the AuNP+IR group compared to 20% in the IR-only groups (p < 0.05).

Conclusions Noninvasive imaging showed that AuNP+IR results in a transient increase and subsequent decline in mean tumor hypoxia, leading to substantial tumor regression and an overall increase in tumor survival. High radiation-induced vascular damage may lead to better tumor reduction and prolonged survival in the human non-small cell lung cancer model.

#86

Pre-existing neurovascular inflammation increases the occurrence of brain metastases.

Dina Sikpa, Lisa Whittingstall, Jérémie P. Fouquet, Luc Tremblay, Réjean Lebel, Martin Lepage. _Université de Sherbrooke, Sherbrooke, Quebec, Canada_.

BACKGROUND: Brain metastases (BM) are the most prevalent intracranial neoplasm. Inflammation is central to the development of cancer. While an intra-tumoral inflammatory microenvironment contributes to the acquisition of malignant phenotypes and leads to the release of circulating tumor cells (CTCs), pre-existing inflammation at distant sites facilitates the adhesion of CTCs to the activated vascular endothelium and the consequent formation of metastases. Cell adhesion molecules expressed by activated endothelial cells contribute to metastatic spread outside of the brain and the vascular cell adhesion molecule-1 (VCAM-1) is a key mediator of inflammation. Herein we assessed if cancer cells entry into the brain is aided by VCAM-1 upregulation with inflammation.

METHODS: Stereotaxic lipopolysaccharide (LPS, 1 µg) injection into the right hemisphere was used to induce neurovascular inflammation in Balb/c mice. The distribution of VCAM-1 was semi-quantified 24 h post LPS injection using molecular magnetic resonance imaging (MRI) with microparticles of iron oxide (MPIOs) functionalized with VCAM-1 antibody (MPIO-VCAM-1). Mice injected with saline served as control. VCAM-1 is also expressed on vessels associated with metastases, MPIO-VCAM-1 were therefore used to detect metastases. Basal level of metastases in animal brains was measured in a group of mice intracardially injected with cancer cells without LPS intra-cortical injection. To study the impact of pre-existing inflammation on tumor cell entry into the brain, mice were injected with LPS (or saline as above) 24 h prior to intracardiac injection of 4T1 breast cancer cells (105 cells in 100 µL PBS). Metastases imaging was performed 18 days post cancer cells injection. To assess if blocking VCAM-1 would affect metastases implantation, an extra LPS-injected group of mice was injected intravenously with MPIO-VCAM-1 4 hours before 4T1 cells injection. In this group, MRI was performed 3 hours after MPIO-VCAM-1 injection (VCAM-1 imaging), and on day 18 post tumor cell injection (metastases imaging). All MRI experiments were conducted on a small animal 7T scanner (Varian Inc.) with a dedicated mouse head-coil (RAPID MR International) using a T2*-weighted sequence. Following the final imaging session brains were extracted for histological analysis.

RESULTS: Both MR and histological data reveal that the metastatic burden significantly increases in the LPS-injected group compared to control conditions. In inflamed animals, blocking VCAM-1 with MPIO-VCAM-1 reduces the metastatic burden back to control values. This suggests that (1) a pre-existing inflammation increases the occurrence of brain metastases and (2) blocking VCAM-1 reduces this effect.

CONCLUSION: We demonstrate that inflammation-induced VCAM-1 contributes to tumor cells adhesion in the brain. Therefore, VCAM-1 may represent an attractive therapeutic target to reduce risks of metastasis.

#87

Recapitulating the orthotopic tumor microenvironment using bioluminescent syngeneic models in immune competent mice for checkpoint inhibitor interrogation.

Joyce Afrakoma Obeng,1 Maya Jetha,1 Jane Wrigley,1 Simon Jiang,1 Jason King,1 Rajendra Kumari,2 Yinfei Yin1. 1 _Crownbio Science UK, Loughborough, United Kingdom;_ 2 _Crownbio Science Inc, Loughborough, United Kingdom_.

BACKGROUND: Checkpoint inhibitors have shown promising therapeutic benefit in various cancer types, but more advanced preclinical models are required to combat resistance and evaluate opportunities for combination treatments. Subcutaneous syngeneic models are routinely used to evaluate the impact of immunotherapies on tumour growth as well as tumour invading leucocytes (TILs). However, the tumour microenvironment of orthotopic models is more comparable to the patient due to the organ-specific location of the tumour which facilitates metastatic spread and also recapitulates the immune and stromal component interactions with the tumour. Bioluminescent imaging (BLI) enables non-invasive longitudinal monitoring of orthotopic tumour burden and end stage quantification of tumour spread. Here we report the generation of a panel of bioluminescent syngeneic cell lines for orthotopic and metastatic modeling with an aim to assess the impact of standard of care agents, immune checkpoint therapies and combinations in a more clinically relevant environment.

METHODS: Bioluminescent variants of syngeneic cell lines were established by lentiviral transduction. Orthotopic models were established via direct implantation into various organs e.g. liver, breast, pancreas and bladder and tumour growth assessed by BLI in-life as well as end stage (Spectrum CT; PerkinElmer). Response to standard of care agents such as sorafenib, gemcitabine, docetaxel and immune checkpoint therapy (anti-CTLA-4, anti-PD-1) was also evaluated and TIL infiltration was assessed by flow cytometry analysis and IHC.

RESULTS: The success rate of tumour transplantation into different organs ranged from 60-100% as confirmed by both in-life imaging and ex vivo imaging at termination. For example, Hepa 1-6 appeared to grow within the parenchyma of the liver whereas 4T1 metastasized to the lungs and bone from the mammary fat pad, which was easily quantified by BLI. Real-time quantification of tumour size could be correlated with biomarkers and end stage assessment, for example, treatment with Sorafenib and check point inhibitors was correlated with alpha-fetoprotein (AFP) and end stage tumour burden, showing significant response in the orthotopic setting (p<0.001, Two way ANOVA). Different responses to check point inhibitors were observed between tumors inoculated at subcutaneous sites and orthotopic sites.

CONCLUSIONS: Bioluminescent syngeneic models enables clinically relevant interrogation of standard of care agents, immunotherapies and combination which is distinct from the subcutaneous setting. The orthotopic microenvironment influences the tumour growth and response to immuno therapy. These models also enable the assessment of disease progression and modelling the metastatic environment.

#88

**Stable luciferase expressing cell lines for** in vivo x **enograft and syngeneic tumor model bioluminescence imaging.**

John Foulke, Luping Chen, Alexei Miagkov, Elizabeth Turner-Gillies, Lysa-Anne Volpe, Fang Tian. _ATCC, Manassas, VA_.

Human and mouse cancer cell lines are used in xenograft and syngeneic models, respectively, for the in vivo study of tumor formation and development, metastases, measuring tumor burden in whole animals and monitoring response to therapeutic treatment. Whole animal in vivo imaging has been widely applied by researchers due to the ease of operation in visualizing in vivo biological events, eliminating the requirement for animal subject sacrifice, allowing for continual monitoring/imaging of a single individual animal and reducing the amount of inter-animal variation. Luciferase reporters provide a relatively simple, robust, and highly sensitive means to measure biological processes and to assess drug efficacy in animal models through in vivo bioluminescence imaging. Here we report on the generation of a large panel of cell lines which express high levels of luciferase and -have broad applications for in vitro and in vivo studies. The panel includes human and mouse cell lines, which represent various common cancer and tissue types, such as breast, colon, lung, pancreas, prostate, and skin. After introduction of a Lenti-LUC2 luciferase reporter into the parental cell lines, single cell cloning was performed to isolate stable clones with high luciferase expression. We quantified both the relative and absolute in vitro bioluminescence signals within the cells and analyzed the signal-to-noise ratio. In addition, the cell lines were characterized and authenticated using cell morphology, growth kinetics, and STR analysis. In addition, luciferase labeled human cell lines were administered into immune-deficient mice either by subcutaneous or orthotropic injection to establish xenograft models. Luciferase labeled mouse cell lines were tested in the associated breed of mice to evaluate tumor growth in the syngeneic mouse model. The bioluminescence live imaging was performed using the Xenogen IVIS imaging system and the in vivo bioluminescent radiance was analyzed. Although growth can be evaluated by both traditional caliper measurement and bioluminescence in subcutaneous mouse models, only live bioluminescent imaging enables continuous monitoring tumor metastasis or analysis of tumor growth of the intracranial brain tumor models. In summary, luciferase expressing cell lines are valuable tools for elucidating mechanisms involved in tumorigenesis, studying tumors in vivo, and screening anti-cancer compounds for drug discovery and development.

#89

Development and characterization of a panel of orthotopic glioblastoma multiforme (GBM) patient-derived xenograft (PDX) mouse models for drug efficacy evaluation.

Carsten H. Nielsen,1 Maria Z. Alfsen,1 Michael J. Wick,2 Melissa Rundle,2 Johann M. Gudbergsson,3 Mette M. Jensen,1 Lotte K. Kristensen,1 Mark U. Juul,1 Kyriakos P. Papadopoulos,2 Andreas Kjaer4. 1 _Minerva Imaging, Copenhagen, Denmark;_ 2 _START, San Antonio, TX;_ 3 _Laboratory of Immunology & Cancer Biology, Department of Biomedicine, Aalborg Universitet, Aalborg, Denmark; _4 _Department of Clinical Physiology, Nuclear Medicine & PET and Cluster for Molecular Imaging, Department of Biomedical Sciences, Rigshospitalet and University of Copenhagen, Copenhagen, Denmark_.

Background

Glioblastoma multiforme (GBM) is an aggressive cancer type with poor prognosis and survival. The lack of effective treatment may be due to the complex molecular composition and the heterogeneity of the tumors. Subcutaneous patient derived xenograft (PDX) mouse models are widely used in drug development. However, the models fail at modeling the complex microenvironment in the brain and the impact of the blood brain barrier on drug bioavailability. Here we report the development and characterization a panel of orthotopic PDX mouse models.

Methods

Low passage subcutaneous tumors from ten different PDX GBM models (ST108, ST112, ST146, ST545, ST610, ST1388, ST2473, ST3537, ST3713, and ST3720) were implanted orthotopically. Tumor development and growth was monitored by T2-weighted magnetic resonance imaging (MRI). Mice were treated with either temozolomide or vehicle when tumor take was confirmed on an individual basis by MRI. Tumor treatment response was evaluated by MRI, and the final end-point was survival by humane endpoints. Whole brains and tumors were formalin fixed or snap frozen for histological evaluation of markers of invasiveness and cancer stem cells (Nestin,, CD44, SOX2, and CXCR4).

In addition, the radiosensitivity of the models was characterized by fractionated external radiation therapy (XRT) delivered as 2 Gy QD x5 or sham in the subcutaneous setting. Expression of EGFR, EGFRvIII, MGMT and mGluR3 were evaluated by qPCR.

Results

The models displayed a wide range in expression levels of EGFR, EGFRvIII, MGMT and mGluR3. Mice treated with XRT showed a variable treatment outcome on both tumor volume and survival. The impact of XRT on prolongation of median survival ranged from 45% to 141%. In the orthotopic setting a variable range of sensitivities was observed in the models ranging from sensitive (ST610 and ST2473) to highly resistant (ST112).

Histologic evaluation showed typical characteristics of GBM tumors such as pseudopalisading cells. Furthermore, cell populations positive for cancer stem cell and invasiveness markers were detected by immunofluorescence imaging.

Conclusion

A panel of GBM PDX models was characterized for several molecular markers and the panel reflected the heterogeneity of human GBM tumors. The models displayed varying sensitivity towards XRT and TMZ.

The established panel of orthotopic GBM PDX models can be used as a platform for testing of new drugs in a setting that more closely mimics the GBM tumor microenvironment and impact of the blood brain barrier.

#90

The "reproducibility crisis" of animal studies in oncology - How did we get here, and how can we resolve it.

Hannes Hentze. _Vivology Consulting, Singapore, Singapore_.

Historically, a large majority of clinical phase III trials fail to gain regulatory approval due primarily to a lack of demonstrated efficacy, demonstrating the challenge of conducting clinically relevant efficacy studies in laboratory animals. In 2012, Amgen researchers published their finding that an astoundingly-high proportion (90%) of more than 50 republished in vivo studies in oncology was not reproducible. This sparked an intense debate about data validity in preclinical sciences, and several reviews have been published in the last two years analysing this issue. A number of best-practice initiatives and resources have since been created along with the development of animal study workflow software applications and databases to address the causes of this significant, global problem in cancer research. Major sources of irreproducibility in animal research such as bias, suboptimal disease models, lack of controls, and most importantly bad documentation practices will be discussed. Several initiatives have resulted in practically-applicable concepts and publicly-available resources to address this issue, such as ARRIVE guidelines, PHISPS protocols and the N3R Design Assistant, will be described. Electronic spreadsheets have been the most prevalently-used tool to plan and conduct preclinical efficacy studies in oncology. The advent of animal study workflow software and database technology, designed specifically for in vivo oncology research, has seen increasingly widespread adoption in recent years in both academia and industry. By employing standardized and more detailed approaches to the processes documenting highly detailed study design, data collection, method and procedure descriptions and adherence, real-time monitoring of animal welfare parameters, standardized graphing, data analyses and report generation, measurement and task scheduling, as well as data preservation approaches, will be explored. Using some real-life examples, the advantages and disadvantages of preclinical study automation and the use of electronic spreadsheets will be discussed in the context of helping to resolve the animal study "Reproducibility Crisis".

#91

Diet-variants and immune characterization of a stage-defined, transgenic immunocompetent mouse model of HCC (ASV-B).

Annemilaï Tijeras-Raballand,1 Christian Hobeika,2 Benoit Rousseau,3 Patricia Hainaud,4 Philippe Bonnin,2 Aurélie Rodrigues,5 Fouad Ladfil,5 Marc Pocard,2 Valérie Paradis,6 Armand de Gramont,1 Eric Raymond,7 Evelyne Dupuy,4 Clarisse Eveno,2 Sandrine Faivre6. 1 _AFR Oncology, Paris, France;_ 2 _Lariboisière University Hospital, Paris, France;_ 3 _Mondor University Hospital, Créteil, France;_ 4 _Institut des Vaisseaux et du Sang, Paris, France;_ 5 _Inserm U955, Innovation and Drug Development Unit, Créteil, France;_ 6 _Beaujon University Hospital, Clichy, France;_ 7 _Saint-Joseph Paris Hospital, Paris, France_.

Background: Non-alcoholic fatty liver disease (NAFLD), especially non-alcoholic steatohepatitis (NASH) is a chronic liver disease commonly associated with hepatic fibrosis. NASH patients have an increased risk for hepatocellular carcinoma (HCC). Due to western way of life, NASH incidence is rising and is predicted to become the leading cause of HCC in the next decades. Therefore, there is an urgent need for robust animal models fully recapitulating the NASH-related HCC carcinogenesis. In this study, we develop and characterize specific diet-induced variants from our transgenic HCC mouse model, focusing on immune landscape.

Methods: To mimic NASH, ASV-B, a transgenic mouse model (C57BL/6J) that spontaneously develops a reproducible stage-defined HCC (hyperplasia at week(W)8, nodular stage at W12, and diffuse carcinoma at W16-20) was exposed to 5 different diets. Ten ASV-B and 5 control mice were fed as follows: classic diet as control (yellow), or a high-fat diet (blue), a diet enriched with saturated fatty acids + 1.25% cholesterol (green), a diet containing 22% of vegetal oil + 0.2% cholesterol (orange), and a 1.25% cholesterol diet containing 21% of milkfat (red). All mice fed with special diets also received 30% fructose in the drink water. RNA was extracted from frozen livers at W20 for 40 immune markers analysis using qRT-PCR (LightCycler, Roche). Immune populations were assessed using automated immunohistochemistry (IHC) (Bond Max, Leica).

Results: ASV-B model shows an increase in liver volume and angiogenesis, ASV-B livers harboring marked arterialization and capillarization as compared to control. Assessing immune markers on 7 evaluable tumor specimens, we observed an increase in CD8, Foxp3, INOS, CD11b, PD-1, PD-L1, IL1β, IFN-γ, TNF-α, IL17A and IL17F mRNA expression, as frequently observed in human inflammatory HCC. In addition, IHC staining showed intratumoral infiltration of lymphocytes (CD8+) and macrophages (F4/80+, a well-characterized and extensively referenced mouse macrophage marker). ASV-B mice receiving yellow, blue, and green regimens showed similar liver volumes and weight. By macroscopic analysis, we observed increased liver steatosis, and fibrosis in the red and orange regimen compared to others. Moreover, we observed a 40% mortality rate in the orange regimen, and a 20% mortality rate in the blue and the green regimens. At the conference, we will show the morphologic changes of the livers using HPS staining and the immune landscape in the livers of the diet-variants.

Conclusion: ASV-B transgenic mouse model mimics several characteristics of human HCC developing on healthy liver including inflammatory reaction and immune cell infiltration. In the ASV-B model, we have been able to develop specific-diets variants aiming at mimicking NASH that could be used for drug testing.

#92

Characterization of hormone-sensitive and castrate-resistant phenotypes in prostate cancer patient-derived PDX models generated from the same patient.

Hervé Lang,1 Claire Beraud,2 Myriam Lassalle,3 Véronique Lindner,1 Eric Potiron,4 Philippe Lluel,5 Thierry Massfelder6. 1 _Strasbourg University Hospital, Strasbourg, France;_ 2 _Urolead, Toulouse, France;_ 3 _Urosphere, Toulouse, France;_ 4 _Clinique Atantis, Nantes, France;_ 5 _Urosphere/Urolead, Toulouse, France;_ 6 _Inserm UMR 1260, Strasbourg, France_.

Prostate cancer (PCa) is a highly heterogeneous and complex disease, with evolving treatment options over the course of disease progression. Preclinical PCa research is hampered by a lack of predictive models fully capturing all phases of this multistage disease. Despite progresses in the development of genetically-engineered animal models, these ones do not recapitulate faithfully (i) human disease and (ii) tumor heterogeneity. Models obtained by xenografting human tumors in immunodeficient animals (PDX models, for patient-derived tumor xenografts) remain unavoidable tools in PCa translational and preclinical research since they closely conserve cancer characteristics observed in patients. PDX models are thus invaluable tools to evaluate new potential therapeutic agents. We are presenting here the characteristics of two PDX models derived from the same patient before and after acquisition of the hormone-resistance status. Samples of PCa were obtained from patients at surgery and then subcutaneously xenografted into immunocompromised mice to establish PDX models. After the first growth in mice, they were serially passaged in vivo, considering a model established from P3. PDX tumors at multiple passages and patients' primary tumors from which they are derived were processed for further analyses. Specifically, we performed histological, genetic (AR, PTEN, P53 and ERG status), transcriptomic (Affymetrix U133 plus 2.0 microarray) and STR profiles analyses. In addition, we also evaluated the responses of the PDX models to androgen deprivation and docetaxel. Since 9 years, 252 prostatic tumors have been collected at all stages. Up to now, 7 PDX models were successfully established (> P3 in mice), i.e. 2.7 % success rate. All histological, genetic and molecular analyses validated the stability of the models compared to the parental tumor. Interestingly, we were able to generate one matched pair of responsive and castration resistant models from the same patient. These two PDX models displayed the major molecular features of the disease in humans including PTEN, TP53 and AR modifications. In addition, in vivo results show heterogeneity of response to androgen deprivation and docetaxel, similar to the responses of patients to these treatments. Considering the scarcity of useful PDX models for PCa and the difficulties to develop such models, the PDX models collection presented here should clearly help to open the road of cure for patients with advanced PCa.

#93

Anti-LAP-TGFb antibodies inhibit tumor growth in a CT26 syngeneic tumor model in combination with radiation therapy.

Kenneth J. Simon,1 Stavros Kopsiaftis,2 Randall Burton,2 Patricia E. Rao,2 Jessie M. English,2 Barbara S. Fox2. 1 _Tilos Therapeutics, Milton, MA;_ 2 _Tilos Therapeutics, Lexington, MA_.

Anti-LAP-TGFβ antibodies inhibit tumor growth in a CT26 syngeneic tumor model in combination with radiation therapy.

TGFβ is a major immunosuppressive cytokine that acts within the TME and is implicated in resistance to checkpoint inhibitors. TGFβ is synthesized as a pro-protein complex in which the mature cytokine is caged within LAP, the latency associated peptide of TGFβ1, and serves to hold TGFβ1 in a latent state. Anti-LAP-TGFβ antibodies have efficacy in mouse models of cancer (Gabriely et al., 2017) and offer a promising new treatment approach in oncology. Radiation is standard of care in many cancer indications and is a known potent inducer of TGFβ. TGFβ is also implicated in resistance to radiation treatment. Thus, combination treatment with anti-LAP antibodies and radiation may yield a novel approach for enhancing therapeutic responses in a population with significant unmet medical need. We have evaluated the anti-tumor effects of targeting TGFβ via an anti-LAP antibody in combination with radiation therapy in a murine syngeneic CT26 colorectal cancer model.

We identified a unique class of anti-LAP antibodies, TLS-01, that specifically target LAP on the surface of cells, but do not bind to LAP-TGFβ in the extracellular matrix. We assessed the effects of combination of TLS-01 and radiation on both tumor efficacy and modulation of immune cell subsets within the TME. CT26 tumor bearing mice (tumor size ~300 mm3) were treated with either TLS-01, an isotype control antibody, 12 Gy or 20 Gy of targeted radiation, or a combination of TLS-01 with 12 Gy or 20 Gy of radiation. Seven days after treatment, 3 animals per group were analyzed for tumor infiltrating immune cell subsets and the remaining animals were followed for tumor growth and survival for up to 19 days. Radiation inhibited tumor growth in a dose dependent fashion. Combination treatment with TLS-01 and radiation inhibited tumor growth to a greater extent than was seen with TLS-01 or radiation treatment alone. Radiation treatment dramatically increased the number of CD8+ T cells in the TME and concomitantly increased both the number and immunosuppressive properties of inhibitory cell populations in the TME, including increases in CD73 expression on M-MDSCs and M2 macrophages. Treatment of irradiated mice with TLS-01 reduced Tregs, increased the CD8 / Treg ratio and reduced CD73 expression on immune suppressive cells.

Combination of TLS-01 with radiation resulted in enhanced efficacy when compared to either agent alone. Our data provides mechanistic insights underpinning this enhanced efficacy. TLS-01 moderated immunosuppression in the TME by radiation via modulation of multiple immunosuppressive cell types. These data support the therapeutic utility of combination treatment of TLS-01 and radiation therapy in the treatment of solid tumors.

#94

Role of Argonaute 2 in oncogene induced senescence in a pancreatic cancer mouse model.

Seema Chugh, Jean C. Tien, Ronald F. Siebenaler, Vijaya L. Dommeti, Sylvia Z. Wang, Sanjana Eyunni, Kristin M. Juckette, Lisha Wang, Sunita Shankar, Arul M. Chinnaiyan. _University of Michigan, Ann Arbor, MI_.

Pancreatic cancer is a highly devastating malignancy with a very poor survival rate of 7%. Mutations in KRAS have been identified in more than 90% of PDAC patients. Previous work from our lab has shown that KRAS directly interacts with Argonaute 2 (AGO2) to promote cellular transformation. To study the involvement of AGO2 in KRASG12D-driven cancers, AGO2 expression was ablated in the KrasG12D/+; p48 Cre model (KC) model of pancreatic cancer. AGO2 floxed mice (AGO2loxP/loxP) were crossed with KC mice, resulting in oncogenic KRAS expression along with knockout of AGO2 in pancreatic acinar cells. Survival and disease progression were compared between wild-type (AGO2+/+; KRASG12D; p48Cre), heterozygous (AGO2fl/+; KRASG12D; p48Cre), and homozygous (AGO2fl/fl; KRASG12D; p48Cre) experimental mice groups. Homozygous knockout of AGO2 in KC mice resulted in significantly increased survival as compared to wild type and heterozygous mice. Pancreatic ductal adenocarcinoma (PDAC) and metastases were restricted to the wild-type and heterozygous mice. Pancreas from AGO2fl/fl; KRASG12D; p48Cre mice develop only early pancreatic intraepithelial lesions (PanINs), which fail to progress to PDAC. Senescence-associated β-galactosidase staining showed strong and significant increase in senescence in PanIN lesions mice lacking AGO2 expression as compared to AGO2+/+; KRASG12D; p48Cre mice. This suggests that AGO2 prevents oncogene induced senescence (OIS) as a result of KRASG12D expression and allows PanIN to PDAC progression.

To gain mechanistic insights of OIS due to AGO2 loss, we evaluated markers for OIS including p16, p53, p21, gamma γH2AX, and RAS-associated signaling (pERK and pAkt). Analysis of PanIN lesions lacking AGO2 showed increased p16 levels and high levels of phospho-ERK, compared to PDAC from pancreas with AGO2 expression. In order to extend these observations in cell line models, we performed AGO2 knockdown in T24 cells harboring HRASG12V. Surprisingly, cells with low AGO2 levels underwent OIS, which was similar to the pancreatic mouse model and was accompanied with increased phospho-ERK signaling and p16 expression. Further studies are underway to determine the contribution of the RAS-AGO2 interaction in the development of OIS.

Additionally, we are using CRISPR/Cas9 technology to screen pancreatic cancer cell lines with AGO2 knockout for their dependence on AGO2 and their ability to undergo OIS in the absence of AGO2 expression. We will present findings from our ongoing studies involving the role of AGO2 loss in the KPC (KRASG12D; p53fl/+; Cre) model, wherein OIS will be assessed in the absence of p53, a canonical inducer of cellular senescence.

#95

Synthetic essentiality of chromatin remodeling factor CHD1 in PTEN deficient cancer.

Di Zhao. _UT MD Anderson Cancer Ctr., Houston, TX_.

Prostate cancer (PCa) is one of the most common cancer types in mem. Advanced PCa, especially metastatic castration resistant prostate cancer (mCRPC), has high morbidity and mortality. ~50% advanced PCa shows PTEN deletion or mutation, therefore, identification of specific therapeutic targets for PCa harboring PTEN deficiency holds hope for patients with advanced PCa. Taking advantage of the vast public available prostate cancer genome database, we recently explored a novel approach to identify potential therapeutic targets by screening for "synthetic essential" genes that were occasionally deleted in cancer but always retained in the context of tumor suppressor gene deficiency. Using this method, we identified the chromodomain-helicase-DNA-binding protein 1 (CHD1) as a synthetic essential gene in PTEN-deficient PCa. Both human PCa xenograft models and genetically engineered PCa mouse models suggested that depletion of CHD1 suppresses the tumor growth and progression of PTEN-deficient PCa, but has minimal effects on normal prostate tissue or PTEN-intact PCa. Importantly, CHD1 inhibition leads to a significantly prolonged overall survival in PTEN-loss PCa mouse model. Mechanistically, we found that PTEN-PI3K-AKT-GSK3βpathway regulates CHD1 phosphorylation, followed by ubiquitination and degradation via β-TrCP mediated proteasome pathway. The ChIP-seq and transcriptional profiling analyses uncovered that, in PCa with PTEN loss, CHD1 is stabilized and interacts with epigenetic marker H3K4me3, resulting in the transcriptional activation of NF-κB network genes. Furthermore, immunophenotyping using Mass Cytometry (CyTOF) and functional validation suggested that CHD1 contributes to the immunosuppressive tumor microenvironment in PCa through stimulating the expansion and activation of myeloid-derived suppressor cell (MDSCs) and reducing tumor-infiltrating CD8+ T-cells. Together, this study demonstrated that CHD1 plays important roles in tumor initiation and progression of PTEN-deficient PCa, and presents a promising context-specific therapeutic target in PCa. In addition, the novel approach of "synthetic essentiality" we proposed provides a good tool to identify context-specific therapeutic targets for undruggable tumor suppressor deficiency in cancer genome.

#96

High iron or high fat diet show similar effects in inducing hepatocellular carcinoma in a mouse model.

Hakim Bouamar,1 Kalyan Kakarla,1 Matyas Cserhati,1 Fatima Ezzahra El Mroussi,1 Francis E Sharkey,1 Francisco Cigarroa,1 Lu-Zhe Sun2. 1 _UTHSC, san antonio, TX;_ 2 _UTHSC, San Antonio, TX_.

Introduction: Hepatocellular carcinoma (HCC) has multiple risk factors such as Hepatitis virus infection and obesity. Iron overload may be another risk factor as patients with hemochromatosis have high levels of liver iron accumulation and higher incidence of HCC than the general population. This study aims to assess the risk of high iron diet in comparison to high fat diet in the development of HCC.

Materials & Methods: We used C3HeB/FeJ male mice, which have been shown to spontaneously develop HCC. Two-month old mice were fed for 15 months with a control diet (10 kcal% from fat; 48 ppm Iron), high iron diet (HID) (10 kcal% from fat; 200 ppm Iron), high fat diet (HFD) (61.1 kcal% from fat; 48 ppm Iron), or high iron and fat diet (HIFD) (61.1 kcal% from fat; 200 ppm Iron). The presence of liver tumor was confirmed by ultrasound imaging and histology analysis. At termination, normal and tumor liver tissues were harvested for tumor size measurement, metal ion quantification, immunohistochemistry, and gene expression with RNA-seq.

Results: HID like HFD induced significant body weight gain. All three diets induced HCC (4 out of 7 by HID, 7 out of 9 by HFD, and 8 out of 10 by HIFD). Yet, HCC was not found in the control diet group. The three diets showed no significantly different effects on tumor burden and grade. The concentrations of iron and copper were increased in the normal liver tissues of HID and HIFD groups. Interestingly, they were significantly lower in the tumor tissues than in the paired adjacent non-tumor tissues. Analyses of RNA-seq data revealed extensive overlap of tumor-specific differentially expressed genes (DEGs) among the three diet groups. The extensive overlap was also observed in the DEG-associated gene otology (GO) terms among the three diet groups. All three diets, but not the control diet, were shown to activate mTORC1 pathway in the tumors according to our RNA-seq data. The activation of mTORC1 pathway in the three diet-induced tumors was also confirmed with Real-time RT-PCR assays revealing upregulation of mTORC1 target genes and with immunohistochemistry showing increased phosphorylation of an mTORC1 substrate.

Conclusions: The spontaneous hepatocarcinogenesis in male C3HeB/FeJ mice requires HID or HFD. The lack of additive effect of HID and HFD on HCC incidence and the extensive overlap of DEGs and GO terms among tumors induced by HID and HFD suggest they may regulate common mechanisms such as mTORC1 in inducing HCC. 

### Drug Targets in the Microenvironment

#97

The effect of hyaluronan degradation on the uptake and cytotoxic activity of liposome-encapsulated doxorubicin in 3D tumor spheroid models.

Feng Gao, Susan Zimmerman, Kelly Chen, Daniel C. Maneval, Chunmei Zhao. _Halozyme Therapeutics, Inc, San Diego, CA_.

The accumulation of hyaluronan (HA) in the solid tumor microenvironment (TME) can be associated with poor prognosis in several human cancers. Accumulation of HA in the TME can increase interstitial pressure that compresses local blood vessels and impairs the access of anti-cancer therapy. In preclinical models, enzymatic degradation of HA by pegvorhyaluronidase alfa (PEGPH20; PVHA), a PEGylated form of recombinant human hyaluronidase PH20 (rHuPH20), is associated with decreased tumor interstitial pressure, improved tumor vascular perfusion, and increased access and anti-tumor efficacy of cytotoxic and immunotherapies.

The functional effect of HA degradation on the TME has been mostly characterized in animal tumor models. Here, we describe the utilization of 3D tumor spheroid models to evaluate the effects of HA degradation on the cytotoxic effect of anti-cancer therapies. A number of commonly used human colorectal cancer (CRC) cell lines were assessed for HA accumulation and tumor spheroid morphology: HCT 116, HT-29 parental, and HT-29/HAS3 (HT-29 cells engineered to over-express hyaluronan synthase 3). CRC 3D tumor spheroid models were developed to evaluate the uptake and cytotoxic activity of liposome-encapsulated doxorubicin (DOX) using the IncuCyte® S3 Live-Cell Analysis System. The effect of HA degradation on DOX uptake and cytotoxic activity was initially tested with rHuPH20 treatment in all three models, and further evaluated with PVHA treatment in HT-29 parental and HT-29/HAS3 models.

A dose-dependent uptake and cytotoxic activity of DOX was observed in HCT 116 tumor spheroids. HA degradation after rHuPH20 treatment enhanced DOX uptake by an average of 6.1% (up to 7.7%) and cytotoxicity by an average of 37.9% (up to 58.2%) over the 5-day duration of the experiment compared with DOX alone. Dose-dependent uptake and cytotoxic activity of DOX in HT-29/HAS3 tumor spheroids were enhanced with rHuPH20 treatment to a greater extent than in HT-29 parental tumor spheroids. Specifically, rHuPH20 treatment led to an average increase in DOX uptake of 9.2% (up to 14.6%) with HT-29/HAS3 spheroids and 2.6% (up to 5.1%) with HT-29 parental spheroids. The addition of rHuPH20 resulted in an average increase in DOX cytotoxicity of 101% (up to 227.6%) with HT-29/HAS3 spheroids and 24.9% (up to 63.9%) with HT-29 parental spheroids. Enhanced uptake and cytotoxic activity of DOX was also observed with PVHA treatment in HT-29 and HT-29/HAS3 models.

These data corroborate our previous findings with in vivo tumor models, in which the combination of PVHA-mediated HA degradation and other anti-cancer therapies display enhanced anti-tumor activity compared with either agent alone. Further, 3D tumor spheroid models may be used to evaluate the effect of extracellular matrix remodeling such as HA degradation and serve as a bridge between 2D cell-based assays and in vivo tumor models.

#98

Stromal-targeting with quercetin in patient-derived models of head and neck squamous cell carcinoma (HNSCC).

Hui Li,1 Wenying Piao,1 Pik Yuk Lau,1 Chin Wang Lau,2 Jason Ying Kuen Chan,1 Yu Xiong Su,3 Leaf Huang,4 Kai Li Hu,5 Vivian Wai Yan Lui1. 1 _The Chinese University of Hong Kong, Hong Kong;_ 2 _Yan Chai Hospital, Hong Kong;_ 3 _The University of Hong Kong, Hong Kong;_ 4 _University of North Carolina at Chapel Hill, NC;_ 5 _Shanghai University of Traditional Chinese Medicine, China_.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive cancer with a high recurrence rate. Recurrences occur in ~25% cases of early -stage patients (Stage I-II) and 50-60% cases of advanced stage (Stage II-IV) patients. Cumulative evidences suggest that tumor stroma, especially cancer-associated fibroblast (CAF) plays a protective role and contributes to drug-resistance in HNSCC, leading to recurrences after treatment. Quercetin is a flavonoid purified from natural plants, such as green tea, onions and berries. It has been reported to suppress CAFs and reduce collagen accumulation surrounding tumors, potentially facilitating drug delivery to tumors. Here, we hypothesize that quercetin targeting of CAFs may enhance the anti-tumor efficacy of cisplatin, and potentially other antitumor therapies in HNSCC. Using a primary co-culture system with primary HNSCC cells (70%) and the paired CAFs (30%) from HNSCC patients, we investigated the effect of quercetin and cisplatin combination treatment in vitro. We found that combination of quercetin and cisplatin elicited a significantly greater growth inhibition (70% of inhibition) as compared to 37% of inhibition with cisplatin and 15% of inhibition with quercetin alone. This result appeared to demonstrate some anti-CAF effects and antitumor effects exerted by this combination strategy.Using patient-derived HNSCC primary tumor cells, stromal-rich xenografts were successfully developed in vivo. The stromal-suppressive effect of quercetin was then examined upon treatment with increasing doses of quercetin (DMF, 2.5, 5 or 10 μM in 5 microliter volume, 2 times injection). Potential alteration of the tumor architecture by quercetin (vs. vehicle treatment) was examined by H&E staining. A marked reduction of stromal component was found in quercetin -treated tumors vs. vehicle-treated tumors. Importantly, we also found that these quercetin-treated tumors have 80.4% reduction in α-SMA protein expression (6.0% in vehicle vs.1.2% in treatment group), strongly indicating a significant stromal suppressive action of quercetin in an HNSCC primary culture-derived xenograft model. Furthermore, quercetin (10 μM) treatment reduced collagen expression by ~80% (vs. vehicle treatment) in the HNSCC primary culture-derived xenograft model by Masson's trichrome staining. In conclusion, quercetin can effectively suppress the activated status of CAFs and may potentially enhance anti-tumor effect of cisplatin in an HNSCC patient-relevant manner. The antitumor efficacy of quercetin combination strategies should warrant further investigations.

#99

Remodeling of calcium influx pathways in models of cancer associated fibroblasts in breast cancer.

Greg Monteith,1 Francisco Sadras,1 Teneale Stewart,1 Melanie Robitaille,1 Priyakshi Kalita-de Croft,1 Patsy Soon,2 Jodi Saunus,1 Sunil Lakhani,1 Sarah Roberts-Thomson1. 1 _The University of Queensland, Brisbane, Australia;_ 2 _The University of New South Wales, Sydney, Australia_.

Calcium (Ca2+) influx pathways are remodelled in a variety of disease states, and alterations in the expression of specific Ca2+ permeable ion channels are a feature of some breast cancer subtypes. However, no studies have yet assessed possible alterations in Ca2+ influx pathways in the context of cancer-associated fibroblasts (CAFs), which are important in the tumor microenvironment and disease progression. The aim of this study was to identify possible changes in the nature of Ca2+ influx in CAFs and to define the role of Ca2+ signaling in the induction of breast CAFs. Levels of mRNA for voltage-gated and store-operated Ca2+ entry (SOCE) components were assessed in paired normal and CAF patient samples using qRT-PCR. The immortalised human mammary fibroblast cell line HMF3S, was treated with transforming growth factor beta (TGFβ) to induce a CAF phenotype (HMF3S-CAF) as confirmed by the expression of alpha smooth muscle actin (αSMA). HMF3S-CAF and HMF3S were also compared for expression of Ca2+ channels using qRT-PCR and functional store operated Ca2+ entry (SOCE) using a Fluorescence Imaging Plate Reader (FLIPR) Ca2+ assay. Specific Ca2+ channel subtypes were pharmacologically inhibited during TGFβ treatment to assess their role in CAF induction. Ca2+ channel levels were altered in CAF patient samples compared to their paired controls and also in TGFβ-treated HMF3S cells. Voltage-gated Ca2+channel inhibitors significantly decreased TGFβ-mediated CAF activation. These data suggest that Ca2+ signaling is remodeled in CAFs and that the Ca2+ signal is important in breast CAF induction.

#100

Novel selective PI3Kγ inhibitor AZD3458 promotes anti-tumor immune responses and reverts resistance to immunotherapy in checkpoint blockade refractory preclinical models.

Larissa S. Carnevalli,1 Danielle Carrol,2 Michele Moschetta,3 Pablo Morentin Gutierrez,4 Cristina Gardelli,5 Molly A. Taylor,1 Antonio Montoya,1 Adina Hughes,1 Matthew King,1 Teresa Klinowska,6 Simon T. Barry1. 1 _Bioscience, Oncology, IMED Biotech Unit, AstraZeneca, Cambridge, United Kingdom;_ 2 _Translational Sciences, Oncology, IMED Biotech Unit, AstraZeneca, Cambridge, United Kingdom;_ 3 _Oncology Translational Medicine Unit, IMED Biotech Unit, AstraZeneca, Cambridge, United Kingdom;_ 4 _DMPK, Oncology, IMED Biotech Unit, AstraZeneca, Cambridge, United Kingdom;_ 5 _Medicinal Chemistry Department, Respiratory, Inflammation and Autoimmunity, IMED Biotech Unit, AstraZenecaIMED Biotech Unit, AstraZeneca, Gothenburg, Sweden;_ 6 _Project Leadership, Oncology, IMED Biotech Unit, AstraZeneca, Cambridge, United Kingdom_.

The PI3Kγ isoform is a key regulator of immune cell proliferation, survival, migration and activation. PI3Kγ inhibitors have the potential to treat a variety of diseases including respiratory, inflammatory, metabolic disorders and cancer. In the context of cancer PI3Kγ inhibition re-polarizes macrophages to an immuno-stimulatory phenotype activating a T-cell mediated tumor immune response. Selective PI3Kγ inhibitors have potential as monotherapy and in combination with checkpoint inhibition to overcome resistance and to enhance efficacy of checkpoint inhibitors. AZD3458 is a highly selective PI3Kγ clinical candidate kinase inhibitor with a cellular IC50 of 8nM with a 100-fold selectivity over PI3Kδ. In vitro, AZD3458 inhibits pAKTS308/S473 in human macrophages and mouse CD11b activation at 32nM and 30nM free IC50, respectively. At these concentrations AZD3458 reverses macrophage polarization increasing IL12/IL-10 ratio and does not impact T cell proliferation and function analysed by gene expression profiling, cytokine quantification and flow cytometry. Oral administration of AZD3458 (20mg/Kg BID) remodeled the tumor microenvironment in 4T1 orthotopic breast tumor model. AZD3458 decreased tumor associated macrophages by 20% compared to vehicle and reduced overall protein expression of immunosuppressive markers CD206 and PD-L1 measured by flow cytometry. In addition, AZD3458 reduced MDSC/neutrophil activation and promoted cytotoxic T-cell activation in vivo, measured by GzmB and Perforin mRNA and protein expression. Consistent to the observed tumor remodeling, combination with checkpoint inhibitors α-PD-1 or α-PD-L1 antibodies (10mg/kg 3x week) had greater anti-tumor effects than checkpoint inhibitor alone in 4T1, LLC, CT-26 and MC-38 mouse syngeneic models. These data demonstrate that AZD3458 can reverse myeloid suppressive tumor microenvironment and revert tumor resistance to immunotherapy in myeloid-enriched immunosuppressive tumor types.

#101

Pentraxin-3 exacerbates glioblastoma invasion and angiogenesis through IL8-VEGF signaling in tumor microenvironment: PTX3 is a potential target for GBM suppression.

Umadevi V. Wesley,1 Paul Clark,1 Ian Sutton,1 John Kuo,2 Robert Dempsey1. 1 _University of Wisconsin, Madison, Madison, WI;_ 2 _University of Wisconsin, Madison, University of Texas at Austin, Madison, WI_.

Background: Glioblastoma (GBM) is highly invasive and resistant to multimodal treatment due to distorted vasculature and aggravated inflammation in a tumor microenvironment rich in dysregulated cytokines and growth factors. This phenomenon is partly attributed to GBM stem cells (GSC) that release angiogenic and inflammatory cytokines. The glycoprotein pentraxin-3 (PTX3), a modulator of inflammation, is correlated with disease severity in some cancers. However, its role in GBM and the molecular mechanisms of PTX3-mediated oncogenesis remain unclear. In this study, we examined the role of PTX3 in GBM growth and invasion, using in vitro and in vivo models.

Methods: Patient derived GSC lines were isolated and validated. Tumor tissue micro arrays (TMA) were used to determine the association of PTX3 expression with patient GBM specimens representing various degree of disease. Intracranial tumor xenograft/orthotopic mouse models, cell culture models, proteomic profiling, immunohistochemistry, molecular and biochemical approaches were used.

Results: Proteomic profiling identified increased PTX3 expression in GSCs as compared to GBM lines, and correlated with the degree of GSC invasiveness in an orthotopic tumor xenograft model. Increased PTX3 correlated with cell cycle progression and survival under hypoxic micro-environment. Conditioned media obtained from PTX3-over-expressing U87 cell line enhanced pro-angiogenic tube formation from endothelial cells. TMA screening showed that PTX3 expression is substantially increased in more than 70% of GBM specimans. Furthermore, PTX3 over-expression significantly increased survival of U87 cells under hypoxic conditions and resulted in increased tumor growth and invasion in an intracranial tumor xenograft mouse model. Upregulation of PTX3 in these tumors was associated with increased levels of inflammatory and angiogenic markers including interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF), but decreased levels of thrombospondin1, a major anti-angiogenic factor. Downstream activation of PI3K/Akt - NFkB signaling pathways was also observed.

Conclusion: PTX3 is dysregulated in GBM, and may augment inflammation and angiogenesis in tumor microenvironment. Thus, PTX3 may serve as an inflammatory marker for GBM microenvironment and also may represent a potential therapeutic target for GBM suppresion.

#102

**Ferrichrome suppresses pancreatic tumor growth** via **targeting TAMs and enhancing CD8+ T-cells infiltration.**

Mehdi Chaib, Bilal Bin Hafeez, Sonam Kumari, Mohammed Sikander, Hassan Mandil, Liza Makowski, Ajeeth Kumar Pingili, Elham Hatami, Advait Shetty, Dan Nirnoy, Murali Mohan Yallapu, Meena Jaggi, Subhash Chauhan. _UTHSC, Memphis, TN_.

Background: Pancreatic cancer (PanCa) is one of the most lethal malignancy with a very poor survival rate in patients due to inadequate treatment options. Accumulating evidences suggest that tumor associated macrophages (TAMs) and reduce infiltration of CD8+ T-cell population provide tumor suppressive environment leading to advanced growth and metastasis of PanCa. Thus, targeting TAMs and enhancing infiltration of CD8+ T-cells to the tumor site by non-toxic agents could be an effective therapeutic approach for the management of PanCa. In this study, we demonstrate that a novel probiotic-derived agent (ferrichrome) inhibits pancreatic tumor growth in syngeneic mouse model via modulating TAMs and increasing infiltration of CD8+ T-cells.

Methods: RAW264.7 cells, murine peritoneal macrophages, mouse pancreatic cancer cells (UN-KC-6141) were used for this study. Effect of ferrichrome on the expression of pro (IL-12, p40, iNOS, IL-6), anti-inflammatory cytokines (MRC1 and Arginase-1) and iron metabolism markers (ferritin and ferroportin) in macrophages and co-culture systems was analyzed by qPCR. Effect of ferrichrome on metastatic phenotypes of PanCa cells was analyzed in a co-culture model of RAW264.7 and UN-KC-6141 cells. Therapeutic efficacy of ferrichrome was determined in syngeneic mouse model. Tumor immune infiltrating cells were analyzed in excised tumors by FACS, double immunofluorescence and immunohistochemistry analyses.

Results: Ferrichrome treatment reversed the polarization of M2 macrophages towards the M1 phenotype as observed by increase expression of IL-12p40, iNOS, IL-6 and decrease expression of MRC1 and Arginase-1. Ferrichrome significantly (P<0.01) reduced the RAW264.7 cells induced migration and invasive potential of UN-KC-6141 cells. We further investigated the molecular mechanisms of ferrichrome induced M1 macrophage phenotype. Our results revealed that ferrichrome-induced M1 polarization of macrophages via upregulation of iron-sequestration protein (ferritin) and downregulation of iron-export protein (ferroportin). Ferrichrome administration (50 µg) intratumorally for 5 days per week for 26 days significantly (P<0.01) inhibited growth of xenograft tumors. We observed an increase of CD8+ T-cell and iNOS+F4/80+ macrophages infiltration and a decrease in the expression of CD163+ macrophages in resected tumors of ferrichrome treated mice.

Conclusion: Our results strongly suggest that ferrichrome inhibits the tumor growth of PanCa via targeting TAMs and enhancing CD8+ T-cell infiltration. Ferrichrome could be used alone or in combination with current therapeutic regimens for the treatment of advanced PanCa.

#103

Disruption of Notch Signaling targeted to the myeloma bone marrow microenvironment simultaneously inhibits tumor growth and prevents bone loss without inducing gut toxicity.

Adam J. Ferrari,1 Kevin McAndrews,1 Jessica H. Nelson,1 James T. Bell,1 Venkatesan Srinivasan,2 Frank H. Ebetino,2 Robert K. Boeckman Jr,2 G. David Roodman,1 Teresita Bellido,1 Jesus Delgado-Calle1. 1 _Indiana University School of Medicine, Indianapolis, IN;_ 2 _University of Rochester, New York, NY_.

Communication between myeloma (MM) cells and cells of the bone marrow via Notch signaling promotes tumor growth/survival and stimulates bone resorption. Systemic inhibition of Notch, using γ-secretase inhibitors (GSIs), decreases MM growth and reduces bone destruction, but the clinical use of GSIs is limited due to dose-limiting severe gut toxicity.

To circumvent GSI side effects, we generated a bone specific Notch inhibitor (BT-GSI) by conjugating GSI-XII to a targeting molecule (BT) with high bone affinity using an acid hydrolyzable linker. In vitro, BT-GSI was inactive unless pre-incubated at low pH, and exhibited equal inhibition of Notch target genes in MM cells as unconjugated GSI. Ex vivo, BT-GSI decreased Notch expression and reduced MM growth in bone organ cultures that reproduce acidic conditions in the MM-bone microenvironment. In vivo, treatment with BT-GSI (5mg/kg/3x/wk, i.p.) for 2 wks decreased Notch signaling in bone more efficiently than unconjugated GSI (10mg/kg/5x/wk, i.p.) in naïve mice. In addition, BT-GSI increased cancellous bone mass (30%) and decreased bone resorption by 40%, without affecting bone formation. In contrast, these parameters remained unchanged by GSI. Next, we examined in vivo the impact of BT-GSI on MM growth and bone disease in a preclinical model of established MM. 8-wk-old immunodeficient mice were injected intratibially with 105 JJN3 human MM (hMM) cells or saline. hMM injected mice exhibited detectable serum levels of the tumor biomarker human K-light chain (40 ng/mL) and visible osteolytic disease (osteolytic area 1.7 mm2) 3 wks after hMM inoculation. Then, hMM-injected mice were randomized based on tumor levels to two subgroups to receive either BT-GSI (10mg/kg/3x/wk) or vehicle (DMSO) for 3 wks. Saline-injected mice received vehicle injections. BT-GSI selectively decreased Notch gene expression in bone, but had no effect in the brain or gut. Further, BT-GSI did not increase the expression of Adipsin in the gut, a biomarker of gut toxicity, nor showed evidence of gut toxicity at necropsy. Mice treated with BT-GSI exhibited a 45% decrease in tumor burden (168 vs 254 ng/mL human K-light chain) and 50% less osteolytic area compared to vehicle treated mice bearing hMM (4.4 vs 10.2 mm2). Moreover, BT-GSI decreased serum CTX by 30%, but did not affect serum P1NP. Importantly, equimolar administration of the unconjugated BT molecule did not alter MM growth nor prevented bone loss in mice with established MM.

In conclusion, these results show that bone-targeted Notch inhibition reduces MM growth and preserves bone mass in mice with established MM. Because BT-GSI shows bone specific Notch inhibition and lacks gut toxicity, it should circumvent the deleterious side effects that limit GSI use in patients. Thus, BT-GSI is a promising approach to inhibit MM growth and to prevent bone loss in MM patients.

#104

Mechanistic insights and dose optimization for AZD3458, a novel selective PI3Kg immuno-modulator, using a quantitative systems approach.

Pablo Morentin Gutierrez,1 Yuri Kosinsky,2 Kirill Peskov,2 Ivan Azarov,2 Lulu Chu,3 Veronika Voronova,2 Martin Johnson,1 Yingxue Chen,3 Larissa Carnevalli,1 Danielle Carroll,1 Michele Moschetta,1 Teresa Klinowska,1 Gabriel Helmlinger3. 1 _IMED BioTech Unit, AstraZeneca, Cambridge, United Kingdom;_ 2 _M &S Decisions, Moscow, Russian Federation; _3 _IMED BioTech Unit, AstraZeneca, Boston, MA_.

Objectives: PI3Kγ inhibition re-polarizes macrophages to an immuno-stimulatory phenotype, thereby activating a T-cell mediated tumor immune response. AZD3458 is a highly selective PI3Kγ inhibitor. Administration of AZD3458 in combination with checkpoint inhibitors such as α-PD-(L)1 antibodies had greater anti-tumor effects (TGI 26-86%) than checkpoint inhibitor alone in 4T1, LLC, CT-26 and MC-38 syngeneic mouse models. In these, AZD3458 remodeled the tumor microenvironment (TME), reducing immunosuppressive markers (e.g in 4T1 model there was a 20% decrease in total macrophages and 50% decrease in markers of immune suppression like CD206 by flow cytometry) and promoting cytotoxic T-cell activation (e.g. in CT-26 model there was a 2-fold increase in gzmB mRNA). We developed a predictive quantitative systems pharmacology (QSP) model, to quantitatively simulate TME effects and delineate mechanistic principles underlying AZD3458 and α-PD-(L)1 synergistic effects.

Methods: The QSP model captures mechanistic, molecular and cellular interactions between PI3Kγ inhibition and checkpoint inhibitors, together with the pharmacokinetics acting on the respective targets. Features such as PI3Kγ inhibition-dependent tumor-associated macrophages, protein expression of immunosuppressive markers, reduction of MDSC activation and promotion of cytotoxic T-cell activation were included in the model. These immuno-changes were then linked to tumor cell death, resulting in macroscopic dynamic effects on tumor size. Some model parameters were taken from the literature and internal studies; some were estimated using NLME modeling of tumor size data.

Results: The model adequately described individual and population tumor size patterns. Inter-animal variability was described using a random effect on a parameter related to the ability of T cells to infiltrate the tumor in response to systemic antigen. Additionally, the model incorporated in one quantitative framework data from 4 syngeneic tumors capturing respective changes in TME conditions. Simulations for the various treatments supported the mechanistic interpretation of the observed AZD3458 and α-PD-(L)1 synergistic effects. The model was further used to simulate treatment scenarios, to infer optimal dosing and scheduling for the combination and given underlying TME conditions.

Conclusions: This study provides quantitative mechanistic insights into the links between PI3Kγ inhibition and anti-tumor immune responses, supporting our understanding of how AZD3458 may alleviate brakes in a myeloid immuno-suppressive TME and revert resistance to immunotherapy. This mechanistic understanding is critical when proceeding with dose escalation in an early clinical trial setting, as it allows to contextualize any potential compound-induced immuno-modulation in patients, for given doses and schedules.

#105

MTH1 promotes mitotic progression to avoid oxidative DNA damage in cancer cells.

Kumar Sanjiv,1 Helge gad,1 Sean G. Rudd,1 Oliver Mortusewicz,1 Ailine Stolz,2 Nuno Amaral,1 Lars Brautigham,1 Linda Pudelko,1 Christina Kaldéren,1 Ann-Sofie Jemth,1 Ingrid Almlöf,1 Torkild Visnes,1 Niklas Schultz,1 Johan Boström,1 José Manuel Calderon Montano1,1 Anna Hagenkort,1 Petra Groth,1 Camilla Gokturk,1 Tobias Koolmeister,1 Prasad Wakchaure,1 Evert Homan,1 Mikael Altun,1 Cecilia Ström,1 Martin Scobie,1 Holger Bastians,2 Ulrika Warpman Berglund,1 Thomas Helleday1. 1 _Karolinska Institutet, Solna, Sweden;_ 2 _Institute of Molecular Oncology, Göttingen, Germany_.

MTH1 prevents incorporation of oxidized deoxynucleoside triphosphates (e.g., 8-oxodGTP) into DNA. Earlier we demonstrated that MTH1 inhibitors TH588 and TH1579 possesses anti-cancer activity in various tumour types, while other structurally distinct MTH1 inhibitors failed to kill cancer cells or elevate the levels of oxidized nucleotides in DNA. In our current study, we found a novel function of MTH1 in mitosis, as it plays an important role in correct spindle assembly in cancer cells and progression through mitosis, and furthermore, we demonstrate MTH1 binds tubulin in vivo. We also observed that structurally distinct MTH1 inhibitors display differential abilities to break the MTH1 tubulin interaction, and also demonstrate that TH588 and TH1579 can directly inhibit tubulin polymerisation in vitro. Consistent with our results, we establish that TH588 and TH1579 kill cancer cells by a mitosis-dependent mechanism and implicate reactive oxygen species (ROS) accumulation and incorporation of oxidised nucleotides into DNA in this process. Furthermore, we found that non-cytotoxic MTH1 inhibitors can become cytotoxic and increase incorporation of oxidised nucleotides into DNA when they are combined with sub-therapeutic concentrations of mitotic inhibitors. In conclusion, MTH1 inhibitors killing cancer cells have a unique and complex mechanism of action: (1) causing a mitotic arrest, induced by targeting MTH1 which is required for mitotic progression, but also through direct inhibition of microtubule polymerisation, in-turn leading to an increase in ROS and likely 8-oxodGTP and (2) MTH1-dependent 8-oxodGTPase inhibitory activity causing incorporation of damaged nucleotides into DNA to kill cancer cells.

#106

Targeting tumor associated pH regulators: Effective therapeutic strategy in ovarian cancer.

Arpita Kulshrestha,1 Gajendra K. Katara,1 Safaa A. Ibrahim,1 Alexandria N. Young,2 Shayna Levine,1 Valerie E. Riehl,1 Mahmood Bilal,1 Alice Gilman-Sachs,1 Kenneth Beaman1. 1 _Rosalind Franklin Univ. of Med. Science, North Chicago, IL;_ 2 _University of Illinois at Chicago, Chicago, IL_.

The cancer cell survival relies on the over-expression of pH regulators that pump out excess protons/lactic acid, in turn acidifying the tumor microenvironment (TME) which further promotes immune suppression and metastasis. Targeting the pH regulatory molecules can neutralize the TME acidification and inhibit cancer growth. Vacuolar ATPase (V-ATPase) proton pump is the primary pH regulator. V-ATPase 'V0a2' subunit, a major pH sensing unit, is over-expressed on cancer cells in a wide array of tumor types including ovarian cancer (OVCA). Here, we examined the clinical relevance of V-ATPase 'V0a2' subunit in OVCA patient samples and its therapeutic potential in controlling tumor growth in pre-clinical mouse model. The gene data from Cancer Genome Atlas (TCGA) showed a high frequency of V-ATPase gene alterations in OVCA patients (74%, 448/606 patients). Specifically, the V0a2 gene was highly expressed in all stages of OVCA patients indicating its importance in a centralized physiological mechanism of pH regulation. Across the different OVCA grades, the V0a2 gene expression significantly increased with the tumor grade (Grade 1< Grade2 <Grade 3). Immuno-histochemical and confocal analysis confirmed V0a2 overexpression in both Type I (IHC score= 5.8±1.6; n=46) and Type II (IHC score=7.3±1.8; n=22) OVCA patient tissues compared to non-cancer control group (IHC score=2.0±1.2; n=10). In addition to cancer cells, V0a2 was also expressed on the immune infiltrated cells recruited to TME using CD68 (macrophages) and neutrophil elastase (neutrophil) markers that co-localized with V0a2 staining. Monoclocal antibody against V0a2 (anti-V0a2; MAb) effectively inhibited the proton pump activity, MMPs activation and migration of OVCA cells in vitro. In vivo anti-V0a2 treatment (300µg in PBS; i.p) resulted in delayed tumor growth (3.8 fold, p< 0.05) compared to control, with no apparent in vivo toxicity in athymic nude mice xenograft model. Histological staining of anti-V0a2 treated tumor tissues revealed higher immune-infiltration and a marked decrease in cancer cell numbers compared to control tissues. We observed an increased total leukocyte population (CD45), macrophages (F4/80) and a higher anti-tumor response in anti-V0a2 treated tumors. These results show that V-ATPase-V0a2 is overexpressed in tumor microenvironment and its inhibition impairs ovarian cancer growth by interfering with tumor acidification and activation of anti-tumor responses. In conclusion, the study demonstrates that targeting V-ATPase pH regulators is an effective treatment strategy in ovarian cancer.

#107

Extracellular vesicles from cancer associated fibroblasts induce drug resistance via integrinβ1/FAK signaling in gastric cancer cells.

Tomoyuki Uchihara, Atsuko Yonemura, Keisuke Miyake, Tadahito Yasuda, Rumi Itoyama, Masaaki Iwatsuki, Yoshifumi Baba, Naoya Yoshida, Hideo Baba, Takatsugu Ishimoto. _Kumamoto University, Kumamoto City, Japan_.

Background: Cancer-associated fibroblasts (CAFs) enhance tumor progression through secretion of soluble factors. Although we demonstrated that direct-interaction between CAFs and cancer cells confer on gastric cancer (GC) cells the ability to invade extracellular matrix (ECM), the molecular mechanism underlying the drug resistance by soluble factors from CAFs has not been determined.

Methods: We have established primary fibroblasts from more than 100 GC patients and collected conditioned medium (CM) of CAFs. Human GC cell lines cultured in normal medium or CAF-CM were examined the drug resistance with or without extracellular matrix (ECM). We isolated extracellular vesicles (EVs) from CAF-CM by ultracentrifugal separation. We performed RNA sequencing of GC cell lines and mass analysis of GC-EVs and CAF-EVs.

Results: GC cells treated with CAF-CM or CAF-EVs did not show morphological change on normal coated plates. Notably, we found that GC cells with CAF-CM or CAF-EVs on ECM-coated plates showed unique network formation and cisplatin resistance. Given that the morphological change appeared within 1-3 hours, we predicted that the stimulation by CAF-EV had an impact on the expression pattern of membrane proteins in GC cells. Mass analysis using isolated CAF-EVs was conducted and revealed that CAF-EVs contained high amount of annexin family protein members. Moreover, we showed that annexin in CAF-EVs is critical for network formation and drug resistance of GC cells. Finally, we identified integrinβ1/FAK signaling was activated in GC cells treated with CAF-EVs during network formation.

Conclusions: These findings highlight the underlying mechanism whereby CAF-EVs in tumor-microenvironment confer GC cells on drug resistance.

#108

Efficiency improvement niche factors in drug susceptibility test using two dimension organoids derived from colorectal cancer.

Kazuhiro Saso, Norikatsu Miyoshi, Masaru Sasaki, Shiki Fujino, Hidekazu Takahashi, Naotsugu Haraguchi, Taishi Hata, Chu Matsuda, Tsunekazu Mizushima, Masaki Mori, Yuichiro Doki. _Osaka University Graduate School of Medicine, Osaka, Japan_.

Colorectal cancer (CRC) is one of the leading cause of cancer-related death of world wide. About one in five patients with CRC has metastatic disease, and it is the major cause of death. Metastasis of the primary tumor is directly related to the patient survival. Systemic chemotherapy is the standard approach to treat the metastatic CRC (mCRC), and there are several protocols currently used for the mCRC, and the median survival time has increased by the systemic chemotherapy such as irinotecan, oxaliplatin, cetuximab and bevacizumab. However, the effect of the chemotherapy depends on the character of the individual tumor; we still have many patients who could not receive any benefit from the systemic chemotherapy. Therefore, we examined the individual drug sensitivity using organoids derived from each patient to predict the clinical efficacy of the chemotherapeutic drugs. We established a novel culture method for the patient's tumor, named "two-dimension organoids (2DO)" using surgically resected specimens harboring phenotypic heterogeneity as an ideal patient's tumor model. The gene expression of these organoids was similar to the each parental tumor, and they constructed a ductal structure of the tumor in the xenograft model. The previous study using 2DO allowed us to predict the clinical efficacy of chemotherapeutic drugs. However, all patients who underwent surgical resection do not need the chemotherapy. We examined the culture method and evaluated additional niche factors to establish 2DO efficiently from small-size samples, such as biopsy samples. Inflammation is a well-known risk factor for the development of colorectal cancer and several cytokines play the important roles in inflammatory reactions. Tumor necrosis factor (TNF)-alpha is a proinflammatory cytokine which is predominantly produced by macrophages as well as tumor cells. It was reported to promote tumor progression in some cancers where TNF-alpha activates the NF-κB pathway, which represents the act in the oncogenesis and cancer cell proliferation. The relationship between TNF-alpha and 2DO growth was examined in surgically resected specimens and endoscopic biopsies (n=15, each). The growth rates of 2DOs with TNF-alpha were significantly faster than the others without TNF-alpha (P=0.039). Using these 2DO cultured with TNF-alfa, the correlations for the chemotherapeutic drugs were observed between the 2DO and clinical outcomes of chemotherapy. In conclusion, TNF-alpha supports the growth of 2DO even from the small-size samples, resulting in the useful material for the precision medicine.

#109

**Myeloid Syk-PI3Kg-HIF axis inhibits anti-tumor adaptive immunity:** In silico **design of a "first in class" novel dual-Syk/PI3K inhibitor, SRX3207, to block the immunosuppressive tumor microenvironment.**

Shweta Joshi,1 Kevin Liu,1 Muamera Zulcic,1 Alok Singh,1 Timothy Pham,1 Christopher K. Glass,1 Andrew Sharabi,1 Guillermo A. Morales,2 Joseph R. Garlich,1 Donald Durden1. 1 _University of California, San Diego, San Diego, CA;_ 2 _Signal Rx, San Diego, CA_.

Introduction: Macrophages (MΘs) play important roles in the initiation and progression of solid tumors and in establishing an immunosuppressive tumor microenvironment (TME) that dampens effective anti-tumor immune responses in cancer. Hence, targeting signaling pathways in MΘs that promote tumor immunosuppression will provide therapeutic benefit. Syk kinase is a well-established cytoplasmic protein tyrosine kinase that has been extensively studied in adaptive immune responses, but its role in macrophage-mediated inflammatory responses and innate immune responses remains poorly understood. The role of PI3Kγ in tumor growth and immunosuppression has been recently established by our and other groups. Our current study examines whether targeting two crucial signaling entities, which promote macrophage-mediated anti-inflammatory responses viz. Syk kinase and PI3K, would maximally activate the anti-tumor immune response.

Methodology: 1) We generated myeloid-specific conditional Syk k/o mice to investigate if the deletion of Syk has any role in a) macrophage polarization in vitro and in vivo; b) tumor growth in various syngeneic mouse models including LLC, B16, CT26, and MC38 carcinoma; c) tumor immunosuppression including polarization of macrophages into immunosuppressive phenotype, infiltration of myeloid-derived suppressor cells, regulatory T cells; and d) infiltration and activation of CD8+T cells in the TME. 2) We used commercially available Syk inhibitor, R788, to study the role of Syk in the control of the aforementioned phenotypes. 3) We used computational chemistry methods to develop a novel chemotype, SRX3207, which inhibits both PI3K and Syk (with a single molecule) as a strategy for combinatorial activation of anti-cancer immunity.

Results: Our results suggest that macrophage, Syk, functions upstream of Rac2 GTPase and PI-3K to modulate integrin (αvβ3/αvβ5&α4β1)-mediated polarization of immunosuppressive macrophages and tumor growth in in vivo syngeneic tumor models. Genetic or pharmacological blockade of Syk in MΘs promotes a pro-inflammatory MΘ phenotype, restores CD8+ T cell activity, destabilizes HIF under hypoxia, and stimulates an antitumor immune response. Moreover, Syk-regulated immune response gene signature predicts survival in cancer patients. Novel dual-Syk/PI3K inhibitor, SRX3207, shows great efficacy in various syngeneic tumor models with no toxicity.

Conclusions: Our results validate the concept of combined Syk and PI3K inhibition as an effective approach to treat macrophage-driven, devastating cancers. This study will open new avenues to explore this chemotype in combination with other immuno-oncologic agents as the majority of cancer patients do not respond to single agent anti-PD1 or other checkpoint inhibitors currently in clinical use.

#110

Modulation of cancer-associated fibrotic stroma reduces pathological progression of PDAC.

Malvika Sharma, Ravi Chakra Turaga, Falguni Mishra, Yi Yuan, Zhi-Ren Liu. _Georgia State University, Atlanta, GA_.

Background: Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with a five-year survival rate of just 9% after diagnosis. Despite large efforts in developing treatments, the outcome of therapies for patient survival only improves marginally. Dense fibrotic stroma and ECM orchestrated by cancer-associated pancreatic stellate cells (CAPaSC) is considered to be one of the major contributors of resistance to anti-tumor therapies in this disease. The presence of dense collagen fibrils forms a physical block to drug delivery and the completely collapsed intratumoral blood vessels lead to poor blood perfusion resulting in treatment failure. CAPaSC also engage in symbiotic "cross-talk" with cancer cells through cytokines, growth factors, and chemokines supporting cancer cell growth, survival, and resistance to apoptosis. In turn, cancer cells provide factors that support pancreatic stellate cells (PaSC) proliferation and survival. PaSC upon activation express high levels of integrin αVβ3. We previously reported the development of a protein, ProAgio that targets integrin αVβ3 at a novel site and induces apoptosis of integrin αVβ3 expressing cells. We demonstrate here that ProAgio specifically induces apoptosis of CAPaSC and reduces collagen in the pancreatic tumor, enabling delivery of drug molecules into the tumor.

Objective: To test whether targeting CAPaSC reduces the pathological progression of PDAC in murine models.

Methods: We examined the effects of ProAgio in vitro and in a xenograft mouse model in vivo wherein Panc-1 cells were co-implanted with immortalized and activated human PaSC. Further, we used two murine PDAC models: a) Genetically engineered mouse (GEM) KPC model, and, b) KP orthotopic mouse model where KPC cells were orthotopically injected into the pancreas. Mice were treated with 20 doses of ProAgio (10mg/kg; i.p.). Tumor histological analysis was performed. Drug diffusion was measured by administering Fluor 595-conjugated goat IgG via tail vein.

Results: PaSC promoted the growth of pancreatic cancer cells in vitro. Integrin αVβ3 is highly upregulated in CAPaSC and ProAgio effectively induced apoptosis of CAPaSC. ProAgio treatment markedly inhibited the tumor growth when Panc-1 cells were co-implanted with activated human PaSC. In more clinically relevant models, such as GEM KPC and KP orthotopic mouse models, ProAgio treatment exhibited around 60-70% decrease in α-SMA positive cells and intratumoral collagen levels. In addition, ProAgio treatment decompressed the intratumoral vessels without an increase in angiogenesis and significantly increased the delivery of drug into the tumor. In conclusion, our findings for the first time demonstrate that ProAgio may potentially be a unique agent that is capable of specifically depleting CAPaSC in PDAC to facilitate treatment.

#111

Tumor microenvironment derived NRG1 promotes antiandrogen resistance in prostate cancer.

Zeda Zhang,1 Wouter Karthaus,1 Jose Mauricio Mota,1 Ping Mu,2 Chao Wu,1 Wassim Abida,1 Eliot Linton,1 Young Sun Lee,1 Eugine Lee,1 Ninghui Mao,1 Elizabeth Adams,1 Danielle Choi,1 Dana E. Rathkopf,1 Brett Carver,1 Anuradha Gopalan,1 Xuejun Jiang,1 Philip Watson,1 Charles Sawyers1. 1 _Memorial Sloan-Kettering Cancer Ctr., New York, NY;_ 2 _UT Southwestern Medical Center, Dallas, TX_.

Despite the improved clinical outcomes for patients with advanced prostate cancer due to the use of second generation antiandrogens, acquired drug resistance inevitably occurs and remains the major challenge for prostate cancer therapy. While several cell-autonomous mechanisms of drug resistance have been elucidated previously, for a large number of patients the mechanism of resistance remains unclear. Recent studies point to the importance of the tumor microenvironment (TME), and cancer associated fibroblasts (CAF) in the TME, in mediating tumor progression and resistance to therapy, but whether CAFs specifically contribute to antiandrogen resistance in prostate cancer is not known. Using a preclinical model that faithfully mimics the typical progression of patients on antiandrogen therapy, we found that antiandrogen resistance develops significantly faster when cells are grown in the presence of their cognate CAFs. By carrying out biochemical purification and mass spectrometry analysis, we identified NRG1 as a CAF secreted factor, and found that it can promote antiandrogen resistance in tumor cells, via activation of HER3 in the tumor. Importantly, blocking either NRG1 or HER3 can re-sensitize tumors to antiandrogen treatment in this model. Moreover, NRG1 expression is up-regulated in CAFs after antiandrogen exposure or in androgen deprivation condition. Clinically, increased stroma-NRG1 expression was observed in patients post androgen deprivation therapy but not in hormonally intact men. Taken together, this work has revealed a novel, NRG1-mediated non-cell autonomous mechanism of antiandrogen resistance in prostate cancer, and suggests that therapeutically targeting NRG1 in the setting of metastatic, antiandrogen-resistant prostate cancer with elevated NRG1 could provide significant benefit to patients.

#112

Patient derived models reveal impact of the tumor microenvironment on therapeutic response.

Ayesha A. Shafi,1 Matthew J. Schiewer,1 Renée de Leeuw,1 Emanuela Dylgjeri,1 Peter A. McCue,1 Neelima Shah,2 Leonard Gomella,1 Costas Lallas,1 Edouard Trabulsi,1 Margaret Centerera,3 Theresa Hickey,3 Lisa Butler,3 Ganesh Raj,4 Wayne Tilley,3 Edna Cukierman,2 Karen E. Knudsen1. 1 _Thomas Jefferson University, Philadelphia, PA;_ 2 _Fox Chase Cancer Center, Philadelphia, PA;_ 3 _University of Adelaide, Australia;_ 4 _University of Texas Southwestern, Dallas, TX_.

Prostate cancer (PCa) is the most common non-cutanenous cancer and the second leading cause of cancer-related death in American men. Androgen receptor (AR) is a hormone-activated transcription factor that plays an important role in both the development and progression of PCa. Androgen deprivation therapy is a common first-line therapy for disseminated disease. However, virtually all tumors become resistant to such therapy and the tumor recurs. Thus, there is a vital need for the development of novel, more effective drugs. One major hurdle in this aspect is the lack of adequate preclinical models. Current models do not effectively recapitulate the heterogeneity and the microenvironment of human PCa tumors, significantly hindering the ability to accurately predict therapeutic response. Our collaborative group has utilized and characterized a method to culture patient tumors ex vivo, termed Patient Derived Explant (PDE). This approach maintains the integrity of the native tumor microenvironment, tumor tissue morphology, and molecular signaling. Importantly, our PDE model can be manipulated both chemically (drugs/compounds) and genetically (shRNA) in order to determine specific reactions and mechanisms of response on individual tumor growth. Furthermore, with this model we can quantitatively assess drug efficacy on numerous parameters (i.e. AR levels, Ki67 staining, apoptosis screening, and desmoplasmic indices). Data to be discussed will assess the variances in response to AR-directed therapeutics and underlying mechanisms of action, while also utilizing TME characteristics as a means to predict response to therapy. In addition, we can potentially identify clinically relevant subpopulations of patients and molecularly profile their cultured tissue to uncover new pathways for therapeutic intervention. Thus, the PDE model allows for a comprehensive evaluation of individual tumors in their native microenvironment to ultimately develop more effective therapies. This study will have transformative clinical impact discerning novel metrics for the inclusion of precision medicine for advanced PCa.

#113

c-Kit-dependent tissue resident macrophage progenitors drive cancer progression.

Paulina Pathria, Hideyuki Takahashi, Megan Kaneda, Minya Pu, Karen Messer, Ryan M. Shepard, Tiani L. Louis, Ann Shih, Mark Bertagnolli, Wolfgang Wrasidlo, David A. Cheresh, Judith A. Varner. _UCSD, La Jolla, CA_.

Macrophages play a key role in promoting tumor growth and resistance to therapy. Here we show that Tissue Resident Macrophages (TRM) as well as Bone Marrow-Derived Macrophages (BMDM) play critical but unique roles in promoting tumor growth. TRM were recently shown to originate in the yolk sac or fetal liver during embryogenesis; these cells self-maintain in post-natal tissues independent of hematopoietic stem cells. We found that BMDM are CD11b+Gr1+F4/80loCX3CR1loCCR2+and are recruited to tumors in a CCR2-dependent manner. In contrast, CD11b+Gr1-F4/80hiCX3CR1hiCCR2\- TRMs accumulate in tumors independently of the trafficking receptor CCR2. Gene expression and functional studies indicate that tumor-derived TRM are highly proliferative, immune suppressive and distinct from BMDM. We show that TRM develop from c-Kit/c-KitL - dependent TRM progenitors that are abundant in tumors but not in normal tissues; purified progenitors form macrophages and potently stimulate tumor growth when adoptively transferred into mice. Tumor cells induce the expansion of TRM progenitors by secreting Stem Cell Factor (SCF/c-KitL). Notably, in vitro and in vivo proliferation of TRM progenitors and tumor growth are significantly inhibited by SCF and c-Kit inhibitors, including a novel, allosteric dual inhibitor of cKit and CDK8/19 that dramatically suppresses tumor growth by targeting both TRM and tumor cells. As cKit inhibitors synergize with other immune therapy regimens to suppress tumor growth, our studies identify cKit as a valuable target for immune therapy of solid tumors.

#114

Aberrant integrin alpha v and alpha 5 expression patterns in prostate adenocarcinomas and bone-metastases from prostate cancer are consistent with a bone-colonizing phenotype.

Brendan Connell, Pavel Kopach, Wenying Ren, Raghav Joshi, Steven Naber, Paul Mathew. _Tufts Medical Ctr., Boston, MA_.

Background: Fibronectin-binding αv and α5 integrins mediate homing, adhesive and survival interactions of prostate cancer cells with bone-marrow mesenchymal stromal cells. Monospecific αv integrin antibody therapy slowed progression of bone metastases but failed to impact overall mortality in prostate cancer. Cross-regulation between αv and α5 integrin is a potential source of adaptive resistance to monospecific blockade of either integrin. We assessed the patterns of expression of these partner integrins in bone metastases and the transition from the physiological gland to the malignant phenotype.

Methods Formalin-fixed paraffin-embedded (FFPE) radical prostatectomy samples (n=25) from patients with a ≥ Gleason grade 4 component and decalcified FFPE samples of prostate cancer bone metastases (n=10) were obtained from institutional tissue biorepository. Optimized immunohistochemistry methods developed in prostate cancer cell suspensions were applied to assess expression patterns of αv and α5 integrins in benign and malignant glandular elements in primary tumors and bone specimens.

Results Integrin αv was universally expressed in the physiological basal layer of benign prostate glands (n=25;100%) but not in the luminal epithelium. With loss of the basal layer in malignant transition, αv expression was recapitulated in 100% of malignant glandular epithelium in distinct patterns including epithelial membranous (24/25;96%), luminal membranous (6/25; 24%), punctate cytoplasmic (14/25;56%), intense foci of membranous staining (single cells or clusters surrounded by αv-negative tumor) (10/25;40%), and rim stromal patterns (14/25;56%). Luminal membranous and rim stromal αv expression patterns were striking in tumors with cribriform morphology. Furthermore, integrin αv was identified in all evaluable bone metastatic samples (7/7:100%). By contrast, integrin α5 was identified infrequently in the physiological basal layer (1/10;10%), was not expressed in malignant glandular epithelium of primary tumors but was paradoxically expressed in malignant epithelium in bone metastases (5/7:71%).

Conclusion Integrin αv expression is universally and exclusively found in the physiological basal layer of the normal gland that harbors stem-functions and is recapitulated in high frequency in prostatic adenocarcinomas in diverse patterns suggestive of distinct biological functions that may contribute to disease progression. Co-expression and enrichment of integrins αv and α5 in osseous metastases is supportive of a proposed cooperative role of these fibronectin-binding integrins toward bone colonization. Given the adaptive cross-regulation we have observed with targeting of either integrin, combined αv and α5 integrin targeting in prostate cancer bone metastases may be required for effective therapy.

#115

A CD40 agonist potentiates the efficacy and immune-stimulatory capacity of chemotherapy in combination with a focal adhesion kinase inhibitor in a mouse model of pancreatic ductal adenocarcinoma.

Meredith L. Stone, Kathleen Graham, Daniel Aldridge, Kanika Jain, Xiaoqing Pan, Jonathan Pachter, Gregory Beatty. _University of Pennsylvania, Philadelphia, PA_.

INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) has proved to be remarkably resistant to immune based therapies. This resistance may be in part due to the PDAC microenvironment that is characterized by an abundance of myeloid cells, a scarcity of effector T cells and a dense fibrotic stroma. Activation of focal adhesion kinase (FAK) signaling promotes the formation of an immune suppressive microenvironment in PDAC, and FAK inhibition (FAKi) can reduce stromal density and improve the sensitivity of tumors to chemotherapy. Likewise, CD40-dependent innate immune responses can disrupt the stromal matrix that surrounds the tumor and sensitize tumors to the cytotoxic and immune-stimulatory effects of chemotherapy. Here, we hypothesize that FAK signaling may limit the efficacy of CD40 agonists and thus, FAK and CD40 may be non-redundant pathways that can be targeted for improving outcomes to cytotoxic chemotherapy.

METHODS: For our studies, we used polyclonal pancreatic cancer cells derived from the KrasG21D/+; Trp53R172H/+; Pdx-1 Cre (KPC) genetic mouse model of PDAC, which recapitulates many of the features of human disease, including a paucity of T cells and a robust infiltration of myeloid cells. KPC cell lines were subcutaneously implanted into syngeneic C57BL/6 mice. Approximately 2 weeks after tumor implantation, mice were block randomized to receive treatment with a CD40 agonist (FGK45) or PBS administered prior to (5 days prior) or after (2 days post) gemcitabine (gem) chemotherapy with or without FAKi.

RESULTS: We first examined the impact of chemotherapy in combination with FAKi to enhance the immune stimulatory capacity of a CD40 agonist. We found that the triple combination (FAKi+gem→FGK45), in which FGK45 is given after FAKi and gem, significantly reduced tumor growth, increased tumor necrosis, and improved survival compared to FAKi alone or gem→FGK45. In this treatment setting, both gem→FGK45 and the triple combination significantly increased the CD8+/Foxp3+ ratio and the proliferation of CD8+ T cells in tumors. We next examined the capacity of a CD40 agonist to enhance the efficacy of chemotherapy, with or without FAKi. The triple combination (FGK45→FAKi+gem), in which FGK45 is given before FAKi and gem, significantly inhibited tumor growth and improved survival compared to all other groups. In this treatment setting, both FGK45→FAKi+gem and FGK45→gem increased the number and proliferation of CD3+ T cells in tumors. Importantly, the triple combination significantly increased the CD8+/Foxp3+ ratio compared to FGK45→Gem, which may account in part for the improvement in tumor response.

CONCLUSION: FAK and CD40 signaling are non-redundant pathways that can be targeted to reverse immune suppressive features in the tumor microenvironment and improve PDAC sensitivity to chemotherapy.

#116

Targeting glutamine addiction in pancreatic cancer.

Xiaoyu Yang, Amanda R. Muñoz, Shih-Bo Huang, Martha Hanes, Roble G. Bedolla, Divya Chakravarthy, Angel Su, Varsha Mulamreddy, Dmytro Kovalskyy, Paul Rivas, Joel Michalek, Rita Ghosh, Addanki P Kumar. _The University of Texas Health Science Center at San Antonio, San Antonio, TX_.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with appallingly poor outcome. Recent years have witnessed the development of a number of combination therapies that have produced modest survival improvement but at the cost of increased adverse effects. A phenomenon that contributes to the dismal prognosis and therapeutic resistance is a unique characteristic of PDAC called desmoplasia. Desmoplasia is initiated by activation of pancreatic stellate cells (PSCs), which through excessive deposition of extracellular matrix components, leads to expansion of tumor stroma that hinders drug penetration. We have recently shown that glutamine mediates communication between PSCs and pancreatic cancer cells (PCCs). Remarkably, a small molecule inhibitor palmatine (PMT) suppresses glutamine-induced PSC-PCC communication and potentiates gemcitabine (GEM) activity in part through downregulation of survivin. Survivin expression is correlated not only with unfavorable overall survival but also therapeutic resistance. In this study we therefore examined the mechanism associated with glutamine-mediated PSC-PCC interaction. We also designed experiments to test the hypothesis that PMT inhibits survivin to disrupt these interactions to potentiate anti-proliferative activity of GEM. Our results indicate that PMT not only interacts with pTyr p705 binding site in the SH2 domain of STAT3 (canonical), but also interacts at the interface of DNA and the linker domain (non-canonical). PMT-STAT3 interaction resulted in decreased phosphorylation with no significant effect on total levels of STAT3 in pancreatic cancer cells following treatment with PMT. Furthermore, PMT treatment attenuated (i) glutamine-mediated increased levels of pSTAT3 and its downstream target survivin; (ii) glutamine-mediated increased survivin reporter activity and (iii) glutamine-mediated enhanced proliferation and clonogenicity in multiple cell lines. Collectively, these data suggest that PMT abrogates glutamine-induced biological outcome through reduced STAT3/survivin signaling. We further tested the preclinical efficacy of PMT in combination with GEM using the syngeneic orthotopic KPC mouse model. Our results show significantly decreased mean log 10 tumor weight in response to treatment with PMT plus GEM (0.08±1.13) relative to controls (1.87±0.21) or single agent treatment (1.48+0.67; p<0.001). Cox regression analysis indicated risk of death decreases with intervention (p<0.001). Taken together, these data show potential clinical utility for the combination of PMT plus GEM in the treatment of pancreatic cancer. Supported by VA-Merit Award I01 BX 003876 (APK).

#117

Pharmacological targeting of activated PSC decreases resistance to gemcitabine and increases survival in murine PDA.

Ravi Chakra Turaga, Malvika Sharma, Falguni Mishra, Zhi-Ren Liu. _Georgia State University, Atlanta, GA_.

Background: Pancreatic ductal adenocarcinoma (PDA) is the most lethal form of cancer owing to its insensitivity to many chemotherapeutic drugs. Gemcitabine remains a cornerstone of PDA treatment despite suboptimal efficacy in clinics. One major mechanism by which PDA becomes resistant to gemcitabine is its conversion to inactive derivative, dFdU by upregulation of the enzyme, cytidine deaminase (Cda) in cancer cells. Activated pancreatic stellate cells (PSC) are the major source of IGF1 in the tumor microenvironment, which may be responsible for an increase in Cda expression in cancer cells. We previously reported a rationally designed protein, ProAgio that targets integrin αVβ3 at a novel site. Here we demonstrate that ProAgio specifically induces apoptosis of integrin αVβ3-expressing activated PSC and therefore circumvents gemcitabine resistance in murine PDA models.

Objective: To test whether targeting activated PSC restores chemotherapeutic sensitivity in previously unresponsive PDA tumors in a murine model.

Methods: To study the effect of ProAgio and gemcitabine combination in PDA, we used two murine PDA models: a) KP orthotopic mouse model where KPC cells were orthotopically injected into the pancreas and, b) LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mouse model. Mice were treated with 20 doses of ProAgio (10mg/kg; i.p.) and 10 doses of gemcitabine twice weekly (50mg/kg; i.p.). IGF1 and Cda levels were analyzed by ELISA and immunohistochemistry. The intratumoral levels of the gemcitabine prodrug 2′,2′-difluorodeoxcytidine (dFdC) and the inactivated and activated metabolites (2′,2′-difluorodeoxyuridine (dFdU) and gemcitabine triphosphate (dFdCTP) respectively were determined by LCMS.

Results: Our data in murine PDA models clearly demonstrate that pharmacological targeting of activated PSC by ProAgio can stabilize PDA, and when combined with gemcitabine significantly increased the survival benefit by 3-fold (p<0.001). Pharmacological targeting of activated PSC by ProAgio consequently decreases IGF1R signaling in cancer cells. As a result, there is a decrease in Cda expression leading to an increase in active metabolite, dfdCTP in the tumor, which contributes to the enhanced efficacy of gemcitabine. In conclusion, our findings for the first time demonstrate the mechanistic approach to target activated PSC, resulting in decreased Cda expression and enhanced gemcitabine delivery, thereby restoring chemotherapeutic sensitivity in murine PDA models.

#118

GPR68, a proton-sensing GPCR that mediates interaction of pancreatic cancer associated fibroblasts and cancer cells, is a potential therapeutic target for pancreatic cancer.

Shu Z. Wiley,1 Krishna Sriram,1 Cristina Salmeron-Salvador,1 Hiroshi Nishihara,2 Randall French,1 Andrew M. Lowy,1 Paul A. Insel1. 1 _University of California, San Diego, La Jolla, CA;_ 2 _Keio University, Tokyo, Japan_.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense fibrotic stroma (desmoplasia) that is generated by pancreatic cancer associated fibroblasts (PCAFs) derived from pancreatic stellate cells (PSCs) and pancreatic fibroblasts (PFs). We discovered that GPR68, a proton-sensing GPCR, has much higher expression in PCAFs compared to either PFs or PSCs. GPR68 activation in PCAFs enhances interleukin-6 (IL-6) expression via a cAMP/PKA/CREB signaling pathway. GPR68 knockdown with siRNA decreased low pH-induced IL-6 production by PCAFs and increased the proliferation of PDAC cells by PCAF conditioned media. Ogerin, a GPR68 positive allosteric modulator, enhanced the pH-dependent increase in cAMP in PCAFs. A pilot screening of 96 GPCR-specific compounds tested with GPR68-overexpressing HEK293 cells identified two compounds (C1 and C2) that decrease intracellular cAMP at pH6.4, but not isoproterenol (β-adrenergic receptor)-induced cAMP, implying their selective antagonism of GPR68. Furthermore, compound C2 decreased low pH-induced IL-6 levels in the conditioned media of GPR68-overexpressing HEK293 cells We conclude that GPR68 in PCAFs detects low pH, contributes to PDAC cell-PCAF interaction and may be a novel therapeutic target for pancreatic cancer.

#119

The influence of CD44/HA signaling on CML survival and resistance to tyrosine kinase inhibitor treatment.

Kylie Humphries, Olga Uchakina, Bryan Hostetler, Robert McKallip. _Mercer University, Macon, GA_.

The development of tyrosine kinase inhibitors (TKIs) such as imatinib has greatly improved the prognosis and survival of patients diagnosed with chronic myeloid leukemia (CML). However, many CML patients relapse due to the presence of minimal residual disease and the development of TKI resistance. It is unknown exactly what causes the relapse, but based on research from our laboratory we hypothesize that hyaluronic acid (HA) produced by fibroblasts in the microenvironment of the bone marrow provide for a protective environment preventing the tumor cells from therapy-induced cell death or senescence. The purpose of this study is to determine the influence that HA and its binding to its receptor (CD44) has on CML survival and resistance development. Initially, we examined the effect that stromal cell derived and/or exogenously added HA has on CML resistance to imatinib. Cell survival, apoptosis, HA production, and signaling events elicited by the interactions of HA with CD44 was examined using methods such as ELISA, western blotting, and MTT analysis. Furthermore, we examined the potential therapeutic use of compounds known to alter HA production and/or signaling for the treatment of CML. Our study demonstrates increased cell survival when the tumor cells are co-cultured with fibroblasts or have exogenous HA added to them after imatinib treatment. Additionally, we show that knocking out hyaluronic acid synthases reduces this protective effect. This data supports our hypothesis and we believe it could help lead to improved therapeutic treatment for CML.

#120

Paracrine TGF-β1 induces tamoxifen resistance in cancer-associated fibroblsts by activation of p44/42 MAPK signaling pathway.

Bikash Chandra Jena,1 Chandan Kanta Das,1 Deblina Bharadwaj,1 Subhayan Das,1 Indranil Banerjee,1 Santosh Gupta,2 Mahitosh Mandal1. 1 _Indian Institute of Technology Kharagpur, Kharagpur, India;_ 2 _Indian Institute of Technology Madras, Chennai, India_.

Introduction: Biochemical crosstalk between tumor and stroma by several secretory factors has evolved as a significant contributor for the development of therapeutic resistance in breast cancer, where cancer-associated fibroblasts (CAFs) in the tumor stroma play a critical role. However, the role of secretory factors of cancer cells in assisting CAF chemoresistance is least apparent. TGF-β is the major cytokine secreted by the cancer cells to the tumor stroma that further imparts resistance in CAFs to the chemotherapy. Here, we hypothesize that tamoxifen-resistant breast cancer cells stimulate surrounding CAFs towards chemoresistant phenotype through TGF-β1 activated p44/42 MAPK signaling pathway.

Methodology: The tamoxifen-resistant (TAM-R) clones of parental MCF-7 cell line were established by treating with sub-lethal doses over several cycles and characterized by MTT assay. Western blot (WB) was performed to quantify the TGF-β1 expression in the parental MCF-7 and the resistant MCF-7 (MCF-7/TAM-R) cells. ELISA was carried out to quantify TGF-β1 in the conditioned media (CM) from MCF-7 and MCF-7/TAM-R. CM from both the MCF-7 and MCF-7/TAM-R was collected and treated to the fibroblasts in dose and time-dependent manner. Initial screening for the expression of CAF markers α-SMA and caveolin-1 was carried out by qPCR and confirmed by WB. Cell cycle analysis was performed by flow cytometry and WB. Scratch assay was performed to evaluate migration and proliferation activity of CAFs. Downstream signaling pathway analysis was carried out by WB. Expression of TGF-β1 and p44/42 was downregulated by appropriate inhibitors.

Results: Tamoxifen treatment to the MCF-7 cells showed a marked increase in TGF- β1 expression both in time and dose-dependent manner. α-SMA was upregulated, and Caveolin-1 was downregulated in CAFs after treatment of CM from MCF-7/ TAM-R cells. MTT result showed a higher IC50 value of TAM in CAFs treated with CM compared to untreated one. WB analysis confirmed the elevated anti-apoptotic and reduced pro-apoptotic protein expressions in CAFs post-treatment. Flow cytometry analysis depicts less number of cells in the sub G0/G1 phage after TAM treatment in CM treated CAFs compared to untreated one. WB results showed increased cyclin D1, and cyclin E in CM treated CAFs. Further, inhibiting TGF- β1 in MCF-7/TAM-R cells and p44/42 MAPK signaling cascade in CM treated CAFs found a strong correlation between the elevated expressions of TGF- β1 in MCF-7/TAM-R cells and p44/42 signaling pathway in CM treated CAFs.

Conclusion: Here we report that chemoresistant breast cancer cells with increased TGF- β1 expression exhibit a greater potential in inducing TAM resistance in CAFs compared to parental cells, with an up-regulated p44/42 MAPK signaling cascade. The mechanism underscores the p44/42 MAPK signaling pathway as an important therapeutic target in tumor stroma.

#121

S100A9 inhibitor Tasquinimod: A novel strategy to inhibit small cell lung cancer progression and metastasis.

Salha E. Sassi, Dinesh Ahirwar, Ramesh K. Ganju. _The Ohio State University, Columbus, OH_.

Small cell lung cancer (SCLC) treatment is a major clinical challenge at present as it is highly refractory to available drugs. The MDSCs/macrophages are known to help SCLC develop resistant to available therapies. S100A9 (Migration inhibitory factor-related protein 14 (MRP14) is an EF-hand calcium-binding protein that has been involved in cell migration, invasion, proliferation, and tumor metastasis in various type of cancers, however not much is known about its role in SCLC. In this study, we found that S100A9 protein is highly up-regulated in various types of pulmonary neuroendocrine carcinomas (NEC) patient tissues compared to normal using tissue microarrays. We also observed that SCLC patients with higher S100A9 expression have significantly increased numbers of macrophage in the stroma. We have also shown that pre-treatment of the cells with S100A9 inhibitor (Tasquinimod) suppressed in-vitro cell migration, invasion, and colony formation. In addition, we analyzed the efficacy of S100A9 inhibitor against SCLC using in vivo mouse models. S100A9 inhibitor significantly reduces tumor growth and metastasis in SCLC in xenograft mouse models. We further observed that S100A9 inhibitor suppressed myeloid-derived suppressor cells (MDSC) populations and TAMs of the M2-polarized phenotype in SCLC. Moreover, we found myeloid cells sequestered from tumors of treated mice expressed were MI type as they showed higher levels of inducible nitric oxide synthase (iNos), and lower levels of arginase-1. Molecular analysis revealed that Tasquinimod decreases expression of IL6, IL10, and TGF-β1 in the cancer cells which helps inhibit macrophage activation to TAMs. Reduced proliferation and vascularization were observed in the tumors obtained from animals treated with S100A9 inhibitor. We also observed S100A9

inhibitor suppressed osteolytic bone formation in ex-vivo resorption assay. Overall, our studies, for the first time, show that Tasquinimod that targets S100A9 signaling could be used as a novel therapeutic strategy against SCLC.

#122

Stromal extracellular matrix is a microenvironmental cue promoting resistance to EGFR tyrosine kinase inhibitors in lung cancer cells.

Ting Zhang,1 Lixia Guo,1 Yuanyuan Wang,1 Tao Ren,2 Yanan Yang1. 1 _Mayo Clinic, Rochester, MN;_ 2 _East Hospital, Shanghai, China_.

The acquisition of resistance to EGFR tyrosine kinase inhibitors (TKIs) remains a critical problem in lung cancer clinic, but the underlying mechanisms have remained incompletely understood. Although the TKI-induced or -selected genetic changes are known to drive resistance, resistance also occurs in tumor cells without genetic changes through poorly-characterized processes. Here, we show that the extracellular matrix (ECM) from various components of the tumor microenvironment, including neighboring tumor cells and fibroblasts, may be the driver of resistance in the absence of genetic changes. Unlike genetic changes, which may evolve during relatively long time of chronic EGFR TKI treatment to drive resistance, briefly culturing on de-cellularized ECM, or co-culturing with the ECM donor cells, immediately confers resistance to tumor cells that are otherwise sensitive to EGFR TKIs. We show evidence that collagen in the ECM may be its primary constituent driving resistance, at least partly through the collagen receptor Integrin-β1. Intriguingly, such effect of ECM and collagen is dose-dependent and reversible, suggesting a potential clinic-relevant application for targeting this effect. Collectively, our results reveal that the stromal ECM acts as a microenvironmental cue promoting EGFR TKI resistance in lung cancer cells, and targeting collagen and Integrin-β1 may be useful for treating resistance, especially the resistance without clearly-defined genetic changes, for which effective therapeutics are lacking.

#123

Evaluating cellular cytotoxicity and potency of antibody-drug conjugates within 3-dimensional tumor models.

Bhaskar S. Mandavilli, Chris Langsdorf, Aimei Chen, Yi-Zhen Hu, Marcy Wickett. _Thermo Fisher Scientific, Eugene, OR_.

3-dimensional tumor spheroids provide biochemical conditions that closely resemble the tumor microenvironment in an intact organism. Noninvasive approaches such as fluorescence microscopy and high content analysis are highly advantageous as they allow for the study of these 3-dimensional systems. Here we investigate the penetration and potency of natural killer cells, cytotoxic T cells, and antibody‐drug conjugates in three‐dimensional models of breast and lung cancer. Unstimulated T cells produced minimal cytotoxicity, similar to untreated spheroids. Activated T cells penetrated and produced significant cytotoxicity throughout cancer spheroids. SKBR3 breast cancer cells form a compact, viable spheroid. Addition of NK cells leads to moderate cytotoxicity, while addition of NK cells and trastuzumab results in substantial cytotoxicity and degradation of spheroid structure. Trastuzumab labeled with iFL pHrodo Red becomes brightly fluorescent following specific endosomal internalization into breast cancer cells, but minimal toxicity is observed. Trastuzumab conjugated with both iFL pHrodo Red and MMAE internalizes into cells and results in cell killing. Fluorescence microscopy combined with novel cell and antibody labeling methods permits investigation of the penetration and potency of natural killer cells, cytotoxic T cells, and antibody‐drug conjugates in three‐dimensional solid tumor models.

### Gene Expression in the Tumor Microenvironment

#124

Evaluating heterogeneity of tumor microenvironment using single cell RNA sequencing.

Untack Cho,1 Yong Sang Song,1 Woong-Yang Park,2 Hae-Ock Lee,2 Youngjin Han1. 1 _Seoul National University, Seoul, Republic of Korea;_ 2 _Samsung Medical Center, Seoul, Republic of Korea_.

Tumor microenvironment interacts with cancer cells, causing tumor progression. Tumor microenvironment consists of cellular and acelluar components. We investigated cellular components of tumor microenvironment and heterogeneity of ovarian cancer. We performed single cell RNA sequencing analysis of cellular components of malignant ascites from a patient with advanced ovarian cancer. Among the various cell types including cancer cells, B cells, macrophages and fibroblasts, macrophage was the most dominant in malignant ascites. To determine the heterogeneity of ovarian cancer, we obtained primary ovarian cancer tissue from ovarian cancer patient. We performed single cell RNA sequencing analysis of cellular components derived from ovarian cancer tissue. We determined intra-heterogeneity of ovarian cancer and cellular components of ovarian cancer tissue. Taken together, we determined cellular components of ovarian cancer tissue and ascites and intra-heterogeneity of ovarian cancer. This study can provide insight of tumor microvironment and help understanding of ovarian cancer heterogeneity.

#125

Digital Spatial Molecular Profiling evidence from H&E stained FFPE that the tumor microenvironment confers gene expression phenotype specificity.

Elliot Imler,1 Raymond Nagle,2 Bruce Seligmann1. 1 _BioSpyder Technologies, Inc., Carlsbad, CA;_ 2 _University of Arizona, Tucson, AZ_.

Identifying the molecular phenotype of cells within the context of their tumor tissue microenvironment may be critical to understanding cancer and improving patient treatments and outcomes. TempO-Seq® (DOI:10.1371/journal.pone.0178302, doi.org/10.1093/toxsci/kfx153) can be used to profile gene expression from lysates of scraped FFPE tissue down to 2 mm2 areas of 5 μm thick sections. We report use of an in situ TempO-Seq transcriptomic assay carried out on slide-mounted FFPE by an automated stainer (Leica BOND), followed by H&E staining and imaging to perform standard histology. Regions of interest (ROIs) down to 20 μm in diameter are selected at the same time. Probes from these ROIs are automatically eluted, obtaining a detailed gene expression profile. This process is called Digital Spatial Molecular Profiling (DSMP). DSMP was used to profile gene expression from histologically discrete areas within tumor sections. A pan cancer assay of 5,207 genes was used, containing biomarkers for cancer, immune responses and interactions, as well as the S1500v2 whole transcriptome surrogate assay (doi.org/10.1371/journal.pone.0191105), permitting all known molecular pathways to be mapped. Data analysis was carried out using the automated TempO-SeqR™ software package. Replicate areas of cancer, high grade PIN (prostatic intraepithelial neoplasia), normal epithelium, and stroma within prostate cancer FFPE sections were profiled and compared to the profiling of a 2 mm2 scraped area of tissue defined as 100% cancer. What was notable from the DSMP data was that key cancer and immune cell biomarkers were not detected at all in normal, key normal biomarkers were not detected at all in cancer, PIN expressed a collection of normal, cancer and immune biomarkers, and stromal biomarkers were not detected in normal, PIN or cancer glands, while housekeepers were detected in all tissue types. In contrast, housekeeper genes and key biomarkers of cancer, normal, stroma, and immune cells, were all expressed in the 100% pure area of cancer, lacking the biomarker specificity afforded by histologically focused DSMP profiling. The DSMP specificity obtained from the FFPE tissue was striking, leading us to hypothesize that factors within the tumor microenvironment (whether it is adjacent cells, the structural effects of tissue, or sequestration/gradients of extracellular modulators within the tissue) exert molecular specificity on cells within the tissue not present in cultured or dissociated cells devoid of context.

#126

Multidimensional gene expression models for characterizing response and metastasis in solid tumor samples.

Walter P. Carney,1 Milan Bhagat,1 Natalie LaFranzo2. 1 _TristarGroup, LLC, Washington D.C., WA;_ 2 _Cofactor Genomics, St Louis, MO_.

Predicting response to standard of care therapies and new immunotherapies is enabled by tools that quantitatively analyze the complex heterogeneity of the tumor microenvironment. Current methods to do so primarily rely either on single gene or protein markers, and often report solely on one feature of the multifaceted immune cycle. Unlike other molecular approaches, the ImmunoPrismTM Immune Profiling Assay has been built using multidimensional gene expression models validated to accurately identify immune cells in a complex mixture with high correlation to flow cytometry measurements, but accessible for formalin-fixed paraffin-embedded (FFPE) tissues. We will report results showing the assay is highly sensitive, capable of detecting immune cells present to as little as 2% of the specimen, important for critical immune cell types such as M1 and M2 macrophages and Tregs. Further, the assay requires as few as two sections of FFPE solid tumor tissue or as little as 20 ng of total RNA which enables the analysis of rare clinical archives. In this study, we apply the ImmunoPrism assay to cohorts of patients with indications including non-small cell lung carcinoma (NSCLC), triple-negative breast cancer (TNBC) and pancreatic adenocarcinoma (ADC) treated with standard of care therapies. Immune profile changes for pre vs. post treatment and primary vs. metastatic tissue are reported in the context of clinical data, such as therapy response. Resulting biomarkers, including a novel machine-learning based multidimensional marker are also reported with predictive accuracy, PPV, NPV, and selection thresholds.

#127

CYP4A11 overexpression increases aggressiveness in glioblastoma and breast cancer.

Thaiz F. Borin,1 Sehar Ali,1 Kartik Angara,2 Mohammad Rashid,1 Stephanie Myers,1 Divya Chawla,1 Roxan Ara,1 Iryna Lebedyeva,1 Bhagelu R. Achyut,3 Ali S. Arbab1. 1 _Augusta University, Augusta, GA;_ 2 _Michigan State University, Grand Rapids, MI;_ 3 _Winship Cancer Institute of Emory University, Atlanta, GA_.

Metastatic breast cancer (BC) and glioblastoma (GBM) are two highly invasive forms of cancer that contribute greatly to patient mortality. Due to the complex mechanisms underlying cancer growth and metastasis, novel therapeutic targets must be found to increase patient survival. One such target is the cytochrome P450 4 (CYP4) metabolic pathway leading to the production of 20-hydroxyeicosatetraenoic acid (20-HETE). 20-HETE is a lipid signaling molecule that promotes invasion, growth, and neovascularization in tumors by inducing the proliferation and migration of both, the endothelial cells and the tumor cells, therefore promoting BC and GBM tumor survival. N-hydroxy-N'-(4-butyl-2 methyl phenyl) formamidine (HET0016) is a selective inhibitor of 20-HETE synthesis and thus impairs tumor growth and neovascularization. In this study, we investigate the functional and phenotypic changes in BC (MDAMB231 and MCF7) and GBM (U251 and U87) cells overexpressing CYP4A11, a human CYP4 enzyme, with and without HET0016 treatment. Cells that overexpressed CYP4A11 were produced using lentivirus infection and confirmed via western blot. Tumor cell aggressiveness was assessed in vitro using migration and proliferation assays and in vivo by ascertaining the Ki67 levels and incidence of lung metastases foci. Tumor growth with and without CYP4A11 overexpression was followed for 21 days in vivo in athymic nude mice for all groups of cells and the acquisition of stem cell and angiogenic phenotypes by the tumor cells were determined by flow cytometry. The results demonstrated that cells overexpressing CYP4A11 showed protein levels 3-6 times higher than the non-modified cells. CYP4A11-overexpressing MDAMB231 and U87 cells demonstrated increased proliferation capacity, motility and significantly increased tumor growth. Interestingly, MCF7 and U251 cells overexpressing CYP4A11 did not show significant alterations in tumor growth compared to non-modified cells. Additionally, all cells treated with HET0016 showed decreased migration after 24 hours. U87 tumors showed increased stem cell and angiogenic phenotypes compared to non-modified U87 tumors. In conclusion, overexpression of CYP4A11 in Triple Negative BC and U87 GBM cells increased tumor aggressiveness proving that this pathway is an important target for therapeutic inhibition to control tumor growth and invasion.

#128

Effects of NSAIDs on gene expression of small leucine-rich proteoglycans in prostate cancer cells.

Niradiz Reyes,1 Juan Rebollo,1 Jan Geliebter2. 1 _Universidad de Cartagena, Cartagena, Colombia;_ 2 _New York Medical College, Valhalla, NY_.

Background: Malignant transformation has been shown to be favored by the interaction between cancer cells and the tumor microenvironment, with a special role attributed to molecular components present in both compartments, such as proteoglycans, including Small Leucine-Rich Proteoglycans (SLRPs). SLRPs are increasingly recognized as powerful signaling molecules involved in cancer growth and inflammation. Recent studies suggest that, among other cancers, prostate cancer (PCa) undergoes changes in the expression patterns of these components of the extracellular matrix to promote their survival, growth, and spread. Growing evidence links inflammation to initiation and progression to advanced metastatic disease of PCa and other human cancers. Non-steroidal anti-inflammatory drugs (NSAIDs) have been associated with reduced risk of several cancers, including PCa. The protective effect of NSAIDs has been attributed to inhibition of cancer metastasis. The aim of this work was to determine the expression profiles of SLRPs in a highly metastatic prostate cancer cell line (PC-3) compared to a non-tumorigenic cell line (PWR-1E) and to identify the effects of NSAIDs in the expression patterns of these molecular components.

Methods: Differences in gene expression were determined by qRT-PCR for a panel of 15 SLRPs (ASPN, ECM2, LUM, PRELP, KERA, OMD, EPYC, OPTC, CHAD, NYX, PODN, OGN, BGN, FMOD, and DCN). Alterations in the expression of these genes in response to treatment with NSAIDs (ibuprofen and nimesulide) were also determined.

Results: Compared to non-tumorigenic PWR-1E cells, highly metastatic PC-3 cells showed higher expression levels of seven genes encoding SLRPs (FMOD, BGN, LUM, ASPN, ECM2, PRELP and EPYC). Of the remaining eight genes, DCN was not differentially expressed while PODN, NYX, KERA, OMD, OPTC, OGN and CHAD had not detectable expression in either PC-3 or PWR-1E cells. Treatment of PC-3 cells with ibuprofen and nimesulide affected the expression pattern of genes encoding SLRPs. Ibuprofen treatment increased the expression of PRELP and BGN and decreased the expression of EPYC and ECM2. On the other hand, nimesulide treatment resulted in increased expression of PRELP and decreased expression of LUM, EPYC and ECM2.

Conclusions: highly metastatic PC-3 cells had increased expression levels of SLRPs compared to non-tumorigenic PWR-1E cells. NSAIDs (Nimesulide and ibuprofen) changed the expression pattern of SLRPGs in metastatic PC-3 cells. Pharmacological effects of NSAIDs in prostate cancer warrants further investigation at the clinical settings. Additional studies are required to determine the clinical usefulness of NSAIDs in prostate cancer.

#129

Tumor-associated macrophages secrete CCL2 and induce the invasive phenotype of human breast epithelial cells through upregulation of ERO1-alpha and MMP-9.

Seungeun Lee,1 Eunhye Lee,1 EunYi Ko,1 Mina Ham,1 Hye Min Lee,1 Eun-Sook Kim,1 Minsoo Koh,1 Hyun Kyung Lim,1 Joohee Jung,1 So Yeon Park,2 Aree Moon*1. 1 _Duksung Women's Univ. College of Pharmacy, Seoul, Republic of Korea;_ 2 _Seoul National University College of Medicine, Seoul, Republic of Korea_.

Tumor-associated macrophages (TAMs) are major components of tumor microenvironment that promote invasion and metastasis of cancer cells. In this study, we investigated the effect of TAMs on phenotypic conversion of non-neoplastic MCF10A human breast epithelial cells using an indirect co-culture system. Co-culture with TAMs induced epithelial-to-mesenchymal transition, invasive phenotype, and MMP-9 upregulation in MCF10A cells. Comparative proteomic analysis revealed that endoplasmic reticulum oxidoreductase (ERO)1-alpha was increased in MCF10A cells co-cultured with TAMs compared to that in mono-cultured cells. ERO1-alpha was crucial for TAMs-induced invasive phenotype and MMP-9 upregulation involving transcription factors c-fos and c-Jun. Cytokine array analysis showed that levels of interleukin (IL)-6, C-X-C motif ligand (CXCL)1, C-C motif ligand (CCL)2, growth-regulated protein (GRO), IL-8, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were increased in conditioned media of co-cultured cells. Among these cytokines increased in conditioned media of co-cultured cells, CCL2 was secreted from TAMs, leading to induction of ERO1-alpha, MMP-9 upregulation, and invasiveness in MCF10A cells. Our findings elucidated a molecular mechanism underlying the aggressive phenotypic change of non-neoplastic breast cells by co-culture with TAMs, providing useful information for prevention or treatment of recurrent breast cancer

#130

Characterization of the tumor microenvironment using single cell transcriptomics of triple negative breast cancer allografts treated with doxorubicin.

Nicholas Hum, Aimy Sebastian, Sean Gilmore, Elizabeth K. Wheeler, Matthew A. Coleman, Gabriela G. Loots. _Lawrence Livermore National Laboratory, Livermore, CA_.

It is currently unclear how stromal components affect drug response and the emergence of drug resistance, in primary tumors. To determine how individual cells within the stroma of triple negative breast cancer (TNBC) allografts respond to chemotherapy, we used single cell sequencing to profile individual cells present in murine tumors with or without exposure to doxorubicin (Dox). Dox is a common chemotherapeutic agent used to treat breast cancer which inhibits breast cancer proliferation by intercalating into DNA and preventing topoisomerase II activity. Several autonomous processes have been implicated in the development of chemoresistance yet the impact of stromal and immune cells on tumor progression is still poorly understood. In this study, TNBC 4T1 cell line were utilized to generate murine allograft tumors in immunocompetent BALB/c mice. Tumor composition was analyzed via single cell RNA sequencing after 3 and 7 days of doxorubicin chemotherapeutic regiment mimicking clinical treatment.

Using Cell Ranger single cell software suite and Seurat R toolkit, single cell transcriptomic analysis identified the cellular composition of tumors through expression of cell-type specific genes. Stromal cell types such as endothelial, fibroblast and epithelial cells were assessed and quantified in the tumor microenvironment. Immune cell types including neutrophils, monocytes, macrophages, T-cells and B-cells were also identified in the stroma and the responses to doxorubicin treatment was determined based on the gene expression changes. In this study, cancer-associated fibroblasts and non-canonical tumor associated macrophage subpopulations are of particular interest. As expected, we found both qualitative and quantitative changes in specific subpopulations of stromal cells in response to Dox exposure.

Identification of stromal and immune cell sub-types could also lead to improved diagnostic capabilities and tumor susceptibilities. Future studies modulating non-cancerous cells in the tumor microenvironment may increase efficacy of chemotherapeutics. Further elucidating the specific cellular subpopulations within the tumor microenvironment that shift in response to drug exposure may provide new therapeutic avenues. Understanding changes in cell populations within the drug exposed tumor microenvironment can aid in future drug development to specifically target cells least sensitive to chemotherapy exposure.

This study received funding from LLNL LDRD grant 19-SI-003. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344).

#131

Concordance of mRNA expression (nCounter) and protein expression (IHC) for the detection of PD-L1 in patients with advanced non-small cell lung cancer (NSCLC).

Cristina Teixido,1 Elba Marin,1 Cristina Aguado,2 Laia Pare,1 Ana Gimenez-Capitan,2 Sandra Lopez-Prades,1 Andres Felipe Cardona,3 Carlos Cabrera,1 Elena Gonzalvo,1 Laura Lopez,4 Ruth Roman,2 Daniel Martinez,1 Ivana Sullivan,4 Pedro Jares,1 Aleix Prat,1 Miguel Angel Molina-Vila,2 Noemi Reguart1. 1 _Hospital Clinic, Barcelona, Spain;_ 2 _Hospital Universitario Quirón Dexeus, Laboratory of Oncology, Pangaea Oncology, Barcelona, Spain;_ 3 _Foundation for Clinical and Applied Cancer Research, Colombia;_ 4 _Hospital Santa Creu i Sant Pau, Barcelona, Spain_.

Background: PD-L1 immunohistochemistry (IHC) staining is currently accepted as the gold-standard biomarker for immune therapy in advanced non-small cell lung cancer (NSCLC). However, the use of various antibodies and cut-offs as well as certain degree of subjectivity in pathological evaluation has overshadowed the clear-cut predictive performance of PD-L1 expression. Multiplexed technologies can be of help in this setting providing an objective measurement of PD-L1 levels. On the other hand, gene expression signatures incorporating not only PD-L1 but also other components of the stroma might better capture the immune-context of the molecular heterogeneity of NSCLC tumors. nCounter gene expression technology is an alternative method to measure PD-L1 gene expression by digital counting proving a direct measurement of mRNA levels.

Methods: A 7-gene 'immune signature' comprising CD4, CD8, programmed cell death-1 (PD-1), programmed death-ligand 1 (PD-L1), interferon gamma (IFNG), granzyme M (GZMM) and forkhead box P3 (FOXP3) were included in a customized nCounter panel (NanoString Technologies), used in our institution on a routine basis to simultaneously screen for relevant oncogenic-drivers (ALK, ROS1, RET, NTRK1 gene fusions and METΔ14 mutation). Total RNA obtained from formalin-fixed paraffin embedded (FFPE) samples was used for PD-L1 digital counting (nCounter) which was normalised with six housekeeping genes (ACTB, MRPL19, PSMC4, RPLP0, SF3A1, GAPDH) and compared with PD-L1 protein IHC evaluation using whole tissue section with 22C3 monoclonal mouse anti-PD-L1 antibody measured on tumor cells.

Results: A total of 425 FFPE samples from advanced NSCLC were analyzed with the nCounter panel. Among them, 25 samples were not evaluable (5.9%). PD-L1 IHC was available for 163 FFPE samples and were compared with nCounter PD-L1 expression results. By IHC, 63/163 samples (38.65%) were scored as negative for PD-L1 protein expression, whereas 100/163 (61.35%) were evaluated as positive. Among positive, 62 (38.04%) and 38 (23.31%) presented a moderate (≥ 1-49%) and high PD-L1 staining (≥50%) respectively. Using an appropriate cut-off value (IHC≥1%), PD-L1 mRNA expression levels correlated with PD-L1 IHC evaluation with a 76% of concordance and a 0.755 Cohen's kappa (confidence interval 95% 0.651- 0.858). Unsupervised clustering across of mRNA expression data from 395 samples using the seven-immune-related genes and correlations between each immune gene were performed and a high correlation was found between PD-1 and FOXP3 (r=0.9) and PD-1 with GZMM (r=0.8).

Conclusions: PD-L1 mRNA gene expression shows promising in predicting PD-L1 protein expression in NSCLC. Further clinical validation is ongoing to confirm if PD-L1 gene expression by nCounter can be an alternative to IHC to select patients' candidates for immune check-point inhibitors.

#132

Direct interactions of oral bacteria and oral squamous cell carcinoma cells.

Stefan Kovac, Tiara Napier, Jessica Scoffield, Hope Amm. _University of Alabama at Birmingham (UAB), Birmingham, AL_.

Head and neck cancers (HNC) are the sixth most common cancer worldwide with more than 90% being squamous cell carcinoma. HNC can occur in the oral cavity, oropharynx, larynx or hypopharynx, or nasal cavities. Oral squamous cell carcinoma (OSCC) is estimated to be diagnosed in 50,000 patients this year with an overall 5-year survival rate of 64%. The tumor microenvironment has been shown to play a vital role in the progression of many human cancers, including OSCC. Given the complexity of the oral microbiome, we hypothesize that OSCC and oral bacteria interact to affect the tumorigenic properties of OSCC.

Methods: OSCC cells were cultured in the presence of oral pathogenic (Streptococcus mutans, Enterococcus faecalis, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans) and non-pathogenic (Streptococcus parasanguinis) bacteria. OSCC cells (A253, Cal27, FaDu, SCC-4, SCC- 9, SCC-25, SCC-152) were assessed for changes in cell proliferation, gene expression, and capacity to invade.

Results: Pathogenic oral bacteria increased proliferation in A253 OSCC cells. Enterococcus faecalis increased proliferation of SCC-4, SCC-25 and SCC-152 cells. No increase in proliferation was seen in FaDu and Cal27 cells. When examining changes in gene expression, oral bacteria increased MMP2 expression in A253 cells. Enterococcus faecalis increased JAG1, markers of hedgehog signaling (GLI1, GLI2), cell proliferation (CCDN1, CDKN1A), and cell invasion (MMP2) in SCC-4. In SCC-25 cells, Enterococcus faecalis increased JAG1, GLI1, and GLI2. Despite having no effect in total proliferation, Enterococcus faecalis increased HES1 (a marker of Notch activity), GLI1, and GLI2 in Cal27 cells. In SCC-9 cells, GLI1 expression was increased with all treatments. Streptococcus mutans increased MMP9 and HES1. Aggregatibacter actinomycetemcomitans increased MMP2 and MMP9. Enterococcus faecalis and Fusobacterium nucleatum also increased HES1.

Conclusions: Both the chemical and spatial interactions between OSCC and oral bacterium may play a role in tumor progression and migration by regulating proliferation of cancer cells and cellular signaling. A direct infection model, as opposed to interactions with bacterial spent media, showed more potential in terms of affecting cell viability and likely gene regulation as well. Future experiments will further explore the interactions, including the complexity of the oral biofilm in cancer patients and the presence of multiple bacteria.

#133

The role of the calcium-sensing receptor (CaSR) in the adaptation of breast cancer cells in high Ca2+ microenvironments.

Heather K. Beasley, Ky'Era V. Actkins, Sarrah E. Widatalla, Diva S. Whalen, Stephen D. Williams, Olga Y. Korolkova, Amos M. Sakwe. _Meharry Medical College, Nashville, TN_.

Cancer-induced hypercalcemia (CIH) is common in cancer patients with metastatic disease and in up to 30% of cases without metastasis. During malignancy, tumor cells secrete factors such as parathyroid hormone related protein (PTHrP) that causes the destruction of bone tissue, or osteolysis. The resulting persistent osteolysis leads to a progressive increase in systemic calcium or CIH. High circulating Ca2+ is sensed by the calcium-sensing receptor (CaSR), which plays a significant role in maintaining Ca2+ homeostasis, and also stimulates the secretion of PTHrP which promotes tumor cell proliferation and motility. Moreover, more than 200 mutations including single nucleotide polymorphisms (SNPs) in the CaSR gene have been described, including the inactivating A986S CaSR at rs1801725 and Q1011E CaSR at rs1801726 SNPs with reduced sensitivity to Ca2+ . Although the CaSR is the major cell surface Ca2+ sensor in most cell types, its function in the Ca2+ rich bone microenvironment remains unclear. We show that high Ca2+ adaptation of MDA-MB-231 cells, did not lead to altered expression of the receptor in the surviving cells. However, the receptor was desensitized to high Ca2+ and the cells were more motile and formed larger colonies in 3D cultures. Meanwhile, gene expression profiling of triple negative breast cancer cells (TNBC) cells transiently exposed to high Ca2+ reveals the expression of early response genes and Ca2+ -inducible genes with known functions in tumor growth and/or motility. Since breast cancer (BC) frequently metastasizes to skeletal tissues, these data suggest that BC cells that successfully colonize the bone microenvironment undergo high Ca2+ adaptation or priming via the expression of high Ca2+ inducible genes that facilitate cell growth and/or motility.

#134

Immunogenomic landscape of neuroendocrine prostate cancer (NEPC).

Alison M. Ferguson,1 Bhavneet Bhinder,1 Vincenza Conteduca,1 Michael Sigouros,1 Andrea Sboner,1 David Nanus,1 Scott Tagawa,1 David Rickman,1 Olivier Elemento,1 Himisha Beltran2. 1 _Weill Cornell Medicine, New York City, NY;_ 2 _Dana Faber Cancer Institute, Boston, MA_.

Background: NEPC is a histological subtype of advanced prostate cancer, predominantly arising clonally from castrate resistant prostate adenocarcinoma (CRPC) as a mechanism of resistance. Given current immunotherapeutic strategies only showing modest clinical benefit in prostate cancer patients and NEPC having clinical features aligned with small cell lung carcinoma (SCLC) we investigated the immune landscape of NEPC in relation to prostate cancer subtypes and SCLC to identify potential targets.

Methods: We evaluated RNA-seq from a 190 patient cohort including benign prostate (n=29; 25 PCa matched), localized prostate adenocarcinoma (PCa; n=68), hormone-naïve metastatic prostate adenocarcinoma (mPCa; n=11), CRPC (n=54), NEPC (n=25; 11 de novo) with follow-up data, and SCLC (n=28) (Rudin et al., Nat Gen 2012). Additionally, 234 prostate cancer patients had tumor mutational burden (TMB) determined by WES. Unsupervised clustering of FPKMs was performed to identify a 232 gene, immune-rich cluster, used to categorize immune status and prioritize validation of targets by IHC.

Results: Prostate cancer is known to have a relatively low TMB. Similarly, the median TMB of NEPC is akin to CRPC (38.0 vs 37.0 p = 0.44) while significantly lower than SCLC (38.0 vs 142.5, p <0.001). Unsupervised assessment identified a predominantly 'cold' immune status across subtypes, with 'hot' tumors (n=8) associated with metastatic tumors of the LN and bone and 'intermediate' NEPC tumors (n=9) associated with de novo cases. Worse overall survival was associated with intermediate vs cold T-cell immune status (66.5 mo vs 101.5 mo; p <0.001). Further analysis of NEPC showed lower expression of cytokines (p<0.01) as well as variation in checkpoint markers. Specifically, NEPC tumors had significantly lower expression of PD1 in relation to CRPC (p = 0.0001) and SCLC (p = <0.0001), higher PDL1 expression than CRPC (p = 0.05) but comparable with SCLC (p = 0.93) and lower PDL2 expression than PCa and SCLC (p = 0.03; <0.001, respectively).

Conclusion: NEPC is characterized by a relatively 'cold' tumor immune microenvironment similar to other metastatic prostate cancer subtypes but higher PDL1 expression comparable to SCLC. Association of colder tumors with treatment-induced disease, inverse correlation between survival outcome and immune infiltration, as well as novel expression changes in cytokines and checkpoint markers support further investigation into the immune landscape and potential targets for NEPC.

#135

Predicting oncogenic viral status using RNA signatures.

Ariane Lozac'hmeur, Denise Lau, Aly Khan. _Tempus Labs, Inc., Chicago, IL_.

Background: Oncogenic cancers, such as human papillomavirus (HPV) and Epstein Barr virus (EBV), account for 10 to 12% of all cancers. Knowing the viral status of a tumor is essential as it may change patients' treatment options. Numerous clinical trials investigating the benefit of radiation or chemotherapy dose reduction for HPV positive head and neck cancers have shown promising results. Additionally, virus-associated tumors are more likely to present higher levels of inflammation and immune infiltration, which make them good candidates for immunotherapy.

We developed a novel viral detection algorithm using the patient's tumor whole transcriptome profiles to predict viral status. As RNA-sequencing of clinical samples becomes increasingly common, the use of gene expression data for viral status and insights about the tumor immune microenvironment may impact clinical treatment decisions for a larger number of patients.

Methods: To develop RNA based classifiers of viral status from tumor transcriptomes, we used the TCGA cervical cancer, head and neck cancer, and gastric cancer cohorts with known viral statuses. To identify predictive genes, we performed a 10-fold cross validation on the TCGA cohorts and trained a Logistic Regression with a L1 regularisation. Since L1 regularization leads to sparse coefficients, only a small subset of genes had non-zero coefficients at each split. Only the genes with non-zero coefficients in more than 80% of the splits were included in the final model. Using this subset of genes, we again trained Logistic Regression classifiers with a L1 regularisation on the TCGA dataset and validated our models on Tempus cohorts.

Results: Our model can predict the HPV subtype with a 99% specificity and 99% sensitivity, and the EBV subtype with a 99% specificity and 100% sensitivity in the TCGA cohorts. In our Tempus validation cohorts, we report 96% specificity and a 88% sensitivity for HPV, and a 97% specificity and a 100% sensitivity for EBV. Subsequent analysis of whole transcriptome data showed a significant increase of the interferon gamma signature, the cytolytic index and the immunotherapy target IDO1 in EBV positive patients in both cohorts. We observed the same pattern in the HPV cohorts, though the increase was not significant in the Tempus cohorts due to the small sample size.

Conclusions: Our models accurately predicts viral infection in tumors using RNA expression data. We confirm viral infections are generally associated with an upregulation of immune responses. Viral detection based on whole transcriptome data could become a useful clinical tool in combination with existing methods, providing insights about the viral status and tumor microenvironment with one test.

#136

Modeling of prognostication and immune profiling, based on genomic analysis in the tumor microenvironment of pancreatic adenocarcinoma via machine learning.

Sunyoung S. Lee,1 Seok Joon Kwon,2 Ahmed Elkhanany,1 Andrew Baird,3 Seongwon Lee,4 Jillian Dolan,5 Stuart Baird,6 Shinyoung Park,1 Renuka Iyer1. 1 _Roswell Park Comprehensive Cancer Center, Buffalo, NY;_ 2 _Korea Institute of Science and Technology, Seoul, Republic of Korea;_ 3 _University of Pittsburgh, Pittsburgh, PA;_ 4 _National Institute for Mathematical Sciences, Daejeon, Republic of Korea;_ 5 _University at Buffalo School of Medicine, Buffalo, NY;_ 6 _St. Lawrence University, Canton, NY_.

The extensive tumor microenvironment (TME) in pancreatic adenocarcinoma (PAD) modulates cancer progression and impact prognosis. Although gene analysis has enhanced understanding of cancer biology, few models exist to model prognosis in association with mRNA expression in the TME. Clinical outcomes data and mRNA-seq of 156 and 64 patients (pts) were obtained from TCGA and Bailey at el. [1] for testing and validation, respectively. Expressivity of 191 genes enriched in cellular and structural elements of TME and clinical data were analyzed by multivariate nonlinear regression aided by machine learning for confined optimization with model-data error minimization. Statistics including Kaplan-Meier (KM), Cox Hazard (CH), and correlation analysis was used. Most pts (85.89% and 85.94%, respectively) were in stage II, and pts in stage I/III/IV were excluded. Prognostication was modeled with higher risk score (RS) representing worse prognosis: RS = -7.6526 x (Age-5.5679) + 0.0813 x (P/G0.3677) + 0.7069, where P/G is a ratio of genes associated with poor to good prognosis (Table 1). Based on RS, pts were clustered into 2 groups (high and low RS) with 2 KM curves showing p<0.0001 and p=0.014 in test and validation sets. Immune profiling of high and low RS groups in both test and validation sets shows that in low RS group, genes related to both immune activation (IA) and inhibition (II) (Table 2) are highly co-expressed, implying that co-expression of IA and II contributes to PAD's poor prognosis even in pts with immune system activation. In high RS group, genes related to cancer stem cells (CD44 and EPCAM) significantly contributed to poor prognosis. Machine learning-assisted modeling of RS and gene analysis suggest that IA genes are suppressed by co-expression of high degree of II, contributing to poor prognosis in PAD. RS enables prognostication of pts encountered in the clinic when genomic profiles are provided. [1] Nature 531, 47-52 (2016).

Table 1 - genes associated with good and poor prognosis out of 191 genes (identification via KM and CH with p<0.05)

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Good prognosis | FCRL3, LILRA4, IL3RA, IL10, CCL22, DOK3, CXCR4, PDGFA, ICOSLG, TNFRSF4

Poor prognosis | TNFSF10, CD44

Table 2 - gene groups of immune activation (IA) and immune inhibition (II)

|

IA gene groups | cytotoxic T, B, NK, T-helper 1 cells, IFN, cytolytic activity, T cell co-stimulation, and antigen presentation

II gene groups | regulatory T cells, desmoplasia, immunosuppressive chemokines, immune checkpoints, angiogenesis, cancer stem cells, epithelial-mesenchymal transition, and neutrophils

#137

Identification of hypoxia regulated genes associated to race and prostate cancer recurrence.

Ingrid Espinoza,1 Carlos S. Moreno,2 Damian Romero,1 Charles R. Pound,1 Christian R. Gomez1. 1 _University of Mississippi Medical Center, Jackson, MS;_ 2 _Emory University School of Medicine, Atlanta, GA_.

The incidence of prostate cancer (PCa) is 70% greater and the mortality rate 137% higher in African-American men (AAM) relative to Caucasian-American men (CAM) (Siegel et al. 2016). AAM also show faster tumor growth, higher levels of PSA, and more aggressive disease relative to CAM (Martin et al. 2013). These data, related to the biology of PCa, suggest that biological mechanisms contribute to PCa health disparities. Because stratification by clinical parameters alone is not sufficient to predict aggressive PCa, there is an unmet need of reliable biomarkers to anticipate outcome and disease progression. This study was designed to identify hypoxia-associated genes with the potential to anticipate the risk of aggressive PCa in a race-specific manner.

Seventy annotated cases with documented follow-up for evidence of biochemical recurrence (BCR) were located. Of these, 22 were AAM (8 with BCR and 14 without BCR) and 48 were CAM (31 with BCR and 17 without BCR). Next, we performed a global sequencing analysis in RNA extracted from formalin-fixed paraffin-embedded blocks. Among 27 hypoxia-associated genes associated with race and PCa by use of Cox proportional hazards regression analysis (p ≤ 0.05), we identified three genes related with BCR and race: regulator of G-protein signaling 1 (RGS1), nuclear receptor subfamily 3, group C, member 1 - glucocorticoid receptor (NR3C1), and myosin light chain kinase (MYLK). A race-independent in silico analysis utilizing TCGA data suggested tumor-specific association between transcripts of RGS1, NR3C1, and MYLK and Gleason score. Likewise, low expression of these transcripts was suggestive of poor survival. Currently, we are analyzing transcripts of these three hypoxia-associated genes by independent techniques. Integration of gene expression data with clinical parameters of prognosis will allow us to develop predictive scores. These studies may enable race-specific differentiation of patients at higher and lower risk of BCR.

Our findings underscore the prognostic value of hypoxia-regulated genes in aggressive PCa. They also warrant studies to establish the relevance of RGS1, NR3C1, and MYLK as race-specific markers.

Funding source: GMaP 2 Pilot Grant (IE)

#138

TNBC induces immune suppressive tumor microenvironments through microRNAs.

Beichu Guo, Melvin Kantono, Wei Sun. _Medical Univ. of South Carolina, Charleston, SC_.

Metastatic breast cancer is the second leading cause of cancer-related deaths for US women. The triple negative breast cancer subtype (TNBC) is one of the major contributors to breast cancer mortality, among all types of breast cancer. There are no effective treatments available for metastatic tumors. Therefore, identifying and targeting novel tumorigenesis pathways will facilitate developing effective therapeutic approaches that could ultimately lead to effective treatment for breast cancer, particularly metastatic cancer and TNBC. Our results demonstrate that miR-146a promotes tumorigenesis and metastasis of breast cancer. We found that miR-146a was markedly overexpressed in TNBC cell lines. Notably, miR-146a deficiency in a spontaneous mammary tumor model leads to reduced tumor growth and metastasis. Notably, miR-146a shifts the polarization of macrophages into immunosuppressive phenotypes. Our studies suggest that miR-146a may contribute to tumor development through modulation of tumor microenvironments.

#139

Single-cell RNA sequencing reveals compromised immune microenvironment in precursor stages of multiple myeloma.

Nicholas J. Haradhvala,1 Oksana Zavidij,2 Tarek H. Mouhieddine,2 Romanos Sklavenitis-Pistofidis,2 Jihye Park,2 Mairead Reidy,2 Abdallah Flaifel,3 Benjamin Ferland,3 Salomon Manier,2 Mark Bustoros,2 Daisy Huynh,2 Marzia Capelletti,2 Brianna Berrios,2 Mahshid Rahmat,2 Chia-Jen Liu,2 Meng Xiao He,2 Esteban Braggio,4 Rafael Fonseca,4 Yosef Maruvka,1 Jennifer Guerriero,2 Melissa Goldman,1 Eliezer Van Allen,1 Steven McCarroll,1 Jamil Azzi,3 Gad Getz,1 Irene M. Ghobrial2. 1 _The Broad Institute, Cambridge, MA;_ 2 _Dana-Farber Cancer Institute, Boston, MA;_ 3 _Brigham and Women's Hospital, Boston, MA;_ 4 _Mayo Clinic Arizona, Scottsdale, AZ_.

In multiple myeloma (MM), despite well-characterized precursor states such as monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM), there is a lack of sufficient biomarkers to predict disease progression. Most genomic analyses have studied the malignant plasma cells, however, cancers form a complex ecosystem with the immune and stromal microenvironment. To characterize the cellular composition and transcriptional programs of each component of the tumor and microenvironment at different stages of MM progression, we employed single-cell RNA sequencing on 48K plasma and 40.8K immune microenvironmental cells from a cohort of 22 patients with varying stages of disease progression and 9 healthy donors.

Expression profiles of plasma cells revealed clear tumor-specific differences in known oncogenic drivers in MM (MMSET/FGFR3, CCND1 and MAFB) as well as other clonally expressed genes (LAMP5, HIST1H1C, and AREG), distinguishing them from healthy plasma cells. We identified a subset of cycling plasma cells in malignant samples, observing a range of proliferative capacity across disease stages. Furthermore, our approach allowed a unique head-to-head comparison of gene expression changes in normal and malignant plasma cells from the same individual, revealing early alterations in genes related to immune modulation (NKBIA) or controlling transcription and differentiation (EID1). Some alterations were patient-specific, while others, such as MHC I overexpression and CD27 loss, were recurrently observed across subsets of the cohort.

Analysis of the BM microenvironment demonstrated significant infiltration of natural killer cells, non-classical monocytes/macrophages, and T cells, even in the earliest stages of the disease. Further investigation revealed upregulation of MHC II expression at the mRNA level in CD14+ monocytes/macrophages and yet, intriguingly, analysis by CyTOF and immunohistochemistry revealed a shift towards intracellular localization of MHC II in these cells. Co-culture with MM cell lines was sufficient to induce the decrease of extracellular MHC II, providing strong evidence for MM-induced compromised antigen presentation by macrophages, and hinting at a mechanism of immune evasion.

Together, our results provide a comprehensive view at the complex interplay of the immune and malignant cells in different stages of the disease. We demonstrate the immune response beginning in premalignant conditions to be heterogeneous, including compromised antigen presentation as well as alterations in cellular composition and signaling. Consideration of the type of immunological response may prove valuable in determination of progression risk, as well as open up potential strategies for therapy.

#140

Comprehensive analysis of immune evasion in breast cancer by single-cell RNA-seq.

Jianhua Yin,1 Hanlin Zhou,1 Chen Yan,2 Ruqian lv,1 Ting Wang,3 Chenyang He,3 Chen Huang,3 Yong Hou,1 Shida Zhu,1 Ling Wang,3 Michael Dean,4 Kui Wu,1 Hong Hu,2 Guibo Li1. 1 _BGI-Research, Shenzhen, China;_ 2 _Shenzhen People's Hospital, Shenzhen, China;_ 3 _Xijing Hospital, Fourth Military Medical University, Xian, China;_ 4 _National Cancer Institute, Gaithersburg, MD_.

The tumor microenvironment is composed of numerous cell types, including tumor, immune and stromal cells. Cancer cells interact with the tumor microenvironment to suppress anticancer immunity. In this study, we molecularly dissected the tumor microenvironment of breast cancer by single-cell RNA-seq. The breast tumor microenvironment was profiled by analyzing the single-cell transcriptomes of 52,163 cells from the tumor tissues of 15 breast cancer patients. The tumor cells and immune cells from individual patients were analyzed simultaneously at the single-cell level. Among more than 40,000 tumor cells, we identified distinct cell clusters with different molecular subtypes and gene signatures in the same tumor , which revealed intra-tumor heterogeneity in breast cancer. We comprehensively profiled the functional state of T cells, macrophages, B cells, CAFs in breast cancer tumor microenvironment, which highlight the immune cell and CAF diversity in the breast cancer TME. Moreover, we analyzed intercellular communication in the breast cancer TME using a dataset of human ligand receptor pairs, which showed distinct cell interaction models across tumors. The evolution of the tumor microenvironment during clinical treatment was dissected by comparing tumor microenvironment of breast cancer pre- and post-chemotherapy. This study investigated the diversity of the cell types in the tumor microenvironment and provided information on the mechanisms of escape from clearance by immune cells in breast cancer, which could be a beneficial resource for immunotherapy development.

#141

**TCR sequencing from tissue micro-regions and single cells utilizing RareCyte CytePicker and Archer** **Immunoverse technologies.**

Laura Johnson,1 Luke Hartje,2 Lance U'Ren,2 Nolan Ericson,2 Rebecca Podyminogin,2 Tad George,2 Steve Daniel1. 1 _ArcherDX, Boulder, CO;_ 2 _RareCyte, Inc., Seattle, WA_.

Background: T-cell receptor (TCR) based immunotherapies are becoming an important cornerstone of immuno-oncology. Complete TCR sequencing requires single-cell resolution to capture both α and β chains. There is great interest in obtaining single-cell TCR sequences from archived tumor tissue. This task requires technology that can not only retrieve single cells but also sequence degraded RNA from archived tissues samples at the single cell level. The RareCyte CyteFinder® platform provides integrated multi-parameter imaging and retrieval capabilities for identification and isolation of rare cells and microscopic regions of interest (ROI) for molecular analyses. Archer has developed the Immunoverse™ platform of targeted Next-Generation Sequencing (NGS) assays to characterize the human immune repertoire from partially degraded RNA inputs. Combining these two technologies affords the unique potential to accelerate engineered cell-based therapeutic by sequencing TCR from individual cells.

Methods: 40 um ROI containing 1 to 10 T-cells were isolated from fresh/frozen (OCT) melanoma or OCT and FFPE tonsil using the CyteFinder® system. In addition, single flu antigen-specific T-cells were retrieved from live cell preparations. OCT tonsil sections were stained with a 3-color panel to discriminate T cell and B cell zones, and OCT tumor sections were stained with a 6-color panel to identify immune infiltrate, while FFPE sections were stained with a nuclear marker. The Archer® Immunoverse™ TCR All Chains library preparation kit was used to generate libraries which were then sequenced on the Illumina NextSeq platform. The resultant library sequences were deduplicated, error corrected, aligned to reference V, (D), J and C regions of TCRs, and assembled to identify clonotypes from α and β chains using the Archer® Analysis tool.

Results: TCR transcripts were observed from RNA isolated from as few as one cell. CDR3 sequences with V and J segments from single flu-targeting T-cells were obtained and found to match previously published sequences. TCR transcripts were observed in all ROI retrieved from OCT and FFPE tonsil when processed with the Immunoverse™ TCR assay. The numbers of transcripts observed correlated with the number of isolated cells analyzed. The sample source also affected the number of observed clones with more clones observed from cells isolated from fresh/frozen tissue.

Conclusions: These results support the utility of the RareCyte platform and Archer® Immunoverse™ TCR assay for profiling RNA derived from low numbers of cells in either fresh/frozen tissue, FFPE tissue, or live cells. A workflow that combines RareCyte and Archer technologies shows promise as a method for pairing α and β chain TCR sequences from RNA isolated from a single cell.

#142

The EGFR signaling modulates in mesenchymal stem cells the expression of microRNAs involved in the interaction with breast cancer cells.

Marianna Gallo,1 Marianeve Carotenuto,1 Cristin Roma,1 Francesca Bergantino,1 Pasqualino de Antonellis,2 Nicola Normanno,1 Antonella De Luca1. 1 _National Cancer Institute, Naples, Naples, Italy;_ 2 _The Hospital for Sick Children, University of Toronto, Toronto, Canada_.

Introduction. We previously demonstrated that the activation of the epidermal growth factor receptor (EGFR) in mesenchymal stem cells (MSCs) modulates the expression of a number of genes coding for secreted proteins that promote breast cancer cell progression. As different microRNAs (miRNAs) have been shown to be involved in the cross-talk between MSCs and tumor cells, we hypothesized that the EGFR might regulate in MSCs the expression of miRNAs that might affect breast cancer progression through paracrine circuits. To this aim, we analysed the whole small RNA transcriptome of MSCs stimulated or not with transforming growth factor α (TGFα), one of the main ligands of the EGFR.

Materials and methods. Small RNA sequencing was performed using the SOLiD 5500xl System. The DAVID (Database for Annotation, Visualization and Integrated Discovery) database was used for pathway analysis. Exosomes containing miRNAs were isolated from conditioned media with the ExoQuick reagent. MiRNA expression levels in conditioned medium were analyzed by Real Time PCR. Anchorage-dependent growth assays and Boyden-chamber based colorimetric migration assays were used to assess the proliferation and the migration of breast cancer cells, respectively.

Results. Following small RNA sequencing, we identified 36 miRNAs differentially expressed between untreated and TGFα-treated MSCs with a fold change (FC) <0.56 or FC ≥1.90 (Confidence Interval, CI, 95%) and a threshold of sequence counts≥150 in at least one sample. In particular, 18 miRNAs resulted up-regulated with FC≥1.90 and 18 miRNAs down-regulated with FC<0.56. Pathways analysis of the target genes of the differentially expressed miRNAs revealed a significant enrichment in different KEGG pathways involved in cancer development and progression, including TGFβ signaling, focal adhesion, Rap1 signaling, Hippo signaling, mTOR and RAS signaling. To study miRNAs involved in the TGFα-mediated interaction between MSCs and breast cancer cells, we collected conditioned media from MSCs and analysed the expression levels of selected miRNAs. Real Time PCR analysis revealed the presence of several miRNAs in the conditioned medium from MSCs, including miR-23c, miR379-3p and miR-432-5p. The biological activity of the identified miRNAs was explored in a panel of breast cancer cell lines. MiR-23c was found to regulate the proliferation and migration of triple negative MDA-MB-468 and MDA-MB-231 breast cancer cells.

Conclusion. Collectively, our data suggest that the EGFR signaling regulate in MSCs a wide number of miRNAs that might be involved in breast cancer progression, providing novel information on the mechanisms that regulate the MSC-tumor cross-talk.

#143

Immune landscape in lung adenocarcinoma drawn by single-cell RNA sequencing.

Bo Mi KU, Nayoung Kim, Kyung Young Lee, Jong-Mu Sun, Se-hoon Lee, Jin Seok Ahn, Keunchil Park, Hong Kwan Kim, Hae-Ock Lee, Myung-Ju Ahn. _Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea_.

Immune cells in the tumor microenvironment from an ecosystem that modulates cancer progression. However, the exact nature and dynamics of tumor microenvironment in lung cancer remain largely unknown. To depict the baseline landscape of the composition of tumor microenvironment at different stages, we conduceted single-cell RNA sequencing of 58 samples from 43 lung adenocarcinoma patients. Here, we analyzed 11 early-stage lung tumor and normal pairs that had been resected before systemic therapy, 11 late-stage lung tumor biopsy, and 5 metastatic pleural-effusion or 10 metastatic brain tumor samples and revealed differential immune profiles encompassing lymphoid and myeloid compartment. In normal lung, large numbers of macrophages and T cells consistently comprised the immune cell repertoire across patients. In paired cancer tissues, decrease in macrophage but increase in B cell proportions were evident indicating the activation of humoral immunity. In addition, diverse effector T cell populations mark the cancer tissues both at the primary and metastatic sites, corroborating the activation of cellular adaptive immunity. These data provide valuable insight of tumor-driven immune changes in lung cancer.

#144

Characterization of glioma subtype-specific macrophage-tumor interactions using single-cell RNA-seq.

Rohit Rao, Ravinder Verma, Jincheng Wang, Qing Richard Lu. _Cincinnati Children's Hospital, Cincinnati, OH_.

Glioblastomas are malignant brain tumors that carry a poor prognosis. The tumor microenvironment has been identified as an important regulator of tumor growth and may represent a novel target for therapy. We have developed mouse models corresponding to two different glioma molecular subtypes, proneural and mesenchymal, to identify changes in microenvironment composition between glioma subtypes. We performed Drop-Seq on endpoint tumors from the proneural glioma model and found that tumor associated macrophages and microglia are the most prominent infiltrating microenvironmental population in proneural glioma. We profiled receptor-ligand interactions between tumor macrophages and tumor cells to identify the mechanisms by which macrophages regulate tumor growth. We identified several regulators previously published in the literature that regulate glioma growth such as pleiotrophin, Igf-1, and osteopontin, as well as some new candidate regulators prosaposin, oncostatin M and thrombospondin. Future directions include validation of tumor-microenvironment interactions using CRISPR activation in vitro, characterization of the tumor microenvironment in mesenchymal-like glioblastoma via single-cell RNA-sequencing, and changes in tumor microenvironment composition and function following treatment with chemotherapy or radiotherapy. A better understanding of tumor-microenvironment interactions in glioma will aid development of new therapies to promote tumor regression

#145

Comparison of whole transcriptome sequencing immune repertoire sequencing using RNA for tumor milieu analysis.

Seulki Song,1 Hyejoo Park,1 Daeyoon Kim,1 Sheehyun Kim,2 Hongseok Yun,2 Sungyoung Lee,2 Youngil Koh,2 Sung-Soo Yoon2. 1 _Seoul National University College of Medicine, Seoul, Republic of Korea;_ 2 _Seoul National University Hospital, Seoul, Republic of Korea_.

Introduction Recent rapid methodological advancements of techniques and reduction of sequencing cost have opened the way for high-throughput immune repertoire sequencing to broaden understanding of the adaptive immune system in cancer. T cell receptor (TCR) repertoire and clonotype analysis are essential for characterizing the tumor-associated T-cells. Current immune repertoire assays identify specific alignments of TCR, B cell receptor (BCR), and CDR3 using both whole transcriptome sequencing (WTS) and immune repertoire sequencing. We tried to compare the characteristics of WTS and repertoire sequencing for assessing immune status in cancer.

Materials and Methods A total of 32 RNA specimen from multiple myeloma was obtained from the Seoul National University Hospital. The mononuclear cell (MNC) layer of bone marrow specimens from multiple myeloma (MM) patients was isolated and stained with CD138 microbeads. By analyzing the degree of fluorescence of CD138-PE, RNA from CD138 negative fraction was extracted from the cells, which represents immune milieu of MM bone marrow. The immune repertoire sequencing from ArcherDX and the WTS was performed with the RNA. We investigated the clone diversity, top 10 clonotype proportion with respect to total clone per reads, and V-D-J region recombination usage analysis from each sample using MiXCR, tcR, and VDJtools.

Results WTS and immune repertoire sequencing of CD138 negative RNAs from the 32 MM patients were compared. First, the mean number of clones was significantly higher in repertoire sequenced data compared to WTS data; mean clone number of WTS was 215.48 and was 139,981 for repertoire sequencing. Furthermore, when the proportion of the clone in each samples was listed by its clonal proportion size, the order of clone count were significantly different according to the sequencing methods. For example, the immune repertoire sequencing result of MM_46 sample showed that it contains the 10th largest clones, but WTS sequencing suggested MM_46 sample have the lowest 5th clone. Also, there was also a significant difference in the results of two sequencing data for the top 10 clonotype proportion related to immune adaptation. Finally, in the results of V-D-J region usage analysis, the ratio was completely different and the usage patterns of TCR genes are different according to the sequencing platform.

Conclusion The paradigm of most recent immune system analysis tools has been the use of high throughput repertoire sequencing methods that can yield unprecedented quantitative insights into lymphocyte diversity. Although there are a number of repertoire analyses performed in WTS, caution is required in future data analysis because it shows inaccurate aspects in comparison with immune repertoire sequencing. Immunoassays are preferably performed using repertoire sequencing data.

#146

Multiple modalities of NanoString GeoMx™ Digital Spatial Profiler allow for spatially-resolved, multiplexed quantification of protein and mRNA distribution and abundance.

Gokhan Demirkan, Elena Viboch, Chris Merritt, Giang Ong, Kristi Zevin, Kristina Sorg, Lindy Irving, Dwayne Dunaway, Gary K. Geiss, Joseph Beechem. _NanoString Technologies, Seattle, WA_.

Characterization of the spatial distribution and abundance of proteins and mRNAs with morphological context within tissues enables a better understanding of biological systems in many research areas, including immunology and oncology. However, it has proven difficult to perform such studies in a highly multiplexed manner. To address this unmet need, we have developed a novel optical-barcode based microscope and tissue-sampling platform designed to simultaneously analyze hundreds of proteins or mRNAs on a single FFPE section from distinct tissue spatial regions (GeoMxTM Digital Spatial Profiler, DSP).

Here, we present a series of modalities and associated applications for the GeoMxTM DSP platform and its integrated software. First, geometric profiling can be utilized by drawing automated circles, squares or even manual hand-drawn polygons as regions of interest (ROI) to characterize tissue heterogeneity. Second, gridded profiling offers high resolution unbiased tumor profiling by placing a grid on tissues and separately analyzing each segment in the grid. Third, contour profiling employs concentric rings, or any custom shape at a growing distance from a site of interest. Finally, segment and rare cell profiling exploits fluorophore-conjugated antibodies to profile specific cell types. These techniques can be used to discover drug mechanism of action or immune activation status, as well as to facilitate prediction of treatment response and disease progression or investigation of specific rare cell populations' molecular profiles.

Using these multiple modalities, we spatially resolve protein and mRNA expression over 30 immune targets on FFPE tissue sections from various organs, including colon and tonsil. We demonstrate multiplexed detection from discrete regions within a tumor (tumor center and immune invasive margin), enabling systematic interrogation of immune activity in FFPE samples. Finally, we present the utility of each modality under different scenarios.

#147

Deletion of the RNA regulator HuR in microglia/macrophages promotes an anti-tumor microenvironment in glioblastoma.

Jiping Wang, Jianmei Leavenworth, Anita Hjelmeland, Ben Borg, Peter H. King. _Univ. of Alabama at Birmingham, Birmingham, AL_.

Glioblastoma is a malignant brain tumor that portends a poor prognosis. Its resilience, in part, is related to a remarkable capacity for manipulating the microenvironment to promote its growth and survival. Microglia/macrophages are a prime target, being drawn into the tumor and stimulated to produce factors that support tumor growth and immune system evasion. Here we show that the RNA regulator, HuR, plays a key role in the tumor-promoting response of microglia/macrophages. Knockout of HuR in microglia/macrophages led to reduced tumor growth and prolonged survival in a murine model of glioblastoma. Analysis of tumor composition by flow cytometry showed that tumor associated macrophages were decreased, more polarized toward an M1-like phenotype, and had attenuated PD-L1 expression. There was a concomitant reduction in tumor-associated polymorphonuclear myeloid-derived suppressor cells, but an increase in Treg cells. In vitro assays showed reduced migration of HuR-/- microglia toward secreted factors derived from glioblastoma cells, and a decrease in migration of glioblastoma cells toward secreted factors derived from HuR-/- microglia. The molecular response of HuR-/- microglia was altered including reduced CXCL1, 2, MMP2, PD-L1 and VEGF expression and an increase in CXCL10 and several MMPs. There was a mixed effect on cytokines and other factors associated with both proinflammatory and alternatively activated phenotypes. In summary, HuR is a key modulator of the tumor microenvironment in glioblastoma, promoting tumor progression through its molecular regulation of factors produced by microglia/macrophages. These findings underscore the relevance of HuR as a therapeutic target in glioblastoma.

#148

Analysis of the tumor microenvironment in seven cancer types by single-cell RNA-seq.

Michael Dean,1 Guibo Li,2 Jianhua Jin,2 Yong Hou,2 Kui Wu,2 Shida Zhu,2 Hanlin Zhou,2 Ruqian Lv,2 Feng Lin,2 Si Liu,2 Shichen Dong,2 Lei Wang,2 Cuijuan Zhang,2 Yi Zhao2. 1 _NCI, Gaithersburg, MD;_ 2 _BGI-Shenzhen, Shenzhen, China_.

The tumor microenvironment (TME) is composed of tumor cells, as well as immune and stromal cells. Cancer cells can interact with the tumor microenvironment to suppress anticancer immunity and host inflammatory cells can modulate that immune response to the lesion. To better understand the TME across cancer types we used single-cell RNA-seq analysis of 224,330 cells from 77 patients with breast, gastric, nasopharyngeal carcinoma (NPC), diffuse large B-cell lymphoma (DLBCL), pancreatic, colorectal and lung cancer. The tumor cells and immune cells from individual patients were analyzed simultaneously at the single-cell level to identify distinct cell clusters and subtypes. Comparison of the profile of T cells, macrophages, dendritic cells (DC), B cells, mast cells, granulocytes and cancer-associated fibroblasts (CAFs) identifies unique profiles by tumor type. Breast tumors have high numbers of macrophages, colorectal tumors are elevated for CD4+-T cells, DLBCLs are low in CAFs, gastric cancers have high levels of mast cells, lung tumors high levels of CD4+-T cells and natural killer (NK) T cells, NPC have elevated levels of B cells and DCs and pancreatic cancers high levels of CAFs and granulocytes and low levels of DC and CD8+-T cells. However, within a tumor type, there is also considerable heterogeneity. Understanding how the TME evolves and changes with therapy or predicts response to treatment and survival may provide insight into tumorigenesis and cancer progression.

#149

Heterogeneity in stromal-fibroblasts determines stemness-status in primary oral tumors.

Sandeep Singh, Ankit Kumar Patel, Kavya Vipparthi. _National Institute of Biomedical Genomics, Kalyani, India_.

Cancer associated fibroblasts (CAFs) often demonstrate the characteristics of myofibroblast differentiation by producing ultrastructure of alpha-smooth muscle actin (αSMA). Clinical data have strongly linked high-αSMA expression in CAFs with poor-prognosis of oral cancer patients; however, it is unclear if the variable levels of αSMA in CAFs are the cause or the effect of aggressive cancer behaviour. Cancer stem cell-model provides a framework to understand the aggressive cancer behaviour; where small population of stem-like cancer cells (SLCCs) is associated with cancer initiation, progression, metastasis and relapse after therapy. Like normal stem cells, SLCCs may retain responsiveness to the microenvironmental signals for their self-renewal and differentiation. Therefore, in this study we tested if the CAFs with variable levels of αSMA in oral tumor-stroma may cause distinct effects on the SLCCs. Immunohistochemical staining of oral tumor tissues (n=46) distinctly demonstrated heterogeneity in levels of αSMA-positive CAFs. Interestingly, proliferation index as measured by percentage of Ki67 positive cells was significantly negatively correlated with αSMA score; whereas, the frequency of ALDH1A1 expressing oral-SLCCs was significantly positively correlated with αSMA score in primary tumor samples. Experiments with in vitro primary cultures of CAFs; established from oral tumors, suggested that the gene expression differences were indeed the basis of heterogeneity among CAFs. This further grouped these CAFs in two distinct clusters, which were termed as C1 and C2. Interestingly, the oral-CAFs belonged to C1 or C2 clusters showed low- or high- αSMA-score respectively. Similar to our results observed with tumor tissues, in vitro co-culture experiments comprehensibly demonstrated that the oral-CAF-subtype with lower score for αSMA (C1-type CAFs) were more supportive for cellular proliferation but suppressive for the self-renewal of oral-SLCCs. Further, data clearly exhibited for the first time the determining role of BMP4 secreted by the C1-type CAFs in restraining the self-renewal growth of oral-SLCCs. Taken together, the observed heterogeneity among CAFs and its role in regulation of self-renewal of oral-SLCCs could be of relevance for understanding the overall biology of oral tumors. By discovering the CAFs-mediated mechanisms which might restrain cancer cells to a less aggressive state, our work has provided an opportunity for prognostic classification of patients, who might get better response to the given treatment as well as opened an avenue for targeting tumor-stroma interactions in aggressive tumors. Further, BMP4 has emerged as an important player and may be explored as differentiating agent against oral SLCCs.

#150

Stromal cell plasticity and immune surveillance revealed by single-cell transcriptomics.

Claire Regan, Yuan Hao, Jonathan Preall. _Cold Spring Harbor Laboratory, Cold Spring Harbor, NY_.

Pancreatic ductal adenocarcinomas are typically characterized by a highly desmoplastic stroma, which is dense in collagen and other extracellular matrix components that limit access to the growing tumor by immune cells and targeted therapies. ECM components are mainly deposited by cancer-associated fibroblasts (CAFs), which are thought to arise primarily from pancreatic stellate cells and have been shown to polarize into at least two distinct subtypes within the tumor microenvironment in response to extracellular ligands. Recent studies have suggested potentially antagonistic roles for the different CAF subtypes in either promoting or restraining tumor growth. While depletion of the restrictive desmoplasia is likely to be a desirable therapeutic avenue, a complete understanding of phenotypic variation and interconversion between CAF populations will be essential for designing targeted therapies.

Using single-cell RNAseq, we have profiled the non-immune stromal cells of pancreatic ductal adenocarcinomas in the KPC mouse model, as well as from orthotopically transplanted organoid-derived pancreatic tumors. In addition to the canonically reported myofibroblastic and inflammatory CAF subtypes, we observe at least one additional distinct type-I collagen expressing fibroblastic cell type in the tumor microenvironment. The high degree of transcriptional divergence suggests that these CAFs may arise from distinct progenitors, whereas myo- and inflammatory-CAFs lie along a continuous pseudotemporal gradient marked by signatures of transcriptional programs driven by extracellular ligand signaling, including Wnt-family genes. Our single-cell transcriptomic results point toward pathway utilization that could be exploited for modulating PDAC desmoplasia and enabling targeted combination therapies.

#151

Comprehensive characterization of gastric cancer at single-cell resolution.

Jiamin Chen, Anuja Sathe, Sue Grimes, Stephanie Greer, Billy Lau, Ann Renschler, George Poultsides, Carlos Suarez, Hanlee Ji. _Stanford University Medical School, Stanford, CA_.

The gastric carcinoma cells have complex interactions with diverse cell types in the associated tumor microenvironment. The tumor microenvironment plays important roles on gastric cancer development, progression and therapy response. In this study, we leveraged high-throughput single-cell RNA sequencing (scRNAseq) to characterize the diverse cell population in gastric tumors.

In total, we analyzed more than 30,000 single cells from eight gastric tumors and matched normal tissues. Cells were sequenced at an average depth of 70000 reads/cell to guarantee sufficient coverage for downstream analysis. Using graph-based cell clustering methods and known cell markers, we identified heterogenous epithelial cells (PGC+, MUC5AC+, TFF1+, EPCAM+, CDH1+), fibroblasts (ACTA2+, THY1+, COLA1+), endothelial cells (VWF+, PECAM1+), and a variety of immune cells including M1 and M2 macrophages (IL1A+, IL1B+, TNF+, MARCO+, MSR1+, CD68+), CD4 T cells (CD3D+, CD4+), CD8 T cells (CD8A+), Gamma Delta T cells (TRGC2+, TRDC+), B cells (CD79+), and plasma cells (CD19+, CD20+, IgG+). Specifically, gastric epithelial cancer cells demonstrated distinct CNVs inferred from single cell transcriptome files.

We established and characterized patient derived gastric cancer organoids from single tumor cells. The organoids recapitulate CNVs found in the original gastric tumors and displayed varied degree of drug responses. We identified specific molecular features of the tumor microenvironment that modulate cell growth and immune responses. For example, we identified heterogenous myofibroblast populations with distinct chemokine secretory profiles compared to normal gastric tissue. We characterized gene signatures associated with immune checkpoints on T cells and targetable molecules in innate cells. Using cell type specific expression profiles, we were able to establish a comprehensive signaling network of tumor-tumor, tumor-stromal, stromal-stromal and stromal-tumor signaling via receptor-ligand pairing.

#152

Gene expression and mutational load in colon tumors from African American patients.

Jenny Paredes,1 Jone Garai,2 Ping Ji,3 Sayed Imtiaz,1 Marzia Spagnardi,1 Maria Munoz-Sagastibelza,1 Mubarak Akadri,1 Raavi Gupta,1 Mohamed Alshal,1 Maksim Agaronov,1 Henry Talus,1 Ellen Li,3 Jennie Williams,3 Jovanny Zabaleta,2 Laura Martello-Rooney1. 1 _SUNY Downstate Medical Ctr., New York, NY;_ 2 _Louisiana State University Health Sciences Center, New Orleans, LA;_ 3 _Stony Brook University, Stony Brook, NY_.

Colorectal cancer (CRC) is the third most common cancer among African Americans (AA) and when compared to Caucasian Americans (CA), they present with more advanced disease and lower survival rates. Our previous findings suggest that this may be related to the differential expression of genes linked to cell recruitment and immune response. Therefore, we aimed to investigate if differences in the cellular anti-tumor immune activity in AA and CA patients play a role in the disparate cancer progression observed between these populations. Our approach includes examining gene expression and immune cell recruitment at the tumor site and secretion of cytokines characteristic of effector T helper cells (Th) subsets in plasma. Lastly, we observed the expression of several proteins involved in apoptosis, stress, and drug resistance in response to treatment with 5-FU in two microsatellite unstable (MSI) CRC cell lines, one from an AA and one from a CA patient, and a microsatellite stable (MSS) CA colon cancer cell line.

Methods: We performed whole transcriptome sequencing in colon tumors, utilizing the NextSeq 500/550 High Output Kit v2.5 (Illumina). ELISA assays (RayBiotech) were used to examine the secretion of cytokines linked to Th subsets (Th1, Th2, Th17) and inflammation in plasma. Using IHC we evaluated the cell recruitment and activation of T and Natural Killer cells in colon tumors and by MetaCore we correlated gene expression to immune-oncology pathways. Western blots were used to evaluate the expression of cleaved caspase 3, phospho-JNK and RRM1 in the in-vitro models.

Results: ELISAs of plasma from CA and AA patients revealed a differential Th cytokines production patterns between early stage (I, II) and late stage (III) disease. Our gene expression results indicate that the immune profiles of AA patients differ from CA in the expression of 36 key genes and cytokines related to cellular anti-tumor activity, including FOXP3, Granzyme B and IL-17A, suggestive of more favorable prognosis in the CA tumors. Lastly, the MSI AA cell line showed sensitivity to 5-FU in terms of protein expression when compared to the CA cell lines.

Conclusions: Our gene expression findings demonstrated the differential expression of immunological pathways involved in immune-surveillance, cancer progression and antigen presentation in colon tumors from these two ethnicities. These results were in accordance with the systemic cytokines' expression patterns observed in plasma and cell recruitment to the tumor sites. Importantly, our data indicates that treatment with 5-FU chemotherapy promotes apoptosis and stress in the MSI AA cell line but fails to produce the same effect in the CA cell lines at the same concentrations. Taken together, the differences in the immunological profiles in AA when compared to CA suggests a deficiency of the appropriate immune defense mechanisms in this population that may contribute to the cancer disparities among CRC patients.

#153

Quantifying tumor heterogeneity and mapping complex immune cell interactions with high-throughput, 7-color multispectral slide scans.

Carla Coltharp, Kent Johnson, Wenliang Zhang, Chichung Wang, Kristin Roman, Daniel Eversole, Clifford Hoyt, Peter Miller. _Akoya Biosciences, Hopkinton, MA_.

Introduction

As the need for multiple biomarker assessment in immune-oncology has become more clear, multiplex fluorescent immunohistochemistry (fIHC) techniques have become integral to immune oncology research.

Applying multispectral approaches to fIHC improves the quantitative performance by ensuring removal of autofluorescence signals and ensuring signal specificity by removing bleed-through between spectrally-adjacent dyes.

We have recently demonstrated a novel high-throughput multispectral scanning approach that allows acquisition of a 7-color multispectral slide scan in 5 - 10 minutes.

Here, we demonstrate the spatial measurements that have now been made possible by this high-throughput, translational workflow. These measurements span the scales related to tumor biology from the distance between nearest cell neighbors to the extent of the invasive margin.

Methods

Formalin-fixed paraffin-embedded samples of primary tumors were immunostained using Opal™ reagents. Tissue sections from primary tumors (lung, melanoma, colorectal, lymphoma)were stained against 5 markers of key interest in the field of immune oncology (CD8, FoxP3, PD-1, PD-L1, CD68) along with a tumor marker (Cytokeratin, Sox10&S100, PAX5 or PAX8) and DAPI counterstain, resulting in 7 colors on each slide.

Conventional and multispectral digital scans were acquired on a Vectra Polaris® automated imaging system and analyzed with inForm® and R software using the phenoptr and phenoptrReports packages.

Results

Multispectral scans showed a wide variety of immunological states among samples and within samples. We observed differences in the overall composition and density of immune cells in the tumor microenvironment, and further distinctions when looking at the local proximities of cell types and cells expressing either PD-1 or PD-L1, based on marker combinations that could not be observed with a conventional 3- or 4-color slide scan.

We developed spatial analyses to visualize and quantify this observed heterogeneity, both across the entire tissue and along the invasive margin. These tools revealed hot spots of immune activity and were further used to compare and categorize the properties of different tumors.

Furthermore, when compared with results from analogous scans analyzed without spectral unmixing, the unmixed imagery showed a marked improvement in the dynamic range of detected positive cells.

Conclusion

These tools for spatial analyses of whole-section 7-color multispectral slide scans provide translational researchers the opportunity to discover and validate biomarkers that capture immuno-biological interactions at microscopic and macroscopic scales, to better understand drug method-of-action and why some patients respond and some don't. 

### Genes That Regulate Migration and Invasion

#154

Inhibition of pancreatic cancer Panc-1 cell migration by omeprazole is dependent on aryl hydrocarbon receptor activation of JNK.

Unho Jin, Keshav Karki, Stephen Safe. _Texas A &M University, College Station, TX_.

Aryl hydrocarbon receptor (AhR)-active pharmaceuticals were screened as inhibitors of pancreatic cancer cell invasion and identified two compounds, omeprazole, that inhibited invasion. Inhibition of highly invasive Panc1 cell invasion by omeprazole involves an AhR-dependent non-genomic pathway, and omeprazolemediated inhibition of Panc1 cell invasion was dependent on Jun-N-terminal kinase and mitogen-activated kinase kinase 7 (MKK7). Results of RNAseq studies indicate that omeprazole induced an AhR-dependent downregulation of several pro-invasion factors including activated leukocyte cell adhesion molecule, long chain fatty acid CoA-synthase, stathmin 3 and neuropillin 2, and the specific functions of these genes are currently being investigated.

#155

Cysteine-rich secretory protein 3 expression leads to invasive prostate cancer by modulating cell motility.

Luc Furic,1 Marianna Volpert,2 Jinghua Hu,2 Anne O'Connor,2 Richard J. Rebello,2 Shivakumar Keerthikumar,1 Jemma Evans,3 Jo Merriner,2 John Pedersen,2 Gail P. Risbridger,2 Peter McIntyre,4 Moira K. O'Bryan2. 1 _Peter MacCallum Cancer Centre, Melbourne, Australia;_ 2 _Monash University, Melbourne, Australia;_ 3 _The Hudson Institute of Medical Research, Melbourne, Australia;_ 4 _Health Innovations Research Institute and School of Medical Sciences, Bundoora, Australia_.

Cysteine-rich secretory protein 3 (CRISP3) is one of the most highly up-regulated proteins during the transition from a healthy human prostatic epithelium to prostate cancer. The role of CRISP3 within this process has not however, been defined. Here we show using a genetically engineered mouse model of prostate cancer, that CRISP3 production greatly facilitates disease progression from carcinoma in situ to invasive prostate cancer in vivo. This observation was further validated using both human and mouse prostate cancer cell lines, which showed that exposure to CRISP3 enhanced cell motility and invasion. Further,using mass spectrometry, we showed that this activity is induced, at least in part, via changes in cell-cell adhesion proteins, including LASP1 and TJP1 both in vivo and in vitro. Collectively, these data identify CRISP3 as being pro-tumorigenic in the prostate and validate it as a bona fide marker of aggressive prostate cancer and a potential target for therapeutic intervention.

#156

Visfatin induces LOX expression and promotes tumor metastasis through the Src/Akt signaling pathway in human chondrosarcoma cells.

Cheng-Chieh Yu,1 Chih-Yang Lin,2 Chih-Hsin Tang1. 1 _China Medical University, Taichung, Taiwan;_ 2 _Mackay Medical College, New Taipei City, Taiwan_.

Chondrosarcoma, a common primary bone malignancy that affects the joints and bones, is associated with metastases in the lung, especially in cases of high-grade chondrosarcoma. The protein lysyl oxidase (LOX) can contribute to tumor growth and metastasis, while the adipokine visfatin is pro-angiogenic and facilitates tumorigenesis as well as metastasis. However, it is unclear as to whether any association exists between visfatin and LOX expression in human chondrosarcoma metastasis. We have discovered that visfatin induces the migration, invasion and increased expression of LOX mRNA and protein in human chondrosarcoma cell lines (JJ012 and SW1353). Pretreatment of these cell lines with Src and Akt inhibitors, or transfecting them with small interfering RNAs (siRNAs), inhibited visfatin-induced chondrosarcoma metastasis and LOX expression. Thus, visfatin promotes chondrosarcoma metastasis by inducing LOX expression via the Src/Akt signaling pathway in the JJ012 cell line. microRNA analysis revealed that visfatin-induced Src/Akt signaling is responsible for downregulating mature miR-26b-5p, which targets the 3' untranslated region (3`UTR) of LOX but does not affect primary and precursor miR-26b-5p expression. This downregulation of miR-26b-5p expression by visfatin was dose-dependent. Pretreatment with Src and Akt inhibitors or transfection with siRNA reversed miR-26b-5p expression. The migration and invasion ability of JJ012 cells was reduced when they were transfected with the miR-26b-5p mimic. In summary, our data demonstrate that visfatin induces LOX expression and promotes chondrosarcoma cell migration and invasion through the Src/Akt signaling pathway, by inhibiting miR-26b-5p.

#157

APE1 upregulates MMP-14 to promote invasion of esophageal adenocarcinoma via redox-sensitive ARF6-mediated recycling.

Heng Lu,1 Ajaz A. Bhat,2 Dunfa Peng,1 Zheng Chen,1 Shoumin Zhu,1 Jun Hong,3 Selma Maacha,3 David Robbins,1 Abbes Belkhiri,3 Wael El-Rifai1. 1 _University of Miami, Miami, FL;_ 2 _Sidra Medicine, Doha, Qatar;_ 3 _Vanderbilt University Medical Center, Nashville, TN_.

The incidence of Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) has increased dramatically in the past three decades. The development of BE and its progression to EAC is highly correlated with exposure to acidic bile salts due to chronic gastroesophageal reflux disease (GERD). A stimulated overexpression of APE1 has been identified in response to bile acids for oxidative DNA repair and oncogenic signal transduction in BE and EAC, but function and mechanism of APE1 in BE and EAC development remains largely unknown. Here, we demonstrate a new function of APE1 in promoting invasion of dysplastic BE and EAC cells that is dependent on its redox function. We observed aberrant overexpression of APE1 in the cell lines and patients' samples of dysplastic BE and EAC. In contrast to wild-type APE1, overexpression of redox-defective mutant, C65A, or treatment with APE1-redox-specific inhibitor, E3330, abrogated the pro-invasive phenotype of APE1. Mechanistically, we found that APE1 upregulated protein levels of the key matrix metalloproteinase MMP-14. Knockdown of APE1 decreased MMP-14 protein levels in dysplastic BE and EAC cells. Zymography assays indicated APE1 silencing reduced MMP-14 activity, subsequently impaired MMP-2 activation, and repressed extracellular matrix (ECM) degradation. Further, endocytosis and recycling assays revealed decreased endocytosis/recycling of MMP-14 protein in APE1-knockdown cells. Interestingly, our immunoprecipitation and proximity ligation assay (PLA) revealed a novel interaction between APE1 and ARF6, a key regulator of MMP-14 recycling. This interaction between APE1 and ARF6 activates ARF6 to regulate MMP-14 recycling through APE1-dependent redox function.

Conclusion: Our findings demonstrate, for the first time, the role of APE1 redox function in upregulating MMP-14 protein levels through activation of ARF6-mediated MMP-14 recycling. We propose APE1/ARF6/MMP-14 as a novel signaling axis in the invasion and progression of dysplastic BE and EAC.

#158

Mutantp53, MAPK and STAT3 promote metastasis to the brain: Demonstrated by protein, gene, and functional analysis.

John V. Wainwright, Jared B. Cooper, Anisha Chandy, Chirag D. Gandhi, Meic H. Schmidt, Meena Jhanwar-Uniyal. _New York Medical College, Valhalla, NY_.

Metastatic tumors to the brain arise from a specific subset of neoplastic cells originating from primary tumors such as lung, breast, colon, etc which disseminate specifically to the cerebral gray/white matter junction. Genetic and epigenetic mechanisms of this complex metastatic transformation are yet to be defined. Inactivation of the tumor suppressor gene p53 is one of the most frequent genetic alterations seen in human cancers. Further, extracellular regulated kinase (ERK), a component of mitogen activated protein kinase (MAPK) and Signal transducer and activator of transcription 3 (STAT3) plays a crucial role in differentiation, proliferation and migration, and is aberrant in many tumors. We hypothesize that aberrant p53, ERK1/2 and STAT3 contribute to metastatic brain tumors. Results demonstrated that: 1. Immunohistochemical analysis (IHC) of mutant p53 (mt-p53), phosphorylated-ERK1/2 Thr202/Tyr204 (pERK 1/2) and phosphorylated-STAT3Tyr705 revealed that 66% of metastatic brain tumors expressed mt-p53, 86% of tumors expressed pERK 1/2, and 75% nuclear expression of pSTAT3Tyr705; 2. Gene expression profiling showed that 17, 44 and 21 differentially expressed genes in metastatic tumors were p53, MAPK and STAT3 associated, respectively with alterations in genes associated with cell cycle regulation, neurogenesis, and differentiation and reprogramming and metastases; and 3. Metastatic breast cancer cells grown in astrocytic media displayed increased cell proliferation and enhanced S-phase cell cycle entry. Migration of primary tumor cells was significantly enhanced in astrocytic media as well. Inhibitors of MAPK (U0126) and STAT3 (STAT3 inhibitor SVI-201) suppressed cell proliferation and migration. Metastatic tumor cells and astrocytes were shown to grow amicably together, forming cell-to-cell interactions. These findings suggest that mt-p53 and aberrant MAPK and STAT3 play a crucial role in brain metastasis, and that the cerebral milieu provides a suitable microenvironment for metastatic cells to grow and disseminate and inhibition of aforementioned pathway can provide suitable therapeutic strategies.

#159

Chemokine receptor expression profiling of lymph node invasive versus lymph node non-invasive melanomas.

David Z. Bushhouse, Kristian M. Hargadon. _Hampden-Sydney College, Hampden-Sydney, VA_.

Regional lymph nodes are one of the first sites of melanoma metastasis during tumor progression, and lymph node invasion by tumor cells is a poor prognostic factor in melanoma patients. We have employed models of lymph node invasive and lymph node non-invasive murine melanomas to investigate the potential role of chemokine/chemokine receptor signaling pathways in the regulation of tumor cell trafficking to regional lymph nodes. PCR array analyses identified several differentially expressed chemokine and chemokine receptor genes in the lymph node invasive B16-F1 melanoma versus the lymph node non-invasive D5.1G4 melanoma. Among these differentially expressed genes, Cxcr3, Cxcr6, Cxcr7, Cx3cr1, Cxcl10, and Cx3cl1 were all significantly upregulated at the RNA level in B16-F1 melanoma. We are currently validating these differential gene expression data at the protein level, and these findings will provide key insights into the potential role of these chemokines and chemokine receptors in the metastasis of melanoma cells to regional lymph nodes.

#160

Role of EMMPRIN in osteosarcoma metastases.

Ilkyu Han,1 Ha Jung Kim,2 Han-Soo Kim1. 1 _Seoul National University College of Medicine, Seoul, Republic of Korea;_ 2 _Seoul National University Hospital, Seoul, Republic of Korea_.

Introduction: Although the survival rate of localized osteosarcoma patients has increased, patients with metastasis carry a poor prognosis. Thus, identification of factors contributing to metastatic progression is required. Extracellular matrix metalloproteinase inducer (EMMPRIN) is a cell-surface glycoprotein which plays multiple roles in physiologic and pathologic conditions. EMMPRIN stimulates adjacent stromal cells or tumor cells to produce matrix metalloproteinase (MMP) and is highly expressed in multiple cancer types. EMMPRIN also stimulates expression of vascular endothelial growth factor (VEGF) which leads to angiogenesis. However, the expression and role of EMMRPIN in osteosarcoma have never been studied. This study was performed 1) to investigate the expression of EMMPRIN in osteosarcoma and 2) to examine if EMMPRIN, via tumor-stroma interaction, is associated with metastatic potential in osteosarcoma. Methods: Level of EMMPRIN mRNA expression was evaluated by RT-PCR in 6 tumor-derived osteosarcoma cell lines and by immunohistochemistry prechemotherapy biopsies from 52 patients. Clinical data of these patients were reviewed to examine the association of EMMPRIN expression and clinical outcome. Transfection of EMMPRIN-targeting siRNA in SaOS-2 cell line was performed to examine the role of EMMPRIN in osteosarcoma progression. To study the role of EMMPRIN in tumor-stromal interaction, co-culture of SaOS-2 with osteoblast (hFOB) was experimented. Functional in vitro assays that reflect metastatic potential of osteosarcoma cells were performed. MMP production, VEGF production, cell invasion and proliferation were studied by gelatin zymography, VEGF ELISA, Matrigel invasion assay and WST-1 assay, respectively. Results: EMMPRIN was expressed in 90% by immunohistochemistry with strong accentuation along the cell membrane. Level of EMMRIN mRNA expression was significantly higher in 5 of 6 tumor-derived cell lines compared to MG63. Patients with high EMMPRIN expression had significantly worse metastasis-free survival. EMMPRIN mRNA levels was significantly down-regulated by siRNA transfection compared to control-siRNA transfected cell. Co-culture of osteoblast and SaOS-2 enhanced stimulation of pro-MMP2. This stimulation was reversed by transfection of SaOS-2 with EMMPRIN-targeting siRNA. Number of cells crossing the chamber was statistically lower in EMMPRIN-siRNA transfected cells. When osteoblast and SaOS-2 were co-cultured, VEGF expression was increased compared to SaOS-2 only culture. EMMPRIN-targeting siRNA transfection resulted in decrease of VEGF expression. SaOS-2 cells transfected with EMMPRIN-siRNA showed decreased proliferation potential compared to control-siRNA transfected cells. Conclusion: Our data suggest that highly expressed EMMPRIN plays an important role in metastatic progression of osteosarcoma. EMMPRIN could serve as a potential therapeutic target in osteosarcoma.

#161

TRIM21 suppresses invasion of breast cancer cells.

Xin Hu, Yue Jin, Yu Zhang, Boyuan Li, Youzhong Wan. _Jilin University, Changchun, China_.

Trim21 is an E3 ligase that is involved in the regulation of metastasis. High-level expression of TRIM21 is associated with good prognosis of breast cancer. After transplantation in immunodeficient mice, TRIM21-depleted MCF7 cells form tumors that show evident local invasion, i.e., extensive infiltration of single tumor cells into nearby muscle fibers. Knockdown of TRIM21 increases the migration and invasion of MCF7 and T47D cells by altering the expression of genes that regulate epithelial to mesenchymal transition(EMT). TRIM21 interacts with Snail, a master regulator of EMT, and the B-Box domain of TRIM21 is essential for the interaction between TRIM21 and Snail. Overexpression of TRIM21 leads to increased ubiquitination and subsequent proteosomal degradation of Snail, while depletion of TRIM21 decreases the ubiquitination and degradation of Snail. Most Importantly, knockdown of Snail suppresses the increased migration and invasion of MCF7 and T47D cells caused by TRIM21 knockdown. Collectively, our study demonstrates that TRIM21 modulates EMT by mediating the stability of Snail in breast cancer cells.

#162

ASB13 inhibits breast cancer progression and metastasis through SNAI2 degradation and transcriptional regulation of YAP.

Huijuan Fan,1 Xuxiang Wang,1 Wenyang Li,2 Minhong Shen,2 Yong Wei,2 Hanqiu Zheng,1 Yibin Kang3. 1 _Tsinghua University, Beijing, China;_ 2 _Princeton University, NJ;_ 3 _Princeton University & Rutgers Cancer Institute of New Jersey, NJ_.

Metastasis is responsible for the majority of cancer-related deaths. The transcription factor SNAI2 promotes metastasis by facilitating tumor cell invasion and tumor initiating activity. However, its post-translational regulation is less studied. We performed a dual luciferase-based, genome-wide E3 ligase siRNA library screening and identified ASB13 as an E3 ubiquitin ligase that targets SNAI2 for ubiquitination and degradation. ASB13 knockout in breast cancer cells leads to increased cell migration and decreased F-actin polymerization; while overexpression of ASB13 suppresses lung metastasis formed by LM2 cells. Furthermore, we discovered that ASB13 knockout decreases YAP expression, which is dependent on increased SNAI2 protein level. YAP functions as a tumor suppressor gene in breast cancer, as YAP knockout increases tumorsphere formation, anchorage-independent colony formation, and cell migration. Clinical data analysis reveals that ASB13 expression is positively correlated with overall survival in breast cancer patients. These findings establish the ASB13-SNAI2-YAP axis as a regulatory mechanism for breast cancer migration and metastasis.

#163

Identification and characterization of TGFBI in circulating tumor cell subline from pancreatic cancer cell line.

Tomoki Muramatsu, Taku Sato, Minoru Tanabe, Johji Inazawa. _Tokyo Medical and Dental University, Tokyo, Japan_.

Distant metastasis to liver, lung, brain, or bone occurs via circulating tumor cells (CTCs). We hypothesized that a subset of CTCs had more malignant features than tumor cells at the primary site. We established a highly malignant cell line, Panc-1-CTC, derived from the human pancreatic cancer cell line Panc-1 (Panc-1-P) using an in vivo selection method. Panc-1-CTC cells exhibited more profound migratory and invasive abilities than Panc-1-P cells in vitro. In addition, Panc-1-CTC cells had a higher tumor-forming ability than Panc-1-P cells in vivo. To investigate whether a difference in malignant phenotypes exists between Panc-1-P and -CTC cells, we performed comprehensive gene expression array analysis. As a result, Panc-1-CTC significantly expressed transforming growth factor beta-induced (TGFBI), an extracellular matrix protein, more abundantly than did Panc-1-P cells. TGFBI is considered to regulate cell adhesion, but its functions remain unclear. In the present study, knockdown of TGFBI reduced cell migration and invasion abilities, whereas overexpression of TGFBI increased both abilities. Moreover, elevated expression of TGFBI was associated with poor prognosis in patients with pancreatic cancer. Taken together, TGFBI might be targeted for cancer therapy and its expression could be a biomarker for the prediction of prognosis in pancreatic cancer.

#164

MALAT1 regulates the transcriptional and translational levels of proto-oncogene RUNX2 and promotes colorectal cancer metastasis.

Qing Ji,1 Guoxiang Cai,2 Xuan Liu,1 Yi Zhang,1 Yan Wang,1 Lihong Zhou,1 Hua Sui,1 Qi Li1. 1 _ShuGuang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China;_ 2 _Fudan University Shanghai Cancer Center, Shanghai, China_.

Ectopic expression of lncRNA-MALAT1 has been discovered in recurrent colorectal cancer (CRC) and metastatic sites in post-surgical patients, however, its biological mechanism remained un-elucidated. Our study firstly revealed the novel mechanisms of MALAT1 in promoting CRC metastasis through at least the following two mechanisms: First of all, MALAT1 binds miR-15 family members, to "de-inhibit" their effect on LRP6 expression, enhances beta-catenin signaling, leading to elevated transcriptional levels of downstream target genes RUNX2. Secondly, MALAT1 binds SFPQ, dissociate SFPQ/PTBP2 dimer to release free PTBP2, which elevates translational levels of RUNX2, through interacting with IRES domain in the 5'UTR of the corresponding RUNX2 mRNAs. Moreover, elevated RUNX2 expression levels were detected in recurrent CRC tumors, which were closely associated with TMN stages and CRC patients' survival. Our data demonstrated that, MALAT1 and RUNX2 may serve as two biomarkers for predicting the recurrence of CRC patients.

#165

CUCDC2 regulates malignant phenotype of triple-negative breast cancer through interacting with â-catenin.

Shuyan Han,1 Hai-Bo Han,1 Yan-Na Jiao,1 Pang-Kuo Lo,2 Yuan Yao,2 Qun Zhou,2 Ping-Ping Li1. 1 _Beijing Cancer Hospital & Inst., Beijing, China; _2 _Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD_.

Triple-negative breast cancer (TNBC) has the worst prognosis among all subtypes of breast cancer. CUE domain-containing protein-2 (CUEDC2) protein has reported to confer endocrine resistance in breast cancer by down-regulation of estrogen receptor α (ERα) expression. However, the function of CUEDC2 in TNBC subtype is unclear. Here, we showed CUEDC2 is expressed in triple-negative breast cancer and associated with its malignant phenotype. Knockdown of CUEDC2 not only inhibits TNBC cell proliferation, clone formation, migration, and invasion, but also suppresses TNBC stem cell mammosphere formation ability and cancer stem cell markers expression. While over-expression of CUEDC2 enhances its cell malignant phenotype. Furthermore, we determined that the ARM domain of β-catenin interacts with CUE domain of CUEDC2 through immunoprecipitation and pull-down assay. In the 34 TNBC samples, the expression levels of CUEDC2 and β-catenin also showed a positive correlation. Taken together, our study revealed CUEDC2 is associated with malignant phenotype and breast cancer stem cell properties of TNBC by interacting with β-catenin, and suggested that CUEDC2 may be a potential promising prognosis and therapy target for TNBC.

#166

MMP12 promotes EMT-mediated tumor aggressiveness in lung cancer cells.

Na-Kyung Han, Yoon-Jin Lee, Hae-June Lee. _Korea Institute of Radiological and Medical Sciences, Seoul, Republic of Korea_.

Epithelial mesenchymal transition (EMT) plays a pivotal role in cancer progression and metastasis. Matrix metalloproteinases (MMPs) have a major role in remodeling the extracellular matrix (ECM) and are implicated in the invasion and metastasis of various malignancies. MMP12 is involved in many pathological processes including cancer, but has not been fully understood yet. To investigate the role of MMP12 in lung cancer cells, we examined the effect of overexpression or knockdown of MMP12 in A549 cells and measured the protein expression level of EMT markers. MMP12 overexpression induced considerable EMT phenotypic changes compared to the control group, both in vitro and in vivo. The mRNA levels of EMT markers, snail2/slug, snail1, zeb1, TGFβ-1, and fibronectin, increased significantly upon MMP12 overexpression. We observed that changes in the expression level of ECM components such as, collagen, lamin, and proteoglycans led to MMP12 overexpression-induced EMT. These alterations promoted the aggressiveness of the A549 cells, evident from changes in functional properties of the cell, such as proliferation, migration, invasion, and adhesion capacity. On the contrary, MMP12 suppression abrogated malignancy of A549 cells. MMP12 knockdown in A549 cells led to decreased mRNA expression of EMT marker proteins associated with adherence, invasion, and migration. Our results suggest that MMP12 is a key regulator of EMT in non-small cell lung cancer (NSCLC). MMP12 overexpression in A549 cells results in changes in the expression of EMT markers and ECM composition. Interestingly, the dynamic changes in the ECM components by MMP12 overexpression cause EMT reprogramming and induce malignant functional properties. Thus, we suggest that the control of MMP12 expression can be a potential target for new therapeutic strategies of NSCLC.

#167

Semaphorin3D signaling in the invasion and metastasis of pancreatic ductal adenocarcinoma.

Noelle R. Jurcak, Stephen Muth, Kenji Fujiwara, Agnieszka Rucki, Kelly Foley, Adrian Murphy, Elizabeth M. Jaffee, Lei Zheng. _Johns Hopkins School of Medicine, Baltimore, MD_.

Pancreatic ductal adenocarcinoma (PDA) is a devastating disease, with the lowest stage-combined 5-year survival rate of any cancer type at 8%. One major attribute for this poor prognosis is the lack of effective treatments in preventing and controlling metastasis. Previously, our lab has identified the secreted protein Semaphorin3D (Sema3D) to be involved in the invasion and metastasis of PDA. Sema3D is part of the axon guidance gene family, recently been reported to be the most frequently altered gene family in PDA. We have previously found Sema3D interacts with its co-receptors PlexinD1 and Neuropilin-1 to cause increased invasion and metastatic capabilities in PDA cells. We found expression of Sema3D and PlexinD1 increases progressively during PDA development. In addition, tumoral knockdown of Sema3D prolongs survival and reduces metastasis of PDA-bearing mice. Also, Sema3D abundance was found to be significantly associated with human metastatic disease formation. However, the molecular signaling of Sema3D in aiding increased PDA cell invasion and metastasis is unknown. In the present study, we found treatment of mutant KrasG12D PDA, KPC cells, with Sema3D increases the activation of ADP-ribosylation factor 6 (Arf6). Interestingly, mutant KrasG12D has recently been reported to signal with Arf6 causing increased glycolytic metabolic changes. We found treating KPC cells with Sema3D increased glycolytic gene expression and increased tumor cell lactic acid secretion. Blockage of PlexinD1, the Sema3D receptor, with neutralizing antibodies inhibits this increase in glycolytic gene expression. Additionally, Panc02 PDA cells, not expressing mutant Kras, intriguingly do not show increased Arf6 activation after Sema3D treatment, suggesting Sema3D signaling could provide a selective role in mutant Kras expressing cells. Also, we investigated the role of Sema3D-induced increased acidity on tumor associated macrophage polarization. Bone-marrow derived macrophages were polarized to an M1, anti-tumor, or M2, pro-tumor phenotype and co-cultured with KPC cells treated with Sema3D or control protein. Macrophages isolated from the co-culture treated with Sema3D had decreased expression of anti-tumor M1 and increased expression of pro-tumoral M2 polarization markers compared to control. Futhermore, to study Sema3D expression in PDA in vivo, we crossed conditional Sema3D knockout mice to KRASG12D TP53R172H PDX-1-CRE+/+ (KPC) mice to create the KRASG12D TP53R172H Sema3D-/- PDX-1-CRE+/+ KPCS mouse model. Current studies are using the KPCS model to evaluate the role of Sema3D in PDA tumor development and metastasis, as well as, examine changes in tumor associated macrophage polarization. Taken together, this study provides enhanced mechanistic understanding of the role of Sema3D in aiding tumor progression and metastasis and, provides support for targeting the axon guidance pathway for PDA metastasis.

#168

Transglutaminase 2 (TGM2) overexpression contributes to triple-negative breast cancer metastasis through AFAP1-dependent pathway.

Mitchell Piacsek,1 Andrea Sand,1 Richard A. Rovin,2 Dmitry Bosenko,1 Santhi D. Konduri,1 Judy A. Tjoe,1 Jun Yin1. 1 _Aurora Research Institute, Milwaukee, WI;_ 2 _Aurora Neuroscience Innovation Institute, Milwaukee, WI_.

Triple-Negative Breast Cancer (TNBC) is a type of breast cancer with absence of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). It represents 10-20% of all breast cancer cases, and is associated with frequent relapse and poor prognosis due to the lack of targeted therapy. Amongst all TNBC, metastatic TNBC is associated with the worse prognosis and has the fewest therapeutic options. Therefore, identifying molecular drivers for TNBC metastasis and developing potential targeted therapies will be highly beneficial for patients with metastatic TNBC.

We classified 8 TNBC cell lines into two groups, more invasive (n = 3) vs. less invasive (n = 5), by assessing their abilities to both invade through 3D Matrigel and migrate distantly, characterized through in vitro cell invasion and migration assays, respectively. We compared the two groups using transcriptome profiles from Cancer Cell Line Encyclopedia (CCLE) and found that transglutaminase 2 (TGM2) was significantly upregulated in more invasive cell lines (fold change = 4.6, p < 0.001). Overexpression of TGM2 at both RNA and protein levels was confirmed experimentally in more invasive TNBC cells. These findings indicated that TGM2 might play a role in TNBC metastasis, but its functionality in invasiveness has not been evaluated by previous studies. In our study, suppressing TGM2 expression either by TGM2 inhibitor, cystamine dihydrochloride (CD), or by silencing TGM2 (siRNA) was found to significantly reduce the amount of invaded cells and inhibit cell migration, suggesting that TGM2 contributes to both cell invasion and cell migration in TNBC.

Our analysis also showed a significant correlation (r = 0.94, p = 0.001) between expression levels of TGM2 and actin filament-associated protein 1 (AFAP-1). AFAP1, which regulates actin cytoskeleton integrity and was found to contribute to tumorigenic growth by regulating focal contacts in other cancer types, was suppressed when TGM2 expression was downregulated, suggesting that TGM2 may potentiate cell metastasis through upregulation of AFAP-1 expression in TNBC. We also observed co-localization of AFAP-1 and actin filaments and that downregulation of TGM2 dramatically reduced actin filament assembly.

These observations identify a novel role of TGM2 in promoting TNBC cell metastasis and a new machinery of TGM2 in regulating microfilaments through AFAP-1. To conclude, TGM2 could act as a powerful biomarker and a molecular target circumventing TNBC metastasis. (Judy A. Tjoe and Jun Yin are considered to be co-corresponding authors for this study)

#169

Uncovering a myosin phosphatase regulator in pancreatic tumor cell mechanics and behavior.

Shantel M. Angstadt. _Johns Hopkins University School of Medicine, Baltimore, MD_.

Pancreatic ductal adenocarcinoma (PDAC) composes more than 80% of all pancreatic cancers and about 95% of exocrine pancreatic cancers. Patients typically present with symptoms post metastatic spread, which contributes to its overall lethality as the 3rd leading cause of cancer-related deaths with a median 5-year survival rate of about 8%. We are investigating the role of the myosin phosphatase targeting subunit 1 (MYPT1) in PDAC. MYPT1 serves as an interaction platform between a catalytic phosphatase subunit and the phosphorylated regulatory light chain (MLC) of myosin, allowing for MLC dephosphorylation and myosin inactivation. MYPT1 has been shown to elicit an antibody response in PDAC patients treated with a cytokine-secreting whole tumor cell vaccine. Additionally, MYPT1 has been shown to be upregulated in PDAC cells through immunohistological staining of surgically resected patient samples and in established PDAC cell lines. As a major regulator of non-muscle myosin II, MYPT1 has implications in cancer cell shape control, adhesion, and migration. To elucidate its implications, we are characterizing tumor cell phenotype with and without MYPT1 expression using the CRISPR genome editing system. We are assessing MYPT1's effect on cell mechanics (e.g. cortical tension), if MYPT1 responds to applied mechanical stresses, and how MYPT1 affects each of the non-muscle myosin II paralogs' ability to respond to these mechanical stresses. We are characterizing MYPT1's impact on cell and tissue behavior, including cell growth, migratory, and invasive phenotypes in culture and in mouse models. Overall, our work will provide insight into MYPT1's role in cancer cell behavior and response to its surrounding environment. The work will also provide insight for early investigations into MYPT1's targetability and immunogenic role in PDAC immunotherapy.

#170

Loss of the ubiquitin-specific peptidase 18 (USP18) reduces lung cancer metastasis.

Zibo Chen, Lin Zheng, Yulong Chen, Masanori Kawakami, Lisa Mustachio, Jason Roszik, Xin Liu, Jonathan Kurie, Xi Liu, Ethan Dmitrovsky. _MD Anderson Cancer Center, Houston, TX_.

Metastasis is a major cause of cancer-related mortality. Yet, current antineoplastic strategies do not effectively repress metastasis. Thus, there is a pressing need to explore targets that can reduce metastasis, especially in lung cancer that is the most common cause of cancer mortality. Our prior work reported that expression of the ubiquitin-specific peptidase 18 (USP18) was augmented in diverse cancers (including lung cancers) and USP18 knock-down suppressed in vitro and in vivo growth of murine and human lung cancers. This deubiquitinase stabilizes target proteins by removing interferon-stimulated gene 15 (ISG15) from substrate proteins. We explored here whether engineered loss of USP18 decreased lung cancer metastasis. Stable USP18 knockdown was independently achieved in human (A549 and H1299) and murine (344SQ and KC2) lung cancer cells by use of small hairpin RNAs (shRNAs). Notably, knockdown of USP18 expression reduced lung cancer cellular proliferation, migration and invasion versus control shRNA transfectants (p < 0.001). Lung cancer cells selected for high metastatic potential (344SQ and KC2) were independently injected via tail vein or subcutaneously to establish metastatic mouse models. USP18 knockdown statistically-significantly decreased lung cancer metastases as compared to control shRNA transfectants. To elucidate engaged mechanisms, Reverse Phase Protein Array (RPPA) analysis was performed to interrogate over 300 growth-regulatory proteins in human (A549 and H1299) and murine (344SQ and KC2) lung cancer cells having engineered USP18 knockdown. Results were compared to control shRNA transfectants. Ingenuity pathway analysis highlighted fatty acid synthase (FASN) as a potential USP18 target that regulated lung cancer metastasis. Independent immunoblot analysis confirmed FASN expression was reduced in cancers once USP18 knockdown was achieved. Functional validation of FASN in lung metastasis is underway. These and other findings that will be presented provide a strong rationale for targeting the deubiquitinase USP18 to reduce lung cancer metastasis and combat lung cancers.

#171

PHIP regulates the focal adhesion dynamics, invasion and motility in glioblastoma.

David De Semir,1 Vladimir Bezrookove,1 Mehdi Nosrati,1 Kara R. Scanlon,1 Eric Singer,1 Jonathon Judkins,1 Christopher Rieken,2 Clayton Wu,1 Julia Shen,1 Christina Schmudermayer,1 Altaf A. Dar,1 James R. Miller,1 Charles Cobbs,3 Garret Yount,4 Pierre-Yves Desprez,1 Robert J. Debs,1 Sean McAllister,1 James E. Cleaver,5 Liliana Soroceanu,1 Mohammed Kashani-Sabet1. 1 _California Pacific Medical Center, San Francisco, CA;_ 2 _Carl Zeiss Microscopy, New York, NY;_ 3 _Swedish Medical Center, Seattle, WA;_ 4 _Institute of Noetic Sciences, Petaluma, CA;_ 5 _University California San Francisco, San Francisco, CA_.

The invasive and migratory potential of glioblastoma multiforme (GBM) is essential to its aggressive behavior. Here we show that pleckstrin homology domain-interacting protein (PHIP), acting through the force transduction layer of the focal adhesion complex, drives GBM motility and invasion. Immunofluorescence analysis unexpectedly localized PHIP to the leading edge of GBM cells. Confocal microscopy specifically localized PHIP to the force transduction layer, along with talin and vinculin. Co-immunoprecipitation revealed a physical interaction between PHIP and vinculin. PHIP gene silencing suppressed expression of focal adhesion proteins and produced profoundly disorganized stress fibers in U251, LN18, U87 GBM cell lines and in 3832 primary GBM cells. Videomicroscopy demonstrated PHIP's regulation of focal adhesion dynamics. PHIP silencing suppressed GBM cell migration and invasion, resulting in substantial reductions in U251 intracranial growth and angiogenesis. PHIP expression was elevated in a subset of GBMs, but not low-grade gliomas. PHIP FISH copy number was enriched in the classical subtype versus the mesenchymal or proneural subtypes of GBM. These results demonstrate a key role for PHIP in regulating the actin cytoskeleton, focal adhesion dynamics, tumor cell invasion and motility in GBM.

#172

BCL11A regulation of extracellular matrix genes may be necessary for invasion of triple-negative breast cancer.

Natasha N. Ingles, Darcie Seachrist, Ruth Keri. _Case Western Reserve University, Cleveland, OH_.

Triple-negative breast cancer (TNBC) represents 15% of all breast cancers, yet conveys the poorest patient prognoses due to the lack of targeted therapies and the high incidence of metastatic recurrence. This prompt recurrence is thought to be due, in part, to the high proportion of cancer stem cells (CSC) within TNBC tumors. Therefore, understanding what regulates CSCs and metastasis will allow us to significantly reduce cancer-related deaths. One of the transcription factors that is upregulated in TNBC is BCL11A, which is critical for CSC biology. We have found that BCL11A is necessary for TNBC invasion and metastasis. However, the genes that BCL11A targets to control invasion and metastasis are unknown. To identify the BCL11A-regulated transcriptome, we performed RNA-seq analysis of MDA-MD-231 TNBC cells transiently transfected with BCL11A-targeted siRNA or a non-silencing control. Gene set enrichment analysis of the differentially expressed genes revealed enrichment of genes involved in extracellular matrix and matrisome genes. Notably, BCL11A silencing reduced expression of several matrix-metalloproteases, with MMP1 being the most significantly differentially expressed gene. Thus, we hypothesize that BCL11A promotes TNBC invasiveness and metastasis through regulation of MMP expression. We confirmed downregulation of MMP1 and MMP9 mRNA and protein in response to BCL11A silencing by both qRT-PCR and western blot in independent MDA-MB-231 cells, as well as in the HCC1143 TNBC cell line. MMP activity was also reduced when assessed by fluorokine assay and gelatin zymography, for MMP1 and MMP9, respectively. Furthermore, both BCL11A and MMP expression is increased in brain-seeking clones of MDA-MB-231 cells and MMP1 and 9 expression is also reduced with BCL11A silencing. Together, these data suggest that BCL11A regulates TNBC invasion by reducing the expression of MMP1 and MMP9. Current studies are directly assessing the functional impact of MMP regulation by BCL11A on invasion and metastatic progression of TNBC.

#173

Upregulated EPHB2 and SRC pathways modulate spatial EGFR dynamics and malignant phenotypes and predict poor prognosis in prostate cancer.

Yen-Liang Liu,1 Aaron M. Horning,2 Che-Kuang Lin,2 Brandon Lieberman,3 Chia-Nung Hung,2 Chih-Wei Chou,2 Chiou-Miin Wang,2 Michael A. Liss,2 Mirae Kim,1 Rohan Vasisht,1 Evan P. Perillo,1 Katherine Blocher,1 Hannah Horng,4 Xi Tian,2 Chun-Lin Lin,2 Andrew K. Dunn,1 Tim H.-M. Huang,2 Hsin-Chih Yeh,1 Chun-Liang Chen2. 1 _The University of Texas at Austin, Austin, TX;_ 2 _UT Health Science Ctr. at San Antonio, San Atnonio, TX;_ 3 _Trinity University, San Atnonio, TX;_ 4 _The University of Maryland, College Park, MD_.

Dysregulated dynamics and trafficking of receptor tyrosine kinases (RTK) have been linked to oncogenesis and metastasis. The mechanisms for disfunction of RTK dynamics are emerging and still not clear. Using single-particle tracking (SPT) techniques, we studied the dynamics and trafficking patterns of epidermal growth factor receptor (EGFR) and underlying mechanisms using prostate cancer cells (PCa) (LNCaP, DU145 and PC3) exhibiting variant metastatic capability. Through SPT and super-resolution imaging, it was revealed that cortical actin disorganization modulates increased dynamics of EGFRs, including high EGFR diffusivity, enlarged EGFR confinement size on the plasma membrane and faster EGFR internalization in advanced invasive cells. The elevated EGFR dynamics were associated with advanced aggressive behaviors, and highly upregulated EGFR, EPHB and SRC signaling as identified using single cell RNA-seq. Strong EGFR activation with relatively low EGFR expression in PC3 implicated some other mechanisms, likely biophysical, beyond EGFR quantity. Moreover, the upregulated EPHB and SRC pathways have been shown to regulate actin organization and metastasis and our in silico analysis indicated that genes in the two pathways predict poor prognosis for disease free and survival statuses in The Cancer Genome Atlas prostate cancer patient cohort. To interrogate the roles of EPHB and SRC pathways in actin organization and EGFR dynamics, a series of drug inhibitions and siRNA knockdown were applied to PCa cells followed by mRNA profiling, evaluations of EGFR dynamics, and cell behaviors. Functional knockdown of EPHB2 and SRC led to decreased EGFR dynamics, cell proliferation, migration and invasion. The loss-of-function effects were more profound in advanced invasive PCa. In this study, we discovered the roles of EPHB and SRC pathways in modulating actin organization and EGFR dynamics and leading to aggressive metastatic phenotypes. Additionally, EGFR dynamics were potential biophysical parameters to differentiate the highly-invasive from the non- and less-invasive PCa. Thus, we believe that the SPT-based EGFR dynamics can serve as a new biophysical assay to probe the metastatic malignancy of cancer cells and to monitor their response to anti-cancer drug treatment.

#174

**Functional characterization of** ZNF750 **in esophageal squamous cell carcinoma.**

Sheyne SA Choi, Valen Zhuoyou Yu, Josephine Mun-Yee Ko, Wei Dai, Maria L. Lung. _The University of Hong Kong, Hong Kong, Hong Kong_.

Background: Esophageal squamous cell carcinoma (ESCC) is a lethal and aggressive malignancy that is a threat worldwide. It is of significant prevalence in regions of Asia including Iran, Korea, Japan and especially China. Epidemiological studies of ESCC reveal genetic variations as a risk-factor to ESCC incidence. Genomic profiling of ESCC primary tumors have already identified candidate genes involved in different cellular processes such as cell cycle and apoptosis. Data from our whole exome sequencing study of primary tumors and lymph node metastases also reveal mutations involved in these pathways; however, we also uncovered an interesting candidate involved in differentiation namely, ZNF750. ZNF750 has been reported to play important roles in other squamous cell carcinomas, and as a candidate tumor suppressor gene in ESCC.

Methods and Results: We exogenously upregulated ZNF750 expression by lentiviral system and used this model to investigate ZNF750 function in ESCC. By MTT 2D cell proliferation assay, we showed that in vitro cell proliferation in the ZNF750 group was hindered compared to controls. In addition, we set up 2D and 3D colony formation assays and found that the ZNF750 groups produced fewer colonies in 2D culture plates and produced significantly smaller colonies, when embedded in Matrigel. All of these findings are consistent with previous studies. We also studied the potential functional role of ZNF750 in 3D invasion/migration. Transwell permeable inserts were used to support a thick Matrigel culture with a nutrient gradient. Cells were embedded in Matrigel and moved along the gradient within the gel. After prolonged incubation (>2 weeks), location and size of the colonies were examined by confocal microscopy scanning. Cells with upregulated ZNF750 were found to exhibit greater mobility compared to the control cells. We also subjected ZNF750 and control groups to cisplatin to test for chemoresistance in ESCC. We found that ZNF750 expression increases chemoresistance, as opposed to the control cells.

Conclusion: From these results, ZNF750 expression inhibits cell proliferation, while enhancing cell mobility and drug resistance, all of which are characteristics of cells undergoing epithelial-mesenchymal transition (EMT). We, therefore, hypothesize that ZNF750 functions in ESSC beyond that of a tumor suppressor, but as a putative EMT regulator.

Acknowledgements: Research Grants Council Collaborative Research Fund grant number 106150246 and Asian Cancer Research Fund to MLL.

#175

Methyltransferase protein A regulates colorectal cancer cell proliferation and metastasis through methylation of protein B.

Songhwa Kang, Sang-Mo Kwon. _Pusan National University, Yangsan, Republic of Korea_.

Post-translational modifications (PTMs) contribute to abnormal cell proliferation, adhesion, and morphology in cancer progression. Methylation is one such PTM that affects many of the protein properties and has a substantial role in tumorigenesis. Here, we identified that the methyltransferase protein A was upregulated in human colorectal cancer (CRC) and was associated with CRC proliferation, migration, and invasion in vitro. Our study showed that Protein B was a novel target of methyltransferase Protein A and that methylated Protein B was upregulated in human CRC. High expression of Protein A and B resulted in a poor prognosis of CRC; moreover, CRC proliferation, migration, and invasion were inhibited when protein A and B were unable to bind through specific residue, as predicted by in silico protein-protein docking analysis. Furthermore, our study showed that methylation of Protein B by methyltransferase protein A plays an important role in CRC proliferation and invasion via enhancement of HIF-1α stabilization. In conclusion, our data suggested that methylated Protein B may be a potentially valuable clinical prognostic marker of CRC and that interference with the binding of Protein B to methyltransferase Protein A may be a new anticancer target in CRC.

#176

Tetraspanins in oral squamous cell carcinoma.

Kiran K. Reddi, Kenneth M. Anderson, Franklin Garcia-Godoy, Yanhui H. Zhang. _College of Dentistry, University of Tennessee Health Science Center, Memphis, TN_.

Objectives: To (1) investigate the expression levels of several tetraspanins in oral cancer cells and tissues; (2) characterize the subpopulation of oral cancer cells with elevated CD82; and (3) investigate roles of the tetraspanins in oral cancer migration, invasion, and metastasis.

Methods: The protein expression levels of tetraspanins CD82(KAI1), CD9, CD81(TAPA), and their associate protein EWI-2(IGSF8) were quantified in normal human gingival epithelial cells (SG) and oral cancer cells (CAL27 and SCC-25 cells) using western blotting and immunofluorescence staining and imaging. CAL27 CD82 knockout cells (CD82KO) were created by CRISPR/CAS-9, sorted by Fluorescent Activated Cell Sorting, and validated by western blotting. A wound healing assay was used to study the role of CD82 in oral cancer cell migration. CD82 perturbation of the CAL27 genome and proteome were evaluated using Microarray (Affymetrix) and TRAQ/TMT Based Differential Protein Expression Analysis (Thermo, mass spectrometer).

Results: So far, we have found (1) CD82 is highly expressed in CAL27 cancer cells, but its expression is low in SG and SCC-25 cells; (2) CD82 is highly expressed in human tongue cancer tissues; (3) CD9 is highly expressed in CAL27 cells; (4) The expression levels of CD81 and IGSF8 are comparable in the three cancer cell lines; (5) CAL27 cancer cells migrate faster than normal SG cells; (6) Knockout of CD82 significantly decreases the migration rate of CAL27 cells, suggesting that CD82 promotes migration of these cancer cells.

Conclusions: CD82 has varied expression in different oral cancer cell lines. Its expression is very high in CAL27 cancer cells. Knockout of CD82 in CAL27 cells significantly inhibits migration of these cells. Our study suggests that CD82 might be used as a biomarker for a subpopulation of oral cancer cells and that CD82 could be a target for therapeutic interventions.

Acknowledgements:

Supported in part by the 2017 UTHSC CORNET Award.

Supported in part by the UTHSC College of Dentistry Alumni Endowment Fund and the Tennessee Dental Association Foundation.

#177

Sulfiredoxin promotes the invasion of human colorectal cancer cells through the activation of fascin.

Hong Jiang, Qi Ying, B. Mark Evers, Qiou Wei. _University of Kentucky College of Medicine, Lexington, KY_.

Sulfiredoxin (Srx) is multifunction enzyme with a primary antioxidant role of reducing the overoxidized inactive form of peroxiredoxins (Prxs). In contrast to the well-studied biochemical function, the biological significance of Srx in human diseases including cancer has not been well studied. Our previous studies show that it plays an important role in tumorigenesis and malignant progression of human skin, lung and colorectal cancers. Fascin homologue 1 (FSCN1) is an actin-binding protein that is critical for cancer cell invasion and metastasis. The purpose of this study is to understand the molecular mechanism of Srx on the invasion and metastasis of human colorectal cancer cells through the regulation of FSCN1. The effects of Srx on the migration and invasion of human colon cancer HCT116 cells were evaluated by wound healing and matrigel invasion assay, respectively. Actin stress fiber was visualized by F-actin fluorescence staining. Western blotting and reverse transcription & real-time PCR were used to examine the protein and RNA levels of FSCN1 in HCT116 cells. The effect of Srx on FSCN1 promoter and 3' UTR were determined using luciferase reporter construct. We demonstrated that (1) knockdown of Srx suppressed HCT116 colony formation and cell invasion in the matrigel invasion assay; (2) knockdown of Srx inhibited FSCN1 expression in HCT116 cells at both RNA and protein level; (3) the inhibition is the result of decreased FSCN1 mRNA stability; (5) Srx stabilizes FSCN1 mRNA 3' UTR mediated reporter expression; (4) knockdown of Srx reduced actin stress-fiber formation; (5) Srx and Prx4 synchronically regulates FASN1 expression. Therefore, we concluded that Srx promotes the invasion and metastasis of human colon cancer cells through the upregulation of FSCN1.

#178

MYO10 aberrant methylation and overexpression in leader cells regulates lung cancer collective cell invasion and fibronectin patterning.

Emily R. Summerbell,1 Jessica Konen,2 Jeanne Kowalski,1 Paula Vertino,1 Adam Marcus1. 1 _Emory University, Atlanta, GA;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Myosin-X (MYO10) is a noncanonical myosin that drives filopodia formation and extension, and is necessary for invasion and metastasis of breast cancer, prostate cancer and melanoma. It is not yet known by what mechanism MYO10 increases cancer cell invasion and metastasis. Two independently published MYO10 knockout mouse models showed severe developmental defects dependent upon collective migration of several cell types, suggesting that MYO10 may also regulate collective invasion of cancer cells. In order to study collective cancer cell invasion, our lab previously developed a technique to isolate and culture purified highly-invasive leader cells and highly-proliferative follower cells from collectively invading lung cancer cell lines. Using this method, we demonstrated distinct gene expression patterns, DNA methylation patterns, and phenotypic properties of leader versus follower cells. MYO10 is highly overexpressed in leader cells but is not expressed in follower cells, and MYO10 overexpression in leaders correlates with highly significant CpG island promoter DNA hypomethylation and gene body DNA hypermethylation. We hypothesize that MYO10 regulates cancer cell collective invasion by driving the formation of long filopodia necessary for leading-edge fibronectin patterning and subsequent leader cell invasion. MYO10 localizes at leader cell filopodia tips but not in follower cell filopodia within both 2D culture and 3D invading spheroids of four non-small cell lung cancer cell lines. MYO10 knockdown in purified leader cells and in these four lung cancer cell lines decreases the number and length of filopodia, 2D cell motility and 3D spheroid invasion; in contrast, MYO10 overexpression in follower cells increases filopodia length, cell motility and spheroid invasion. In addition, proteomic analysis shows that leader cells produce and secrete fibronectin (FN1), unlike follower cells. Immunofluorescence within invading spheroids shows the formation of long FN1 fibrils, i.e. elongated parallel bundles of FN1 that extend far past the leader cell body in multiple cell lines. FN1 fibrils preferentially localized with MYO10+ filopodia. Knockdown of MYO10 disrupts the formation of these fibronectin fibrils. We previously reported that knockdown of fibronectin completely abrogates lung cancer spheroid collective invasion. Therefore, our data suggest that MYO10 regulates cancer cell collective invasion by driving the formation of long filopodia that then regulate fibronectin architecture and subsequent invasion by leader cells.

#179

M6A demethylation enzyme FTO upregulates LDHA and promotes glycolysis and invasion and migration in oral squamous cell carcinoma.

Jinsong Hou. _Sun Yat-sen Univ, Guangzhou, China_.

Objective: To investigate the role of lactate dehydrogenase A (LDHA) in the glycolysis and invasion and metastasis of oral squamous cell carcinoma (OSCC), and to explore the molecular mechanism of fat mass and obesity-associated protein (FTO) regulating LDHA on the glycolysis and invasion and metastasis of OSCC.

Methods: Immunohistochemistry and RT-qPCR were performed to detect the expression of LDHA protein and gene in OSCC and adjacent non-cancerous tissues, and their correlation with clinical prognosis and pathological parameters were analyzed. Lentivirus-mediated shRNA silencing of LDHA and lactate dehydrogenase inhibitor (oxamate) were used to establish stable OSCC Cell lines HSC3 and SCC15, Cell Counting Kit-8 and colony formation assay were applied to evaluate cell proliferation, scratch test and Transwell assay were performed to detect cell migration and invasion, Glucose utilization assay and Lactate production assay were carried out to assess cell uptake of glucose and lactate production respectively. The molecular mechanism of FTO regulating LDHA on glycolysis, invasion and metastasis of oral squamous cell carcinoma was further explored, OSCC cell lines HSC3 and SCC15 transfected with siRNAs to silence FTO expression, EpiQuik™ m6A RNA Methylation Quantification Kit was applied to assess the m6A% in total RNA, the effect of FTO on LDHA expression was detected by Western blot and RT-qPCR. The biological function of FTO on oral squamous cell carcinoma cells was demonstrated by cell experiments in vitro.

Results: Compared with adjacent non-cancerous tissues, LDHA was highly expressed in OSCC (P<0.001), which was closely related to the differentiation, clinical stage and T stage of OSCC (P<0.05). Silencing LDHA, FTO or treating oxamate in HSC3 and SCC15 cells reduced glucose consumption, lactate production and cell proliferation, invasion and migration (P<0.05). In addition, after FTO silencing, the m6A level of total RNA increased (P<0.05), while the expression level of LDHA protein and mRNA decreased (P<0.01).

Conclusion: FTO may promote the expression of LDHA protein and mRNA in a m6A-dependent manner and enhance the ability of glycolysis, proliferation, invasion and

metastasis of OSCC.

#180

Defining the metastasome in colorectal cancer: Novel hypotheses on tumor progression, metastasis evolution and therapy by whole genome sequencing.

Heike Allgayer. _Univ. Heidelberg, Mannheim, Germany_.

Metastasis poses the highest challenge for personalized therapy, partly due to incomplete understanding of its molecular evolution. This work presents the most comprehensive whole-genome study of colorectal metastasis vs. matched primary tumors. The cohort presented supports both early and late dissemination models. 65% of somatic small mutations originated from a common ancestral truncal node, with 15% of small mutations being tumor- and 19% metastasis-specific, implicating a higher mutation rate in metastases. In tumor- and metastasis-specific mutations we observed elevated levels of BRCA-ness signatures. A multistage progression model was confirmed with new components ARHGEF7/ARHGEF33, and ploidy dependent co-occurring SOX9 and HK3 mutations. We found previously undescibed recurrently mutated non-coding elements including specific ncRNAs and 3' UTRs which will be presented at the meeting, some of them known as potential dual protagonists in metastasis and efferocytosis-/PD-L1 mediated immunosuppression. Furthermore, we discovered genomic aberrations explaining microRNA expression changes during metastasis. Moreover, we found actionable metastasis-specific lesions (which will be reported at the meeting) which would not have been detected in whole genome sequencing of the corresponding primary tumor, suggesting a reconsideration and modifications of current personalized therapy concepts to prevent metastasis. Metastasis-specific small mutations were enriched in PI3K-Akt signaling, cell adhesion, extracellular matrix but also hepatic stellate activation pathway genes, suggesting the initiation of genetic programs for site-specific colonization during metastasis. Taken together, the results presented here reveal novel components of disease progression and metastasis evolution with evidence for metastasis-specific genomic lesions relevant for personalized therapy.

#181

Prolyl-4-hydroxylase α subunit 3 promotes renal cancer progression and metastasis.

Larry E. Broome,1 Bingzhi Wang,2 Menghuang Zhao,3 Junhua Yang,1 Hakim Bouamar,1 Matyas Cserhati,1 Lu-Zhe Sun1. 1 _University of Texas Health Science Center San Antonio, San Antonio, TX;_ 2 _Xiangya School of Medicine, Changsha,, China;_ 3 _Second Affiliated Hospital of Wenzhou Medical University,, Wenzhou, China_.

Collagen is the major component of the extracellular matrix, its overexpression has been shown to facilitate tumor development and progression. Prolyl-4-hydroxylase α subunit 3 (P4HA3) plays a crucial role in the synthesis of collagen, as it is involved in the catalysis to convert proline residues to 4 hydroxyproline residues, which are essential for the conformational stability of mature collagen. It has been reported that P4HA3 functions as a tumor promoter in gastric cancer. However, the role of P4HA3 in renal cell carcinoma (RCC) has not been established. To identify the role of P4HA3 in RCC, we analyzed the expression of P4HA3 in RCC patients and knocked down the expression of P4HA3 in two renal cancer cell lines with different P4HA3 siRNAs and P4HA3 shRNAs. We performed cell proliferation, migration and invasion assays in vitro and in vivo. Our results demonstrated that expression of P4HA3 was upregulated in RCC patients and knockdown of P4HA3 significantly inhibited renal cancer cell proliferation, migration and invasion. Mouse xenograft studies demonstrated the tumor promoting role of P4HA3 in renal cancer. Therefore, P4HA3 appears to be a potential therapeutic target for the treatment of RCC.

### Targeting Tumor-Host Interactions

#182

Cancer stem cells and tumor angiogenesis in serous adenocarcinoma of ovary.

S Krishnapriya, C Sidhanth, P Manasa, S Sneha, S Bindhya, R P. Nagare, T S. Ganesan. _Cancer Institute (WIA), Chennai, India_.

The traditional view of tumour vascularization is that tumours acquire blood supply from the neighbouring normal stroma. However, recently the origin of tumour endothelial cells or pericytes in part has been shown to be derived from cancer stem cells (CSCs) in glioblastoma. In high grade serous ovarian cancer (HGSOC), the origin of endothelial cells is not known. Our objective was to determine if components of a tumour blood vessel and lymphatic vessel are derived from CSCs in ovarian cancer. Using spheroids as an in vitro model, we have evaluated the role of CSCs in primary malignant cells (PMCs) from patients with serous adenocarcinoma of ovary cultured under specific conditions. The expression of endothelial, pericyte and lymphatic endothelial markers was evaluated by flow cytometry. In addition, functional assays were performed to assess the endothelial phenotype. Further, the ability of CSCs to express endothelial markers under appropriate growth conditions was also evaluated with Bevacizumab which antagonize VEGF. PMCs grown in endothelial growth medium (EGM) showed significantly higher expression of CD105 (n=32, p = 0.002) and CLEC14A (n=10, p = 0.012) and co-expression of CD105/CLEC14A (n=10, p= 0.012) than that of spheroids. Primary malignant cells when grown in pericyte and lymphatic endothelial specific conditions, showed significantly higher expression of desmin (n=10, p=0.03), Smooth muscle actin (SMA) (n=10, p=0.017) and VEGFR3 (n=10, p=0.028) than that of the spheroids. When the PMCs were grown as spheroids in endothelial conditions in the presence or absence of Bevacizumab (1 μg/μl), there was a reduction in the co-expression of CD105 and CLEC14A (P=0.04). The cells grown in endothelial conditions showed formation of tubes, uptake of Dil-ac-LDL and expressed eNOS, confirming their endothelial phenotype. GFP transduced spheroids from PMCs formed tumours in mice and the blood vessels in the tumour co-expressed CD31, SMA, VEGFR3 and GFP, suggesting that these cells are derived from CSCs. These results prove that a proportion of endothelial cells, pericytes and lymphatic endothelial cells are derived from CSCs in serous ovarian carcinoma and the VEGF pathway has a key role. This property of CSCs to contribute to tumour angiogenesis can be inhibited.

#183

N-myc downstream regulated gene 1 (NDRG1) is indispensable for VEGF-A-induced tumor angiogenesis through PLCγ/ERK signaling activation in vascular endothelial cells.

Kosuke Watari,1 Tomohiro Shibata,1 Ai Shinoda,1 Hideyuki Abe,2 Akihiko Kawahara,2 Yuichi Murakami,3 Eiji Oki,1 Jun Akiba,2 Yoshihiko Maehara,4 Michihiko Kuwano,3 Mayumi Ono1. 1 _Kyushu University, Fukuoka, Japan;_ 2 _Kurume University Hospital, Kurume, Japan;_ 3 _St. Mary's Institute of Health Sciences, Kurume, Japan;_ 4 _Kyushu Central Hospital, Fukuoka, Japan_.

[Background] Vascular endothelial growth factor (VEGF)-A is a key regulator of tumor angiogenesis that is essential for tumor growth and progression. The unraveling of the precise mechanisms behind VEGF-A-induced tumor angiogenic process will further contribute to development of novel and potent anti-cancer therapeutics. N-myc downstream regulated gene 1 (NDRG1) has been shown to play essential roles in multiple biological processes including embryogenesis, tissue development, cell growth, differentiation, and tumorigenesis. We previously reported that NDRG1 expression levels in cancer cells are closely correlated with tumor angiogenesis and growth (Hosoi et al., Cancer Res., 2009; Murakami et al., J Biol Chem., 2013), and also that NDRG1 promotes tumor angiogenesis through enhanced VEGF-A production by tumor-associated macrophages (Watari et al., Sci Rep., 2016). However, it remains unclear whether NDRG1 expression in vascular endothelial cells (ECs) plays any crucial role in VEGF-A-induced tumor angiogenesis. In our present study, we further ask whether and how NDRG1 in ECs could specifically regulate tumor angiogenesis. We also present our finding of intrinsic importance that NDRG1 functions as an essential factor for VEGF-A-induced angiogenesis.

[Methods] NDRG1 deficient mice: The NDRG1 deficient mice on C57BL6 background were purchased from Laboratory Animal Resource Bank, National Institutes of Biomedical Innovation, Health and Nutrition (Osaka, Japan). Isolation of mouse endothelial cells: CD31+ endothelial cells were isolated from mouse lung by magnetic sorting using CD31 MicroBeads. Aortic ring assay: 1 mm mouse aortic rings were embedded in 3-dimensional growth factor reduced Matrigel, treated with or without FGF-2 (50 ng/mL) or VEGF (25 ng/ml), and incubated at 37°C. Vascular length and branching point were measured at day 7.

[Results] [1] Analysis of TCGA datasets revealed that NDRG1 expression was positively correlated with VEGF-A expression in patients with various cancer types. [2] In breast cancer patients in Kurume University hospital, NDRG1 was expressed in both cancer cells and ECs. In NDRG1 deficient mice, we observed following experimental results. [3] Both tumor growth and angiogenesis were all suppressed in syngeneic tumors. [4] VEGF-A-induced angiogenesis was specifically impaired in corneal micropocket assay and aortic ring assay, whereas FGF-2 could induce angiogenesis in both assays. [5] NDRG1 formed a complex with PLC-γ, and this complex formation was requisite for the VEGF-A-induced PLCγ/ERK activation in ECs.

[Conclusion] We first present an indispensable role of NDRG1 in VEGF-A-induced angiogenesis through PLCγ/ERK activation in ECs. The NDRG1 could be a novel candidate target for development of therapeutics for VEGF-A-induced tumor angiogenesis and other vascular diseases.

#184

**The role of matrix metalloproteinases (MMPs) and** CTNNB1 **mutations in endometrial carcinoma.**

Amnon A. Berger,1 Douglas A. Levine,1 Emily Kawaler2. 1 _NYU Langone Health, New York, NY;_ 2 _New York University, New York, NY_.

Introduction: Advanced endometrial carcinoma (EC) carries a poor prognosis, especially for tumors with CTNNB1 somatic mutations. GOG-86P recently demonstrated that treatment of CTNNB1-mutated tumors with the anti-VEGFA antibody bevacizumab (bev) resulted in a better outcome, however, the mechanism for this response is unknown. We tested the hypothesis that overexpression of MMP proteins increases angiogenesis through VEGFA downstream signaling leading to improved clinical response in CTNNB1-mutated tumors.

Methods: Data from the endometrial TCGA and CPTAC projects were used to examine the relationship between exon 3 CTNNB1 mutations and VEGFA expression. We used 3 EC cell lines with wildtype (WT) CTNNB1, Ishikawa, HEC59 and JHUEM7, and a single EC cell line with a mutated CTNNB1, HEC108. We compared our results to a colorectal cancer (CRC) model by using the WT cell line RKO and mutated cell line HCT116. We measured VEGFA, CTNNB1, MMP2, MMP7, MMP9 RNA and protein levels in these cell lines using RT-PCR, western blots and ELISA. We transiently and stably overexpressed CTNNB1 in WT CRC and EC cell lines and downregulated CTNNB1 using shRNA transduction in mutated cell lines.

Results: An analysis of TCGA microsatellite stable, TP53-wildtype, endometrioid EC showed a 1.4-fold increase in VEGFA gene expression in tumors with CTNNB1 mutations compared to those without (P=0.01). CPTAC endometrial cancer proteomic data did not identify greater VEGFA protein expression in CTNNB1-mutated tumors. An analysis of known β-catenin targets in TCGA and CPTAC revealed significantly increased MMP7 and MMP9 gene and protein expression in CTNNB1-mutated tumors. Transient overexpression of CTNNB1 in WT EC and CRC cell lines increased VEGFA gene expression of 2-3 fold. Stable overexpression of physiologic levels of mutated CTNNB1 in WT EC and CRC lines resulted in significantly increased VEGFA and MMP7 gene expression and increased levels of secreted MMP7. Knockdown of CTNNB1 induced a significant decrease in VEGFA gene expression in HCT116, but not HEC108. MMP7 gene expression was paradoxically increased in CTNNB1-knockdown HCT116, but decreased in CTNNB1-knockdown HEC108.

Conclusions: We have shown in silico and in vitro that CTNNB1 exon 3 mutations are associated with increased VEGFA and MMP7 expression. Knockdown experiments in mutated tumors in EC and CRC models suggest that control of angiogenesis and mechanism of action for bev may vary between tumor types supporting the role of CTNNB1 mutations as a biomarker in EC. Ongoing experiments will study the role of MMP7 in CTNNB1-mutated tumors. These findings are being translated into biomarker stratified clinical trials and individualized treatment for women with advanced EC.

#185

Direct cytotoxic effect of a novel anti-angiogenic drug F16 towards U87MG glioblastoma cell line.

Mohammad Algahtani,1 Khalid Alhazzani,2 Thiagarajan Venkatesan,3 Ali Alaseem,4 Sivanesan Dhandayuthapani,3 Appu Rathinavelu1. 1 _College of Pharmacy, Nova Southeastern University, Fort Lauderdale, FL;_ 2 _College of Pharmacy, King Saud University, Riyadh, Saudi Arabia;_ 3 _Rumbaugh-Goodwin Institute for Cancer Research, Nova Southeastern University, Fort Lauderdale, FL;_ 4 _College of Medicine, Al Imam Mohammad Ibn Saud Islamic University, Riyadh, Saudi Arabia_.

Glioblastoma multiforme (GBM) is one of the most aggressive and lethal types of brain cancers with low 5-year survival rate. Therefore, search for better drugs and effective treatment strategies for GBM to improve patient outcomes is continuing. Since GBM is one of the most highly vascularized solid tumors and its growth is angiogenesis-dependent, antagonizing tumor angiogenesis by using angiogenesis inhibitors seems to be one of the promising approaches. In this context, intensive pre-clinical evaluation of our patented anti-angiogenic compound, code-named F16, has exhibited potent anti-angiogenic and anti-tumor activities via selectively antagonizing VEGFR-2 in both in vitro and in vivo models. More importantly, assessment of biochemical parameters that are reflecting the safety of F16 in the in vivo system showed no alterations in the toxicological parameters of heart, liver, kidney, and pancreas, after 30 days of treatment, at the dose of 100 mg/kg body weight. Hence, we tested the direct effects of F16 for inhibiting the growth, angiogenesis and the migratory abilities of the U87MG glioblastoma cells, which are known to express high levels of VEGFR. Our in vitro studies have confirmed potent inhibitory effects of F16 towards the migration and invasion of U87MG cells, and also revealed potent cytotoxic effects (IC50 26 µM) against U87MG cells in comparison to Temozolomide (IC50 430 µM) treatment. In addition, F16 inhibited the phosphorylation of VEGFR-2 through competitive binding and also induced cell cycle arrest and apoptosis by activating p53 pathway in U87MG cells. Furthermore, our in vivo results with ectopically implanted xenograft model confirm the fact that F16 can significantly inhibit tumor growth in the mice implanted with U87MG glioblastoma cell line. Based on our preliminary results, we are proposing that F16 could be a potential candidate for treating GBM, either in monotherapy or in combination with a cytoreductive drug. (This research was supported by the generous funds provided by the Community Foundation of Broward, Florida and also by the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida)

#186

Androgen deprivation promotes neuroendocrine differentiation and angiogenesis through CREB-EZH2-TSP1 pathway in prostate cancers.

Yan Zhang,1 Dayong Zheng,1 Ting Zhou,1 Mohit Hulsurkar,1 Michael Ittmann,2 Long Shao,2 Martin Gleave,3 Wenliang Li1. 1 _University of Texas Health Science Center at Houston, Houston, TX;_ 2 _Baylor College of Medicine, Houston, TX;_ 3 _University of British Columbia, Vancouver, British Columbia, Canada_.

The incidence of aggressive neuroendocrine prostate cancers (NEPC) related to androgen-deprivation therapy (ADT) is rising. NEPC is still poorly understood, such as its neuroendocrine differentiation (NED) and angiogenic phenotypes. Here we reveal that NED and angiogenesis are molecularly connected through EZH2 (enhancer of zeste homolog 2). NED and angiogenesis are both regulated by ADT-activated CREB (cAMP response element-binding protein) that in turn enhances EZH2 activity. We also uncover anti-angiogenic factor TSP1 (thrombospondin-1, THBS1) as a direct target of EZH2 epigenetic repression. TSP1 is downregulated in advanced prostate cancer patient samples and negatively correlates with NE markers and EZH2. Furthermore, castration activates the CREB/EZH2 axis, concordantly affecting TSP1, angiogenesis and NE phenotypes in tumor xenografts. Notably, repressing CREB inhibits the CREB/EZH2 axis, tumor growth, NED and angiogenesis in vivo. Taken together, we elucidate a new critical pathway, consisting of CREB/EZH2/TSP1, underlying ADT-enhanced NED and angiogenesis during prostate cancer progression.

#187

Trastuzumab induced myeloid cell alterations identifies mechanistic properties of vascular normalization.

Meghan J. Bloom, Angela M. Jarrett, Todd A. Triplett, Anum K. Syed, Thomas E. Yankeelov, Anna G. Sorace. _University of Texas, Austin, TX_.

Introduction: The purpose of this study is to quantify longitudinal changes in myeloid cell infiltration after trastuzumab treatment to reveal mechanistic properties of improved tumor vascularization in human epidermal growth factor receptor type 2 positive (HER2+) breast cancer. We have previously presented in vivo data showing that trastuzumab increases vascular maturation leading to better delivery of therapeutics. The balance of myeloid cells in the tumor microenvironment (TME) can impact neovascularization. Specifically, M2 macrophages (MACs) promote angiogenesis causing irregular vascularization and M1 MACs exhibit anti-angiogenic properties counteracting the M2 cells and improving tumor perfusion. We present results revealing macrophage polarization towards an M1 phenotype after trastuzumab therapy in HER2+ tumors.

Experimental Design: 107 BT474 breast cancer cells were implanted subcutaneously into athymic nude mice. After tumors reached 250 mm3, mice were treated with trastuzumab (10 mg/kg) or saline over one week and were excised for analysis on days 0, 4 and 7. Half of each tumor was fixed in 10% neutral buffered formalin for immunohistochemical staining (IHC) of the TME and half was digested for analysis of myeloid cell infiltration with flow cytometry. The flow cytometry panel used consists of ten markers, two specifically for the identification of M1 and M2 MACs; CD38, and CD206, respectively. A non-parametric Wilcoxon rank sum test was used to determine statistical differences between control and treated groups.

Results and Discussion: Initial studies revealed an increase (P = 0.03) of MACs in day 4 and day 7 treated tumors. Of these MACs, there was an increase (P = 0.03) in co-expression of M2 and M1 phenotype markers CD206 and CD38 in day 4 treated tumors indicating a phenotypic switch occurring. After a third dose of trastuzumab, there was an increase (P = 0.03) in M1 MACs (CD38+/CD206-) in day 7 treated tumors. In day 4 and 7 treated tumors, there was a decrease (P = 0.02) in non-differentiated MACs (CD38-/CD206-) supporting evidence that a phenotypic switch is occurring. Ongoing studies are evaluating spatial variations in MAC infiltration (CD206, F4/80), hypoxia (pimonidazole), and vascular maturation index (ratio of alpha-smooth muscle actin to total vessel counts as stained with CD31) through IHC. Cytokine detection assays are being done to evaluate changes in cytokines contributing to macrophage polarization and angiogenesis.

Conclusion: These results offer strong evidence supporting the hypothesis that MACs in the TME are switching to an M1 phenotype after trastuzumab treatment and contributing to trastuzumab induced vascular alterations. Analyses of IHC will validate vascular changes occurring simultaneously with myeloid infiltratration.

#188

The oncometabolite d-2-hydroxyglutarate induces angiogenic activity through the vascular endothelial growth factor receptor 2 signaling pathway.

You Mie Lee, Jiyoon Seok, Soo-Hyun Yoon, Sun-Hee Lee, Jong Hwa Jung. _Kyungpook National Univ., Daegu, Republic of Korea_.

Mutation of isocitrate dehydrogenase (IDH) 1 (R132H) and IDH2 (R172K) and induction of hypoxia in various solid tumors results in changes to metabolic profiles, including production of the d- or l-forms of 2-hydroxyglutarate (2HG) from α-ketoglutarate in aerobic metabolism in the TCA cycle. However, it is unclear whether the oncometabolite d-2HG increases angiogenesis in endothelial cells. Therefore, in this study, we analyzed the levels of various metabolites, including d-2HG, under hypoxic conditions and in IDH2R172K mutant breast cancer cells by mass spectrometry. We then further evaluated the effects of this metabolite on angiogenesis in breast cancer cells. Our results showed that treatment with d-2HG increased the levels of secreted vascular endothelial growth factor (VEGF) in cancer cells and enhanced endothelial cell proliferation in a concentration-dependent manner. Wound healing and transwell migration were significantly increased by d-2HG to a level similar to that induced by VEGF. Tube formation was significantly stimulated by d-2HG, and chick chorioallantoic membrane angiogenesis was also enhanced by d-2HG. d-2HG activated VEGF receptor (VEGFR) 2 and VEGFR2 downstream signaling, extracellular signal-regulated kinase 1/2, focal adhesion kinase, AKT, and matrix metalloproteinase (MMP) 2. Taken together, our results suggested that d-2HG induced angiogenic activity via VEGFR2 signaling and increased MMP2 activity.

#189

EGFR TKI resistance via role VEGFR2.

Chike Osude, Leo Lin, Neelu Puri. _University of Illinois College of Medicine at Rockford, Rockford, IL_.

The treatment paradigm for late stage non-small cell lung carcinoma (NSCLC) has shifted towards molecular targeted therapy like epidermal growth factor receptor (EGFR). Tyrosine kinase inhibitors (TKIs) against EGFR have proven to be effective; however, TKI resistance is commonly acquired by the T790M mutation in the EGFR kinase domain. Very few studies suggest the role of vascular endothelial growth factor receptor (VEGFR) in mediating erlotinib resistance. We suggest that overexpression of VEGFR-2 in tumors creates an autocrine loop that mediates TKI resistance. Thus, our purpose is to investigate the VEGF/VEGFR autocrine loop as a potential mechanism of TKI resistance via alternative signaling.

H2170, H358, H1975 and H3255 NSCLC parental cells were obtained from ATCC. Parental cells were then treated with erlotinib to create resistant cells. DNA sequencing was used to exclude the presence of the T790M mutation. Quantitative real-time PCR (qPCR) was conducted to code for VEGF, VEGFR-2, and neuropilin-1 (NP-1) genes and immunoblotting (WB) was used to detect their protein expression. Fluorescence-activated cell sorting (FACS) analysis and immunofluorescence (IF) analysis was performed with antibodies against VEGFR-2 and NP-1. Secretion of VEGF from NSCLC cells was studied at 24/48 hours by ELISA. Cell proliferation assay (MTT) was conducted to assess the viability of resistant cells after treatment with VEGFR-2 inhibitor and VEGF neutralizing antibody in media with/without erlotinib. 48 and 31 tumor biopsies were immunostained (IHC) with anti-VEGFR-2 and anti-NP-1 antibodies, respectively.

qPCR showed a 1.4 to 3.5-fold increase in expression of VEGF, VEGFR-2 and NP-1 in H2170 resistant cells and a 2.2 to 3.6-fold increase in expression in H358 resistant cells in comparison to their parental cells. WB results showed an increase in protein expression in resistant cells compared to parental cells. FACS analysis showed a 1.3 to 6-fold increase in expression of VEGFR and NP-1 in resistant cells which was confirmed by IF, showing higher expression of both VEGFR-2 and NP-1 in the resistant cells. ELISA studies showed that VEGF secretion increases over time. MTT assays showed that the VEGFR-2 inhibitor (ZM HCl 323-881) lowered cell death to 10-57% (p=0.05) with erlotinib in resistant cells compared to the erlotinib alone (72-75%). Kaplan-Meier analysis revealed shorter median survival with high vs low expression of total VEGFR-2 expression (14mo vs 21mo, p=0.05) in patients NSCLC tumors.

Our results suggest that by upregulating the VEGF/VEGFR autocrine system as an alternative signaling pathway to promote proliferation, tumor cells may bypass the inhibitory effect of TKIs on the EGFR pathway, ultimately overcoming resistance. Furthermore, IHC demonstrated that high expression of VEGFR-2 is associated with poor prognosis in tumors, suggesting its potential as a prognostic biomarker for lung cancer.

#190

Lenvatinib induced cell death of cultured human hepatocellular carcinoma (HCC) under nutrient-starved condition through inhibition of FGFR signaling pathway.

Taisuke Hoshi,1 Saori Watanabe Miyano,1 Regina Mikie Kanada Sonobe,1 Hideki Watanabe,1 Yuki Seki,1 Etsuko Ohta,1 Kenichi Nomoto,2 Junji Matsui,2 Yasuhiro Funahashi1. 1 _Eisai Co., Ltd., Tsukuba-shi, Ibaraki, Japan;_ 2 _Eisai Inc., Woodcliff Lake, NJ_.

Introduction: HCC is a one of the leading cause of cancer death worldwide. However systemic therapeutic option for unrespectable HCC was limited. Lenvatinib (LEN) is a multi-targeted tyrosine kinase inhibitor that mainly inhibits VEGFR1-3 and FGFR1-4. In a recent phase 3 clinical trial in unresectable HCC (REFLECT study), LEN showed statistical non-inferiority of OS compared to sorafenib (SOR) and clinically meaningful outcome in ORR, PFS, and TTP. We previously showed LEN decreased in vitro proliferation of HCC cells with activated FGF signaling pathways, and inhibited tumor FGF signal and angiogenesis. However, it is largely still unknown that the contribution of FGFR inhibition on tumor cells to the antitumor activity of LEN in addition to antiangiogenic activity. In this study, we investigated the role of tumor FGF signaling pathway in human HCC cells under nutrient-poor condition to mimic tumor environment induced by anti-angiogenesis.

Methods: LEN, SOR or the corresponding vehicle were given orally to mice bearing Hep3B2.1-7 xenograft tumors for 7 days. The tumor sections were stained with H&E, and analyzed histologically. To evaluate the roles of FGF signaling pathway in cell deaths of HCC in vitro, HCC cells were treated with LEN, FGFR1-3 specific inhibitors E7090, or MEK inhibitor PD0325901 under nutrient-starved condition, and cell death induction was assessed. The phosphorylation status of downstream molecules of FGFRs and PARP cleavage were evaluated by Western blot analysis.

Results: LEN (10 and 30 mg/kg) and SOR (30mg/kg) significantly increased focal necrotic area compared to vehicle treated Hep3B2.1-7 xenograft tumors by histological analysis. LEN increased focal necrotic area in a dose dependent manner. 10 mg/kg LEN increased focal necrotic area more than 30 mg/kg SOR, although anti-angiogenic activity was not different. Under nutrient-starved condition in vitro, LEN and E7090 significantly increased death of Hep3B2.1-7 and HuH-7 cells, harboring FGF19 overexpression, but not SOR, which does not inhibit FGFR. LEN and E7090 clearly decreased the phosphorylation of downstream molecules (FRS2 and Erk) in HCC cell lines, and then LEN and E7090 also increased PARP cleavage under nutrient-starved condition. MEK inhibitor PD0325901 induced cell death and PARP cleavage in Hep3B2.1-7 and HuH-7 under nutrient-starved condition.

Conclusion: FGF signaling pathway plays a key role in survival of HCC cells under nutrient-starved condition, and inhibition of FGF signaling pathway by LEN or E7090 induced HCC cell death through FGFR-MAPK-PARP cleavage. The dual inhibition of VEGFR and FGFR by LEN may provide enhanced antitumor activity against HCC with activated FGF signaling pathways by induction of death of HCC cells, which escape from angiogenesis inhibition.

#191

Extracellular signaling linked to PI3K/AKT triggers the formation of vasculogenic mimcry in ovarian cancer.

Andres Valdivia,1 Gabriel Mingo,1 Varina Aldana,1 Alejandra Sandoval,1 Alejandro Corvalan,1 Francisco Nualart,2 Gareth I. Owen1. 1 _Pontificia Universidad Catolica de Chile, Santiago, Chile;_ 2 _Universidad de Concepcion, Santiago, Chile_.

Introduction: The presence of an endothelial cell-free perfusion pathway, known as Vasculogenic Mimicry (VM), strongly correlates with reduced patient survival, however the mechanisms by which a tumor can create a self-generated irrigation system remain unknown. Currently there is no mechanism of VM formation reported in fluid conducting tubular structures. Herein, using a standardized in vitro model, we sought to understand the pathways required for VM formation.

Methods: We utilized confocal microscopy and Imaris reconstruction to demonstrate that cancer cells can create an internal tubular network in cultured cancer cell lines, cancer spheres and primary cancer cultures of ovarian cancer. Matrigel and 3D matrices containing specific protein components allowed the deciphering of the extracellular matrix proteins involved in VM formation. Intercellular pathways were resolved using chemical inhibitors.

Results: VM only occurs when cells are grown in 3D culture and only 30% of primary cancer cultures undergo this process. The presence of laminin 1 is sufficient to trigger the formation of tubular structures and this may involve the alpha2beta1 integrin and metalloproteases. Downstream FAK and PI3K/AKT intercellular pathways are required to form functional tubular networks. Microarray data elucidated the presence of the mRNA transcripts that were increased in cells that undergo VM.

Discussion: We demonstrate that Laminin 1, a component of the extracellular matrix (ECM), can promote the process of VM, which further involves the downstream signaling of the potentially druggable targets of the PI3K pathway. As VM is associated with reduced patient survival, elucidation of the mechanisms of formation may deliver new cancer therapies.

Funding: FONDECYT 1180241, CONICYT FONDAP 15130011, IMII P09/016F

#192

The extracellular vesicles from lung cancer cells activates Stat3 and tumor angiogenesis.

Chien-Chung Lin,1 Yu-Ting Huang,2 Chun-Hua Hung,3 Mei-Ling Tsai,2 Wu-Chou Su3. 1 _National Cheng Kung University, College of Medicine, Tainan, Taiwan;_ 2 _National Kaohsiung University of Science and Technology, Kaohsiung, Taiwan;_ 3 _National Cheng Kung University, Tainan, Taiwan_.

Current research suggests that on tumor-related extracellular vesicles (EV) play a significant role in paracrine signaling pathways, thus potentially influencing cancer progression via multiple mechanisms including angiogenesis. Our previous study demonstrated that IL6 induced secretion of VEGF and contributing angiogenesis via activation of Stat3. To further clarify the biological activities of the lung cancer derived EVs, we analyzed the content of IL6 and VEGF in exosome using ELISA kit. We found that IL-6 and VEGF were more enriched in EVs than non-EVs in AS2 and CL1-5 cells. And the isolated EV from AS2 and CL1-5 cells increased vascular permeability in miles assay and promotes angiogenesis in tubular formation assay. We further investigated if the inhibition of exosome secretion affects the autocrine IL6/Stat3/VEGF pathway. GW4869 inhibits secretion of exosome by playing an inhibitor of neutral sphingomyelinase (nSMase). Treatment of either cell with GW4869 significantly decreased the levels of released exosomes in AS2 but not in CL1-5 cell. Consistent with the result, the treatment GW4869 dampened autocrine IL6/Stat3/VEGF pathway in AS2 but not in CL1-5 cell. Bevacizumab, the recombinant anti- VEGF monoclonal antibody, were approved for treating patients with advanced NSCLC, has been shown to be efficient in suppressing the accumulation of pleural fluid (malignant pleural effusion). To investigate if the exosomal VEGF has a different affinity for bevacizumab, we treated AS2, CL1-5 and MPE-derived cancer cells with bevacizumab and EV or non-EV part from cell-free medium. We found VEGF dimer form were more abundant in EV part and cannot be neutralized by bevacizumab. Conversely, VEGF mono-form was more abundant non-EV part and can be neutralized by bevacizumab. In animal model, AS2 was injected intraperitoneal and then malignant ascites developed as follow. We found the administration of bevacizumab after AS2 injection i.p. inhibited peritoneal seeding and ascites formation compared to control group. But the inhibition was reversed when we injected EV as well as bevacizumab in combination group. In breast cancer cell, microvesicles (MVs) activates VEGF receptors through a unique 90 kDa form of VEGF (VEGF90K) which has a weakened affinity for Bevacizumab, causing Bevacizumab to be ineffective in blocking MV-dependent VEGF receptor. And an Hsp90 inhibitor (17-AAG) was demonstrated to release VEGF90K from MVs without interaction with VEGF restoring the sensitivity of VEGF90K to Bevacizumab. However, our study found 17-AAG alone inhibited VEGF secretion in EV as well as the activity of upstream Stat3, and Akt pathway in lung cancer. Our preliminary data implied EV-VEGF and the existence of different isoform of VEGF in EV contribute to tumor angiogenesis and limit the effectiveness of bevacizumab therapy which is not dependent on Hsp90.

#193

**Antiangiogenic effect of conditioned media derived from triple negative breast cancer cells in brain microvascular endothelial cells** in vitro **.**

Nehad M. Ayoub, Ahmed Alhusban, Laila Alhusban. _Jordan Univ. of Science & Technology, Irbid, Jordan_.

Breast cancer is the most common malignancy among females and is a leading cause of cancer mortality. Brain metastasis is associated with poor prognosis and reduced survival among breast cancer patients. For brain metastasis to occur, cancer cells are required to remain in direct contact with endothelial cells that line brain blood vessels. Data regarding the interaction between brain endothelial cells and breast cancer is limited. The aim of this work was to understand the interaction between breast cancer cells and brain endothelial cells. Human brain microvascular endothelial cells (hBMECs) were treated with conditioned media from MDA-MB-231 cell line and their angiogenic characteristics were evaluated. The involvement of the glycogen synthase kinase-3β (GSK-3β), vascular endothelial growth factor (VEGF), brain derived neurotrophic factor (BDNF), and thrombospondin-1 (TSP-1) was evaluated using release levels and inhibitor-based approaches. Levels of secreted proteins were assayed using ELISA. Results of the study showed that conditioned media from MDA-MB-231 breast cancer cells reduced angiogenic potential of hBMECs. Conditioned media significantly suppressed migration and tube formation of hBMECs compared to control group. In these experiments, control group represents hBMECs which were incubated with regular serum-free RPMI-1640 culture media. Inhibiting GSK-3β in conditioned media-treated hBMECs by its selective inhibitor (SB 216763; 10nM), restored the migratory potential of endothelial cells. In addition, MDA-MB-231 cells released VEGF at a rate that exceeded VEGF release from endothelial cells (6342 vs. 67.6 pg/ml). The release of VEGF from conditioned media-treated hBMECs was not altered by the high levels of VEGF presented in MDA-MB-231 cells conditioned media. GSK-3β inhibition in hBMECs significantly increased VEGF release from endothelial cells by about 1000 pg/ml compared to conditioned media-treated endothelial cells. MDA-MB-231 cells also released high levels of BDNF into the media and treating hBMECs with conditioned media from MDA-MB-231 cells did not alter the endogenous release of total BDNF from endothelial cells. GSK-3β inhibition did not alter BDNF release from endothelial cells. BDNF inhibition using TrkB-Fc (4µg/ml) did not restore the migratory potential of endothelial cells and the anti-angiogenic effect of conditioned media was maintained despite the inhibition of BDNF. Breast cancer cells released high levels of TSP-1 into the media. hBMECs treated with TSP-1 (5µg/ml) reduced wound healing by 40%, an effect that was restored by GSK-3β inhibitor. In conclusion, conditioned media from breast cancer cells induced anti-angiogenic effects in hBMECs which was found to be GSK-3β-dependent. The anti-angiogenic effect was further illustrated in terms of increased TPS-1 levels released by breast cancer cells.

#194

Overcoming adaptive resistance to anti-VEGF therapy by targeting CD5L.

Christopher J. LaFargue,1 Paola Amero,1 Kyunghee Noh,1 Selanare Mangala,1 Chunhua Lu,1 Minsoon Cho,1 Sunila Pradeep,1 Yihong Wan,2 Wei Hu,1 Rajesha Rupaimoole,1 Robert L. Coleman,1 Ningyan Zhang,3 Zhiqiang An,3 Gabriel Lopez-Bernstein,1 Vittorio de Franciscis,4 Anil K. Sood1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _The University of Texas Southwestern Medical Center, Dallas, TX;_ 3 _The University of Texas Health Science Center, Houston, TX;_ 4 _Istituto di Endocrinologia ed Oncologia Sperimentale, Naples, Italy_.

Purpose: To identify mechanisms responsible for the development of adaptive resistance to anti-vascular endothelial growth factor (VEGF) therapy, and to develop novel agents to reverse its occurrence.

Methods: Gene expression profiling was performed on endothelial cells isolated from tumors sensitive or resistant to anti-VEGF therapy in a syngeneic ovarian cancer mouse model. A series of in vitro (lentiviral transfection, tube formation, siRNA knockdown, luciferase promoter assays) and in vivo (including Tie2; PPARγ KO mice) experiments were then used to elucidate the upstream and downstream effectors of CD5L, as well as the efficacy of CD5L inhibition in pre-clinical orthotopic mouse models.

Results: We identified substantially increased CD5L expression in endothelial cells isolated from anti-VEGF therapy resistant vs. sensitive tumors. Ectopic expression of CD5L in RF24 endothelial cells resulted in increased tube formation, cell migration, and cell proliferation, whereas siCD5L treatment resulted in an overall decrease. Observing that a PPARγ binding site was present in the CD5L promoter, we found that ectopic expression of PPARγ into RF24 cells results in increased CD5L expression, whereas siPPARγ treatment decreased it. Ectopic PPARγ expression increased luciferase activity but decreased activity with siPPARγ treatment when using a reporter assay for CD5L promoter. Conversely, mutation of the PPARγ binding site in the promoter construct lacked any increase in activity after ectopic PPARγ expression. IPA analysis identified HIF1α as playing an integral role in the PPARγ /CD5L activation pathway. Indeed, hypoxic conditions resulted in an increase of both PPARγ and CD5L in RF24 cells. HIF1α stabilizers (cobalt chloride) and inhibitors (YC-1 and topotecan) also showed an increase and decrease in PPARγ and CD5L expression, respectively. RPPA analysis of anti-VEGF resistant tumor endothelial cells implicated an upregulation of the PI3K pathway. While exogenous CD5L treatment led to increased p-AKT, the addition of the PI3K inhibitor LY294002 negated this effect and also decreased tube formation and cell migration. Next, we injected both WT B6 and Tie2:PPARγ KO mice with the ID8 mouse ovarian cancer cell line and treated with B20 until moribund. Mice harboring the PPARγ KO had significantly increased survival as well as decreased tumor weight, number of nodules, Ki67 expression, and blood vessel density. Our non-syngeneic model was then treated with B20 and either CD5L RNA aptamer or vehicle. The aptamer group treated with CD5L aptamer displayed significantly decreased tumor weight, number of nodules, Ki67 expression, and blood vessel density.

Conclusions: This study identifies a pathway centered on CD5L as an important driver of adaptive resistance to anti-angiogenic therapy.

#195

SPARCL1 is an angiocrine inhibitor of tumorigenesis in colorectal carcinoma.

Clara Tenkerian,1 Daniela Regensburger,1 Victoria Langer,1 Anika Klingler,1 Anne Borau,1 Heinrich Sticht,1 Andreas Ramming,1 Thomas Wohlfahrt,1 Benjamin Schmid,1 Valérie Méniel,2 Robert Grützmann,1 Nathalie Britzen-Laurent,1 Vera Schellerer,1 Michael Stürzl,1 Elisabeth Naschberger1. 1 _University Hospital Erlangen, Erlangen, Germany;_ 2 _European Cancer Stem Cell Research Institute, Cardiff, United Kingdom_.

Colorectal carcinoma (CRC) is the second leading cause of cancer-related mortality worldwide. The contribution of the tumor microenvironment (TME) to CRC pathogenesis is well established, whereby the dominance of one immune response (Th1) over another (Th17) can yield opposite effects on patient prognosis. We recently reported on TME-dependent vascular plasticity in CRC and identified the secreted protein, acidic and rich in cysteine-like 1 (SPARCL1) as a marker thereof. SPARCL1 is a matricellular protein expressed and secreted exclusively by the vascular system (endothelial and mural cells). In previous studies, gene expression analyses in CRC tumor tissues of different patient cohorts congruently showed a significant loss of SPARCL1 in colon and rectum adenocarcinomas as compared to uninvolved colon tissues and indicated that this loss is TME-dependent. Specifically, high SPARCL1 expression was associated with a Th1-TME, reduced incidence of distant metastasis and increased cancer-related survival. In contrast, SPARCL1 expression was lost in aggressive CRC with a non-Th1-TME. Here we show in functional analyses that SPARCL1 inhibits angiogenesis in different in vitro (endothelial cell proliferation, capillary formation on matrigel, 3D spheroid sprouting), ex vivo (fetal mouse metatarsal explant) and in vivo (FITC-dextran vascular permeability) assays. Moreover, increased tumor cell colonization is detected in the lungs of SPARCL1-knockout mice, further consolidating the anti-tumorigenic function of SPARCL1. To characterize the molecular mechanism of SPARCL1 anti-angiogenic function, we identified the TGF-β co-receptor Endoglin as a cellular receptor for SPARCL1 and found that SPARCL1 regulates ERK1/2 activity. The RAS-ERK pathway is amplified in more than half of all CRC tumors and as such is considered an important therapeutic target. Interestingly, SPARCL1 regulates ERK1/2 not only through phosphorylation but also through its subcellular localization. We show that SPARCL1 induces ERK1/2 phosphorylation preferentially in the cytoplasm and activates specifically cytoplasmic substrates downstream of it. Finally, we show that the regulation of ERK1/2 by SPARCL1 is not limited to endothelial cells but extends to epithelial CRC cells, indicative of autocrine and paracrine functions of SPARCL1. In summary, our study indicates that SPARCL1 is a TME-dependent angiocrine tumor suppressor in CRC acting through the regulation of ERK1/2 activity. This finding opens novel perspectives elucidating the crosstalk between the TME and the ERK signaling pathway and accordingly may enable new strategies to overcoming drug resistance and/or therapy-oriented patient stratification.

#196

Antitumor activity of lenvatinib against acquired resistance tumor to anti-VEGF therapy in mouse syngeneic tumor models.

Kenji Ichikawa, Saori Watanabe Miyano, Yukinori Minoshima, Junji Matsui, Yasuhiro Funahashi. _Eisai Co., Ltd., Tsukuba Research Laboratories, Tsukuba-shi, Ibaraki, Japan_.

Anti-vascular endothelial growth factor (VEGF) therapies have been in clinical use to treat patients with multiple types of cancers, but benefits of anti-VEGF therapy are still limited because of the acquired resistance to the treatment. In this study, we developed a novel resistance model to anti-VEGF therapy by expressing VEGFR2-Fc in mouse Renca and B16F10 tumor models. We investigated activated signaling pathways in established resistant tumors by differentially expressed gene analysis and using pathway analysis using Reactome based on RNA-Seq data. Differentially expressed gene analysis identified 293 genes, which are altered commonly in both resistant tumors, and pathway analysis showed that those genes were enriched in FGF2-FGFR2 signaling pathway. IHC analysis by staining with anti-CD31 and αSMA antibodies demonstrated that tumor vessels in VEGFR2-Fc expressing resistant tumors were covered with pericytes, and further IHC analysis with anti-FGF2 antibody indicated that FGF2 was stained in pericytes, which covered endothelial cells in VEGFR2-Fc expressing tumor. Moreover, we isolated CD31 positive cells as endothelial cells, CD90.2 positive cells as pericytes, and CD45 positive cells as tumor infiltrated lymphocytes, respectively. Expression level of Fgf2 and Fgfr2 mRNA in these subsets from Mock tumors and VEGFR2-Fc expressing tumors were analyzed. As a result, Fgf2 and Fgfr2 mRNA expressions in VEGFR2-Fc expressing tumors were upregulated in pericytes and endothelial cells, respectively compared with Mock tumors. These results suggested that there is a spatially effective activation of FGF signaling pathway between endothelial cells and pericytes in tumor vessels that are resistant to anti-VEGF therapy. Indeed, by expression of FGFR2-Fc, in vivo tumor growth of anti-VEGF therapy-resistant tumors was suppressed as well as angiogenesis. Lenvatinib mesilate (lenvatinib) is an oral multiple receptor tyrosine kinase (RTK) inhibitor that suppresses the kinase activities of VEGF receptors (VEGFR1-3), in addition to other proangiogenic and oncogenic pathway-related RTKs including fibroblast growth factor receptors (FGFR1-4), the platelet-derived growth factor receptor (PDGFR) α, KIT, and RET. Lenvatinib suppressed phosphorylation of FGFR2 in VEGFR2-Fc expressing tumors, and showed antitumor activity accompanied with further decrease of tumor vessels. But, sorafenib and aflibercept, which are selective inhibitors against VEGF signaling pathway, didn't show antitumor activity against anti-VEGF therapy resistant tumors. In conclusion, our findings revealed key roles of pericytes expressing FGF2 in anti-VEGF therapy acquired resistant tumor models, where lenvatinib showed antitumor activity, indicating an importance of inhibition of FGF signaling pathway to suppress escape from anti-VEGF therapy.

#197

Molecular interactions of Globo H ceramide with translin-associated factor X (TRAX) underlie its proangiogenic activities.

John Yu,1 Sheng-Hung Wang,1 Jing-Yan Cheng,1 Jung-Tung Hung,1 Chun-Cheng Lin,2 Fei-Yun Lo,1 Chien-Wei Lee,1 Alice L. Yu1. 1 _Chang Gung Memorial Hospital, Taoyuan, Taiwan;_ 2 _National Tsing Hua University, Taiwan_.

Glycosphingolipids (GSLs) are ubiquitous glycolipids in eukaryotic plasma membranes that consist of a polar glycan chain and a hydrophobic sphingosine- containing ceramide tail. Although GSLs display various diversities and functions, the mechanistic details of GSL interactions with membranous or intracellular components are poorly understood. Globo H ceramide (GHCer), a tumor-associated GSL overexpressed in most types of epithelial cancers, is a potential target of cancer immunotherapy. Previously, we showed that GHCer is shed by cancer cells to mediate immune suppression and promote angiogenesis in the tumor microenvironment. At the cellular level, GHCer from cancer cells binds to translin-associated factor (TRAX), with consequent release of PLCB1 from TRAX to trigger Ca2+ mobilization. Herein, we found that Gb5-ceramide (Gb5Cer), the pentasaccharide precursor of GHCer, could not promote angiogenesis nor Ca2+ mobilization. At the molecular level, ELISA and biacore analysis revealed that GHCer binds to TRAX with a distinct dissociation constant of 4.09×10-8 M, while its precursors, galactosyl-, lactosyl-, Gb4-, and Gb5Cer- showed negligible binding to TRAX. Molecular docking and molecular dynamics indicated that the fucose residue of Globo H is an important contributor to a favorable glycan conformation for interaction with TRAX, consistent with the finding that Gb5Cer had no proangiogenic activities. In addition, GHCer competed with recombinant C-terminus region of phospholipase C β1 (PLCB1) for binding to TRAX, thus providing a molecular mechanism for the proangiogenic effect of GHCer on tumor microenvironment. This study provides the first evidence for interaction of TRAX with a non-protein GSL, deciphered distinct regions of TRAX for interacting with glycan and lipid portions of GHCer, and illustrated the crucial contribution of fucose moiety on GHCer for its interaction with TRAX and angiogenic activity.

#198

The T3R domain of thrombospondin-1 delays tumor growth and improves tumor response to chemotherapy by remodeling the tumor vasculature.

Denise Pinessi,1 Andrea Resovi,1 Lavinia Morosi,2 Patrizia Borsotti,1 Alexander Berndt,3 Raffaella Giavazzi,2 Massimo Zucchetti,2 Giulia Taraboletti1. 1 _Mario Negri Institute for Pharmacological Research IRCCS, Bergamo, Italy;_ 2 _Mario Negri Institute for Pharmacological Research IRCCS, Milano, Italy;_ 3 _Institute of Forensic Medicine, Jena University Hospital, Jena, Germany_.

The disorganized and inefficient nature of the tumor vasculature affects the delivery and activity of antineoplastic treatments. Certain antiangiogenic agents induce a transient normalization of tumor vessels, hence representing a promising strategy to improve the delivery and efficacy of cytotoxic therapy. Our previous studies demonstrated the antiangiogenic activity of the type III repeats (T3R) domain of thrombospondin-1 (TSP-1), a major endogenous inhibitor of angiogenesis. Aim of this study was to investigate the role of the T3R domain in tumor growth and tumor response to chemotherapy. Ectopic tumor cell expression of T3R delayed in vivo growth in two tumor models, the human ovarian carcinoma A2780-1A9 and endometrial adenocarcinoma HEC-1B. This was associated with vascular remodeling, decreased cell proliferation and increased apoptosis. T3R was active in preventing tumor growth also when expressed by the host cells (rather than the tumor cells), following pretreatment of mice with adeno-associated virus (rAAV)-T3R before tumor cell implantation. Expression of T3R also resulted in improved tumor response to paclitaxel (PTX 10 mg/kg Q4x3, i.v.) and cisplatin (DDP 4 mg/kg Q4x3, i.v.). Through the pharmacokinetic study, conducted by HPLC and MALDI imaging mass spectrometry analysis, a higher concentration and a more homogeneous drug distribution were observed in tumors expressing T3R 4h after a single administration of PTX (60 mg/kg, i.v.). This was associated with a marked reorganization of the tumor vasculature that presented decreased vessel area and vessel diameter and increased number of CD31-positive vessels. These findings indicate the ability of T3R to reduce tumor growth and improve tumor response to chemotherapy through the remodeling of tumor vasculature. This sets the basis for the development of new therapeutic strategies based on T3R to be used in combination therapies.

Supported by AIRC. DP is recipient of the AIRC fellowship "Lina e Giovanni Giasini"

#199

TGF-β in exosomes facilitates HNSCC progression by accelerating tumor angiogenesis.

Nils Ludwig,1 Saigopalakrishna S. Yerneni,2 Cynthia S. Hinck,3 Monika Pietrowska,4 Andrew P. Hinck,3 Theresa L. Whiteside1. 1 _University of Pittsburgh Cancer Institute, Pittsburgh, PA;_ 2 _Carnegie Mellon University, Pittsburgh, PA;_ 3 _University of Pittsburgh School of Medicine, Pittsburgh, PA;_ 4 _Maria Sklodowska-Curie Institute, Gliwice, Poland_.

TGF-β is a key regulator for tumor initiation and progression in head and neck squamous cell carcinoma (HNSCC). Tumor-derived exosomes (TEX) contain TGF-β and accumulate in the tumor microenvironment (TME). This study characterizes the TGF-β content of HNSCC-derived exosomes and evaluates in vitro and in vivo TGF-β signaling by exosomes that results in promotion of angiogenesis. TEX were isolated from supernantants of 5 different HNSCC cell lines by mini size exclusion chromatography (mini-SEC) and characterized by electron microscopy, nanoparticle tracking analysis and mass spectrometry (LC-MS/MS). TGF-β content in exosomes was evaluated by immunoblotting. Proliferation and migration of SVEC4-10 lymphendothelial cells in response to TEX were investigated in vitro and results were confirmed in vivo, using a matrigel plug model in mice. In these experiments a novel trivalent TGF-β receptor trap (mRER) was used to inhibit TGF-β signaling. TGF-β levels and activity were similarly measured in exosomes isolated from plasma of 20 HNSCC patients. TEX carried high levels of TGF-β and were found to be potent inducers of angiogenesis in vitro and in vivo through functional reprogramming and phenotypic modulation of endothelial cells. Proliferation (p<0.01) and migration (p<0.01) by SVEC4-10 were stimulated by TEX and effects were inhibited by mRER treatment of SVEC4-10 (p<0.05). TEX promoted formation of defined vascular structures in vivo, and increased (p<0.001) vascularization in matrigel plugs relative to control. Those effects were inhibited by mRER treatment (p<0.05). Exosomes in plasma of HNSCC patients carried varying levels of TGF-β, and patients with nodal metastases had elevated TGF-β levels (p<0.01) relative to patients with no meastasis.The data show that TGF-β signaling by TEX in HNSCC promotes angiogenesis and drives tumor progression. Future efforts should focus on silencing TEX, thereby adding new options to existing anti-angiogenic therapies.

#200

The MCP-1/CCR2 axis enhances VEGF-A-dependent angiogenesis by directly downregulating miR-29c expression in oral squamous cell carcinoma.

Ming-Yu Lien,1 Chih-Hsin Tang2. 1 _China Medical University Hospital, Taichung, Taiwan;_ 2 _China Medical University, Taichung, Taiwan_.

Oral squamous cell carcinoma (OSCC), the sixth most common malignancy worldwide, has a poor prognosis due to its aggressiveness and potential for metastasis. Activation of monocyte chemoattractant protein-1 (MCP-1) and its receptor, CC chemokine receptor 2 (CCR2), induces angiogenic activation of endothelial cells, inflammatory responses and aberrant MCP-1/CCR2 expression in malignant tissues. However, the effect of MCP-1 and CCR2 in OSCC angiogenesis is unclear. Our study sought to determine how the MCP-1/CCR2 axis mediates VEGF-A expression in OSCC and results in angiogenesis. Immunohistochemistry (IHC) analysis identified higher concentrations of VEGF-A protein expression in tissue obtained from patients with OSCC compared with tissue from healthy controls; the degree of VEGF-A tumor expression was significantly associated with the clinical disease stage. Our in vitro investigations found that MCP-1 dose-dependently regulated VEGF-A expression through the CCR2, ILK and MEK signal transduction pathways. Following MCP-1 application, conditioned medium from the human OSCC cell line SCC4 significantly promoted endothelial progenitor cell (EPC) migration and tube formation; these events were attenuated and VEGF-A production was suppressed when we stimulated the OSCC cells with the pathway inhibitors RS102895, KP392, and PD98059. We also showed that microRNA (miR)-29c expression was negatively regulated by MCP-1 via the CCR2/ILK/MEK cascade, tying in with an analysis of the cancer genome atlas (TCGA) database showing lower levels of miR-29c expression in tissues from patients with oral cancer compared with samples from healthy controls. According to our results, the MCP-1/CCR2 axis promotes VEGF-A expression in OSCC via the ILK and MEK signal transduction pathways and downregulates miR-29c expression. Thus, MCP-1/CCR2 mediation of VEGF-A mobilizes EPCs required for OSCC and is important for tumor angiogenesis. MCP-1/CCR2 may be an appropriate target in the treatment of OSCC angiogenesis.

#201

The mechanism of vascular mimicry in clear cell renal cell cancer.

Jun-Ping Yang, Xing-Si Peng, Chao-Nan Qian. _Sun Yat-sen University Cancer Center, Guangzhou, China_.

Although anti-angiogenic drugs were used as first-line treatment in metastatic clear cell renal cell cancer (ccRCC), some patients with metastatic ccRCC still had limited survival benefit from anti-angiogenic therapy. The main reason was resistance to anti-angiogenesis treatment. Tumor blood vessel could be divided into endothelial cell-derived or tumor cell-derived vessels. Tumor cell-derived vascular channel was called as tumor vascular mimicry (VM). PAS-CD34 staining in ccRCC tissues revealed that the expression status of VM was different in ccRCC patients. And the progression-free survival and overall survival were significantly shorter in ccRCC patients with high VM expression level. To explore the effect of VM on the resistance to anti-angiogenesis treatment and the molecular mechanism of VM, we successfully established a three-dimensional culture model for the formation of tumor cell-derived vessel-like structures. We found the formation of blood vessel-like structures in kidney cancer cells was not affected by the anti-angiogenic drug, such as Sunitinib, suggesting that VM was closely associated with resistance to anti-angiogenic therapy. Moreover, we had discovered the "molecular signature of vascular mimicry in renal cancer". Using this signature, 531 ccRCC patients in TCGA could be divided into two groups with different prognosis. More importantly, we found that HGF/c-MET signaling pathway regulated the expression of Serpinb2 through the downstream AKT pathway, and thus affected the formation of vascular mimicry in SN12C, 786-O cells. In addition, we also had found that HGF could promote the formation of blood vessel-like structure in lung and bladder cancer cells, and the " molecular signature of vascular mimicry in renal cancer " also could differentiate patients with renal papillary carcinoma, bladder cancer, endometrial cancer, lung adenocarcinoma cancer, and pancreatic cancer. These finding suggested that the molecular mechanism of vascular mimicry in renal cancer was universal, to some extent. This project partially elucidated the molecular mechanism of vascular mimicry in renal cancer and its clinical significance, laying a theoretical foundation for the development of more effective anti-angiogenic drugs.

#202

STAT1 regulates B7H3 mediated angiogenesis in medulloblastoma.

Ian J. Purvis,1 Janardhan Avilala,1 Maheedhara R. Guda,1 Sujatha Venkataraman,2 Rajeev Vibhakar,2 Andrew J. Tsung,1 Kiran K. Velpula,1 Swapna Asuthkar1. 1 _Univ. of Illinois College of Med. at Peoria, Peoria, IL;_ 2 _Univ. of Colorado, Aurora, CO_.

B7-H3 (CD276), an immune checkpoint member of the B7/CD28 family, plays a key role in the repression of the immune response by cancer cells. B7-H3 is overexpressed on tumor and tumor-associated cells, making it an interesting therapeutic target. B7-H3 also appears to play a role outside of immune evasion, contributing to the progression of cancer via invasion/migration, angiogenesis, and metastasis. Our previous data has confirmed the presence of B7-H3 in highly aggressive Myc+ medulloblastoma (MB) tumors. Data-mining studies revealed that B7-H3 is correlated with Myc expression in Group 3 and 4 MBs, and is associated with worse patient outcomes. miR-29, a tumor suppressor, has been shown downregulating the expression of B7-H3 in various cancers. We have shown that miR-29 can reduce the angiogenesis abilities of D283 and D425 cells by downregulating B7-H3 in vitro. Subsequent RNA-sequencing analysis of miR-29-transfected D283 cells revealed upregulation of STAT1 activity. Immunoblot analysis and real-time PCR of D283 and D425 cells demonstrated increased expression of p-STAT1, when transfected with miR-29 plasmid. Treatment with all-trans retinoic acid induced p-STAT1 expression, with a concomitant decrease in the expression of B7-H3 and MMP-9. This preliminary data revealed a novel role for STAT1 in potentially regulating B7-H3, and consequently, angiogenesis and metastasis in MB. To date, the role of STAT1 in MB progression has not been fully investigated, and so our results provide a new avenue for developing potential treatments for aggressive MB tumors.

#203

Pseudosemiglabrin inhibits tumor angiogenesis and tumor growth of human colon cancer in xenograft mouse models by downregulating endothelial functions, Notch and VEGF pathways.

Loiy E. Ahmed Hassan,1 Azman Seeni,2 Amin M. Abdul Majid3. 1 _Omdudman, Khartoum, Sudan;_ 2 _National Institutes of Biotechnology Malaysia, Penang, Malaysia;_ 3 _Universiti Sains, Khartoum, Malaysia_.

Many antineoplasic agents were identified from natural product. One such agent is (-)-pseudosemiglabrin extracted from aerial parts of Tephrosia apollinea, while significant progress has been made in understanding the antineoplasic and anti-inflammatory of prenylated flavone (-)-pseudosemiglabrin (SSG), little is known about its antiangiogenic and antitumor in vivo. Using rat aortic ring assay and zebrafish model, SSG dramatically suppressed angiogenesis in vitro, ex vivo and in vivo models, SSG profoundly inhibited the outgrowth of new vessels from explants rat aortic ring, moreover it impaired human endothelial cells (HUVEC) migration and formation of capillary-like structure in vitro in none toxic doses. In this study we hypothesize that SSG can hampered tumor angiogenesis and tumor growth of colorectal cancer in ectopically and orthotopically implanted in nude mice, oral administration of SSG (25- 100mg/kg) significantly inhibited colon tumors growth in nude mice. Histological and Immunohistochemistry analysis further revealed that the stained blood vessels and expression of CD31 in xenograft were remarkably decreased in tumors excised from animals treated with SSG. Mechanistically the compound inhibits tumor angiogenesis by downregulation of Notch downstream (NCID); also SSG was found to inhibit VEGF secretion from human cancer cells as well as endothelial cells. In conclusion, the results demonstrated an original role of (-)-pseudosemiglabrin in inhibiting tumor angiogenesis and tumor growth via inhibition Notch and VEGF pathways. This study also validates the central role of Notch in tumor angiogenesis and suggests that (-)-pseudosemiglabrin could be a potential therapeutic candidate for cancer and other angiogenesis related diseases.

#204

Integrated phosphoproteomics, transcriptomics, and phenotypic analyses unveil distinct endothelial kinase pathways differentially altered by cancer conditioning.

Or Gadish, Sabrina E. Ibarra, Elazer R. Edelman. _Massachusetts Institute of Technology, Cambridge, MA_.

Extending the successes of cancer cell-targeted kinase inhibitor therapies, we sought to identify endothelial kinases altered in cancer conditioning by combining phenotype assays, phosphoproteomics, and transcriptomics. Human umbilical vein endothelial cells (EC) cultured in conditioned media from MDA-MB-231 human breast cancer cells, display a marked and robust transformation in cellular morphology. Furthermore, scratch test assays revealed that cancer-conditioned ECs have significantly impaired wound healing responses, closing 25% less of a 750 µm-wide scratch than control ECs by 48 hours. Since regulation by phosphorylation is an important component of changes to cell shape and migration, we predicted that kinase inhibitors could affect cancerous processes in ECs. To identify specific kinases of interest, mass spectrometry-based phosphoproteomics was run to characterize changes in phosphorylation, and then combined with KinomeXplorer analysis to predict the associated kinases. To refine the predictions and overcome the limited depth of analysis inherent to the phosphoproteomic technique, we matched kinase predictions to RNA sequencing analysis of the same cultures. Based on the strongest combined predictions, a set of kinase inhibitors was chosen and tested by measuring the effects on morphology and wound healing, compared to cancer conditioning. First, inhibitors were added to control ECs: using a Src-family kinase inhibitor, for example, in control ECs resulted in a phenotype strongly resembling the cancer phenotype in both wound healing and elongation, consistent with the decreased phosphorylation of Src targets; however, a Bmx/Btk kinase inhibitor, while also predicted to have a similar impact, showed little effect in either assay. Then, a simple model of prevention and rescue was tested with inhibitors to increased predicted activity (e.g. Akt- and Pdh-family kinases and several phosphatases): inhibitors were added to EC cultures before, concurrent with, or after cancer conditioning. The inhibitors resulted in a range of phenotypic changes, depending both on the kinase as well as the prevention/rescue model. By using a combination of different assays on a well-controlled culture model we successfully developed a comprehensive library of kinase pathways in cancer-conditioned ECs, characterized by multimodal molecular data (phosphoproteomics and transcriptomics) and directly associated with functional effects of inhibitors using phenotype analysis. We hope this provides the beginning for the development of a tumor EC-targeted therapy based on kinase inhibitors, while also shedding light on the potential effects of existing cancer-targeting kinase inhibitors on the tumor endothelium.

#205

BBI-608 enhances the activity of chemoradiotherapy in colorectal cancer pre-clinical models.

Ganji Purnachandra Nagaraju,1 Rajitha Balney,2 Shipra R. Bethi,1 Sneha Govardhanagiri,1 Gregory B. Lesinski B. Lesinski,1 Bassel F. El-Rayes1. 1 _Emory University, Atlanta, GA;_ 2 _Wellstar Healthcare, Marietta, GA_.

Background: Chemoradiotherapy is a standard treatment modality for early stage rectal cancer (CRC). The pathologic complete response rate after chemoradiation in CRC remains low at 15%. Given the potential for BBI608 to elicit effects on multiple oncogenic cellular pathways, we hypothesized that BBI608 can sensitize colorectal cell lines to the effects of chemoradiotherapy. The aim of this study was to evaluate if BBI-608 enhances the response of chemoradiotherapy against CRC.

Methods: The combined effects of BBI-608 and chemoradiotherapy [5-FU + ionizing radiation (IR)] were evaluated in CRC cell lines (HCT 116 and HT-29) using Br-dU cell proliferation and clonogenic assays. Effects on the expression of DNA damage and repair (pATM, pATR, Rad51 and γ-H2AX) molecules and angiogenesis (pSTAT-3 and VEGF) pathways were examined by Western blot. Egg CAM assay was also performed to evaluate effects of the drugs on angiogenesis (quantification was performed by AngioQuant software). The in vivo efficacy of BBI-608 alone or combined with 5-FU + IR, was evaluated in nude mice bearing subcutaneous human HCT116 xenograft tumors

Results: The combination of BBI-608 and chemoradiotherapy significantly decreased cell proliferation (p<0.001), colony formation (p<0.001) and inhibited angiogenesis (p<0.001) in CRC cell lines as compared to chemoradiotherapy. BBI-608 decreased angiogenesis as determined by both matrigel plug and egg CAM assays. Immunoblot analysis indicated BBI-608 and chemoradiotherapy also decreased expression of pSTAT-3, MDM2, Rad51 and VEGF while increasing pATM, γ-H2AX and p53, in comparison to chemoradiotherapy alone. In animal models, combined therapy with BBI-608 and 5-FU + IR led to significant inhibition of longitudinal tumor growth as compared to chemoradiotherapy alone (p<0.001). No overt signs of systemic toxicity were evident and animals showed no loss of body weight in any of the treatment groups, indicating the combination was well-tolerated.

Conclusion: These observations provide preclinical proof-of-principle data that BBI-608 can enhance the anti-tumor effects of chemoradiotherapy. BBI608 plus chemoradiotherapy is a rational approach for potential future development in early stage rectal cancer.

#206

miR551b-mediated tumor-specific angiogenesis in ovarian cancer.

Deepak Parashar,1 Anjali Geethadevi,2 Jasmine George,2 Gopakumar Gopinathan Nair,2 Changliang Chen,2 Sunila Pradeep,2 Pradeep Chaluvally-Raghavan2. 1 _Medical College of Wisconsin, Milwaukee, WI;_ 2 _Medical College of Wisconsin, Wauwatosa, WI_.

Background: Ovarian cancer is the most lethal gynecological cancer. Tumor angiogenesis is essential for the progression and metastasis of ovarian cancer. Our data shows that STAT3, HIF-1α and VEGFA are the key regulators of tumor-specific angiogenesis. Our studies have provided evidence that VEGF is directly regulated by STAT3 in miR551b-3p expressing cells, which indicates that STAT3 is an important regulator of angiogenesis in a subset of ovarian cancers. Recently, we showed that miR551b-3p acts as an upstream regulator of STAT3 in ovarian cancer cells. We identified that anti-miR551b-3p inhibit the expression of miR551b-3p disrupt tumor-specific angiogenesis by preventing the expression of HIF-1α and VEGFA through direct inhibition of STAT3.

Methods: In this study, we explored the role of miR551b on angiogenesis in ovarian cancer cells by performing cell proliferation, western blot and qPCR of angiogenic markers, in vitro capillary tube formation, in vivo matrigel plug assay and ex-vivo aortic ring assays.

Results: We found that miR551b-3p upregulates STAT3, HIF-1α, VEGFR and VEGF in ovarian cancer cells and increases the abilities of cell proliferation, migration and tube formation of human umbilical vein endothelial (HUVEC) cells. In conjunction, knockdown of miR-551b decreased the level of STAT3, HIF-1α and VEGFR2 proteins as well as the angiogenic activity; also increased cellular apoptosis. Altogether, our data suggest critical roles of miR-551b on STAT-3–mediated angiogenesis in ovarian cancer cells. In complement to our in vitro results, the delivery of anti-miR551b inhibited angiogenesis in the matrigel plug injected in nude mice. Furthermore, the treatment of anti-miR551b reduced the angiogenic sprouting from rat thoracic aorta in our ex vivo culture. Taken together, our findings provide the first evidence that miR-551b is a pivotal regulator of tumor angiogenesis and inhibiting miR551b could be used to inhibit tumor-specific angiogenesis in ovarian cancer.

Conclusions: miR551b markedly augmented the pro-angiogenic effects in vitro, in vivo and ex-vivo models. Our data suggest that miR551b is an actionable target to disrupt tumor-specific angiogenesis in ovarian cancer. In our pre-clinical model, we are using anti-miR551b encapsulated in nanoliposomes for in vivo delivery to inhibit and sensitize anti- angiogenic therapy and chemotherapy. We expect that the completion of our studies will identify novel mechanisms regulate tumor-specific angiogenesis and novel targets to treat ovarian cancer patients.

#207

CMG2 regulates angiogenesis through interactions with extracellular matrix.

Samuel R. Garrard,1 Mark Gold,1 Tsz Ming Tsang,1 Sai Lun Lee,1 David Fogg,1 Rachael Dannenberg,1 Michael S. Rogers,2 Kenneth A. Christensen1. 1 _Brigham Young University, Provo, UT;_ 2 _Children's Hospital Boston, Harvard Medical School, Boston, MA_.

Angiogenesis is a critical process in cancer progression, facilitating initial tumor growth and allowing for metastasis into other tissues. Though several therapeutic agents are now available that reduce angiogenesis in vivo, all exhibit undesirable side effects, and none target more than one signaling pathway. This minimizes the potential to block additional signaling pathways and hence therapeutic applications, especially in cancers, which often utilize multiple signaling pathways. Capillary morphogenesis gene protein 2 (CMG2) is an integrin-like cellular receptor whose gene is highly upregulated during angiogenesis. Previously, we have demonstrated that CMG2 function is critical to neovascularization in vivo, and that CMG2 regulates angiogenesis through multiple signaling pathways, marking CMG2 as an effective target in development of multi-pathway antiangiogenic therapeutic agents.

CMG2 is also known to function as an anthrax toxin receptor, the mutation target in the rare genetic disease hyaline fibromatosis syndrome (HFS), and an interactor with extracellular matrix. Regarding HFS, it was recently shown that CMG2 knockouts lead to accumulation of extracellular matrix, especially collagen VI (Col-VI). To investigate a link between HFS and angiogenesis, we probed CMG2 affinity for several intact matrix proteins by ELISA, including laminin-111, fibronectin, and collagens I, IV, and VI. We discovered that CMG2 bound all these matrix proteins with similar affinity (Kd = 500-1000 nM). We then constructed a peptide array, containing the entire sequences of the collagen IV α1/α2 chains; fibronectin; and laminin-111 to identify CMG2-binding regions, using a 10-amino acid sliding window. We thereby identified a small peptide, designated S16, which binds CMG2 with high affinity and which inhibts angiogenic processes ex vivo. Since the integrin family of proteins regulate cell signaling and angiogenesis and are homologs of CMG2, we suspected that CMG2 may also function to regulate angiogenesis through matrix interactions. Consequently, we ran a higher resolution peptide array (2-amino acid sliding window) to identify specific matrix epitopes. In this expanded peptide array, we covered the sequences of the collagen IV α1 and α2 chains, collagen IV α3- α6 non-collagenous domains, collagen VI α1 and α2 chains, and portions of the collagen VI α3 chain and fibronectin. We noted several hits in each matrix protein on the array, including several in Col-VI, whose sequences map to specific vWFA domains in that matrix protein. We have now confirmed that CMG2 binds these collagen VI-derived peptides and their respective domains with high affinity. This has significant implications for the role of these domains in angiogenic phenotypes such as cell migration and adhesion. We anticipate that the interactions between these peptides and CMG2 can be exploited to develop multi-pathway inhibitors of angiogenesis.

#208

Transcriptional profiling of the microenvironment in pediatric osteosarcoma.

Laurie Sorenson, Troy A. McEachron. _USC Keck School of Medicine, Los Angeles, CA_.

Osteosarcoma (OS) is the most common bone tumor in pediatric and adolescent/young adult patients. Over the past three decades, significant improvements in the survival rates or therapeutic approaches for these patients have not been made, especially in the context of metastatic disease. While immune checkpoint blockade has revolutionized the therapeutic landscape in various adult malignancies, its impact in OS has been largely underwhelming. Currently, it is unknown whether the lack of therapeutic benefit of immune checkpoint inhibition observed in patients with OS is truly due to treatment inefficacy rather than a limited understanding of the tumor microenvironment that supports this aggressive disease. To address this knowledge gap, we have performed targeted gene expression profiling of metastatic and non-metastatic osteosarcoma specimens. Our profiling experiments have revealed that metastatic OS specimens are immunologically "colder" than non-metastatic OS specimens. Our data also demonstrates that T cells are largely excluded from the metastatic specimens and that this exclusion significantly correlates with markers of vascular instability. In a pathological setting, such as that of cancer, VEGF and ANG2 signaling promote vascular instability which limits leukocyte extravasation and subsequent tumor infiltration. Our data suggests that vascular destabilization mediated by VEGF/ANG2 signaling impedes T cell infiltration specifically in metastatic OS and identifies these molecules as potential targets for therapeutic intervention.

#209

Metastatic colonization in the perivascular niche: A mechanotransduction perspective.

Ekrem Emrah Er, Maria Tello-Lafoz, Jing Hu, Morgan Huse, Joan Massagué. _Memorial Sloan Kettering Cancer Center, New York, NY_.

Metastases are responsible for majority cancer related deaths. Disseminated tumor cells (DTCs) gain a diverse array of metastatic traits in order to adapt to and survive within different microenvironments, making them very difficult to therapeutically target. Remarkably, regardless of the secondary organ, DTCs survive and thrive in perivascular niches. Using xenograft and syngeneic models of metastasis in mouse, we found that upon extravasation, neuronal cell adhesion molecule L1 (L1CAM) expressed by DTCs engages the vascular basal lamina in order to amplify integrin β1 and integrin linked kinase (ILK) signaling in DTCs. ILK promotes actin polymerization for cell spreading and subsequent activation of mechanotransduction effector transcription factors YAP and MRTF. We found that activation of YAP and MRTF coincides with DTCs ability to dislodge resident pericytes from the perivascular niche and is an essential step in the metastatic colonization regardless of dormancy state. Therefore, these results provided us a unifying molecular mechanism that governs cancer progression from metastatic seeding to overt colonization. Ongoing studies will determine how mechanotransduction and pericyte-DTC interactions can be manipulated to prevent metastatic colonization by potentiating the immune response and exposing their biophysical vulnerabilities. 

## EXPERIMENTAL AND MOLECULAR THERAPEUTICS

### Antibody-Drug Conjugates: New Agents and Technologies

#210

Novel antibody drug conjugate to inhibit mesothelioma tumor growth.

Jorge Marquez, Chun Dong, Jianping Dong, Binbin Yue, Ginette Serrero. _A &G Pharmaceutical, Inc., Columbia, MD_.

Mesothelioma is an aggressive but rare form of cancer with a poor prognosis. 1-year survival is ~40% and 5-year survival remains in single digits. Treatment involves a combination of surgery, chemotherapy, and radiation therapy. Mesothelioma is a disease with unmet medical needs and deserving development of novel targeted therapies. Our laboratory is focused on developing novel antibodies useful in treating rare cancers such as mesothelioma. We have raised a mouse antibody library against human cancer cell surface proteins (CSPs). Antibodies were screened against several cancer cells including mesothelioma to identify antibodies specific for cancer CSP as druggable candidates. Screening identified antibodies that bound to and internalized into cancer cells. Such candidates were further characterized. One such antibody (AG02) was identified as having superior binding and internalization characteristics to the other antibodies. The therapeutic potential of AG02 was established as follows; 1) the cell surface protein named CSP-1 against which AG02 bound on the cell surface of selected cancer cells was identified by immunoprecipitation followed by mass spectrometry. Identified CSP-1 was demonstrated as being associated with metastatic potential thus emphasizing its potential as an important target for development of an anti-cancer agent; 2) We demonstrated that upon binding to CSP-1, AG02 antibody internalized the CSP-1 target; 3) We confirmed that AG02 antibody was first-in-class as no other internalizing antibody to this CSP-1 had been developed; 4) Since AG02 is an internalizing antibody, we developed ADC by conjugating AG02 to a generic cytotoxic drug saporin, routinely used as a cost-effective payload in ADCs for proof-of-concept (POC) study. Our POC AG02ADC was tested against a panel of cancer cells and found to bind strongly to CSPs in several cancers with orphan status and unmet needs including mesothelioma. In vitro and in vivo studies established that: (1) AG02 conjugated to saporin at 10nM was able to kill cells expressing CSP while it did not kill non-CSP-1 expressing cells, natural or transfected. (2) AG02-ADC acted in a dose dependent fashion, (3) when tested against different know cancer cell types, AG02-ADC killed >50% of cancer cells while a non-specific mouse IgG-ADC or Drug alone had no effect. (4) Using AG02-ADC treatment in nude mice, we demonstrated tumor growth inhibition >50% in several xenografts. While saporin was used in these studies as payload and shows efficacy, we expect more potent drugs such as MMAE or DM1 to have considerably more efficacy. In summary, we have demonstrated that: AG02 is specific for a CSP-1 overexpressed in certain cancers and involved in metastasis; AG02 is internalized in CSP-1 expressing natural cancer cells; AG02-ADC kills CSP-1 expressing cancer cells. Targeting mesothelioma may enable AG02-ADC to benefit of the FDA's Orphan Drug Program as it nears drug and clinical development.

#211

**A new class of sequence-selective DNA cross-linking ADC payloads with increased** in vivo **tolerability.**

George Procopiou,1 Jennifer Auer,1 Daniella di Mascio,2 Keith R. Fox,2 Paolo Andriollo,1 Ilona Pysz,1 Francesco Cascio,1 Nicolas Veillard,1 K. Miraz Rahman,1 Paul J. Jackson,1 David E. Thurston1. 1 _Femtogenix, Welwyn Garden City, United Kingdom;_ 2 _University of Southampton, Southampton, United Kingdom_.

Although five Antibody-Drug Conjugates (ADCs) have been approved and over eighty others are in development, the majority contain payloads belonging to two classes: tubulin inhibitors and DNA interactive agents. Most DNA cross-linking payloads (e.g., the PBD dimers) have potent cytotoxicity but ADCs containing them have high hydrophobicity and a narrow therapeutic window. Thus, there is interest in developing novel payloads which benefit from a potency similar to the PBD dimers but that possess lower hydrophobicity and produce ADCs with a wider Therapeutic Index (TI). The pyridinobenzodiazepines (PDDs) are a new class of guanine-alkylating payloads, and these have been coupled to an adenine-alkylating CXI/duocarmycin pharmacophore to generate molecules that can form G-A DNA cross-links. The lead PDD-CXI payload (FGX-8-46) has a sequence-selectivity profile that differs from other DNA cross-linking agents in that it spans seven to eight base-pairs compared to six to seven for a typical PBD dimer. DNA cleavage experiments have indicated that it cleaves at discrete Adenine-containing sequences of the type 5'-XGXWWWW-3' (X is any base; W is A/T, and the underlined bases show the cleavage points), and Transcription Factor (TF) Array studies show that it is a potent TF inhibitor, down-regulating several key oncogenic TFs (e.g., NF-κB). In in vitro cell line studies, the G-A cross-linkers have low pM cytotoxicity comparable to the PBD dimers in a wide and diverse range of cell lines, including those from both solid and haematological cancers (e.g., IC50 of ~2 pM in SW-48). This payload class is also compatible with a wide variety of linker technologies, and attachment can be made through either the PDD or CXI units. Importantly, these payloads are significantly less hydrophobic than other equivalent payload classes. ADCs have been generated by conjugating these new payloads to the EGFR-targeting antibody Cetuximab with DARs of between 1.8 and 2.2. The ADCs exhibit potent cytotoxicity in vitro, significant in vivo efficacy and substantially increased tolerability compared to other DNA cross-linking payloads (e.g., the PBD dimer Tesirine). While the G-A cross-linkers retain the cytotoxic potency of the PBD dimers, the "softer" cross-link formed compared to the G-G cross-linking PBD dimers may contribute to the enhanced tolerability profile of this molecular class. The favourable hydrophobicity profile of the PDD-CXI payloads and their ease of conjugation to antibodies, along with their significant in vitro cytotoxicity, in vivo efficacy and tolerability of ADCs produced from them, suggest that they represent a promising new class of ADC payloads.

#212

**CD166-DM4 probody** **drug conjugate (CX-2009) treatment of 198 patient-derived xenograft models (PDX) in a mouse clinical trial format.**

Bob Y. Liu, Joel Shen, Matthias Will, Sreeni Yalamanchili, Judi Gordon, Mark Stroh, Jennifer Richardson, Annie Weaver, Luc Desnoyer, Marcia Belvin, Michael Kavanaugh, Siew Schleyer. _CytomX, South San Francisco, CA_.

Ideal targets for antibody drug conjugates (ADC) are highly and homogeneously expressed on tumor cells in a wide variety of tumor types and in a high proportion of patients. Such targets should also be efficient cellular internalizers and have limited expression on normal tissues. Unfortunately, targets that meet all these criteria are rare. Probody™ drug conjugates (PDCs) are masked antibody prodrugs designed for activation by tumor-associated proteases. PDCs are designed to direct drug activity to the tumor microenvironment by exploiting the dysregulation of tumor protease activity that is the hallmark of most cancers. The Probody platform is designed to minimize interaction with healthy tissues, mitigate "on-target" toxicity regardless of expression outside of the tumor, and greatly expand the number of potential ADC targets. CD166 (ALCAM) is one such novel target. It is expressed at high levels in a high proportion of patients across multiple cancer types, but it is also on healthy tissues, including lung, gastro-intestinal tract, and liver. CX-2009, an investigational SPDB-DM4 PDC targeting CD166, is currently being evaluated in PROCLAIM CX-2009, a clinical phase I/II trial (NCT03149549). CX-2009 exhibited robust preclinical activity against cell-line derived xenograft (CDX) models across various solid tumor indications. Because the DM4 mechanism of action is similar to that of taxane-based chemotherapy, CX-2009 is being evaluated in the clinic for cancer types that respond to microtubule inhibitors and have prevalent CD166 expression. To help further inform patient selection for clinical studies, CX-2009 is being evaluated in 198 PDX models using a murine clinical trial format in collaboration with South Texas Accelerated Research Therapeutics (START). Each mouse is engrafted with a patient's resected tumor sample that has not been passaged in vitro, and when kept minimally passaged in vivo, may more faithfully recapitulate the tumor biology and response to therapeutics than CDX models. The PDX models are dosed with 5mg/kg of CX-2009 every 2 weeks for 6 weeks via i.v. (q2wX3), and tumor volume is measured twice per week. Currently, we have dosed 66 breast, lung, and ovarian PDX models, obtaining an end-of-study response rate of 21% using clinically defined criteria, e.g. complete response (CR) plus partial response (PR), and a 29% disease-control rate (DCR). Relative to untreated controls, 43% of CX-2009-treated tumors yielded tumor growth inhibition (TGI) of greater than fifty percent. Because of the large cohort data set, this effort can potentially inform patient selection for clinical studies based on correlating efficacy with CD166 expression, tumor subtype, tumor growth rate (doubling time), and taxane sensitivity. PROBODY is a trademark of CytomX Therapeutics, Inc.

#213

Development of a probody-drug conjugate targeting EpCAM for the treatment of solid tumors.

Yimao Liu,1 Rui Wu,1 Cristina Gavrilescu,1 Jason Sagert,2 Kimberly Tipton,2 Shouchun Liu,2 Chanty Chan,2 Steven Boulé,1 Alan Wilhelm,1 Jacquelynn Lucas,1 Bahar Matin,1 Jean-Michel Lecerf,1 Marian Themeles,1 Ashley Morneault,1 Tara Drake,1 Sadiqa Yancey,1 Neeraj Kohli,1 Christopher Espelin,1 John Follit,1 Kerry A. Donahue,1 Tom Chittenden,1 Cynthia Guidi,1 Stuart W. Hicks1. 1 _ImmunoGen Inc., Waltham, MA;_ 2 _CytomX Therapeutics, Inc., South San Francisco, CA_.

Epithelial Cell Adhesion Molecule (EpCAM) is a glycosylated, 40-kDa type I transmembrane protein that plays a role in cell adhesion and cell signaling. EpCAM is an attractive target for antibody drug conjugate (ADC) development due to its overexpression on a variety of tumors of epithelial origin, including lung, colon, breast, ovarian, prostate and pancreatic cancers. In addition, EpCAM is enriched on tumor-initiating cells (TICs), which are often resistant to conventional cancer therapies. As such, EpCAM-targeted therapies may lead to more durable responses. However, EpCAM is also expressed on a variety of normal epithelia, thus limiting its utility as an ADC target due to potential toxicity. We aim to overcome this limitation by developing an EpCAM-targeting Probody™ drug conjugate (PDC). A Probody therapeutic is an antibody engineered with a mask that blocks the antigen binding site. Probody therapeutics can be selectively activated by tumor associated proteases releasing an active antibody with restored antigen binding activity. Therefore, an EpCAM-targeting PDC could have the anti-tumor potency of an ADC, while limiting binding to healthy tissues and minimizing toxicities.

Here, we describe the development of EpCAM-targeted PDCs, based on a novel human/cynomolgus cross-reactive anti-EpCAM antibody. Probody molecules were successfully conjugated to either the maytansine-derived microtubule disruptor, DM4, linked via a hindered disulfide hydrophilic linker (sulfo-SPDB) or the ultra-potent DNA alkylating payload, DGN549. The antigen binding and in vitro cytotoxicity of intact PDCs were dramatically reduced compared to the corresponding ADC, but could be restored following in vitro proteolytic activation. Furthermore, EpCAM-targeting PDCs displayed compelling and specific anti-tumor activity in xenograft mouse models. In addition, the tolerability and pharmacokinetics (PK) of a selection of the EpCAM PDCs were compared to the EpCAM-targeting ADC in cynomolgus monkeys. EpCAM-targeting PDCs were better tolerated than the corresponding EpCAM-targeting ADC even at higher dose levels and displayed longer half-lives and greater exposure. Therefore, EpCAM PDCs showed greatly improved tolerability and PK profiles compared to the EpCAM ADC.

The studies presented herein support an anti-EpCAM PDC as a promising novel therapeutic to target a wide range of EpCAM-expressing cancers with the potential to overcome the associated on-target toxicities.

PROBODY is a trademark of CytomX Therapeutics, Inc.

#214

Preclinical evaluation of a novel MET antibody-drug conjugate in uveal melanoma models.

Oliver Surriga,1 John DaSilva,2 Gary K. Schwartz1. 1 _Columbia University Irving Medical Center, New York, NY;_ 2 _Regeneron Pharmaceuticals, Inc., Tarrytown, NY_.

Uveal melanoma is the most common primary intraocular malignant tumor in adults. These tumors are characterized by mutations in G-proteins (GNAQ and GNA11) and high expression of MET, a receptor tyrosine kinase. While inhibition of MET signaling delays the development of uveal melanoma metastases in preclinical models (possibly by inhibiting cell migration), primary tumor growth is not inhibited, indicating a lack of dependence on the MET pathway. Thus, treatment of established metastatic uveal melanoma with MET signal blockers is unlikely to be effective. However, the high expression of MET protein in metastatic uveal melanoma presents an opportunity to directly kill tumor cells with a MET-targeted antibody drug conjugate (ADC). METxMET-ADC is a novel bispecific antibody that binds to two distinct epitopes on MET and is conjugated to a maytansinoid payload via a cleavable linker. The METxMET-ADC potently induces receptor internalization and mitotic arrest of cancer cells. Our in vitro studies show that the METxMET-ADC significantly and specifically decreases the viability of MET expressing uveal melanoma cell lines in a dose-dependent manner with an IC50 of less than 1 nM. A longer 7-day cell viability assay shows significantly enhanced inhibition of proliferation of several MET expressing cell lines (Mel270, Mel202, OMM1.3 and MP65) but not of OCM3 cells which express low levels of MET. Apoptosis is also induced up to 40% in the MET-expressing cell lines when treated with 10 nM of the METxMET-ADC for 48 hours. Cell cycle analysis shows a significant increase of OMM1.3 and Mel202 cells in mitosis after 6-24 hours of treatment and appearance of a sub-G1 population after 24-48 hours. Western blots confirm a decrease of MET protein levels, presumably due to receptor internalization and trafficking to lysosomes. Finally, treatment with the METxMET ADC is associated with induction of PARP cleavage and phosphorylation of histone H3 after 24 hours in OMM1.3 and Mel202 cells, but not in OCM3 cells. In summary, conjugation of a METxMET bispecific antibody with a cytotoxic payload allows for potent and selective killing of MET-expressing uveal melanoma cells. The METxMET-ADC demonstrates promising therapeutic potential for the treatment of uveal melanoma.

#215

Generation of site-specific DARPin® drug conjugates using EGFR as a model system.

Laura A. Laviolette,1 Cynthia J. Guidi,1 Christian Reichen,2 Qifeng Qiu,1 Luke Harris,1 Patricia Schildknecht,2 Stefanie Fischer,2 Zita Arany,2 Tanja Hospodarsch,2 Anna Skaletskaya,1 Megan Fuller,1 Rebecca McCarthy,1 Jenny Lee,1 Katherine Francisco,1 Kerstin Sinkevicius,1 Sharlene Adams,1 Christopher Espelin,1 Emily Reid,1 Wei Li,1 Carla Marashio,1 Kerry Donahue,1 Stuart Hicks,1 Dan Snell2. 1 _ImmunoGen, Inc, Waltham, MA;_ 2 _Molecular Partners AG, Zurich-Schlieren, Switzerland_.

DARPin® molecules are small engineered proteins, derived from natural ankyrin repeat proteins that are selected to bind to specific targets with high affinity. Individual DARPin® molecules can be linked together genetically in order to create multi-specific drug molecules. This versatility of DARPin® molecules makes them an attractive alternative to antibodies for the development of drug conjugates. We have developed DARPin® drug conjugates (DDCs) using a model EGFR multi-specific DARPin® molecule, consisting of four DARPin® domains linked together. DARPin® constructs were conjugated to the indolinobenzodiazepine mono-imine DGN549, a potent DNA alkylating payload. Biophysical characterization showed the DDCs to be well behaved in stability and solubility assays. The in vitro binding and cytotoxicity of these DDCs were evaluated. The DARPin® conjugates displayed in vitro potency in direct cytotoxicity assays across a panel of cell lines expressing EGFR. The impact of conjugation on PK parameters and in vivo efficacy is currently under evaluation. DARPin® drug conjugates combine the potency observed with antibody drug conjugates and the modular design of DARPin® molecules to create designer therapeutics.

#216

pro **-Pyrrolobenzodiazepine (** pro **-PBD) bioconjugates, the legacy: Design and synthesis of** pro **-PBD conjugates containing self-immolative substituted disulfide linkers.**

Iontcho Vlahov, Longwu Qi, Hari K. Santhapuram, Garth Parham, Kevin Wang, Jeremy F. Vaughn, Spencer J. Hahn, Marilynn Vetzel, Melissa Nelson, Joseph A. Reddy, Christopher P. Leamon. _Endocyte, W. Lafayette, IN_.

Naturally occurring pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a group of highly potent antitumor antibiotics, isolated from various streptomycin species. PBDs covalently bond and cross-link with discrete sequences of base pairs in the minor groove of DNA. A wide range of synthetic PBDs and PBD dimers have been shown to be highly potent cytotoxic agents, and have been used as payloads in antibody drug conjugates (ADCs). However, all PBDs as stand-alone agents or as delivered via small molecule drug conjugates (SMDCs), cause undesired off-target toxicity. Recently, we designed folate conjugated pro-PBDs, masking the imine moiety with its synthetic precursors: namely, 1,3-oxazolidine carbamate and an aromatic amine. The 1,3-oxazolidine carbamate provided a conjugation site for attachment of pro-PBD to a targeting ligand. After delivery to the targeted cell and internalization, reductive cleavage of the linker system results in the generation of a 1,3-oxazolidine. The oxazolidine reacts with water to form an aldehyde which subsequently reacts intramolecularly to ultimately form the PBD diazepine ring. Herein, we report the design and synthesis of folate conjugates of pro-PBDs and self-immolative sterically hindered disulfide linker systems, which pave the way toward novel, highly-specific therapeutics which have the potential to minimize off-target toxicities.

One of the so designed conjugates, EC2629, satisfied all of our criteria for selection for pre-clinical development:

• EC2629 shows high affinity towards FR mediated binding.

• Cross-links DNA only following self-immolative release of free drug.

• Picomolar potency against FR-expressing cells.

• Curative activity with no weight loss in animal models.

• Active against drug resistant tumor xenograft models in-vivo.

• EC2629 is active against patient derived xenograft models in-vivo.

• Based on high cytotoxicity and minimal off target toxicity (in-vitro & in-vivo), EC2629 is selected for pre-clinical development.

• Biological evaluation of EC2629 was presented at World ADC, Berlin, Germany (March, 2018).

#217

Development of a rapid and efficient methodology to quantitate thiol detection in antibody-drug conjugates (ADCs).

Ilona Pysz, Paolo Andriollo, Paul J. Jackson, K M. Rahman, David E. Thurston. _Femtogenix, Welwyn Garden City, United Kingdom_.

Antibody-Drug Conjugates (ADCs) are comprised of three distinct components, an antibody, a payload (usually a cytotoxic agent) and a linker (a synthetic chemical chain joining the antibody and payload). Payloads can be conjugated to antibodies in either a stochastic or site-specific manner, with stochastic conjugations usually accomplished through either amine groups of lysine residues or through thiol groups in disulfide bonds located in the hinge region of antibodies. In the latter case, an electrophilic Michael acceptor grouping (e.g., maleimide) of a linker-payload construct reacts with a nucleophilic thiol group of the antibody, forming the ADC. In order for this to occur, disulfide bonds need to be reduced to free thiols. However, knowing the concentration of reactive thiols at different stages during the reduction process can be challenging, and so a more accurate assessment of thiol concentration may allow greater control of the desired average Drug-Antibody Ratio (DAR). Currently, different spectrophotometric (i.e. Ellman's reagent) and fluorometric assays are used to monitor thiol concentration, but these methods can be expensive, time consuming and inaccurate.

To address these issues, a rapid and efficient fluorescence-based method has been developed to allow the reduction of thiols to be monitored. The method is based on a fluorescein derivative produced via a four-step synthesis starting from commercially available fluorescein sodium salt. In its unreacted form, this novel probe exists in a lactone form. However, once it reacts with a free thiol group, it converts stoichiometrically via opening of the lactone ring to a fluorescent form, thus allowing quantitative determination of the concentration of thiol groups present in solution.

The three antibodies used to trial this procedure (i.e., Trastuzumab, Cetuximab and Rituximab) were reduced using TCEP over a 120 minute period. Aliquots of solution were collected at various time intervals, and incubated with the fluorescein probe for 5 minutes, monitoring fluorescence with a simple fluorimeter. Using this approach, it was possible to accurately assess thiol concentration at each time interval, and relate the overall reduction profile of each antibody to the concentration of reducing agent used. Payloads were then conjugated to the three antibodies at specific timepoints (selected through monitoring thiol reduction) in an effort to generate species with a defined DAR. Overall, the resulting ADCs were found to have average DAR profiles consistent with the measured number of thiols in the reduced antibodies.

When compared with other assays, the developed method gave results with improved reproducibility. Furthermore, the fluorescein probe was less complex and faster to use than other fluorometric kits, and was easy to synthesize, making it a useful new tool in the generation of ADCs.

#218

AVID100 is an anti-EGFR ADC that promotes DM1-meditated cytotoxicity on cancer cells but not on normal cells.

Michael J. Thwaites,1 Rene Figueredo,2 Gilles Tremblay,1 James Koropatnick,2 Victor Goldmacher,1 Maureen O'Connor-McCourt1. 1 _Forbius, Montréal, Quebec, Canada;_ 2 _University of Western Ontario, London, Ontario, Canada_.

AVID100 is a novel rationally designed antibody drug conjugate (ADC) that specifically targets the epidermal growth factor receptor (EGFR). EGFR is highly expressed on a variety of cancers making it a promising target for ADCs. However, due to the presence of EGFR on normal skin cells, on-target off-tumor toxicity is a concern. AVID100 is an anti-EGFR-DM1 conjugate exhibiting high potency against cancer cells relative to unconjugated antibodies, while not exhibiting increased toxicity on normal cells. In a series of in vitro and in vivo experiments, we provide mechanistic evidence rationalizing why AVID100 shows higher cytotoxicity on tumor cells compared to normal cells.

The antibody moiety of AVID100, denoted MAB100, was shown to have a high affinity for EGFR (approximately 2nM) and to compete with EGF for binding to the EGFR. Also, MAB100 prevented downstream signalling from EGFR. In addition to exhibiting full antagonist activity, MAB100 effectively delivered the microtubule inhibitor DM1 to tumor cells, as demonstrated by an increase in apoptosis in AVID100-treated tumor cells relative to MAB100-treated tumor cells. AVID100 was very effective on tumor cell lines derived from breast, head and neck, and lung cancers with the cytotoxic IC50 values generally correlating with the number of EGFR molecules on the cell surface. Importantly, on keratinocytes, AVID100 did not exhibit increased cytotoxicity relative to MAB100. We hypothesized that this lack of increase in cytotoxicity on keratinocytes is due to the antagonistic effect of MAB100 on EGFR signalling. Blocking the EGFR pathway in keratinocytes strongly inhibits their proliferation, which should protect them against the cytotoxic action of DM1. To address this hypothesis, we compared the effect of AVID100 to that of a non-antagonistic anti-EGFR ADC, huML66-DM1. As expected, both ADCs were highly effective at killing MDA-MB-468 cancer cells, confirming their ability to deliver DM1. In contrast, keratinocytes exhibited a significantly higher level of apoptosis in response to huML66-DM1 as compared to AVID100. These results confirm that the antagonistic nature of the antibody moiety of AVID100 plays a critical role in protecting keratinocytes from the cytotoxic effect of DM1.

Finally, the activity of AVID100 was investigated in multiple mouse xenograft studies, including in MDA-MB-468 human breast adenocarcinoma, H292 NSCLC, and FaDu SSCHN models. AVID100 treatment significantly reduced tumor growth and caused tumor regressions in some of the mice, even when administered as a single dose. Toxicology studies in cynomolgus monkeys demonstrated that AVID100 was well tolerated at up to 4 weekly doses of 10mg/kg. AVID100 has completed a Phase 1 clinical trial with the recommended Phase 2 dose having been determined. The Phase 2 trial of AVID100 is ongoing.

#219

**The potential benefit of lower drug-antibody ratio (DAR) on antibody-maytansinoid conjugate** in vivo **efficacy.**

Kerstin W. Sinkevicius, Leanne Lanieri, Jenny Lee, Steven Boule, Nicholas C. Yoder, Stuart W. Hicks, Jan Pinkas, Jose F. Ponte, Richard J. Gregory. _ImmunoGen, Waltham, MA_.

The majority of antibody-drug conjugates (ADCs) with maytansinoid payloads in development are linked via lysine residues and target an average drug-antibody ratio (DAR) of 3.5. These ADCs demonstrate substantial preclinical potency while maintaining good biophysical and pharmacological properties. However, some targets are expressed in normal tissues at levels that result in target-mediated drug disposition (TMDD), which can impact systemic and tumor ADC exposure and tumor penetration. For targets with TMDD, lowering the DAR of the ADCs may be advantageous. Since ADC tolerability is generally determined by the payload dose, lower DAR ADCs can be dosed at a higher antibody concentration, resulting in an increased conjugate exposure which may improve efficacy by saturating TMDD and/or increasing tumor delivery and penetration of the conjugate. In preclinical cell line xenograft studies, lysine-conjugated 2.0 and 3.5 DAR ADCs had comparable efficacy when dosed by payload concentration. However, the effect of TMDD cannot be captured with these models because the antibody in the ADCs does not cross-react with murine target antigen expressed on normal tissues. To supplement our understanding of the factors that influence ADC activity, we generated a cross-reactive model system that utilizes a chimeric anti-murine folate receptor α (FRα) antibody that binds with similar affinity to mouse and human FRα. Using this cross-reactive system, where the target is also expressed in normal tissues, 2.0 DAR conjugates were more efficacious than 3.5 DAR conjugates when dosed at matched payload concentrations in multiple xenograft models, suggesting that lower DAR can be an effective strategy to compensate for TMDD. Additional studies using xenograft models with varying antigen expression levels are ongoing, and studies designed to assess different conjugation methods of generating lower DAR ADCs are planned. This work highlights some of the many factors that impact ADC activity and demonstrates that the DAR of an ADC should be optimized for each target.

#220

U3-1402, a novel HER3-targeting ADC, inhibits the tumor growth of colorectal cancer xenografts.

Shigehiro Koganemaru,1 Yasutoshi Kuboki,1 Yoshikatsu Koga,1 Takashi Kojima,1 Mayumi Yamauchi,1 Naoyuki Maeda,2 Takashi Kagari,2 Kenji Hirotani,2 Masahiro Yasunaga,1 Yasuhiro Matsumura,1 Toshihiko Doi1. 1 _National Cancer Center Hospital East, Kashiwa-shi, Chiba, Japan;_ 2 _Daiichi Sankyo Co., Ltd., Tokyo, Japan_.

Background: HER3 (human epidermal growth factor receptor 3) is a member of the HER family and overexpressed in a broad range of cancer types including colorectal cancer. HER3 targeted therapies such as anti-HER3 antibodies have been investigated in clinical trials; however, no HER3-targeting therapy has been approved to date. U3-1402 is a novel antibody drug conjugate (ADC) composed of a fully human anti-HER3 antibody (patritumab) and a novel topoisomerase I inhibitor (DXd) which is conjugated via an enzymatically cleavable peptide-linker with high drug antibody ratio (DAR: 7-8 to 1).

Purpose: To investigate the antitumor efficacy of U3-1402 in HER3-expressing colorectal cancer xenograft models.

Material and methods: The following cell lines were used in this study; WiDr, Colo320DM, and DiFi with KRAS wild type, DLD-1, HCT15, HCT116, LoVo, SW480, and SW620 harboring KRAS mutation. The sensitivities of 9 colorectal cancer cell lines to DXd were evaluated by in vitro cell killing assay. Cells were treated with DXd at different concentrations (0.5pM to 500nM) over 144 hours, and the 50% inhibitory concentration (IC50) values were determined. Cell surface HER3 antigen was quantified by flow cytometry. In vivo screening assay and tumor growth inhibition assay were performed in tumor xenograft models exhibiting a range of HER3 expression levels. In addition, the efficacy of U3-1402 was compared to that of parental antibody patritumab, CPT-11, control-ADC, and saline in SW620 tumor xenograft

model harboring KARS mutation. All results were expressed as mean ± standard deviation (SD) and statistical significance was analyzed using ANOVA with Dunnet multiple comparison.

Results: In in vitro study, the range of IC50 value of DXd for each cell line was 2.16 ± 0.11 nM to 16.04 ± 0.10 nM. Most of the cell lines were sensitive to DXd. In in vivo screening assay, the maximum regression from U3-1402 was observed in the tumor xenograft model of both DiFi (high HER3 expression; KRAS wild type) and SW620 (high HER3 expression; KRAS mutation). In in vivo tumor growth inhibition assay, the antitumor efficacy was dependent on the HER3 expression level. Notably, SW620 tumor growth was significantly suppressed with U3-1402 compared with that of saline group (p < 0.001) and had improved potency compared with CPT-11. Of note, no significant difference in tumor growth inhibition was observed amongst the treatments with the same dose of patritumab or control-ADC and saline. Tumor regression was only observed in U3-1402 treated group, and some of which achieved complete regression.

Conclusions: The antitumor efficacy of U3-1402 in xenograft models was dependent on HER3 expression and did not show a clear relationship to KRAS mutation status. These results support further investigation of development strategies for U3-1402 in patients with HER3-expressing colorectal cancer.

#221

**Tisotumab vedotin induces anti-tumor activity through MMAE-mediated, Fc-mediated, and Fab-mediated effector functions** in vitro **.**

Stephen C. Alley,1 Jeffrey R. Harris,2 Anthony Cao,1 Elke Gresnigt-van den Heuvel,3 Jyoti Velayudhan,1 David Satijn,3 Sandra Verploegen,3 Teresa Dominguez,1 Esther C. Breij3. 1 _Seattle Genetics, Inc., Bothell, WA;_ 2 _Genmab, Princeton, NJ;_ 3 _Genmab, Utrecht, Netherlands_.

Tisotumab vedotin is an antibody-drug conjugate composed of a human IgG targeting tissue factor (TF), a valine citruline linker, and the microtubule disrupting agent monomethyl auristatin E (MMAE). TF is aberrantly expressed in solid tumors and is thought to contribute to tumor progression by enhancing tumor growth, neo-angiogenesis and metastatic potential through local activation of coagulation and protease-activated receptor-2 (PAR-2) signaling. Tisotumab vedotin was previously shown to induce cytotoxicity through MMAE- and Fc-dependent mechanisms. In addition, tisotumab vedotin inhibited PAR-2 signaling in TF-positive tumor cells. Here we aimed to elucidate additional effector mechanisms by assessing the capacity of tisotumab vedotin to induce immunogenic cell death (ICD), bystander cytotoxicity, and antibody-dependent cellular phagocytosis (ADCP) in vitro. ICD was assessed by incubating A431, MDA-MB-231, and HPAFII cells with tisotumab vedotin, and measuring ER stress by western blot, ATP secretion by reporter assay, and HMGB1 release by ELISA. Bystander cytotoxicity was assessed by incubating mixed cultures of TF+ (MDA-MB-231) and TF- (A549) tumor cells with tisotumab vedotin and quantifying viable cells by flow cytometry. To assess ADCP, PKH26-labeled BxPC-3 or A431 cells were incubated with human monocyte derived macrophages in presence of tisotumab vedotin. ADCP activity was analyzed by measuring the fraction of macrophages that double stained with PKH26 and anti-CD11c antibody using flow cytometry. Tisotumab vedotin induced key hallmarks of ICD including induction of ER stress, ATP secretion, and release of HMGB1, which have been shown to facilitate antitumor immunity. In co-cultures of TF+ and TF- tumor cells, tisotumab vedotin induced cytotoxicity in both cell types, even at a target-positive to target-negative cellular ratio of 1:15. This was mediated through bystander cytotoxicity, as demonstrated by the lack of cytotoxicity in monocultures of TF- cells. Finally, ADCP was observed with macrophages derived from multiple donors, with double-positive macrophages comprising up to 50% of total macrophages. In summary, ICD, bystander cytotoxicity, and ADCP were identified as novel effector mechanisms of tisotumab vedotin in vitro. Combining the present study with previous results, tisotumab vedotin induces TF-dependent anti-tumor activity through 1) MMAE-mediated effector mechanisms, including MMAE-mediated direct and bystander cytotoxicity, and induction of ICD; 2) Fc-mediated effector mechanisms, including ADCC and ADCP; and 3) Fab-mediated inhibition of PAR-2 dependent signaling. Tisotumab vedotin is currently being investigated in a variety of solid tumors (NCT03245736, NCT03485209, NCT03438396, NCT03657043).

#222

Novel protein drug conjugates targeting ROR1 through the development and exploitation of a drug discovery platform based on small, engineered VNAR domains.

Graham Cotton,1 Jennifer Thom,1 Paul Trumper,1 Stacey Bell,1 Andrei Kamenski,1 Mark Wappett,1 Caroline Barelle,2 Marina Kovaleva,2 John Steven,2 Andy Porter,2 Estelle McLean,3 Chiara Saladino,3 Aidan McCann,3 Aaron Cranston,3 Tim Harrison3. 1 _Almac Discovery, Edinburgh, United Kingdom;_ 2 _Elasmogen Ltd, Aberdeen, United Kingdom;_ 3 _Almac Discovery, Belfast, United Kingdom_.

Whilst antibody drug conjugates (ADCs) have been successfully used to target highly potent cytotoxic agents to tumours, there is a continued emphasis on developing technologies for improving the therapeutic effectiveness of this class of agents. The vast majority of ADCs that have entered the clinic have been based on large intact immunoglobulins, however, there is significant interest in exploiting small protein domains as the payload delivery vehicle. These offer a number of potential benefits over full length antibodies including increased tumour penetration, amenability to protein engineering and site-specific conjugation, and improved tolerability. To this end, we have developed a therapeutics platform based on shark Variable New Antigen Receptor (VNAR) proteins. Shark VNAR domains are the smallest naturally occurring antigen binding domain (~11 kDa), with a binding mechanism distinct from traditional antibodies. Here we report the application of this platform to the development of homogenous VNAR-drug conjugates targeting the onco-embryonic receptor tyrosine kinase ROR1.

ROR1 expression has been observed across a broad range of solid tumours and haematological malignancies, and is reported to correlate with poor clinical outcome for a number of cancer indications, but is largely absent from normal adult tissue. As such, the expression pattern of ROR1, coupled with its functional role in tumourogenesis and disease progression, make it an attractive PDC target. Using a combination of direct immunisation and synthetic VNAR library screening, we have generated high affinity binders to the extracellular domain of ROR1 which show species cross-reactivity, do not bind the closely related family member ROR2, and can target distinct non-overlapping regions of the protein. By exploiting the ability of VNAR domains to be flexibly reformatted, the lead sequences have been configured as a series of bivalent Fc fusions, VNAR multimers and novel bi-paratopic binders, and constructs identified that show further enhanced binding to ROR1 and which can induce internalisation and lysosomal trafficking in ROR1 expressing cancer cell-lines. Subsequent attachment of highly potent DNA-damaging cytotoxic payloads yielded homogenous VNAR-drug conjugates which demonstrated extremely potent (sub-nanomolar) in vitro cytotoxicity against selected cancer cell-lines in a receptor mediated fashion. Studies to assess the in vivo efficacy of these conjugates are in progress. These ROR1 VNAR constructs have also been successfully fused to sdAbs and scFv directed to other cell-surface protein targets, demonstrating the power of the platform and opening the way for novel bi-specific approaches for targeting both solid and haematological tumours.

#223

Antibody intracellular activated drug conjugate (AiADC) as novel ADC technology.

Atsushi Suwa, Kazuo Komiya, Kenta Kadotsuji, Shoko Ochiai, Yosuke Takanashi, Eiji Sugaru, Hitoshi Ban. _Sumitomo Dainippon Pharma Co., Ltd., Osaka, Japan_.

Antibody drug conjugate (ADC) consisting of antibody and modifier (potent cytotoxic drug with chemical linker) is expected to selectively eliminate cancer cells expressing corresponding cell-surface antigen. More than 60 ADCs are in clinical trials and about 75 percent of its modifiers are based on three series (auristatin, maytansine, and pyrrolobenzodiazepine). Whereas some ADCs show promising clinical benefits, several ADCs may not have shown maximum value of ADC due to their narrow therapeutic windows. The function of modifiers impacts on total characteristics of ADC such as the efficacy, tolerability, and pharmacokinetics (PK). Therefore, further exploration of new types of modifier for ADC technology platform is needed. We designed hydrophilic modifiers which can be activated only inside of target cells. The ADC consisting of certain IgG1 monoclonal antibodies and this modifier (DSP-ADC) showed selective and potent cytotoxicity to target cells with IC50s at picomolar range. The DSP-ADC with the average drug-to-antibody ratio (DAR) of 7-8 was stable in blood circulation and showed favorable PK profiles in mice. Additionally, administration of the DSP-ADC led to dose-dependent suppression of cancer cell proliferation in mice xenograft models and achieved complete remissions at 3 mg/kg without body weight reduction. The toxicity studies of DSP-ADC in rats showed no effect on hematologic parameters up to 100 mg/kg. These modifiers are applicable to various monoclonal antibodies. Taken together, our modifiers can be fundamental component for ADC technology. We present here the novel ADC technology named as antibody intracellular activated drug conjugate (AiADC).

#224

Antibody-drug conjugates (ADCs) of a new class of N-10 amino linked DNA alkylating indolino-benzodiazepines (IGNs).

Michael L. Miller, Emily E. Reid, Katie E. Archer, Luke Harris, Erin K. Maloney, Laura M. Bartle, Olga Ab, Alan J. Wilhelm, Jose F. Ponte, Rajeeva Singh, Thomas A. Keating, Ravi V. Chari. _ImmunoGen, Waltham, MA_.

We have previously reported on our antibody-drug conjugates (ADCs) that incorporate a highly potent novel DNA alkylating indolino-benzodiazepine (termed IGN) dimer. ADCs of these DNA alkylating IGNs were found preclinically to demonstrate better tolerability and an improved overall therapeutic index (TI) compared with those of the corresponding DNA crosslinking IGNs (Miller, et al., Mol. Cancer Ther. 2016, 2018). IGN-containing ADCs that have reached the clinical stage bear DNA alkylating IGN compounds linked to the antibody via a cleavable linker, incorporated at the amino group of a centrally located anilino spacer moiety. In our ongoing effort to further explore the structure-activity relationship (SAR) of DNA alkylating effector molecules for ADCs, we disclose here a new class of IGNs possessing a self-immolative peptide linker attached at the N-10 amine of the imine-reduced IGN monomer subunit. We explored the impact of modifying the central spacer group connecting the IGN monomers, and site of linkage on in vitro potency and the interaction with DNA. A set of IGN molecules that met our potency criteria were identified and linkable forms of these compounds were prepared and conjugated to a folate receptor-α (FRα)-binding antibody. These ADCs displayed potent, antigen-specific in vitro activity across a panel of FRα-expressing cell lines. A lead ADC selected for in vivo studies demonstrated high stability in plasma and potent efficacy in various xenograft models, at doses well below the maximum tolerated dose. Thus, IGNs modified with an N-10 self-immolative peptide linker are promising DNA alkylating effector molecules for use in ADCs.

#225

TR1801-ADC, an optimized anti cMet PBD ADC with high efficacy in solid tumors of the GI tract and head & neck cancer.

Marco Gymnopoulos,1 Oscar Betancourt,1 Vincent Blot,2 Ryo Fujita,3 Diana Ly,1 Sophie Nguyen,1 Jeanette Snedden,1 Jose Villicana,1 Jon Wojciak,1 Eley Wong,4 Neki Patel,5 Francois D'Hooge,6 Balakumar Vijayakrishnan,5 Conor Barry,5 John A. Hartley,7 Phil W. Howard,5 Roland Newman,1 Julia Coronella1. 1 _Tanabe Research Laboratory USA, Inc., San Diego, CA;_ 2 _AbbVie Inc., North Chicago, IL;_ 3 _Mitsubishi Tanabe Pharma Corporation, Toda-shi, Japan;_ 4 _Neurocrine Biosciences, Inc., San Diego, CA;_ 5 _Spirogen Ltd, London, United Kingdom;_ 6 _GamaMabs Pharma SA, Toulouse, France;_ 7 _University College London, London, United Kingdom_.

cMet is a well-characterized oncogene that is the target of many drugs including small molecule and biologic pathway inhibitors, and more recently, antibody drug conjugates (ADCs). However, clinical benefit from c-Met targeted therapy has been limited up to this point. We developed an optimized c-Met targeted antibody drug conjugate TR1801-ADC that utilized optimization at all steps including specificity, stability, internalization, toxin linkers, conjugation site, PK, and in vivo efficacy. The results were a highly potent c-Met ADC that was superior in comparison to a conventional MMAE (monomethylauristatin E) cMet ADC in potency, efficacy and duration of response. TR1801-ADC is site-specifically conjugated to a PBD-toxin linker and has low picomolar activity in multiple cancer cell lines derived from different solid tumors including lung cancer, colorectal cancer and gastric cancer. The potency of our cMet ADC is independent of MET gene copy number and its activity was high not only in high cMet cell lines but also in medium to low cell lines (100,000 to 40,000 cMet/cell). We identified potential Phase 1 cancer indications based on IHC and cMet H-score from tissue micro arrays (TMAs). 3 cancer indications (gastric, colorectal and head & neck cancer) were chosen based on high abundance of high cMet expression (>20% abundance of H-Score higher than 150) and were used to assess the efficacy of our cMet ADC in PDX models. For each indication 10 PDX (patient-derived xenograft) models were chosen based on their cMet expression. TR1801-ADC was administered in all models as single i.v. dose at 1, 0.5, 0.25 and 0.125 mg/kg. Control ADC was administered at 1mg/kg. Tumor growth inhibition varying from >100% to around 40% was seen in all gastric cancer PDX models with single dose injections of 1 mg/kg and 0.5 mg/kg. Complete tumor regression was seen in 70% of the 1 mg/kg group, in 50% of the 0.5 mg/kg group and in 40% of the 0.25 mg/kg group. TR1801-ADC produced statistically significant growth inhibition in comparison to untreated controls in 9 colorectal cancer PDX models. Complete tumor regression was observed in 40% (4/10) PDX models when treated with 1 mg/kg TR1801-ADC. The further 50% showed partial regression (5/10 models) and one showed no significant tumor response. The head & neck cancer PDX models were in general least sensitive to TR1801-ADC. TR1801-ADC produced statistically significant growth inhibition in comparison to untreated controls in 8/10 head & neck cancer PDX models. Complete tumor regression was observed in 30% (3/10) PDX models when treated with 1 mg/kg TR1801-ADC. 50% showed statistical significant partial regression while two models (20%) showed no significant tumor inhibition. Altogether, TR1801-ADC is a novel highly potent cMet ADC that shows very promising efficacy in PDX models of gastric, colorectal and head & neck cancer with long lasting anti-tumor effect with a single dose.

#226

Optimization of folate receptor alpha-targeting tunable drug conjugates.

Sara C. Sigler, Vinayak Gupta, Hanh N. Nguyen, Michael K. Rood, Jutta Wanner, Doug S. Werner. _BlinkBio, Inc, Jupiter, FL_.

We have integrated a family of innovative technologies with an aligned set of properties into Tunable Drug Conjugates (TDCs). TDCs are made up of a targeting moiety (e.g. small molecules, peptides, antibodies or antibody fragments), combined with our proprietary Silicon-based linkers (SiLinkers) and novel Payload Cassette (PC) designs which allow for delivery of multiple payloads, preferentially Conditionally Active Payloads (CAPs). CAPs are cytotoxic moieties which cannot readily permeate into normal cells due to their charged state at physiological pH of 7.4; however, at the more acidic pH of the endosome or the tumor microenvironment, the charge is neutralized enabling the CAP to readily permeate into and accumulate within the cancer cells. Our goal is to establish a beneficial Therapeutic Index (TI) and dosing flexibility by creating TDCs around a rapid payload release/rapid systemic clearance approach. Therefore, SiLinker/CAP constructs are designed to concentrate payload in the targeted tumor cell and associated tumor microenvironment while minimizing systemic payload release and rapidly clearing the parent TDC. Folate Receptor alpha (FRα) is overexpressed in multiple cancers, most notably, ovarian, endometrial, and NSCLC. Our optimized FRα-targeting TDCs use folic acid as the ligand and deliver various triple- and hexa-CAP cassettes. The various CAPs were selected from different target classes in order to allow for optimized treatment of different cancer types. We will present comparative cellular data, in vivo xenograft studies in KB, OV90 and IGROV1 models, as well as DMPK parameters of our lead FRα-TDCs. Using our triple- and hexa-PCs, we have achieved cures in in vivo xenograft studies which were durable and lasted even after treatment was stopped. We also demonstrate that in vivo our hexa-PC shows superior efficacy compared to our triple-PC in the moderate FRα expressing models. Based on our data, we believe that the combined properties of our innovative CAP-TDCs and rapid payload release/rapid systemic clearance approach will further increase TI, differentiate from other Drug Conjugates, and ultimately provide a compelling addition to existing Drug Conjugate technologies.

#227

BPRDP056, a novel small molecule drug conjugate specifically targeting phosphatidylserine for cancer therapy.

Chia-Yu Hsu, Yun-Yu Chen, Chen-Fu Lo, Tai-Yu Chiu, Ching-Ping Chen, Chen-Lung Huang, Chung-Yu Huang, Min-Hsien Wang, Yu-Sheng Chao, Joe C. Shih, Teng-Kuang Yeh, Lun K. Tsou, Chiung-Tong Chen. _Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli County, Taiwan_.

Zinc(II)-dipicolylamine (Zn-DPA) have been described to specifically complex with phosphatidylserine (PS), which has a higher level at the external surface of cancer cells in tumorigenic condition. BPRDP056 is a Zn-DPA-SN38 conjugate designed to provide a PS-targeting drug delivery of cytotoxic payload SN-38 at the tumor microenvironment, thereby, decrease the dosage of SN38, while induce apoptosis in cancer cells. The in vivo therapeutic efficacy of BPRDP056 against the growths of human tumors has been shown significant in mice subcutaneously bearing a tumor type of pancreas, prostate, colon, liver, breast and glioblastoma, as well as in mice with an orthotopic pancreatic tumor. BPRDP056 shrunk tumors at a lower dosing intensity (~20%) of SN38 compared to CPT-11 in all models tested. Micro-Western assays showed that BPRDP056 exhibited apoptotic cell death signal levels similar to those of CPT-11 in the treated tumors in mice. Furthermore, pharmacokinetic and preliminary toxicology studies showed that BPRDP056 has a good stability in circulation with an acceptable therapeutic safety window in mice. BPRDP056 has been demonstrated with a tumor targeting ability and thus increases the cytotoxic payload SN38 concentration in situ for improved efficacy. Its therapeutic spectrum against malignant neoplasm will be expected to cover the PS-rich tumor microenvironment of all cancer types. BPRDP056 is a first-in-class Small Molecule Drug Conjugate for anti-cancer therapy.

#228

KSPi-ADCs: ADCs with novel kinesin spindle protein inhibitor payloads and a tailor-made linker chemistry.

Hans-Georg Lerchen,1 Beatrix Stelte-Ludwig,1 Anette Sommer,2 Sandra Berndt,2 Anne-Sophie Rebstock,3 Sarah Johannes,1 Leo Marx,4 Christoph Mahlert,1 Simone Greven,1 Lisa Dietz,1 Hannah Joerissen,1 Hilmar Weinmann2. 1 _BAYER AG, Wuppertal, Germany;_ 2 _BAYER AG, Berlin, Germany;_ 3 _BAYER AG, Lyon, France;_ 4 _Debiopharm, Martigny, Switzerland_.

Inhibitors of kinesin spindle protein (KSP/Eg5) have raised great interest because of their high antitumor potency which, however, could not be transferred into highly efficient clinical regimens due to dose limiting side effects such as neutropenia. We have developed a new, highly potent pyrrole subclass of KSPis as a novel payload class in ADCs. To increase the therapeutic window, a tailor-made effector chemistry has been designed. The tumor associated protease legumain (LGMN) is utilized for ADC cleavage to provide active metabolites with high and long-lasting exposure in tumors, thus matching the KSPi mode of action. LGMN is a lysosomal asparaginyl endopeptidase overexpressed in solid tumors which is associated with invasion, metastasis and poor survival. In our studies, we show an efficient LGMN mediated linker cleavage and payload release from different ADCs. In addition, the unique LGMN substrate sequence allows for high cleavage specificity and discrimination versus other proteases such as elastase, which has been associated with an undesired extracellular cleavage of e.g. vc-MMAE ADCs resulting in neutropenia. Variations of the linker chemistry were performed to retain high potency of the KSPi-ADCs against tumor cells while decreasing the activation of KSPi-ADCs in cells of healthy organs such as the liver. From this approach, highly potent KSPi-ADCs with improved tumor/organ ratios of active metabolites were obtained. In conclusion, ADC metabolism under legumain control is a versatile strategy to provide KSPi-ADCs with high potency and an improved safety profile.

#229

Utilizing a mouse cross-reactive model system to better understand antibody-drug conjugate pharmacokinetics, biodistribution and efficacy.

Leanne Lanieri, Rassol Laleau, Bahar Matin, Jenny Lee, Steven Boule, Paulin Salomon, Luke Harris, Michael Miller, Nicholas C. Yoder, Yulius Setiady, Neeraj Kohli, Thomas A. Keating, Jan Pinkas, Richard Gregory. _ImmunoGen, Inc., Waltham, MA_.

Antibody-drug conjugates (ADCs) are designed to deliver a potent cytotoxic payload directly to tumors, thus limiting exposure in normal tissues. However, target antigen expression on normal tissues can lead to lower systemic ADC exposures, resulting in sub-efficacious concentrations at the tumor site as well as heterogeneous distribution within tumors. Traditional preclinical efficacy studies performed in rodent models using ADCs with non-cross-reactive antibodies have been of limited translational relevance, and a better understanding of the factors that impact ADC dose and activity remains to be elucidated. To examine relationships between variables that could influence ADC efficacy, we generated a cross-reactive model system that utilized a chimeric anti-murine folate receptor α (FRα) antibody (designated rmFR1-12) that binds both mouse and human FRα, and can be conjugated to either maytansinoid (DM) or indolinobenzodiazepine (IGN) payloads. This model system was predicted to have substantial target-mediated drug disposition (TMDD) due to normal tissue expression of FRα. An rmFR1-12-s-SPDB-DM4 ADC was made using tritium-labeled DM4 and administered to tumor-bearing mice in order to assess ADC pharmacokinetics (PK), biodistribution, and efficacy. This approach allowed tracking of the ADC at multiple levels – whole animal, organ, tumor, and cell. Studies were undertaken that assessed the impact of xenograft antigen expression, ADC dose, and ADC drug-to-antibody ratio (DAR) on the PK, biodistribution, and efficacy of the rmFR1-12-s-SPDB-DM4 conjugate. The results showed that TMDD significantly impacted the PK, biodistribution, and activity of the conjugate relative to a non-cross-reactive ADC, with lower ADC doses being more severely impacted than higher doses. Antigen expression positively correlated with local ADC exposure and efficacy. Decreasing the DAR (by co-dosing naked antibody with the ADC) increased systemic exposure. A positive correlation between systemic exposure and dose of naked antibody was observed. Factors that impacted local exposure included: the type of tissue (normal vs tumor), presence or absence of FRα expression on normal tissue, and dose of naked antibody. Of note, in the naked antibody plus ADC study, efficacy did not correlate with local exposure - suggesting that ADC distribution within tumors is of similar importance as the amount of ADC delivered. Overall, these findings underscore the importance of accounting for site, and extent of, normal tissue target expression with respect to ADC PK/PD, and the data generated from these studies are currently being used to build a multiscale physiologically based PK model of a cross-reactive ADC.

#230

Antibody-drug conjugates (ADCs) with indolinobenzodiazepine dimer (IGN) payloads: DNA-binding mechanism of IGN catabolites in target cancer cells.

Rajeeva Singh, Luke Harris, Paulin Salomon, Emily E. Reid, Michael L. Miller, Ravi V. Chari, Thomas A. Keating. _ImmunoGen, Inc., Waltham, MA_.

A DNA-interacting indolinobenzodiazepine dimer (IGN) payload was designed with a single reactive imine group towards the goal of eliminating DNA cross-linking and avoiding related toxicities, while conferring a strong binding of the IGN scaffold to duplex DNA. Several IGN ADCs, wherein the payload is linked via a peptide or hindered disulfide, are currently being evaluated in the clinic. In contrast to our lead IGNs, DNA-interacting pyrrolobenzodiazepine (PBD)-based ADC payloads, such as talirine and tesirine, contain two reactive imine groups that can cross-link DNA.

Here, we investigated the mechanism of binding of IGN catabolites with DNA in target cancer cells, and with model duplex DNA or hairpin oligonucleotides. Hairpin and duplex oligonucleotides, designed for high melting temperatures (around 50-60 °C), were custom synthesized with labels. Model IGN catabolites bearing a single imine (mono-imine) or two imine groups (di-imine) were synthesized with a biotin label. Sensitive assays were developed to measure IGN-DNA binding in cells at sub-cytotoxic concentrations (lower than IC50) to allow studies of DNA adduct stability and repair.

The mono-imine IGN molecules bind readily to oligonucleotides, generating stable adducts as determined by gel filtration and reversed phase HPLC analysis. To investigate the binding of unconjugated IGNs with cellular DNA, cancer cells were incubated with mono- and di-imine IGNs for a short-term, followed by wash and further incubation in fresh media. Both mono-and di-imine IGN molecules remained bound to genomic DNA even at 2 days, suggesting a potent interaction with cellular DNA. The time course of binding of IGN to DNA in cells was slower than that observed with model oligonucleotides, as expected because the tightly coiled cellular DNA presumably binds IGN only after the unwinding of DNA during cell cycle or transcription.

Upon DNA cleavage by an added nuclease, free IGN was released from IGN adducts of model oligonucleotides and from genomic DNA of cells that had been treated with unconjugated IGN or IGN ADC. This dissociation of IGN from IGN-DNA adducts only upon cleavage with nuclease suggests that a strong non-covalent interaction between IGN and duplex DNA stabilizes the adduct. The amount of free IGN released from cellular DNA adduct upon nuclease treatment was about 2-fold greater for mono-imine IGN than di-imine IGN, presumably because di-imine IGN was partly cross-linked to cellular DNA. Mono-imine IGN-DNA adducts could potentially be repaired by cellular endonucleases via a DNA cleavage mechanism. In conclusion, the mono-imine IGN payload molecules form highly stable adducts with DNA, which dissociate upon DNA cleavage at physiological temperature.

#231

Optimizing lysosomal activation of antibody-drug conjugates (ADCs) by incorporation of novel cleavable dipeptide linkers.

Paulin Salomon, Luke Harris, Emily E. Reid, Erin K. Maloney, Alan J. Wilhelm, Michael L. Miller, Ravi V. Chari, Thomas A. Keating, Rajeeva Singh. _ImmunoGen, Inc., Waltham, MA_.

Peptides constitute a major linker class in antibody-drug conjugates (ADCs), designed to connect antibodies to cytotoxic drug molecules. An optimal linker for an ADC should be stable in circulation and be cleaved efficiently in lysosomes upon binding and internalization of the ADC in target cells, releasing drug molecules that exit lysosomes and inhibit key cellular functions. Previous studies have focused on a limited selection of peptide linkers and specific biochemical tools, such as a cathepsin B cleavage assay, leading to the selection of a valine-citrulline dipeptide linker with a p-aminobenzyloxycarbonyl (PABC) spacer, which is now often used in candidate ADCs.

In this study, we screened a panel of dipeptide linkers for efficient lysosomal proteolysis. Dipeptide linkers bearing different amino acids (typically both L, with D used as a control) were synthesized with a fluorogenic leaving group, 7-amino-4-methylcoumarin, which provided a convenient model system for screening of peptide cleavage using lysosomal extracts from cancer cells as well as several individual cathepsins. The linkers were also tested for stability in mouse, rat, cynomolgus, and human plasma.

Based on these screens, we identified several novel, previously unreported peptide linker designs and incorporated them into ADCs bearing a DNA-alkylating indolinobenzodiazepine (IGN) payload. These ADCs with different peptide linkers were assayed for in vitro cytotoxicity in multiple cancer cell lines, in vivo efficacy in human tumor xenograft models in mice, and ex vivo plasma stability. In addition, we measured the impact of peptide linkers on the kinetics of proteolytic processing of ADCs in cancer cell lines. We observed that several dipeptide linker designs were superior in rates of lysosomal processing compared to a reference standard L-Ala-L-Ala dipeptide linker.

#232

An antibody-drug conjugate carrying a microtubule inhibitor and a DNA alkylator exerts both mechanisms of action on tumor cells.

Jeremy R. Duvall, Marc Damelin, Mariya V. Kozytska, Barrett J. Nehilla, Marina Protopopova, Patrick R. Conlon, LiuLiang Qin, Mark Nazzaro, Josh D. Thomas, Qingxiu Zhang, Dorin Toader, Timothy B. Lowinger . _Mersana Therapeutics, Inc., Cambridge, MA_.

Antibody-drug conjugates (ADCs) consist of a tumor-targeted antibody, a drug (payload) with specified mechanism of action, and the chemical framework for attaching them to each other. By selective delivery of the payload to the tumor and not to normal tissues, ADCs can provide greater efficacy and tolerability than systemic chemotherapies, which can translate to longer duration of treatment and response, as well as more options for combination therapies without the concern of overlapping toxicity. To preempt potential resistance to therapy, we have engineered a dual-payload ADC (DP-ADC) that delivers two mechanistically distinct payloads to a single target cell.

To build a precision dual payload-ADC (DP-ADC), we leveraged our Synthemer platform, which enables us to chemically attach the payloads to a synthetic scaffold in a defined manner, and then to chemically attach the loaded scaffold to the antibody. This approach allows for unambiguous control of architecture and ratio of the payloads: the payloads are incorporated in a fixed ratio into the scaffold and subsequently into the ADC. Moreover, the synthetic design also allows for selection of solubilizing groups and charge compensation for each payload, allowing for optimal properties of the ADC.

Our prototype DP-ADC combines the microtubule inhibitor auristatin F hydroxypropyl amide (AF-HPA) with the DNA monoalkylator I-BiP (related to pyrrolobenzodiazepine), at a payload ratio of 3:1. The drug-to-antibody ratio (DAR) was 12 AF-HPA and 4 I-BiP (16 total), achieved by conjugating 4 of the loaded dual-payload scaffolds to the antibody. The DP-ADC exhibited comparable antigen binding to the unconjugated antibody, and cytotoxic potency comparable to both single-payload ADCs alone. Using cell-based assays that specifically probe the mechanism of action of each payload, we demonstrated that the DP-ADC exerted both expected mechanisms of action on the tumor cell in a target-dependent manner.

Considerations for the selection of payload combinations will also be presented. While synergistic action is typically assumed to be preferred if not required, a recent study demonstrated that the observed clinical benefit of most combinations can be explained by the drugs' independent modes of action, not synergy. Indeed, AF-HPA and I-BiP did not exhibit synergy in cytotoxicity studies in cancer cell lines, yet their combination in a DP-ADC is still expected to confer potential clinical benefit over either single-payload ADC across a patient population.

#233

Bispecific HER2 ADC: Making more potent HER2 ADC by improving target internalization.

Andres Perez Bay, Anuj Kalsy, Shiwani Tiwari, Bo Luan, Art Kunz, Zhaoyuan Chen, Li Zhang, Terra Potocky, Thomas Nittoli, Gavin Thurston, Christopher Daly, Julian Andreev. _Regeneron Pharmaceuticals, NY_.

Trastuzumab-emtansine (T-DM1) targets the well-characterized breast cancer oncogene HER2, and has shown success in the clinic, but tumors expressing intermediate levels of HER2 remain resistant to T-DM1 therapy, apparently due to insufficient lysosomal trafficking of T-DM1. Here, we engineered bispecific HER2-DM1 conjugates that bridge HER2 with the rapidly internalizing receptor APLP2. The high affinity arm of HER2xAPLP2 bispecific antibodies mediates surface binding to HER2+ tumor cells (but not to HER2- normal cells). After binding to HER2, the low affinity APLP2 arm promotes antibody internalization and lysosomal degradation. The strength of the APLP2 arm allows for modulation of the degree of internalization, degradation and killing. In in vitro killing assays, HER2xAPLP2-DM1 were significantly more potent than T-DM1 in cell lines expressing high (IHC3+) and intermediate (IHC 2+) levels of HER2, albeit induced little or no killing in cell lines expressing low HER2 levels (IHC 1+). Monovalent APLP2-ADCs induced little or no killing in all cell lines tested. Selected HER2xAPLP2-DM1 conjugates had acceptable PK profile in APLP2 humanized mice while significantly outperforming T-DM1 in inhibiting growth of IHC2+ tumor xenografts. These data pave the way to generate clinical candidate ADCs that will improve on T-DM1. In addition, our study opens up a possibility to exploit internalizing proteins such as APLP2 in combination with bispecific antibodies to enhance efficacy of ADCs.

#234

Pre-clinical characterization of 3A4-PL1601, a novel pyrrolobenzodiazepine (PBD) dimer-based antibody-drug conjugate (ADC) directed against KAAG1-expressing malignancies.

Francesca Zammarchi,1 Francois Bertelli,2 Karin Havenith,1 Ian Kirby,1 Simon Chivers,1 Patrick H. van Berkel1. 1 _ADC Therapeutics, London, United Kingdom;_ 2 _Spirogen/MedImmune, London, United Kingdom_.

Kidney-associated antigen 1 (KAAG1) is an 84 amino acid protein encoded by the reverse strand of a housekeeping gene called DCDC2 which was identified from a cDNA library derived from a histocompatibility leukocyte antigen-B7 renal carcinoma cell line as an antigenic peptide presented to cytotoxic T lymphocytes. More recently, KAAG1 has been identified by a sensitive subtractive cloning technology called STAR as a novel tumor-associated antigen expressed in a high percentage of ovarian tumors and triple-negative breast cancers (TNBCs), while it has restricted normal tissue expression. 3A4 is a humanized antibody raised against human KAAG1, which binds to KAAG1 expressed on the surface of cancer cells, rapidly internalizes and co-localizes with LAMP-1, a lysosomal marker. Altogether, KAAG1 represents an attractive target for an antibody-drug conjugate (ADC) approach based on its high and selective expression in various malignancies and its restricted expression in healthy tissues. 3A4-PL1601 is an ADC composed of the 3A4 antibody, site-specifically conjugated using GlycoConnectTM technology to PL1601, which contains HydraspaceTM, a valine-alanine cleavable linker and the PBD dimer cytotoxin SG3199. The purpose of this study was to characterize the in vitro and in vivo anti-tumor activity of 3A4-PL1601 in human cancer cell lines and xenografts models and to determine its safety and tolerability in the cynomolgus monkey. In vitro, the 3A4-PL1601 demonstrated potent cytotoxicity in a panel of human cancer cell lines of different origin and levels of KAAG1, while its potency was strongly reduced in KAAG1-negative cell lines. In vivo, 3A4-PL1601 showed potent and specific anti-tumor activity in the TNBC-derived MDA-MB-231 xenograft compared to the vehicle- and isotype control ADC-treated mice. A single dose of 3A4-PL1601 at 0.6 mg/kg resulted in 4/8 partial responders (PR) and 3/8 complete responders, two of which were tumor-free survivors at the end of the study on day 59, while none of the mice treated with the vehicle or the isotype-control ADC showed any activity. Moreover, 3A4-PL1601 showed potent and dose-dependent anti-tumor activity in the KAAG1-expressing, human renal cell carcinoma-derived SN12C xenograft when tested as single dose at 0.3, 0.6 or 1 mg/kg. At the highest dose tested, 3A4-PL1601 resulted in sustained tumor growth control and 2/8 PRs at the end of the study on day 60. 3A4-PL1601 was stable, well tolerated (MTD at least 0.8 mg/kg) and showed a favorable pharmacokinetic profile (t1/2 ~ 6 days) in the cynomolgus monkey (cross-reactive species). In conclusion, 3A4-PL1601 demonstrated potent and specific in vitro and in vivo anti-tumor activity in KAAG1-expressing cancer-derived models and it was stable and well tolerated in cynomolgus monkey, warranting further development of this ADC into the clinic.

#235

Preclinical activity of an antibody drug conjugate targeting tumor specificmuc1 structural peptide-glycotope.

Marc Trombe,1 Anne Caron,1 Alexia Tellier,1 Chantal Carrez,1 Stephane Guérif,1 Severine Clavier,1 Nathalie Karst,1 Juhani Saarinen,2 Tero Satomaa,2 Virve Pitkänen,2 Olli Aitio,2 Annamari Heiskanen,2 Matteo Fassan,3 Jan Pinkas,4 Raffaele Baffa,5 Veronique Blanc,1 Celine Nicolazzi1. 1 _Sanofi, Vitry Sur Seine, France;_ 2 _Glykos, Helsinki, Finland;_ 3 _University of Padova, Padua, Italy;_ 4 _ImmunoGen, Waltham, MA;_ 5 _Sanofi, Cambridge, MA_.

SAR566658 antibody drug conjugate (ADC) is a humanized DS6 (huDS6) antibody conjugated through a cleavable linker to the cytotoxic maytansinoid derivative DM4 that has been evaluated in the clinical setting. The purpose of our work was; 1) characterize the epitope targeted by anti DS6 on mucin1, 2) determine the prevalence of antigen expression in a patient population and 3) further explore preclinical activities of the ADC. Murine DS6 monoclonal antibody (muDS6) was generated from serous ovary adenocarcinoma immunization of immunocompetent mice. It specifically recognizes a MUC-1 tandem repeat domain in the context of cancer associated glycosylation. CA6-positive MUC-1 carries mucin-type O-linked glycans with α2,3-sialylated and β1,4-galactosylated termini, and antibody binding was abrogated by treating MUC-1 with specific glycosidases that remove either one of these glycan structures. However, the antibody did not bind to synthetic glycans modeled according to the major O-glycans of MUC-1. Our characterization of the peptide-glycotope leads us to conclude that tumor associated glycosylation is essential for the formation of the epitope on the peptide sequence of the MUC-1 tandem repeat domain. CA6 expression was evaluated by immunohistochemistry in paraffin embedded tumor tissue samples: 35.2% of breast cancer patients, 70,1% of ovarian cancer patients and 58,5% of bladder cancer patients have at least 30% of CA6 positive cells with an intensity of 2+/3+ in a multinational population-based study. In pre-clinical in vivo models, SAR566658 induced anti-tumor activity against CA6 positive tumor models of human pancreas, cervix and bladder cancer as well as and 3 Breast Patient-Derived Xenografts (PDX). Efficacy was correlated with MUC1-CA6 expression levels. In 3 additional models, SAR566658 showed anti-tumor activity that was more potent when compared to 3 conventional tubulin cytotoxic agents, docetaxel, vinorelbine and vinblastine.

#236

CLDN18.2 ADC for gastric and pancreatic cancer.

Guoyun Zhu,1 Davide Foletti,1 Jody Melton-Witt,1 Adela Hasa-Moreno,1 Mathias Rickert,1 Charles Holz,1 Laura Aschenbrenner,2 Eugenia Kraynov,3 Leslie Obert,4 Timothy M. Coskran,4 Shobha Potluri,1 Pavel Strop,1 Shu-Hui Liu1. 1 _Pfizer Cancer Immunology Discovery / Oncology Research, South San Francisco, CA;_ 2 _Pfizer Drug Safety Research and Development (DSRD), La Jolla, CA;_ 3 _Pfizer BMD/PPD / Oncology Research, La Jolla, CA;_ 4 _Pfizer Drug Safety Research and Development (DSRD), Groton, CT_.

The claudin multigene family encodes tetraspan membrane proteins that are crucial structural and functional components of tight junctions. Human CLDN18.2, a splice variant of claudin 18, is highly expressed in significant proportions of primary and metastatic gastric cancer. It is also upregulated in pancreatic and esophageal adenocarcinoma. An antibody targeting CLDN18.2, claudiximab, in combination with chemotherapy, epirubicin, oxaliplatin, and capecitabine (EOX), showed promising results in clinical trials of gastric cancer patients by increasing PFS from 4.8 to 7.9 months and OS from 8.4 to 13.2 months in a phase II study. Antibody-Drug Conjugate (ADC) represents a promising therapeutic modality for improving the clinical management of cancer. We described here a hCLDN18.2 ADC, site-specifically conjugated to auristatin-0101 and cross-reactive to human, mouse and rat CLDN18.2, displayed potent cytotoxicity in engineered CLDN18.2 over-expressing tumor cell lines and xenograft models. The anti-CLDN18.2 ADC exhibited in vitro cytotoxicity to BxPC3/hCLDN18.2 (IC50 =1.52nM) and KATO-III/hCLDN18.2 (IC50 =1.60nM) tumor cells. In addition, The ADC (up to 6mg/kg tested) dose-dependently inhibited tumor growth of pancreatic PDX model Pan-1907 and gastric PDX model CTG-1010. In a rat ETS study, CLDN18.2 ADC was tolerated to 10 mg/kg, potentially presenting a suitable therapeutic window. Taken together our data indicated that targeting CLDN18.2 with ADC can be another promising clinical approach for gastric and pancreatic cancer patients.

#237

Preclinical evaluation of anti-HER2 Antibody Targeted Amanitin Conjugates (ATACs) on HER2low breast cancer with chromosome 17p deletion.

Christian Breunig, Anikó Pálfi, Michael Kulke, Christian Lutz, Christoph Müller, Torsten Hechler, Andreas Pahl. _Heidelberg Pharma Research GmbH, Ladenburg, Germany_.

Background:

Triple negative breast cancer (TNBC) is the most difficult to treat subtype of breast cancer with limited therapeutic options. At least 50% of TNBC patients have low epidermal growth factor receptor 2 (HER2; ERBB2) expression with the majority harboring hemizygous loss of POLR2A/chromosome 17p. For these patients the treatment with antibody-targeted amanitin conjugates (ATACs) targeting HER2 is a new promising approach. ATACs comprise a new class of antibody-drug conjugates (ADCs) using amanitin as toxic payload and are able to kill antigen low expressing cells. Amanitin binds to the eukaryotic RNA polymerase II and thereby efficiently inhibits the cellular transcription process. In the current study, in vitro and in vivo data of ATACs targeting human HER2low as well as tolerability studies are presented. HER2low TNBC is considered an interesting target for amanitin-based ADCs.

Material and methods:

Different HER2 expressing cell lines were treated with anti-HER2 ATACs. Cysteine reactive amanitin-linkers were conjugated site-specifically to engineered cysteine residues of an anti-HER2 antibody yielding ATACs with a DAR of 2.0. Quantitative determination of cell viability was analyzed by BrdU ELISA assay. Subcutaneous mouse xenograft models with HER2-positive cell lines were performed with single-dose treatments. In addition, ATAC efficacy was tested in HER2low heterogeneous TNBC patient derived xenograft (PDX) models with and without POLR2A deletion. Tolerability of ATACs was assessed in mice and non-human primates (NHP).

Results:

Anti-HER2 ATACs showed in vitro cytotoxicity on HER2+ high and low cell lines in low nanomolar to picomolar range. In mouse xenograft models, the anti-HER2 ATACs caused dose-dependent tumor regression independent of Her2 expression level. In HER2low heterogeneous TNBC PDX models anti-HER2 ATACs caused dose-dependent tumor regression. The efficacy of anti-Her2 ATACs was more pronounced in PDX models with hemizygous loss of TP53 and POLR2A reflecting a 17p deletion. Safety profiling of an optimized anti-Her2 ATAC in cynomolgus monkeys revealed a good tolerability indicating a good therapeutic window for 17p deleted TNBC.

Conclusions:

Targeted cytotoxic drug delivery to HER2 positive cell lines was achieved by using anti-HER2 ATACs. The mode of action of the payload amanitin led to an efficient anti-tumor potential in vitro and in vivo with good tolerability in NHP studies. TNBC PDX models with HER2low expression were sensitive to ATAC treatment. Loss of POLR2A/chromosome 17p increased susceptibility to anti-HER2 ATAC making 17p del TNBC a suitable indication for optimized anti Her2 ATACs.

### Brain Cancers

#238

Therapeutic activity of the non-internalizing antibody drug conjugate 1959-sss/DM3 targeting galectin3-binding protein in human neuroblastoma.

Emily Capone,1 Sara Ponziani,2 Francesco Giansanti,2 Roberta Gentile,2 Giulia Di Vittorio,2 Vincenzo De Laurenzi,1 Michele Sallese,1 Sandra Bibbò,1 Arturo Sala,3 Rodolfo Ippoliti,2 Jean Frederic Sauniere,4 Stefano Iacobelli,4 Gianluca Sala1. 1 _University of Chieti-Pescara, Chieti, Italy;_ 2 _University of Aquila, L'Aquila, Italy;_ 3 _Brunel London University, London, United Kingdom;_ 4 _Mediapharma srl, Chieti, Italy_.

Neuroblastoma is a solid tumour affecting the peripheral nervous system accounting for ∼13% of all pediatric cancer mortality. Despite immunotherapy with the GD2 antibody has improved the clinical outcome of a fraction of patients with metastatic neuroblastoma, the majority of patients with relapsing, high risk disease cannot be successfully treated. Recently, it has been shown that non-internalizing, linker-less antibody drug conjugates (ADCs) targeting the tumor extracellular environment can exert a potent therapeutic activity against different tumors.

We identified LGALS3BP (aka Mac-2 BP or 90K) as an important target for non-internalizing ADC development, as the protein is largely secreted by the majority of human tumors, while being virtually undetectable in normal adult tissues.

In this study, we show that a variant of the humanized 1959 antibody, targeting human LGALS3BP, coupled to the maytansinoid drugs DM3 by means of disulfide linker, bound human neuroblastoma cell lines harboring or not N-myc amplification, although with different affinity. Importantly, normal human fibroblasts were not decorated by the antibody. The ADC potently inhibited metastatic lesions in the liver and lungs of NSG mice injected intravenously with the neuroblastoma cell line SKNAS. Our findings offer a preclinical proof-of-concept for the development of a non-internalizing ADC endowed with potent therapeutic activity for neuroblastoma treatment through an innovative mechanism of action.

#239

Aurora kinase inhibition to enhance Tumor Treating Fields efficacy in glioblastoma treatment.

Paula Bartmann,1 Silvia Roosz,1 Achim Temme,1 Moshe Giladi,2 Eilon D. Kirson,2 Yoram Plati,2 Uri Weinberg,2 Adrian Kinzel,3 Verena Leidgens,4 Dietmar Krex1. 1 _Department of Neurosurgery, Dresden, Germany;_ 2 _Novocure Ltd., Haifa, Israel;_ 3 _Novocure, Germany;_ 4 _Novocure, Munich, Germany_.

Tumor Treating Fields (TTFields) have shown to be effective in prolonging progression-free and overall-survival and increasing the rate of two- and five-year survivors of patients with primary glioblastoma. However, the two-year survival rate is still below 50%. A promising approach to enhance the efficiency of TTFields is the use of drugs which extend metaphase-anaphase transition and telophase. In a previous study we tested the efficacy of the combined treatment of TTFields and the Aurora B kinase inhibitor AZD1152 in different established glioma cell lines: U87-MG, U87-MGshP53 and U-251. We found that the combined treatment of TTFields and AZD1152 led to a significant reduction in the number of glioma cells in all three cell lines as compared to each treatment alone.

In the present study we analyzed primary tumor-cell-lines to validate these data. In addition, we tested MLN8237, an Aurora A kinase inhibitor, to confirm that Aurora kinase inhibition is a valuable target for a combination therapy with TTFields.

Primary tumor-cell lines were established from glioblastoma tissue taken intraoperatively. TTFields (1.6 V/cm RMS, 200 kHz) were applied for 72 hours using the inovitro system. AZD1152 was added to the media in concentrations of up to 100 nmol/l. Cell counts, cell cycle and clonogenic potential were determined at the end of treatment. Formation of multinuclear cells was determined using microscopic images of cells stained with crystal violet. MLN8237 was used in concentrations up to 50 nmol/l for the treatment of U87 MG cells.

The combined treatment of TTFields and AZD1152 led to a significant reduction in the number of primary glioblastoma cells (Mann-Whitney-U-test, p<0.001) as compared to each treatment alone. Microscopy images of glioblastoma cells stained with crystal violet after treatment, revealed high prevalence of multi nuclear cells in cells exposed to TTFields and AZD1152 (25nM) as compared to cells treated with AZD1152 (25nM) alone. Cells treated with TTFields and higher doses of AZD1152 (50-100nM) demonstrated increased rates of pyknosis. The combined treatment of MLN8237 and TTFields also resulted in a significant decrease of U87 MG cell numbers compared to each treatment alone (Mann-Whitney-U-test, p<0.01).

The results presented in this work demonstrate that the combination of TTFields and aurora kinase inhibition can be an effective treatment against glioma cells. Based on the above, there is a strong rational to continue exploring the potential of combining TTFields and aurora kinase inhibition in early clinical trials.

#240

Therapeutic targeting of protein kinase ck2 in mycn-driven neuroblastoma.

Haiwei Lian,1 Ning Shen,2 Yun Zhou,1 Dun Li,2 Hui Feng2. 1 _Wuhan University Renmin Hospital, Wuhan, China;_ 2 _Boston University, Boston, MA_.

Amplification of the MYCN oncogene occurs in about 30-40% of advanced neuroblastoma cases and is the most reliable predictor of poor prognosis. Protein kinase CK2 (casein kinase II) is a highly conserved serine/threonine kinase that can be effectively inhibited by small molecules. In fact, CX-4945, a potent, specific, ATP-competitive CK2 inhibitor, is currently tested in clinical trials to treat multiple cancers. However, the role of CK2 in MYCN-driven neuroblastoma remains unknown. To determine the relevance of CK2 in MYCN-driven neuroblastoma pathogenesis, we analyzed published microarray data and compared the expression of the CK2 subunits in primary neuroblastoma patients samples. We found that both CK2α and CK2α' are upregulated in neuroblastoma patients samples with MYCN amplification, compared to the ones without MYCN amplification. To determine the effect of CK2 inhibition on neuroblastoma cells, we treated a panel of human neuroblastoma cell lines with the CK2 inhibitor CX-4945 and short hairpin RNA specifically targeting CK2α. CK2 inhibitor CX-4945 suppressed human neuroblastoma cell growth, mainly through inducing apoptosis, especially in the MYCN-amplified cells. More over, CK2α knockdown also induced cell apoptosis. In-vitro assay indicated that CK2 inhibition through either genetic or pharmacological approaches effectively reduced cell growth of human neuroblastoma cells primarily through induction of apoptosis. 14 days in-vivo treatment, CX-4945 significantly decreased tumor growth in mouse xenografts by subcutaneously implanting human KELLY neuroblastoma cells, but didn't affect their body weight. These studies characterize the role of CK2 in neuroblastoma development, and provide valuable information about the relevance of CK2 as a target in MYCN-driven neuroblastoma.

#241

An efficient cell-based assay for quantification of cellular polysialic acid in neuroblastoma.

Xiaoxiao Guo, Francis Mprah Barnieh, Jodie Malcolm, Amanda D Race, Goreti Ribeiro Morais, Steven D Shnyder, Paul M Loadman, Laurence H Patterson, Robert A Falconer. _University of Bradford, Bradford, United Kingdom_.

Introduction Polysialic acid (polySia) decorates the surface of NCAM (neuronal cell adhesion molecule) on neuroendocrine tumors, notably neuroblastoma and small cell lung cancer, and is strongly associated with poor prognosis and aggressive disease in patients in the clinic [1]. PolySia modulates tumor cell-cell and cell-matrix adhesion, migration, invasion and metastasis. SiRNA knockdown of polysialyltransferase (polyST) ST8SiaII, the enzyme primarily responsible for polySia synthesis in tumors, abrogates tumor cell migration and invasion. PolyST is a selective and largely unexplored therapeutic target for neuroblastoma dissemination [1].

Methods and Results We describe the development of a cell-based assay that quantifies the formation of polySia on the cell surface of neuroblastoma cells. This is a sensitive assay that detects the polySia that is released after pH adjustment, organic precipitation and acid hydrolysis. PolySia was first separated from cell lysate by mild acid hydrolysis and acetone precipitation. It was then purified by ethanol precipitation. We further analysed the role of lactonisation of polySia during organic precipitation. The purified polySia is further hydrolysed into sialic acid monomers, followed by DMB labelling and RPLC-fluorescence analysis. This method was validated by analysis of polySia in C6-ST8SiaII cells, following treatment with Endo-NF in a various concentrations. This method proved to be useful to assess polySia changes after treatment of cells with novel polyST inhibitors. The effect of lactonisation of polySia during organic precipitation was also investigated. It was found that lactonised polySia was mainly soluble in 90% acetone/90% ethanol, while natural polySia (non-lactonised) can be precipitated by ethanol in the presence salts. Based on the different solubility of polySia, a cost-efficient assay was designed to purify polySia released from cells with a low level of non-polySia-derived sialic acid contamination before further acid hydrolysis. With this method we quantified the different levels of cell-surface polySia-derived sialic acid between polySia-positive and polySia-negative cell lines. Furthermore, we successfully validated this method by Endo-NF treatment in C6-ST8SiaII cells: the EC50 of Endo-NF digestion of polySia in C6-ST8SiaII cells was approximately 5.4 pM. The assay has been successfully utilised to evaluate novel polyST inhibitors in vitro. [1] Falconer, R.A. et al., Curr. Cancer Drug Targets, 2012, 12, 925-939; [2] Al-Saraireh YMJ et al., PLoS ONE, 2013, 8:e73366.

#242

Analysis of temozolomide-loaded CLENs for treatment of human glioblastoma multiforme in vitro.

Alexander Arau, Ilia Pidroutchniak, Woong Jae Song, Robert Campbell. _MCPHS University, Worcester, MA_.

Introduction: Glioblastoma multiforme (GBM) is the most prevalent neoplasm of the central nervous system, and has a 100 percent mortality rate despite current standard of care. We previously reported the use of cell membrane lipid-extracted nanoliposomes (CLENs) for tumor targeting. The approach involved the extraction of lipids directly from breast cancer cell membranes to develop nanoliposomes to efficiently target the same cell population from which the lipids were derived. This current study involves the development of CLENs for treatment of drug resistance in GBM, based on optimized formulation properties from our research involving breast cancer.

Methods: Three liposome preparations were formulated with either U87-MG (human GBM) cell line membrane lipid extract (LE), DSPE-PEG5000 (PEG), or both, in addition to conventional components. The specific formulations contain DOPC, Cholesterol, LE, and PEG at ratios of 60/25/10/5 (Prep 1), 65/25/10/0 (Prep 2), and 70/25/0/5 (Prep 3), respectively. Temozolomide (TMZ) drug loading studies were determined by UV-spectroscopy. Cellular uptake of liposome preparations by U87-MG was determined with fluorescence intensity values using rhodamine-labeled liposomes in vitro. Cytotoxicity analysis of TMZ-loaded liposome preparations was evaluated using a sulforhodamine B (SRB) cytotoxicity assay.

Results: Liposome preparations were characterized for mean particle size (nm) and surface charge (mv) in the presence and absence of incorporated TMZ. Average particle size for formulations loaded with TMZ were 148 nm (Prep 1), 221 nm (Prep 2), and 153 nm (Prep 3), which were not significantly different from preparations without incorporated drug, 149 nm, 217 nm, and 148 nm, respectively. Average zeta potential values for formulations loaded with TMZ were 3.3 mV (Prep 1), -2.7 mV (Prep 2), and -7.9 mV (Prep 3). For preparations without TMZ, values were -5.1 mV (Prep 1), -3.1 mV (Prep 2), and -5.8 mV (Prep 3). Cellular uptake studies suggested that the inclusion of LE had no effect on uptake of the liposomes by U87-MG target cells, with minimal effect on cytotoxicity. Moreover, the cytotoxicity conferred by the preparation containing PEG in the absence of LE (Prep 3) was consistent with a greater drug-incorporation efficiency than Prep 1.

Conclusion: The role of LE incorporation for selective tumor targeting involving CLENs developed for GBM remains unclear, and seems to confer diminished cytotoxicity and formulation properties, when compared to favorable findings involving use of breast cancer cell lines published elsewhere. It is thus possible that the inclusion of LE in nanoliposomes for brain cancer may require more personalized optimization in relation to the brain tissue environment over the use of more standard or shared formulation considerations studied in other organ tissues. Optimization studies involving CLENs to test the theory are currently underway.

#243

Differential effects of combination treatment of biochanin A and statins on glioblastoma multiforme cell proliferation and cell metabolism.

Vilas S. Desai,1 Eric Buchhalter,1 Max Cabanzo,1 Arushi Tiwari,1 Gagan Kaushal,1 James C. Lai,2 Alok Bhushan1. 1 _Jefferson College of Pharmacy, Thomas Jefferson University, Philadelphia, PA;_ 2 _College of Pharmacy, Division of Health Sciences, Idaho State University, Pocatello, ID_.

Glioblastoma Multiforme (GBM) is a deadly brain cancer and represents the most common central nervous system (CNS) tumors in adults. Although GBM does not metastasize, its aggressive growth and invasive nature are responsible for poor patient prognosis. The current standard therapy for newly diagnosed GBM patients involves surgical resection, followed by radiation and chemotherapy with Temozolomide. However, its rapid rate of infiltration into normal brain tissue ultimately renders the therapy ineffective. Epidemiological studies on dietary isoflavones (e.g., genistein, biochanin A) have shown their anti-cancer potential in different cancers. Though primarily used in management of hyperlipidemia and cardiovascular diseases, statins are known to exert anti-proliferative effects. We therefore hypothesized that a combination treatment of biochanin A & statins (e.g., Atorvastatin, Lovastatin, Simvastatin, Fluvastatin and Pravastatin) exerts enhanced anticancer effects on GBM U-87 MG and T98 G cells. Our studies showed statins induced differential effects on viability of GBM cells in combination with biochanin A, with U-87 MG being more susceptible than T98 G cells. The combination treatment of biochanin A and atorvastatin also decreased invasion in U-87 MG cells. Additionally, cell metabolism studies using seahorse XFp analyzer showed a switch in their metabolic phenotype with an increase in mitochondrial respiration and a decrease in glycolytic activity with the combination treatment. We also performed metabolite extraction on GBM cells for a global unbiased profiling of metabolites using a single extraction procedure and dual separation analysis by LCMS. A differential analysis of alternative GBM treatment indicated statistically significant changes in carbohydrate and amino acid metabolism as well as alterations to various degradation pathway intermediates. Together, the combination treatment-induced effects on GBM cell lines are differential and our results may have potential implications in developing combination therapies with biochanin A in vivo and support the design of new and better therapies for the treatment of a lethal cancer like GBM.

#244

Inhibition of atypical PKC signaling enhances the sensitivity of glioblastoma cells towards Temozolomide therapy.

Avijit Dey, Rekha Patel, Tracess Smalley, Wishrawana Sarathi Ratnayake, Anisul Islam, Mildred Acevedo-Duncan. _USF, tampa, FL_.

Glioblastoma is a highly aggressive brain cancer which has a mean survival rate of only 12 months. Current treatment options of glioblastoma include chemotherapy and limited surgical resection. Temozolomide (TMZ) is the current therapeutic choice for chemotherapy but has severe limitations due to development of resistance that occurs by genetic modification and constitutive activation of several other pathways. Therefore, to overcome Temozolomide resistance and to prevent recurrence of the disease a more effective therapeutic approach is required. One of the signaling pathways that contribute to the aggressive behavior of glioma cells is the activation of PKC signaling. Of the various PKC isoforms, the atypical PKCs, PKC ζ and ι were found to be over-expressed in glioblastoma tissue samples compared with normal brain tissue and high atypical PKC levels were correlated with increased cellular proliferation and invasiveness of glioma cells. [4-(5-amino-4-carbamoylimidazol-1-yl)-2, 3-dihydroxycyclopentyl] methyl dihydrogen phosphate also known as ICA-1 was used as a specific inhibitor of PKC-ι and [8-hydroxynaphthalene-1,3,6-trisulfonic acid] also known as ζ-Stat was used as a specific inhibitor of PKC- ζ. T98G and U87 glioblastoma cells were treated with ICA-1 monotherapy (7.5µM), ζ-stat monotherapy (10 µM), TMZ monotherapy at varying doses, TMZ combined with ICA-1 (7.5µM) and TMZ combined with ζ-stat (10 µM) for five consecutive days and analyzed for cell viability by WST assay. Results exhibited that ICA (7.5µM) when combined with TMZ showed significantly increased cytotoxicity compared to TMZ monotherapy. Annexin-V/PI assay revealed that combination treatment with TMZ and ICA nucleotide (7.5µM) significantly increased the number of dead cells and the number of cells undergoing late apoptosis when compared to TMZ monotherapy. This study offers the first evidence for the novel combination of ICA-1 with TMZ to induce robust apoptosis in a Caspase-3 mediated mechanism. Scratch assay results also showed that inhibition of PKC-ζ by ζ-Stat lead to decreased invasion compared to control. Our studies suggest that atypical PKCs particularly PKC-ι might be an important therapeutic target in the treatment of glioblastoma.

#245

Characterization and classification of glioblastoma multiforme using the novel multiparametric cyclic immunofluorescence analysis system MACSima.

Sandy Reiß,1 Stefan Tomiuk,1 Jutta Kollet,1 Jan Drewes,1 Wolfgang Brück,2 Melanie Jungblut,1 Andreas Bosio1. 1 _Miltenyi Biotec GmbH, Bergisch Gladbach, Germany;_ 2 _Universitätsmedizin Göttingen, Institut für Neuropathologie, Göttingen, Germany_.

Glioblastoma multiforme (GBM), a highly malignant, non-curative brain tumor of the primary central nervous system, has been subclassified in the past years in several distinct subtypes using a multitude of analysis methods. Here, we introduce the MACSima™ imaging platform which allows for fully automated, multiparametric, cyclic immunofluorescence analysis of specimens with hundreds of antibodies. We apply this method to characterize and classify glioblastomas according to published classification schemes and identify novel glioblastoma specific markers. Glioblastoma xenografts derived from primary tumors were dissociated to single cells using the Tumor Dissociation Kit, human and the gentleMACS™ Octo Dissociator (Miltenyi Biotec). Single cells were analyzed for cell surface marker expression by flow cytometry (MACSQuant Analyzer 10) including 371 directly conjugated antibodies (MACS Marker Screen, Miltenyi Biotec). A ranking was applied according to the percentage of positive cells and the stain index, leading to a selection of 96 markers for characterization of eight primary glioblastoma using the MACSima™ imaging platform. Cryosections were fixed by acetone and each specimen was exposed to 96 fluorescent labeled antibodies by cycles of antibody reaction, image acquisition and erasure of signal. The 2D image stacks were analyzed for antigen quantification and pattern recognition using both, pixel and segmented single-cell data. The detection of previously published markers, such as PDGFRα, Olig2, p16, p53, Synaptophysin, CD44, Nestin, Podoplanin, GFAP, MET, Hes-1 and EGFR was used to subclassify the glioblastomas according to i) Motomura (Motomura et al., 2012) into Oligodendrocyte Precursor (OPC), Differentiated Oligodendrocyte (DOC), Astrocytic Mesenchymal (AsMes) or Mixed subtype, and ii) Verhaak (Verhaak et al., 2010) into Proneural, Neural, Classical, or Mesenchymal subtype. The analysis of well-established glioblastoma marker partially already used in CAR T cell based clinical trials such as EGFRvIII, HER2, or IL-13Rα2 revealed a broad inter- and intratumor diversity of expression. Infiltrating immune cells were present in most of the tumors, but showed varying percentages. Finally, segmentation, clustering, and correlation analysis allowed for identification of new marker which might be used for a more robust classification of glioblastomas. In summary, our analysis using the MACSima™ imaging platform reveals a high heterogeneity of protein expression in glioblastomas along with the ability to deeper classify the diverse tumors and identify novel markers that allow selective detection of tumor cells for their potential use in immunotherapy.

#246

Glioblastoma cell-modified cell membrane lipid-extracted nanoparticles (CLENs) for imaging and treatment: Influence of magnetic nanoparticle on physiochemical properties of CLENs, cellular uptake, and cytotoxicity.

Woongjae Song, Robert Campbell. _MCPHS, Worcester, MA_.

Background: Glioblastoma multiforme (GBM), located in the cerebral hemispheres of the brain, is the most aggressive form of cancer. Surgery associated with chemotherapy with radiation treatment is the defined way to treat the disease. The delivery of chemotherapeutic agents frequently result in serious side effects. To address this issue we have taken the first steps in the development of CLENs (cell membrane lipid-extracted nanoliposomes) to enhance selective delivery of drugs to the brain.

Method: The CLENs used for the experiments were composed of various ratios of DOPC, DOTAP, cholesterol, and lipid extract (LE) material derived from the target cells (i.e., U87-MG), and DPPE-PEG5000 (PEG). Studies include the determination of drug (cisplatin) loading, magnetite (MAG-C) loading in CLENs, quantitative studies for particle size, zeta potential, SQUID (superconducting quantum interference device) measurements, and fluorescence detection to study cellular interactions.

Results: The average size for the CLENs fell within the range of 130 to 190 nm. The zeta potential reported for CLENs was negatively-charged. The inclusion of 15mol % LE resulted in the most significant cellular uptake by target cell U87-MG. Overall, the uptake by these cells was less compared to CLENS previously published for other organ tissue environments such as with breast tissue. The inclusion of cationic lipid DOTAP in CLENs did not alter the size distribution; however, the values for zeta potential increased from -8 ± 5 to 12 ± 6 mv. The additional inclusion of DOTAP in CLENs increased cellular uptake approximately 4-fold compared to CLENs without DOTAP (fluorescence intensity (A.U) from 145 ± 52 to 525 ± 49)). The inclusion of DOTAP increased the efficiency of loading Cisplatin in CLENs by approximately 30%. Studies involving the incorporation of MAG-C in U87-MG CLENs are currently underway, as well magnetization studies involving SQUID.

Conclusion: All CLENs demonstrated practical physicochemical properties similar to conventional liposomes. The cellular uptake results suggested that cellular uptake of CLENs in vitro, although relatively low overall, varied according to the total LE content. Furthermore, the inclusion of DOTAP in CLENs significantly increased uptake and the efficiency of drug loading.

#247

**Identification of the GABA** A **receptor in melanoma brain metastases patient tumors and demonstration that it is a viable drug target using benzodiazepine-derivatives.**

Milota Kaluzova,1 Tahseen Nasti,1 Hiao-Rong Chen,1 Lindsey Lowder,1 Robert Press,1 Havi Rosen,1 Manali Rupji,1 Laura Kallay,1 Rikesh Patel,1 Andre Burnham,1 Maxwell Xu,1 Alexandra Ross,1 Havva Keskin,1 Erin Connelly,1 Benjamin Izar,2 Cory Adamson,1 Jeffrey Olson,1 Jing Su,3 Walter Curran,1 Ragini Kudchadkar,1 Matthew Schniederjan,1 Stewart Neill,1 David Lawson,1 Michael Chan,3 Jeanne Kowalski,1 Mohammad Khan,1 Daniel Pomeranz Krummel,1 Soma Sengupta1. 1 _Emory University, Atlanta, GA;_ 2 _Harvard Medical School, Cambridge, MA;_ 3 _Wake Forest, School of Medicine, Wake Forest, NC_.

Primary cutaneous melanoma is often successfully treated in early stages in patients with regional disease. While distant metastatic disease, including brain metastasis, that develops in most stage IV melanoma patients, carries a particularly poor prognosis with median overall survival of only 4-5 months. Standard-of-care treatment of brain metastases includes surgery, radiation, and chemotherapy. The fact that melanoma is a radio-resistant cancer, significantly limits treatment options for melanoma that has metastasized to the brain. There is clearly a significant demand for new therapeutic approaches. We have conducted a whole transcriptomic analysis of melanoma brain metastases to provide insight into molecular changes that may contribute to metastasis as well as to identify potential therapeutic targets in the metastasized cancer. Total RNA was extracted from 29 formalin-fixed paraffin-embedded (FFPE) melanoma brain metastatic samples, libraries constructed and enriched for transcript fragments with coding regions. Libraries were subjected to Transcriptome Capture (TCap) targeting 21,415 genes, which represents more than 98% of the total RefSeq exome. Sequencing was performed on the Illumina HiSeq platform. Gene expression analysis of melanoma brain metastatic samples reveals high expression levels of ion channels, including subunits of the ligand-gated neurotransmitter GABAA receptors. We will present differential expression analysis between recent melanoma transcriptomic studies [1, 2] and melanoma brain metastases samples. More than 20% of FDA approved drugs target ion channels. We report that repurposing of one such class of drugs targeting GABAA receptors can impair melanoma cell viability in vitro and reduce tumor volume in vivo. GABAA receptors can serve as a potential therapeutic target for treatment brain metastasis.

References 1. Akbani, et al. (The Cancer Genome Atlas Network, TCGA). Genome classification of cutaneous melanoma. Cell 2015; 161(7): 1681-1696. 2. Tirosh, et al. Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq. Science 2016; 352 (6282): 189-196.

#248

Dynamic multi-OMICS analysis of glioblastoma cells reveals context of vulnerability to neddylation inhibition by pevonedistat.

Shayesteh R. Ferdosi, Brett Taylor, Nanyun Tang, Rita Bybee, Sen Peng, Victoria David-Dirgo, Krystine Garcia-Mansfield, Ritin Sharma, Patrick Pirrotte, Michael Berens, Harshil Dhruv. _Translational Genomics Research Institute, phoenix, AZ_.

As the most lethal primary brain tumor, glioblastoma multiforme (GBM) calls for novel therapeutic development. Global over activation of neddylation (a post-translational modification) has recently been found in GBM patients and has correlated with shorter patient survival. Significant accumulation of neddylation in recurrent GBM tissues indicates its importance in tumorigenesis and tumor progression. Analogous to the ubiquitination pathway, neddylation is essential to many protein regulation and biological processes. Although most well-characterized substrates of neddylation are the cullin subunits of Cullin-RING ligases (CRLs), non-cullin NEDD8 substrates have been investigated in recent years. Neddylation and subsequent degradation of PARC, p53, MDM2 and EGFR exemplify the broader functional role of neddylation. The neddylation inhibitor MLN4924 targets NEDD8 Activating Enzyme (NAE), an upstream activator of neddylation, and, as a result, induces cell cycle arrest, apoptosis and senescence in cancer cells. In this work, we investigated the context of vulnerability to Pevonedistat (MLN4924) in GBM by comparing the dynamic response of sensitive and non-sensitive cells using transcriptomics and proteomics profiling, using long-established and patient derived glioma cell lines. Efficacy of MLN4924 in glioma cell models was evaluated by measuring cell viability (CellTiterGlo®), colony formation efficiency, and cell cycle progression (flow cytometry with propidium iodide staining). GB1 (IC50= 0.28 μM), LN18 (IC50 = 0.19 μM), and GBM43 (IC50= 0.45 μM) were established as sensitive and M059K (IC50 = 5.5 μM), SNU1105 (IC50 = 20.9 μM), and GBM39 (IC50= 10.3 μM) as non-sensitive cell lines based on IC50 values. Western blot analysis of known cell cycle regulatory pathways and DNA damage response pathway did not show significant dynamic differences between sensitive and non-sensitive glioma cell models. To discover genomic and/or proteomic markers of differential response we collected RNA and protein for LN18 (sensitive) and SNU1105 (Non-sensitive) cells after 0, 2, 8 and 24 h treatment with MLN4924 at 100 nM and 500 nM concentration for transcriptomics and proteomics analysis. RNA sequencing was utilized for dynamic transcriptomic analysis. Cell lysates were processed using bottom-up proteomics workflow and the data was acquired on a Thermo Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer. Proteins were identified by querying spectral data against canonical and RNA-Seq predicted proteins and differential analysis was carried out to identify candidate determinats of vulnerability. An understanding of determinants of vulnerability to MLN4924 will expand knowledge of neddylation's role in cancer and may point to signatures of GBM patients most likely to respond to this targeted intervention.

#249

Combination of ONC201 with radiation exhibits synergistic efficacy in high grade gliomas and other advanced cancers.

Rohinton S. Tarapore,1 Sachin Jhawar,2 Mark Stein,3 Andrew Zloza,2 Sabine Mueller,4 Jie Zhang,4 Francesca Amoroso,5 Ian Mills,5 Wolfgang Oster,1 Joshua Allen1. 1 _Oncoceutics Inc, Philadelphia, PA;_ 2 _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ;_ 3 _Columbia University, New York, NY;_ 4 _University of California San Francisco, San Francisco, CA;_ 5 _Queens University, Belfast, United Kingdom_.

Background: Radiation is widely administered in many treatment settings for solid tumors. ONC201 is the first small molecule DRD2 antagonist for oncology that is being investigated in advanced cancer clinical trials as a single agent. ONC201 has exhibited preclinical and clinical anti-tumor activity in high grade gliomas and its immunostimulatory activity involving natural killer (NK) cells has been recently reported. Given that ONC201 exhibits broad synergy with anti-cancer drugs, excellent safety, and single activity in tumor types where radiation is used routinely, we evaluated the combination of ONC201 with radiation.

Methods: Cell viability was evaluated in human and/or mouse breast, prostate and high grade glioma cell lines in response to ONC201 (1 - 10uM), radiation (2- 10 Gy), or the combination. Incubation times ranged from 24 to 96 hours and the sequence of the two agents in combination was varied.

Results: Cell viability assays for ONC201 in combination with radiation in breast or prostate cancer cell lines revealed a cytotoxic response to the combination that was superior to either single agent. Western blot analysis of PC3 cells showed a synergistic induction of CHOP and ATF6 that are components of the integrated stress response. In glioblastoma or diffuse intrinsic pontine glioma (DIPG) cell lines, combination indices computed from cell viability experiments indicated modest synergy (~0.7 CI) for the combination ranging between 1-5 µM ONC201 and 2-10 Gy. In MDA-MB-468 breast cancer cells, Western blot analysis demonstrated a striking induction of PARP cleavage, a marker of caspase-mediated apoptosis, with 2 µM ONC201 in combination with 2 Gy radiation, whereas either single agent produced minimal PARP cleavage. In 4T1 murine triple-negative breast cancer subcutaneous tumors, the combination of oral ONC201 and radiation produced antitumor effects at subtherapeutic doses that required CD4+ T cells and NK cells.

Conclusions: ONC201 combined with radiation produced additive or synergistic anti-cancer effects in high grade glioma, breast and prostate cancer in vitro and increased anti-tumor efficacy in an in vivo model of breast cancer that involved immune cells.

#250

Tumor treating fields increases membrane permeability in glioblastoma cells.

Edwin Chang,1 Chirag Patel,2 Christoph Pohling,2 Caroline Young,1 Jonathan Song,1 Thomas A. Flores,3 Yitian Zeng,4 Lydia Marie Joubert,5 Hamed Arami,1 Arutselvan Natarajan,1 Robert Sinclair,4 Sanjiv S. Gambhir1. 1 _Stanford University, MIPS, Palo Alto, CA;_ 2 _Stanford University, MIPS, Stanford, CA;_ 3 _Stanford University, Stanford, CA;_ 4 _Stanford University, Palo Alto, CA;_ 5 _Stellenbosch University, Stellenbosch, South Africa_.

INTRODUCTION: Glioblastoma multiforme (GBM) is the most lethal primary brain cancer (median survival: 15-17 months, 5-year survival: 5%). Standard interventions consist of aggressive surgical resection, radiotherapy, and chemotherapy; however, GBM is heterogeneous and present therapies are often ineffective. Novel approaches need consideration. We investigated two such novel approaches: Withaferin A and Tumor Treating Fields (TTFields). Withaferin A is a steroidal lactone originating from extracts of the winter cherry plant (Withania somnifera). Extracts such as AshwaMAX can contain up to 4.3% (w/w) Withaferin A. Recently, the U.S. Food and Drug Administration approved another approach to treating glioblastoma: tumor treating fields (TTFields). TTFields involves the delivery of alternating electric fields to the tumor but mechanisms of action are not fully understood.

METHODS: To investigate these two therapies, we used human glioblastoma cells (GBM2, GBM39, and U87-MG) isolated from primary tumors. Cells were modified to express firefly luciferase. Proliferation was monitored by: bioluminescent imaging, cell counting via hemocytometer, or cell titer blue viability assay. TTFields were imposed on cell cultures by the inovitroTM device from Novocure Ltd.

RESULTS: Both AshwaMAX and Withaferin A inhibited growth of GBM cell lines (IC50 ~0.25 µM). In exploring Withaferin A and TTFields interventions in GBM cell culture, we discovered that the steroid worked synergistically with TTFields (p<0.01, n=3 experiments) to inhibit GBM proliferation. Current theories about TTFields' action involve it disrupting mitosis through interference with mitotic spindle assembly. In the present studies, we show that TTFields also alters cellular membrane structure thus rendering it more permeant to chemotherapeutics. In GBM, increased permeability was indicated through increased bioluminescence with TTFields exposure (p<0.0001, TTFields vs no TTFields), as well as with increased binding and ingress of membrane-associating reagents such as Dextran-FITC (p<0.0001, TTFields vs no TTFields). Increased permeability was observed and with scanning electron microscopy showing augmented number (54+19 vs. 24+11 per ROI, TTFields vs no TTFields, p=0.0002) and sizes of holes (241+92 vs. 130+32 nm2, TTFields vs no TTFields, p=0.0005) in the GBM cellular membrane. Such phenomena were reversible after cessation of TTFields.

CONCLUSIONS: In summary, increased membrane permeability by TTFields helps to explain previous reports of additive vs. synergistic effects between TTFields and chemotherapies, including Withaferin A. These findings have implications for the design of combination therapies in glioblastoma and other cancers and may significantly alter standard of care strategies.

#251

Anti-malarial drug Pyronaridine modulates microenvironment leading to potent anti-cancer activity against GBM.

Jeevan D. Ghosalkar,1 Vinay R. Sonawane,1 Siddhika R. Raut,1 Mariamma Thekkumpurath,1 Radha Pujari,2 Padma Shastri,2 Geena Malhotra,1 Kalpana S. Joshi1. 1 _Cipla Ltd, Mumbai, India;_ 2 _NCCS, Pune, India_.

Glioblastoma multiforme (GBM) is a commonly diagnosed primary brain tumor with median survival of about one year. GBM is highly aggressive malignancy with limited treatment options. The standard of care for GBM is Temozolomide (TMZ), given as first-line therapy, however, 90% of recurrent gliomas acquire TMZ resistance. Treatment to GBM remains greatest challenge in the management of cancer patients worldwide due to inherent tumor heterogeneity and drug resistant nature leading to high unmet medical need.

New drug development is challenging and takes an enormous amount of time, money and effort. Drug repurposing, the application of an existing therapeutic to a new disease indication holds the promise of rapid clinical impact at a lower cost than de novo drug development.

This study reports the anti-cancer efficacy of Pyronaridine (PYR), a benzonaphthyridine derivative, used as an agent for the treatment of malaria. Anti-malarial action of PYR therapy is known to target haematin formation and inhibit activity of P. falciparum DNA topoisomerase II. PYR was earlier reported to show in vitro anticancer activity in combination with other drugs in breast and haematological cancer. However, in this study we report its anticancer activity in GBM with novel mechanism of action selective against GBM resistant cells.

In our studies, anticancer activity of PYR was confirmed in panel of cancer cell lines comprising of kidney, pancreatic, ovarian, glioma and bladder cancer (IC50: 0.907 - 11 µM). Interestingly, PYR demonstrated dose dependant anti-proliferative and pro-apoptotic activity in both TMZ sensitive and TP53 and PTEN mutated resistant GBM cells (IC50: 1.82-4.2 µM). The 3D culture multicellular spheroid model (MCS) is important and relevant as it is predictive of in vivo antitumor efficacy, PYR inhibited MCS developed from TMZ resistant cells. Additionally, PYR also showed potent activity against glioma stem cell line and primary culture derived from high grade glioma. Cell cycle analysis indicated G1 and G2/M phase arrest and increased sub G0 population when GBM cells were treated with PYR.

While deciphering the mechanism of action of PYR, we astoundingly invented a novel role for PYR as an inducer of Prostate Apoptosis Response-4 (PAR-4), a pro-apoptotic tumor suppressor protein. The expression of PAR-4 is known to be downregulated in most tumors including GBM. Here, we demonstrate that PYR modulates microenvironment by inducing PAR-4 in normal fibroblast cells in a dose dependent manner. The conditioned medium from these cells induced profound cytotoxicity against cancer cells. PYR is relatively safe to use in humans and our data clearly indicates its anti-cancer potential and hence is a promising therapeutic candidate. The detailed studies focusing on its novel mechanism of action alone and in combination in drug resistant models will be discussed.

#252

**Tumor treating fields (TTFields) affect blood brain barrier (BBB) integrity** in vitro **and** in vivo **.**

Almuth F. Kessler,1 Clara M. Schaeffer,1 Malgorzata Burek,1 Ursula Ruschig,1 Catherine Tempel-Brami,2 Tali Voloshin,2 Moshe Giladi,2 Ellaine Salvador,1 Ralf-Ingo Ernestus,1 Mario Löhr,1 Carola Förster,1 Carsten Hagemann1. 1 _University Hospital Würzburg, Würzburg, Germany;_ 2 _Novocure, Haifa, Israel_.

Background: The blood-brain barrier (BBB) may impede application of drugs to the brain for treatment of malignant brain tumors, in particular glioblastoma multiforme (GBM). Alternating electric fields with intermediate frequency and low intensity, called Tumor Treating Fields (TTFields), are an established novel adjuvant treatment modality for GBM. Here, the effect of TTFields on BBB permeability is analyzed.

Material and Methods: After TTFields treatment with a frequency of 100-300 kHz for up to 72 h, immortalized murine brain capillary endothelial cells (cerebEND) grown on cover slips and transwell inserts were stained for immunofluorescent assessment of the tight junction proteins Claudin-5 and ZO-1. Transendothelial electrical resistance (TEER) was applied to investigate BBB integrity. Moreover, BBB permeability was determined by fluorescein isothiocyanate (FITC) staining followed by flow cytometry. For in vivo analysis, the increase in vessel permeability was quantified by utilizing i.v. injected Evans Blue (EB) in rats during TTFields application to the brain (100 kHz, 72 h).

Results: The BBB was disturbed by treatment with TTFields as tight junction proteins were delocalized from the cell boundaries to the cytoplasm with maximal effects at 100 kHz. TTFields application significantly reduced the BBB integrity by 65% and significantly increased the BBB permeability for 4 kDa large molecules. Initial recovery of the cell morphology was observed 48 h post-treatment and a complete recovery could be detected after 96 h, indicating a reversibility of the TTFields effect on the BBB. Average accumulation of EB in the rat brain was significantly increased by TTFields application to the rats head.

Conclusion: In the future, TTFields could be utilized to deliver drugs generally unable to cross the BBB to the central nervous system as TTFields at a frequency of 100 kHz are potentially able to disrupt the BBB. The data presented on in vitro and in vivo application of TTFields to permeabilize the BBB may be a rationale for a phase I clinical trial and clinical application in the future.

#253

Protein-tyrosine phosphatase 1B (PTP1B) as a therapeutic target in glioblastoma.

Ruben Bartolomé,1 Marta Jaén,1 Miranda Burdiel,1 Irina V. Balyasnikova,2 Ignacio Casal1. 1 _Centro de Investigaciones Biológicas (CIB), Madrid, Spain;_ 2 _Feinberg School of Medicine, Northwestern University, Chicago, IL_.

New targeted therapies are urgently needed in glioblastoma (GBM). IL13Rα2 receptor is considered a highly specific receptor for GBM cells and has been proposed for therapeutic purposes. Here, we aimed to identify the protein interaction network of IL13Rα2 and its mediators in GBM searching for novel associated therapeutic targets.

We investigated the IL13Rα2 protein interaction network in U251 GBM cells using immunoprecipitation followed by mass spectrometry. Different cell lines were used for validation. Two glioblastoma cell lines were used for testing proliferation, adhesion, migration and invasion. Claramine, a PTP1B inhibitor, was tested to study inhibition of GBM cells growth in cultured cells and in animal experiments. In vivo experiments with NSG mice were performed after treatment with PTP1B inhibitors.

Using a proteomic approach, we identified the association of the PTP1B phosphatase with IL13Rα2 in U251 GBM cells. Although GBM cell lines were positive for PTP1B, the highest PTP1B expression was found in GBM clinical samples. Moreover, "in silico" dataset analysis showed PTP1B association with poor outcome in GBM patients. PTP1B silencing inhibited the IL-13-promoted activation of Src (Tyr419), AKT and ERK1/2 in all the cell lines tested causing a significant effect on IL-13-mediated migration and invasion, and proliferation at a lower extent, of GBM cells. Therefore, PTP1B-silencing reverted most of the pro-metastatic capacities of the IL-13/ IL13Rα2 signaling axis. Further immunoprecipitation of PTP1B showed its association with a large number of NF-κB and NOTCH-related proteins in GBM cells. Both pathways are highly relevant for cancer proliferation and invasion. Claramine, a selective inhibitor of PTP1B, caused similar results to PTP1B silencing, inhibiting the dephosphorylation of Src Tyr527, migration and invasion in GBM. Moreover, treatment with Claramine caused a complete regression of GBM xenografts in NSG mice and total inhibition of liver metastatic growth in nude mice inoculated with colorectal cancer cells.

In conclusion, our results provide new insights into the mechanisms underlying IL13Rα2-driven GBM aggressiveness after identifying PTP1B as a critical mediator of IL-13/IL13Rα2 signaling. A PTP1B inhibitor such as Claramine showed a great value as a therapeutic candidate in GBM. PTP1B inhibitors might be highly effective to prevent GBM progression and invasion.

#254

Discovery of dual inhibitors of NQO1 and GSTP1 for malignant glioblastoma treatment.

Kecheng Lei,1 Shilin Luo,1 Lingjing Jin,2 Keqiang Ye1. 1 _Emory University, Atlanta, GA;_ 2 _Tongji Hospital,Tongji University, Shanghai, China_.

Glioblastoma (GBM) is a highly aggressive form of neoplasia due to its frequently overexpressed or mutant epidermal growth factor receptor (EGFR). NADPH quinone Oxydoreductase1 (NQO1) and glutathione-S-transferase Pi 1 (GSTP1) are commonly upregulated in GBM. NQO1 and GSTP1 are enzymes with dual roles that decrease the formation of reactive oxygen species (ROS), which mediates the oxidative stress and GBM cell proliferation. To search for a small molecule that blocks the malignant cancer proliferation, we conducted a high-throughput Screening (HTS) and identified a small molecule inhibitor (5-methyl-N-(nitro-1,3-thiazol-2-yl)-3-phenylisoxazole-4-carboxamide) as "HIT" that binds to both NQO1 and GSTP1 with high affinity and selectivity. The "HIT" blocks NQO1 and GSTP1 enzyme activity and mediates apoptosis in GBM via caspase activation. Inactivation of NQO1 and GSTP1 with siRNA or HIT resulted in imbalanced redox homeostasis, leading to mitigated cancer cell proliferation in vitro and in vivo. Thus, optimization of "HIT", the dual NQO1 and GSTP1 inhibitor, may provide a novel strategy to antagonizing GBM.

#255

Antagonizing Na-K-Cl cotransporter NKCC1 impairs medulloblastoma cell viability.

Andre Burnhamm,1 Milota Kaluzova,1 Laura Kallay,1 Gaunguan Li,2 Taukir Ahmed,2 Farjana Rashid,2 Andrew Jenkins,1 James Cook,2 Danial Pomeranz Krummel,1 Soma Sengupta1. 1 _Emory University, Atlanta, GA;_ 2 _University of Wisconsin-Milwaukee, Milwaukee, WI_.

Medulloblastoma, a common pediatric brain cancer, is comprised of four molecular subgroups: Wnt-activated; Shh-activated; group 3; and group 4. Group 3 subgroup patients have the lowest survival rate and the highest rates of relapse and metastasis [1]. The standard-of-care for medulloblastoma consists of surgical removal of the tumor, radiation, and chemotherapy [1]. However, the standard-of-care poses many risk factors including hearing loss, permanent damage to endocrine and neurocognitive function, and secondary tumors [1]. There is a high demand for new treatment options for medulloblastoma. Interestingly, group 3 medulloblastoma tumors have high expression of GABRA5 and SLC12A2, which code for the α5-subunit of the ligand-gated ionotropic γ-aminobutyric acid type A receptor (GABAAR) and the sodium-potassium-chloride cotransporter isoform-1 (NKCC-1), respectively [2]. Several recent studies have provided evidence that ion channel activity contributes to brain cancer progression [3]. If so, then modulating ion channel(s) function may impair cancer cell viability. We have already shown that use of a positive allosteric modulator of the GABAAR can impair viability of group 3 medulloblastoma cells in vitro and in vivo and with greater specificity and potency than standard-of-care chemotherapeutic [4, 5]. Others have shown that inhibition or down-regulation of NKCC-1 can reduce cell viability, increase apoptosis, and decrease migration and invasion of cells derived from various cancer types [3]. We have examined how modulating NKCC-1 and GABAAR alone and simultaneously impacts the viability of group 3 medulloblastoma cells. We find that there is a synergistic effect in the inhibition of NKCC-1 and positive modulation of GABAAR. We will report on these observations and our model for the mechanism of action. References 1. Sengupta, S., Pomeranz Krummel, D., Pomeroy, S. (2017) The evolution of medulloblastoma therapy to personalized medicine. F1000Research 6, 490. 2. Cho, Y.J., Tsherniak, A., Tamayo, P., Santagata, S., Ligon, A., Greulich, H., et al. (2011) Integrative genomic analysis of medulloblastoma identifies a molecular subgroup that drives poor clinical outcome. J. Clin. Oncol. 29, 1424-1430. 3. Cong, D., Zhu, W., Kuo, J.S., Hu, S., Sun, D. (2015) Ion transporters in brain tumors. Curr. Med. Chem. 22, 1171-81. 4. Sengupta, S., Weeraratne, S.D., Sun, H., Phallen, J., Rallapalli, S.K., Teider, N., et al. (2014) α5-GABAA receptors negatively regulate MYC-amplified medulloblastoma growth. Acta Neuropathol. 127, 593-603. 5. Jonas, O., Calligaris, D., Methuku, K.R., Poe, M.M., Francois, J.P., Tranghese, F., et al. (2016) First in vivo testing of compounds targeting group 3 medulloblastomas using an implantable microdevice as a new paradigm for drug development. J. Biomed. Nanotechnol. 12, 1297-1302.

#256

Repurposing neuroleptic drug for brain tumor therapy.

Iti S. Kaushik, Alok Ranjan, Blake Schwettmann, Sanjay Srivastava. _Texas Tech University, Amarillo/Abilene, TX_.

Brain tumor is considered as one of the most aggressive and incurable form of cancer. Majority of the brain tumors have a median survival rate of only 12%. Even though advanced treatments such as surgical removal, chemotherapy and radiotherapy are available, brain tumor persists to be lethal. Temozolomide (TMZ) is the main therapy for brain tumor but resistance to TMZ is the major cause of treatment failure. This resistance in tumor cells has frequently been linked to the expression of O6-methylguanine-DNA methyltransferase (MGMT). Pimozide (PMZ) is a neuroleptic drug used for the treatment of schizophrenia and chronic psychosis. In our study, we observed that pimozide significantly reduced the viability of U87MG, U251MG, DAOY and GBM28 cell lines with an IC50 ranging from 11µM to 20µM after 24 h of treatment. Pimozide induced apoptosis in these cell lines as evaluated by FITC/Annexin assay and further validated by the cleavage of caspase 3 as well as PARP. Western blot analysis resulted in the concentration dependent decrease in the expression of STAT3 signaling mainly targeting the anti-apoptotic markers BCl-XL, BCl-2 and Survivin etc in U87MG, U251MG, DAOY and GBM28 brain cancer cell lines. PMZ alone suppressed the growth of U87MG cells with an IC50 of 10µM, after 96 hours of treatment. On the other hand, treatment of U87MG cells with 750 µM of TMZ in combination with 10µM PMZ for 96 hours increased the killing of U87MG cells by 50% as compared to 10% when treated with TMZ. In addition, PMZ treatment reduced the survival of TMZ resistant U251R cells with an IC50 ranging from 15-20 µM after 24 h of treatment. Our results further demonstrated that oral administration of PMZ (25mg/kg) significantly inhibits the growth of brain tumors in an intracranial tumor model without any apparent signs of toxicity. It is important to note that PMZ is an FDA approved drug with no considerable toxicity. Overall, this study depicts that PMZ is a potential candidate to target brain tumors.

### Cell Death and DNA Repair Pathways

#257

Targeting AML through apoptosis activation using Bcl-2/Mcl-1 or Bcl-2/Hdm2 inhibitor combination therapies.

Youzhen Wang,1 Shumei Qui,1 Sneha Sanghavi,1 Iain Mulford,1 Gaëlle Lysiak,2 Maïa Chanrion,2 Prakash Mistry,3 Ulrike Pfaar,3 Marie Schoumacher,2 Audrey Claperon,2 Laurence Kraus-Berthier,4 Sébastien Banquet,4 Alix Derreal,4 Claire Fabre,3 Heiko Maacke,3 Frédéric Colland,2 Olivier Geneste,2 Erick Morris,1 Ensar Halilovic1. 1 _Novartis Insts. for BioMedical Research, Cambridge, MA;_ 2 _Servier Oncology R &D Unit, Croissy-sur-Seine, France; _3 _Novartis Insts. for BioMedical Research, Basel, Switzerland;_ 4 _Servier Oncology R &D Unit, Suresnes, France_.

Acute myeloid leukemia (AML) is an aggressive and heterogeneous hematologic malignancy, characterized by uncontrolled proliferation and impaired differentiation of myeloid cells. With the exception of certain subtypes, the average long-term survival rate remains low, thus underlining the need to further improve the outcome of AML patients. Since AML is one of the least mutated cancer types, the majority of AML patients may not carry targetable genetic alterations. However, the anti-apoptotic proteins of the Bcl-2 family, such as Bcl-2 and Mcl-1, are often overexpressed in AML, allowing deregulated survival; hence pro-apoptosis priming with small molecule inhibitors of Bcl-2 and Mcl-1 may provide a broader therapeutic benefit across the disease. In addition, a majority of AML patients carry wild-type p53, providing therapeutic opportunity for Hdm2 inhibitors to stabilize p53 and lead to expression of pro-apoptotic molecules (e.g., PUMA & BAX). Therefore, targeting the combined apoptosis mechanisms by inhibiting different anti-apoptotic Bcl-2 family of proteins and activating p53 concomitantly may synergistically enhance apoptotic cell death of AML tumor cells.

We tested the combination of Bcl-2 inhibitors (BCL201/S55746 or venetoclax) with either MIK665/S64315, a novel and selective inhibitor of Mcl-1 or HDM201, a selective small molecule inhibitor of p53:Hdm2 interaction, in a series of in vitro and in vivo studies in AML. In vitro, strong combination synergy was observed with a remarkable induction of cell death for both combinations. In vivo, the combination of Bcl-2 inhibitors with MIK665/S64315 or HDM201 lead to complete and durable antitumor responses in a variety of p53wt AML patient-derived xenograft models of heterogeneous genetic profiles. Notably, lowering the dose of HDM201 by 4 fold from its most efficacious dose, resulted in a high degree of tumor regressions while mitigating the toxicity effects on platelets. Taken together, these data demonstrate that a combination of Bcl-2 inhibitor (BCL201/S55746 or venetoclax) with MIK665/S64315 or HDM201 provide therapeutic benefit over the monotherapy, and support a rationale for testing these apoptosis enhancing combination approaches in AML patients.

#258

Recombinant human TRAIL or a DR5 agonistic antibody convert the response of non-triple negative breast cancer cells to ONC201 from anti-proliferative to apoptotic.

Marie D. Ralff,1 Jocelyn E. Ray,2 Avital Lev,2 Lanlan Zhou,2 David T. Dicker,2 Wafik S. El-Deiry2. 1 _The Lewis Katz School of Medicine at Temple University, Philadelphia, PA;_ 2 _Fox Chase Cancer Center, Philadelphia, PA_.

ONC201 was initially identified as an inducer of cell death through the tumor necrosis factor related apoptosis inducing ligand (TRAIL) pathway. The compound is being tested in patients with a variety of tumor types, including those of the breast. Both triple negative breast cancer (TNBC) cells and non-TNBC cells are sensitive to ONC201. In a subset of TRAIL-sensitive TNBC cells, ONC201 treatment leads to the induction of apoptosis in a TRAIL-dependent manner. In the majority of TRAIL-resistant non-TNBC cells, ONC201's effects are anti-proliferative rather than apoptotic. In vivo, the apoptotic response to ONC201 leads to efficacy of the compound while the anti-proliferative response does not (Ralff et al., Mol Cancer Ther., 2017). Here we worked to identify strategies to convert the response of cancer cells to ONC201 from anti-proliferative to apoptotic. ONC201 treatment primed TRAIL-resistant non-TNBC cells to undergo TRAIL-dependent cell death. Increases in the mRNA and surface protein levels of death receptor 5 (DR5) as well as decreases in the expression of multiple anti-apoptotic proteins occured in ONC201-treated cells. We also observed small but significant increases in TRAIL mRNA and surface protein in non-TNBC cells following treatment with ONC201. Despite apoptotic priming and the induction of TRAIL, the effect of the compound in the non-TNBC cells remains anti-proliferative but not apoptotic. We hypothesized that the level of TRAIL induced by ONC201 in these cells, known to be TRAIL-resistant, was insufficient. Remarkably, the addition of exogenous recombinant human TRAIL (rhTRAIL) converted the response of TRAIL-resistant non-TNBC cells to ONC201 from anti-proliferative to apoptotic. Cleaved PARP, caspase-3, caspase-9, and caspase-8 were observed in cells treated with ONC201 followed by rhTRAIL, but not in cells treated with ONC201 or rhTRAIL alone. Propidium iodide staining and quantification of the subG1 population via flow cytometry further confirmed these findings. Similar observations about the ability of rhTRAIL to convert the cellular response to ONC201 from anti-proliferative to apoptotic were made in cancer cells from other tumor types. Importantly, rhTRAIL did not convert the response of normal fibroblasts to ONC201 from anti-proliferative to apoptotic. Addition of a DR5 agonistic antibody to ONC201 treated non-TNBC cells also converted the response of the non-TNBC cells to ONC201 from anti-proliferative to apoptotic. Ongoing experiments are currently testing the combination of ONC201 and rhTRAIL in vivo. These findings describe a strategy for potently converting the response of a cancer cell to ONC201 from anti-proliferative to apoptotic. Clinical application of this combination therapy may result in tumor regressions in patients with decreased sensitivity to ONC201 as a single agent.

#259

Interplay between Bcl-2 and cFLIP in lymphoma disease progression is a function of an altered redox milieu.

Jayshree L. Hirpara, Shazib Pervaiz. _National Univ. of Singapore, Singapore, Singapore_.

Bcl-2 is associated with chemoresistance and despite the preponderance of evidence linking its anti-apoptotic function to hematologic cancers, it's now emerging that overexpression of Bcl-2 is a feature shared by a host of cancers. Canonically, the death inhibitory activity of Bcl-2 is associated with its ability to prevent mitochondrial outer membrane permeabilization. However, recent evidence has highlighted a novel facet in the biology of Bcl-2, whereby enforced expression of Bcl-2 resulted in an increase in mitochondrial oxygen consumption and elevated levels of the reactive oxygen species (ROS), superoxide (O2-.). Alleviating the levels of intracellular O2-. sensitized Bcl-2 overexpressing cells to death receptor induced apoptosis via a robust increase in upstream caspase 8 activation. Using a variety of functional mutants of the NOX-activating small GTPase Rac1, we show a clear link between Bcl-2 induced apoptosis resistance and intracellular O2-.. In the search for the molecular target of this inhibitory signal, we now show that Bcl-2 overexpression is associated with an increase in the transcription and protein levels of the receptor inhibitory protein, cFLIP. We employed pharmacological and genetic approaches to modulated intracellular O2-. and present evidence cFLIP is transcriptionally regulated by O2-.; increase in cFLIP promoter activity and mRNA levels. Interestingly, inhibiting glycolysis also resulted in downregulation of cFLIP in a ROS-dependent manner. Notably, primary cells derived from patients with lymphoma also showed a significant downregulation of cFLIP when subjected to conditions that decrease O2-. and elicited enhanced sensitivity to TRAIL-induced apoptosis. Importantly, TCGA and Affy meta-cohorts' analyses revealed consistent correlation trend: Bcl-2 and cFLIP (CFLAR) were positively correlated; Bcl-2 and SOD1 (superoxide dismutase 1) were negatively correlated. A strong negative correlation was also observed between CFLAR and SOD1. Furthermore, Kaplan-Meier analyses of overall survival (OS) were performed in the in-house High expression of either Bcl-2 or CFLAR conferred poor OS outcome. When stratified by CFLAR and Bcl-2 expressions, cases with higher expression of the two genes had the worst prognosis with the shortest median survival. Notably, DLBCL with high Bcl-2 and low SOD1 had the worst OS outcome, compared against expression levels of Bcl-2 and SOD1. Together, these findings point to a concordance between cFLIP and Bcl-2, which negatively correlates with SOD1 in driving lymphoma progression.

#260

Identifying cancer drug sensitivity using live cell imaging dynamic BH3 profiling of solid tumor core biopsies.

Rebecca German, Elizaveta Lavrova, Timothy Hagan, Otari Chipashvili, Ewa Sicinska, James Cleary, Kimmie Ng, Anthony Letai, Patrick Bhola. _Dana-Farber Cancer Institute, Boston, MA_.

Given the rapid development of new small molecule cancer therapeutics, there is a growing need for predictive diagnostics to match cancer patients with optimal therapies. We previously developed a precision medicine technology with a functional phenotypic readout called dynamic BH3 profiling (DBP). DBP exposes cancer cells to drugs and measures induction of apoptotic cell death signaling after 24 hours ex vivo. Nonetheless, the application of DBP to core biopsies from metastatic tumors or other limited samples remains a technical challenge. Here, we adapt the DBP protocol for use on samples with small numbers of cells such as core biopsies. We maximize information returned per cell by imaging mitochondrial integrity in response to BH3 peptide exposure over time. We first show that the adapted protocol works in limited numbers of cancer cell lines, and in limited cells from the MMTV-PyMT genetically engineered mouse model of breast cancer. Specifically, we show that our ex vivo DBP predictions of the MMTV-PyMT mouse tumor matches known in vivo response. Finally, we apply our modified protocol to patient derived xenografts of colon cancer and primary patient colon tumors. We expect that our adapted protocol will find utility as a clinical biomarker, and as a method to optimize pre-clinical drug testing.

#261

**L1-** MET **transcription silencing modulates** MET **and** EGFR **gene and their protein expression and induces apoptosis and cell-death in different types of cancer cells.**

Enrico Berrino,1 Umberto Miglio,2 Valentina Miano,3 Letizia Lanzetti,2 Silvia Benvenuti,2 Carla Debernardi,2 MIchele De Bortoli,3 Caterina Marchio,1 Tiziana Venesio,2 Anna Sapino1. 1 _University of Turin, FPO-IRCCS Candiolo Cancer Institute, Turin, Italy;_ 2 _FPO-IRCCS Candiolo Cancer Institute, Candiolo, Italy;_ 3 _University of Turin, Turin, Italy_.

The activation of the LINE-1 sequence located within the second intron of MET leads to the onset of L1-MET transcript. We recently characterized the full L1-MET structure in breast cancer and showed that high levels of this transcript recognize a subset of more aggressive breast carcinomas, mainly of triple negative phenotype. However, at present, the relationship between L1-MET and MET is still poorly understood. In order to elucidate this function, we silenced L1-MET transcription using cells expressing different levels of L1-MET/MET, including lung cancer (A549 and EBC1), gastric cancer (GTL16), and breast cancer (MDA-MB231), that were transiently transfected with Gapmers-LNA (Exiqon), specifically targeting L1-MET sequence. Cell viability and apoptosis were evaluated after 24 h by cell count, Cell Titer Glow (Promega) and propidium iodide/annexin based-cytofluorimeter assays. RNA was purified from sample and control cells to assess the L1-MET silencing by qRT-PCR and to evaluate the gene-expression of a subset of cancer-related genes using Nanostring Technology, whereas western blot analyses were carried out to measure the protein expressions. A significant decrease of cell viability was detected in A549, EBC1 and GTL16, but not in MDA-MB231 cells, characterized by the lowest level of L1-MET overall. In parallel, the highly expressing L1-MET cells showed an increased rate of early and late apoptosis together with a strong reduction of MET gene and its protein expression. On the contrary, in MDA-MB231 cells L1-MET silencing induced only a slight MET gene and protein impairment. Overall, L1-MET knock-down caused a decrease expression of a conserved gene cluster, including a marked reduction of EGFR protein expression. Moreover, L1-MET silenced cells showed lower MET and EGFR phosphorylation, with a downstream silencing effect on pERK and pAKT. Results of cell treatment with the inhibitors of lysosome and proteasome activity bafilomycin and MG-132 ruled out the interaction of L1-MET silencing with protein degradation pathway. This is the first study investigating the function of the L1-MET transcript in cancer models. Our results show that although L1-MET is unable to encode for a protein, its silencing exerts a strong phenotypic effect on different tumor cell types, suggesting potential regulations at the transcriptional level.

#262

Anti-tumorigenic effect of ONC201 is enhanced by combination treatment with TRAIL or a DR5 agonist in endometrial cancer in vitro.

Jocelyn Ray, Marie Ralff, David Dicker, Wafik El-Deiry. _Fox Chase Cancer Center, Philadelphia, PA_.

ONC201 is a well-tolerated, orally active small molecule in the novel imipridone class that has anti-tumorigenic properties in a number of solid tumors, but not in non-neoplastic cells. ONC201 has demonstrated promising activity as a single agent in patients with advanced endometrial cancer in the first-in-human clinical trial with Phase II trials in endometrial cancer patients now underway. Although the mechanism of action in endometrial cell pathology has not been well studied, ONC201's anti-tumor effect has been shown in other model systems to be p53-independent and mediated through activation of the integrated stress response (ISR) leading to DR5 activation and through induction of cell death or growth arrest. ONC201 inhibits ERK and AKT leading to upregulation of TRAIL in many systems. We hypothesized that ONC201 upregulation of DR5 could sensitize tumor cells to TRAIL and that TRAIL could convert growth arrest to cell death in ONC201-treated cells. Three endometrial cancer cell lines AN3CA, HEC1A and KLE were treated with ONC201 alone or in combination with TRAIL, or a DR5 agonist. Effects on cell viability were assessed by the Cell Titer-Glo cell viability assay, colony formation assays and cell cycle analysis by propidium iodide staining. QPCR and Western blot analysis were used to evaluate mRNA and protein expression, respectively. Assessment of TRAIL and DR5 cell surface expression was evaluated by surface staining using flow cytometry and FlowJo data analysis. ONC201 decreased the cell viability of all three endometrial cancer cell lines at clinically achievable low micro-molar concentrations. ONC201 activated the ISR and an anti-proliferative effect involving a G1 phase arrest and little cell death as indicated by increased Sub-G1 analysis and PARP cleavage. DR5 mRNA and protein expression at the cell surface were induced by ONC201. All three endometrial cancer cell lines were resistant to TRAIL alone, however, pre-treatment with ONC201 sensitized the AN3CA and KLE to TRAIL and a DR5 agonist, leading to potent cell death induction. The combination of ONC201 plus TRAIL did not cause appreciable cell death in normal human fibroblast. ONC201 decreases cell viability in endometrial cancer cells lines primarily through growth arrest while the combination of ONC201 and TRAIL or a DR5 agonist promotes cell death in AN3CA and KLE cells but not in HEC1A endometrial cancer cells. Our results suggest a novel cancer therapeutic strategy that can be exploited in the clinic.

#263

**KIT** low **cells mediate Imatinib resistance and disease persistence in gastrointestinal stromal tumor.**

Sudeep Banerjee,1 Chih-Min Tang,1 Mayra Yebra,1 Kwat Medetgul,1 Adam M. Burgoyne,1 Pablo Tamayo,1 Robert Wechsler-Reya,2 Jason K. Sicklick1. 1 _UCSD, La Jolla, CA;_ 2 _Sanford-Burnham Medical Research Institute, La Jolla, CA_.

INTRODUCTION: Gastrointestinal stromal tumor (GIST) is commonly driven by oncogenic KIT mutations that are effectively targeted by Imatinib (IM). However, IM does not cure GIST; adjuvant therapy only delays recurrence in high-risk tumors. Therefore, we hypothesized that GIST contains cells with primary IM resistance, representing a critical target for overcoming disease persistence.

METHODS: Gene Set Enrichment Analysis (GSEA) was performed with publicly available data (GSE1596636). Human GIST cell lines (GIST-T1 and 882) and primary KIT-mutant GIST (IRB #090401) were analyzed by fluorescence-activated cell sorting (FACS) to sort KITloCD34+ (KITlo) vs KIThiCD34+ (KIThi) subpopulations using anti-human KIT and CD34 antibodies. RNA was extracted from cell lysates for analysis by quantitative RT-PCR. Cell viability was assessed by CellTiter-Glo or MTT assays following drug treatments.

RESULTS: We performed GSEA on 27 matched GISTs comparing pre- and post-neoadjuvant IM treatment (RTOG S0132). Post-IM samples had 50% lower KIT expression (P=0.002) and were enriched in "cancer stem cell" and Axl/Gas6/NF-κB signaling gene signatures. Similarly, in vitro IM treatment of GIST cell lines resulted in 2-fold reduction of KIT expression and 1.9 to 4.4-fold increased expression of stem-associated transcription factors (SATFs: OCT4, SOX2, KLF4, NANOG). Parallel FACS analysis of IM-treated GIST cell lines (125 nM, 72-h) demonstrated the presence of a KITlo subpopulation (GIST-T1: 0.75%; GIST882: 4%) while untreated cells had 3- to 11-fold fewer KITlo cells. Primary human GISTs also had KITlo cells by FACS (4-8%; N=4), suggesting that the population is not an artifact of in vitro culture. KITlo GIST882 cells were IM-resistant (IC50: KITlo 3641 nM vs KIThi 180 nM) and significantly overexpressed all SATFs by qRT-PCR (2.0 to 4.1-fold; P<0.001) consistent with RTOG S0132 analysis. Moreover, RNAseq confirmed that KITlo cells (relative to KIThi) are enriched in factors present in the "cancer stem cell" gene signature identified by GSEA (KLF4, CCL5, ATF3, JUN, IFIT1, PMP22). Lastly, we tested candidate drugs against targets overexpressed in IM-treated tumors. Unsorted cells were pre-treated with IM (185 nM, 48-h) to enrich for KITlo cells. Subsequent treatment with R428 (AXL inhibitor) or bardoxolone (NF-κB inhibitor) resulted in 70% (R428, 5 μM) and 80% (bardoxolone, 5 μM) cell death. Finally, FACS-sorted KITlo cells were sensitive to both drugs (50% and 88% killing, respectively).

CONCLUSIONS: KITlo cells are a distinct subpopulation in human GIST with intrinsic IM-resistance and may represent a novel mechanism of GIST persistence. These cells overexpress stem cell transcriptional programs, including the Axl/Gas6/NF-κB pathway, which represent novel therapeutic targets in vitro. Further studies are needed to explore the in vivo efficacy of combination or sequential targeting of KITlo cells in GIST.

#264

Differential expression of HPRT in prostate cancer leads to investigation of its ADCC effects.

Michelle H. Townsend, Kelsey A. Bennion, Zac E. Ence, Eliza E. Bitter, Abi M. Felsted, John E. Lattin, McKay D. Reese, Stephen R. Piccolo, Kim L. O'Neill. _Brigham Young University, Provo, UT_.

As 1 in 41 American men will die of prostate cancer, this study proposes hypoxanthine- guanine phosphoribosyl transferase (HPRT) as another therapeutic target in the repertoire of prostate cancer immunotherapeutic targets. We evaluated the HPRT enzyme because of its role in the purine salvage pathway, nucleotide synthesis, and cell cycle progression. To evaluate the role of HPRT in prostate cancer, PC3 and DU145 prostate cancer cells were used because of their markedly different levels of HPRT expression. Flow cytometry and scanning electron microscopy showed the surface localization of HPRT while immunohistochemistry revealed HPRT upregulation within tissue. We consistently found a significant association between HPRT and the plasma membrane of DU145 cells, but found no HPRT presence on the surface of PC3 cells. Flow cytometry showed insignificant (p = 0.14) changes in fluorescence when PC3 cells were exposed to HPRT antibodies, while there was a significant increase in fluorescence on DU145 cells (p = 0.0004). To confirm this finding, we used gold conjugated antibodies to determine the distribution of HPRT across the membrane with an electron microscope. This analysis further supported the surface presence of HPRT on DU145 cells as the gold weight percentage of DU145 cells increased significantly when exposed to HPRT antibodies (p < 0.0001). In addition to its surface expression on DU145 cells, elevated levels of HPRT were found in 47% of prostate patient tissue samples compared to healthy controls (n = 25), while 53% of patients had no HPRT upregulation (n = 28). Additionally, RNA-seq data from The Cancer Genome Atlas (TCGA) was used to evaluate general HPRT levels in patients with prostate cancer (n = 502) and healthy individuals (n = 52). This data revealed a significant (p < 0.0001) increase in HPRT levels upon malignancy. While some patients' levels were consistent with healthy control levels, there was a significant number of patients with increased protein expression upon cancer development. Because of the consistent and notable presence of HPRT on the surface of DU145 cells and in the tissue of prostate cancer patients, we began to investigate HPRT as a therapeutic target. Through MTS assays and live cell imaging, we assessed the potential of HPRT antibodies in an ADCC format. We observed increased DU145 cell death in wells treated with HPRT antibody when compared to wells treated with an isotype antibody and untreated wells (p < 0.01 and p < 0.0001, respectively). Furthermore, the amount of PC3 cell death in HPRT treated wells was insignificant when compared to isotype wells and untreated wells (p > 0.05). These results strongly indicate a unique relationship between prostate cancer cells and HPRT and supports the use of an HPRT antibody to harness the patient's immune system in treating subsets of prostate cancer.

#265

YWHAZ **amplification/overexpression defines aggressive bladder cancer and contributes to chemo-/radio-resistance via deregulation of caspase-mediated apoptosis.**

Jim Jinn-Chyuan Sheu,1 Chia-Cheng Yu,2 I-Hsuan Chen,2 Praveen Korla,1 Grace Ko,1 Chee-Yin Chai,3 Chien-Fong Li4. 1 _National Sun Yatsen University, Kaohsiung, Taiwan;_ 2 _Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan;_ 3 _Kaohsiung Medical University, Kaohsiung, Taiwan;_ 4 _Chi-Mei Foundation Medical Center, Tainan, Taiwan_.

The objective of this study is to characterize the oncogenic roles of a recently identified cancer-associated gene called YWHAZ (also known as 14-3-3 α/ζ ) in urothelial carcinomas of urinary bladder (UCUB). Based on a genomewide study, YWHAZ was found located at the 8q22.3 amplicon and its genetic amplification was predominantly detected in tumors at more advanced stages, muscle-invasive bladder cancer (MIBC). IHC staining confirmed the association of YWHAZ overexpression with higher tumor stages, lymph node/vascular invasion, and mitotic activity. Univariate and multivariate analyses further indicated the prognostic potential of YWHAZ for more aggressive cancer types. Both gene set enrichment analysis (GSEA) and STRING network studies suggest the involvement of YWHAZ in regulating caspase-mediated apoptosis. Ectopic expression of YWHAZ in bladder cells with low YWHAZ levels boosted cells becoming more tolerated toward chemical and ionizing radiation treatments. Gene knockdown by specific shRNA in cells with high YWHAZ levels attenuated the survival activity, resulting in cell growth suppression and cell death. Our data confirmed an essential role of YWHAZ in providing survival signals to sustain cell proliferation during chemo- or radio-therapy. Treatments based on anti-YWHAZ strategies may be beneficial for patients with YWHAZ overexpression.

#266

Relationship between BAG3 expression and the cytotoxicity of laromustine in HL60 cells.

Stanley R. Clarke, Allie H. Naccara, Amanda J. Loya, Sam W. Marchant, Xiaoou Wang, Thomas J. LaJoie, Jordyn N. Smith, Kevin P. Rice. _Colby College, Waterville, ME_.

Laromustine is an experimental sulfonylhydrazine prodrug with clinical potential against acute myelogenous leukemia and glioblastoma multiforme. Cytotoxicity is largely due to DNA crosslinking activity from an electrophilic subspecies of laromustine generated in situ. However, methylisocyanate, another electrophile cogenerated upon base-catalyzed activation of laromustine, induces acute toxicity in cultured leukemia cells. Isocyanate-mediated cytotoxicity likely involves apoptotic processes revealed in experiments presented herein showing PARP cleavage, caspase 3/7 activation, and annexin-V staining. We demonstrate that compounds that yield methylisocyanate, including laromustine, cause substantial dysregulation of gene transcription patterns that are not observed with a derivative of laromustine that possesses the DNA crosslinking activity but not the methylisocyanate. Purified mRNA from promyelocytic HL60 cells treated with either 100 µM agent or an equivalent volume of DMSO was measured using real-time RT-PCR experiments and GeneChip analyses. Among the nearly 3,000 genes dysregulated upon exposure to both laromustine and its methylisocyanate-bearing analog, BCL2-associated athanogene 3 (BAG3) emerged as a promising candidate for further investigation. This anti-apoptotic gene was significantly upregulated in these conditions. So as to assess the significance its gene product in cell death or survival in cultured cells treated with laromustine, a stable cell line expressing an shRNA construct to knock down BAG3 was engineered from HL60 cells using lentiviruses. Preliminary evidence suggests that these cells are more susceptible to the cytotoxic effects of laromustine. These data suggest that BAG3 may be part of a defensive strategy for promyelocytic leukemia cells to survive treatment with laromustine.

#267

Targeting the alt-NHEJ DNA repair pathway selectively sensitizes KRAS-mutant cancer cells to chemotherapy.

Patricia S. Haehnel,1 Sarah Swoboda,1 Nadine Lehmann,1 Sebastian Rosigkeit,1 Hernike Gothe,2 Danial I. Sasca,1 Dennis Strand,1 Matthias Theobald,1 Vassilis Roukos,2 Ernesto Bockamp,1 Thomas Kindler1. 1 _University Medical Center of Mainz, Mainz, Germany;_ 2 _Institute of Molecular Biology, Mainz, Germany_.

Genomic instability is considered as a hallmark of human cancer. Whereas the oncogene-induced emergence of genetic lesions caused by increased levels of reactive oxygen species (ROS) and/or replicative stress is well characterised, the impact of defined driver mutations on individual DNA damage response and repair pathways is poorly understood. Recently, we have shown that oncogenic Kirsten-rat sarcoma (KRAS) dysregulates the repair of DNA double-strand breaks (DSB) and shifts the balance from canonical non-homologous end-joining (c-NHEJ) towards the highly error-prone alternative-NHEJ (alt-NHEJ) repair pathway by upregulation of XRCC1, PARP1 and DNA ligase 3α (Lig3α).

In this study, we wanted to investigate, whether KRAS-mutant (KRASmut) lung cancer cells rely on alt-NHEJ to repair genotoxic stress-induced DSBs. In addition, we addressed the question whether targeting essential component of the alt-NHEJ pathways sensitises KRASmut cells toward genotoxic agents.

The presence of oncogenic KRAS associates with increased expression of essential components of the error-prone alt-NHEJ pathway and causes enhanced alt-NHEJ activity compared to KRAS-wild-type (KRASwt) lung cancer cells as revealed by in vivo host cell reactivation assays. Pharmacologic inhibition of PARP, which has been shown to promote end joining by alt-NHEJ, resulted in increased apoptosis upon combined treatment with standard genotoxic agents in KRASmut cells but not KRASwt cells. Moreover, mice bearing tumor xenografts derived from KRASmut lung cancer cell lines but not KRASwt tumors experienced attenuated tumor growth and longer overall survival upon treatment with the PARP-inhibitor Olaparib and chemotherapy compared to single agent therapy, respectively. Similar results were obtained using a genetically modified mouse model of inducible, KRASmut-dependent lung cancer. Mice bearing KRASmut lung tumors showed a significantly reduced tumor burden when treated with the combination therapy of Olaparib and the topoisomerase II inhibitor VP-16 compared to animals treated only with single agents alone. Mechanistically, the DNA damage sensor protein KU70 is trapped in the cytoplasm in a KRASmut-dependent manner. siRNA-mediated knockdown of KRAS caused a shift of KU70 into the nucleus allowing access of KU70 to DSBs and to initiate DNA repair via the c-NHEJ repair pathway.

Our data provide evidence for a novel synthetic lethal interaction between oncogenic KRAS and DNA damage repair. Targeting components of the alt-NHEJ represents a so far not recognized therapeutic strategy to induce synthetic vulnerability in cells harbouring otherwise non-druggable KRAS mutations.

#268

Engineering of transgenic mice expressing human mesothelin for investigation of mesothelin-targeted therapeutics.

Xianyu Zhang,1 Brendan Hagerty,1 Theresa Guerin,2 Norene O'Sullivan,2 Serguei Kozlov,2 Christine Alewine1. 1 _National Cancer Inst., Bethesda, MD;_ 2 _National Cancer Inst., Frederick, MD_.

Most monoclonal antibody-based therapeutics are specific for the human isoform of their targets and do not cross-react with orthologous mouse isoforms. This frequently limits pre-clinical modeling to human-derived tumors growing in immune compromised mice and precludes combination studies with immune modulating agents which rely on an active immune system for activity. Although introduction of human cDNA into mouse tumor cells will make these cells susceptible to targeting, a syngeneic mouse may reject or partially reject cells bearing the foreign transgene, impacting anti-tumor efficacy studies. Mesothelin (MSLN) is the target of many cell- and antibody-based therapies that have reached clinical trials due to its lack of expression in critical normal tissues and robust expression in many solid tumors. We have generated two distinct transgenic C57/Bl6 mouse models with compartment-limited expression of human MSLN (hMSLN) for use in pre-clinical testing of MSLN-targeted therapeutics: 1) TPO/hMSLN mice express full-length hMSLN from an insulated rat thyroid peroxidase gene promoter. Analysis by RT-PCR, immunoblot and IHC demonstrate strong thyroid-specific expression hMSLN. 2) In Nor/hMSLN mice, the hMSLN coding region was inserted as a full-length cDNA into the native mMSLN locus via a homologous recombination knock-in technique such that hMSLN is expressed in place of the mouse isoform under the control of endogenous murine MSLN transcription regulatory elements. IHC studies show expected expression of hMSLN in mouse pleura, pericardium and peritoneum. Shed hMSLN can be readily detected in the serum of both transgenic lines using ELISA, similar to human patients. Treatment of either model with MSLN-targeted immunotoxin LMB-100 at the maximum dose tolerated by non-transgenic C57/Bl6 mice resulted in no gross toxicity. Histologic analysis revealed subclinical pericarditis in Nor/hMSLN mice. No post-treatment thyroid damage was observed in TPO/hMSLN mice even upon detailed histologic examination of thyroid tissue. Syngeneic mouse pancreatic cancer cells expressing hMSLN transgene implanted orthotopically into TPO/hMSLN or Nor/hMSLN grew as well or better as those grown in non-transgenic C57/Bl6 mice. We have established two immunologically proficient transgenic mouse models expressing hMSLN that can be used for evaluating immune effect of hMSLN-targeted therapeutics as well as their on-target/off-tumor toxicities.

#269

DNA-PK inhibitor, M3814, as a new combination partner of Mylotarg in the treatment of acute myeloid leukemia.

Astrid Zimmermann,1 Michael Carr,2 Frank T. Zenke,1 Andree Blaukat,1 Lyubomir T. Vassilev2. 1 _Merck KGaA, Darmstadt, Germany;_ 2 _EMD Serono, Billerica, MA_.

Despite significant advances in the development of new treatments for acute myeloid leukemia (AML) the therapeutic success remains relatively poor. There remains an urgent need for improved therapies with increased tumor potency and selectivity. Mylotarg is the first AML drug from a new generation of antibody drug conjugate (ADC) therapies targeting the acute leukemia cell compartment with increased specificity. This agent targets leukemia cells for apoptosis with a cytotoxic warhead, calicheamicin, carried by a CD33-specific antibody. Calicheamicin induces DNA double strand breaks (DSB) which, if left unrepaired, lead to cell cycle arrest and apoptosis in cancer cells. However, repair of DSBs by the non-homologous end joining pathway driven by DNA-PK (DNA-dependent protein kinase) can reduce the efficacy of calicheamicin. M3814 is a novel, potent and selective inhibitor of DNA-PK protein kinase. This compound effectively blocks DSB repair, strongly potentiates the antitumor activity of ionizing radiation and DSB-inducing chemotherapeutics and is currently under clinical investigation. Suppressing DSB repair with M3814 synergistically enhanced the apoptotic activity of calicheamicin in cultured AML cells. Combination of M3814 with Mylotarg in two AML xenograft models, MV4-11 and HL-60, demonstrated increased efficacy and significantly improved survival benefit without elevated body weight loss. Our results support a new application for pharmacological DNA-PK inhibitors as enhancers of Mylotarg efficacy and a potential new combination treatment option for AML patients.

#270

Temozolomide resistant MGMT negative/MMR proficient cancer cells rely on ATR signaling and homologous recombination for DNA repair and survival.

Lara El Touny,1 Erik Harris,1 Curtis Hose,1 John Connelly,1 Diana Vengsarkar,1 Anne Monks,1 Ralph E. Parchment,1 James H. Doroshow,2 Beverly A. Teicher,2 Annamaria Rapisarda1. 1 _FNLCR/Leidos Biomed. Research, Inc., Frederick, MD;_ 2 _NCI, NIH, Bethesda, MD_.

MGMT loss is used to predict response to temozolomide (TMZ), however, some patients do not benefit, suggesting that additional criteria for selection remain to be identified. Using patient derived and well-established cancer lines cancer lines we observed a 100% association between mismatched repair (MMR) defects and TMZ resistance; however, only 16% of MGMT-/MMR+ lines were sensitive to TMZ at clinically relevant concentrations. Activation of DNA damage and repair signaling was noted in both sensitive and resistant bladder cancer cell lines in response to TMZ but protracted DNA damage, accompanied by persistent G2/M arrest, was observed only in the sensitive models. ATR inhibition sensitized MGMT-/MMR proficient resistant models to TMZ resulting in measurable DNA damage, prolonged G2/M arrest and persistent growth inhibition: an effect that was mediated by Chk1. Homologous recombination and repair has been shown to play an important role in repairing TMZ-induced DNA damage. We demonstrate that ATR inhibition abrogated homologous recombination (HR) activity in a MGMT-/MMR proficient TMZ resistant bladder model and that a TMZ sensitive bladder model had minimal HR (assessed by pDR-GFP/SceI assay). Moreover, HR -deficiency (mediated by BRCA1 defects or RAD51 inhibition) conferred sensitivity to TMZ in combination with an ATR inhibitor. Our in vitro data suggest that a MGMT-/MMR+/HR-Deficiency profile identifies models that respond to TMZ/ATR inhibitor combination. Indeed, one Patient-derived Xenograft (PDX) model (available in the Patient Derived Models Repository), selected by this criterion (MGMT-/MMR+ and LOH>40%) showed delayed tumor growth and increased survival in the TMZ-VX970 combination arm, compared to single agents. Overall, our data suggest that MGMT-/MMR+ cells rely on ATR-dependent signaling for repair of TMZ-induced DSBs and that HR defects should be evaluated as criteria for selecting patients that will benefit from an ATR inhibitor/TMZ combination regimen. Funded by NCI Contract No. HHSN261200800001E. This research was supported, in part, by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute.

#271

The role of ATM and DNA-PK in responding to AZD6738-induced damage in pancreatic ductal adenocarcinoma cells.

Charles R. Dunlop,1 Yann Wallez,2 Sandra Bernaldo de Quirós Fernández,1 Saadia A. Karim,3 Alan Lau,2 Frances M. Richards,1 Duncan I. Jodrell1. 1 _Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, United Kingdom;_ 2 _Bioscience, Oncology, IMED Biotech Unit, AstraZeneca, Cambridge, United Kingdom;_ 3 _Cancer Research UK Beatson Institute, Glasgow, United Kingdom_.

Therapeutic targeting of the DNA damage response (DDR) has the potential to improve the poor survival outcomes of pancreatic ductal adenocarcinoma (PDAC). Many common genetic alterations in PDAC augment replication stress, which activates Ataxia-telangiectasia and Rad3-related kinase (ATR). We have demonstrated previously, efficacy of the ATR inhibitor (AZD6738) in PDAC models, particularly when combined with gemcitabine (gem) (Wallez et al, MCT, 2018). This combination strongly induces activation of Ataxia-telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK), indicating the induction of double-strand breaks. It has been suggested that ATM-deficiency can sensitize cancer cells to ATR inhibitors. This study sought to assess the relevance of this finding to PDAC and to interrogate the distinct roles of ATM and DNA-PK in response to ATRi/gem in PDAC cell lines.

The ATM inhibitor, AZD0156, sensitized human pancreatic cancer cell lines (MIA PaCa-2, HPAF-II, AsPC-1) to AZD6738, as determined by SRB assays (e.g. MIA PaCa-2; AZD6738 GI50 = 3.8 µM, versus 1.3 µM in the presence of 30 nM AZD0156) and in longer term colony forming assays (surviving fraction (SF) (1 µM AZD6738); 78 +/- 0.9%, SF (1 µM AZD6738 + 30nM AZD0156); 11 +/- 4.2%). However, ATM knockdown with siRNA did not sensitize to AZD6738, nor to the combination of AZD6738/gem. This suggests that the presence of kinase-inhibited ATM at sites of DNA damage is more deleterious to PDAC cells than deficiency of ATM protein expression.

We established that AZD6738/gem could induce phosphorylation of the canonical targets of ATM (Chk2, KAP1) in MIA PaCa-2 when ATM was knocked down or inhibited. DNA-PK was also activated in response to exposure to ATRi/gem and we postulated that it was responsible for the Chk2/KAP1 activation. We then demonstrated that Chk2/KAP1 activation could be abrogated by DNA-PK inhibition with NU7441. Furthermore, in an ATM-null pancreatic cancer cell line, AZD6738/gem-induced KAP1 phosphorylation was also abrogated by inhibition of DNA-PK. Thus, DNA-PK appears to be responsible for the downstream activation of Chk2 and KAP1, induced by AZD6738/gem in PDAC cells.

Therefore, as well as revealing that inhibition of ATM using the novel inhibitor AZD0156 is highly deleterious to ATR inhibited PDAC cells, we have identified a compensatory mechanism via DNA-PK, which may be relevant for maximizing the therapeutic potential of DDR inhibitors in PDAC. Since DNA-PK clearly plays a dominant role in the DDR signaling pathways, we are now assessing whether DNA-PK inhibition synergizes with AZD6738+/- gem.

#272

Synergistic activity of the ATR inhibitor BAY1895344 in combination with immune checkpoint inhibitors in preclinical tumor models.

Antje Margret Wengner, Dennis Kirchhoff, Lars Roese, Sandra Berndt, Gerhard Siemeister, Bertolt Kreft, Dominik Mumberg. _Bayer AG, Berlin, Germany_.

The DNA damage response (DDR) consists of complex signaling pathways that secure the integrity of the genome in eukaryotic cells. Numerous lines of evidence suggest that enhanced DNA damage caused by intrinsically or acquired defects in DDR, increases tumor immunogenicity, potentially impacting anti-tumor immune responses and sensitivity to immune checkpoint inhibition. The ataxia telangiectasia and Rad3-related (ATR) kinase plays a central role in DDR by activating essential signaling pathways of DNA damage repair and is the key mediator of the replication stress response being indispensable for cellular proliferation and viability. Inhibition of ATR has been shown to lead to impaired cell cycle progression, increased DNA damage and replication stress ultimately resulting in rapid cellular lethality. The potential of combining inhibition of ATR with immune checkpoint blockade was studied in preclinical tumor models. We assessed efficacy of the novel ATR inhibitor (ATRi) BAY 1895344 in monotherapy and in combination with anti-PD-1 (Programmed Death 1) or anti-PD-L1 (Programmed Death 1 Ligand 1) antibody in syngeneic tumor models in immunocompetent mice. Interestingly, depletion of CD8+ T cells strongly reduced single agent activity of BAY 1895344 in the C57BL/6 mouse colorectal cancer (CRC) model MC38. Along this line, synergistic anti-tumor activity of BAY 1895344 and anti-PD-1/ PD-L1 could be demonstrated in MC38 tumors as well as in the BALB/c CRC model CT26 (both anti-PD-1/ PD-L1 sensitive), and the lymphoma model A20 (anti-PD-1/ PD-L1 insensitive). Remarkably, the synergistic activity was only achieved by sequential dosing of combination partners in the schedule: 1) anti-PD-1/PD-L1 Q2W (days 1 and 4 each week) followed by 2) BAY 1895344 BID 3 days on/ 4 days off (days 5, 6, 7 each week). Concomitant application or sequential dosing in the reverse order did not result in synergy or was even antagonizing single agent-mediated anti-tumor effects. Potential mechanisms underlying the interplay of ATRi and immune system activation will be discussed. Overall, our data suggest that the combination of immune checkpoint inhibitors and ATRi BAY 1895344 may enhance the anti-tumor efficacy of each single agent and potentially re-sensitize tumors to immune oncology treatments. BAY 1895344 is currently under clinical investigation in patients with advanced solid tumors and lymphomas as single agent (NCT03188965). Clinical exploration of a combination with anti-PD-1 is warranted.

#273

KDM1A inhibition enhances chemotherapy response in glioblastoma via downregulation of DNA repair pathways.

Bridgitte E. Palacios,1 Prabhakar Pitta-Venkata,1 Yihong Chen,1 Suryavathi Viswanadhapalli,1 Uday P. Pratap,1 Aleksandra Gruslova,1 Takayoshi Suzuki,2 Ratna K. Vadlamudi,1 Andrew Brenner,1 Gangadhara R. Sareddy1. 1 _UT Health San Antonio, San Antonio, TX;_ 2 _Kyoto Prefectural University of Medicine, Kyoto, Japan_.

Background: Glioblastomas (GBM) have dismal survival rates (1 year-34.6% and 5 year-4.75%) and affect 13,000 patients yearly. Standard of care treatment consists of surgical resection, external beam radiation therapy, adjuvant chemotherapy with temozolomide (TMZ), and tumor treating fields. Despite heavy investment in therapy, all patients will eventually succumb to their disease. Oncogenic and epigenetic signaling mechanisms may modulate DNA damage response (DDR) in tumors and contribute to chemo and radiation therapy resistance. Recently, we have shown that lysine-specific histone demethylase 1A (KDM1A), an epigenetic regulator, is overexpressed in GBM. In this study, we tested the hypothesis that KDM1A is essential for DDR, and that inhibition of KDM1A induces DNA repair deficiency and sensitizes GBM to TMZ therapy.

Methods: To study the role of KDM1A in GBM cells, we have generated KDM1A knockout (KDM1A-KO) cells using the CRISPR/Cas9 system and KDM1A-shRNA transfected primary GBM cells (KDM1A-KD). Effect of KDM1A-KO, -KD or KDM1A inhibitor (NCD-38) on TMZ sensitization was studied using cell viability and survival assays. Mechanistic studies were conducted using RNA-seq, RT-qPCR and western blot analysis. The blood-brain barrier (BBB) permeability of NCD-38 was studied by pharmacokinetic (PK) and brain bioavailability studies. Further, the in vivo efficacy of KDM1A inhibitor was studied using orthotopic models of GBM.

Results: Cell viability and survival assays showed that knockout or pharmacological inhibition of KDM1A sensitized GBM cells to TMZ treatment. KDM1A expression is increased in GBM cells after treatment with TMZ. RNA-seq analysis revealed a decreased expression of several genes involved in DNA repair, including MGMT, RAD51, and MRE11A in NCD-38 treated cells compared to control. PK and brain bioavailability studies following a single intravenous dose (IV, 1 mg/kg) and per oral administration (PO, 10 mg/kg) of NCD-38 demonstrated that NCD-38 has favorable PK properties and exhibited a significant penetration of the BBB, with a total brain/plasma ratio of more than two at all tested time points. Further, KDM1A inhibition significantly reduced the in vivo tumor progression in established and primary GBM orthotopic models.

Conclusions: Our results provide evidence that KDM1A contributes to chemotherapy resistance in GBM by modulating DNA repair pathways and the use of KDM1A inhibitor in conjunction with standard chemotherapy will serve as novel therapy for GBM patients.

#274

The ATR inhibitor BAY 1895344 shows strong preclinical activity in lymphomas and appears associated with specific gene expression signatures.

Eugenio Gaudio,1 Chiara Tarantelli,1 Filippo Spriano,1 Luciano Cascione,1 Alberto Arribas,1 Emanuele Zucca,2 Anastasios Stathis,2 Antje Margret Wengner,3 Francesco Bertoni1. 1 _Università della Svizzera italiana, Institute of Oncology Research, Bellinzona, Switzerland;_ 2 _Oncology Institute of Southern Switzerland, Bellinzona, Switzerland;_ 3 _Preclinical Research, Research & Development, Pharmaceuticals, Bayer AG, Berlin, Germany_.

Introduction. Targeting the DNA repairing pathways represents an intriguing new approach for cancer treatment. BAY 1895344 is a potent, highly selective and orally available ATR inhibitor (Luecking et al, AACR 2017) in its early clinical development in patients with advanced solid tumors and lymphomas (NCT03188965). We performed evaluated its antitumor activity in a large panel of lymphoma cell lines.

Methods. IC50s and caspase 3/7 activation were obtained in cell lines derived from human B (n=50) or T cell (n=9), murine (n=2) and canine (n=1) lymphomas exposed to increasing doses of BAY 1895344 for 72h. Apoptosis activation was defined by at least 1.5-fold increase in caspase 3/7 signal activation vs controls. Transcriptome data (Illumina HumanHT 12 Expression BeadChips, HTG Biomarker Panel) were analyzed with GSEA (statistical significance: absolute NES >1.5, FDR < 0.01). In vivo efficacy was evaluated in xenograft studies of human lymphoma in mice.

Results. BAY 1895344 showed anti-tumor activity with a median IC50 of 60 nM (95%C.I.; 3-500 nM) across the 62 cell lines, comprising mainly germinal center B-cell diffuse large B-cell lymphoma (GCB-DLBCL, n.=17), activated B-cell DLBCL (8), mantle cell lymphoma (MCL, 10), marginal zone lymphoma (6), T cell lymphomas (9). BAY 1895344 was mostly cytotoxic with apoptosis induction in 38/62 (61%) of the cell lines. Sensitivity was not affected by lymphoma histology or DLBCL cell of origin, TP53/BCL2/MYC status or ATM/ATR loss.

A xenograft experiment (MCL Rec-1 cell line) demonstrated complete tumor remission with BAY 1895344 (50 mg/kg, twice daily, 3 days on and 4 days off).

Comparing the gene expression profile of 4 less (IC50>200nM) vs 3 very sensitive (IC50<10nM) GCB-DLBCL cell lines, cell cycle regulation and DNA repair genes were positively associated with higher sensitivity (E2F targets; G2M checkpoint; DNA repair), while survival and inflammation transcripts with lower (TNFA signaling; IL2/STAT5 signaling). Similar results for E2F targets, G2M checkpoint, TNFA signaling and IL2/STAT5 signaling were confirmed in MCL cell lines. Finally, we compared the transcriptome obtained using an NGS-based platform in 30 B cell lymphoma cell lines divided by the median IC50. Genes involved in BRCA1 and/or ATM networks, RB1 and/or TP53 targets, WNT signaling were higher in the most sensitive cell lines, while the opposite was true for genes taking part to apoptosis, cytokine interaction pathway, inflammatory response and TNFA signaling via NFκB, and MAPK signaling.

Conclusion. A strong anti-tumor activity, associated with specific gene expression signatures, was seen with BAY 1895344 in lymphoma models, providing further support for the on-going phase I study.

#275

**A possible relationship between gene mutation profiles and activity of E7386 in** in vitro **models of colorectal cancer organoids.**

Toshio Imai,1 Mie Naruse,1 Masako Ochiai,1 Yoichi Ozawa,2 Takashi Owa,2 Atsushi Ochiai3. 1 _National Cancer Center Research Institute, Tokyo, Japan;_ 2 _Eisai Co., Ltd., Tokyo, Japan;_ 3 _EPOC, National Cancer Center, Kashiwa, Japan_.

A novel beta-catenin/CBP binding modulator E7386 is expected to affect colorectal carcinoma (CRC) cells particularly those with aberrant activation of Wnt/beta-catenin signaling pathway. The Wnt signaling pathway is recently reported to play important roles for activation of not only CRC cells but also cancer-associated fibroblasts (CAFs). Here, we established an in vitro model of CRC-derived organoids as well as a co-culture method of the CRC organoids with CAFs, and effects of E7386 on the growth of the organoids were examined. Fragments of CRC tissues from a total of 45 patients who underwent surgery were obtained, dissociated into single cells, which were seeded on polymerized Matrigel (Corning), and cultured with serum-free media containing EGF and Noggin optimized for organoids using 12-well plates. Fourteen CRC organoids from 45 cases were successfully established. For 2D culture of CAFs from CRC tissues of each patient, RPMI medium containing 10% FBS was used. A co-culture method of the CRC organoids with CAFs was applied using a double chamber system with transwell inserts in 24-well plates. Cell viabilities of the organoids and CAFs were measured using CellTiter-Glo Luminescent Cell Viability Assay reagent (Promega). Gene aberrations in original CRC tissues were analyzed using NCC Oncopanel, and gene expression profiles in established CRC organoids and CAFs were analyzed using SurePrint G3 Human GE microarray (Agilent). [Effects of CAFs on cell viabilities and gene expressions of organoids] Cell viabilities of organoids and CAFs appeared to increase at 72 hours after beginning of the culture by 2 to 8 and 1 to 2-fold, respectively. When organoids were co-cultured with CAFs, cell viabilities of organoids were increased and epithelial-mesenchymal transition-related gene expressions were dramatically changed in several cases, but not in all cases. The organoids co-cultured with CAFs appeared to be more sensitive to E7386 as compared to those cultured alone. [Possible relationship between gene mutations in CRCs and activity of E7386 in CRC organoids] Cell viabilities of organoids without APC mutations (n=1) and organoids with APC and TP53 mutations alone (n=2) were less than those of organoids with other oncogene mutations, e.g., KRAS or PIK3CA (n=4). Efficacy of E7386 on the cell viabilities of organoids with APC and TP53 mutations alone was found at lower concentrations at 30-100 nM, as compared to those of organoids without APC mutations. From the results, CRC organoids were established from 14 patients, and activity of E7386 appeared to be related to gene mutation profiles in the established CRC organoids.

#276

Targeting AXL sensitizes non-small cell lung cancer to ATR inhibitors by enhancing replication stress.

Kavya Ramkumar, Pan Tong, You-Hong Fan, David Peng, John V. Heymach, Don L. Gibbons, Jing Wang, Lauren A. Byers. _UT MD Anderson Cancer Center, Houston, TX_.

Therapeutic resistance limits effective treatment of non-small cell lung cancer (NSCLC) and a better understanding of mechanisms contributing to resistance and strategies to overcome these are urgently needed. AXL, a TAM family receptor tyrosine kinase, has emerged as a key determinant of intrinsic and acquired resistance to chemotherapy, radiation and targeted therapies in NSCLC and other cancers, through its roles in mediating epithelial-mesenchymal transition (EMT) and immune escape. We previously showed that AXL may also play a role in DNA damage repair and that AXL overexpression mediated primary as well as acquired resistance to inhibitors of WEE1, a replication stress response kinase, in small cell lung cancer. In the present study, we further investigated the role of AXL in replication stress response.

We found that, in NSCLC cell lines, AXL inhibition with the selective small-molecule AXL inhibitor BGB324 caused replication stress checkpoint activation, as indicated by increased RPA32 hyper-phosphorylation and ATR-mediated CHK1 phosphorylation. We further screened ATR inhibitors, VX-970 and AZD6738, in a panel of 20 NSCLC cell lines and correlated drug sensitivity with baseline expression of over 200 phosphorylated and total proteins, measured by reverse phase protein array. Notably, AXL was one of the biomarkers of resistance to the ATR inhibitor VX-970 (rho=0.52, p<0.05). Therefore, we hypothesized that AXL plays a unique role in regulating replication stress and targeting AXL will sensitize NSCLC cells to ATR inhibitors. Combination of BGB324 and ATR inhibitors (VX-970 and AZD6738) significantly decreased cell proliferation in a panel of human and GEMM-derived NSCLC cell lines as compared to single agents alone. In NSCLC cells with primary resistance to ATR inhibition, co-targeting AXL and ATR significantly increased RPA32 hyper-phosphorylation, concomitantly with increased DNA double strand breaks and inactivated G2/M checkpoint, resulting in mitotic catastrophe. AXL knockdown in a GEMM-derived Kras/Trp53 mutant NSCLC model also showed similar results. Notably, NSCLC cell lines with low levels of SLFN11 (a DNA/RNA helicase that induces replication arrest following DNA damage independently of ATR) were more sensitive to AXL/ATR co-targeting.

In conclusion, these findings suggest that AXL may play a novel and unexpected role in regulating replication stress. Furthermore, our results show that targeting AXL sensitizes NSCLC cell lines with primary resistance to ATR inhibitors and that AXL/ATR inhibitor combinations could be useful in treating platinum- and PARP inhibitor-resistant SLFN11-low tumors.

#277

Inhibition of protein arginine methylation alters RNA metabolism and DNA damage response providing a new therapeutic strategy in pancreatic ductal adenocarcinoma.

Virginia Giuliani,1 Alessandro Carugo,1 Meredith Miller,1 Lionel Sanz,2 Chiu-Yi Liu,1 Christopher A Bristow,1 Erika Suzuki,1 Caleb A Class,1 Stella R. Hartono,2 Guang Gao,1 Ningping Feng,1 Jason P Gay,1 Bhavatarini Vangamudi,1 Joseph R Marszalek,1 Jeffrey Kovacs,1 Maria Emilia Di Francesco,1 Frederic Chedin,2 Philip Jones,1 Giulio Draetta,1 Timothy Heffernan1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _University of California, Davis, CA_.

With limited therapeutic options, poor overall 5-year survival rates, and increasing incidence, pancreas cancer is estimated to become the second leading cause of cancer deaths by 2030. Recognizing the need for transformative advances in pancreas cancer management, we developed an in vivo target discovery platform to uncover molecular vulnerabilities in patient-derived pancreatic ductal adenocarcinoma (PDAC) xenografts to identify and rapidly translate novel therapeutic concepts to the clinic. We identified protein arginine methyltransferase 1 (PRMT1) as a dependency in PDAC required for disease maintenance and progression. Extensive genetic and pharmacological studies support PRMT1 as a novel vulnerability, which prompted our design and synthesis of proprietary series of potent, selective PRMT Type I inhibitors (PRMTi) with compelling in vivo activity. While advancing the project in drug discovery, we deployed a comprehensive approach to elucidate the mechanism of action of PRMTi. We characterized the PRMT1 interactome via PRMT1 immunoprecipitation followed by LC/MS and observed that PRMT1 binding partners were significantly enriched in RNA-binding and -processing genes. In addition, because methylation of arginine residues is a common post-translational modification regulating protein function, we identified substrates differentially methylated upon PRMT inhibition. Integrating these results with the PRMT1 interactome confirmed a strong correlation between PRMT1 substrates and complexes that are physically associated and linked to RNA metabolism. Transcriptome assays demonstrated that PRMT inhibition globally impaired RNA metabolism, including but not limited to RNA splicing, transcription termination, and R-loop formation. In addition, PRMTi caused a profound down-regulation of multiple pathways involved in the DNA damage response (DDR) promoting genomic instability. Taken together, these data support PRMT1 as a compelling target in an area of high unmet medical need and inform a mechanism-based translational strategy for future clinical development.

#278

**System x** c- **inhibition shares features of necroptosis and ferroptosis in hepatocellular carcinoma cells.**

Md Abdullah, Do Hyung Kim, Seung Jin Lee. _College of Pharmacy, Chungnam National University, Daejeon, Republic of Korea_.

Ferroptosis is characterized by iron-dependent non-apoptotic cell death involving the accumulation of lipid peroxides. Inhibition of system xc- which uptake L-cystine with the exchange of L-glutamate decreases glutathione synthesis, leading to ferroptosis. However, molecular interplay between ferroptosis and necroptosis has not been clearly identified. In this study, we investigated molecular process for cell death by SLC7A11 inhibition in hepatocellular carcinoma cell lines. The sensitivity for SLC7A11 inhibition by sulfasalazine was in the order of HepG2, Huh7 and Huh6, and then PLC/PRF/5. The decrease of cell viability by sulfasalazine and erastin was prevented by co-treatment of β-mercaptoethanol in all kinds of cell lines, indicating that the limited supply of L-cystine may contribute to cell death. Both ferrostatin and necrostatin, an inhibitor of ferroptosis and necroptosis, respectively, rescued sulfasalazine-induced growth inhibition by 60% in Huh7 and Huh6 and by 20% in HepG2. However, they failed to restore cell survival in PLC/PRF/5 cells. Sulfasalazine at the dose inducing equivalent level of cell death significantly increased lipid peroxidation in Huh7 and Huh6, but not in HepG2 and PLC/PRF/5 cells, as determined by C11-BODIPY dye. RIPK activation in necroptosis pathway by sulfasalazine was monitored in these cell lines. These results suggest that SLC7A11 inhibition triggers mixed-type of cell death via ferroptosis and necroptosis in a context-dependent manner in hepatocellular carcinoma cells (MD Abdullah and DH Kim are equally contributed to this work).

#279

c-Jun N-terminal kinase is involved in an oxidative stress- and vacuole-associated cell death pathway in triple negative breast cancer.

Milad Soleimani, Ritvik Bhattacharjee, Uma Giri, Tamer S. Kaoud, Kevin N. Dalby, S. Gail Eckhardt, Carla L. Van Den Berg. _The University of Texas at Austin, Austin, TX_.

Breast cancer is the most prevalent type of cancer among women. Triple negative breast cancer (TNBC) is a highly aggressive and heterogeneous subtype of breast cancer. It is the most challenging subtype to treat mainly due its aggressive nature and lack of targets such as HER2 (human epidermal growth factor 2), PR (progesterone receptor), and ER (estrogen receptor) found in other subtypes. We have shown that inhibiting both EGFR (epidermal growth factor receptor)/HER2 with lapatinib and JNK with JNK-IN-8 causes synergistic cell death in 11 out of 12 TNBC cell lines. EGFR is expressed in up to 90% tumors and JNK activation is linked to TNBC phenotype, metastasis, and stem cell character, among others. Synergism was achieved in the resistant, low-EGFR expressing cell line when a FGFR inhibitor (BGJ398) was used instead of lapatinib in combination with JNK-IN-8. Combination drug effects were accompanied by elevated oxidative stress. Suppression of oxidative stress restored cell viability in cells treated with the drug combination. JNK-IN-8 treatment alone or in combination with lapatinib induced massive cytoplasmic vacuolization with little apoptotic blebbing. The vacuoles were shown to be membrane-derived and acidic using Lucifer Yellow and Acridine Orange, respectively. Pre-treatment of cells with bafilomycin A1 (lysosome inhibitor), 5-(N-Ethyl-N-isopropyl) amiloride (macropinocytosis inhibitor), or N-acetylcysteine (antioxidant) reduced vacuolization. A similar effect was observed when cells were serum-starved. JNK1 knockout mouse mammary tumor cells, in comparison with JNK2 knockout, were resistant to drug synergism and displayed significantly less oxidative stress and vacuolization. Our studies demonstrated that concomitant blockade of JNK and individual tumor-appropriate targets induce synergistic cell death in TNBC cells in response to oxidative stress. Moreover, JNK blockade resulted in the formation of serum-dependent, acidic vacuoles that were reversible by lysosome or macropinocytosis inhibition. Loss-of-function studies indicated that JNK1 was the key isoform responsible for the drug response in TNBC cells. These findings suggest that JNK is involved in a novel cell death pathway and is a robust target in combination therapies against TNBC. 

### Combination Approaches to Novel Therapies

#280

Synthetic lethality of KDM1A and mTOR inhibitors: A novel combination therapy for endometrial cancer.

Prabhakar Pitta-Venkata,1 Bridgitte Palacios,1 Yihong Chen,1 Suryavathi Viswanadhapalli,1 Uday P. Pratap,1 Yiliao Luo,1 Mengxing Li,1 Kristin Altwegg,1 Xiaonan Li,1 Takayoshi Suzuki,2 Rajeshwar Rao Tekmal,1 Edward Kost,1 Gangadhara Reddy Sareddy1. 1 _UT Health San Antonio, San Antonio, TX;_ 2 _Kyoto Prefectural University of Medicine, Kyoto, Japan_.

Background: Endometrial cancer (EC) is the sixth most common cancer in women. Annually 63,230 new cases are diagnosed with 11,350 deaths estimated in the USA. Currently, advanced EC therapies remain palliative and new therapeutic strategies are urgently needed. Common risk factors include exposure to high levels of estrogen, obesity, and alterations in genetic and epigenetic factors. The lysine-specific demethylase-1A (KDM1A/LSD1) regulates gene expression programs by changing the epigenetic histone marks at the gene promoters. Emerging studies provided the evidence that KDM1A is overexpressed in EC. In studies examining the synthetic lethality of KDM1A inhibition on chemotherapy drug sensitivity, we made an unexpected discovery that KDM1A inhibition potentiate activity of mTOR inhibitors. In this study, we tested the hypothesis that inhibition of KDM1A could sensitize EC to mTOR inhibitor therapy.

Methods: To study the significance of KDM1A inhibition on chemotherapy drug sensitivity, we performed MTT assays to screen 119 FDA approved drugs using KDM1A knockdown HEC1A and RL95 EC cell lines. Effect of KDM1A knockdown or KDM1A inhibitor (NCD-38) therapy on EC cells was examined using MTT cell viability assays and clonogenic survival assays. The effect on cell migration was examined using scratch wound healing assay. Mechanistic studies were conducted using RNA-seq, western blot, qRT-PCR, and IHC analysis. The in vivo efficacy of NCD-38 and sirolimus on EC progression was studied using mouse xenograft models.

Results: Studies using 119 FDA approved drugs identified that mTOR inhibitors sirolimus and temsirolimus has potent synthetic lethality on KDM1A knockdown cells compared to control cells. Cell viability and survival assays demonstrated that KDM1A knockdown or inhibition in combination with sirolimus synergistically reduced cell viability and the survival of EC cells. Further, combination of KDM1A inhibitor and sirolimus reduced the migration of EC cells. Western blot analysis demonstrated that knockdown or inhibition of KDM1A attenuated the activation of mTOR signaling cascade in EC cells. RNA-seq and gene set enrichment analysis identified the down regulation of E2F pathway and DNA replication pathways in KDM1A and mTOR inhibitor treated cells compared to control cells. Further, combination of NCD-38 and sirolimus significantly reduced the in vivo tumor progression in xenograft models. IHC analysis of tumors revealed the downregulation of proliferation marker Ki67 and phosphorylation of mTOR signaling molecules in combination treated tumors compared to vehicle treated tumors.

Conclusions: The results from these studies provide compelling evidence that KDM1A inhibition sensitizes EC cells to mTOR inhibitors, and the use of KDM1A inhibitor in conjunction with mTOR inhibitors may be an attractive therapy for advanced EC patients.

#281

Sensitivity to splicing modulation of BCL2 family genes reveals cancer therapeutic strategies for splicing modulators.

Daniel Aird, Teng Teng, Chia-Ling Huang, Ermira Pazolli, Deepti Banka, Kahlin Cheung-Ong, Cheryl Eifert, Craig Furman, Jeremy Wu, Michael Seiler, Silvia Buonamici, Peter Fekkes, Craig Karr, James Palacino, Eunice Park, Peter Smith, Lihua Yu, Yoshiharu Mizui, Markus Warmuth, Agustin Chicas, Laura Corson, Ping Zhu. _H3 Biomedicine, Cambridge, MA_.

Dysregulation of RNA splicing by spliceosome mutations or in cancer genes is increasingly recognized as a hallmark of cancer. Small molecule splicing modulators have been introduced into clinical trials to treat solid tumors or leukemia bearing recurrent spliceosome mutations. Nevertheless, further investigation of the molecular mechanisms that may enlighten therapeutic strategies for splicing modulators is highly desired. Here, using unbiased functional approaches, we report that the sensitivity to splicing modulation of the anti-apoptotic BCL2 family genes is a key mechanism underlying preferential cytotoxicity induced by the SF3b-targeting splicing modulator E7107. While BCL2A1, BCL2L2 and MCL1 are prone to splicing perturbation, BCL2L1 exhibits resistance to E7107-induced splicing modulation. Consequently, E7107 selectively induces apoptosis in BCL2A1-dependent melanoma cells and MCL1-dependent NSCLC cells. Furthermore, combination of BCLxL (BCL2L1-encoded) inhibitors and E7107 remarkably enhances cytotoxicity in cancer cells. These findings inform mechanism-based approaches to the future clinical development of splicing modulators in cancer treatment.

#282

Minnelide improves the efficacy of the nab-paclitaxel plus gemcitabine plus cisplatin chemotherapy in mouse models of PDAC.

Liang Ng, Pawan Noel, Stephanie Mou, Daniel D. Von Hoff. _Translational Genomics Research Institute, Phoenix, AZ_.

Background: There have been some recently developed regimens which have improved the survival of patients with advanced pancreatic cancer. In a recent Phase Ib/II pilot trial in patients with advanced stage IV PDAC, nab-paclitaxel/gemcitabine/cisplatin regimen (triple combination, TC) exhibited promising clinical activity with a response rate of 71% and overall median survival of 16.5 months associated with manageable adverse effects (Jameson, et al., J. Clin. Oncol. 2017 35:4 suppl, 341). Minnelide (Min), a novel water soluble pro-drug of triptolide, a potent antitumor agent isolated from a Chinese medicinal herb, has demonstrated efficacy against several human cancers implanted in mouse models. Min is currently in Phase II trial in patients with refractory pancreatic cancer. In this study we evaluated the synergy between the TC and Min in various pancreatic cancer models (including the treatment sequence of the agents).

Methods: The drug combination efficacy was evaluated in athymic nude mouse models generated with MIA PaCa-2 cells and patient derived xenografts (PDX) implanted subcutaneously. The mice were treated with vehicle control, Min (QDx21), TC (QWx3) or TC (QWx3) plus Min(QDx21). In the PDX models Min (QDx21) given before or after the TC (QWx3) was also evaluated. To evaluate the effects of the combination treatment on tumor immune microenvironment, the KPC transgenic mice (K-RasG12D/+, p53R172H/+) were also treated with same combinations. Tumor volumes were assessed by ultrasound and immune cell infiltration was assessed by immunohistochemistry (IHC).

Results: The TC plus Min shows the greatest effect in tumor growth inhibition which remains persistent even after the treatment is discontinued in both the cell line xenograft model and the PDX models with several of the mice exhibiting complete remissions. In the MIA PaCa-2 cell line xenograft model, comparing to the vehicle control the tumor growth inhibition after drug treatment (21 days) was 97%, 93%, and 100% for Min, TC, and Min + TC, respectively. The animal survival at 45 days was 0%, 73%, 67%, and 90% for vehicle, Min, TC, and Min + TC, respectively. In the PDX model, the tumor growth inhibition after drug treatment was 86%, 84%, and 96% comparing to the vehicle, whereas the animal survival at 45 days was 0%, 89%, 100% and 100% for vehicle, Min, TC, and Min + TC, respectively. Min administered prior to or after the TC treatment also exhibited improved efficacy versus TC alone, with tumor growth inhibition similar to those treated with the agents concurrently. These results provide preclinical insights for the design of clinical trials that may further improve outcome in patients with advanced PDAC. (This work was supported by the SU2C-CRUK-Lustgarten Dream Team grant (Grant number: SU2C-AACR-DT-20-16))

#283

Combination treatment of PARP and SRC inhibitors in BRCA2 mutated prostate cancer.

Nabeela Khan,1 Goutam Chakraborty,1 Subhiksha Nandakumar,1 Ying Z. Mazzu,1 Mohammad Atiq,1 Yuki Yoshikawa,1 Gwo-Shu Mary Lee,2 Philip Kantoff1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Dana Farber Cancer Institute, Boston, MA_.

Background: Recently, poly (adenosine diphosphate [ADP]-ribose) polymerase inhibitors (PARPi) have shown promise in metastatic castration resistant prostate cancers (mCRPC) patients harboring DNA repair defects due to mutations of BRCA1/2 and ATM. Despite responses, resistance is common and treatment modalities for PARPi-resistant patients are limited. We hypothesized that combining PARPi with other agents in BRCA2 altered tumors would create synergy and greater response rate and durability.

Methods: We established a transcriptomic profile associated with genomic deletion of BRCA2 (homozygous and heterozygous) using publicly available datasets from mCRPC patient tumors. To identify the significantly unregulated oncogenic signaling pathways associated with BRCA2 loss, we used gene-set enrichment analysis (GSEA). We also analyzed this transcriptomic profile by Toppgene suite to identify potential drug targets for BRCA2 deleted tumors. We used experimental human prostate cancer cell lines to validate our current observations from human clinical datasets and pathway analysis.

Results: Our GSEA analysis showed significant enrichment of the SRC signaling pathway in BRCA2-altered tumors, and based on the Toppgene suite, we also identified dasatinib (pharmacological inhibitor of SRC) as a potential agent for BRCA2-deleted tumors. Similarly, we observed significant up regulation of SRC phosphorylation in human prostate cancer cells (LNCaP-abl and PC3M) that harbor genomic deletion of BRCA2. We performed cell growth assays in these BRCA2 deleted cells treated with PARPi (olaparib, talazoparib), and dasatinib alone or in combination and calculated drug synergy based on the Chou-Talay Method. PC3M and LNCaP-abl prostate cancer cell lines showed different sensitivities to olaparib and talazoparib. We used olaparib for PC3M and talazoparib for LNCaP-abl cells. Co-administration of the PARPi and SRCi showed significant synergy in both cell lines compared to either inhibitors alone. We will develop the rationale for combining PARPi and SRCi in CRPC patients who harbor defects of BRCA2 after completion of our study in xenograft models.

Conclusion: Our study reveals the synergistic effect of PARPi and SRCi in prostate cancer cell lines. We believe this shows great potential for future clinical trials in patients with mCRPC harboring BRCA2 deletions.

#284

Combination therapies to prevent resistance to androgen deprivation therapies in prostate cancer.

Zhenghong Li,1 Carrie Qi Sun,2 Rebecca Arnold,2 John A. Petros,2 Carlos S. Moreno1. 1 _Emory University School of Medicine, Atlanta, GA;_ 2 _Emory Hospital, Atlanta, GA_.

Androgen receptor (AR) signaling is a distinctive feature of prostate cancer (PCa) and represents a major therapeutic target for treating metastatic prostate cancer (mPCa). Thus, androgen deprivation therapy (ADT) is a first-line treatment for mPCa. Although initially highly effective as a treatment for mPC, ADT is characterized by the frequent emergence of resistance, a disease state termed castration-resistant prostate cancer (CRPC) and is generally incurable after progression to metastatic disease. Therefore, understanding the mechanisms underlying CRPC and subsequent progression to metastatic disease is critical. In our previous study, which was mainly focused on how transcriptional networks change in response to ADT and lead to metastasis, we analyzed matched pre-ADT and post-ADT tissue samples via RNAseq analysis of 40 formalin-fixed paraffin-embedded (FFPE) patient-matched pre-ADT biopsy (Bx) and post-ADT radical prostatectomy (RP) prostate cancer samples. We observed strong upregulation of components of the MAPK pathway including FOS, FOSB, and JUN, as well as downstream targets of MAPK signaling. These data suggest that ADT may induce a compensatory increase in MAPK signaling in response to the decrease in androgen signaling. Thus, we hypothesize that combination therapies targeting AR and the MAPK pathway may synergistically kill prostate cancer cells and prevent recurrence and progression to CRPC. In the current study, we have tested the effects of the MEK inhibitors PD0325901 and GSK1120212, ERK1/2 inhibitor GDC-0994, and the JNK inhibitor AS602801 alone and in combination with enzalutamide in androgen-sensitive LNCaP and MDA-PCa-2b cells. Cell viability assays indicated that enzalutamide combined with MEK and JNK inhibitors synergistically killed LNCaP and MDA-PCa-2b cells, and decreased migration and invasion of LNCaP cell more than any of the drugs alone. We therefore propose that combination therapy targeting AR and MEK and/or JNK signal pathways may be an effective treatment for recurrent prostate cancer. We are currently investigating the most promising combinations of enzalutamide with JNK inhibitors for anti-tumorigenic effects in vivo using a mouse xenograft model.

#285

Ixazomib combined with the nuclear export inhibitors selinexor or eltanexor for the treatment of multiple myeloma.

Joel G. Turner,1 Jana Dawson,1 Alexis Bauer,1 Juan Gomez,1 Erkan Baloglu,2 Yosef Landesman,2 Daniel M. Sullivan1. 1 _Moffitt Cancer Center, Tampa, FL;_ 2 _Karyopharm Therapeutics, Newton, MA_.

Introduction In the past decade, response and survival rates in multiple myeloma (MM) patients have been greatly increased by newer therapies. Nevertheless, most patients with MM eventually die from relapsed/refractory disease. Previous studies in our lab and in others have shown that exportin 1 inhibitors (XPO1i) are potent anti-MM drugs, whether used as single agents or in combination with proteasome inhibitors (PIs), immunomodulator drugs (IMiDs), anthracyclines, or alkylating agents. Ixazomib (IXA), an orally available PI, has been shown to be effective when combined with IMiDs, dexamethasone, alkylating agents, and/or prednisone for the treatment of patients with MM. We investigated whether the clinical XPO1i selinexor (SEL) or eltanexor (ELT) combined with IXA could overcome PI resistance in MM cell lines and in ex vivo bone marrow aspirates from PI-refractory patients.

Materials and Methods PI-resistant MM cell lines 8226B25 and U226PSR and their respective parental cell lines, 8226 and U266, were treated in vitro and in vivo (mice) with SEL/IXA or ELT/IXA combinations or with each drug as a single agent. Bone marrow aspirates from newly diagnosed and PI-refractory MM patients were treated ex vivo with SEL/IXA or ELT/IXA and assayed for apoptosis. Mechanistic studies included NFkB and IkBα protein assays, immunofluorescence microscopy, ImageStream flow-cytometry, and proximity-ligation assay. IkBα knockdown and NFkB transcriptional activity were measured in SEL/IXA- or ELT/IXA-treated MM cells.

Results SEL and ELT each sensitized parental and, to a lesser extent, PI-resistant MM cells to IXA, as measured by apoptosis. XPO1i/IXA treatments inhibited U266 and 8226 MM tumor growth and increased survival in mice; however, PI-resistant tumors were not sensitized by XPO1i/IXA treatment. Myeloma cells isolated from both newly diagnosed and PI-refractory MM patients were sensitized by XPO1i to IXA, as compared to untreated and single agent-treated MM cells, without affecting non-MM cells. Immunofluorescence microscopy, Western blot, and ImageStream analyses of MM cells showed increased nuclear IkBα with the XPO1i/PI treatments. Proximity ligation found increased IkBα-NFkB complexes in treated MM cells. IkBα knockdown abrogated XPO1i/PI-induced cytotoxicity in MM cells. XPO1i/PI treatment decreased NFkB transcriptional activity.

Conclusions In previous studies, we found that SEL sensitized PI-resistant cell lines to carfilzomib and bortezomib; however, IXA was less effective in these same MM cells. SEL or ELT used in combination with IXA have the potential to overcome PI drug resistance in MM patients. Sensitization may be due to inactivation of the NFkB pathway by IkBα. Further investigation is underway to examine these mechanisms. The results presented in this study support combinatorial clinical trials in relapsed and refractory MM patients who use PI therapies.

#286

Synergistic efficacy of CK2 inhibitor with common chemotherapy drugs by restoring Ikaros function in high-risk ALL.

chunhua song,1 Zheng Ge,2 Chandrika Gowda,1 Yali Ding,1 Jonathon Payne,1 Bihua Tan,1 Nathalia M. Cury,1 Elanora Dovat,1 Zhijun Zhao,1 Xiaoguang Lyu,1 Mary McGrath,1 Dhimant Desai,1 Soumya lyer,1 Pavan K. DhanyamRaju,1 Kimberly J. Payne,3 Sinisa Dovat1. 1 _Pennsylvania State University College of Medicine, Hershey, PA;_ 2 _Zhongda Hospital, Medical School of Southeast University, Nanjing, PA;_ 3 _Loma Linda University, Loma Linda, CA_.

Objective: IKZF1 gene-coding protein, Ikaros functions as a leukemia suppressor. Casein Kinase II activity is overexpressed in acute lymphoblastic leukemia (ALL) and CK2-mediated-dysfunction of Ikaros is one of the key reason for high-risk ALL and CK2 inhibitor -CX4945 treatment shows high therapeutic efficacy on high-risk ALL. The anti-apoptotic factors are highly expressed in leukemia and the commonly-used 1st-line chemotherapy drugs exerts the anti-tumor effect by suppression of anti-apoptosis signaling. Ikaros binding peaks was identified in the promoter of anti-apoptotic genes by ChIP-seq, suggesting Ikaros regulation on their expression. These observations also suggest the synergistic effect of restoring Ikaros function with common chemotherapy durgs in ALL.

Methods: The ChIP-seq and qChIP assays were performed to determine the enrichment of Ikaros and H3K4me3 in promotor of the genes. Lentiviral Ikaros or IKZF1 shRNA were used for functional analysis. WST-1 cell proliferation assay, Annexin-V staining plus flow cytometry and Patients-derived xenograft mouse (PDX) model were used for observing the anti-tumor effect in vitro and in vivo, respectively.

Results: ChIP-seq and qChIP assays identified Ikaros binding peaks in the promoter of anti-apoptotic genes in cell-lines and patients' samples. Ikaros overexpression suppresses but IKZF1 knockdown promotes the gene expression. CX-4945 suppresses the expression of the genes by decreasing the H3k27me3 enrichment in an Ikaros and HDAC1-dependent manner in B-ALL cells. The anti-apoptotic gene is significantly up-regulated in ALL patients. CX-4945+chemoterhapy drugs significantly induces the cell proliferation arrest and apoptosis compared to single drugs in vitro and also show the synergistic effect analyzed by CalcuSyn software. CX-4945+chemotherapy drugs significantly reduced the total leukemia cells and % leukemic cells in the three high-risk B-ALL Patient Derived Xenograft (PDX) mice model compared to that of single drugs, which indicated that their synergistic therapeutic efficacy on leukemia development.

Conclusion: Ikaros suppressed anti-apoptotic gene expression through histone modification in ALL. CK2 inhibitor, CX-4945 by restoring Ikaros function have synergistic efficacy with common chemotherapy drugs on high-risk B-ALL.

#287

A novel class III receptor tyrosine kinase inhibitor synergizes with olaparib by inhibition of DNA repair-related genes in non-small cell lung cancer cells.

Hwani Ryu, Jie-young Song, Sang-Gu Hwang, Jiyeon Ahn. _Korea Inst. of Radiological & Medical Sci., Seoul, Republic of Korea_.

The platelet-derived growth factor receptors (PDGFRs), which are classified with class III receptor tyrosine kinase (RTK) family, play critical roles in cell proliferation, differentiation, and malignancy in various types of human cancers. To develop novel class III RTK inhibitors, dozen novel oxazole amine derivatives were designed and synthesized. Among the synthesized compounds, KM201 showed inhibitory activities of pan-class III RTK by an in vitro kinase activity assay and potent anti-proliferative activities in variety of cancer cell lines including leukemia and solid cancer cells. Genome-wide RNAseq profiling revealed a significant decrease of DNA repair-related genes in non-small cell lung cancer A549 cells treated with KM201. Moreover, KM201-induced DNA damage was detected by H2AX phosphorylation, which led to cell cycle arrest in the G2/M phase. We hypothesized that KM201 in combination with a PARP inhibitor may exhibit synergistic effect on inhibition of proliferation since PARP inhibitors are primarily effective against cancers with loss-of-function mutations in BRCA1 and BRCA2. Combination of KM201 and olaparib (a PARP inhibitor) markedly more effective in anti-proliferation and apoptotic cell death in A549 and H1299 cells harboring wild type BRCA1/2, compared with either drug alone. Our results show that the novel class III RTK inhibitor KM201 inhibits proliferation of leukemia and solid tumor cells by potentiating DNA damage and exerts synergistic cytotoxicity in combination with olaparib. Further detailed studies could help to investigate the full potential of KM201 as a class III RTK inhibitor and a combination adjuvant of PARP inhibitors.

#288

RAGE inhibitors make pancreatic tumor responsive to gemcitabine therapy by reducing autophagy and facilitating apoptosis.

Priyanka Swami,1 Estelle Leclerc,1 Prakash Radhakrishnan,2 Ayrianne Crawford,2 Prathamesh Patil,2 Simon Shin,2 Tom Caffrey,2 James Grunkemeyer,2 Kelly O'connell,2 Michael Hollingsworth2. 1 _North Dakota State University, Fargo, ND;_ 2 _University of Nebraska Medical Center, Omaha, NE_.

Pancreatic cancer (PC) is a lethal disease with the current 5-year survival of only about 8%. Despite decades of research, gemcitabine remains the standard of therapy for PC. However, after an initial good response, eventual development of chemo-resistance is a major challenge faced by gemcitabine therapy. One principal cause of chemo-resistance is the release of high mobility group box 1 (HMGB1) protein from dying tumor cells, upon treatment with gemcitabine. When released, HMGB1 interacts with the receptor for advanced glycation end-products (RAGE), stimulating distinct signaling pathways leading to increased autophagy and tumor cell proliferation along with a reduction in apoptosis. RAGE activation by HMGB1 thereby supports tumor cell survival, making the tumor less sensitive towards gemcitabine. We hypothesized that inhibiting RAGE-HMGB1 interaction, in combination with gemcitabine therapy could reduce autophagy and tumor cell proliferation as well as facilitate apoptosis. This would ultimately reduce chemo-resistance and make pancreatic tumor more sensitive towards gemcitabine therapy.

In our study, we intended to determine the efficacy of RAGE inhibitors (anti-RAGE antibody IgG2A11 and small molecule RAGE inhibitor FPS-ZM1) in combination with gemcitabine in PC. We employed an orthotopic mouse model of PC to test our hypothesis. In this model, the murine #5508 KPC pancreatic cancer cell line was implanted in the pancreas of C57BL/6 mice. The implanted mice received different treatments (saline, RAGE inhibitor, gemcitabine or gemcitabine with RAGE inhibitor). At the end of the study, tumors obtained from different treatment groups were assessed for their weight. Additionally, these tumors were analyzed for the expression of markers of autophagy, cell proliferation and apoptosis by western blot.

We observed smaller tumors in mice treated with the combination of IgG2A11 and gemcitabine as well as FPS-ZM1 and gemcitabine, as compared to mice treated with gemcitabine alone, supporting our hypothesis. When the tumors from the mice treated with IgG2A11 and gemcitabine were examined, smaller tumors were associated with an increase in p62 levels, implying a reduction in autophagy. We also noticed lower levels of phosphorylated ERK in the group that received IgG2A11 and gemcitabine, indicating a reduction in tumor cell proliferation. Additionally, we observed higher levels of cleaved PARP in the group treated with the combination of IgG2A11 and gemcitabine suggesting higher levels of apoptosis in tumor cells. In conclusion, our data suggests that inhibiting RAGE in combination with gemcitabine could be a valid therapeutic approach for the treatment of pancreatic adenocarcinoma.

#289

MET activation mediates lapatinib resistance in experimental esophageal adenocarcinoma.

Md Sazzad Hassan,1 Fiona Williams,2 Lucia Petrova,1 Niranjan Awasthi,1 Margaret Schwarz,1 Roderich Schwarz,1 Urs von Holzen3. 1 _Indiana University School of Medicine-South Bend, South Bend, IN;_ 2 _University of Notre Dame, South Bend, IN;_ 3 _Indiana University School of Medicine-South Bend and Goshen Center for Cancer Care, South Bend and Goshen, IN_.

Introduction: A major target for esophageal adenocarcinoma (EAC) therapies is the human epidermal growth factor receptor 2 (HER2). Unfortunately, patients treated with lapatinib, a dual EGFR and HER2 inhibitor, frequently develop resistance. Lapatinib fails to improve patient survival in HER2-postive EAC, and the mechanisms contributing to resistance remain largely unknown. Recent studies have demonstrated that HER2 and MET receptor tyrosine kinases are co-overexpressed in a subset EAC. In this study, we therefore studied the role of MET activation in lapatinib resistance mechanisms in experimental EAC.

Methods: We first characterized MET and HER2 activation in a panel of human EAC cell lines, and the differential susceptibility of these EAC cell lines to single agent or combination of MET and HER2 inhibitions by foretinib and lapatinib, respectively. We then explored the antitumor efficacy with survival advantage following foretinib and lapatinib monotherapy and in combination in murine subcutaneous xenograft and peritoneal metastatic survival models of human EAC. We also tested MET and HER activation status with sensitivity to their inhibitors in newly generated lapatinib resistant OE19 EAC cells (OE19-LPR).

Results: The OE33 EAC cell line with phosphorylation of both MET and HER2, demonstrated reduced sensitivity to foretinib and lapatinib when used as single agents. The co-administration of foretinib and lapatinib effectively inhibited both MET and HER2 phosphorylation, synergistically inhibited cell proliferation and xenograft tumor growth, induced apoptosis, and significantly enhanced overall mouse survival, thus overcoming single agent resistance. In the OE19 EAC cell line with only HER2 phosphorylation and the ESO51 EAC cell line with only MET phosphorylation, profound cell growth inhibition with induction of apoptosis was observed in response to single agent with lack of enhanced growth inhibition when the two agents were combined. Interestingly, OE19-LPR cells showed significant upregulated expression of phosphorylated MET compared to parent OE19 cells, detected by both Activesignal assay and Western blot analysis. In addition, OE19-LPR cells showed significantly reduced sensitivity to lapatinib compared to parent OE19 cells, and the co-administration of foretinib and lapatinib synergistically inhibited cell proliferation in OE19-LPR cells.

Conclusion: These data suggest that resistance to HER2 targeted therapies in HER2 and MET overexpressed EAC cells can be due to MET activation. Thus MET and HER2 targeted therapy appears to be a sensible treatment strategy for HER2 positive MET-overexpressing EAC.

#290

Formulation of tolerable protocols for combination regimens to control triple-negative breast cancer cell-derived xenograft tumor development in animals.

Pawat Pattarawat, Shelby Wallace, Agricola Odoi, Bianca Pfisterer, Hwa-Chain R. Wang. _Univ. of Tennessee, College of Veterinary Medicine, Knoxville, TN_.

Cumulative research and clinical results suggest an advantage to use a multiple drug combination over a single drug to control tumor growth and metastasis. In general, combination therapy may result in lower treatment failure rates, lower case fatality ratios, slower development of resistance, and fewer side effects than monotherapy, as well as reducing cost. Triple-negative breast cancer (TNBC) is aggressive, lethal, and heterogeneous. The molecular heterogeneity may account for the differential responses of TNBC to chemotherapies. TNBC is sensitive to conventional chemotherapies with initially high response rates; however, the prognosis is not optimal due to cancer recurrence and metastasis. Although recent advancements in drug development of targeted therapeutics and immunotherapy were somewhat effective in treating TNBC, these therapeutics still need extensive study. Although combination regimens are thought to have better therapeutic outcomes than single drug treatments, drug combinations may also come with overlapped toxicities. Thus, it is important to identify combination regimens that are safe and effectively treat cancers. Based on our in vitro results of combination regiments effective in the synergistic induction of TNBC cell death, we systemically investigated the toxicity and efficacy of combined triple and double agents at various doses and administration protocols to assess the treatment of mice bearing TNBC cell-derived xenograft tumors. We analyzed the adverse effects of combination regimens on the bone marrows, livers, and kidneys, as well as the blood cell profiles in immune-competent and -deficient mice. Our studies successfully identified tolerable protocols for triple and double combination regimens of gemcitabine, romidepsin, and cisplatin, which are FDA-approved anticancer agents, as well as the efficacy of these combination regimens in controlling TNBC xenograft tumors in immune-deficient mice. Our results indicated that the triple combination of gemcitabine plus romidepsin and cisplatin regimen was more effective than double combination regimens in the control of TNBC tumor development in vivo. In conclusion, our studies suggested that the triple combination regimens should be promptly considered as an advanced treatment of TNBC over the conventional gemcitabine and cisplatin regimen or the clinical trial romidepsin plus cisplatin regimen.

#291

Synergistic inhibition of cancer invasion and metastasis by combined anti-PD1-TRC105-mediated Endoglin targeting on cancer-associated fibroblasts and endothelial cells.

Mark JA Schoonderwoerd,1 Madelon Paauwe,1 Ricardo Angela,1 Maaike Koops,1 Tom Harryvan,1 Cornelis Sier,1 Marieke Fransen,1 Charles Theuer,2 Lukas JAC Hawinkels1. 1 _Leiden Univ. Medical Ctr., Leiden, Netherlands;_ 2 _Tracon Pharmaceuticals, San Diego, CA_.

Cancer-associated fibroblasts (CAFs) are the major component of the tumor microenvironment in colorectal cancer (CRC) and play an important role in tumor progression and metastasis, partly through the transforming growth factor-β (TGF-β) signaling pathway. Recently, we showed that a subset of CAFs characterized by the expression of the TGF-β family co-receptor endoglin, selectively localized at the invasive borders of CRC tissues. Moreover, CAF-specific endoglin expression increased with higher tumor stage and predicted metastasis-free survival of stage-II CRC patients (n= 140, p<0.05). Targeting endoglin on CAFs using TRC105 (carotuximab), an endoglin neutralizing antibody currently in clinical development, inhibits endoglin dependent signaling and metastatic colonization when CRC cells and CAFs are co-injected into mice. We further investigated these anti-tumor effects in four different models of CRC (chemically-induced model of early CRC, subcutaneous MC38 and CT26 syngeneic mouse CRC models, and orthotopic MC38 model). TRC105 treatment inhibited tumor initiation (early stage CRC), growth, and progression. Therapeutic effects of TRC105 therapy were dependent on antibody dependent cytotoxicity (ADCC) and CD8+ T-cells, as no therapeutic benefit from TRC105 was observed in Fc-y I-IV receptor knockout mice or CD8+ cell depleted mice. Since therapeutic TRC105 activity was immune cell dependent, we combined TRC105 with anti-programmed cell death (PD-1 immunotherapy). We observed synergistic inhibition of tumor initiation and growth in all four tumor models, leading to delayed tumor outgrowth, increased survival and cure rates up to 60% in the combination treated mice bearing palpable tumors. Combined treatment also induced memory responses, as shown by resistance to repeated tumor challenge. Anti-tumor responses were accompanied by enhanced immune cell activation including an increased number of tumor-specific T-cells, NK cells, decreased number of tumor-infiltrating neutrophils and increased granzyme B production. CD8+ cytotoxic T-cells were instrumental in these synergistic therapeutic responses, as their depletion abolished the effects. Taken together, our data indicate significant therapeutic effects of targeting endoglin by affecting signaling and multiple immune related components in the tumor microenvironment. These results are currently being translated in a clinical trial combining TRC105 with nivolumab in patients with non-small cell lung cancer (NCT03181308).

#292

PTC596 combination therapy for sarcoma.

Marla Weetall,1 Art Branstrom,1 John Baird,1 Liang Cao,1 Josephine Sheedy,2 Kylie O'Keefe,2 Matthew Ingham,3 Gary K. Schwartz,4 Robert Spiegel,1 Edward O'Mara1. 1 _PTC Therapeutics, South Plainfield, NJ;_ 2 _PTC Therapeutics, Morristown, NJ;_ 3 _Columbia Universtity, New York, NY;_ 4 _Columbia University, New York, NY_.

Sarcomas are rare cancers derived from mesenchymal tissues. Although rare, there are approximately 15,000 new cases of sarcoma diagnosed each year in the United States. Certain soft tissue sarcomas are treated with doxorubicin and olaratumabas first line, gemcitabine as second line, and often dacarbazine (DTIC) as third line. PTC596 is a first-in-class, oral investigational new drug that reduces levels of BMI1, a protein required for cancer stem cell survival. PTC596 acts by binding to tubulin causing a G2M cell cycle arrest. PTC596 lowers levels of BMI1 in preclinical tumor models and is generally well tolerated as a monotherapy at doses that result in preclinical target plasma concentrations. Efficacy of PTC596 was evaluated in a series of preclinical models. PTC596 delayed tumor growth as monotherapy and significantly enhanced, in combination, the activity of standard chemotherapeutics including gemcitabine, doxorubicin/ doxil, dacarbazine (DTIC), and other tubulin binding agents. Based on these results, a Ph1b clinical trial in patients with relapsed refractory leiomyosarcoma is to be initiated. It is a dose escalation study of a combination regimen of dacarbazine and PTC596 with the dose-limiting toxicity (DLT) to be determined using a continuous reassessment methodology. Previously, a Phase I multi-center dose escalation study was conducted in patients with advanced solid tumors. PTC596 was administered as an oral monotherapy in 4-week cycles using bodyweight-adjusted twice-per-week (biw) dosing. A maximum tolerated dose (MTD) was not determined though a well-tolerated recommended Phase 2 dose of 7mg/kg biw was identified. The most relevant toxicities observed were neutropenia, nausea, and vomiting which were generally mild to moderate, manageable and reversible.

#293

Inhibition of BET proteins augments nab-paclitaxel-gemcitabine-based chemotherapy response in preclinical models of pancreatic cancer.

Niranjan Awasthi, Sandeep Singh, Ross D. McCauley, Johann RE Schwarz, Margaret A. Schwarz, Roderich E. Schwarz. _Indiana University School of Medicine, South Bend, IN_.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) often presents with metastatic activity, leading to an extremely poor prognosis. Nanoparticle albumin-bound paclitaxel (NPT) in combination with gemcitabine (NPT+GEM) is a standard treatment for PDAC patients, resulting in a survival of ~8.5 months. Bromodomain and extraterminal domain (BET) proteins are epigenetic regulators of gene expression and are involved in cancer pathogenesis. Targeted inhibition of BET protein is currently under investigation for several cancers. We hypothesize that BET protein pathway inhibition by iBet-762 will enhance nab-paclitaxel-gemcitabine-based standard chemotherapy response in PDAC.

METHODS: In vitro cell proliferation assays were performed using WST-1 reagent. Protein expressions were determined by Western Blot analysis. In vivo animal survival and tumor growth experiments were performed in NOD-SCID mice.

RESULTS: BET inhibitor (iBET-762) and standard chemotherapy (NPT+Gem) had a dose-dependent in vitro growth inhibitory effect on several PDAC cell lines tested. Inhibition in cell proliferation at 1 μM drug concentration in NPT+Gem, iBET-762 and NPT+Gem+iBet-762 treatment groups was 64%, 27%, 76% in AsPC-1; 43%, 13%, 69% in Panc-1; and 42%, 51%, 75% in MIA PaCa-2 cells. Immunoblot analysis demonstrated that iBET-762 decreased the expression of oncogenic proteins c-Myc, β-catenin, vimentin and phospho-AKT, while increasing the expression of apoptosis-related proteins such as cleaved PARP-1 and cleaved caspase-3 and cell cycle inhibitor proteins P21 and P27. In subcutaneous xenografts, compared to controls, NPT+Gem and iBet-762 decreased tumor growth by 72% and 57%, respectively (p<0.02). Importantly, the combination therapy group (NPT+Gem+iBet-762) had an additive effect on tumor growth inhibition (98%, p<0.0001). In a peritoneal dissemination model, median animal survival compared to controls (21 days) was increased by NPT+Gem (33 days, a 57% increase) and iBet-762 (30 days, a 43% increase) therapy. This was further increased in the combination therapy group NPT+Gem+iBET-762 (44 days, a 110% increase).

CONCLUSION: These findings demonstrate that nab-paclitaxel-gemcitabine-based standard chemotherapy response can be enhanced through specific inhibition of BET proteins by iBET-762 in preclinical models of PDAC. The data support the potential of this combinatorial therapeutic strategy for clinical PDAC therapy.

#294

ChemoINTEL: A high-throughput, multi-parametric compound screening platform for intelligent lead compound and therapeutic combination identification.

Kellye C. Kirkbride,1 Kevin J. Polach,1 Samantha J. Braxton,1 Megan D. Hoeksema,1 Dustin C. Rogers,1 Patricia Ladd-Ward,1 Durdica Vojnic Zelic,1 Santosh Putta,2 Matt Westfall,1 Norman Purvis1. 1 _Pierian Biosciences, Franklin, TN;_ 2 _Qognit, Inc., San Carlos, CA_.

Pierian Biosciences has developed the ChemoINTEL platform for high-throughput, semi-automated quantification of cellular responses to chemotherapeutics. ChemoINTEL relies on a microscopy-based process that captures and analyzes single cell behavior to reflect responses within a population. The platform is customizable to a variety of small molecule panels, either specific to certain tumor types or for titration screening of small molecules in development. Automated imaging of each well of a 384-well plate over time provides real-time kinetic response data under a variety of treatment conditions. Brightfield and fluorescent images are used to determine quantitative changes in morphology and molecular metrics reflecting induced apoptosis and cell death. Unique to ChemoINTEL as a drug screening platform is the quantification of intensity values, at the single cell level versus whole well fluorescence, for each fluorescent probe at each timepoint through a high-throughput analysis routine. These data are further processed through an internally developed algorithm to compare a treated population's response relative to an untreated control and reports a sensitivity score. By combining different treatment conditions, the platform provides intelligent design of single agent or combination treatment approaches. By analyzing individual cells in the population, ChemoINTEL aids in better understanding response of tumor cell sub-populations to different chemotherapeutic agents. As a compound library screening tool or potential clinical diagnostic, Pierian Biosciences' quality-controlled processes ensure all its equipment, reagents and processes follow ISO17025 guidelines to ensure the quality of all data generated. Each assay is internally controlled to include a standard cell line, whose response is monitored for accuracy of the reported results. In collaboration with several biorepositories and through the development of a standardized dissociation approach resulting in purification of viable primary tumor cells, the ChemoINTEL platform is also under development to assess chemotherapeutic response within patient populations. In conjunction with the ChemoINTEL platform, Pierian Biosciences has also developed a sophisticated multi-color flow cytometry platform (ImmunoINTEL) that when used in conjunction with ChemoINTEL provides information on the purity of isolated primary tumor cells prior to plating, as well as information on the cell populations present within the tumor microenvironment. These two platforms can be further complexed to evaluate immunotherapies in combination with front-line therapies. In summary, Pierian Biosciences has developed a high-throughput, multi-parametric platform for both drug development and diagnostic applications.

#295

Synergism between oxaliplatin or irinotecan with the PARP inhibitor niraparib in a preclinical model of KRAS/BRAF mutated colorectal cancer is associated with MSI status.

Pietro Paolo Vitiello,1 Davide Ciardiello,1 Claudia Cardone,1 Giulia Martini,2 Valentina Belli,1 Nunzia Matrone,1 Luca Poliero,1 Carola Borrelli,1 Pasquale Vitale,1 Nicoletta Zanaletti,1 Teresa Troiani,1 Davide Melisi,3 Fortunato Ciardiello,1 Erika Martinelli1. 1 _University of Campania Luigi Vanvitelli, Napoli, Italy;_ 2 _Vall d'Hebron Institute of Oncology (VHIO), Barcelona, Spain;_ 3 _University of Verona, Verona, Italy_.

DNA damage response (DDR) is crucial in a variety of tumors and several new drugs that interfere with this mechanism are already available or in advanced clinical testing. RAS-MAPK pathway activation, induced by either RAS or BRAF mutation, is a frequent feature of colorectal cancers (CRCs) and is strongly associated to mitotic stress and dependency upon DDR. Among the drugs used for CRC, oxaliplatin and irinotecan are known to induce DNA damage in form of single or double strand breaks (SSB/DSBs) and thus require active DDR systems in cancer cells to be tolerated. The poly-ADP-Ribose Polymerase (PARP) inhibitor Niraparib (MK-4827) is an FDA-approved orally bioavailable drug with strong PARP1-trapping activity, a feature ofted associated with synergism in combination with platinum salts, while synergism with other DNA damaging agents is independent of the PARP1-trapping activity. We tested the activity of niraparib used in combination with oxaliplatin or irinotecan (in the form of its active metabolite SN38) in a panel of 8 KRAS or BRAF mutated CRC cell lines (LOVO, HCT15, SW1116, LS1034, SW948, WiDr, SW480, HCT116) with different microsatellite and CIMP status. Combination index (CI) analysis was performed in order to evaluate the synergism between niraparib and the chemotherapeutics. Cell cycle distribution and apoptosis assays were performed in order to further characterize the effects of the combinations.

The combination between niraparib and oxaliplatin resulted synergistic in 6 out of 8 cell lines, with strong synergism (CI < 0,5) in 2 cell lines, while the combination between niraparib and irinotecan (SN38) resulted synergistic in 7 out of 8 cell lines, with strong synergism in 5 cell lines and very strong synergism (CI < 0,1) in 3 cell lines. Interestingly, the only non-synergistic cell line to the combination with irinotecan is also non-synergistic to the combination with oxaliplatin; on the other hand, the cell lines that exhibit strong synergism to the combination with irinotecan do not present strong synergism to the combination with oxaliplatin and vice versa. Moreover, all the microsatellite instable (MSI) cell lines tested resulted synergistic for the two combinations. Finally, the synergistic combinations showed an increased induction of apoptosis and cell cycle arrest compared to non-synergistic combinations.

Taken together, these data investigate the combination between the PARP inhibitor niraparib and the antiproliferative agents oxaliplatin and irinotecan in KRAS/BRAF mutated CRC cell lines, showing how MSI status predicts good synergism with the drug combinations, while MSS status is not a good predictor of synergism. Further studies are needed to identify better predictive biomarker beyond MSI status, in order to optimize the use of these combinations for future development.

#296

**Trastuzumab sensitizes HER2+ breast cancer to increase efficacy of chemotherapy** in vitro **and** in vivo **.**

Alay Shah,1 Angela Jarrett,1 Anum Syed,1 Thomas Yankeelov,1 Anna Sorace2. 1 _UT Austin, Austin, TX;_ 2 _University of Alabama at Birmingham, Birmingham, AL_.

Purpose: Optimization of drug synergy has potential to improve efficacy of combination treatments. Breast cancer that overexpresses the human epidermal growth factor receptor (HER2) has a higher chance of metastasis and recurrence compared to HER2- patients. Standard-of-care treatment typically includes chemotherapy (i.e., paclitaxel, doxorubicin) followed by or in combination with targeted antibody treatments (i.e., trastuzumab, pertuzumab). The purpose of this study is to investigate, the timing, order, and dosage of standard-of-care combination therapies and how they affect the overall efficacy of treatment in HER2+ breast cancer in vitro and in vivo.

Experimental Procedure: BT474, HER2+ breast cancer cells were longitudinally evaluated with time resolved microscopy to measure changes in cell confluence (Incucyte). Cells were treated with combinations of trastuzumab (10-50 ug/ml) and paclitaxel (10-150 nmol/L) with alternating sequencing and timing (24, 48 hrs) of the therapies (N=8 replicates per combination). Cellular confluence was measured over 4 days and normalized percent changes were calculated. BT474 tumor-bearing mice (N=42) were treated singularly or in combination with alternating sequencing of trastuzumab (10 mg/kg) and doxorubicin (1.5 mg/kg). Tumors were evaluated for changes in tumor size and survival. An unpaired t-test was used for statistical comparisons for in vitro and in vivo studies. Assessment of cellular and tumor microenvironment influences on drug synergy are ongoing.

Results: Experimental outcomes indicate that alternating order and dose play a crucial role in the anticancer efficacy and synergistic drug effects. Trastuzumab (25 ug/ml) dosing prior to paclitaxel (50 nmol) significantly decreased cell confluence compared to the same total therapeutic dose where paclitaxel was introduced first in vitro (p < 0.001). Similarly, trastuzumab dosing in vivo prior to a one dose of chemotherapy achieves the same tumor response as a two doses of chemotherapy administered simultaneously with trastuzumab (p < 0.001).

Discussion and Conclusions: Sequencing of targeted and chemotherapies altered treatment response both in vivo and in vitro, resulting in differences in overall cancer cell death. In vitro longitudinal imaging can provide numerous combinations of treatments can be tested to ultimately find optimal treatments to test in vivo. Recent evidence also shows that targeted anti-HER2 therapy can alter vascularity, oxygenation, and immune cell infiltration in HER2+ breast cancer tumors; therefore, further investigations in the temporal effects of the tumor microenvironment are warranted. Improved temporal windows of anticancer effects for standard-of-care combination therapies could improve breast cancer treatment regimens and potentially minimize toxic side effects.

Funding was provided by CPRIT RR160005 and ACS-RSG-18-006-01-CCE.

#297

Activity of carfilzomib (CFZ) alone and in combination with cytarabine and chloroquine diphosphate (CHQ) in acute myeloid leukemia (AML).

Mao Yu Peng, Yasmin Abaza, Dylan Conklin, Erika Von Euw, Monica Mead, John Timmerman, Dennis Slamon, Sarah Larson. _UCLA, Los Angeles, CA_.

Background: Despite the recent advances in AML with the emergence of multiple targeted therapies, patient outcomes remain poor, particularly in the relapsed/refractory setting underscoring the need for novel therapeutic strategies. Proteasome inhibitors (PIs) have antitumor activity in AML through inhibition of the nuclear factor κB pathway and induction of apoptosis with multiple clinical trials showing promising activity of bortezomib particularly when combined with other cytotoxic agents (Csizmar 2016). CFZ, a second-generation PI, has preferential preclinical activity in AML compared to bortezomib and is therefore an agent of interest in AML therapy (van der Helm 2015). Here we assessed the activity of CFZ alone and in combination with cytarabine and the autophagy inhibitor CHQ in AML cell lines.

Methods: After screening 18 AML cell lines, 3 sensitive: KASUMI1, ML2, and MV411, and 3 resistant cell lines: AML193, THP1, and NOMO1, were selected for further analysis using an IC50 cutoff for CFZ of 10 nM. Apoptosis, cell cycle, and cell senescence analysis were performed after 72 hours of CFZ exposure at 10 nM. Combination assays using CFZ 10 nM, cytarabine 200 nM, and CHQ 20 µM were performed to evaluate for potential interaction in the form of antagonism or potentiation. Proteomic analysis was performed at baseline using reverse phase protein assay (RPPA) and confirmed with western blot analysis.

Results: Single-agent CFZ induced apoptosis in sensitive cell lines with apoptotic rates increasing by 85% to 95%; while apoptotic rates remained unchanged in resistant cell lines. Similarly, CFZ resulted in G0/G1 cell cycle arrest in sensitive but not resistant AML cell lines. Lack of difference in cellular senescence confirmed apoptosis and cell cycle arrest as the major mechanisms of CFZ-induced growth inhibition in AML cell lines. No antagonism was noted when CFZ 10 nM was combined with cytarabine (200 nM). RPPA revealed that AML cell lines with low expression of autophagy related proteins are more sensitive to proteasome inhibition with CFZ treatment compared to resistant cell lines. This was confirmed with western blot which showed upregulation of the autophagy related proteins in resistant cell lines upon exposure to CFZ. However, sensitive cell lines did not upregulate autophagy pathways which may lead to cell cycle arrest and apoptosis. Inhibiting autophagy with CHQ sensitized CFZ resistant lines to CFZ treatment.

Conclusion: CFZ demonstrated single agent activity in human AML cell lines and upregulation of autophagy may contribute to resistance to CFZ which can be overcome using autophagy inhibitors such as CHQ. These results support the development of CFZ and autophagy inhibitor-based combinations for AML.

#298

Identification and evaluation of novel drug combinations with aurora kinase inhibitor CCT137690 for enhanced efficacy in 2D monolayer and 3D spheroid model of oral cancer cells.

Muhammad Furqan, Zainab Ashfaq, Apsra Nasir, Zille Huma, Rahim Ullah, Maheen Iqbal, Irshad Hussain, Amir Faisal. _Lahore university of management sciences., Lahore, Pakistan_.

Background: Oral cancer is the most prevalent subtype of head and neck cancers with a high incidence among men in South-Central Asia and Central and Eastern Europe. The mortality rate is disproportionately high in developing countries, presumably due to late diagnosis and limited access to quality treatment. Despite the advances in the understanding of the molecular basis and treatment of oral cancer during the last decade, the survival rates remain poor for patients with the advanced metastatic disease. Deregulated expression of Aurora family of mitotic kinases has also been associated with pathogenesis and poor prognosis in many cancers including oral cancers. Here we have evaluated the efficacy of pan-Aurora inhibitor, CCT137690, in oral cancer cell lines and identified that it synergizes with EGFR and PI-3 kinase inhibitors in 2D monolayer and 3D spheroid models. Methods: Inhibition of aurora kinases followed by induction of apoptosis in control and CCT137690 treated samples were analyzed through western blotting and immunofluorescence. We assessed proliferation of oral cancer cells through SRB assay, while cell cycle profile following inhibitor treatment was analyzed through flow cytometry. We also synthesized polyethylene glycol based Nanocapsules (P-NCps) containing combinations of CCT137690 with GDC-0941 (CG-L-P-NCps) or ZD1839 (CGe-L-P-NCps) and compared their efficacy with free drug combinations through proliferation assay in 2D monolayer and 3D spheroid model of oral cancer cells. Results: CCT137690 treatment inhibited proliferation and induced apoptosis in both types of oral cancer cell lines (ORL-48 and ORL-115). Such inhibition was accompanied by significant morphological changes in cells such as aberrant mitotic spindle formation, endoreduplication and increased cell size. Combining CCT137690 with inhibitors of EGFR (ZD1839) or PI3K (GDC-0941) synergistically reduced proliferation and increased apoptosis induction in the oral cancer cells. Furthermore, Nanocapsules harboring these drug combinations also showed a comparable inhibitory effect in 2D monolayer and 3D spheroid model of oral cancer cells. Conclusion: Our results suggest that the combination of CCT137690 with ZD1839 or GDC-0941 can be further evaluated for the treatment of oral cancer. Moreover, these combinations can be delivered through nanocapsules to confer additional advantages of reduced cytotoxicity and sustained release for better therapeutic index and efficacy.

#299

Synergistic activity of mebendazole and chemotherapeutic agents for rare cancers.

Mahta Samizadeh, Debashree Chakrabarti, Yuhan Zhu, Robert Louis Treuting, Johanne Kaplan, William Siders, Jamie D. Barber. _SHEPHERD Therapeutics, Hanover, MA_.

Depending upon the definition used, as many as half of all cancers diagnosed are considered rare cancers, and most rare cancer patients would greatly benefit from additional therapies. Repurposing of drugs for use in oncology represents an attractive strategy for the rapid development of therapies that address the significant unmet medical need that remains for rare cancers. The FDA-approved anti-helminthic drug mebendazole (MBZ) has been reported to affect several biological pathways involved in tumor growth and metastasis. Based on this activity, multiple rare cancer tumor cell lines including neuroblastoma, carcinoid tumors, esophageal adenocarcinoma, gastrointestinal stromal tumors, leiomyosarcoma, pheochromocytoma, and mesothelioma were analyzed for their susceptibility to MBZ treatment alone or in combination with standard chemotherapeutic agents in vitro. Cultured cells exposed to increasing concentrations of MBZ alone showed a significant decrease in cell viability with IC50s ranging from 0.09-2.2 μM. To evaluate efficacy of MBZ in an additional model system, an ex vivo 3D assay using gastric cancer primary cells isolated from two patient-derived xenograft (PDX) models was performed. Cells were seeded in 3D cultures and treated for seven days with MBZ at nine different doses in triplicate. IC50s were determined to be 0.74 and 0.76 μM indicative of significant potency. These results along with the in vitro data generated using cell lines suggest that MBZ may represent an effective anti-cancer agent across multiple rare cancer indications. However, since tumor cells can often evade or develop resistance to single agent therapy, the potential for synergistic combinations with chemotherapeutic agents was also explored in vitro. Cells were plated in triplicate and treated with a range of concentrations of MBZ and relevant chemotherapeutic agents in a complete Latin square. Combination indexes (CI) were determined based on the method described by Chou and Talalay. Several synergistic drug combinations were identified with CI values ranging from 0.008 to 0.428 (CI < 0.9 indicates synergy). Overall, our results indicate that synergistic combinations of MBZ and chemotherapy agents represent a promising strategy for the treatment of rare cancers.

#300

A new combination therapy to treat metastatic breast cancer.

Bahram Sharif-Askari, Raquel Aloyz, Lawrence Panasci. _McGill Univ. Lady Davis Inst., Montreal, Quebec, Canada_.

Poly (ADP-ribose) polymerase (PARP) resulting poly (ADP-ribosyl)ation is an early player in modification of proteins detected at single strand breaks and double strand breaks contributing to DNA repair. PARP1, 2 and 3 are involved in this process. However, PARP3 is unique in that, it is also required for efficient mitotic progression, specifically in stabilization of the mitotic spindle. The vinca alkaloids such as vinorelbine, arrest cells in metaphase due to inhibition of the assembly and dynamics of microtubules. Nontoxic concentrations of 2 PARP3 inhibitors, ME-0328 and olaparib, sensitized various her-2 negative and triple negative breast cancer cell lines greater than or equal to 10 fold to vinorelbine associated with potentiation of vinorelbine's interaction with tubulin and also vinorelbine-induced PARP3 inhibition, mitotic arrest, and apoptosis without evidence of a DNA repair mechanism involved in this sensitization (Sharif-Askari B et al. Breast Cancer Res Treat. 2018 Nov;172(1):23-32).

Eribulin is a Halichondrin B analogue, an antitubulin drug, with a unique mechanism of action; it inhibits microtubule dynamics via a novel mechanism of action which seems to involve binding to a unique site on tubulin resulting in the suppression of microtubule polymerization without affecting depolymerization along with sequestration of tubulin into nonfunctional aggregate. After impressive results in phase III trials (including one of the rare trials to show prolongation of survival in comparison to standard of care in heavily pretreated metastatic breast cancer patients) the U.S. Food and Drug Administration approved eribulin for treatment of patients with metastatic breast cancer. PARP3 inhibitors again sensitized at least 5 fold her-2 negative and triple negative breast cancer cell lines to eribulin. We will do similar studies with eribulin as we have done with vinorelbine including in-vivo studies. These studies could define a new therapy in the treatment of metastatic triple negative and ER+/her-2 negative breast cancer. The inhibition of PARP3 may increase the sensitivity of tumor cells to mitotic spindle poison chemotherapies. Our results should stimulate development of specific PARP3 inhibitors for clinical use.

#301

Concurrent targeting of BMI1 and CDK4/6 inhibited breast cancer tumorigenicity in vitro and in vivo.

Nehad M. Alajez. _Qatar Biomedical Research Institute, Doha, Qatar_.

Despite recent advances in cancer management and therapy, resistance to cytotoxic medications remains a major clinical challenge. Intratumoral heterogeneity appears to play crucial role in drug resistance; therefore, combination-based approaches are currently gaining momentum. In the current study, we investigated the ramifications of concurrent targeting of tumor-initiating cells (using BMI1 specific inhibitor, PTC-209) and cell cycle progressiontargeting CDK4/CDK6 using palbociclib on breast, colorectal and prostate cancer cell growth in vitro and in vivo. PTC-209 reduced BMI1 protein expression, while palbociclib (PB) inhibitedCDK4, Rb, and PRb Ser795 phosphorylation. PTC-209 and palbociclib exhibited dose-dependent cytotoxic effects against MDA-MB-231, HCT-116, and PC-3 cell models, which was more profound in the combination group. Global gene expression profiling in MDA-MB-231 treated with PTC-209 revealed multiple affected cellular pathways including insulin signaling, focal adhesion, DNA damage response, and Wnt/pluripotency. On the other hand, PB treatment ofMDA-MB-231 led to substantial changes in gene expression affecting mainly cell cycle progression. PTC-209 and palbociclib reduced colony formation potential, sphere formation, cell migration, and cell viability, which was further enhanced in the combination group. Consistent with our results in vitro, combination treatment of PTC-209 and palbociclib reduced tumor growth more profoundly than either of these drugs alone in in vivo model of MDA-MD-231 cells. Therefore, our data suggest concurrent targeting of BMI1 and CDK4/CDK6 might provide novel therapeutic opportunity for breast, colorectal, and prostate cancer.

#302

The combined inhibition of Plk1 and Notch1 results in a synergistic anti-proliferative response in human melanoma cells.

Shengqin Su, Gagan Chhabra, Chandra K. Singh, Mary A. Ndiaye, Nihal Ahmad. _University of Wisconsin-Madison, Madison, WI_.

Melanoma is the most aggressive form of skin cancer, and its incidence and mortality have been increasing over the past thirty years. Therefore, additional therapeutic strategies are needed for an effective management of this neoplasm. We previously demonstrated that polo-like kinase 1 (Plk1, a serine/threonine kinase involved in mitotic regulation) and Notch1 (a type I transmembrane protein deciding cell fate during development) are strongly correlated in melanoma (Cancer Res 2018; 78 (13 Suppl): Abstract nr 2530). In addition, a number of studies from our lab and elsewhere have suggested the important functions of Plk1- as well as Notch- signaling in melanoma progression. Based on these observations, in this study, we determined the potential efficacy of a combined inhibition of Plk1 and Notch1 against melanoma cells. Employing Plk1 inhibitor volasertib (BI6727) and Notch1 inhibitor MK-0752, we determined the effects of concomitant targeting of these pathways in A375 (wild type TP53) and SK-MEL-2 (mutant TP53) human melanoma cell lines. Employing RealTime-Glo and trypan blue exclusion assays, we found that volasertib (10 and 20 nM) and MK-0752 (50 and 100 μM) resulted in a significant decrease (60-80%) in viability and growth of human melanoma cells A375 and SK-MEL-2. The Combination Index (CI), as calculated using the Chou-Talalay theorem, was less than 1 when volasertib at 10 nM was given with MK-0752 at 50 and 100 μM, indicating a synergism between these two drugs. As a result of the synergism, the melanoma cells treated with combined drugs showed decreased colony formation ability compared to individual drugs. In addition, the combined treatment also resulted in enhanced apoptosis of melanoma cells. Interestingly, cell cycle analysis by flow cytometry showed that combined inhibition of Plk1 and Notch1 caused a G2-M phase arrest of melanoma cells. Furthermore, our data demonstrated that volasertib and MK-0752 combination caused a marked increase of cleaved PARP (poly ADP ribose polymerase) and cleaved caspase-3 in both melanoma cell lines, as well as increased expression of tumor suppressor protein TP53 and its target p21 (CDKN1A) in A375 melanoma cells. Taken together, our data demonstrated that a combined targeting of Plk1- and Notch1- signaling pathways imparts a synergistic anti-proliferative response against melanoma. However, additional experiments are needed i) to validate our in vitro data to in vivo situations in human relevant melanoma models, and ii) to define the interactive mechanism(s) of the observed synergistic response.

#303

The efficacy of the combined treatment of 150 kHz Tumor Treating Fields (TTFields) and FOLFOX in gastric cancer in vitro.

Tali Voloshin, Einav Zeevi, Karnit Gotlib, Rosa S. Schneiderman, Mijal Munster, Yaara Porat, Shiri Davidi, Anna Shteingauz, Noa Kaynan, Moshe Giladi, Eilon D. Kirson, Uri Weinberg, Adrian Kinzel, Yoram Palti. _Novocure, Haifa, Israel_.

Background: Gastric cancer is the fourth most common cancer and the second most common cause of cancer death worldwide. Despite systemic therapies improvement in recent era, long‐term survival rates for patients with advanced gastric cancer remains poor. FOLFOX (Oxaliplatin, 5-FU and Leucovorin) is an approved chemotherapy regimen for treatment of gastric cancer. Tumor Treating Fields (TTFields) therapy is an effective anti-neoplastic treatment modality delivered via noninvasive application of low intensity, intermediate frequency, alternating electric fields. The aim of this work is to explore the potential of the use of TTFields alone and in combination with FOLFOX as a treatment for gastric carcinomas.

Methods: AGS and KATO III cells were treated for 72 hours with TTFields (1.1 and 1.7 V/cm, respectively) at various frequencies, using the inovitro system. Efficacy of the combined treatment of TTFields and FOLFOX was tested by applying TTFields at the optimal frequency together with various drug concentrations. Cell counts, induction of apoptosis, clonogenic potential and overall effect were determined at the end of treatment.

Results: The optimal TTFields frequency leading to the highest reduction in cell counts was found to be 150 kHz for both cell lines resulting in 55% and 52% reduction in cell counts for AGS and KATO III, respectively. In addition, clonogenic potential of both cell lines was reduced by more than 70%. The combined treatment of TTFields with each chemotherapy (Oxaliplatin, 5-FU or Leucovorin), led to a significant reduction in the survival of AGS and KATO III cells (2-way ANOVA, p<0.001 for both cell lines) as compared to each treatment alone. The combined treatment of TTFields with FOLFOX led to further reduction in the overall effect (cytotoxic and clonogenic) of AGS (79%) compared to TTFields alone (65%) and FOLFOX alone (34%). Similar results were observed for the combined treatment of TTFields and FOLFOX in KATO III cells.

Conclusions: The results presented in this work demonstrate that TTFields can be an effective treatment against gastric carcinoma and that the combination with FOLFOX may further enhance treatment efficacy. Based on the above, there is a strong rational to continue exploring the potential of the use of TTFields together with standard of care for the treatment of gastric cancer in the clinical settings.

#304

Combination therapy of cisplatin with cilastatin enables to increase the dose of cisplatin for enhancing its antitumor effect by suppressing nephrotoxicity.

Masashi Arita,1 Satoshi Watanabe,1 Nobumasa Aoki,1 Miho Takahashi,1 Satoshi Shoji,1 Koichiro Nozaki,1 Kosuke Ichikawa,1 Rie Kondo,1 Shoji Kuwahara,2 Junta Tanaka,1 Toshiyuki Koya,1 Akihiko Saito,1 Toshiaki Kikuchi1. 1 _Niigata University, Niigata City, Japan;_ 2 _The University of Shiga Prefecture, Hikone City, Japan_.

Cisplatin is an anticancer drug widely used in the treatment of many cancers, including lung cancers. Although cisplatin causes various types of adverse events, the main dose-limiting toxicity of cisplatin is nephrotoxicity. Megalin is an endocytic receptor expressed at the apical membranes of proximal tubules. We previously demonstrated that cisplatin was reabsorbed through megalin and caused kidney injury. Cilastatin, an inhibitor of renal dehydropeptidase-I and used with imipenem, blocked the binding of cisplatin to megalin and reduced the nephrotoxicity induced by cisplatin. In the current study, we precisely evaluated the effect of cilastatin-mediated suppression of cisplatin nephrotoxicity to safely enhance the antitumor activity of cisplatin. BALB/c mice were administrated cisplatin with or without cilastatin. Tubular dilation or atrophy, brush border loss, tubular cell lysis and cast formation were observed in mice treated with cisplatin alone. However, these kidney injuries were decreased or disappeared in mice treated with cisplatin and cilastatin. Cilastatin also decreased the urinary levels of N-acetyl-β-D-glucosaminidase and neutrophil gelatinase-associated lipocalin, proximal tubular injury markers. Next, SCID mice were injected s.c. with A549, a human lung cancer cell line, and treated with cisplatin with or without cilastatin. Cilastatin did not affect the antitumor activity of cilastatin. Notably, A549 did not express megalin. Combined with cilastatin, the mice were successfully treated with 1.5 times dose of cisplatin with enhanced antitumor effects of cisplatin but without nephrotoxicity. In conclusion, cilastatin effectively suppressed nephrotoxicity of cisplatin by blocking the binding of cisplatin to megalin. These findings indicated that we could administer cisplatin into cancer patients without nephrotoxicity if we used cilastatin. Moreover, we might increase the dose of cisplatin and improve the outcome of cancer patients.

#305

Combination analysis for the potential chemomodulatory effects of mansorin-A and its naphthoquinone derivative (mansonone-G) to 5-fluorouracil against liver cancer cells.

Hanadi G. Aljohani,1 Gehan A. Hegazy,1 Ali M. El-Halawany,2 Aliaa A. Alamoudi,1 Ghada M.A. Agabnoor,1 Ahmed M. Al-Abd3. 1 _King Abdulaziz University, Jeddah, Saudi Arabia;_ 2 _Cairo University, Cairo, Egypt;_ 3 _Gulf Medical University, Ajman, United Arab Emirates_.

Mansorin-A and its naphthoquinone derivative, mansonone-G are naturally occurring compounds isolated from the trunk of Mansonia gagei, family Sterculariaceae. In a previous work, they showed potential anticancer effects besides their antioxidant properties. Herein, we investigated their potential chemomodulatory effects to 5-fluorouracil (5FU) against two liver cancer cell lines (Huh7 and HepG2). Despite the significant cell killing effect of 5FU to Huh7 and HepG2 cells (IC50's of 2.6±0.2 µM and 0.82 ± 0.2 µM, respectively), the resistance fractions of both cell lines were considerably high (53.2±1.7% and 39.4±2.7%, respectively). Mansorin-A and mansonone-G induced moderate cytotoxicity against both liver cancer cell lines with IC50's ranging from 25.8±3.2 µM to 36.3±2.7 µM. In Huh7, mansorin-A and mansonone-G synergized cell killing effect of 5FU and decreased its IC50's to 0.9±0.1 and 1.0±2.1 µM, respectively. On the other hand, mansorin-A and mansonone-G showed apparent antagonism by combination with 5FU (CI values of 6.2 and 9.4, respectively) against HepG2 cells. However, both compounds significantly abolished the resistant fraction of HepG2 to 5FU (3.3±0.3% and 8.4 ± 2.6%, respectively). Further investigation showed that mansonone-G alone showed antiproliferative effect and arrested cells in G0/G1-phase. Both mansorin-A and mansonone-G enhanced 5FU induced cell cycle arrest at G0/G1-phase with reciprocal decrease in cells entering S-phase and G2/M-phase. In addition, apoptosis due to 5FU treatment alone and in combination with mansorin-A and mansonone-G was assessed using annexin-V/FITC staining coupled with flowcytometry. Yet, both mansorin-A and mansonone-G enhanced 5FU induced apoptotic effect against liver cancer cells. Mansonone-G alone induced significant apoptosis in both HepG2 and Huh7 cells. Besides apoptosis, autophagy induction properties of mansorin-A and mansonone-G were assessed using flowcytometry after acridine orange staining. Mansonone-G aborted autophagy in both HepG2 and Huh7 cell lines and indirectly forced liver cancer cells to undergo apoptotic cell death. On the other hand, both mansorin-A and mansonone-G significantly induced the production of reactive oxygen species within both HepG2 and Huh-7 cells which might directly induce apoptosis in both cell lines. In conclusion, mansonone-G and to a lesser extent mansorin-A, potentiated the anticancer properties of 5FU against liver cancer cells via potentiating its cell cycle arrest and potentiating its apoptotic influence due to ROS production.

#306

The efficacy of the combined treatment of 150 KHz tumor treating fields (TTFields) and Sorafenib in hepatocellular carcinoma in vitro and in vivo.

Tali Voloshin,1 Shiri Davidi,1 Catherine Tempel-Brami,1 Mijal Munster,1 Karnit Gotlib,1 Einav Zeevi,1 Rosa S. Schneiderman,1 Moshe Giladi,1 Adrian Kinzel,2 Eilon D. Kirson,1 Uri Weinberg,1 Yoram Palti1. 1 _Novocure, Haifa, Israel;_ 2 _Novocure, Minuch, Germany_.

Hepatocellular carcinoma (HCC) is the third cause of cancer related mortality and the primary cause of cancer death. Sorafenib, an oral multikinase inhibitor is approved for patients with advanced HCC, yet its survival benefit is still limited. Tumor Treating Fields (TTFields) therapy is an effective anti-neoplastic treatment modality delivered via noninvasive application of low intensity, intermediate frequency, alternating electric fields. The aim of this work is to explore the potential of the use of TTFields alone and in combination with Sorafenib as a treatment for HCC. HepG2 and Huh-7D12 cells were treated with various TTFields frequencies for 72 hours using the inovitro system. Efficacy of the combined treatment of TTFields and Sorafenib (36-3000 nM) was tested by applying TTFields at the optimal frequency together with various drug concentrations. Cell counts, induction of apoptosis, cell cycle and clonogenic potential were determined. N1S1 HCC cells were injected to the left lobe of the liver of SD rats. After 1 week, TTFields (1.2 V/cm) and Sorafenib (10 mg/kg) were applied for 6 days and tumor growth was followed using MRI. Healthy rats were used to study safety of the use of TTFields (150 kHz) applied to the abdomen. The optimal TTFields frequency was found to be 150 kHz for both cell lines. TTFields application (1.0 - 1.7 V/cm, 72 hours) at 150 kHz led to 53-64% reduction in cell counts and to additional reduction of over 70% in the clonogenic potential. The combined treatment of TTFields and Sorafenib led to a significant reduction in the number of cells (2-way ANOVA, p<0.001) as compared to each treatment alone. Tumor growth was significantly reduced by the combined group compared to the control group (student t test, p<0.01). Moreover, the averaged tumor volume fold increase of the combination treatment group (1.6) was significantly lower than the one observed in the : control group (5.9, p <0.0001), the TTFields group (3.3, p <0.01) and the Sorafenib group (2.3, p <0.05). Safety studies did not reveal any adverse event associated with TTFields application to the rat abdomen. The results presented in this work demonstrate that TTFields can be a safe and effective treatment against HCC cells and that the combination with Sorafenib may further enhance treatment efficacy.

#307

The combined treatment of 150 kHz Tumor Treating Fields (TTFields) and Cisplatin or Pemetrexed inhibit mesothelioma cells in vitro.

Mijal Munster, Rosa Schneiderman, Yaara Porat, Tali Voloshin, Shiri Davidi, Anna Shteingauz, Noa Kaynan, Einav Zeevi, Karnit Gotlib, Moshe Giladi, Eilon Kirson, Uri Weinberg, Adrian Kinzel, Yoram Palti. _Novocure, Haifa, Israel_.

Malignant pleural mesothelioma (MPM) is a rare thoracic solid tumor cancer that has been strongly linked to asbestos exposure. The standard of care treatment for unresectable mesothelioma is cisplatin plus pemetrexed chemotherapy which offer short and insufficient efficacy. Furthermore, no validated treatment beyond first-line therapy is available. Thus, there is an urgent need to identify more effective treatments for mesothelioma patients. Tumor Treating Fields (TTFields) therapy is an effective anti-neoplastic treatment modality delivered via noninvasive application of low intensity, intermediate frequency, alternating electric fields. TTFields are employed as a local treatment with the intent to target dividing cells by disrupting microtubules leading to mitotic catastrophe, abnormal chromosome segregation and the induction of different forms of cancer cell death. The aim of this work is to explore the potential of the use of TTFields alone and in combination with cisplatin or pemetrexed as a treatment for Mesothelioma. MSTO-211H and NCI-H2052 cells were treated with various TTFields frequencies for 72 hours using the inovitro system. Efficacy of the combined treatment of TTFields and Cisplatin or Pemetrexed was tested by applying TTFields at the optimal frequency together with various drug concentrations. Cell counts, induction of apoptosis, and clonogenic potential were determined at the end of treatment. The optimal TTFields frequency leading to the highest reduction in cell counts was found to be 150 kHz for both MSTO-211H and NCI-H2052 cells. TTFields application (1.1 V/cm, 72 hours) at 150 kHz led to 51%, 65% reduction in cell counts, 40%, 55% reduction in the clonogenic potential in NCI-H2052 and MSTO-211H cells, respectively. The combined treatment of TTFields and Cisplatin or Pemetrexed led to a significant reduction in cell count, induction of apoptosis and reduced clonogenic potential as compared to each modality alone. (2-way ANOVA, p<0.0001). Safety studies did not reveal any adverse event associated with 150 kHz TTFields application to the rat torso. The results presented in this work demonstrate that TTFields can be an effective treatment against Mesothelioma cells and that the combination with cisplatin or pemetrexed may further enhance treatment efficacy. In accordance with these results, it was recently reported that patients treated by the combined treatment of TTFields with pemetrexed and cisplatin experienced improved overall survival as compared to historical control with no increase in systemic toxicity.

#308

In-vitro combination analysis for colossolactone-G with gemcitabine and 5-fluoruracil against colorectal cancer cells.

Rinad A. Aljohani,1 Riham S. Eldine,2 Gehan A. Hegazy,1 Aliaa A. Alamoudi,1 Ghada A. Agabnoor,1 Ali M. El-Halawany,2 Ahmed M. Al-Abd3. 1 _King Abdulaziz University, Jeddah, Saudi Arabia;_ 2 _Cairo University, Cairo, Egypt;_ 3 _Gulf Medical University, Ajman, United Arab Emirates_.

Naturally occurring terpenoids isolated from Ganoderma colossum showed potential chemotherapeutic properties. In the current work, we assessed the potential chemomodulatory effects of colossolactone-G to gemcitabine (GCB) and 5-fluorouracil (5FU) against colorectal cancer cells. Colossolactone-G induced moderate cell killing effects against both HT29 and HCT116 cells with IC50's of 90.5±1.7 µM and 22.3±3.9 µM, respectively. By equitoxic combination, colossolactone-G synergized the cytotoxic effects of GCB and 5FU against both HT29 and HCT116 cells resulting in combination index ranging from 0.22 to 0.67. By assessing cell cycle distribution, both GCB and 5FU induced moderate cell cycle arrest at G0/G1-phase and S-phase. Despite colossolactone-G lacks any influence on cell cycle distribution, it significantly potentiated GCB and 5FU induced cell cycle arrest in S-phase and G2/M-phase. Longer exposure (48 h) of cells to colossolactone-G alone induced antiproliferative effect and accumulation of cells at G0/G1-phase. In addition, cell cycle arrest attributed to GCB, 5FU, colossolactone-G and their combinations was accompanied by significant accumulation of cells at Pre-G phase which is indicative of cell death. Further assessment for cells undergoing apoptosis was measured using annexin-V/FITC and PI staining coupled with flowcytometric analysis. Similarly, colossolactone-G treatment alone did not induce any significant apoptosis in both cell lines. However, 5FU and GCB induced significant apoptosis which was further potentiated by colossolactone-G combination. Besides, autophagic death within HCT116 and HT29 cells was assessed using acridine orange lysosomal stain coupled with flow-cytometric analysis in response to single and combination treatments of 5FU, GCB, colossolactone-G. In HCT116, colossolactone-G significantly suppressed the process of autophagy and decreased acridine orange lysosomal fluorescence signal to 52.7% of control cells. On the other hand, colossolactone-G significantly increased autophagic response in HT29 cells. GCB treatment alone decreased autophagic signal within HT29 cells. However, colossolactone-G combination with 5FU or GCB resulted in significant increase in autophagy by 8.7% and 82.7% of control cells, respectively. In conclusion, colossolactone-G represents promising chemomodulatory adjuvent to GCB and 5FU via potentiating their apoptotic, autophagic and cell cycle interference properties.

#309

FAK blockade enhances antitumor effect of BTK inhibitor in esophageal squamous cell carcinoma via EGFR-ERK-Akt pathway inhibition.

Qiu-Yun Luo,1 Su-Na Zhou,1 Wen-Tao Pan,2 Meng-xian Pan,2 Lu-ping Yuan,1 Lin Zhang,1 Xiang-Lei Yan,1 Yu-xin Zhang,1 Jian Sun,1 Miaozhen Qiu,1 Da-Jun Yang2. 1 _Sun Yat-sen University Cancer Center, China;_ 2 _Suzhou Ascentage Pharma Inc, China_.

Background: Esophageal squamous cell carcinoma (ESCC) is a fatal disease with poor prognosis and lack of effective targeted therapy. Recently, some researches have shown that BTK can be a novel therapeutic target for esophageal cancer, while the mechanism remains unknown. In addition, FAK is one of the important factors to promote the metastasis of esophageal cancer. In this study, we sought to explore the synergistic antitumor effect and underline mechanism of combining BTK inhibitor Ibrutinib with a novel FAK inhibitor APG-2449 for esophageal squamous cell carcinoma.

Methods: Cell viability and growth inhibition were determined by CCK-8 assay and colony formation assay. Migration ability was detected by Transwell assay. EMT markers and ki67 were measured by RT-qPCR. Cell cycle analysis and apoptosis were confirmed by flow cytometry. Western Blot analysis and siRNA interference for mechanism exploration. ESCC xenograft models were established to evaluated the synergistic antitumor effect in vivo.

Results: We found that ESCC cell lines harbor EGFR or c-myc-amplified exhibited more sensitive to Ibrutinib inhibition. Moreover, Ibrutinib combined with APG-2449 showed synergistic inhibition of cell proliferation in ESCC cell lines. By analyzing data from the Gene Expression Profiling Interactive Analysis database, we found that EGFR expression positively correlated with FAK expression in ESCC samples. The combination of Ibrutinib and APG-2449 exerted obviously synergistic apoptosis induction and colony formation inhibition. Furthermore, we found that Ibrutinib combined with APG-2449 could induce more cancer cells arrested in G1/S phase. In addition, the co-treatment of Ibrutinib and APG-2449 could significantly suppress ESCC cells migration by upregulating E-cadherin. Ibrutinib alone could down-regulate the protein expression of p-EGFR and p-ERK. Moreover, combination treatment significantly reduced the expression of p-Akt and p-c-myc, and upregulated P21 and PUMA expression. In ESCC xenotransplantation models, single therapy with either Ibrutinib or APG-2449 was equivalent in delaying tumor growth, while the combination therapy demonstrated more significantly tumor growth suppression.

Conclusions: Taken together, our data strongly suggest that the combination of FAK inhibitor APG-2449 and Ibrutinib provide an effective and precise therapeutic strategy for treating esophageal squamous cell carcinoma.

### Drug Resistance 1

#310

Hyperleptinemia in obese state renders luminal breast cancers refractory to tamoxifen coordinating a crosstalk between Med1, miR205 and Erb B kinases.

Arumugam Nagalingam, Nethaji Muniraj, Sumit Siddharth, Dimiter. B Avtanski, Sheetal Parida, Panjamurthy Kuppusamy, Balázs Győrffy, Neeraj Saxena, Dipali Sharma. _Johns Hopkins Sidney Kimmel Comp. Cancer Ctr., Baltimore, MD_.

Background and Aim: Obese state is associated with increased breast cancer growth, metastatic progression, poor overall survival and poor response to therapy. Analyses of clinical trial population showed that obesity associates with poor outcomes in patients with estrogen receptor (ER)-positive breast cancer treated with standard chemohormonal therapy. A characteristic feature of obesity is hyperleptinemia (high leptin levels) which mediates the biological effects of obesity. In this study, we seek to decipher the underlying molecular mechanisms by which obesity/hyperleptinemia reduces the efficacy of tamoxifen.

Results: Obese mice models exhibiting hyperleptinemia supported larger tumors and responded poorly to tamoxifen therapy. Kaplan meier analyses showed that high leptin and high leptin receptor levels associates with poor prognosis in ER positive breast cancer. Indeed, leptin treatment abrogated tamoxifen-mediated inhibition of clonogenicity, and soft-agar colony formation. Leptin treatment increased breast tumor growth and significantly reduced the efficacy of tamoxifen. At the molecular level, leptin induced nuclear translocation of pER (ser 118 and ser 167) and increased the expression of ER-responsive genes while reducing tamoxifen-mediated gene repression. Tamoxifen is known to recruit corepressors to ER-responsive genes. ChIP analysis revealed that tamoxifen-induced recruitment of NCoR, SMRT and Mi2 corepressors was abrogated in the presence of leptin. We found that Med1, anchor subunit of human mediator complex, interacts with various key effector molecules of leptin-signaling network and potentially associates with 48 (out of 75) obesity-signature genes indicating its functional importance. Further, we found that leptin upregulated Med1 via decreasing miR-205 and increased its functional activation via phosphorylation, nuclear translocation and promoter-recruitment. Med1 phosphorylation was mediated by activation of Her2 and EGFR in response to leptin. It is important to note that silencing of Med1 abrogated the negative effects of leptin on tamoxifen efficacy. Bioactive strategies using honokiol or adiponectin treatment effectively inhibited leptin-induced Med1 expression and increased the efficacy of tamoxifen even in the presence of leptin.

Conclusion: In conclusion, these studies show the molecular mechanisms by which obese state may contribute to poor response to endocrine therapy. We show that activation of Med1 via leptin-induced EGFR and Her2 kinases act as the key node mediating the negative effects of leptin on tamoxifen efficacy. A combination regimen of honokiol and adiponectin with tamoxifen may increase its efficacy in obese state.

#311

Diverse biological mechanisms drive resistance to Lorlatinib in ALK-rearranged Lung Cancer.

Gonzalo Recondo,1 Laura Mezquita,1 David Planchard,1 Anas Gazzah,1 Francesco Facchinetti,1 Ludovic Bigot,1 Ahsan Z. Rizvi,1 Jean-Paul Thiery,2 Jean-Yves Scoazec,1 Rosa L. Frias,1 Tony Sourisseau,1 Linda Mahjoubi,1 Justine Galissant,1 Aurelie Abou-Lovergne,1 Gilles Vassal,1 Rastislav Bahleda,1 Antoine Hollebecque,1 Claudio Nicotra,1 Maud Ngocamus,1 Stefan Michiels,1 Ludovic Lacroix,1 Catherine Richon,1 Nathalie Auger,1 Thierry De Baere,1 Frederic Deschamps,1 Eric Solary,1 Ken A. Olaussen,1 Eric Angevin,1 Alexander Eggermont,1 Fabrice André,1 Christophe Massard,1 Jean-Charles Soria,1 Benjamin Besse,1 Luc Friboulet1. 1 _Institut Gustave Roussy, Villejuif, France;_ 2 _Yong Loo Lin School of Medicine, National University of Singapore, Singapore_.

Background: ALK rearrangements occur in 3-6% of patients (pts) with lung adenocarcinoma. Lorlatinib, is a novel third generation ALK tyrosine kinase inhibitor (TKI) with proven efficacy for patients previously treated with second generation ALK TKI.

Methods: The MATCH-R study is a prospective single-institution trial aiming to identify mechanisms of resistance to targeted agents and immunotherapy in pts with advanced cancer (NCT02517892). Patients that achieve an initial partial or complete response or stability of disease for at least 6 months with selected agents are included upon disease progression. Tumor biopsies are performed and serial blood samples are collected. Extensive molecular profiling with panel next-generation sequencing (NGS), whole exome sequencing (WES) and RNA sequencing (RNAseq) is performed on tumor samples. Patient-derived xenografts (PDX) in NOD scid gamma (NSG) or nude mice and patient-derived cell lines are developed. We report mechanisms of resistance in a cohort of pts with ALK-rearranged lung cancer treated with lorlatinib.

Results: From June 29th 2015 to November 15th 2018, 113 pts treated with a TKI were included in the MATCH-R study, of which 14 (12%) received treatment with ALK TKI, 6 pts treated with lorlatinib and with adequate tumor biopsies for molecular analysis were included. Tumor types studied were lung adenocarcinoma (n=4), anaplastic thyroid carcinoma (ATC, n=1) and myofibroblastic inflammatory tumor (MIT, n=1). An NF2 frame-shift deletion was detected by NGS in the ATC sample and a TNIK Q674 missense mutation was detected in the MIT sample. In the four pts with lung cancer treated with lorlatinib, we identified novel ALK G1202R/F1174L compound mutations from the tumor biopsy in one case and characterized them with Ba/F3 models (ctDNA analysis will be presented). Induction of epithelial mesenchymal transition (EMT) with lorlatinib exposure was responsible for resistance in one patient-derived model and susceptible to combined ALK/SRC inhibition. This cell line also had ALK C1156Y/G1269A compound mutations, not contributing to lorlatinib resistance. In a third case, double deleterious events in NF2 were identified in temporo-spatial distinct tumor biopsies on progression to lorlatinib. We further validated the effect of these events in patient-derived cell lines developed from two different biopsies. Downstream mTOR pathway activation conferred resistance to lorlatinib, and was reversible with mTOR inhibitors. We performed NF2 knockout in H3122 cells using Crispr-Cas9 gene editing to validate these findings. The resistance mechanism to lorlatinib treatment is yet to be elucidated in one patient-derived model.

Conclusions: Mechanisms of resistance to lorlatinib can be diverse and complex, involving compound mutations, EMT and bypass activation. The present evidence could provide new insights for the development of tailored treatments for patients.

#312

Hypoxia induces EGFR inhibitor resistance in lung cancer cells via fibroblast growth factor receptor 1 (FGFR1) by promoting epithelial-mesenchymal transition (EMT).

Yuhong Lu,1 Gary J. Zhang,2 Peter M. Glazer1. 1 _Yale Univ. School of Medicine, New Haven, CT;_ 2 _Tufts University, Medford, MA_.

The development of small molecule tyrosine kinase inhibitors (TKIs) specific for epidermal growth factor receptors (EGFRs) with activating mutations has led to a new paradigm in the treatment of non-small cell lung cancer (NSCLC) patients. However, most patients eventually develop resistance. Hypoxia is a key micro-environmental stress in solid tumors that is associated with poor prognosis due in part to acquired resistance to conventional therapy. In this study, we show that long-term, moderate hypoxia promotes resistance to the EGFR TKI, gefitinib, in the NSCLC cell line, HCC827, which harbors an activating EGFR mutation. We also found that hypoxia induces resistance to the third generation EGFR TKI, osimernitib, in the NSCLC cell line, H1975, which developed resistance to first and second EGFR TKI resistance by harboring a second EGFR mutation, T790M. Following growth in hypoxia, HCC827 and H1975 cells show increased N-cadherin expression with downregulation of E-cadherin, characteristics of an epithelial-mesenchymal transition (EMT), which prior studies have linked to EGFR TKI resistance. Mechanistically, we show that hypoxia increases fibroblast growth factor receptor 1 (FGFR1) expression in both HCC827 and H1975 cells, accompanied by down regulated BIM expression. ZEB1, an EMT activator, was also increased by hypoxia in HCC827 H1975 cells. Knockdown of the FGFR1 by shRNA can attenuate gefitinib resistance in HCC827 cells and osimerinitib resistance in H1975 cells, respectively. Inhibition of FGFR1 function by the small molecular inhibitor, ponatinib, resensitizes hypoxia-induced resistant HCC827 clones to gefitinib treatment, and also attenuates osimerinitib resistance in hypoxic H1975 cells. These results suggest that hypoxia is a driving force for acquired resistance to EGFR TKIs through increased FGFR1 expression and coordination of EMT in NSCLC. The combination of EGFR TKIs and FGFR1 inhibitors may offer an attractive therapeutic strategy for NSCLCs.

#313

AXL confers intrinsic resistance to osimertinib and the emergence of tolerant cells.

Hirokazu Taniguchi,1 Tadaaki Yamada,2 Rong Wang,3 Keiko Tanimura,2 Akihiro Nishiyama,3 Azusa Tanimoto,3 Yuta Adachi,3 Shinji Takeuchi,3 Hiroyuki Yamaguchi,1 Minoru Fukuda,1 Koichi Takayama,2 Hiroshi Mukae,1 Seiji Yano3. 1 _Nagasaki University, Nagasaki, Japan;_ 2 _Kyoto Prefectural University of Medicine, Kyoto, Japan;_ 3 _Kanazawa University, Kanazawa, Japan_.

A novel EGFR-tyrosine kinase inhibitor (TKI), osimertinib, has marked efficacy in patients with EGFR-mutated lung cancer. However, some patients show intrinsic resistance and an insufficient response to osimertinib. This study showed that osimertinib adversely stimulated AXL by inhibiting a negative feedback loop. Activated AXL was associated with EGFR and HER3 in maintaining cell survival and inducing the emergence of cells tolerant to osimertinib. AXL inhibition reduced the viability of EGFR-mutated lung cancer cells overexpressing AXL that were exposed to osimertinib. The addition of an AXL inhibitor during either the initial or tolerant phases reduced tumor size and delayed tumor re-growth compared to osimertinib alone. AXL was highly expressed in clinical specimens of EGFR-mutated lung cancers and its high expression was associated with a low response rate to EGFR-TKI. These results indicated pivotal roles for AXL and its inhibition in the intrinsic resistance to osimertinib and the emergence of osimertinib-tolerant cells.

#314

Acquired FGF and FGFR alterations confer resistance to estrogen receptor (ER) targeted therapy in metastatic ER+ breast cancer.

Pingping Mao,1 Ofir Cohen,2 Kailey Kowalski,1 Justin Kusiel,3 Jorge Buendia,2 Pedro Exman,1 Seth A. Wander,1 Adrienne G. Waks,1 Eric P. Winer,1 Nancy U. Lin,1 Nikhil Wagle1. 1 _Dana Farber Cancer Institute, Boston, MA;_ 2 _Broad Institute, Cambridge, MA;_ 3 _Albany Medical Center, Albany, NY_.

In estrogen-receptor positive (ER+) breast cancer, therapies that target the ER have led to reductions in recurrence and mortality. However, in the metastatic setting, resistance to ER-targeted therapies is near universal. Various resistance mechanisms have been proposed, including ESR1 activating mutations and upregulation of alternative signal transduction pathways, yet clinically relevant resistance mechanisms have not been clearly defined. We conducted a genome-scale gain-of-function screen in ER+ breast cancer cell lines, spanning 17,255 overexpressed open reading frames (ORFs), to investigate genes whose overexpression is sufficient to confer resistance to selective estrogen receptor degraders (SERDs). Among several validated resistance genes, the fibroblast growth factor receptor (FGF/FGFR) pathway was among the top pathways, with several FGF ligands scoring as top resistance genes in this screen. In parallel, we performed whole exome sequencing (WES) in paired pre-treatment and post-resistance biopsies from 60 patients with ER+ metastatic breast cancer (MBC) who had developed resistance to ER-targeted therapy. The FGFR pathway was altered in 40% (24 of 60) of the post-resistance biopsies; alterations included FGFR1 amplifications (9 patients), FGFR2 amplifications and activating mutations (4 patients) , and FGF3 amplifications (17 patients). In 12 out of the 24 cases (5 FGFR1, 4 FGFR2, 3 FGF3), the FGF/FGFR pathway alterations were present in the resistant tumors and not detected in the pre-treatment tumors, suggesting that these alterations were acquired under the selective pressure of ER-directed therapy. Overexpression of wildtype FGFR1, FGFR2, FGF3 in ER+ breast cancer cells (T47D and MCF7) led to resistance to fulvestrant as well as the combination of fulvestrant and the CDK4/6 inhibitor palbociclib. This resistance phenotype was reversed by the FGFR inhibitor PD173074, and, to a lesser extent, the MEK inhibitor trametinib and mTOR inhibitor everolimus, suggesting several potential treatment strategies. The three FGFR2 mutations (M538I, N550K and K660N) identified in these patients induced hyperactive FGFR signaling and conferred resistance to fulvestrant, which was abrogated by the irreversible FGFR inhibitors FIIN-2 and FIIN-3. Together, these results offer new insights into endocrine resistance and suggest new clinical approaches to overcome or preempt therapeutic resistance.

#315

Identification of distinct and targetable mechanisms of acquired resistance to CDK4/6 inhibitors.

Stephen Parsons. _Eli Lilly and Company, Indianapolis, IN_.

Recently, three CDK4/6 inhibitors were approved by FDA and became effective treatments for ER+ breast cancer patients. However, most if not all patients eventually progress on treatment . To identify mechanisms associated with acquired resistance to CDK4/6 inhibitors, we derived 15 CDK4/6i-resistant variants from human cancer cell lines highly sensitive to CDK4/6 inhibition with abemaciclib or palbociclib. Transcriptome and proteomic profiling of the derivatives revealed four general classes of alteration: decreased RB1, increased CCNE1 or CCNE2, increased ERK phosphorylation , and increased Aurora-A expression. A drug screen was employed to identify drugs or abemaciclib-drug combinations that could suppress growth of the resistant variants. Inhibitors of Aurora-A, Chk1 and the Ras-MAPK pathway were effective for the treatment of the respective classes of resistant derivative. Importantly, Rb loss, Aurora-A amplifications and Ras mutations were all observed as recurrent alterations in biopsies from ER+ breast cancer patients after progression on CDK4/6 inhibitors (1,2). Together these data demonstrate that cancer cells can escape from suppression by CDK4/6 drugs via multiple mechanisms and suggest clinically available therapeutic strategies to treat different classes of resistance.

1. S Wander et al, AACR, 2018

2. R Condorelli et al, Annals of Oncology, vol 29, issue 3, 640-645, 2017

#316

Parallel activation of MEK as a mechanism of resistance to ALK inhibitor therapy.

Vincent Huang,1 Yingjun Yan,2 Huan Qiao,2 Yunkai Zhang,2 Christine M. Lovly2. 1 _Vanderbilt University, Nashville, TN;_ 2 _Vanderbilt University Medical Center, Nashville, TN_.

Chromosomal rearrangements of ALK can result in a fusion protein that serves as an oncogenic driver in non-small cell lung cancer (NSCLC). Although patients with ALK-positive (ALK+) lung cancer have significant response rates when treated with tyrosine kinase inhibitors (TKIs), resistance against ALK targeted therapy consistently develops. Here, we modeled acquired resistance to ALK TKI therapy using 5 different FDA-approved ALK TKIs, including crizotinib, ceritinib, alectinib, brigatinib, and lorlatinib, using two distinct ALK+ lung cancer cell lines, H3122 and STE-1 (10 resistant cell lines total). Both of these cell lines harbor an EML4-ALK E13;A20 fusion and were derived from patients prior to ALK directed therapy. An ALK kinase domain mutation (F1174L) was detected in the H3122 ceritinib resistant cells, but none of the other resistant cell lines harbored mutations in the ALK kinase domain known to confer TKI resistance. For each of the cell lines tested, we found that ALK TKI treatment in TKI-resistant cells failed to inhibit MAP kinase pathway activity. We hypothesized that inhibition of the MAP kinase pathway, via MEK inhibition, could restore drug sensitivity. The allosteric MEK inhibitors, trametinib and binimetinib, did not effectively reduce cell viability as single agents. However, ALK TKI co-treatment with a MEK inhibitor resensitized these cells by reducing cell viability in a synergistic manner across all ALK TKI-resistant cell lines tested. Whole transcriptomic analysis of ALK TKI-resistant cell lines also revealed an upregulation in EGFR and HER2 mRNA levels. These results provide rationale for combined ALK+MEK inhibitor therapy in patients who have relapsed on first line ALK TKI therapy.

#317

Vecabrutinib inhibits C481 mutated Bruton's tyrosine kinase and its downstream signaling in vitro.

Burcu Aslan,1 Mikhila Mahendra,1 Michael D Peoples,1 Joe R. Marszalek,1 Christopher P Vellano,1 Xiaofeng Zheng,1 Jing Wang,1 Pietro Taverna,2 Varsha Gandhi1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Sunesis Pharmaceuticals, South San Francisco, CA_.

Inhibition of Bruton's Tyrosine Kinase (BTK) by ibrutinib, an irreversible inhibitor, has dramatically improved the outcomes in both previously treated and naïve chronic lymphocytic leukemia (CLL) patients. Ibrutinib inactivates BTK through covalently binding to the cysteine 481 residue (C481) which then leads to the inhibition of autophosphorylation of BTK and the inactivation of downstream survival nexus. Although ibrutinib demonstrated >90% overall and event-free survival, about 25% of patients discontinue ibrutinib due to leukemia progression and intolerance. Patients that initially respond to treatment may develop resistance and the most prevalent resistance mechanism of ibrutinib is described as the point mutation affecting the C481 residue of BTK that results in disruption of ibrutinib binding and acquired ibrutinib resistance. Most common mutations are C481S and C481R. Vecabrutinib is a potent reversible BTK inhibitor that binds to BTK through noncovalent interactions. As vecabrutinib does not require binding to C481 residue, it retains its efficacy with mutant BTK in vitro. To better understand differential biology of Wild-Type (WT) and serine and arginine substituted BTK, we labelled MEC-1 cell line with green fluorescence protein (GFP) and overexpressed either BTKWT, BTKC481S or BTKC481R that contributes to ibrutinib resistance. We selected MEC-1 cell line as it represents CLL disease and the phenotype of the cells share several characteristics of ex vivo CLL cells. We performed Reverse Phase Protein Array (RPPA) and mRNA-sequencing to determine and compare the proteomic and transcriptomic profiles of the MEC-1 cells harboring WT and mutant BTK. Ingenuity Pathway Analysis (IPA) of RPPA data revealed that overexpression of BTK WT leads to the enrichment of protein alterations involved in cell cycle regulation, B cell receptor signaling, PI3K/AKT signaling, PTEN signaling and ERK/MAPK signaling. IPA of RNAseq data upon BTK WT overexpression unraveled the top canonical pathways that include signaling through axonal guidance, ephrin receptor, c-AMP mediated, CXCR4 signaling and PTEN signaling. Comparative analysis of MEC-1 cells with mutant BTK (C481S vs C481R) using IPA distinguished the activated pathways in BTKC481S harboring cells from cells that express BTKC481R. These results are being validated by western blot and qRT-PCR assays. Immunoblotting results showed that following 24, 48, and 72 hour of vecabrutinib (at 1 µM) treatment reduced p-BTK (Tyr223), p-PLCG2 (Tyr759) and p-ERK (Thr 202/Tyr 204) levels in MEC-1 cells with mutant BTK. These data indicate that vecabrutinib effectively inhibits BTK and its downstream signaling in the presence of mutant BTK, suggesting that vecabrutinib treatment may be a rational approach to overcome ibrutinib resistance.

#318

Mechanisms of acquired resistance to FGFR inhibitors in molecularly-selected solid tumors: A prospective cohort from the MATCH-R study.

Francesco Facchinetti,1 Rastislav Bahleda,1 Antoine Hollebecque,1 Yohann Loriot,1 Gonzalo Recondo,1 Ludovic Bigot,1 Ken A. Olaussen,1 Gilles Vassal,1 Stefan Michiels,1 Rosa L. Frias,1 Justine Galissant,1 Tony Sourisseau,1 Claudio Nicotra,1 Maud Ngo-Camus,1 Linda Mahjoubi,1 Ludovic Lacroix,1 Etienne Rouleau,1 Catherine Richon,1 Aurélie Abou-Lovergne,1 Olivier Deas,2 Nathalie Auger,1 Thierry De Baere,1 Frederic Deschamps,1 Eric Solary,1 Jean-Yves Scoazec,1 Eric Angevin,1 Alexander Eggermont,1 Fabrice André,1 Benjamin Besse,1 Jean-Paul Thiery,3 Jean-Charles Soria,4 Christophe Massard,1 Luc Friboulet1. 1 _Gustave Roussy Cancer Campus, Villejuif, France;_ 2 _XenTech, Evry, France;_ 3 _Yong Loo Lin School of Medicine, Singapore, Singapore;_ 4 _MedImmune, Gaithersburg, MD_.

Background: Molecular alterations involving FGFR family genes (FGFR 1-4) are emerging driver events in a variety of solid tumors, mainly represented by urothelial carcinoma (UC) and intrahepatic cholangiocarcinoma (CC). Several tyrosine kinase inhibitors (TKI) are in clinical development to counteract FGFR-driven diseases, being especially active against activating gene mutations and rearrangements. Progression on these targeted agents eventually appears and the understanding of molecular mechanisms of resistance is crucial to develop novel strategies.

Methods: In the MATCH-R prospective study (NCT02517892), patients with unresectable or metastatic cancer are included upon acquired resistance to targeted therapies or immunotherapy, defined as progressive disease after complete/partial response or stable disease for six months. Serial blood samples are collected and tumor biopsy is performed upon progression. Targeted NGS, CGH, WES and RNAseq are performed on the tissue samples. PDX models and patient-derived cell lines are developed to fully investigate the underlying mechanisms of resistance. Only patients receiving TKI for FGFR-mutated or -rearranged tumors were included (i.e. FGFR amplifications were excluded) in the analysis.

Results: From June 2015 to November 2018, 113 patients treated with a TKI were included in the MATCH-R study, of which 17 (15%) had received an FGFR inhibitor. Tumor types and corresponding molecular aberrations were as follows: 8 CC (n=6 FGFR2-rearranged, n=1 FGFR2:C383R, n=1 FGFR3:S249C), 7 UC (n=5 FGFR3:S249C, n=1 FGFR3:R248C, n=1 FGFR3:Y373C), 1 breast (FGFR3-rearranged) and 1 ovarian (FGFR2-rearranged) cancers. Evaluable tumor biopsies were taken upon progression to treatment with erdafitinib (n=12), pemigatinib (INCB54828) (n=3) or TAS-120 (n=4). Two patients underwent multiple biopsies as progressing on sequential FGFR inhibitors. Resistance mechanisms consisted of polyclonal secondary mutations (n=5), bypass pathways activation (n=3) and the remaining nine cases are still under investigation. PDX models/patient-derived cell lines were obtained in eight cases and extensively characterized in three. Adaptive treatment with novel FGFR TKI or combinatorial strategies aiming to block the bypass pathways allowed to restore sensitivity in both cell lines (readouts: IC50 and Western Blots) and PDX (readout: median tumor growth). Novel mutations potentially implicated in resistance to FGFR TKI were characterized by infecting Ba/F3 cells with respective lentiviral vectors, as well as the inhibitory potential of the differential FGFR inhibitors.

Conclusions: Novel mechanisms of resistance to FGFR inhibitors in solid tumors were identified and consequent treatment strategies allowed to regain sensitivity in both patient-derived cell lines and PDX. Updated results will be presented at the Meeting.

#319

Integrated ATAC-seq and single-cell synergistic chemosensitivity profiling identifies rational drug combinations in ibrutinib treated CLL patients.

Gregory I. Vladimer,1 Christoph Schmidl,2 Andre Renderio,2 Susanne Schnabl,3 Medhat Shehata,3 Giulio Superti-Furga,2 Ulrich Jäger,3 Christoph Bock2. 1 _Allcyte, Vienna, Austria;_ 2 _CeMM, Vienna, Austria;_ 3 _Medical University of Vienna, Vienna, Austria_.

Chronic lymphocytic leukemia (CLL) is characterized by proliferation and accumulation of malignant B cells, where this process is associated with constitutively activated B cell receptor (BCR) signaling, and interference with BCR signaling provides therapeutic benefit. Specifically, the BTK inhibitor ibrutinib prevents BTK tyrosine phosphorylation and thereby interferes with the pathway. It has shown high clinical response rates in patients with relapsed and refractory CLL, including patients with adverse cytogenetic profiles. Despite high responses achieved by ibrutinib, it has important limitations such as inducing CLL cell redistribution from protected niches to the periphery, and the clinical response to ibrutinib is slow and often incomplete. Further, there is no evidence that a cure can be achieved, even among patients that tolerate long-term treatment with ibrutinib, a considerable percentage develops drug resistance, BTK independent disease progression, or Richters transformation, indicating drug synergies with ibrutinib may increase prognosis. Recent studies have explored combined use of ibrutinib with inhibitors for the proteasome (carfilzomib), BCL2i (venetoclax), and HDAC (abexinostat) in preclinical models, which has shown promising initial results. However, these approaches were largely empirical, and do not have systematic rational.

We charted the ibrutinib-induced chromatin regulatory landscape of CLL, and in parallel mapped targetable pathways for combination therapies that could potentially improve disease control. Peripheral blood from 24 CLL cases were collected before and during therapy with ibrutinib. Chromatin accessibility was measured by ATAC-seq to gather the genome-wide regulatory landscape, and ex vivo chemosensitivity to >130 drugs on paired CLL samples was measured using Pharmacoscopy, a translatable method for ex vivo single-cell drug cytotoxicity profiling in primary samples (Snijder et al. Lancet Haemato, 2017).

We integrated these datasets, establishing a comprehensive picture of the cellular responses to ibrutinib, and to prioritize targets, we performed secondary combination screen of drugs with Ibrutinib in 8 CLL patient samples taken prior to clinical Ibrutinib treatment, with the goal to visualize changes in targeted sensitivity of key drugs with and without Ibrutinib.

Specifically we observed gained sensitivity to proteasome, PLK1, and mTOR inhibitors during ibrutinib treatment. Validations of both combination screening through image based screening and also classical CLL co-culture models confirmed the findings. More generally, our study establishes a broadly applicable method for investigating treatment-specific vulnerabilities by integrating the complementary perspectives of epigenetic cell states and phenotypic drug responses in primary patient samples.

#320

**Met/Ror1-β-catenin-Bcl-xL axis contributes to the emergence of drug-tolerant cells that evolve into resistant tumors in** EGFR **-mutant lung cancer.**

Masanori Fujii, Sohei Nakayama, Naoki Akanuma, Gilbert Pan, Ikei S. Kobayashi, Hisashi Takei, Kohei Shimizu, Mariko Ando, Eunyoung Heo, Carol Gergis, Hiroyuki Inuzuka, Wenyi Wei, Daniel B. Costa, Susumu S. Kobayashi. _Beth Israel Deaconess Medical Ctr., Boston, MA_.

Despite of the success of targeted therapy with tyrosine kinase inhibitors (TKI) in epidermal growth factor receptor (EGFR) -mutant lung cancer, the duration of response is limited due to the inevitable development of acquired resistance. Previous studies have revealed that TKIs may induce survival in drug-tolerant cells (DTC) following initial treatment, leading to acquired resistance from further evolution over time. Moreover, recent studies have also shown that Bcl-xL is involved in the survival of DTC; however, little is known about the mechanism of how Bcl-xL regulates DTC in the presence of EGFR TKIs. Therefore, it is crucial to elucidate pathways which promote DTC emergence in order to develop more effective therapeutic protocols. In this study, we found that the Met/Ror1-β-catenin-Bcl-xL axis may contribute to the emergence of DTC in the presence of EGFR TKIs. We previously demonstrated that β-catenin has an essential role in lung tumorigenesis driven by EGFR-mutants, particularly EGFR-T790M. We found that genetic deletion of β-catenin gene reduced Bcl-xL expression both in vitro and in vivo. Secondly, in the presence of EGFR TKI, we found that Met-induced Ror1 activation led to tyrosine phosphorylation of β-catenin via Src. Our experiments further suggested tyrosine phosphorylation of β-catenin by EGFR and the Met-Ror1-Src cascade, whose sites were identified by mass spectrometry, plays a critical role in the interaction between β-catenin and a transcriptional factor, TBX5. Contrary to known transcriptional interaction with TCF/LEF, tyrosine phosphorylated β-catenin was found to form a complex with TBX5 and the transcriptional regulator, YAP1. This complex may regulate anti-apoptotic genes such as BCL2L1, which encodes Bcl-xL. Combination treatment with osimertinib and stable knockdown of Ror1 (or TBX5) effectively reduced Bcl-xL expression. Furthermore, treatment with osimertinib in lung cancer mouse model in which Bcl2l1 can be conditionally knocked out inhibited completely the emergence of resistant tumors compared to Bcl2l1 wild-type mouse model treated with osimertinib. These observations suggest that the Met/Ror1-β-catenin-Bcl-xL axis may serve to explain the underlying mechanism for the emergence of DTC. Our findings thus identify this axis as a promising target for EGFR-mutant lung cancer.

#321

Genome-wide CRISPR screens identify combination strategies for Capivasertib (AZD5363; AKT) and AZD8186 (PI3Kβ/δ) in PTEN-null breast cancer.

Shanade Dunn,1 Jason Yu,2 Albert Gris-Oliver,3 James Pilling,1 Philip Hopcroft,1 Urs Yelland,4 Natalie Cureton,4 Anna Staniszewsla,1 Yan Zi Au,2 Swee Hoe Ong,2 Beverley Isherwood,1 Violeta Serra,3 Simon Barry,1 Barry R. Davies,1 James T. Lynch,1 Kosuke Yusa2. 1 _AstraZeneca, Cambridge, United Kingdom;_ 2 _Wellcome Trust Sanger Institute, Cambridge, United Kingdom;_ 3 _Vall d'Hebron Institute of Oncology, Barcelona, Spain;_ 4 _AstraZeneca, Alderley Park, United Kingdom_.

The PI3K-AKT-mTOR pathway is frequently hyper-activated in breast cancer and several inhibitors targeting the PI3K pathway, including AKT (Capivasertib; AZD5363) and PI3Kβ/δ (AZD8186), are currently in clinical development (phase I/II) targeting tumours with mutational activation of the pathway including those with loss of PTEN. A better understanding of the mechanisms of resistance/sensitization to Capivasertib and AZD8186 in PTEN-null breast cancer is critical to fully exploit the anti-tumour activity of these compounds and to develop combination strategies for more effective therapy. Here we performed genome-wide CRISPR-Cas9 knockout screens to identify genes that when targeted promote resistance (gRNA enrichment) or sensitivity (gRNA depletion) to Capivasertib and AZD8186 in three PTEN-null breast cancer cell lines. Our screens identified five genes (NPRL2, DEPDC5, DDIT4, HNRNPD and ZC3H4) whose inactivation promoted resistance to Capivasertib; two resistance genes (PIK3R2 and INPPL1) for AZD8186 and three resistance genes (TSC2, TSC1 and FIBP) for both compounds across all three cell lines. While this result strongly suggests that mTOR pathway reactivation is the major resistance mechanism, we also identified novel resistance genes such as FIBP. Our screens also identified a number of genes that when inactivated sensitize cells to Capivasertib and AZD8186 treatment. Eighteen of these sensitizers have inhibitors in clinical development and we have performed a combination screen to identify compounds that give the best synergy with Capivasertib/AZD8186. The most striking combination effect was detected with the Mcl-1 inhibitor, AZD5991. Mechanistic work is ongoing and our data suggest that Capivasertib/AZD8186 prime cells for apoptosis by a previously undescribed mechanism and combined inhibition with AZD5991 drives a rapid apoptotic response. Importantly, the combination with AZD5991 remains effective in breast cancer cells with acquired resistance to Capivasertib/AZD8186. These combinations showed synergistic tumour suppressive effect in a PTEN-null TNBC breast cancer xenograft model (AZD5991 + AZD8186/Capivasertib) and activity in a patient-derived xenograft model (AZD5991 + Capivasertib). Overall, our CRISPR screening data provide new insights into the genes and pathways that drive resistance to inhibitors of the PI3K pathway in PTEN-null breast cancer and have identified novel synergistic combinations to maximize therapeutic response.

#322

Discovery of synergistic PI3K inhibitor combination therapies using high throughput approaches in HNSCC.

Nicole Lynn Michmerhuizen, Elizabeth Leonard, Caitlin Heenan, Vivek Nair, Chloe Matovina, Micah Harris, Gabrielle Herbst, Daniel Kim, Amanda Bachand, Jingyi Zhai, Susan K. Foltin, Aditi Kulkarni, Thomas E. Carey, Carol R. Bradford, Hui Jiang, John C. Brenner. _Univ. of Michigan, Ann Arbor, MI_.

Recent head and neck squamous cell carcinoma (HNSCC) sequencing studies have identified the phosphatidylinositol 3-kinase (PI3K) pathway as the most frequently mutated, oncogenic pathway in this cancer type. Despite frequent activating mutation or amplification in PIK3CA (the gene encoding the catalytic subunit of PI3K), targeted PI3K inhibitors have shown limited clinical efficacy as monotherapies. To identify factors that might predict sensitivity and resistance to PI3K inhibitors, we tested a panel of more than 40 patient-derived HNSCC cell lines and observed varying sensitivity to PI3K inhibitors. While all cell lines were much more sensitive than fibroblasts to these drugs, responses varied widely; for pan-PI3K inhibitor BKM120, IC50 values ranged from 0.2 to 4.2 μM across the OSCC cell line panel. Responses to BKM120 do not correlate with PIK3CA mutation status copy number, RNA expression, p110α protein levels, and AKT phosphorylation at baseline and after inhibitor treatment, among other genetic or phenotypic features.

In order to characterize potential mechanisms of PI3K inhibitor resistance in HNSCC, we have also developed and optimized an unbiased, high-throughput screening approach. We have used this assay to test ~1400 inhibitors as monotherapies and in combination with PI3K inhibitors HS-173 and BKM120 in ten HNSCC models. Our initial screening data suggested that combinations of PI3K inhibitors and ALK inhibitors are among the most synergistic in a subset of cell lines. We have evaluated the effects of these drug combinations on apoptosis, cell cycle, and downstream signaling in several in vitro HNSCC models, observing synergy in response to FDA-approved agents PI3K inhibitor pictilisib and ALK inhibitor brigatinib in various systems using a range of techniques. We next plan to expand these analyses to organoid and xenograft models to evaluate their effects in more clinically relevant settings.

Future studies will also examine additional factors that may predict PI3K inhibitor responses and will seek to better understand promising combination treatments and advance them for effective use in HNSCC patients.

#323

Combined inhibition of STAT-3 & DNA repair in palbociclib resistant breast cancer.

Nicole M. Kettner, Smruthi Vijayaraghavan, Merih Guray Durak, Tuyen Bui, Mehrnoosh Kohansal, Min Jin Ha, Bin Liu, Xiayu Rao, Jing Yang, Min Yi, Jason P. Carey, Xian Chen, T. Kris Eckols, Akshara S. Raghavendra, Nuhad K. Ibrahim, Meghan Karuturi, Stephanie S. Watowich, Aysegul A. Sahin, David J. Tweardy, Kelly K. Hunt, Debu Tripathy, Khandan Keyomarsi. _UT MD Anderson Cancer Center, Houston, TX_.

The CDK4/6 inhibitor palbociclib is currently being used in combination with endocrine therapy to treat advanced ER positive breast cancer patients. While this treatment has shown great promise in the clinic, about 25-35% of the patients do not respond initially, and almost all patients eventually acquire resistance. Hence, understanding the mechanisms of acquired resistance to CDK4/6 inhibition is crucial to devise alternate treatment strategies.

To identify mechanisms of resistance to CDK4/6 inhibition we developed MCF-7 and T47D palbociclib resistant cells in a step-wise manner by gradually increasing concentrations of palbociclib. These cells are not only resistant to palbociclib, but exhibited resistance to the other approved CDK4/6 inhibitors; ribociclib and abemaciclib. Additionally, we assessed if these resistant cells have an altered response to endocrine therapy and observed that these cells are also resistant to treatment with tamoxifen or fulvestrant by about 16-fold. Multi-omics analyses revealed enrichment of pathways known to regulate EMT and promote stem-like properties, as well as, downregulation of estrogen response and DNA repair pathways.

Palbociclib resistant cells exhibited mammosphere formation and CD44high/CD24low population indicating the presence of increased breast cancer stem cell-like cells (B-CSC-L). Given the recently elucidated role of IL-6/STAT-3 mediated B-CSC-L phenotypes in drug resistance, we examined IL-6 mRNA levels, which increased by >12-fold in the resistant cells. Treatment with STAT-3 inhibitors, napabucasin and C188-9, significantly decreased the B-CSC-L population and mammosphere formation, indicating a crucial role for the IL-6/STAT-3 pathway in driving B-CSC-L phenotype and palbociclib resistance.

Since DNA repair pathways were collectively downregulated in the palbociclib resistant cells, we examined their sensitivity to DNA damaging agents. Results showed that resistant cells were more sensitive to olaparib (PARP inhibition), with no effect on B-CSC-L population. Next, we examined if combined treatment with agents targeting STAT-3 and PARP would be synergistic in palbociclib resistant cells. Results show that combined treatment with olaparib and napabucasin or C-1889 significantly decreased B-CSC-L population, colony formation and increased cell death via apoptosis, when compared to no-treatment or single treatment controls of the palbociclib resistant cells.

Lastly, we interrogated matched tumor samples from breast cancer patients who progressed on palbociclib for deregulation of estrogen receptor, DNA repair, and IL-6/STAT3 signaling and found that these pathways are altered as compared to the pre-treatment samples.

Taken together, the results show that targeting IL-6/STAT-3 mediated cancer stem cells and DNA repair deficiency by PARP inhibitors in combination can effectively treat acquired resistance to palbociclib.

#324

BRAF/MEK inhibitors selectively prime melanoma for Mcl-1 dependence and sensitize to Mcl-1 inhibition.

Karson J. Kump, Lei Miao, Nurul Ansari, David B. Lombard, Zaneta Nikolovska-Coleska. _University of Michigan, Ann Arbor, MI_.

Frontline treatment for patients diagnosed with metastatic melanoma harboring a V600 BRAF mutation is a combination of BRAF/MEK inhibitors. Despite advances in targeted therapies used to treat melanoma, the majority of patients will relapse due to acquired resistance. The intrinsic apoptotic pathway is frequently dysregulated in cancer, promoting cell survival and resistance mechanisms. The anti-apoptotic Mcl-1 protein was identified as a key resistance factor to vemurafenib, a mutant BRAF inhibitor, and likely plays a role in preventing apoptosis in the context of dual BRAF/MEK inhibitor resistance. Activation of apoptosis by using small molecule inhibitors of the anti-apoptotic Bcl-2 proteins has become a validated therapeutic approach and can re-sensitize cancers to standard of care treatments. We found that melanoma cell lines display a heterogeneous dependency on anti-apoptotic proteins. In this study, we systematically investigated the role of Mcl-1 in vemurafenib resistance in melanoma cell lines using genetic and pharmacological inhibition of Mcl-1. Using the functional BH3 profiling assay, we discovered that melanoma cell lines resistant to vemurafenib are increasingly primed for Mcl-1 dependence. Additionally, applying dynamic BH3 profiling we demonstrated that melanoma cell lines subjected to short term treatment with vemurafenib showed greater dependency upon Mcl-1 for survival, thus serving as a selective priming agent. Consistent with these findings, both vemurafenib resistant and short-term vemurafenib pre-treated melanoma cell lines displayed increased sensitivity to Mcl-1 genetic silencing using siRNA and direct Mcl-1 inhibition using small-molecule inhibitors developed by our group. We further demonstrate significant synergy in the induction of apoptosis induced by the combination of vemurafenib and Mcl-1 inhibitors such as UM-101. In a similar way, Mcl-1 priming and increased sensitivity to Mcl-1 inhibitors was shown in melanoma cells pre-treated with MEK inhibitors. We further studied the mechanism of apoptosis induced by Mcl-1 inhibitors and validated their cellular target engagement. These studies provide evidence that targeting Mcl-1 represents a promising therapeutic strategy for treating patients with resistant melanomas and aids in the design of rational combination therapies before resistance is acquired.

#325

A genome-wide CRISPR screen on AML cells reveals the TP53 apoptotic network is a primary mediator of resistance to BCL2 inhibition.

Tamilla Nechiporuk, Stephen E. Kurtz, Olga Nikolova, Amanda d'Almeida, Kevin Watanabe-Smith, Mara Rosenberg, Brian Druker, Jeffrey W. Tyner, Shannon K. McWeeney. _OHSU, Portland, OR_.

Therapeutics targeted to specific proteins known to drive or modulate progression of acute myeloid leukemia (AML) have shown limited effectiveness as resistance frequently develops during their clinical use. Similar to many cancers, a stalled process of programmed cell death in apoptosis-primed cells is one of the prevalent mechanisms of drug resistance in AML. The intrinsic mitochondrial apoptotic pathway is controlled by a rheostat of anti-apoptotic and pro-apoptotic proteins and perturbing the balance toward pro-apoptotic responses remains a focus of many small molecular therapeutics. Venetoclax, an inhibitor of anti-apoptotic gene BCL2, approved for treatment of a certain subtypes of chronic lymphocytic leukemia (CLL), shows limited success as a monoagent in AML clinical trials due to an inherent resistance in approximately 37% of all patients (IC50 > 5μM). To identify mechanisms underlying intrinsic and acquired resistance to the BCL2 inhibitor, venetoclax, we used a genome-wide CRISPR/Cas9 screen to identify genes whose inactivation results in loss of venetoclax sensitivity in MOLM13 cells, derived from an AML patient that harbored mutation in FLT3-ITD, one of the major genetic landscapes of AML. Interrogation with two independently derived genome-wide libraries yielded identical top candidates TP53, BAX and PMAIP1. We also observed other gene targets that implicate the mitochondrial intrinsic, TP53-controlled pro-apoptotic pathway in the acquisition of venetoclax resistance. Correlatively, in a large AML cohort (Beat AML dataset), patients with loss of function mutations in TP53, or either low expression of TP53 or BAX exhibited statistically significant lower levels of responses to venetoclax ex vivo. Resultant venetoclax resistant cells with inactivation of TP53 or BAX had significantly reduced capacity for apoptosis when treated with venetoclax and exhibited elevated pro-survival signaling exemplified by increased levels of MAPK and AKT. In addition, loss of TP53 led to changes in the ratio of anti-apoptotic proteins BCL2, BCL-xL and MCL1 implicating TP53 as a major transcriptional regulator of the apoptotic rheostat in MOLM13 cells. Evaluation of the TP53 and BAX knockout cells for sensitivities to a panel of small molecule inhibitors revealed loss of sensitivities to a wide range of additional inhibitors including FLT3, AKT, multi-kinase inhibitors as well as a surprising gain of sensitivity to NTRK inhibitors relying on alternative pathways of cell death. Our results provide an independent confirmation of TP53 and its apoptotic network involvement to the venetoclax response in AML cells and suggest strategies to overcome resistance.

#326

Enhanced efficacy of Sitravatinib (MGCD516) in metastatic models of antiangiogenic therapy resistance.

Melissa Dolan,1 Michalis Mastri,1 Amanda Tracz,1 James G. Christensen,2 Gurkamal Chatta,1 Yuhao Shi,1 John M. Ebos1. 1 _Roswell Park Comprehensive Cancer Center, Buffalo, NY;_ 2 _Mirati Therapeutics, San Diego, CA_.

Tyrosine kinase inhibitors (TKIs) that primarily target vascular endothelial growth factor receptors (VEGFRs) are approved to treat several cancers in the metastatic setting. However, improvements in survival for most patients are short-lived and resistance is common. Sequential treatment or 'switching' from one VEGFR TKI to another has proven effective following failure in many instances. This may be due to broad drug-specific secondary TKI target profiles that can block metastasis- and angiogenesis-promoting processes. Here we examined sitravatinib (MGDC516), a novel small molecule TKI with activity against VEGFRs, that also can block Met, Axl, and several ephrin receptors (Eph), amongst several others. Transcriptomic and proteomic analysis showed multiple sitravatinib targets upregulated in VEGFR TKI-resistant mouse and human tumor cell lines derived from metastasis in mice. In vitro, sitravatinib treatment decreased proliferation in VEGFR TKI-resistant cells compared to untreated controls, suggesting prior RTKI resistance may enhance sitravatinib anti-tumor potency, while cabozantinib, a multi-targeted RTKI clinically approved after antiangiogenic therapy failure did not demonstrate enhanced anti-proliferative activity in resistant cells. In vivo, sitravatinib treatment significantly reduced orthotopically-implanted primary kidney and breast tumors in mice, with increased effects observed in VEGFR TKI-resistant tumors. Sitravatinib treatment was also found to suppress metastatic disease progression and improve survival in mice when continued after primary tumor removal, indicating metastasis-inhibiting effects of sitravatinib may be enhanced in VEGFR TKI-resistant settings. Together, these studies suggest that broad-spectrum RTKI inhibitors such as sitravatinib that target multiple metastasis-promoting mechanisms improve efficacy after antiangiogenic treatment failure and improve outcomes in a second-line setting.

#327

The impact of PIK3CA/PTEN/AKT1 genes in advanced NSCLC patients with acquired EGFR-TKI resistance and clinical response to EGFR-TKI plus everolimus combination therapy.

Wenfeng Fang,1 Yihua Huang,1 Jiadi Gan,1 Yunpeng Yang,1 Yifen Wu,2 Jingbin Huang,3 Zhiyong Xu,4 Wenjing Wang,5 Li Zhang1. 1 _State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China;_ 2 _Dongguan people's hospital, Dongguan, China;_ 3 _Shunde District Hospital of Traditional Chinese Medicine, Foshan, China;_ 4 _Southern Hospital of Chinese People's Liberation Army, Guangzhou, China;_ 5 _OrigiMed, Shanghai, China_.

Background EGFR-TKIs have shown remarkable effect in non-small cell lung cancer (NSCLC) patients with sensitive EGFR mutations. Nevertheless, several mechanisms including activation of PI3K/AKT/mTOR pathway have been proved to generate acquired assistance to EGFR-TKIs. In this study, we investigated the genomic characteristics of PI3K pathway in NSCLC patients with acquired resistance to EGFR-TKIs and whether both targeting EGFR and mTOR could reverse resistance.

Methods A total of 605 NSCLC cases who received prior TKI treatment were reviewed, in which 328 patients harboring EGFR-mutants were confirmed to have progressed on one or more TKI and finally enrolled. FFPE tumor or blood samples were collected at the onset of TKI resistance for NGS based panel assay (PIK3CA/PTEN/AKT1 genes and NSCLC driver genes are included). Six patients with co-mutations in EGFR and PI3K pathway who had been heavily treated and progressed on EGFR-TKI received EGFR-TKI plus mTOR inhibitor everolimus combination therapy.

Results In a total of 328 patients, 294 (89.6%) of the patients were adenocarcinomas, 4(12.2%) were small cell lung cancer, while the rest were squamous carcinoma or NSCLC not otherwise specified (30, 9.2%). Forty nine (14.9%) patients with TKI resistant have genomic alterations in PI3K pathway. PIK3CA, PTEN and AKT1 variations were detected in 31(9.5%), 18(5.5%) and 3(0.9%) of patients respectively. Three patients had both PIK3CA and PTEN mutations and one patient had co-mutation of PIK3CA/PTEN/AKT1. PIK3CA hotspot mutations of E545, H1047 and E542 accounted for 60.5% of PIK3CA mutations. Six patients were treated with everolimus (5mg) plus EGFR-TKI (2 patients with gefitinib, 250mg; 1 patient with afatinib, 30mg; 3 patients with osimertinib 80mg, daily) combination therapy. One (1/6, 16.7%) patient achieved partial response and the rest (5/6, 83.3%) achieved stable disease. The most common adverse events were dental ulcer (6/6), rash (2/6), pruritis (1/6) and diarrhea (1/6). One patient withdrew combination treatment due to grade 3 dental ulcer.

Conclusions Our study revealed that PI3K pathway was activated in approximately 15% of EGFR-TKI resistant patients. Combination therapy of EGFR-TKIs and everolimus revealed limited antitumor activity in EGFR-TKI resistant NSCLC patients with dysregulation of PI3K pathway.

#328

ADAM17-induced activation of HER receptors mediate resistance to trastuzumab in a subset of moderate HER2-expressing breast cancer cells.

Katharina Feldinger,1 Vasanthy Vigneswara,2 Gillian Murphy,3 Anthony Kong2. 1 _Deloitte Digital, London, United Kingdom;_ 2 _Univ. of Birmingham, Birmingham, United Kingdom;_ 3 _Univ. of Cambridge, Cambridge, United Kingdom_.

Currently anti-HER2 therapies are indicated for HER2-positive breast cancer patients that are IHC3+ or FISH positive. However, there have been studies suggesting that some HER2-negative patients may also respond to these treatments. Previously, our laboratory showed that trastuzumab upregulates ADAM17 and ADAM10 levels; and the resultant ligand release and the activation of HER receptors mediates trastuzumab acquired resistance in HER2-positive breast cancer. The aim of this study is to further understand the mechanisms of response and resistance of HER2 targeting agents such as trastuzumab and neratinib in low and moderate HER2-expressing breast cancer cells.

The HER2 expression of a panel of eight breast cell lines was assessed by IHC, FISH, western blot, and qRT-PCR. Using cell viability studies we found that in comparison with high (IHC 3+) and low HER2 expressing (IHC 0 or 1+) breast cancer cells, moderately expressing HER2 (2+) cells showed an intermediate response to trastuzumab and neratinib. Three moderate HER2-expressing breast cancer cell lines (IHC 2+), MDA-MB-361, MDA-MB-453 and HCC1569 were shown to respond to increasing doses of neratinib (with an IC50 less than 30nM). However, increasing doses of trastuzumab (up to 120 μg/ml) was unable to decrease the cell viability of these cells to below 50% of the control. The response to trastuzumab and neratinib correlated with basal ADAM17 but not ADAM10 expression. Trastuzumab treatment led to an upregulation of ADAM17 and the shedding of HER ligands in the media, as well as the phosphorylation of HER members. ADAM17 inhibition, using a specific anti-ADAM17 antibody or knockdown of the protein, decreased activation of HER members as well as downstream markers, correlating with reduced cell viability in moderate HER2-expressing cell lines. In addition, the combination of trastuzumab with neratinib was more effective than either single agent in moderate HER2-expressing cell lines.

Thus, ADAM17 may play a key role in the resistance to trastuzumab in moderate HER2-expressing breast cancer cells. Combining trastuzumab with an ADAM17 inhibitor or an irreversible pan-HER inhibitor such as neratinib may be an effective therapeutic strategy for targeting breast cancers with moderate HER2 expressing tumors.

#329

Hyperactivation of mTORC1 drives acquired resistance to the pan-HER tyrosine kinase inhibitor neratinib in HER2-mutant cancers.

Dhivya R. Sudhan,1 Angel Guerrero-Zotano,2 Helen Won,3 Paula Gonzales Ericsson,2 Qi Liu,2 Teresa Dugger,2 James Koch,2 Alison Schram,3 Alberto Servetto,1 Richard Cutler,4 Alshad Lalani,4 Richard Bryce,4 Alan Auerbach,4 Ariella Hanker,1 Carlos L. Arteaga1. 1 _UT Southwestern Medical Center, Dallas, TX;_ 2 _Vanderbilt University Medical Center, Nashville, TN;_ 3 _Memorial Sloan Keterring Cancer Center, New York, NY;_ 4 _Puma Biotechnology Inc., Los Angeles, CA_.

Background: The HER2 tyrosine kinase inhibitor (TKI) neratinib has exhibited clinical activity in patients with metastatic HER2-mutant cancers. However, responses are heterogeneous across tumor types and not generally prolonged, suggesting mechanisms of de novo and acquired drug resistance.

Methods: Neratinib-resistant 5637 (HER2S310F) bladder cancer and OVCAR8 (HER2G776V) ovarian cancer cells were developed after gradual dose escalation. Candidate pathways associated with drug resistance identified by RNA sequencing were validated in a panel of HER2-mutant cell lines and in the SUMMIT basket trial in patients with HER2-mutant cancers.

Results: Neratinib-resistant 5637 and OVCAR8 cells were cross-resistant to the HER2 TKIs afatinib and lapatinib. Immunoblot analysis showed that neratinib was still able to suppress HER2, EGFR and HER3 phosphorylation. Gene Set Enrichment and Connectivity Map analyses of RNA-seq data suggested mTORC1 signaling as a druggable pathway driving neratinib resistance. Immunoblot analysis of drug-resistant cells revealed a striking increase in S6K and S6 phosphorylation compared to parental cells. P-S6 levels and viability of drug resistant cells/tumors were ablated upon combining neratinib with the TORC1i everolimus both in vitro and in vivo. Similar results were obtained in cells transfected with Raptor or Rheb siRNAs. Further, neratinib resistance was induced by TSC2 knockdown and resultant TORC1 hyperactivation in parental 5637, OVCAR8, and MCF7 cells expressing L755S or V777L HER2 mutations. RNA-seq also revealed significant enrichment of RAS pathway in neratinib resistant cells which was confirmed by RAS-GTP pulldown. Pharmacological inhibition of RAS signaling using the PI3Ki buparlisib and the MEKi trametinib, or genetic suppression using H-, K-, and N-RAS isoform-specific siRNAs, ablated P-S6 and viability of neratinib resistant cells, suggesting RAS is causally associated with TORC1 hyperactivity and drug resistance. Further, intrinsically neratinib-resistant HER2-mutant cell lines with KRAS or PIK3CA co-mutations [DV90 (ERBB2V842I, KRASG13D), SNUC2A (ERBB2R678Q, KRASG12D, TSC2P1521T), MCF7 (HER2L755S/V777L, PIK3CAH1047R)] were sensitized to neratinib upon the addition of everolimus. Finally, DNA sequencing of tumors (MSK-IMPACT panel; 410 genes) from 141 patients enrolled in the SUMMIT trial showed enrichment of somatic alterations associated with aberrant activation of TORC1 pathway (KRAS, NRAS, NF1, PIK3CA, PIK3R1, AKT1/2, PTEN) in patients exhibiting primary resistance to neratinib.

Conclusions: These data suggest that hyperactivation of TORC1 pathway promotes de novo and acquired resistance to neratinib across histologically distinct HER2-mutant cancers. Thus, we propose the combination of neratinib with TORC1 inhibitors is worthy of investigation in patients with HER2-mutant cancers.

#330

High-throughput drug screens in osimertinib-resistant NSCLC cell lines, using continuous high-content imaging, identify potential co-targets to overcome acquired resistance.

David Westover,1 Yun-Kai Zhang,2 Yingjun Yan,2 Stephen Himmelberg,2 Darren R. Tyson,2 Joshua A. Bauer,1 Christine M. Lovly2. 1 _Vanderbilt University, Nashville, TN;_ 2 _Vanderbilt University Medical Center, Nashville, TN_.

INTRODUCTION: Oncogenic mutations in EGFR are found in 15-30% of non-small cell lung cancers (NSCLC). Patients with EGFR-mutant NSCLC derive clinical benefit from EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib and afatinib. Recently, a 3rd-generation EGFR TKI, osimertinib, was approved for first-line treatment of metastatic EGFR-mutant NSCLC based on improved progression-free survival compared to earlier EGFR TKIs. Unfortunately, resistance to osimertinib and other EGFR TKIs invariably develops. While secondary mutations in EGFR are a common resistance mechanism, resistance also frequently arises independent of EGFR alterations. We hypothesize that "bypass" signaling pathways are responsible for much of this off-target drug resistance. Moreover, we hypothesize that these bypass signaling pathways are pharmacologically targetable.

DESIGN: Osimertinib-resistant cell lines were developed for 6 months and characterized via Western blot, CellTiter Blue, and RNA-seq. Drug screens were conducted with nine matched isogenic osimertinib-sensitive and -resistant cell lines, using continual imaging and cell counting on an automated high-content imaging microscope, in the presence and absence of osimertinib. The drug-induced proliferation (DIP) rate metric was used to generate time-resolved analyses of drug effect while minimizing effect of deviations in plating density. A library of 1176 compounds was selected based on: (1) published bioactivity/safety data; (2) prior clinical investigation; (3) target diversity; (4) minimized chemical redundancy.

RESULTS: No de novo secondary EGFR alterations were found in any osimertinib-resistant cell line. Clear evidence of epithelial-to-mesenchymal transition was apparent in 4 of 9 resistant lines, which correlated with decreased EGFR and ErbB-family protein expression. FGFR1 mRNA expression was elevated in 8 of 9 resistant lines, and protein expression increased in 4 of 9 osimertinib-resistant lines. Initial drug screens have identified candidate "hits" which are more efficacious in an osimertinib-resistant cell line than its matched parental line.

CONCLUSION: These results suggest that acquired osimertinib resistance, relying on certain "bypass" signaling pathways, arise independently in multiple cell culture models. Secondly, these results suggest that such "bypass"-mediated resistance can be identified and is pharmacologically targetable. Lastly, screening data demonstrates the utility and robustness of the DIP rate metric in image-based high-throughput drug screens.

#331

Dynamic kinome targeting reveals kinases involved in acquired resistance to tyrosine kinase inhibitors in EGFR-driven glioblastomas.

Abby Shelton,1 Erin Smithberger,1 Madison Butler,2 Alex Flores,1 Ryan Bash,1 Steve Angus,1 Noah Sciaky,1 Harshil Dhruv,3 Gary L. Johnson,1 Michael E. Berens,3 Frank Furnari,4 C. Ryan Miller1. 1 _UNC Chapel Hill, NC;_ 2 _NCI, Bethesda, MD;_ 3 _Translational Genomics Research Institute, AZ;_ 4 _UC San Diego, CA_.

Glioblastoma (GBM) is a devastating primary brain tumor with limited treatment options. Extensive molecular characterization has revealed two particularly frequent mutations: CDKN2A deletion (50-60%) and EGFR (40-50%). EGFRvIII (~35%) is a constitutively active truncation mutant with exons 2-7 deleted. EGFR is a particularly attractive therapeutic target due to frequent activating mutations, such as EGFRvIII, and ready availability of multiple targeted inhibitors. Several EGFR tyrosine kinase inhibitors (TKI) have failed clinically, due in part to acquired resistance. To mechanistically examine this type of resistance, we used genetically-engineered mouse astrocytes harboring homozygous deletions of Cdkn2a, as well as EGFRvIII (CEv3). CEv3 astrocytes were made intrinsically resistant to the EGFR TKI gefitinib or erlotinib via long-term exposure, both in vitro and in vivo. We found that long-term gefitinib or erlotinib exposure conferred variable levels of cross resistance to a panel of second- and third-generation EGFR TKI (ΔIC50 1.12-36.1-fold), relative to non-resistant parent lines. We have previously shown that dynamic kinome reprogramming may be responsible for TKI resistance in glioblastoma. Therefore, we used a chemical proteomics method, multiplexed inhibitor beads and mass spectrometry (MIB-MS), to examine changes in the expressed and functional kinome, in both the presence or absence of one of several EGFR TKI known to penetrate the blood-brain barrier. Additionally, we performed RNA sequencing (RNA-seq) to inspect transcriptomic alterations in response to these drugs. RNA-seq showed that resistant CEv3 mouse astrocytes clustered separately from their non-resistant in vitro and in vivo counterparts. Acquired resistance also induced transcriptome alterations governing cellular metabolism, including upregulation of metabolic pathways and downregulation of RNA processing genes. Importantly, the kinase transcriptome was rewired, as 67 kinases were differentially expressed across parental and resistant cell lines (Q<0.001). Probing the dynamic kinome response to afatinib, an EGFR TKI, using RNA-seq identified two potential kinases involved in acute, adaptive resistance to afatinib, Bmx and Ntrk3. Integrated kinome profiling using RNA-seq and MIB-MS in murine models of GBM with defined mutational profiles provides a powerful framework to define novel therapeutic targets that could significantly alter current treatment paradigms.

#332

Development of a clinically relevant cellular model of acquired resistance to savolitinib using CRISPR/Cas9 methods.

Cheryl M. Koh,1 Daniel J. O'Neill,2 Ian L. Dale,2 Aleksandra Markovets,1 Barrett Nuttall,1 Daniel Stetson,1 Gavin W. Collie,2 Christopher J. Stubbs,2 Puneet Khurana,2 Alice Eddershaw,2 Arjan Snijder,3 Fredrik Mauritzson,3 Louise Barlind,3 Joseph Shaw,2 Christopher Phillips,2 Edward Hennessy,1 Tony Cheung,1 Ana J. Narvaez2. 1 _AstraZeneca, Waltham, MA;_ 2 _AstraZeneca, Cambridge, United Kingdom;_ 3 _AstraZeneca, Gothenburg, Sweden_.

The cMET receptor tyrosine kinase plays a key role in cell proliferation, survival, motility and angiogenesis. cMET dysregulation, including overexpression, gene amplification and activating mutations, has been reported in multiple cancer types, and results in aberrant pro-oncogenic signaling. As a result, cMET has received considerable attention as a therapeutic anti-cancer drug target, with a significant number of cMET-targeted small molecule inhibitors being assessed in the clinic. Several studies have indicated the clinical emergence of c-MET kinase domain A-loop resistance mutations in NSCLC following treatment with type I c-MET inhibitors. There is therefore a need to understand specific cMET mutations at the molecular level, particularly concerning small molecule recognition, in order to guide future inhibitor design. Single-point alterations that have previously been described include cMET Y1230H/C, D1228N/H and D1231Y. Recently, the D1228V mutation was reported in a single NSCLC patient treated with savolitinib and osimertinib (Bahcall et al. 2016). To generate a clinically-relevant in vitro model, we used CRISPR/Cas9 methods to knock in the cMET D1228V mutation into NCI-H1993 cells, a heavily MET-amplified NSCLC cell line. A single cell clone was chosen for further characterization within the context of small molecule inhibition. Growth inhibition studies showed the parental (i.e. wild-type) NCI-H1993 cells to be sensitive to savolitinib (type-I) (GI50 = 9.4 nM)and foretinib (type-II) (GI50 = 61.2 nM) . Modulation of signaling components downstream of cMET was consistent with the anti-proliferative response, as phospho-cMET (pMET) and phospho-AKT (pAKT), a key downstream effector of cMET signaling, were both reduced by savolitinib and foretinib treatment. In contrast, NCI-H1993 cMET D1228V cells demonstrated the expected loss in growth inhibition sensitivity to savolitinib (GI50 = 8.4 µM) compared to the parental cell line, but maintained sensitivity to foretinib (GI50 = 53.1 nM). Accordingly, savolitinib showed no reduction in pMET or pAKT levels in the D1228V background, while these markers remained sensitive to foretinib. To expand the range of MET inhibitors tested and quantitively determine inhibition of MET activity in cells, we used a cellular homogenous time-resolved FRET (HTRF) system, and found that savolitinib and crizotinib were much more potent in wild-type NCI-H1993 cells than NCI-H1993 cMET D1228V cells. In contrast, foretinib and BMS-777607 were seen to be equally active against both cell lines. Collectively, these findings confirm the consequence of acquired D1228V cMET mutation leading to resistance to type-I, but not type-II, MET inhibitors. However, as both foretinib and BMS-777607 are not currently in active development, there remains a need for clinically viable candidates with activity against on-target cMET resistance mutations.

#333

BRD4 inhibitor-resistant acute myeloid leukemia cells develop new sensitivities and resistances to small-molecule inhibitors identifiable through high-capacity drug screening.

Kyle A. Romine, Stephen Kurtz, Jeffrey Tyner. _Oregon Health & Science University, Portland, OR_.

Acute Myeloid Leukemia (AML) has a poor prognosis for patients who cannot tolerate conventional chemotherapy, which has prompted the development of many small-molecule inhibitors now in clinical trials. Recently, inhibitors targeting bromodomain containing protein 4 (BRD4i), an epigenetic regulator of many proliferative genes, have shown promise in AML based on in vitro and ex vivo activity. Mechanisms through which AML cells develop resistance to epigenetic modifying drugs such as BRD4i has not been thoroughly evaluated and its elucidation will provide insight into the epigenetic states of AML as well as resistance mechanisms that could lead to combination therapies with broader and more durable activity. The aim of this study is to 1) Develop AML cells resistant to 3 BRD4i (JQ1, OTX-015, and CPI-0610) with similar mechanisms-of-action, 2) Screen a panel of small molecules to identify inhibitors that are differentially effective in BRD4i-resistant lines compared to parental, 3) Identify changes in signaling pathways by evaluating the phosphorylation state of proteins in resistant versus parental cells. Results: We developed BRD4i-resistant versions of the OCI-AML2 (DNMT3a) and MOLM13(FLT3-ITD) cell lines by passaging the cells for 3 months under increasing selective pressure of the three inhibitors, reaching a minimum of 5 fold resistance to the BRD4i. We found that these cell lines were pan-resistant to the BRD4i, such that a cell line treated with JQ1 was also more resistant to OTX-015 and CPI-0610. Drug sensitivity changes in BRD4i-resistant OCI-AML2 show a 90-fold increased resistance to the BCL-2 inhibitor, venetoclax. BRD4i-resistant MOLM13 cells also have a 10-fold increased resistance to venetoclax. To evaluate the phosphorylation status of many oncogenic proteins simultaneously, phosphokinase arrays were performed on parental and resistant cells with and without treatment of drug. JQ1-resistant OCI-AML2 cells have significantly reduced phosphorylation of CREB and p38α. Additionally, these cells have reduced sensitivity to the CREB inhibitor 666-15. In contrast, MOLM13 CPI-0610-resistant cells showed a dramatic increase in CREB phosphorylation compared to parental cells, indicating dysregulation of CREB pathway activation state may be important in developing BRD4i resistance. This may also explain resistance to venetoclax, as activated CREB mediates the transcription of BCL-2. Conclusions: Our work demonstrates the potential of AML cell models of BRD4i-resistance to evaluate changes in oncogenic kinase signaling as well as the rapid identification of alternative therapeutic strategies that may have immediate clinical benefit. In addition, we have begun to understand at a mechanistic level how cancer cells evade apoptosis induced by BRD4 inhibition.

#334

Proteostasis by HSP70/STUB1 complex regulates androgen receptor variants expression and confers resistance to enzalutamide and abiraterone.

Chengfei Liu, Wei Lou, Joy C. Yang, Liangren Liu, Cameron M. Armstrong, Alan P. Lombard, Ruining Zhao, Onika DV Noel, Clifford G. Tepper, Hong-Wu Chen, Marc Dall'Era, Marc Dall'Era, Christopher P. Evans, Allen C. Gao. _University of California, Davis, Sacramento, CA_.

INTRODUCTION AND OBJECTIVES: Proteostasis is a complementary process by which cells control the protein biosynthesis, folding, trafficking and degradation. Evidence suggests that proteomic instability, such as protein misfolding and aggregation plays pivotal roles in cancer cell survival and progression. AR-V7 lacking the ligand binding domain confers resistance to enzalutamide and abiraterone in prostate cancer, targeting AR-V7 is an logical strategy to overcome the resistance. The mechanisms of AR-V7 proteostasis haven't been fully studied so far. The objectives of this project are to investigate the roles of proteostasis in regulating AR-V7 expression and sensitivity to enzalutamide and abiraterone treatment.

METHODS: Expression of HSP70 and STUB1 was determined by qRT-PCR and western blot. Expression of HSP70 and STUB1 was downregulated using specific siRNA. HSP70/STUB1 and AR-V7 interaction was determined by co-immunoprecipitation and dual immunofluorescence. The gene regulating mechanisms underlying the HSP70 inhibition in drug resistant prostate cancer cells was determined by RNA sequencing analyses. The effects of HSP70 inhibition on enzalutamide sensitivity were examined in vitro and in vivo. The correlation between HSP70 and AR-V7 in high Gleason score prostate tumors was determined by qRT-PCR.

RESULTS: In the present study, we analyzed enzalutamide and abiraterone resistant prostate cancer cells and found ubiquitin mediated proteolysis pathway was suppressed, and E3 ubiquitin ligases STUB1 is downregulated in enzalutamide and abiraterone resistant prostate cancer cells. STUB1 binds to AR-V7, degrades AR-V7 expression and suppresses its activity. HSP70, the STUB1 binding protein, also binds to AR-V7 and enhances AR-V7 transcriptional activity. Mechanistically, STUB1 disassociates HSP70 from AR-V7 binding and increases AR-V7 degradation. Targeting HSP70 by siRNA or small molecular inhibitors (Apoptozole and Ver155008) significantly suppressed prostate cancer growth and improved enzalutamide and abiraterone treatment through AR-V7 inhibition in vitro and in vivo. Additionally, HSP70 expression is upregulated in mCRPC tumors and correlates with AR-V7 levels in high Gleason score and metastatic prostate tumor specimens.

CONCLUSION: Enzalutamide and abiraterone treatment induces the imbalance of AR-V7 proteostasis through the ubiquitin-proteolysis alteration. STUB1/HSP70 complex controls AR and AR variants proteostasis. Targeting HSP70 could be a valuable strategy to overcome the next generation anti-androgen resistance and improve their therapy.

#335

Efficacy of FGFR inhibitors and combination therapies for acquired resistance in FGFR2-fusion cholangiocarcinoma.

Melanie A. Krook,1 Alexandria M. Lenyo,1 Maxwell J. Wilberding,1 Hannah D. Barker,1 Mikayla Dantuono,1 Hui-Zi Chen,1 Julie W. Reeser,1 Michele R. Wing,1 Jharna Miya,1 Eric Samorodnitsky,1 Amy M. Smith,1 Thuy Dao,1 Dorrelyn M. Martin,1 Kristin Dittmar,1 Kristen K. Ciombor,2 John L. Hays,1 Aharon G. Freud,1 Sameek Roychowdhury1. 1 _The Ohio State University, Columbus, OH;_ 2 _Vanderbilt University Medical Center, Nashville, TN_.

Background. The fibroblast growth factor receptor (FGFR) signaling pathway is aberrantly activated in approximately 20% of cases of cholangiocarcinoma through various genomic alterations including gene fusions, point mutations, and copy number amplifications. Currently, several FGFR kinase inhibitors are being assessed in clinical trials for patients with FGFR-altered cholangiocarcinoma. Despite evidence of initial responses and disease control, virtually all tumors eventually develop drug resistance. Overcoming acquired drug resistance to FGFR inhibitors has not been comprehensively studied.

Methods. Apatient with metastatic cholangiocarcinoma and an activating FGFR2-KIAA1598fusion was enrolled on a Phase 2 clinical trial for the oral FGFR inhibitor, BGJ398. BGJ398 treatment was initially effective with radiographic and tumor marker response; however, after eight months of BGJ398 therapy, the patient developed disease progression. Sequencing of a post-progression liver biopsy revealed a novel secondary mutation (E566A) in the FGFR2 kinase domain, which was absent in the pre-treatment biopsy suggesting that this mutation may be responsible for the resistance to BGJ398. To confirm this, we conducted in vitrostudies to characterize the sensitivity of the FGFR2 secondary mutation to FGFR inhibitors.

Results and Conclusions. In vitrocharacterization of the fusion and putative resistance mutation demonstrated that FGFR2-KIAA1598 WT cells were sensitive to BGJ398 with (IC50value of 10.95 nM) whereas the FGFR2-KIAA1598 E566A cells were resistant (IC50values of 422.4 nM). We investigated additional selective (AZD4547 and JNJ42756493), non-selective (dovtinib and ponatinib), and irreversible (TAS120) FGFR inhibitors. FGFR2-KIAA1598 WT cells were sensitive to AZD4547, JNJ42756493, and TAS120, whereas FGFR2-KIAA1598 E566A cells were resistant to these inhibitors. Interestingly, neither the FGFR2-KIAA1598 WT cells nor the FGFR2-KIAA1598 E566A cells were sensitive to dovitinib, a nonspecific tyrosine kinase inhibitor. Lastly, both populations of cells were equally sensitive to ponatinib, a non-selective FGFR kinase inhibitor. Proteomic analysis by reverse phase protein array (RPPA) revealed upregulation of the mTOR/PI3K/AKT pathway in fusion cells compared to empty controls (FDR: 0.00618), and this expression was further potentiated in FGFR2-KIAA1598 E566A (FDR: 1.96e-19). Furthermore, combination studies with the above FGFR inhibitors and the mTOR inhibitor, INK128, yielded synergistic antitumor effects suggesting that combination therapy may be able to overcome resistance to FGFR inhibitors.

Significance. In conclusion, further elucidation of the mechanisms driving resistance to FGFR inhibitors will support the development of next generation FGFR inhibitors as well as combination therapeutic strategies.

#336

A recombinant human protein targeting HER2 overcomes drug resistance in HER2-positive breast cancer.

Lu Yang, yun Li, Arup Bhattacharya, yuesheng Zhang. _Roswell Park Comprehensive Cancer Center, Buffalo, NY_.

Background & Goals: HER2 is an oncogenic receptor tyrosine kinase (RTK) implicated in several types of human cancer. It is strongly expressed in about 20% of breast cancer (BC), known as HER2-positive BC (HER2-BC), due to gene amplification. HER2 amplification or overexpression is a strong predictor of poor disease prognosis. Several HER2-targeting drugs are available for treating HER2-BC, including monoclonal antibodies trastuzumab (Ttzm) and pertuzumab, T-DM1 (Ttzm coupled to a microtubule inhibitor), and tyrosine kinase inhibitors (TKIs) lapatinib and neratinib. While these agents have greatly improved disease outcomes, primary and acquired drug resistance, including cross-resistance, is common. Overcoming drug resistance remains a major unmet medical need in HER2-BC. Notably, HER2 remains overexpressed in drug-resistant HER2-BC cells. We recently found that recombinant human peptidase D (PEPD), also known as prolidase, strongly downregulates HER2 and EGFR in cancer cells in vitro and in vivo via its binding to HER2 and EGFR. The enzymatic activity of PEPD plays no role in its modulation of HER2 and EGFR, and we subsequently focused on recombinant PEPDG278D, an enzymatically inactive mutant (point mutation at codon 278). Our objective in the present study is to determine whether PEPDG278D is active in preclinical models of HER2-BC resistant to clinically available HER2 inhibitors and if so, its mechanism of action.

Results: PEPDG278D was exceedingly effective against HER2-BC cells that are resistant to Ttzm and other HER2 inhibitors. The cells carried activating PIK3CA mutations, low expression of PTEN, and/or cyclin E overexpression. All the cell lines also overexpressed MUC4 which shields HER2 from Ttzm and stabilizes HER2. However, PEPDG278D rapidly bound to and freed HER2 from MUC4, and rapidly disrupted the interaction of HER2 with other RTKs, including MET and IGF1R. PEPDG278D subsequently induced persistent downregulation of HER2 via internalization and lysosomal degradation. PEPDG278D also downregulated EGFR. Thus, PEPDG278D suppresses multiple RTKs either directly or indirectly in HER2-BC cells. In contrast, Ttzm cannot perform any of these functions of PEPDG278D. PEPDG278D was highly active in mouse tumor models of HER2-BC resistant to Ttzm and also enhanced the therapeutic efficacy of paclitaxel. However, adverse effects of PEPDG278D were not detected in the animal studies.

Conclusions & Therapeutic Relevance: The therapeutic activity of PEPDG278D highlights that HER2 remains a critical target in drug-resistant HER2-BC. The strong therapeutic activity of PEPDG278D in preclinical models of drug-resistant HER2-BC, which carry clinically relevant molecular changes that confer resistance to current HER2 inhibitors provides the scientific

premise for potential evaluation of this recombinant human protein in patients with drug-resistant HER2-BC.

#337

Degradation of AXL overcome acquired cross-resistance to gefitinib and osimertinib in non-small cell lung cancer cells.

Donghwa Kim, Duc-Hiep Bach, Yan-hua Fan, Thi-Thu-Trang Luu, Ji-Young Hong, Hyen-Joo Park, Sang Kook Lee. _Seoul National Univ. College of Pharmacy, Seoul, Republic of Korea_.

Epidermal growth factor receptor (EGFR) mutation is one of the major driver oncogenes in non-small cell lung cancer (NSCLC) and most frequently found in Asian patients. Although the first generation of EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib have led to improved prognoses for NSCLC patients, acquired resistance has been a major obstacle in the treatment of non-small cell lung cancer (NSCLC) patients. Recently, AXL has been reported to play a role in drug resistance mechanisms for many anti-cancer drugs, including erlotinib and cetuximab, as well as in ionizing radiation therapy for multiple cancers. In the present study, we demonstrate the involvement of AXL in the acquired resistance to gefitinib and osimertinib of EGFR-mutant NSCLC cells, then show the combination effect of an AXL degrader and EGFR-TKIs to overcome EGFR-TKIs-driven resistance in EGFR-mutant NSCLC cells. We found that AXL was overexpressed and its degradation was delayed in EGFR-TKI-resistant NSCLC cells. Yuanhuadine (YD), a natural antitumor agent, effectively suppressed the expression of AXL by accelerating protein degradation. YD combined with gefitinib or osimertinib synergistically inhibited the growth of resistant cells in vitro and suppressed tumor growth in a nude mouse xenograft model. Moreover, administration of YD with gefitinib effectively delayed gefitinib-driven acquired resistance in a long-term xenograft model. Our results suggest that the combination of YD with either gefitinib or osimertinib is a potentially effective treatment strategy for overcoming and delaying acquired resistance in NSCLC by targeting AXL degradation.

#338

Inhibition of phosphatidylinositol 3-kinase signaling antagonizes paclitaxel-mediated resistance in cervical cancer.

Youn Jin Choi, Yoo Yeon Jung, Jing Jing Liu. _Catholic Univ. of Korea, Seoul, Republic of Korea_.

Paclitaxel is used as an effective treatment against advanced cervical cancer. However, acquired resistance limits its success and results in cancer progression. We investigated whether the inhibition of phosphatidylinositol 3-kinase (PI3K) signaling overcomes paclitaxel resistance in cervical cancer. Two human cervical cancer cell lines (Me180 and HeLa) were used. We established paclitaxel-resistant cell lines (PTX-R Me180/PTX-R HeLa) and confirmed inhibitory concentrations (IC)50 and cell death rates by tetrazolium dye (MTT) and apoptosis assays, respectively, after exposure to a combination of paclitaxel with PI3K inhibitors (LY294002 and BYL-719). Migration and invasive abilities were explored by wound healing and transwell assays, respectively. Western blots were performed to determine the mechanism underlying the antitumor effects of PI3K inhibitors.PTX-R Me180 and PTX-R HeLa cells were gradually established from parental cell lines by stepwise exposure to paclitaxel over 7 months and with stable resistance at under 20 nM. We found that the activation of the PI3K pathway in paclitaxel-resistant lines increased tumor growth and invasiveness, and impeded cell apoptosis. The combination of LY294002 or BYL-719 with paclitaxel significantly enhanced drug sensitivity and synergistic apoptosis through the activation of Bax, caspase-3, and cleaved poly ADP ribose polymerase compared with paclitaxel alone. In addition, PI3K inhibition also suppressed tumor migration and invasive ability by targeting vimentin, matrix metalloproteinase-9 and vascular endothelial cell growth factor.We suggest that a combination of a PI3K inhibitor with paclitaxel may enhance antitumor activity through a cascade of PI3K signaling events.

#339

Mechanisms of resistance to poly (ADP-ribose) polymerase inhibitors in prostate cancer.

Mohammad Atiq,1 Goutam Chakraborty,1 Subhiksha Nandakumar,1 Ying Z. Mazzu,1 Konrad Stopsack,1 Yuki Yoshikawa,1 Nabeela Khan,1 Gwo-Shu Mary Lee,2 Philip W. Kantoff1. 1 _Memorial Sloan Kettering Cancer Center, New York City, NY;_ 2 _Dana-Farber Cancer Institute, Boston, MA_.

Background: Poly (ADP-ribose) polymerase inhibitors (PARPi) have demonstrated promise in treating cancers with DNA damage repair (DDR) gene abnormalities. As a result, olaparib and more recently, rucaparib have been given Breakthrough Therapy designation by the FDA for use in metastatic castration resistant prostate cancer (mCRPC) with BRCA1/2 and ATM mutations. One of the problems encountered with PARPi is drug resistance which generally limits drug efficacy. Mechanistic studies on PARPi resistance have shown one of the main mechanisms of acquired resistance to be a reversion mutation of BRCA1/2. However, this is likely more applicable to cancers in which impaired BRCA1/2 function is due to a mutation rather than in prostate cancer (PC) where BRCA2 tends to be frequently deleted. Therefore, we hypothesize that mechanisms of resistance to PARPi in PC may involve alternative molecular mechanisms rather than a reversion mutation.

Methods: We used human castration-resistant PC cell lines that harbor genomic deletions of BRCA2, PC-3 and LnCaP-Abl, and performed cell viability (MTT) assays to determine the inhibitory growth (IG) concentrations of these cell lines with talazoparib and olaparib. We cultured parental PC-3 cells in sublethal concentrations (IG 50% and IG 90%) of talazoparib-supplemented media to develop talazoparib-resistant cells. RNA sequencing followed by gene-set enrichment analysis (GSEA) of hallmark gene sets was performed on the talazoparib-resistant PC-3 cells to understand the underlying molecular mechanisms.

Results: We observed that the talazoparib-resistant PC-3 cells exhibited significantly enhanced cell growth compared to parental cells when cultured in the IG 90% concentration of olaparib. However, interestingly, the talazoparib-resistant cells grew much slower in 2D compared to parental PC-3 cells when cultured in the PARPi-free media. Our transcriptomic analysis showed significant enrichment of various inflammatory response pathways, including TNF-α and IFNα/γ signaling pathways, in the talazoparib-resistant cells and even in the parental PC-3 cells transiently treated with talazoparib.

Conclusion: We hypothesize that resistance to PARPi in PC may be related to upregulation of inflammatory signaling. Therefore, further exploration of TNF-α and IFNα/γ and their role in PARPi resistance mechanisms may lead to the identification of targets that allow for overcoming PARPi resistance in PC. 

### New Anticancer Agents

#340

Development of high affinity anti-CLDN18.2 antibody to treat gastric cancers.

Zhenhao Zhou. _Genbase, Shanghai, China_.

Claudins are tight junction proteins that regulate epithelial-cell barrier function and polarity. Gastric lineage-specific isoform 2 of CLDN18 (CLDN18.2) has been proposed as therapy target in primary/metastatic gastric cancers and CLDN18.2-ectopically-activated cancers, such as pancreatic, esophageal and lung adenocarcinomas. As demonstrated by Zolbetuximab, a monoclonal chimeric antibody directing CLDN18.2, the median overall survival was prolonged to 16.7 months when treated with Zolbetuximab+EOX (OS of EOX group: 9.0 months) in pre-screened CLDN18.2 highly expressing patients (2+/3+ staining in ≥70% tumor cells). Expression of CLDN18.2 in gastric tumors in Chinese population, however, showed diverse levels of frequency with low-frequency expression in a significant portion of tumor cells. Antibody of high affinity is expected to have advantage in targeting antigen of low-frequency, thus clearing tiny lesions clinically. We developed a high affinity anti-CLDN18.2 antibody, GB7004-09. GB7004-09 binded to human/mouse CLDN18.2 while sparing CLDN18.1. It displayed significantly better binding/ADCC/CDC potency against CLDN18.2 expressing tumor cells comparing to leading clinical drug entity. And its in vitro superiority translated to greater in vivo efficacy. Humanization version of GB7004-09, GB7004-09hu15, has been put into preliminary toxicity and PK study. As we are observing safety, stability and activity of GB7004-09hu15, GB7004-09hu15 is a promising agent for extended clinical exploration in cancer indications mention above

#341

Synergy of FLT3 inhibitors and a small molecule inhibitor of LIM kinase1/2 in FLT3-ITD positive acute myeloblastic leukemia (AML).

Thorsten Braun,1 Jeannig Berrou,1 Hanane Djamai,1 Mélanie Dupont,1 Anna Kaci,1 Jan Erik Ehlert,2 Holger Weber,2 André Baruchel,1 Fabrice Paublant,3 Renaud Prudent,3 Claude Gardin,1 Hervé Dombret1. 1 _LTL Saint Louis, Paris, France;_ 2 _Proquinase, Freiburg, Germany;_ 3 _CELLIPSE, Grenoble, France_.

Introduction: LIM kinases 1/2 are downstream effectors of signalization pathways implicated in cytoskeleton dynamics via phosphorylation of Cofilin family proteins,matrix degradation and in activity control of Aurora kinase A. Recently, Rho kinases (ROCK) were identified to be constitutively activated by FLT3-ITD, BCR-ABL and KIT in hematologic malignancies via PI3 kinase and Rho GTPase mediated phosphorylation. Upon its activation by upstream kinases (ROCK and PAK) LIMK1/2 inactivates Cofilin by phosphorylation, leading to enhanced polymerization of Actin. Here we investigated the potential therapeutic role of LIMK1/2 inhibition in FLT3-ITD mutated AML.

Materials and methods: Expression of LIMK1/2 was determined by RQ-PCR and WB. A small molecule inhibitor of LIMK1/2 (LIMKi) was tested alone or in combination with FLT3 inhibitors Midostaurin, Quizartinib and Crenolanib or the hypomethylating agent Azacitidine in FLT3-ITD driven AML cell lines MOLM-13 and MV-4-11. Cell viability and IC50 was assessed by MTT assays. In combination experiments, compounds were added simultaneously and relative cell numbers were determined at 72h with MTT assays and combination index (CI) was calculated with the Chow and Talalay model. Cell-cycle distribution was determined by cytofluorometric analysis detecting nuclear propidium iodide (PI) intercalation. Apoptosis was evaluated in cell lines and patient cells by outer Annexin V exposure and PI incorporation.Cells from healthy donors were obtained after informed consent and enriched for CD34+ cells by immunomagnetic selection and seeded in methylcellulose, FCS and cytokines with or without LIMKi. For in vivo experiments we used a bone marrow engraftment tumor model with MOLM13-LUC cells using bioluminescence imaging in NOD-SCID mice treated either with LIMKi, Midostaurin or LIMKi+Midostaurin.

Results:Expression of LIMK1/2 in MOLM-13 and MV-4-11 cells could be detected by QT-PCR and at the protein level.IC50 after LIMKi exposure was 440 nM in MOLM-13 cells and 420 nM in MV-4-11 cells. Combination experiments with the LIMKi and either the FLT3 inhibitors Midostaurin, Quizartinib, Crenolanib or the hypmethylating agent Azacitidine were synergistic for treatment of MOLM-13 cells. Exposure of MOLM-13 cells to increasing doses of LIMKi induced cell cycle arrest in the G1/S transition and dose dependent apoptosis. No significant toxicity of increasing doses of LIMKi after exposure of CD34+ cells from healthy donors could be detected. In NOD-SCID mice engrafted with MOLM13-LUC cells Midostaurin and LIMKi delayed MOLM13 engraftment as detected by in vivo bioluminescence imaging and LIMKi+Midostaurin prolonged significantly survival of mice as compared to Midostaurin alone.

Conclusion: LIMK1/2 inhibition seems to be promising in combination with various FLT3 inhibitors or Azacitidine in vitro as well as in vivo with Midostaurin.

#342

S63845, a novel BH3 mimetic Mcl-1 inhibitor synergizes with midostaurin to induce potent apoptosis in acute myeloid leukemia cells carrying FLT3-ITD mutations.

Anna Skwarska,1 Qi Zhang,1 Shelley M. Herbrich,1 Natalia Baran,1 Ensar Halilovic,2 Peter Ruvolo,1 Vivian Ruvolo,1 Erick Morris,2 Andrew Wei,3 Donia Moujalled,3 Michael Andreff,1 Marina Konopleva1. 1 _MD Anderson Cancer Center, The University of Texas, Houston, TX;_ 2 _Novartis Institutes for BioMedical Research, Cambridge, MA;_ 3 _The Alfred Hospital and Monash University, Melbourne, Australia_.

Myeloid cell leukemia 1 (Mcl-1) is one of the key anti-apoptotic Bcl-2 family proteins that binds and neutralizes pro-apoptotic BIM, BAX and BAK at the mitochondrial outer membrane, preventing cytochrome c release and caspase activation. Selective upregulation of Mcl-1 functionally contributes to resistance of acute myeloid leukemias (AML) with FMS-like tyrosine kinase-3-internal tandem duplications (FLT3-ITD) to chemotherapy (Kasper S. et al. 2012, Blood Cancer J. 2:e60, doi:10.1038/bcj.2012.5). Here we show that a novel Mcl-1 inhibitor S63845 (Kotschy A. et al. 2016, Nature 538, 477-482), has synergistic proapoptotic activity in combination with FLT3-ITD kinase inhibitor midostaurin in pre-clinical models of AML. Our studies demonstrate that S63845 has potent single agent activity in AML cell lines and primary AML samples harboring FLT3-ITD with IC50 values in low nanomolar range. Co-targeting of Mcl-1 and FLT3-ITD with S63845 and midostaurin, respectively, significantly increased apoptosis in FLT3-ITD cells with caspase-3 activation and PARP cleavage occurring rapidly within 6 hours of treatment. Consistent with markedly reduced cell growth and viability, analysis of drug combinations efficacy using Bliss independence model revealed strong synergistic interactions between S63845 and midostaurin in FLT3-ITD cell lines and primary AML samples. Midostaurin caused de-phosphorylation of FLT3-ITD and its downstream targets such as STAT5, AKT and MAPK. This was accompanied by significant downregulation of MAPK-mediated phosphorylation of Mcl-1 at Thr163 required for Mcl-1 stability. Consequently, midostaurin reduced Mcl-1 protein levels, with no major changes in antiapoptotic Bcl-2 or Bcl-XL. Importantly, midostaurin increased expression of pro-apoptotic Bim, which could in turn bind and negate residual Mcl-1 pro-survival activity. Elevated Bim was sustained upon S63845 co-treatment, suggesting that Bim plays functional role in midostaurin/S63845-mediated lethality. Dynamic BH3 profiling showed that midostaurin primed FLT3-ITD cells to Mcl-1 and Bcl-2 inhibitors and facilitated general apoptosis priming in response to Bim peptide. Importantly, given that Mcl-1 is a major contributing factor to resistance of AML to Bcl-2 selective BH3-mimetic venetoclax, S63845/midostaurin treatment induced cell death in venetoclax-resistant FLT3-ITD mutants. In summary, S63845/midostaurin is highly synergistic in FLT3-ITD mutated AML cells including those resistant to venetoclax. In vivo experiments of tolerability and efficacy are ongoing and will be reported.

#343

A potent and selective cfms inhibitor EI-1071 inhibits CSF1R signaling and regulates the tumor-associated macrophages.

Hung-Kai Chen,1 Huey-Wen Hsiao,1 Li-Ming Lu,1 Chih-Lun Hsiao,1 Yin-Ping Wang,1 Jing-Yi Huang,1 Chu-Bin Liao,2 Shao-Zheng Peng,2 Daw-Tsun Shih1. 1 _Elixiron Immunotherapeutics, Taipei, Taiwan;_ 2 _Development Center for Biotechnology, Taipei, Taiwan_.

We have found that tumor-associated macrophages (TAMs) isolated from metastatic tumors displayed a predominant M2 phenotype. Preclinical studies show that M2-polarized TAMs forge an immunosuppressive microenvironment at metastatic niches to facilitate colonization. Colony-stimulating factor 1 receptor (CSF-1R) signaling is known to regulate the survival and maintenance of M2 TAMs, suggesting targeting CSF-1R might have therapeutic potential in halting cancer progression. To this end, we developed an orally active and selective small-molecule CSF-1R inhibitor EI-1071.

Experimental procedures: EI-1071 was evaluated in kinase and cell-based bioassays, including macrophage proliferation and osteoclast differentiation. In vivo activity was verified using murine syngeneic models of colorectal cancer and breast cancer. EI-1071 was administered individually or in combination with an anti-PD1 antibody. Tumor infiltrating leucocytes were profiled using flow cytometric analysis.

Results: EI-1071 exhibited nanomolar potency for inhibition of CSF-1R but not other 453 kinases tested. In cell-based bioassays, EI-1071 inhibited CSF-1R activity in human monocytes/macrophages, AML-5 leukemia, MNFS-60 leukemia, and human osteoclasts. In preclinical studies, EI-1071 displayed a favorable ADME properties, including hepatocyte stability, high Caco-2 permeability, and pharmacokinetic profiles. Oral administration of EI-1071 reduced TAM infiltration in the MC38 colorectal cancer and EMT6 breast cancer models. Moreover, EI-1071 dosed with an anti-PD-1 antibody showed further enhanced antitumor activity in vivo .

Conclusions: Preclinical characterization of EI-1071 demonstrated that it is a potent and selective CSF-1R inhibitor and has a favorable drug like properties for further clinical evaluation. Therefore, EI-1071 is undergoing IND-enabling activities for the First-In-Human (FIH) Phase 1 study.

#344

SJP1604, a novel targeted therapeutic agent for AML (acute myeloid leukemia) including standard therapy-resistant AML.

Jihyun Um,1 Dohyeong Lee,1 Jisun Oh,1 Yongbin Park,1 Sung Hwan Moon,2 Su Jin Lee,2 Min-Hyo Ki,1 Eui Hwan Cho1. 1 _Samjin Pharm co., Ltd, Seongnam, Republic of Korea;_ 2 _Aptabio, Yongin, Republic of Korea_.

Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are cancers characterized by the rapid growth of abnormal white blood cells. Generally, cytarabine (Ara-C), azacitidine, and decitabine are treated as the standard first-line chemotherapy for AML/MDS. Although the therapy leads to high rates of remission, approximately 30% of the treated patients are refractory and more than 50% of them face a relapse because of the occurrence of drug resistance and high toxicity. Therefore, there is a strong unmet need for the next-generation targeted therapy to overcome drug resistance and reduce toxicity in AML/MDS. Here, we designed an aptamer-nucleoside drug conjugate (SJP1604) via utilizing easy and continuous synthesis specific for nucleolin, which is highly expressed only on the cell membrane of cancer cells. In particular, AML, CLL (chronic lymphoblastic leukemia) and ALL (acute lymphoblastic leukemia) cells are known to express the highest mRNA level of nucleolin among various cancer cells, relatively. SJP1604 can be delivered into cancer cells with its high targeting ability and plasma stability via its unique conformational property. In this study, we verified that SJP1604 selectively targeted cancer cells by nucleolin-binding on the cancer cell membrane in the drug uptake assay. Owing to the selectivity, SJP1604 demonstrated less cytotoxicity on murine bone marrow cells in vitro and mouse white blood cells in vivo. SJP1604 also exhibited a long-lasting plasma stability in 50% human plasma up to 48 hours in contrast to unstability of general aptamers. Furthermore, SJP1604 inhibited the growth of various human AML/MDS cancer cells. Interestingly, SJP1604 showed the significantly reduced IC50 values with the decreased expression level of nucleolin in cytarabine-, azacitidine- and decitabine-resistant MOLM-13 while cytarabine caused no effect on the cell growth and the expression of nucleolin of the same resistant cell lines. Injection of SJP1604 led to tumor regression and improved survival rate in MOLM-13 xenograft mouse model and C1498 syngeneic mouse model. In ex vivo study, we figured out that the expression level of nucleolin is highly up-regulated in AML patient-derived bone marrow cells and the level is much higher in relapsed/refractory (R/R) AML patient-derived bone marrow cells. Moreover, SJP1604 also significantly reduced the colony forming unit (CFU) of R/R AML patient-derived bone marrow cells compared to that of AS1411 treatment. In conclusion, these findings suggest that SJP1604 could be developed as a first-in-class drug for novel targeted therapy of AML/MDS with less drug toxicity as well as as a remarkable orphan drug for overcoming drug resistance of AML/MDS in which distinctive solutions do not exist up until recently.

#345

**Novel RAS inhibitor, MCI-062, potently and selectively inhibits the growth of** KRAS **mutant pancreatic tumor cells by blocking GTP loading of RAS.**

Tyler E. Mattox, Xi Chen, Jacob Valiyaveettil, Yulia Maxuitenko, Bing Zhu, Antonio Ward, Veronica Ramirez-Alcantara, Kristy Berry, Michael Boyd, Adam Keeton, Gary A. Piazza. _USA Mitchell Cancer Institute, Mobile, AL_.

Oncogenic mutations in RAS genes result in an intracellular elevation of active RAS and increased cellular propagation through downstream signaling pathways responsible for tumor cell growth and survival. Mutations in KRAS drive over 90% of pancreatic ductal adenocarcinomas (PDACs), presenting promising therapeutic potential for a RAS inhibitor. However, many have deemed RAS "undruggable" as a result of its relatively smooth structure and its high affinity for guanine nucleotide substrates. We have identified a novel series of indene derivatives that potently and selectively inhibits the growth of tumor cells with high levels of active RAS, while having minimal effects on tumor cells lacking constitutively active RAS, or cells derived from normal tissues. Here we report that our lead compound, MCI-062, potently and selectively inhibits the growth of KRAS mutant MIA PaCa-2 PDAC cells harboring oncogenic KRAS with an IC50 value of approximately 5 nM and greater than 300-fold selectivity over BxPC-3 PDAC cells that lack constitutively active RAS. MCI-062 also completely inhibits colony formation of a panel of PDAC cell lines with various KRAS mutations at low nanomolar concentrations. MCI-062 treatment of MIA PaCa-2 cells in basal and EGF-stimulated conditions depletes RAS-GTP levels in a dose-dependent manner. Consequently, MCI-062 treatment leads to inhibition of downstream MAPK and AKT signaling, cell cycle arrest, and induction of apoptosis in MIA PaCa-2 cells. Further investigation of the mechanism of action in cell-free systems demonstrated that this class of compounds inhibits GTP loading of RAS. MCI-062 inhibits binding of MANT-GTP to recombinant K-RAS in a dose-dependent manner. In a functional assay, MCI-062 reduces RAS-RAF-RBD binding when recombinant K-RAS is treated in a nucleotide-free state, but not when K-RAS is treated in a nucleotide-bound state. Testing in 3D spheroid models involving MIA PaCA-2 tumor cells revealed that MCI-062 potently inhibits growth of non-adherent cells. MCI-062 also has anti-tumor activity in a KRAS mutant CT26 mouse tumor xenograft model with no discernable toxicity. Western blotting of tumor lysates from mice treated with MCI-062 showed a reduction of active, GTP-bound RAS when compared to tumor lysates from control mice. These results provide in vitro and in vivo evidence that MCI-062 inhibits RAS-driven tumor cell growth by blocking GTP loading of RAS, supporting further evaluation of this novel class of RAS inhibitors for the treatment of pancreatic cancer, as well as other RAS-driven cancers. Funding provided by NCI grants R01CA131378, R01CA148817, R01CA197147, and R01CA155638.

#346

E2F8 induces proliferation and invasion through the epithelial-mesenchymal transition and notch signaling pathways in ovarian cancer.

Kyung Jin Eoh,1 Jong Woo Lee,2 Young Tae Kim,1 Peter Jaseok Koo2. 1 _Yonsei University Health System, Seoul, Republic of Korea;_ 2 _Yale Cancer Center, New Haven, CT_.

Purpose: Recent research has investigated the role of E2F8 in cancer progression. However, whether the progression of ovarian cancer involves E2F8 remains to be elucidated. In this study, the bio-functional consequences of E2F8 knockdown in vitro and in vivo were explored. Experimental

Design: E2F8 expression was compared between ovarian cancer and non-cancer tissues, and the association between E2F8 expression and progression-free survival in ovarian cancer patients was analyzed. To demonstrate the function of E2F8 in cell proliferation, migration, and invasion, RNA interference was employed to suppress E2F8 expression in ovarian cancer cell lines. Finally, the effect of E2F8-knockdown was investigated in an ovarian cancer xenograft mouse model.

Results: Ovarian cancer tissue exhibited significantly higher E2F8 expression than normal ovarian tissue. Clinical data confirmed that E2F8 was a significant predictor of progression-free survival, and ovarian cancer patients with high E2F8 expression exhibited poorer prognosis than patients with low E2F8 expression. In vitro experiments using E2F8-knockdown ovarian cancer cell lines showed that E2F8 knockdown inhibited cell proliferation, migration, and tumor invasion. Additionally, E2F8 was a potent inducer and modulator of the expression of epithelial-mesenchymal transition (EMT) and Notch signaling pathway-related markers. We confirmed the function of E2F8 in vivo, demonstrating that E2F8-knockdown was significantly correlated with reduced tumor size and weight.

Conclusions: In conclusion, our study suggests that E2F8 is highly correlated with ovarian cancer progression; hence, E2F8 may be a prognostic marker and therapeutic target for ovarian malignancy.

#347

Identification of poziotinib alone or in combination with TDM1 as a pan-HER2 inhibitor.

Jacqulyne P. Robichaux, Monique B. Nilsson, Fahao Zhang, Limei Hu, Junqin He, Marlese Pisegna, Alissa Poteete, John V. Heymach. _UT MD Anderson Cancer Ctr., Houston, TX_.

Clinical studies of HER2 targeting agents have shown mutational variant & cancer-specific differences in patient outcomes. Furthermore, the drug sensitivity profiles of HER2 mutational variants have not been fully characterized. To this end, we expressed the 16 most frequently detected HER2 mutations across exons 19-21 into Ba/F3 cells & determined activating potential & drug sensitivity to 13 HER1/2 targeting agents including afatinib, neratinib, dacomitinib, tarloxotinib-TKI, poziotinib, pyrotinib, TDM-1, & trastuzumab. Third generation EGFR TKIs, osimertinib, ibrutinib, & nazartinib were not effective at inhibiting cell viability in cells expressing exon 20 mutations; however, 3rd generation TKIs demonstrated activity against cells expressing D769 exon 19 variants & exon 21 variants. By comparison, covalent, quinazoline-based TKIs, afatinib, neratinib, dacomitinib, tarloxotinib-TKI, & poziotinib, differentially inhibited HER2 mutants across all three exons. Across all HER2 mutation variants & TKIs tested, poziotinib had the lowest average IC50 (1.73nM) & was significantly more effective in reducing cell viability than neratinib & tarloxotinib-TKI (p<0.001 & p=0.018). In addition, while poziotinib was more efficacious than either neratinib or tarloxotinib-TKI against HER2 exon 19 & 20 mutations, there was no significant difference in average IC50 for exon 21 mutants, suggesting that mutation location impacts drug binding. Furthermore, within exon 19, L755S & L755P variants had significant differences in drug sensitivity across all TKIs tested, & L755P & exon 20 insertion mutations were refractory to most inhibitors, but remained sensitive to poziotinib. We next tested the anti-tumor activity of HER2 targeting agents in human breast (MCF10A), colorectal (CW-2), & kidney (TUHR14TKB) cell lines with HER2 mutations in exons 19-20 & observed that poziotinib was the most potent inhibitor of the 13 TKIs tested. Furthermore, in a CW-2 xenograft model, poziotinib treatment resulted in a 58% reduction in tumor volume compared to the control group (p=0.011). In comparison, neratinib & afatinib treatment did not reduce tumor growth compared to control animals. Lastly, in a HER2 mutant NSCLC PDX model (HER2 Y772dupYVMA), the combination of low dose poziotinib (2.5mpk) & a single dose of TDM1 (10mpk) resulted in complete tumor regression in 9/9 mice, compared to 2/9 mice receiving TDM1 alone or 0/9 mice receiving low dose poziotinib (p<0.0001). In conclusion, HER2 exon 20 insertions & L755P mutations are resistant to the majority of HER2 TKIs, but remain sensitive to poziotinib. Combination of low dose poziotinib & TDM1 caused complete tumor regression in a HER2 exon 20 mutant PDX model. These data suggest that poziotinib alone & in combination with TDM1 has activity against the most common HER2 variants across diverse malignancies & that clinical studies testing these agents in HER2 mutant cancers are warranted.

#348

ABI-009 (nab-Sirolimus) improves tumor accumulation and antitumor activity over oral mTOR inhibitors.

Shihe Hou, Anita Schmid, Neil Desai. _Aadi Bioscience, Inc., Pacific Palisades, CA_.

Background: mTOR pathway has been implicated in cell survival and proliferation and is an attractive target for cancer therapy. ABI-009 (nab-sirolimus) is an injectable nanoparticle form of human albumin-bound sirolimus with a mean particle size of approximately 100 nm developed with a proprietary nanoparticle albumin-bound (nab®) technology. ABI-009 is currently in phase 1 and 2 clinical studies for the treatment of multiple cancer types, including bladder cancer, soft-tissue sarcomas, neuroendocrine tumors, colorectal cancer, glioblastoma, and various childhood cancers, and a registrational phase 2 study for malignant perivascular epithelioid cell carcinoma (PEComa). In nonclinical and clinical studies, ABI-009 demonstrated a PK profile distinct from oral mTOR inhibitors. This study compared the antitumor activity, tissue penetration, and pharmacodynamics of ABI-009 with equal doses of oral mTOR inhibitors at clinically relevant doses in a mouse tumor xenograft model.

Methods: Athymic mice bearing subcutaneous UMUC3 bladder cancer xenografts were treated with saline, ABI-009 (IV, 7.5 mg/kg, 2x/wk), sirolimus and everolimus (PO, 3 mg/kg/day, 5 days/wk). Body weights, tumor measurements, and clinical signs were recorded 3x/wk for up to 5 wks. Tumor and blood sirolimus concentrations were also obtained (tumor: D1 1h and 24h, D4 1h, D7; blood: D4 1h, D7). Markers for mTOR pathway activation (pS6, p4EBP1) will also be analyzed.

Results: Overall, all treatments were well tolerated with no significant body weight loss in any group. ABI-009 IV resulted in significantly higher sirolimus concentrations in the tumor compared with oral mTOR inhibitors at all time points tested. Tumor AUC with ABI-009 IV over 1 week were significantly higher vs equal weekly dose of oral sirolimus (43 fold) and everolimus (12 fold) (p < 0.0001). The enhanced tumor drug delivery by ABI-009 corresponded with significantly greater tumor growth inhibition (TGI: ABI-009 69.6% vs oral sirolimus 24.3%, p < 0.00001; vs oral everolimus 36.2%, p = 0.0023) and longer animal survival (median OS: ABI-009 not reached vs oral sirolimus 21 days, p < 0.05; vs oral everolimus 19 days, p < 0.05). mTOR pathway inhibition in tissues is being assessed.

Conclusions: Compared with equal weekly dose of oral mTOR inhibitors, ABI-009 at clinically relevant dose of administered IV demonstrated significantly greater tumor drug delivery, stronger antitumor activity, and prolonged animal survival. These advantages observed in this nonclinical study support the clinical investigation of ABI-009 in numerous oncology indications.

#349

Oncolytic reovirus variants induce direct oncolysis in human prostate cancer.

Arjanneke F. van de Merbel,1 Geertje van der Horst,1 Maaike H. van der Mark,1 Diana J. van den Wollenberg,1 Jack A. Schalken,2 Anne Collins,3 Norman J. Maitland,3 Rob C. Hoeben,1 Gabri van der Pluijm1. 1 _Leiden University Medical Center, Leiden, Netherlands;_ 2 _Radboud University Medical Center, Nijmegen, Netherlands;_ 3 _University of York, York, United Kingdom_.

The acquisition of therapy resistance and the formation of distant metastases represent major problems in prostate cancer. To date, immunotherapeutic approaches have been relatively disappointing in the treatment of prostate cancer. Therapies based on oncolytic viruses are being exploited for several tumor entities and have been studied in multiple clinical trials. Mammalian orthoreovirus is a non-enveloped double stranded RNA virus that has not been linked to serious disease in humans. Reovirus is relatively safe to use and several clinical trials are currently ongoing to study the oncolytic effect of reovirus in various tumor types. Type 3 Dearing (T3D) is a promising candidate for the use as an oncolytic agent. Besides wildtype T3D reovirus (R124), spontaneous mutants Jin1, 2 and 3 were generated which harbor mutations in spike protein Sigma-1. In this way, Jin mutants are able to infect cells independently of the Junction Adhesion Molecule-A (JAM-A) entry receptor on the tumor cell surface, which is often downregulated in different types of cancer.

In this study, the direct oncolytic effect of wildtype (R124) and Jin mutant reovirus was examined in human prostate cancer cells in vitro and in our 'near-patient' model of ex vivo cultured human prostate cancer tissue slices. Both R124 and Jin3 dose-dependently induced prostate cancer cell death in PC-3M-Pro4luc2, DU145 and 22Rv1 cells in vitro. Interestingly, JAM-A negative cancer cells were insensitive for R124, but were effectively killed by Jin1 mutant, indicating the beneficial effects of the Jin mutant in JAM-A low/negative tumors. Moreover, tumor tissue slices were generated from explanted patient prostate cancer tissues and patient-derived xenografts (PDX) and cultured with reovirus. Oncolytic effects were observed in ex vivo cultured tumor slices upon viral replication and subsequent stimulation of apoptosis. Therefore, ex vivo culture of patient-derived tumor tissue allows the assessment of individual anti-tumor response of the oncolytic virotherapy (i.e. personalized therapeutics). Taken together, our results indicate that both wildtype R124 virus and mutant Jin reovirus can infect and replicate in human prostate cancer cells and patient-derived prostate cancer tissues. The Jin mutants are of particular interest in JAM-A negative tumors. The indirect immunomodulatory effects of reovirus in human prostate cancer are currently ongoing.

In conclusion, our selected oncolytic reoviruses may represent a promising novel treatment strategy in human prostate cancer alone or combined with immunostimulatory agents.

#350

Investigating the mechanisms of cellular uptake and metabolism of ICT2588, an MT-MMP-activated prodrug.

Francis Mprah Barnieh, Amanda D. Race, Steven D. Shnyder, Paul M. Loadman, Robert A. Falconer. _University of Bradford, Bradford, United Kingdom_.

ICT2588 is a non-toxic MT1-MMP-activated peptide prodrug of the potent vascular disrupting agent (VDA) azademethylcolchicine [1]. ICT2588 produces significant tumor-selective delivery (≥10-fold) of the toxic and potent azademethylcolchicine, as compared to the administration of azademethylcolchicine alone, in murine models of human colorectal, breast, lung and prostate cancers. In addition, ICT2588 exhibited reduced potential for toxicity, and cardiotoxicity in particular (a phenomenon associated with most VDAs) as a result of its metabolic stability in plasma and normal tissues [2]. ICT2588 is progressing towards clinical evaluation in the UK in 2019. In this study we aim to further investigate the cellular uptake and metabolism of ICT2588 and related compounds in MT1-MMP positive (HT1080) and MT1-MMP negative (MCF-7) cells in vitro. ICT2588 and related compounds were synthesized in-house and purified by preparative HPLC in excellent yield and purity. Activation of ICT2588 by recombinant MT1-MMP and its preferential metabolism in MT1-MMP positive tumor xenograft homogenates has been shown previously [1]. We are currently studying the mechanism of cellular uptake and metabolism of these peptide prodrugs by incubating these compounds in HT1080 and MCF-7 cell cultures. Cellular concentrations of ICT2588 and all metabolites identified were determined using RP-HPLC, mass spectrometry and immunofluorescent microscopy. We describe the first data on the cellular mechanism of uptake and metabolism of ICT2588 and related peptide prodrugs in cancer cells. Our findings have important implications in the future design of effective peptide-based tumor-targeted prodrugs. Reference: [1] Atkinson, J.M et al "Development of a tumor-targeted vascular disrupting agent activated by Membrane-type Matrix Metalloproteinases (MT-MMPs)." Cancer Research, 2010, 70, 6902-12. [2] Gill, J.H et al "Tumor-targeted prodrug ICT2588 demonstrates therapeutic activity against solid tumors and reduced potential for cardiovascular toxicity". Mol Pharm. 2014,11:1294-300.

#351

A targeted nanocomplex of the IKBKE siRNA inhibits invasiveness and growth of triple negative breast cancer cells.

Zhen Zhao, Yuanke Li, Kun Cheng. _University of Missouri-Kansas City, Kansas City, MO_.

IκB kinase ε (IKBKE, or known as IKKε), an important mediator in the activation of NF-κB pathway, was recently identified as an oncogene in breast cancer and overexpressed in approximately 30% of breast carcinomas. It is also highly expressed in TNBC cells, which is correlated with angiogenesis process, high metastasis in NF-κB pathway of TNBC. We found that silencing IKBKE in TNBC cells using siRNA significantly inhibits the proliferation, migration and invasion of TNBC cells by specific silenced 71% of IKBKE in vitro. In vivo study indicated that IKBKE siRNA can inhibit TNBC tumor growth after peritumoral injection at a IKBKE siRNA dose of 0.3 mg/kg. We next developed a CD44-targeting, cholesterol-peptide based nanocomplex to co-deliver the IKBKE siRNA and cabazitaxel to TNBC cells. The nanocomplex showed the synergistic effect of the IKBKE siRNA and cabazitaxel on inhibiting the growth of TNBC tumors in vivo. Modification of the nanocomplex with CD44 further improved tumor uptake and anti-tumor efficacy of the nanocomplex. Our results suggest that IKBKE siRNA is a promising anti-tumor agent for TNBC therapy, and co-delivery of IKBKE siRNA and cabazitaxel using a CD44-targeting nanocomplex is a potential strategy for TNBC treatment. Moreover, this multifunctional delivery system provides a platform for other combination therapy including an siRNA and a chemotherapy drug.

#352

The effect of anti-TNF-alpha Ab in AOM/DSS-induced colitic cancer mouse model.

Shinobu Ohnuma,1 Wang Dang Yang,1 Hideyuki Suzuki,1 Keigo Kanehara,1 Hideaki Karasawa,1 Atsushi Kohyama,1 Kazuhiro Watanabe,1 Koh Miura,2 Takashi Kamei,1 Takeshi Naitoh,1 Michiaki Unno1. 1 _Tohoku Univ. Hospital, Sendai, Japan;_ 2 _Miyagi Cancer Center, Sendai, Japan_.

Aim: To reveal whether anti TNF-α antibody (Ab) induces colon carcinogenesis or not in colitic cancer models.

Methods: The effects of TNF-α and anti TNF-α Ab were analyzed with cell-proliferation, migration, and invasion assays in human colon cancer cell lines, in vitro. Then, the effects of anti TNF-α Ab, infliximab (10 mg/kg, i.p.) were investigated with drug-induced colitis-associated colon cancer model by azoxymethane (AOM: 10 mg/kg, i.p.)/dextran sodium sulfate (DSS: 2.5%, oral) in vivo.

Results: 50 ng/ml of TNF-α inhibited cell proliferation, migration, and invasion of HCT8 and COLO205, however, anti TNF-α Ab offset the TNF-α mediated-inhibition in vitro. Infliximab significantly attenuated the development of colon cancers in AOM/DSS treated mice. The microarray analyses revealed that mast cell related genes (mast cell protease1, mast cell protease 2 , chymase 1, etc) were down-regulated in cancer tissues of the infliximab-treated AOM/DSS mice. The results of microarray were validated by real-time RT-PCR. Finally, the number of mast cells were also reduced in those mice.

Conclusions: Mast cell may have a pivotal role in the development of colitic cancer. Anti TNF-α Ab may prevent colitic cancer in the patients with inflammatory bowel diseases.

#353

A novel molecule with profound tumor killing activity.

Ling Liu, Aaron D. Wrobleski, Yin Yin, Wei Zeng, Xianming Chen, David J. Stokell, Sheng-bin Peng, Amita Datta-Mannan, Gregory P. Donoho, Philip W. Iversen, Philip Hipskind, Yiqing Feng. _Eli Lilly and Company, Indianapolis, IN_.

LY3343544: A novel MET antibody drug conjugate that shows profound pre-clinical in vivo anti-tumor activities, irrespective of MET pathway dependence

MET is over-expressed in many types of human tumors. Due to the heterogeneity of human tumors, MET antibodies or small molecule inhibitors have benefited only small subsets of patients with tumors driven by signaling through the c-Met pathway. The patient selection strategies to identify those tumors with MET activation dependence are helpful in predicting sensitivity to many of these inhibitors. It was reported previously that Lilly's MET antibody, emibetuzumab, showed clinical activity in selective NSCLC patients with high MET IHC staining (90% to 100% 3+ positive) when it was combined with erlotinib in Phase II clinical Trials. In searching for a better treatment for patients carrying the MET overexpression tumors regardless other co-existing mutations, we developed LY3343544, a novel antibody drug conjugate (ADC) molecule that consists of emibetuzumab conjugated with the potent microtubule inhibitor MMAE using a unique lysine conjugation approach. Upon binding to MET, LY3343544 is internalized via receptor-mediated endocytosis. LY3343544 maintains the similar binding and internalization activities to the cell surface MET as compared to emibetuzumab in the competitive cell binding assay and the internalization assay. We reported here that LY3343544 showed profound anti-tumor activity in a preclinical mouse models, and overcome intrinsic resistance mechanisms including KRAS, BRAF, PI3K and TP53 mutations. LY3343544 kills tumor cells expressing a wide range of MET levels on the cell surface and is capable of killing a variety of MET-overexpressing tumor cells including pancreatic, cholanglocarcinoma, colorectal, NSCLC, gastric, head and neck tumor cells in vitro. In contrast, LY3343544 does not kill human normal endothelial cells and normal epithelial cells, no activity on human peripheral blood mononuclear cells with or without activation as well as in cell-based assays. Moreover, LY3343544 is more stable in rodent PK studies than typical inter chain Cys VC-MMAE conjugates and showed tumor regressions in colorectal, NSCLC, gastric and pancreatic mouse xenograft models. Furthermore, LY3343544 shows profound tumor regression in >50% of PDAC PDX models (n=40): 20% complete response (CR); 22.5% partial response (PR); and 17.5% stable disease (SD); overall disease control rate (DCR) is 60%. In addition, LY3343544 shows tumor growth inhibition in cholangiocarcinoma PDX model that is resistant to emibetuzumab. In summary, LY3343544 is highly potent in killing a variety of tumor cells in cell-based killing assays. It demonstrated good stability in vivo and profound anti-tumor efficacy in multiple mouse xenograft models and patient-derived xenograft models thus is a promising agent to treat many types of cancers.

#354

BBI-608 modulates stemness, angiogenesis and enhances the efficacy of chemoradiotherapy in pre-clinical models of pancreatic cancer.

Ganji Purnachandra Nagaraju, Matthew R. Farren, Sneha Govardhanagiri, Shipra Reddy Bethi, Batoul Farran, Gregory B. Lesinski, Bassel F. El-Rayes. _Emory Univ., Atlanta, GA_.

Background: The transcription factor signal transducer and activator of transcription 3 (STAT-3) is constitutively activated in pancreatic ductal adenocarcinoma (PDAC) and signaling via this pathway regulates a diverse set of cellular processes including stemness, angiogenesis and sensitization to chemo or radiotherapy. Given the potential for BBI608 to elicit effects on multiple oncogenic cellular pathways including STAT-3, we hypothesized that BBI608 can sensitize pancreatic cell lines to the effects of 5FU and ionizing radiation.

Methods: The combined effects of BBI-608 and chemoradiotherapy (5-FU + IR) were evaluated in human (MIA PaCa-2, PANC-1) and murine (PANC-02) PDAC cell lines using a clonogenic assay. Effects on the expression of cell surface markers associated with stemness (EGFR, CD24, and CD44) in MIA PaCa-2 cell lines in vitro and in vivo were examined by flow cytometry. Modulation of pSTAT3 and VEGF, key pro-angiogenic factors, were studied via Western blot. Using media from MIA PaCa-2 and PANC-1 cell lines following treatment, egg CAM assays were also performed to evaluate the effects of the tested drugs on angiogenesis (quantification was performed by AngioQuant software). The MIA PaCa-2 cell line was tested for activity to BBI-608, 5-FU + IR, alone and in combination, using an in vivo tumor xenograft model.

Results: The combination of BBI-608 and chemoradiotherapy significantly (p<0.001) decreased colony formation, stemness and angiogenesis in PDAC cell lines as compared to chemoradiotherapy alone. BBI-608 and chemoradiotherapy also decreased pSTAT-3 and VEGF expression. Flow cytometric analysis revealed that combined treatment of MIA PaCa-2 cell lines with BBI-608 and chemoradiotherapy significantly (p<0.001) reduced the expression of stemness markers (EGFR, CD44 and CD24) in vitro and in vivo. In animal models (MIA PaCa-2), BBI-608 potentiated the effects of chemoradiotherapy, as measured by tumor volume. There was no evidence of systemic toxicity or loss of body weight in any of the treatment groups, indicating the combination was well-tolerated. Matrigel plug implantation results revealed that BBI-608 and chemoradiotherapy significantly (p<0.0001) decreased blood vessel formation as compared to chemoradiotherapy alone, with similar results seen in the egg CAM assay.

Conclusion: These observations provide preclinical proof-of-principle that combinatorial BBI-608 and chemoradiotherapy could be a promising therapeutic strategy for PDAC.

#355

A novel anti-HER2 antibody that displays superior synergetic effects in Trastuzumab mediated HER2-overexpressing tumor arrest.

Haomin Huang, xuesai zhang, jianhe Chen, Le Zhao, qingrou Li, zhenping zhu. _3sBio, shanghai, China_.

The majority of patients with metastatic breast cancer on Trastuzumab, an anti-HER2 monoclonal antibody, generally develop resistance to the drug within a year after initiation of the treatment. Therefore, there is an unmet need for a novel agent to treat HER2 overexpressing breast cancers. Here we described a novel anti-HER2 humanized monoclonal antibody, 19H6-hu, which binds to HER2 ECD with high affinity and could inhibit the proliferation of multiple HER2-overexpressing cancer cell lines as a single agent, though, to a lesser degree when compared to Trastuzumab alone. Interestingly, when in combination with Trastuzumab, 19H6-hu exhibited much stronger potency in inhibition of BT474, N87 and SKBR3 cell growth relative to that of Perjeta in combination with Trastuzumab as well as in suppression of tumor growth in vivo when compared to Trastuzumab alone. Alanine scanning demonstrated that the antibody binds to an alpha-helix located within the domain III and in proximity to the domain IV of HER2 ECD, which is different from the known binding sites of Trastuzumab and Perjeta. Western blotting further confirmed that 19H6-hu in combination with Trastuzumab is more efficacious in blocking phosphorylation of ERK and phosphorylation of HER2 at Y1248 when compared to Trastuzumab in combination with Perjeta or applied alone. Thereby, our results highlight the functional variability of HER2 domains and provide a new insight into the design of HER2-targeting agents.

#356

Novel conditionally active biologic (CAB) antibody targeting EpCAM demonstrates anti-tumor efficacy in vivo.

Cathy Chang, Gerhard Frey, Leslie Sharp, William (Bill) J. Boyle, Jing Wang, Charles Xing, Haizhen Liu, Christina Wheeler, Marlena Walls, Jay M. Short. _BioAtla LLC, San Diego, CA_.

Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein mediating Ca2+-independent homotypic cell-cell adhesion during cell signaling, migration, proliferation, and differentiation. EpCAM is expressed at high levels exclusively in epithelia and epithelial-derived neoplasms, making it a suitable target for many important solid tumor types and cancer stem cells. EpCam expression on normal tissues limits its utility as target for therapeutic antibodies and ADCs due to the potential effects on normal epithelial throughout the body. Conditionally Active Biologics (CAB) technology is a proprietary platform that selects antibodies that bind to target antigens in the context of diseased tissues, but not normal tissues, by taking advantage of the unique conditions in the tumor microenvironment. Using our CAB technology, we have developed CAB antibodies to EpCAM that reversibly bind to recombinant EpCAM and EpCAM expressing cells under select conditions that are present in the tumor microenvironment but not in normal tissues. In vitro and in vivo efficacy data for several anti-EpCAM antibodies and ADCs will be presented and suggest that conditionally active EpCam ADCs generated using the CAB technology will provide drug candidates that have an increased safety margin and therapeutic index in the clinic.

#357

Pan-HER, an antibody mixture simultaneously targeting EGFR, HER2, and HER3, is highly effective in triple negative breast cancer patient-derived xenografts.

Roberto R. Rosato,1 Don S. Choi,1 Wei Qian,1 Wen Chen,1 Johan Lantto,2 Ivan D. Horak,2 Michael Kragh,2 Jenny C. Chang1. 1 _Houston Methodist Research Institute, Houston, TX;_ 2 _Symphogen A/S, Ballerup, Denmark_.

As a heterogeneous disease, breast cancer is clinically classified taking into account the expression of estrogen receptor alpha (ERα), progesterone (PR), and the presence/amplification status of the oncogenic human epidermal growth factor receptor 2 (HER2). Triple negative breast cancer (TNBC) represents ~10-20% of all cases, and displays poor prognosis and aggressive clinical course. EGFR is more often overexpressed in TNBC than in other breast cancer subtypes, providing cancer cells with compensatory signals that greatly contribute to the development of resistance and survival in response to therapy. This characteristic has made HER family members potentially important therapeutic targets. The present study was designed to investigate the effects of Pan-HER, a novel mixture of six monoclonal antibodies directed against members of the human epidermal growth factor receptor (HER) family EGFR, HER2, and HER3, in a preclinical trial of TNBC patient-derived xenografts (PDXs). Fifteen low passage TNBC PDX tumor samples were transferred and engrafted into the right mammary fat pad of mice for engraftment. When tumors reached an average size of 150-200 mm3, mice were randomized (n ≥ 3 per group), and treated following three, one-week cycles, consisting of 3 times/week intraperitoneally (IP) injection of either formulation buffer (Vehicle control) or Pan-HER (50 mg/kg). At the end of treatment, tumors were collected for western blot, RNA and immunohistochemistry analyses. All 15 TNBC PDXs were responsive to Pan-HER treatment, showing significant reductions in tumor growth consistent with Pan-HER-mediated tumor down-modulation of EGFR and HER3 protein levels and significantly decreased activation of associated HER family signaling pathways including AKT, ERK and NF-κB. Tumor regression was observed in five of the models, which corresponded to those PDX tumor models with the highest level of HER family activation. This observation may provide a plausible explanation for the response of TNBC tumors to Pan-HER as an indication of the potential use of EGFR and associated signaling expression as biomarkers for selecting patients that may benefit of targeting these proteins. In summary, this preclinical TNBC PDX trial with Pan-HER provides strong justification for further biomarker-guided studies in TNBC. The finding that tumors were affected rapidly and effectively, with long-lasting results, offers exciting perspectives to treat this aggressive form of breast cancer, for which available treatment options are currently limited.

#358

Inhibition of AKT phosphorylation in acute myeloid leukemia by ISC 4.

Shreya Thakur,1 Charyguly Annageldiyev,2 Srinivasa Ramisetti,3 Trupti Patel,4 Saumya Iyer,3 Pavan Dhanyamraju,3 Shantu Amiin,3 Arun Sharma,3 David Claxton,2 Arati Sharma2. 1 _University of Pittsburg, Pittsburgh, PA;_ 2 _Penn State Cancer Institute, Hershey, PA;_ 3 _Pennsylvania State University College of Medicine, Hershey, PA;_ 4 _VIT, Vellore, India_.

Acute myeloid leukemia (AML) is a heterogeneous disease with percent remission of 35-40% for younger patients and 5-15% for older patients and a survival of 5-10 months if left untreated. This emphasizes the urgent unmet need for new therapeutics in AML. The activation of PI3K/AKT signaling pathway plays an important role in cellular survival, proliferation and regulation of apoptosis in cancer cells including leukemia. Recent studies showed that activation of AKT may correlate with poor prognosis in AML. Furthermore, mutations such as FLT3-ITD in the PI3K-AKT pathway causing constitutive activation of AKT, are very common in AML, therefore, it remains as an important target for AML treatment. Phenylbutyl isoselenocyanate (ISC-4), a small drug-like molecule that is developed by our group through optimization of naturally occurring isothiocyanates, targets the P13K-AKT pathway in melanoma and other gastrointestinal malignancies. In this study, we demonstrated potent activity of ISC-4 against AML. We tested the cytotoxicity of ISC-4 in various human AML cell lines (n=6) and in samples collected from AML patients (n=8). AML cell lines exhibited dose-dependent sensitivity to ISC-4 in the low micromolar range (IC50: 2-5 uM) as did most primary AML cases. Western blotting showed a dose dependent decrease in AKT phosphorylation in MV-4-11 and AML-OCI3 cell lines, and lead to an increase in cleaved PARP levels, indicating the induction of apoptosis. Furthermore, ISC-4 treatment reduced the clonogenicity of primary AML cells at relatively low concentrations. Future studies will aim to further develop ISC-4 as a single agent and in combination with anti-leukemic agents and demonstrate the preclinical efficacy in AML animal models including AML patient derived xenografts (PDXs). Together, these data demonstrates the therapeutic potential of ISC-4 that can be a promising drug candidate for further development as a novel therapy for AML.

#359

Dual BET/kinase inhibitors as a novel strategy for the treatment of multiple myeloma.

Marilena Tauro, Muhammad Ayaz, Harshani R. Lawrence, Nicholas J. Lawrence, Kenneth H. Shain, Ernst Schonbrunn, Conor C. Lynch. _Moffitt Cancer Center Research Institute, Tampa, FL_.

The bromodomain and extra-terminal (BET) family of proteins are important epigenetic regulators of critical oncogenes, including c-Myc. JQ1 is the most potent BRD4 inhibitor, currently used in several clinical trials and effective in preventing multiple myeloma progression in vivo.

Currently, clinical data is showing that patients often develop acquired resistance, indicating that single agent therapies targeting BRD4 such as JQ1 may not provide durable therapeutic responses. To overcome this limitation, we have developed a new class of dual inhibitors, capable to simultaneously target BRD4 and a panel of tyrosine kinases highly expressed in cancer including JAK2, FLT3, RET, FGFR1, ULK1 and ULK3. A preliminary in vitro screening identified SG3-014 as the lead compound, showing BRD4 and c-Myc inhibition activity similar to JQ1, but additionally, it is capable of inhibiting kinases that are over-activated in cancer and responsible for cytokine-mediated drug resistance pathways.

In vitro assays on MM cell lines reveals higher SG3-014 sensitivity (5TGM1, 0.85μM; U266, 0.99μM) compared to JQ1 (5TGM1, 4.7μM; U266, 16μM). In vivo studies using 1x106 5TGM1-Luc cells demonstrate that SG3-014 treatment (25mg/Kg) significantly contributes to overall survival compared to JQ1 and the vehicle control cohort (n=10/group) (median survival CTRL=40.5; SG3-014=50.5; JQ1=46 days). Post-study analyses demonstrated a significant reduction in myeloma induced bone disease in the SG3-014 treated mice as measured by X-ray/ μCT/ histomorphometry.

Clinical datasets identified ULK3 as the candidate kinase of SG3-014 targeting activity and correlated its expression with MM disease stages. Since ULK3 is involved in cellular senescence and autophagy regulation, a key mechanism by which several cancer cell lines protect themselves against apoptosis and become resistant to standard treatments, we explored the role of SG3-014 inhibition in the autophagy pathway.

Preliminary data confirm that SG3-014 is an autophagy inhibitor and, as opposed to JQ1, downregulates ATG proteins level, leading to dysfunctional autophagosome formation. We noted that within 6 h, SG3-014 completely shut down autophagy with decrease levels of ULK1, beclin-1, ATG16L-1, ATG12, ATG3, p62 and increased LC3I/II ratios (additionally confirmed by microscopy and flow cytometry). These effects were not noted with JQ1. Further investigation is clarifying the mechanism by which SG3-014 inhibitory activity on ULK3 is responsible for autophagy mediated cancer cell death.

In order to validate this target in the progression of the human disease, our future directions will analyze ex vivo multiple myeloma patient samples and examine the efficacy of the inhibitor in CD138 isolated multiple myeloma cells derived from patient biopsies.

We propose dual BRD4/autophagy inhibition as a resilient strategy to prevent multiple myeloma by-pass of single targeted therapies.

#360

Therapeutic potential of EC-18 as a chemotherapy adjuvant for 5-fluorouracil-induced neutropenia.

Yong-Jae Kim,1 Jinseon Jeong,2 Ki-Young Sohn,1 Do Young Lee,1 Sun Young Yoon,1 Jae Wha Kim2. 1 _Enzychem Lifesciences, Jecheon-si, Republic of Korea;_ 2 _Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea_.

Chemotherapy-induced neutropenia (CIN) is a complication that arises during cancer treatment and necessitates dose reduction. Preventing CIN and maintaining absolute neutrophil counts (ANC) is critical for successful chemotherapy because a rapid decline of neutrophils increases susceptibility to infection. Here, we investigated whether administration of EC-18 has therapeutic effects on the treatment of CIN in 5-fluorouracil (5-FU)-induced neutropenia mouse model. A single injection of 5-FU 100mg/kg reduced the ANC in the control, EC-18 125 and EC-18 250mg/kg-treated cohort from pre-injection values to <500 cells/μL by 5.2±0.45, 5.8±0.45 and 5.8±0.45 days, respectively. The administration of EC-18 in 5-FU-injected mice resulted in significant reduction in the duration of neutropenia and the time to recovery of ANC >1000 cells/μL. EC-18 125 or 250mg/kg significantly reduced the duration of neutropenia from 7.4±1.14 days to 2.6±0.55 or 3.0±0.71 days, respectively. Moreover, the ANC of all individuals in the control cohort fell to severely neutropenic range (ANC <100 cells/μL), while only 20% of individuals in both EC-18 125 and 250mg/kg-treated cohorts experienced severe neutropenia. EC-18 also reduced the duration of severe neutropenia from 5.2±1.48 days to 2 days. EC-18 125 or 250mg/kg administration significantly increased the mean nadir after 5-FU injection from 2.0±4.47 cells/μL to 236±4.47 or 158±11.32 cells/μL, respectively. The time of recovery to an ANC ≥500 or 1000 cells/ μL was significantly reduced in EC-18 125 and 250 mg/kg-treated cohorts. Besides neutropenia, a single treatment of 5-FU induced the reduction of blood monocytes and eosinophils, similar to the pattern of the decrease of neutrophil counts. The administration of EC-18 125 or 250mg/kg in 5-FU-injected mice remarkably prevented the reduction of blood monocytes and eosinophils. In this study, thrombocytopenia is defined as a 50% or greater reduction in platelet count from baseline, and 2-fold or greater increase of platelet count from baseline for thrombocytosis. 5-FU treatment induced the moderate thrombocytopenia from 4 to 6 days and followed by a more pronounced and prolonged rebound thrombocytosis. EC-18 significantly reduced the extreme change in platelet counts, thus preventing 5-FU-induced thrombocytopenia and thrombocytosis. Moreover, EC-18 effectively prevented a constant reduction of red blood cell (RBC) count induced by 5-FU treatment. Based on the observations in this study, we concluded that EC-18 has therapeutic potential as a chemotherapy adjuvant for the treatment of 5-FU-induced CIN as well as chemotherapy-associated other hematologic disorders.

#361

NT1195, a novel biguanide, exhibits superior uterine penetration and anti-tumorigenic efficacy as compared to metformin/phenformin in endometrial cancer.

Katherine Tucker,1 Allison Staley,1 Yali Fan,1 Xiaoling Zhao,1 Yajie Yin,1 Ziwei Fang,1 Wenchuan Sun,1 Kenneth Batchelor,2 Nick Livingston,2 Chunziao Zhou,1 Victoria Bae-Jump1. 1 _University of North Carolina, Chapel Hill, NC;_ 2 _NovaTarg Therapeutics, Durham, NC_.

Objectives: Obesity and diabetes are associated with increased risk and worse outcomes in endometrial cancer (EC). Anti-diabetic biguanide drugs such as metformin/phenformin may have anti-tumorigenic effects by behaving as AMPK activators. Metformin requires organic cation transporters (OCTs) for entry into cells whereas phenformin is not as reliant on OCTs for cell entry. NT1195 (NovaTarg) is a novel biguanide designed to have greater affinity for the OCT1/3, PMAT and MATE1 transporters. Thus, we compared head-to-head the pharmacokinetic properties and anti-tumorigenic potential of metformin vs phenformin vs NT1195 in a genetically engineered mouse model of endometrioid EC (LKB1fl/flp53fl/fl).

Methods: Pharmacokinetic parameters, including half-life, AUC, bioavailability and uterine concentration were measured in female control mice after oral dosing of metformin, phenformin and NT1195 (5 mg/kg oral for all drugs). AdCre was injected at 6 wks of age to induce invasive EC in LKB1fl/flp53fl/fl mice. Following tumor onset, mice were treated with placebo vs metformin vs phenformin vs NT1195 for 4 wks (50 mg/kg, oral for all drugs).

Results: NT1195 (> 24 hrs) had a longer half-life when compared to metformin (3.7 hrs) and phenformin (7.2 hrs). Drug bioavailability was heightened in the NT1195-treated mice when compared to metformin and phenformin (149 vs 83.5 vs 8.31%, respectively). The AUC (h*kg*ng/ml/mg) for NT1195 was significantly higher at 1143 than that of metformin (26) and phenformin (184). Drug accumulation in uterine tissue at 24 hrs post dose demonstrated markedly increased presence of NT1195 (45.4 ng/g), compared to metformin and phenformin, which yielded levels below the limits of quantification. NT1195 had increased anti-tumorigenic efficacy in LKB1fl/flp53fl/fl mice, inhibiting tumor growth by 88% compared to 78% for metformin and 80% for phenformin (p<0.01).

Conclusions: When compared to metformin/phenformin, NT1196 had superior bioavailability, a longer half-life and greater uterine tissue penetration, culminating in improved efficacy in an EC mouse model. Thus, NT1195 may be a promising novel biguanide therapy in EC, particularly in women who want to avoid hysterectomy and preserve fertility or are poor surgical candidates.

#362

Depolymerized fucosylated chondroitin sulfate in pancreatic cancer, metastasis and cancer-associated thrombosis.

Yiming Yao,1 Kavitha Godugu,2 Yongsheng Jin,1 Yi Jiang,3 SHAKER A. MOUSA2. 1 _Ronnsi Pharma Co., Ltd, bioBay, JiangSu, China;_ 2 _Albany College of Pharmacy, Rensselaer, NY;_ 3 _Suzhou Yihua Biotechnology Co., Ltd, bioBay, JiangSu, China_.

The Depolymerized fucosylated chondroitin sulfate (dHG) prepared from natural fucosylated chondroitin sulfate, which was isolated from sea cucumber, showed remarkable anti-cancer efficacy in suppressing primary tumor growth, tumor angiogensis and metastasis. The anti-metastasis properties were demonstrated in multiple mouse cancer models, including B16F10 melanoma-induced lung metastasis model, 4T1 breast cancer induced lung metastasis model, Luciferase transfected SUIT-2 pancreatic cancer induced lung metastasis model as well as surgical removal SUIT-2 pancreatic tumor induced metastasis. Additionally, dHG demonstrated potent anti-angiogenesis efficacy against various growth factors-mediated angiogenesis. Cancer metastasis is a multiple process and regulated by a complex signaling network. Our In vitro studies demonstrated that dHG is a multiple targets agent. dHG inhibits angiogenesis and P-selectin, both are involved in cancer metastasis. dHG also induces TFPI release, especially TFPI-2 release. TFPI-2 has been recognized as a potent tumor suppressor. It inhibits the migration and invasion of human cancer cells. Low expression of TFPI-2 results in enhanced growth and metastasis of a variety of human tumors. Thrombelastography (TEG) showed that mixing of dHG and 1-10 picogram of TFPI-1 or TFPI-2 generated significant anti-thrombotic effect while dHG itself has very low anticoagulant activity. Thrombosis is a significant problem for cancer patient. dHG potentially inhibits thrombosis and normalizes hemostasis. Our studies demonstrated that dHG is a promising therapeutic agent for metastatic cancer and cancer-associated thrombosis management.

#363

CYT01B, a novel RAD51 inhibitor, acts synergistically with PARP inhibitors.

Tyler Maclay, Melinda Day, Kevin Mills. _Cyteir Therapeutics, Lexington, MA_.

Genomic instability is recognized as a driver of tumorigenesis and cancer progression. Loss of tumor suppressors or activation of oncogenes can induce DNA damage stress, promoting genomic instability and creating dependencies upon key DNA repair pathways. These dependencies can be targeted therapeutically to induce synthetic lethality. We have developed a novel RAD51 inhibitor, CYT01B, which we have previously shown to be preferentially active in Activation Induced Cytidine Deaminase (AID) expressing cells, and in other tumor derived cell lines with elevated levels of DNA damage and/or reduced DNA repair capabilities. CYT01B acts by reducing RAD51 focus formation, and depleting the nuclear pool of RAD51. We have observed anti-tumor effects with CYT01B in preclinical models, but have yet to determine if RAD51 could act as a sensitizer to current therapeutics. A main resistance mechanism to PARP inhibitors is the overexpression of RAD51, making it an attractive potential combination for our RAD51 inhibitor. To determine potential drug synergy, a matrix study was performed with CYT01B (concentration range of 20nM to 5µM) and 5 different PARP inhibitors including olaparib (78nM to 5µM), niraparib (78nM to 5µM), veliparib (780nM to 50µM), rucaparib (156nM to 10µM), and talazoparib (39nM to 2.5µM). The combination matrix was tested in cell lines of varying AID expression and PARP inhibitor sensitivity: ARPE19/HPV16 (HPV immortalized epithelial cell line), KYSE-70 (head and neck cancer cell line), Daudi (Burkitt's Lymphoma cell line), HCC1143 (TNBC), and BT20 (TNBC). We used both the Loewe Additivity model and the Bliss Independence model to determine drug interaction (synergistic, independent, or antagonistic). In general, we observed synergy with all the combinations in the tumor derived cell lines. In the transformed epithelial cell line, synergy was observed only in the olaparib combination. The strength of the synergistic effect differed amongst the PARP inhibitors, with olaparib giving the strongest synergy scores. From this data we hypothesize that the synergistic activity with CYT01B is related to the PARP trapping efficiency of the PARP inhibitors; greater trapping efficiency the greater the synergy. These data suggest that CYT01B may be active as a combinatorial therapy with PARP inhibitors. Overall, we conclude that there is significant potential for combining RAD51 and PARP inhibition in future cancer treatment strategies, and warrants future exploration in vivo.

#364

The dopamine receptor D2 antagonist ONC201 has anti-tumorigenic activity in obesity-driven epithelial ovarian cancer.

Allison Staley, Katherine Tucker, Ziwei Fang, Yali Fan, Wenchuan Sun, Yajie Yin, Yingao zhang, Varun Prabhu, Chunxiao Zhou, Victoria Bae-Jump. _University of North Carolina, Chapel Hill, NC_.

Objectives: ONC201 is an orally bioavailable dopamine receptor D2 antagonist that induces tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and TRAIL has been implicated in both the physiopathology of ovarian cancer (OC) and obesity. In addition, DRD2 is a G protein-coupled receptor that activates the trimeric G-protein complex Ras, modulating downstream metabolic pathways such as Akt-signaling which is highly dysregulated in both obesity and OC. ONC201 has demonstrated anti-tumorigenic activity in solid tumors, including uterine serous carcinoma, without significant toxicity in phase 1/2 trials. Thus, we sought to evaluate the anti-tumorigenic effects of ONC201 in human ovarian cancer (OC) cell lines and a high grade serous OC mouse model (K18-gT121+/-;p53fl/fl;Brca1fl/fl; KpB), using both obese and lean mice.

Methods: The human epithelial OC cell lines (OVCAR3, SKOV3, IGROV, OVCAR433, and OVCAR5) were treated with ONC201 at doses of 0.01 to 250 μM. Cell proliferation and apoptosis were assessed by MTT and Annexin V assays, respectively. Adhesion and invasion were assessed by laminin and wound healing assays, respectively. For our animal study, we utilized the K18-gT121+/-; p53fl/fl;Brca1fl/fl (KpB) high grade serous OC mouse model. KpB mice were fed either a low fat diet (LFD, lean) with 10% calories from fat, or a high fat diet (HFD, obese) with 60% calories from fat in order to mimic diet-induced obesity. Following tumor onset, obese and lean mice were treated with vehicle or ONC201 (130 mg once a week, oral gavage) for 4 weeks (n = 8-10 mice/group).

Results: ONC201 significantly decreased cell proliferation in a dose-dependent manner in all five cell lines, with a range of IC50s between 1 and 10μM. Moreover, ONC201 increased Annexin V expression in a dose-dependent manner in both the SKOV3 and OVCAR5 cell lines (p<0.05). Treatment with ONC201 reduced cell adhesion by 25-30% as well as invasion by 20-25% in both OC cell lines when compared to controls (p<0.05). HFD-fed mice (obese) had tumors that were significantly greater in size as compared to LFD-fed mice (lean) (p<0.05). ONC201 decreased tumor weight/size by 65% in the obese mice and 61% in the lean mice (p<0.01).

Conclusions: ONC201 inhibited cell growth, induced apoptosis and decreased cellular adhesion and invasion in human OC cells. ONC201 was efficacious in reducing ovarian tumor growth in both obese and non-obese KpB mice. These results suggest that ONC201 holds promise as a novel targeted therapy in the treatment of OC.

#365

Indotecan (LMP400), indimitecan (LMP776) and LMP744, a new class of non-camptothecin topoisomerase I inhibitors selective for schlafen11-positive and BRCA-deficient cells that synergize with olaparib.

Laetitia Marzi,1 Ludmila Szabova,1 Zoe Weaver Ohler,1 Shyam Sharan,1 Mike Beshiri,1 Junko Murai,2 Kathy Kelly,1 Yves Pommier1. 1 _NCI-NIH, Bethesda, MD;_ 2 _Keio University, Keio, Japan_.

To overcome the limitations of topoisomerase inhibitors, irinotecan and topotecan (chemical instability, drug efflux substrates, short half-life, dose-limiting bone marrow and gastrointestinal toxicity), we have developed the indenoisoquinolines (LMP400, LMP776 and LMP744). To rationally select patients for phase 2 clinical trials, we have taken two approaches. First, we mined the cancer cell lines genomic databases with CellMinerCDB and found that a dominant response determinant to the indenoisoquinolines is Schlafen 11 (SLFN11), a recently identified executor of cells undergoing replication stress. We validated this finding in isogenic cell lines. Second, because the major lesions generated by the trapping of TOP1 are replication-induced DNA double-strand breaks that are repaired by homologous recombination (HR), we determined whether the indenoisoquinolines exhibit a "synthetic lethality" in cells presenting BRCA1, BRCA2 or PALB2 deficiency. In addition, we tested wether this selectivity could be enhanced when combined with the PARP inhibitor, olaparib. Survival and cell cycle alterations were tested after treatment with the indenoisoquinolines as single agents in isogenic DT40, DLD1 and OVCAR cell lines, with BRCA1, BRCA2 or PALB2 deficiencies, and organoids cultured from patient-derived xenografts with BRCA2 loss, as well as in combination with olaparib. We found that BRCA1-, BRCA2- and PALB2-deficient cells are 3 to 5 times more sensitive to the indenoisoquinolines compared to isogenic cell lines. Moreover, combination treatments showed high synergy between all three indenoisoquinolines and olaparib. We also established the synergy between LMP400 (Indotecan) and olaparib in orthotopic allograft models derived from genetically engineered mouse models for serous epithelial ovarian cancer harboring BRCA1 loss. Better efficacy was observed with the combination over single agent treatments of LMP400 or olaparib. Our results provide a rationale for Phase 2 indenoisoquinoline clinical trials with the indenoisoquinolines in HR-deficient cancers as single agents and in combination with PARP inhibitors, and for measuring Schlafen 11 (SLFN11) as a clinical response determinant.

#366

Arctin and arctigenin as a potential treatment for solid tumors.

Mani Maheshwari,1 Joseph Media,1 Qi Jia,2 Frederick A. Valeriote1. 1 _Henry Ford Health System, Detroit, MI;_ 2 _Unigen Pharmaceuticals, Tacoma, WA_.

Arctigenin (AR) and its glucoside arctiin are two major active ingredients of the lignin-containing plant Arctium lappa. Traditional Chinese Medicine has demonstrated the utility of these lignin-containing compounds for a wide spectrum of activity. These compounds have been shown to possess various biological activities like anti-inflammatory, anti-viral, and anti-cancer activity. Our interest in arctiin/AR relates to their antiproliferative/antitumor activity. The studies conducted in the past have not been structured in a well-defined manner for anti-cancer potential. We focused on the anti-tumor activity of these compounds against a set of human solid tumor cell lines (Pancreatic-PANC-1, colon-H116, lung-H125, liver- HepG2, OVC-5 and brain-U251N). Further, our study is based on our developmental paradigm; determination of the IC50 value; an in vitro clonogenic assessment of the drug as a function of both concentration and duration of exposure; preparation of an intravenous formulation of the drug followed by a determination of the maximum tolerated dose. To carry out our study, we first did an in-vitro disk diffusion assay which defines the differential cell killing against the solid tumor cell lines examined. When compared with murine CFU-GM and human leukemia CEM; we found that arctiin and AR were selective against H116, PANC-1, HepG2 and U251N cell lines. Following this, IC-50 determinations were performed against H116 and we found that IC50 values were 2.5µg/ml for arctiin and 0.31µg/ml for AR. Colonogenic (colony forming) studies were done for these compounds for H116 cell line at 2 hr, 24hr and 7days. The clonogenic assay defines tS10 which is 10% survival of the colonies after treatment with the compounds for time "t". We found that for arctiin, 2hS10 was >100µg/ml, 24hS10 was >100µg/ml and 7dS10 was 1.5µg/ml whereas for AR, 2hS10 was >100µg/ml, 24hS10 was >100µg/ml and 7dS10 was 0.3µg/ml. Maximum Tolerance Dose (MTD) was done in mice for these compounds. Arctiin and AR were administered to the mice both via oral and intravenous (iv) route. For arctiin, MTD was >313 mg/kg for both oral and iv routes and for AR, MTD was above 50mg/kg (oral) and 6.25mg/kg (iv) route. Pharmacokinetic studies are being done to understand the kinetics and the metabolism of these drugs. Based on the studies mentioned above, our data suggests that AR is more potent than arctiin. Additionally, arctiin can likely be used as a prodrug since it is less toxic than arctigenin and is converted to AR. In conclusion, Arctiin and AR have a potential to be developed as drugs for the treatment of solid tumors in humans.

#367

**The connection of vitamin D** 3 **induced cytotoxicity with the vitamin D** 3 **receptor.**

Takaya Hoshi, Yuta Abe, Ikumi Sugiyama, Yasuyuki Sadzuka. _Iwate medical university, Iwate, Japan_.

[Purpose] This study aims to evaluate the possible antitumor effect of vitamin D. Vitamin D is a group of lipophilic compounds comprising of vitamin D2 and vitamin D3. Vitamin D3 has been shown to play an important role in humans. It has been previously reported that the onset risk of malignant melanoma and leukemia can be decreased by maintaining high vitamin D3 levels in the body. In addition, the cytotoxic effect of vitamin D on breast cancer cells has also been shown. In this study, the relationship between the cytotoxicity of vitamin D3 and vitamin D3 receptor (VDR) expression on melanoma and leukemia cells was examined.

[Method] B16F10 melanoma (B16F10) and P388 leukemia (P388) cells were used in this study. Western blot analysis was performed to determine VDR expression in each cell type. To evaluate cytotoxicity, B16F10 or P388 cells were seeded at 1×105 cells/mL, and calcitriol as vitamin D3 (range 9 ng/mL-90 μg/mL) was added at once. After two days, cytotoxicity was evaluated by colorimetric analysis using Cell Counting Kit-8. Additionally, the cytotoxicity of P388 cells was determined following the combined treatment of vitamin D3 and retinoic acid.

[Results and Discussion] Western blot analysis revealed that both B16F10 and P388 cell types, expressed VDR with a higher expression in P388 cells. In P388 cells, there was a significant reduction in cell viability following vitamin D3 treatment, which decreased to 10% at 90 μg/mL. In contrast, the viability of B16F10 cells following vitamin D3 treatment only slightly decreased. Interestingly, these results suggest that the cytotoxicity of vitamin D3 correlates with VDR expression. VDR reportedly forms a heterodimer with the retinoid X receptor. However, the presence of retinoic acid did not alter vitamin D3 cytotoxicity. Therefore, it was considered that cytotoxicity induced by vitamin D3 was not connected with the formation of the VDR-retinoid X receptor heterodimer. In conclusion, there was a higher expression of VDR in P388 cells compared with B16F10 cells. This higher expression correlated with a higher level of vitamin D3 cytotoxicity, suggests an antitumor effect of vitamin D3. This study proposes the possibility of using vitamin D3 as a novel treatment for leukemia and other cancers.

#368

Next generation DNA aptamers with prolonged stability and antitumor activity.

Paola AMERO, Cristian Rodriguez-Aguayo, Rajan R. Chaudhari, Shuxing Zhang, Anil K. Sood, Gabriel Lopez-Berestein. _MD ANDERSON, Houston, TX_.

Aberrations in Gas6/AXL signaling is associated with many human diseases including ovarian cancer, where in patients expressing high levels of AXL have shorter overall survival than patients expressing low levels. Aptamers are short synthetic oligonucleotides (DNA or RNA) that can bind with highest specificity to various other macromolecules such as peptides, proteins and carbohydrates, based on their tertiary structures. Aptamers are similar in their functionality to antibodies in their specificity;

easier to manufacture than antibodies; have minimal side effects; and thus hold a high promise as therapeutics. The typical issues with the use of aptamers as therapeutic agents are their selectivity, stability, and bioavailability. We present the development of the next generation DNA aptamers, targeting p-AXL specifically to as a therapeutic in ovarian cancer (OC) by chemical modifications to their primary sequence. Improved bioavailability and stability, based on the comparative analysis the library of 17 DNA aptamers that differed in the position and number of fluoro- and thio- modifications on their nucleotide sequence, were used as the criteria for the selection and characterization of the best aptamer candidates to target p-AXL. Two of the best candidates, GLB-G25 and GLB-A04 thus chosen with the highest bioavailability and stability, when tested in ovarian cancer cell lines, decreased the migration and invasion of cells in vitro. Treatment of ovarian cancer orthotopic murine model animals with modified DNA aptamer candidates chosen above significantly reduced tumor growth and the number of metastases. The candidates, GLB-G25 and GLB-A04 show highest potential to be developed into therapeutics to target p-AXL that is elevated in several cancers including ovarian cancer.

### New Molecular Targets

#369

Antitumor activity of M4344, a potent and selective ATR inhibitor, in monotherapy and combination therapy.

Frank T. Zenke,1 Astrid Zimmermann,1 Heike Dahmen,1 Brian Elenbaas,2 John Pollard,3 Philip Reaper,3 Andree Blaukat1. 1 _Merck KGaA, Darmstadt, Germany;_ 2 _EMD Serono, Billerica, MA;_ 3 _Vertex Pharmaceuticals Limited, Abingdon, United Kingdom_.

The protein kinase ataxia telangiectasia mutated and Rad3-related ATR is one of the key mediators of the DNA damage response. ATR is recruited to regions of single-stranded DNA, which most commonly arise during replication stress (RS). RS occurs during S-phase when the cell's DNA replication machinery encounters problems such as unresolved DNA lesions. In addition, treatment of cells with DNA-damaging agents can lead to RS as cells progress to S-phase without resolving damage incurred by such agents. Elevated levels of RS are evident in some cancer cells, even in the absence of a DNA-damaging agent resulting from expression of oncogenes that drive dysregulated replication, a hypoxic environment, or from defects in other repair pathways. RS in cancer cells can drive reliance on ATR for survival and, accordingly, ATR inhibitors may have benefit as monotherapy. M4344 was determined to be an adenosine triphosphate (ATP)-competitive, highly potent, and tight-binding inhibitor of ATR with a Ki of < 150 pM. Minimal inhibitory activity was observed against a large panel of unrelated protein kinases, with 308 of 312 kinases tested having a measured Ki corresponding to more than 100-fold selectivity. M4344 potently inhibits ATR-driven phosphorylated checkpoint kinase-1 (P-Chk1) phosphorylation with an IC50 of 8 nM. Profiling on a selected set of cancer cell lines showed synergy with several types of DNA damaging chemotherapeutics as well as PARP1/2 and CHK1 inhibitors. In monotherapy efficacy studies M4344 showed tumor stasis to regression in tumor models with alternative lengthening of telomeres (ALT). In combination with PARP inhibitors, tumor regression could be observed in triple-negative breast cancer xenograft models. A dose-escalation phase 1 study in patients with advanced solid tumors is currently ongoing.

#370

The role of DLX1 as a biomarker and novel target in poor prognostic prostate cancer.

Nicholas Forsythe, Cathal McKinney, Nuala McCabe, Richard D. Kennedy. _Queen's University Belfast, Belfast, United Kingdom_.

Introduction: Previous work from our lab has identified a molecularly distinct subgroup of primary prostate cancers with biology similar to metastatic prostate cancers. These "met-like" cancers can be identified by a 70 gene classifier and are statistically more likely to result in distant metastases than their "non-met like" counterparts. This study aimed to identify exploitable biology common to both "met-like" primary tumours and metastatic prostate cancer.

Experimental procedures: Differential gene expression and Ingenuity pathway analysis (IPA) was used to identify nodal genes upregulated in both "met-like" primary prostate cancers and metastatic prostate cancers. siRNA screening was performed on 40 nodal genes in a panel of normal, primary and metastatic prostate cancers. Cell viability was assessed using cell titer-glo luminescent Assay. Relative levels of apoptosis were assessed using PARP, caspase-8 and caspase-3 cleavage Western Blots. siRNA mediated knockdowns were confirmed by Western Blot and rtPCR.

Results: We have identified upregulation of the homeobox transcription factor DLX1 in "met-like" primary prostate cancers and metastatic prostate cancers. High levels of DLX1 expression were found to be independently predicative of biochemical recurrence and metastatic disease in primary prostate cancers. DLX1 knockdown resulted in significantly decreased cell viability in primary and metastatic prostate cancer cell lines with minimal reduction of viability in normal prostate cells. Additionally DLX1 knockdown was shown to result in increased PARP, caspase-8 and caspase-3 cleavage providing evidence of apoptotic cell death. Furthermore knockdown of DLX1 resulted in significantly increased transcript levels of the pro-apoptotic BH3 protein BIK, suggesting its involvement in the observed apoptotic cell death.

Conclusion: DLX1 has previously been identified as a marker of prostate cancer metastasis with increased expression being observed in both primary and metastatic prostate cancers. However we have observed increased DLX1 expression in a subset of primary prostate cancers that developed metastatic diseased compared to primary tumors that did not metastasise. The elucidation of apoptotic mechanisms following DLX1 knockdown may uncover potential exploitable biology that may be used for the treatment of prostate cancer.

#371

Synthetic lethal and resistance pathways in midostaurin-treated AML.

Lindsey T. Brinton, Katie Williams, Shelley Orwick, Larry Beaver, Daniel Canfield, Casey Cempre, Jordan Skinner, Ronni Wasmuth, Quais Hassan, John C. Byrd, Rosa Lapalombella, James S. Blachly. _Ohio State University, Columbus, OH_.

Introduction: Mutations in fms-like tyrosine kinase 3 (FLT3) are common genomic abnormalities in adult acute myeloid leukemia (AML). When FLT3 is present as a dominant internal tandem duplication (ITD) AML clone, it is associated with poor treatment outcome and short overall survival (OS). A phase 3 study with the multi-kinase (including FLT3) inhibitor midostaurin improved OS of FLT3+ AML prompting widespread use, yet little is understood about resistance pathways and synergistic pharmacologic targets. We conducted a genome-wide CRISPR-KO (loss-of-function) screen in a FLT3+ AML cell line treated with midostaurin, validated results in vitro and in vivo, and identified distinct findings relative to limited CRISPR screening previously reported (Cancer Res 77:4402, 2017) with a very selective FLT3-ITD inhibitor (quizartinib).

Methods: CRISPR pooled screens (GeCKOv2 library, >500 cells/sgRNA) of midostaurin versus vehicle were analyzed via MAGeCK. Gene essentiality was derived from guide abundance after 7-day selection of transduced cells compared to plasmid levels. In vitro validation included shRNA knockdown and/or inhibition followed by proliferation and clonogenic assays in cell lines and primary AML blasts. NSG mice (n=7/group) engrafted with MOLM-13-luciferase cells were treated with vehicle, single agents, or combination and evaluated for disease burden (IVIS) and survival (Kaplan-Meier).

Results: We first confirmed our screening approach correlated well with previously reported essential genes (Cell Rep 17:1193, 2016; Cancer Cell 33:386, 2017;Genome Biol 16:281, 2015). We then identified >500 genes (FDR<2%) with significant depletion (synergistic) or enrichment (resistance) after midostaurin treatment. Top synergistic genes included BCL2, MEF2D, CCNG1, and RPS14, with no overlap to reported quizartinib results. We selected BCL2 for immediate validation based on demonstrated efficacy targeting this gene in AML. Indeed, shRNA knockdown of BCL2 sensitized FLT3+ AML cell lines to midostaurin. Similarly, combination treatment of FLT3+ cell lines and AML patient tumor samples with midostaurin and venetoclax displayed synergy at clinically relevant doses. In a xenograft model, combination therapy decreased disease burden and improved survival over single agents supporting synergy of these agents in vivo.

Conclusion: FLT3 inhibitors represent the first targeted therapeutic to impact AML survival and herein we use genome-wide CRIPSR screening to demonstrate that resistance mechanisms of broad-spectrum kinase inhibitors are distinct from those previously reported with specific inhibitors (quizartinib). Furthermore, we validate the select target BCL2 genetically and with the therapeutic venetoclax demonstrating in vitro and in vivo synergy, thereby justifying pursuit of this combination in future clinical trials. Ongoing efforts include validation of other targets that will be presented.

#372

VGF is functionally responsible for chemotherapy resistance in non-small cell lung cancer.

Wataru Nogami,1 Yumi Tona,1 Soichi Tofukuji,1 Yoshino Ishioka,1 Mitsunobu Matsumoto,1 Hajime Yamada,1 Kenji Kuwabara,1 Hidekazu Tanaka,1 Shigeki Adachi,2 Yoko Yamamoto,3 Ryu Kanzaki,3 Soichiro Funaki,3 Yasushi Shintani,3 Meinoshin Okumura,4 Taisei Nomura2. 1 _Shionogi & Co., Ltd., Osaka, Japan; _2 _National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, Japan;_ 3 _Osaka University Graduate School of Medicine, Osaka, Japan;_ 4 _National Hospital Organization Toneyama Hospital, Osaka, Japan_.

Despite the success of immune checkpoint therapy, most non-small cell lung cancer (NSCLC) patients still receive conventional chemotherapy. Overcoming chemotherapy resistance by identifying specific targets should improve cancer treatment and patient survival. In this study, we examined a prospective chemotherapy-resistant subpopulation in NCI-H1975 NSCLC cells and identified a novel target, neurosecretory protein VGF (VGF). Using flow cytometry, we first analyzed the expression profile of cancer stem cell markers in 100 cancer cell lines and found that NCI-H1975 cells could be divided into three subpopulations, CD44-low (CD44-L), CD44-middle (CD44-M) and CD44-high cells based on the expression pattern of CD44. CD44-M cells account for less than 5% of the NCI-H1975 cells under normal conditions, but only this subpopulation can proliferate in vitro and in vivo after treatment with cytotoxic agents, such as paclitaxel, pemetrexed and 5-fluorouracil. Affymetrix microarray analysis revealed that VGF is specifically expressed in CD44-M cells and we hypothesized that VGF would play a critical role in chemotherapy resistance. We next employed patient-derived xenograft (PDX) models of NSCLC to confirm this hypothesis with human clinical samples. NOD/SCID mice were subcutaneously transplanted with PDX and treated with either vehicle or paclitaxel (12 mg/kg, Q3Dx3, i.v.). VGF immunohistochemistry on resected PDX revealed that the number of VGF-expressing cells increased 5.6-fold in the paclitaxel-treated group compared to the vehicle control. We also performed VGF immunohistochemistry on NSCLC surgical specimens which were obtained from 127 patients, consisting of 42 neoadjuvant chemotherapy treated cases and 85 non-treated cases. The frequency of VGF-expressing specimens was higher in neoadjuvant chemotherapy treated cases (50%) than non-treated cases (18%). These results suggest that VGF-expressing tumor subpopulation is paclitaxel-resistant even in clinical settings. We also analyzed the molecular functions of VGF. Knockdown of VGF expression by siRNAs could signifıcantly suppress paclitaxel resistance in CD44-M cells. On the other hand, overexpression of VGF could significantly induce paclitaxel resistance in CD44-L cells. These results suggest that VGF is functionally responsible for chemotherapy resistance in NSCLC patients and combined administration of an anti-VGF drug and chemotherapy could be effective for preventing cancer recurrence and prolonging cancer free survival.

#373

Dysregulated RPB1 is a key driver of overgrowth and drug resistance in acute myeloid leukemia.

Qingfeng Yu, Shu Zheng, Ying Xu, Rongzhen Xu. _The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China_.

Acute myeloid leukemia (AML) is a group of highly aggressive malignancies with a 5-year overall survival (OS) of <40%. Cell overgrowth with apoptosis resistance is a hallmark of AML, but little is known about how it occurs. In this study, we show that dysregulated the largest subunit of RNA polymerase II (RPB1) encoded by POLR2A gene is critically involved in this hallmark in AML. qRT-PCR and RNA-seq results showed that POLR2A mRNA were significantly overexpressed in 76.87% (113/147) compared with normal hematopoietic cells(1.5526 versus 0.8447, P<0.001). Western blotting analysis revealed that RPB1 protein is also aberrantly activated in 68.14%(67/113) of AML patients but rare or even absent in normal hematopoietic cells, (0.7889 versus 0.0509, P<0.01). TCGA data analysis indicated that aberrant activation of POLR2A mRNA occurred predominantly in AML, CHOL and THYM, but not in most other types of cancers. Importantly, we observed that RPB1 expression levels were closely associated with tumor burden and poor prognosis. Notably, we found that POLR2A inhibition induced potent apoptosis and growth inhibition of refractory AML cells. Global transcriptomic analysis reveals that POLR2A knockout selectively depleted an array of AML-related oncogenic and anti-apoptosis genes such as c-MYC, RUNX2, MEIS1, FLT3, CDC25A and BCL-2. Conversely, enforced overexpression of RPB1 exhibits robust enrichment of oncogenes and anti-apoptosis genes, leading to cell overgrowth and drug resistance. Of particular interesting, targeting RPB1 by both genetic and pharmacological molecules could lead complete regression of refractory human AML in mouse models. In conclusion, our findings for the first time reveal that dysregulated RPB1 is a central oncogenic hub that drives overgrowth and drug resistance by coordinately hijacking an array of oncogenic genes and anti-apoptosis factors. Importantly, RPB1 is not absolutely required for the survival of normal hematopoietic cells, which is contrary to the commonly accepted belief that RPB1, the heart of the cellular transcription machinery, is required for all protein-coding genes. These data suggest that targeting RPB1 might be a potential therapeutics for treating refractory/relapse AML.

#374

Discovery and targeting MKK3-MYC protein-protein interaction for therapeutic interrogation in cancer.

Xuan Yang, Yuhong Du, Haian Fu, Andrey A. Ivanov. _Emory University, Atlanta, GA_.

Protein-protein interactions (PPIs) dictate the signal transduction and regulate diverse physiological programs, including cell death and survival. Recently we have developed a PPI high-throughput screening platform to detect the PPIs between cancer-associated proteins in the context of cancer cells. Characterization of ~3,500 PPIs tested for a set of lung-cancer related proteins resulted in a network of high-confidence direct PPIs, termed OncoPPi. To enable streamlined and integrated analysis of PPI datasets, we have developed the OncoPPi Portal, a web-based resource that integrates the network of experimentally detected cancer-associated PPIs with cancer genomics, pharmacological, and protein structural data to support cancer research.

The analysis of the OncoPPi network revealed a new connectivity between transcription factor MYC and Mitogen-Activated Protein Kinase Kinase 3 (MKK3) known an activator of p38 signaling pathway. MYC is frequently amplified across the vast majority of cancer types, and extensive clinical and biological studies have established MYC as a highly attractive therapeutic target in cancer. However, currently, there are no FDA-approved MYC inhibitors, and novel therapeutic strategies to control MYC-dependency are urgently needed.

We found that in contrast to other MYC-regulating kinases, such as ERK, MKK3 enhances MYC protein stability and transcriptional activity in a phosphorylation-independent manner. The biochemical studies guided by computational molecular modeling have revealed specific domains of MKK3 and MYC that are responsible for their interaction. Furthermore, we found that MKK3-MYC PPI can be disrupted by a short 27 amino acid residue fragment derived from MYC helix-loop-helix domain. Overexpression of this peptide was correlated with the cell growth inhibition and induction of apoptosis in multiple cancer cell lines.

To identify small molecule chemical probes to test MKK3-MYC dependency in cancer, we have developed a time-resolved fluorescence resonance energy transfer (TR-FRET) assay to monitor the interaction of MKK3 and MYC in mammalian cells. We have further optimized the assay for ultra-high-throughput screening (uHTS) in 1536-well plate format. A pilot screening with a library of 3,280 compounds has identified several positive hits that were further validated in orthogonal affinity pull-down PPI inhibition assays.

In summary, the integration of experimentally determined OncoPPi network with bioinformatics approaches has established a new biological role for MKK3 as a direct regulator of MYC-driven cell growth and proliferation. Our HTS PPI inhibitor platform has revealed first small molecule inhibitors for MKK3-MYC PPI. Together, this data defines the MKK3/MYC interface as a new potential therapeutic target to control MYC-driven tumorigenesis.

#375

Integrin β4-targeted cancer immunotherapies.

Shasha Ruan,1 Ming Lin,1 Yongshun Chen,2 Elaine Hurt,3 Alfred E. Chang,1 Max S. Wicha,1 Qiao Li1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _Renmin Hospital of Wuhan University, Wuhan, China;_ 3 _Medimmune LLC, Gaithersburg, MD_.

Integrins are heterodimeric cell surface receptors that mediate interaction of cells with extracellular matrix components. Integrin β4 (ITGB4) has been reported by other investigators as a marker of cancer stem cells (CSCs). Immunological targeting of CSCs utilizing ITGB4 as a biomarker represnets a novel therapeutic opportunity to improve the efficacy of current cancer treatment. We previously reported the use of ALDH as a marker to isolate CSCs from multiple tumor types, including both human and animal tumors. In this study, we used two murine tumor models: triple-negative breast cancer (TNBC) 4T1 tumor syngeneic to Balb/c mice and SCC7 tumor which is a spontaneously arising head and neck squamous cell cancer syngeneic to C3H mice. 4T1 cells highly express ITGB4 (about 90%) in unsorted, ALDHhigh and ALDHlow cell populations. In contrast, SCC7 cells preferentially express ITGB4 on ALDHhigh CSCs because ITGB4 cell-surface abundance on ALDHhigh SCC7 cells is approximately 2-fold higher than that on ALDHlow SCC7 cells. We tested two different approaches for ITGB4-targeted immunotherapy: using ITGB4 protein as antigen to pulse dendritic cells (DC) to prepare ITGB4-DC for vaccination, or coating tumor-draining lymph node (TDLN) T cells with anti-CD3 x anti-ITGB4 bispecific antibody (ITGB4BiAb) to prepare ITGB4BiAb-T cells for adoptive transfer. We found that for the tumor which expresses ITGB4 on all the tumor cells at high level (4T1), ITGB4-targeted immunotherapies could inhibit both local tumor growth and metastasis; for the tumor which preferentially expresses ITGB4 on ALDHhigh CSCs (SCC7), ITGB4-targeted immunotherapies inhibited the tumor growth in the lung, with partial effect on local tumor. Co-current administration of anti-PD-L1 mAb significantly enhanced the therapeutic effectiveness of both approches. Our immune monitoring studies in vitro revealed that splenetic T cells harvested from mice treated with ITGB4-DC vaccine or ITGB4BiAb armed T cells mediated significant cytotoxicity against 4T1 cells and SCC7 cells, especially to ALDHhigh CSCs. In addition, ITGB4-DC vaccine or ITGB4BiAb armed T cells conferred significant host anti-ITGB4 humoral immunity as evident by produced antibodies that specifically bind and kill 4T1 cells and SCC7 cells, especially the ALDHhigh CSC population. As a result, both immunotherapeutic approaches significantly reduced the number of ALDHhigh CSCs in the residual tumor subjected to immunotherapies. In conclusion, ITGB4-targeted immunotherapies alone or combined with PD-L1 blockade demonstrated significant antitumor immunity via targeting CSCs. This may provide novel therapeutic strategies for the treatment of ITGB4-positive cancers, such as breast cancer, head and neck squamous cell carcinoma and other soilid tumors.

#376

**Therapeutic role of** ESR1 **-** CCDC170 **gene fusion in breast cancer endocrine resistance.**

Li Li,1 Jamunarani Veeraraghavan,2 Yiheng Hu,1 Xian Wang,1 Ying Tan,2 Rachel Schiff,2 Xiaosong Wang1. 1 _Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA;_ 2 _Lester & Sue Smith Breast Center, Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX_.

Background: Gene fusions resulting from genomic rearrangements are important drivers for cancer initiation and progression. Estrogen receptor-positive (ER+) breast cancer is the most common type of breast cancer, and luminal B confers a more aggressive phenotype and higher risk of early relapse and prone to resistance to endocrine therapy. Identify new druggable genetic driver will be critical to improve clinical outcome of aggressive luminal B tumors. Our lab has identified a recurrent rearrangement between the estrogen receptor gene ESR1 and its neighbor gene CCDC170, in 6-8% of luminal B tumors. The exon 2 of ESR1 fused with the exon 6, 7, 8 or 10 of CCDC170, which enables the expression of different-sized N-terminally truncated CCDC170 (ΔCCDC170) under ESR1 promoter. Ectopic expression of ∆CCDC170 led to malignant transformation phenotypes in ER+ breast cancer cells, such as increase the cell migration, invasion and anchor-independent cell growth, as well as enhance xenograft tumor growth in mice.

Methods: The effect of ESR1-CCDC170 on endocrine resistance was examined by cologenic assay in vitro, and by xenograft mouse model in vivo. Bioinformatics approaches, Reverse Phase Protein Array (RPPA) analysis and Bimolecular Fluorescence Complementation assay were used to elucidate the potential molecular mechanism.

Results: This study aimed to determine the role of ESR1-CCDC170 in breast cancer endocrine resistance and elucidate the potential mechanisms as well as the therapeutic effect thereof. Our results show that ectopic expression of fusion variants in ER+ T47D cell line sustains the cell proliferation ability under estrogen deprivation (ED), and decreases the sensitivity to 4-OH Tamoxifen treatment. While silencing of ESR1-CCDC170 in fusion positive HCC1428 cell line increases the sensitivity to 4-OHTamoxifen and Fulvestrant treatment. Results from T47D xenograft tumor models show that ESR1-CCDC170 fusion induces endocrine resistance in vivo. Further mechanism studies show that ESR1-CCDC170 possesses a potential ATP binding pocket, has the ability to form homodimers.

Conclusion: These results demonstrate the important role of ESR1-CCDC170 fusions in breast cancer endocrine resistance, and suggest that ESR1-CCDC170 fusion could be a potential therapeutic target for the treatment of breast cancer.

#377

Anti-tumor effects in combination with PARP inhibitor and DNMT inhibitor in ovarian cancer.

Jeong-Won Lee, Soo Young Jeong, Ji Yoon Ryu, Yoo-Young Lee, ESUN Paik. _Samsung Medical Center, Seoul, Republic of Korea_.

Objective: Olaparib is one of the first PARP inhibitors and has been used for maintenance therapy in BRCA-mutant patients with platinum-sensitive, recurrent, high-grade serous ovarian cancer. However, there are some results of failure to show impressive clinical benefit with PARP inhibitor only. So, new strategies to maximize the efficacy for using these agents were developed. DNA-demethylating agents, inhibitors of DNA methylation and DNA methyltransferases(DNMTs), increase PARP1 tightly bound into chromatin in acute myeloid leukemia (AML) and breast cancer cells. The purpose of this study is to investigate the anti-cancer effect of combination therapy, olaparib with DNA demethylating agents(5-azacytidine) in ovarian cancer cell models including in vitro and in vivo - cell line xenografts and patient-derived tumor xenografts (PDX) - experiments.

Methods: In vitro experiments, we treated olaparib only, 5-azacytidine only, and combination with olaparib and 5-azacytidine in ovarian cancer cell lines (A2780, SKOV3ip1, HeyA8) to evaluate the effect on cell proliferation using MTT assay. To check the human active caspase3, MMP2, and MMP9 level and the apoptosis according to each treatment, we performed the Western blot, ELISA, and apoptosis assay in ovarian cancer cell line. With wound-healing assay and migration assay, invasive ability of cancer cell was evaluated and compared after few hours of treatment with inhibitors. In addition, in vivo therapy experiments for this co-treatment in established cell line xenografts and PDX models of epithelial ovarian cancer were done. For cell line xenografts, A2780 were injected into the peritoneal cavity of mice. For the PDX model, surgical patient tumor specimens (less than 2-3mm) implanted into the subrenal capsule of left kidney and propagated by serial transplantation. We recorded body weight, tumor weight, and number of tumor nodules.

Results: Both olaparib and 5-azacytidine inhibited the cell survival and increased the apoptosis in ovarian cancer cells. In this study, combination treatment of cells with olaparib and 5-azacytidine significantly inhibited the cell growth and increased the apoptosis compared to the single agent treatment in ovarian cancer cells. In in vivo experiments, combination treatment with olaparib and 5-azacytidine significantly decreased weight and nodule numbers of tumor in cell-line xenograft models with A2780 cells and PDX model compared with control, olaparib alone, or 5-azacytidine alone group.

Conclusions: We found that combination treatment with olaparib and DNA-demethylating agent (5-azacytidine) has synergetic effect to attack ovarian cancer cells compared with control or single agent treatment through in vitro and in vivo tests.

#378

HDAC inhibition enhances MEK antagonist therapy in uveal melanoma through combined blockade of YAP, AKT and RTK signaling.

Fernanda Faiao-Flores,1 Michael Emmons,1 Michael Durante,2 Biswarup Saha,1 Bin Fang,1 John Koomen,1 Srikumar Chellappan,1 Silvya Maria-Engler,3 Jonathan Licht,4 William Harbour,5 Keiran Smalley1. 1 _The Moffitt Cancer Center & Research Institute, Tampa, FL; _2 _Bascom Palmer Eye Institute, Miami, FL;_ 3 _University of São Paulo, Brazil;_ 4 _University of Florida, Gainesville, FL;_ 5 _University of Miami Miller School of Medicine, Miami, FL_.

Around 85-90% of all uveal melanomas harbor driver mutations in GNAQ or GNA11 leading to constitutive activation of numerous signaling pathways, including the MAPK pathway. MEK inhibitors have been evaluated clinically for metastatic uveal melanoma, but the responses are short-lived and the mechanisms of adaptation are poorly understood. In the current study, we performed RNA-seq and activity-based protein profiling (ABPP) to define the adaptive response of uveal melanoma cells to MEK inhibition and to design more effective combination therapy strategies. These analyses showed that MEK inhibition caused cytoskeleton remodeling driven by cortactin/Rho-GTPases with an increase in YAP activity, which in turn allowed therapeutic escape. Cortactin knockdown decreased YAP activity in response to MEK inhibition, increased cell death in vitro and was associated with tumor shrinkage in vivo. The proteomic data showed that MEK inhibition increased HDAC activity and an increase in global protein deacetylation. Co-targeting of HDACs and MEK was associated with increased apoptosis, decreased survival in 2D and 3D cell culture assays and suppression of YAP signaling. As YAP was unlikely to be the only escape pathway, we performed kinome and RTK arrays and demonstrated MEK inhibition also increases ROR1/2 and IGF-1R phosphorylation, leading to downstream PI3K/AKT signaling. At a signal transduction level, the combination of a pan-HDAC inhibitor (panobinostat) with a MEK inhibitor (trametinib) blocked all of the adaptive signaling pathways we identified, including RTKs, AKT, YAP and cortactin. In vivo xenograft studies revealed the MEK/HDAC inhibitor combination to outperform either agent alone, leading to long-term decrease of tumor growth and the suppression of adaptive PI3K/AKT, cortactin and YAP signaling. These findings identify HDAC inhibitors as a promising combination partner for MEK inhibitors in advanced uveal melanoma that may lead to improved systemic responses.

#379

Supraphysiological testosterone inhibits tumor growth and is associated with inhibition of ARV7 signaling and DNA damage response in preclinical models of enzalutamide-resistant prostate cancer.

Hung-Ming Lam,1 Mark P. Labrecque,1 Holly M. Nguyen,1 Lisha G. Brown,1 Ilsa M. Coleman,2 Roman Gulati,2 Bryce Lakely,1 Daniel Sondheim,1 Brett Marck,3 Alvin M. Matsumoto,1 Elahe A. Mostaghel,3 Michael T. Schweizer,1 Peter S. Nelson,2 Eva Corey1. 1 _University of Washington, Seattle, WA;_ 2 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 3 _Veterans Affairs Medical Center, Seattle, WA_.

Background: Anti-androgen therapies suppress castration-resistant prostate cancer (CRPC) but CRPC cells develop resistance. One of the mechanisms of resistance is through overexpression of androgen receptor (AR) and AR splice variants. In contrast to AR pathway inhibition therapies, recent clinical studies using bipolar androgen therapy demonstrated CRPC inhibition using supraphysiological levels of testosterone (SPT). The objective of this study was to investigate the mechanisms driving SPT-mediated tumor growth inhibition using CRPC patient-derived xenografts (PDX).

Methods: PDXs were implanted in castrated SCID mice and randomized to control or SPT arms. For enzalutamide-resistant (ENZR) PDX studies, mice with established tumors were treated with enzalutamide and randomized to control or SPT upon development of resistance. Tumors were monitored for growth and collected for analyses.

Results: In a SPT preclinical trial using thirteen LuCaP CRPC PDX models, four PDXs responded to SPT treatment while nine demonstrated de novo resistance. Our analysis revealed that responding PDXs had intrinsically higher AR and ARV7 expression compared to non-responding PDXs. Moreover, ARV7 expression was negatively correlated with E2F signaling and proliferation only in responding PDXs, suggesting that the ARV7 program functions differently in responder and non-responder phenotypes. Another PDX trial using ENZR PDXs determined that SPT inhibited the growth of LuCaP 35CR ENZR and LuCaP 96CR ENZR (responders), but not LuCaP 77CR ENZR (non-responder). Serum and intratumoral T were increased in both responders and the non-responder, suggesting that differential T delivery and tumoral retention were not the cause of differential tumor responses. Tumor analyses determined that SPT decreased AR transcript levels, however, nuclear AR protein levels and canonical AR signaling remained high in both responders and the non-responder. Conversely, ARV7 transcript was consistently decreased but the ARV7 program was downregulated only in responders. Additionally, an unbiased pathway analysis of RNASeq revealed that SPT drastically decreased genes associated with E2F-mediated cell cycle progression and proliferation and the DNA damage response (DDR) exclusively in responders. Further support for these pathways driving SPT-mediated tumor inhibition was demonstrated through the resolution of the suppressed ARV7/E2F1/DDR pathways in LuCaP 35CR ENZR upon acquiring SPT resistance, whereas the pathways remained suppressed in LuCaP 96CR ENZR, which exhibited a durable response to SPT.

Conclusion: Our data indicates that SPT therapy inhibits progression of a unique subset of ENZR CRPC and highlights critical roles for ARV7 signaling, DDR and E2F1-mediated proliferation in tumor inhibition.

#380

Tumor suppressor retinoblastoma status as a predictor of ATR inhibitor sensitivity.

Caterina Mancusi, John Hartley, Sibylle Mittnacht. _University College London - UCL, London, United Kingdom_.

Inhibitors of the Ataxia Telangiectasia and Rad3 related protein kinase (ATR) are increasingly entering clinical trials as single agents or in combination with inhibitors of poly (ADP-ribose) polymerases (PARPs), DNA-damaging chemotherapy or radiotherapy. However, and despite promise of broad utility and cancer selectivity, compelling results have remained an exception, underscoring the need to identify responsive patient population and tumour types. Based on in vitro testing in an extended cancer cell line panel we found that deleterious mutation of RB1 is significantly associated with hypersensitivity to multiple clinically relevant ATR inhibitors. Cancer cell lines with deleterious RB1 mutation displayed significantly enhanced cell death following ATR inhibitor treatment compared to a histotype matched cancer cell line groups with RB1 normal status and depletion of RB1 gene in RB1 normal cancer cell lines using siRNA, increased sensitivity to ATR inhibition accompanied by increased cells death. Deletion or deleterious mutation of RB1 is frequent in small cell lung cancers, cancers of neuroendocrine origin, and a considerable portion of triple negative breast and urinary tract cancers, cancers of the nervous system, sarcoma and chronic lymphatic leukemia. The data obtained document a link between RB1 status and ATR inhibitor sensitivity and argue that focus on cancer types with RB1 mutation or 13q14 involvement, where RB1 is encoded, should be considered in the design and analysis of trials involving ATR inhibitors.

#381

Combined inhibition of Bcl-2 and MCL-1 in small cell lung cancer (SCLC) is most effective in tumors with low Bcl-xL expression.

Benjamin J. Drapkin,1 Sneha Sanghavi,2 David T. Myers,1 Jun Zhong,1 Sarah Phat,1 Youzhen Wang,2 Ensar Halilovic,2 Javad Golji,2 Anna Farago,1 Erick Morris,2 Nicholas J. Dyson1. 1 _Massachussetts General Hospital, Boston, MA;_ 2 _Novartis, Boston, MA_.

Introduction: SCLC is an aggressive high-grade neuroendocrine malignancy in which targeting anti-apoptotic regulators such as Bcl-2 and Bcl-xL has shown efficacy in pre-clinical models but has not resulted in successful clinical trials (Rudin et al., Clin Cancer Res. 2012). Although SCLC cell-lines do not reflect the clinical impact of these inhibitors, patient-derived xenograft (PDX) models may more accurately recapitulate Bcl-2 family expression profiles and BH3 mimetic efficacy. One promising hypothesis is that the fellow anti-apoptotic protein MCL-1 rescues viability in the presence of Bcl-2/Bcl-xL antagonists. Here we evaluate the efficacy of the MCL-1 inhibitor S63845 in combination with a novel specific inhibitor of Bcl-2, BCL201/S55746, in SCLC patient-derived xenografts. Methods: BH3 mimetic compounds were tested for synergy in vitro in SCLC cell lines. A set of ten cell lines was chosen based on relative expression of BCL2, MCL1, and BCL2L1 (Bcl-xL) mRNA. Single agent and and pair-wise combinations of Bcl2 family inhibitors were compared in three-day growth inhibition assays. Loewe synergy scores were plotted versus Bcl2 family mRNA expression to identify the determinants of drug sensitivity. Based on the cell line synergy assays, a combination of BCL201/S55746 and S63845 was selected to test in PDX models of SCLC. Bcl-2 family expression was profiled across a panel of 37 SCLC PDX models generated at MGH by quantitative western blot, and standardized to the most sensitive SCLC cell line, NCI-H211. Ten models were selected based on absolute expression of Bcl-2, Bcl-xL and MCL-1. Mice were treated when subcutaneous tumors reached a volume of 400-800 cc, enabling precise measurement of tumor regression and time to tumor regrowth. Findings: Bcl-2 family dependency in SCLC cell lines was profiled with selective inhibitors as single agents or combinations. Maximum synergy was found between BCL201/S55746 and S63845 in cell lines with the highest Bcl-2:Bcl-xL expression ratio. Bcl-2 family expression was profiled across a panel of 37 PDX models of SCLC, and a representative set of 10 models was selected for in vivo testing. Consistent with cell line results, the two most sensitive models to BCL201/S55746+S63845 demonstrated the highest Bcl-2:Bcl-xL ratios, with moderate to high expression of MCL-1. In these models BCL201/S55746+S63845 resulted in a 44-70% tumor regression that was stable throughout 4 weeks of treatment. Efficacy was not dependent on MCL-1 expression, and was not strongly correlated with PDX sensitivity to platinum-etoposide. Conclusions: Combined inhibition of Bcl-2 with BCL201 and MCL-1 with S63845 is effective in SCLC tumors with relatively low Bcl-xL expression. This combination overcomes MCL-1 mediated resistance to Bcl-2 inhibitors, and represents a promising strategy to target anti-apoptotic dependency in SCLC.

#382

Development and pre-clinical assessment of a first-in-class small molecule inhibitor of FLIP.

Catherine A. Higgins,1 Jennifer Fox,1 Joanna Majkut,1 Greti E. Fiedler,1 Jamie Roberts,1 Luke Humphreys,1 Ray J. Boffey,2 Trevor R. Perrior,2 Timothy Harrison,1 Daniel B. Longley1. 1 _Queen's Univ. Belfast, Belfast, United Kingdom;_ 2 _Domainex Ltd., Saffron Walden CB10 1XL, United Kingdom_.

Evasion of cell death is a major cause of resistance to anti-cancer therapies, making proteins that regulate cell death clinically relevant therapeutic targets. The anti-apoptotic protein FLIP is frequently overexpressed in a number of cancers and leukemias and has been shown to be a major mediator of resistance to chemo- and radio-therapies and to cell death induced by immune effector cells. FLIP and procaspase-8 form complexes with the adaptor protein FADD in response to a variety of clinically relevant stimuli, including ligation of death receptors, such as TRAIL-R1 and R2, and treatment with chemotherapeutic agents. In these complexes, FLIP modulates the activation of procaspase-8, and thereby regulates induction of apoptosis and necroptosis - two major cell death mechanisms. Herein, we report the development and pre-clinical characterization of first-in-class inhibitors of FLIP.

Molecular modelling identified a putative drug-binding pocket on FLIP against which a virtual small-molecule screen was carried out. Subsequent biochemical screening of selected compounds using protein-protein interaction assays identified hits with on-target activity. Medicinal chemistry optimization of these hits identified a series of compounds that are able to disrupt FLIP's interaction with the TRAIL-R2 DISC and display nanomolar activity in NSCLC cell-based assays in line with their binding affinity in an orthogonal biophysical assay (isothermal calorimetry). The pro-apoptotic effects of these FLIP inhibitors were enhanced upon addition of death ligands, such as TRAIL, and lead-molecules have been shown to potentiate the effects of standard-of-care chemotherapeutics, ionizing radiation and targeted anti-cancer agents. FLIP overexpression and CRISPR-mediated deletion of procaspase-8 and FADD abrogated the effects of these novel inhibitors consistent with their expected mechanism-of-action. Recent studies have also demonstrated that lead FLIP inhibitors target Treg cells, which are key immuno-suppressive, tumor-promoting immune cells. Lead molecules have also demonstrated in vivo efficacy in NSCLC cancer models both as a single agent and in combination with a multivalent TRAIL agonist.

The first-in-class inhibitors of FLIP developed in this study have the potential for broad application in treatment of several cancers, including solid tumors and haematological malignancies, either as monotherapy or in combination with other agents.

Acknowledgements: This work was supported by a Seeding Drug Discovery award from the Wellcome Trust.

#383

APOBEC3B is induced by activation of DNA repair pathway and modulates the survival and treatment response in human multiple myeloma.

Lijie Xing, Jiye Liu, Liang Lin, Shih-Feng Cho, Kenneth Wen, Tengteng Yu, Gang An, Ligui Qiu, Kenneth Anderson, Yu-Tzu Tai. _Dana-Farber Cancer Inst., Boston, MA_.

Constitutive genomic complexity, ongoing DNA damage, and accumulating mutations are observed with progression from monoclonal gammopathy of undetermined significance (MGUS) to active multiple myeloma (MM) to relapsed/refractory disease. Apolipoprotein B mRNA editing catalytic polypeptide-like 3B (APOBEC3B), a DNA cytosine deaminase in cancer mutagenesis, is recently linked to sub-clonal diversification, intra-tumor heterogeneity, and tumor evolution in MM. We here studied the upstream mechanisms of APOBEC3B dysregulation and further defined functional consequences of molecular manipulation of APOBEC3B in MM cells. We first asked whether APOBEC3B levels are altered in MM cells upon treatments with Melphalan (Mel), an alkylating agent used to treat MM which is known to induce replication stress, or with ionizing radiation (IR). Sub-lethal doses of Mel or IR induce APOBEC3B transcript and protein expression in a dose- and time-dependent manner in MM cell lines (n=7), associated with phosphorylation of γH2AX. Significantly, Bortezomib (btz), even at sub-lethal doses triggering DNA damage signaling, induced APOBEC3B expression in multiple MM cell lines. Next, inhibition of ATR or ATM activation pathway significantly decreased Mel- or IR or btz-induced APOBEC3B, suggesting that replication stress induced by Mel, IR, or btz activates ATM/ATR-dependent APOBEC3B induction. We used gene-specific CRISPR knock out (KO), shRNA knockdown (KD), and inducible-shRNA KD to study the functional impact of perturbation of APOBEC3B in MM cells. Both KO and KD of APOBEC3B decreased growth and survival in multiple MM cell lines sensitive or resistant to dexamethasone or lenalidomide. APOBEC3B inhibition significantly enhanced growth arrest followed by apoptosis in MM cells, suggesting that increased APOBEC3B levels contribute to MM cell survival. We next analyzed available data sources for MM cell lines from Cancer Cell Line Encyclopedia and the Genomics of Drug Sensitivity in Cancer, which include microarray gene expression and drug sensitivity information. APOBEC3B expression negatively correlates with MM cell sensitivity to JQ1, a BET inhibitor which inhibit MM cell growth and survival in vitro and in vivo. Importantly, in MM cell lines which are relatively resistant to pomalidomide and JQ1 than other cell lines, APOBEC3B KD by its shRNA enhances sensitivity to both drugs. Taken together, our findings provide new insights into the role of APOBEC3B in triggering cytidine deaminase-induced mutagenesis associated with progression of disease. Furthermore, DNA replication stress triggered by Mel, IR, or btz upregulates APOBEC3B expression, which in turn confers drug resistance. The role of APOBEC in disease pathogenesis and progression, coupled with its role mediating drug resistance, suggest potential utility of targeting APOBEC3B in novel MM therapies.

#384

Identification of a lung adenocarcinoma cell line addicted to ERBB4, an actionable target for cancer therapy.

Noritaka Tanaka,1 Kanami Yamazaki,1 Yuko Uno,2 Yoshimi Ohashi,1 Yumiko Nishimura,1 Masaaki Sawa,2 Shingo Dan1. 1 _Japanese Foundation for Cancer Research, Tokyo, Japan;_ 2 _Carna Biosciences, Kobe, Japan_.

Cancer cells often survive by getting addicted to specific genes. In most cases, such genes function as an enzyme, especially kinases that are specifically activated in cancers by genetic aberrations; e.g., BCR-ABL fusion kinase found in chronic myelogenous leukemias and epidermal growth factor receptor (EGFR) with activating mutations found in non-small cell lung cancers (NSCLCs). These activated kinases would be an ideal target for cancer therapy and indeed imatinib and gefitinib displayed good clinical benefits to the patients in these diseases. However, only a limited population of cancer cells harbor such well-characterized driver genes which could be attacked by targeted drugs developed so far and there remain unmet medical needs to identify novel therapeutic targets and drugs for the rest of cancers. To this end, we exploited our original database of drug efficacy of >100 kinase inhibitors across a panel of 39 human cancer cell lines (JFCR39) to characterize each of the cell line in terms of susceptibility to these kinase inhibitors. Expectedly, cell lines harboring PIK3CA hotspot mutation and PTEN mutation were susceptible to PI3K and mTOR inhibitors, whereas those harboring KRAS or BRAF mutation to MEK inhibitors, respectively. Of note, we found four of the cells responding to a series of EGFR family tyrosine kinase inhibitors (TKIs) including gefitinib and lapatinib. Gefitinib exert selective effect on NSCLCs addicted to activated EGFR mutant; however, none of the four gefitinib-sensitive cell lines possessed such EGFR mutation. Of these, an NSCLC cell line NCI-H522 hardly expressed EGFR but overexpressed ERBB4 instead, while an ovarian cancer cell line SK-OV3 overexpressed ERBB2 as reported previously. Actually, treatment with a certain TKI in this class efficiently dephosphorylated ERBB4 in NCI-H522 along with inhibition of cell growth and suppression of downstream factor ribosomal S6 protein. Moreover, knockdown of ERBB family kinases revealed that cell growth was dependent on overexpressed ERBB4 in NCI-H522 cells. These results indicate that NCI-H522 cells exhibited susceptibility to EGFR family TKIs via inhibition of ERBB4. Of note, NCI-H522 cells exhibited hypersensitivity to three of these TKIs, sapitinib, PF-7775 and GW583340, as compared to gefitinib. Especially, GW583340 suppressed growth of NCI-H522 cells at the same or lower concentration than in HCC827 cells harboring an EGFR active mutation. Consistent with this observation, in vitro kinase assay revealed that sapitinib and PF-7775 exhibited ~10-fold higher ERBB4-inhibitory effect than gefitinib did while GW583340 displayed selective inhibition on ERBB4, with ~10-fold higher activity than that on activated EGFR. In conclusion, we identified NCI-H522 as an ERBB4-dependent cell line, and found ERBB4 as a novel therapeutic cancer target that could be attacked by ERBB4-directing TKIs.

#385

MTAP gene deficiency creates vulnerability to anti-folate therapy in urothelial bladder carcinoma.

Jianfeng Chen,1 Omar Alhalabi,1 Guangchun Han,1 Wei-Lien Wang,1 Xin-Qiao Zhang,1 Jian H. Song,1 Lidia P. Lopez,1 Sumankalai Ramachandran,1 Anh G. Hoang,1 Tyrone Garnett,1 Matthew Campbell,1 Amishi Y. Shah,1 Jennifer Wang,1 Arlene O. Siefker-Radtke,1 Shi-Ming Tu,1 Mark Titus,1 Charles C. Guo,1 Gary E. Gallick,1 Eleni Efstathiou,1 William F. Benedict,1 Christopher J. Logothetis,1 Thai H. Ho,2 Linghua Wang,1 Jianjun Gao1. 1 _The UT MD Anderson Cancer Center, Houston, TX;_ 2 _Mayo Clinic Arizona, Phoenix, AZ_.

Background: The methylthioadenosine phosphorylase (MTAP) gene encodes an essential enzyme for the salvage pathway of adenosine synthesis and is frequently lost in different types of cancer including urothelial bladder carcinoma. Therefore, MTAP-deficient tumors are theoretically very sensitive to anti-folate agents such as pemetrexed that can effectively block the de novo pathway of adenosine synthesis and as a result, create a state of synthetic lethality. We thus hypothesize that tumor MTAP gene deficiency is associated with response to pemetrexed therapy in bladder cancer.

Methods: In this study, we investigated MTAP gene deficiency rates in the TCGA database and confirmed MTAP protein loss by immunohistochemistry using a tumor tissue microarray containing bladder tumor tissues from 151 patients. We then performed in vitro and in vivo studies using MTAP-proficient and MTAP-deficient human bladder cancer cell lines. Functional loss of MTAP was verified with mass spectrometry, which detects its substrate methylthioadenosine (MTA) levels. We also correlated these pre-clinical studies with clinical response data on patients with metastatic bladder cancer treated with pemetrexed.

Results: We identified that 27.8% bladder cancer patients have MTAP protein deficiency, which is consistent with exome sequencing data from the TCGA database. In vitro data showed MTAP-deficient human bladder cancer cell lines were significantly more sensitive to pemetrexed, with IC50 at least 40 times lower than MTAP-proficient cell lines. Subsequent knockdown of the MTAP gene in MTAP-proficient cell lines increased sensitivities to pemetrexed treatment. Consistent with the in vitro data, pemetrexed significantly inhibited the growth of MTAP-deficient or knockdown xenograft tumors but not MTAP-proficient tumors. Furthermore, 4 of 4 (100%) patients with MTAP-deficient metastatic bladder cancer responded to pemetrexed treatment, whereas only 1 of 11 (9%) patients with MTAP-proficient metastatic bladder cancer responded to pemetrexed.

Conclusion: Our data demonstrate that MTAP gene loss in urothelial bladder cancer leads to a metabolic state of synthetic lethality with pemetrexed therapy. Therefore, bladder tumor MTAP loss should be further investigated as a potential biomarker for selection of patients for anti-folate therapy.

#386

Targeting MDM4 to reactivate p53 signaling in neuroblastoma.

Zhenghu Chen, Sarah E. Woodfield, Roma H. Patel, Aryana M. Ibarra, Sanjeev A. Vasudevan. _Baylor College of Medicine, Houston, TX_.

Introduction: Despite aggressive therapeutic regimens, long-term survival of patients with high risk neuroblastoma (NB) is less than 50%. The p53 tumor suppressor gene is mutated in more than 50% of human cancers, however, unlike in most adult tumors, p53 is wild-type and functional in 99% of NB cases at diagnosis. Reactivation of p53 tumor suppressor function by pharmacological inhibition of MDM4, a negative regulator of p53, is a novel strategy in NB treatment. We hypothesized that targeting MDM4 to reactivate p53 signaling would have effective anti-tumor effects on NB.

Methods: Levels of gene expression of MDM4 and its association with event-free and overall survival in NB patients were analyzed using the publically available patient datasets in the R2 Genomics Analysis and Visualization Platform. MDM4 gene and protein expression were also examined in six NB cell lines with qPCR and immunoblotting assays. The effects of MDM4 inhibitor NSC207895 on p53 wild-type (p53wt) and mutated cell lines were analyzed for cytotoxicity, growth in soft agar, and expression of apoptosis markers and p53 downstream targets. Moreover, an orthotopic xenograft NB mouse model was used to test the in vivo efficacy of NSC207895.

Results: MDM4 expression is significantly correlated with both event-free and overall survival of NB patients in the Kocak (n=476) and Oberthuer (n=251) datasets in the R2 Genomics Analysis and Visualization Platform, with higher expressions correlated with decreased survival (*** p <0.001). All six NB cell lines showed gene and protein expression of MDM4. NSC207895 had strong cytotoxic effects at relatively low concentrations (IC50 from 0.45-2.46 μM) after 48 hours of treatment with p53wt IMR-32, NGP, NB-19 and SH-SY5Y cells, but not with p53 mutant SK-N-AS (IC50:10.55 μM). NSC207895 significantly inhibited the colony formation ability of several NB cells. NSC207895 exposure also led to upregulation of p53 targets p21, Puma, and Bax in p53wt cells but not in p53 mutant cells. In addition, NSC207895 is more potent in p53wt SH-SY5Y (IC50=0.46 uM) than in the p53 knock out SH-SY5Y cells (IC50=0.98 uM). Moreover, NSC207895 significantly enhanced the cytotoxicity of doxorubicin on p53wt cells but not in p53 mutant cells. Above all, NSC207895 inhibited the tumor growth in an orthotopic xenograft NB mouse model (Mann-Whitney test, ** p <0.01) with no significant side-effects. In addition, compared with the placebo group, NSC207895 treated NB tumors demonstrated elevated expression levels of cleaved PARP, together with p53 downstream targets p21, Puma, and Bax, suggesting that NSC207895 induced p53-mediated apoptosis in vivo.

Conclusion: Novel MDM4 inhibitor NSC207895 shows anti-tumor efficacy in p53 wild-type NB by reactivating p53 tumor suppressor signaling and inducing p53-mediated apoptosis, both in vitro and in vivo. Inhibition of MDM4 by small molecules is a promising treatment strategy for high risk patients with wild-type p53.

#387

Pyrimethamine inhibits STAT3 transcriptional activity via dihydrofolate reductase.

Lisa N. Heppler,1 Sarah R. Walker,2 Sanaz Attarha,3 Brent D. Page,3 David A. Frank4. 1 _Harvard University, Cambridge, MA;_ 2 _University of New Hampshire, Durham, NH;_ 3 _Karolinska Institute, Sweden;_ 4 _Dana-Farber Cancer Institute, Boston, MA_.

Cancer is often characterized by aberrant gene expression patterns that alter cellular function. Such alterations are commonly caused by the inappropriate activation of transcription factors. STAT3, in particular, is a key transcriptional regulator of many pro-tumorigenic processes, including inflammation, proliferation, and survival, and is persistently phosphorylated and activated in more than 70% of breast tumors. Accordingly, it is a promising therapeutic target for the treatment of breast cancer. However, like many transcription factors, STAT3 has proven difficult to target clinically. To address this unmet clinical need, we performed an unbiased, high-throughput screen of the Prestwick collection, a library of 1,120 small molecules known to be biologically active in humans. We identified the antimicrobial drug pyrimethamine as a novel and specific inhibitor of STAT3 transcriptional activity at concentrations known to be safely achieved in humans. Intriguingly, unlike STAT3 phosphorylation inhibitors and STAT3 SH2 domain inhibitors, pyrimethamine does not affect STAT3 phosphorylation, nuclear translocation, or DNA binding at concentrations sufficient to inhibit STAT3 transcriptional activity, suggesting a potentially novel mechanism of STAT3 inhibition. To elucidate this mechanism and identify the direct molecular target of pyrimethamine, we performed thermal proteomic profiling. We detected 4,462 proteins, four of which demonstrated a statistically significant shift in thermal stability in the presence of pyrimethamine. We further characterized these hits by assessing the effect of siRNA-mediated knockdown on STAT3 transcriptional activity. Of the four hits, only knockdown of dihydrofolate reductase (DHFR) consistently reduced STAT3 transcriptional activity. Likewise, treatment with methotrexate, a known inhibitor of DHFR, also reduced STAT3 activity. The combination of pyrimethamine and methotrexate had no additive effect on STAT3 inhibition, suggesting that both compounds were acting through a common mechanism. Finally, we tested whether folinic acid, a reduced form of folate, could rescue pyrimethamine-dependent loss of STAT3 activity. Strikingly, folinic acid, but not folic acid, eliminated the effect of pyrimethamine on STAT3 transcriptional activity in a dose-dependent manner. Together, these results suggest that DHFR is the molecular target responsible for the STAT3-inhibitory effects of pyrimethamine. Moreover, these findings implicate folate metabolism in the regulation of STAT3 transcriptional activity, revealing a previously unknown node within the STAT3 pathway that may be important for the development and treatment of STAT3-driven breast cancer.

#388

SS18-SSX modulates YAP/TAZ-TEAD transcriptional activity in synovial sarcoma.

Ilka Isfort,1 Magdalene Cyra,1 Sandra Elges,1 Sareetha Kailayangiri,2 Bianca Altvater,2 Claudia Rossig,2 Jan-Henrik Mikesch,3 Agnieszka Wozniak,4 Patrick Schöffski,4 Eva Wardelmann,5 Marcel Trautmann,1 Wolfgang Hartmann1. 1 _University Hospital Muenster, Gerhard-Domagk-Institute of Pathology, Division of Translational Pathology, Muenster, Germany;_ 2 _University Children´s Hospital Muenster, Departments of Pediatric Hematology and Oncology, Muenster, Germany;_ 3 _University Hospital Muenster, Department of Medicine A, Hematology, Oncology and Respiratory Medicine, Muenster, Germany;_ 4 _KU Leuven, Department of Oncology and University Hospitals Leuven, Department of General Medical Oncology, Leuven, Belgium;_ 5 _University Hospital Muenster, Gerhard-Domagk-Institute of Pathology, Muenster, Germany_.

Introduction: Synovial sarcoma (SySa) is a rare soft-tissue malignancy characterized by a specific reciprocal translocation t(X;18). The resulting chimeric SS18-SSX fusion protein acts as transcriptional dysregulator representing the major oncogenic driver in SySa tumorigenesis. Since targeting the fusion protein itself remains a particular challenge, it appears reasonable to therapeutically address signaling pathways which are functionally dependent on the SS18-SSX fusion protein. As different tumor entities were recently shown to harbor aberrant Hippo signaling patterns leading to increased activity of the transcriptional coactivators YAP/TAZ, the aim of this study was to analyze the role of YAP/TAZ in SySa and to decipher the functional link to the SS18-SSX fusion protein.

Experimental procedures: YAP/TAZ expression was analyzed by immunohistochemistry in a large cohort of SySa tissue specimens (n=65). Five SySa cell lines and mesenchymal SCP-1 stem cells stably expressing the SS18-SSX fusion protein were employed for in vitro analyses. We set out to analyze whether YAP/TAZ-TEAD transcriptional activity is dependent on the SySa-specific fusion protein, if this dependency is mediated by IGF-IR signaling (known to be activated in SySa) and to understand the biological function of YAP/TAZ in SySa. To modulate YAP/TAZ-TEAD transcriptional activity, RNAi-mediated knockdown and the small molecule inhibitor verteporfin were applied. Finally, the therapeutic effect of YAP/TAZ inhibition was tested in vivo using SySa cell line-based and patient-derived xenografts.

Results: SySa tissue specimens and cell lines strongly expressed nuclear YAP/TAZ. RNAi-mediated knockdown of SS18-SSX fusion protein led to significant reduction of YAP/TAZ-TEAD transcriptional activity while SS18-SSX overexpression in SCP-1 cells induced aberrant YAP/TAZ signals. This regulatory connection was at least partly realized through an IGF-II/IGF-IR loop, in which the SS18-SSX fusion protein drives IGF2 expression causing dysregulation of the Hippo effectors LATS1 and MOB1, eventually leading to YAP/TAZ activation. Inhibition of YAP/TAZ-TEAD transcriptional activity by RNAi or verteporfin resulted in a significant induction of apoptosis and significant reduction of SySa cell growth in vitro and in vivo.

Conclusions: Our study reveals SS18-SSX fusion protein-driven YAP/TAZ-TEAD signals to play an elementary role in SySa. Given the high efficacy of YAP/TAZ-directed pharmacological approaches in SySa xenografts, this preclinical study may constitute the basis for a novel therapeutic strategy to inhibit SS18-SSX-driven tumorigenesis.

#389

Role of her3 signaling pathways in er+ and her2+ breast cancers.

Rosalin Mishra, Samar Alanazi, Hima Patel, Long Yuan, Joan T. Garrett. _Univ. of Cincinnati College of Pharmacy, Cincinnati, OH_.

HER3 is an important molecule that may regulate oncogenic activity in estrogen receptor (ER)+ breast cancers, which accounts for about 70-80% of all breast cancers. HER3 gene expression is highest in ER+ or luminal tumors. Treatment with the ER degrader fulvestrant induces protein expression of HER3 in ER+ clinical breast tumors, suggesting that HER3 may have therapeutic value in luminal breast cancers. About 20% of all breast cancers diagnosed are HER2 positive (+). HER3 is as essential as HER2 for maintaining cell viability in HER2-overexpressing breast cancer cells. We aim to identify proteins that bind to HER3 upon therapeutic inhibition of HER2 or anti-endocrine therapy.

We have found that ERα is upregulated in ER+ T47D and MCF-7 breast cancer cell lines expressing the activating hotspot T355I HER3 mutation compared to HER3 wild-type (WT) cells. We noted that ERα co-immunoprecipitated to HER3-T355I but not to HER3-WT T47D cells. Knocking down HER3 resulted in decreased ERα expression in both HER3-WT and HER3-T355I expressing T47D cells. We are currently analyzing the nuclear expression of endogenous HER3 in MCF-7, T47D, BT474, MDA-MB-361, MDA-MB-453 and SK-BR3 breast cancer cells. We are determining if stimulation with the HER3 ligand heregulin alters the subcellular location of HER3 or the ability to co-immunoprecipitate with ERα in cells expressing exogenous HER3-WT, exogenous HER3-T355I or endogenous HER3.

We are using a proteomics approach to identify HER3-interacting proteins to determine how HER2 inhibition affects the repertoire of HER3 binding partners in HER2+ breast cancer cells. We immunopurified HER3 from BT474 cells treated with DMSO or with neratinib (24 hour treatment). We then identified enrichment of specific proteins by mass spectrometry. MALDI tandem mass spectrometry experiments identified non-muscle myosin-II heavy chain from HER3 immunoprecipitates treated 24 hours with neratinib treatment but not 24 hours of DMSO treatment in BT474 cells. Furthermore, our immunoprecipitation data indicated that there is more myosin-IIa bound to HER3 in response to neratinib treatment. Myosin-IIa has a key role in cytokinesis, cell migration, polarization, adhesion and metastasis. Thus, we aim to decipher the mechanism of action by which myosin-IIa interacts with HER3 and determining if downstream myosin/actin signaling could be a possible target in HER2 over-expressing breast cancer patients who show resistance to HER2 targeted therapy.

In conclusion, we have identified two potential novel HER3 binding partners in breast cancer: ERα and myosin-IIa. Deciphering the function of HER3 binding partners has potential implication for understanding the invasive properties of ER+ and HER2+ breast cancers.

#390

Prostate cancer progression depends on the activity of the mitotic kinase citron kinase.

Salma Ben-Salem,1 Salam Bachour,1 Varadha Balaji Venkadakrishnan,1 Yixue Su,1 Eduardo Cortes Gomez,2 Qiang Hu,2 Eric Klein,1 Nicolas Marlo,1 Cristina Magi-Galluzzi,1 Liu Song,2 Hannelore V. Heemers1. 1 _Cleveland Clinic, Cleveland, OH;_ 2 _Roswell Park Comprehensive Cancer Center, Buffalo, NY_.

Androgen receptor (AR) is the major driver of prostate cancer (CaP) progression. Despite initially inducing remissions, androgen deprivation therapy (ADT) does not cure CaP and the majority of CaP that recurs under ADT continues to rely on AR. In a subset of patients, administration of novel, more potent ADT drugs leads to emergence of a neuroendocrine (NE) CaP phenotype that is AR-indifferent and even harder to treat. The molecular mechanisms by which AR regulates cell cycle progression and that can be developed into alternative targets to inhibit CaP growth, overcome acquired resistance to ADT, or prevent NE progression, however, remain largely unknown. Here, we identify citron kinase (CIT), which controls cell division, as a novel drugabble target that acts downstream of AR. In multiple CaP models, CIT protein expression was stimulated selectively by low doses of androgens that promote CaP cell proliferation and decreased by AR silencing or short-term administration of AR-antagonist enzalutamide, confirming its AR dependence. Silencing of CIT significantly reduced CaP cell viability and cell proliferation, delayed cell progression, increased the number of multinucleated cells in ADT-naïve and -recurrent CaP cells, and attenuated CaP growth in xenograft models. Overexpression of CIT, transiently or stably, stimulated CaP cell proliferation in androgen-supplemented conditions and under ADT, and this depended entirely on an intact CIT kinase domain. In 2 independent clinical CaP datasets, CIT mRNA levels increased during CaP progression and higher CIT expression correlated with shorter disease-free and overall survival. Using CaP tissue microarrays that contain more than 200 patient specimens, CIT protein expression was significantly higher in primary CaP when compared to adjacent non-neoplastic prostate tissues and correlated with increasing Gleason scores, validating the relevance of CIT overexpression to CaP aggressiveness. Integrated RNA-Seq, MSigDB and GSEA analyses confirmed that the AR- and CIT-dependent transcriptome preferentially controlled CaP cell cycle progression and proliferation. Mechanistically, the use of inhibitors of gene transcription, mRNA translation and proteasome indicated that the androgen-dependence of CIT expression is regulated at the post-transcriptional level. Specifically, CIT protein expression was controlled by the E2F family of cell cycle regulators, with androgen-responsive E2F2 as the major determinant of CaP CIT expression. Our work, thus, isolated a novel role for the mitotic kinase CIT in AR-dependent CaP cell proliferation and clinical progression and identified CIT's kinase moiety as a novel target for CaP therapy.

Funding: NIH/NCI, Case Comprehensive Cancer Center Pilot Research Award

#391

Serine/threonine kinase LASK2 as a biomarker and therapeutic target for lung cancer.

Yataro Daigo,1 Atsushi Takano,1 Yusuke Nakamura2. 1 _Univ. of Tokyo Inst. of Medical Science, Tokyo, Japan;_ 2 _Cancer Institute, Japan_.

Since the number of lung cancer patients responding well to standard chemotherapies is still small, further development of new anti-cancer drugs with minimum risk of adverse effects and highly sensitive cancer biomarkers for precision medicine is eagerly required. We have been developing new molecular therapies targeting oncogenic proteins with their companion diagnostics by screening system as follows; i) To identify overexpressed genes in 120 lung cancers by our original gene expression profile database, ii) To confirm the candidate genes for their low expression in 23 normal tissues, iii) To validate the clinicopathological significance of their protein expression by tissue microarray covering hundreds of non-small cell lung cancers (NSCLCs), iv) To verify whether they are essential for the growth/invasion of cancer cells by siRNAs. During this process, we selected dozens of druggable oncoproteins with various enzymatic activities, and detected higher expression of serine/threonine kinase LASK2 (lung cancer-associated kinase 2) in the majority of various types of lung cancers, but scarce expression in normal tissues except testis. We further observed much higher expression patterns of LASK2 in brain metastasis as well as advanced primary lung tumors, compared to those in earlier-stage primary lung tumors. Immunohistochemical analysis using tissue microarray showed that strong LASK2 positivity was an independent prognostic factor for NSCLC (P<0.0001). Suppression of LASK2 expression with its siRNAs inhibited growth of lung cancer cells, whereas enhanced cellular proliferation by exogenous expression of LASK2 in mammalian cells also supported its oncogenic function in vitro and in mice model. Invasion of mammalian cells transfected with LASK2-expression vector through Matrigel was significantly enhanced, compared to the control cells transfected with mock or LASK2-kinase dead vector, suggesting that LASK2 could also contribute to the highly malignant phenotype of lung-cancer cells. Induction of LASK2 appeared to activate oncogenic signals for lung cancer such as phosphorylation of MAPK and PCNA. Our data indicate that LASK2 is a promising diagnostic biomarker and therapeutic target for developing precision medicine of lung cancer.

#392

LKB-inactivated lung cancer cells exhibit dependency on INSL4 signaling.

Rongqiang Yang, Steven W. Li, Zirong Chen, Xin Zhou, Wei Ni, Dongtao A. Fu, Jianrong Lu, Frederic J. Kaye, Lizi Wu. _University of Florida, Gainesville, FL_.

Lung cancer is the leading cause of cancer deaths worldwide. The LKB1 (STK11) tumor suppressor gene is commonly inactivated in non-small cell lung carcinomas (NSCLC), a major form of lung cancer. Currently, no effective treatments are available for LKB1-inactivated lung cancer. Elucidation of critical signaling downstream of LKB1-inactivation has the potential to uncover rational therapeutic targets for this unique and prevalent molecular subset of lung cancers. In this study, we identified INSL4 (insulin-like 4), a member of the insulin/IGF/relaxin superfamily, as a novel downstream target of LKB1-deficiency through gene expression profiling analysis. We observed that INSL4 expression was highly induced through aberrant CRTC-CREB activation subsequent to LKB1 inactivation. INSL4 was highly expressed in human LKB1-deficient NSCLC cells and primary tumors while undetectable in all normal tissues except in the placenta. High INSL4 expression was associated with more advanced lung cancer and worse patient survival. INSL4 depletion inhibited the growth and viability of LKB1-null NSCLC cells in vitro and blocked the growth of lung cancer xenografts. Moreover, expression profiling identified an INSL4-regulated gene program that is associated with the regulation of cell cycle, growth, and survival. In conclusion, LKB1 deficiency induces an autocrine INSL4 signaling that critically supports the growth and survival of lung cancer cells. Therefore, aberrant INSL4 signaling is a promising therapeutic target for LKB1-deficient lung cancers.

#393

Deoxymab: A targeted biologic that is synthetically lethal to TNBC brain metastases.

Shenqi Zhang,1 Christopher May,1 Anupama Shirali,1 Valentina Dubljevic,2 James A. Campbell,2 Jiangbing Zhou,1 James E. Hansen1. 1 _Yale School of Medicine, New Haven, CT;_ 2 _Patrys Ltd, Australia_.

Brain metastases in triple-negative breast cancer (TNBC) harbor defects in homology-directed DNA repair that may be exploited to develop new therapeutic strategies. However, these tumors are protected by the blood-brain barrier (BBB), and mechanisms of DNA repair are sequestered in the nucleus. Methods to cross the BBB and target intranuclear processes are needed.

3E10 is a nuclear-penetrating lupus anti-DNA autoantibody that inhibits the DNA damage response to selectively kill cancer cells with defects in DNA repair. 3E10 localizes to DNA in tumor environments, and penetrates cells via the ENT2 equilibrative nucleoside transporter. ENT2 is expressed in the BBB, and 3E10 has previously delivered cargo proteins to the brain. We believe 3E10 can be turned against TNBC brain metastases, and used MDA-MB-231-BR brain-seeking human TNBC cells and tumors as a model system to test this hypothesis.

We re-engineered 3E10 into an optimized fragment, called Deoxymab-1 (DX1), to maximize effect on cancer cells and minimize toxicity. DX1 expressed in CHO cells was purified over a HiTrap Capto S column by FPLC, and purity and quality was confirmed by SDS-PAGE and SEC-HPLC. Immunostaining and colony formation assays demonstrated that DX1 penetrated the nuclei of ~100% of MDA-MB-231-BR cells in culture and was synthetically lethal to the cells, with doses of 5 and 10 μM reducing surviving fractions to 0.47±0.15 (P<0.03) and 0.29±0.07 (P<0.01), respectively.

TNBC brain metastases were established in nude mice by intracardiac injection of 1.75x105 MDA-MB-231-BR cells transfected with luciferase. One week later the presence of brain metastases was confirmed by IVIS. Mice were treated with tail vein injection of vehicle control (n=7) or DX1 (20 mg/kg) (n=7) 3x/week for 3 weeks. Tumor burden was monitored by weekly IVIS. DX1 significantly suppressed growth of the brain metastases (Table 1). These results establish proof of the novel concept of use of a modified lupus autoantibody against TNBC brain metastases.

Table 1: Brain Radiance Efficiencies (x105)

---

Treatment | Week 0 | Week 1 | Week 2 | Week 3

Control | 1.2±0.2 | 2.5±0.4 | 5.1±0.7 | 20±5

DX1 | 1.3±0.2 | 1.4±0.3 | 2.0±0.2 | 3.8±0.8

P | ns | <0.01 | <0.01 | <0.02

#394

In vivo **shRNA screens under treatment pressure by BRAF and MEK inhibitors to identify novel combination treatment strategies for** BRAF **-mutant colorectal cancer.**

Hyo-eun C. Bhang,1 Matthew T. DiMare,1 David P. Kodack,1 Lujian Tan,1 Grainne Kerr,2 Viveksagar Krishnamurthy Radhakrishna,1 Javad Golji,1 David A. Ruddy,1 Tina Yuan,1 Matthew J. Niederst,1 Joshua M. Korn,1 Diana Graus Porta,2 Peter S. Hammerman,1 Jeffrey A. Engelman,1 Tinya Abrams,1 Juliet Williams1. 1 _Novartis Insts. for BioMedical Research, Cambridge, MA;_ 2 _Novartis Insts. for BioMedical Research, Basel, Switzerland_.

Approximately 10% of patients with colorectal cancer (CRC) harbor the BRAF V600E driver mutation. Unlike melanoma, the response rate of BRAF-mutant CRC to the combination of BRAF and MEK inhibitors is limited. In order to target the MAPK signaling pathway more effectively by blocking EGFR-mediated re-activation of the pathway, triple combination trials of BRAF, MEK and EGFR inhibitors are on-going, but the response is underwhelming.

To find alternative combination strategies that could deepen therapeutic responses driven by a BRAFi and MEKi combination, we performed pooled shRNA screens under the treatment pressure of the dual combination of the BRAF inhibitor dabrafenib and MEK inhibitor trametinib. In some of the BRAF-mutant CRC models, we observed marked discrepancies in the therapeutic responses between in vitro and in vivo conditions. Therefore, shRNA screens were conducted in cancer cell lines grown both in vitro (i.e. 2D and 3D culture conditions) and in vivo in xenograft tumor models. The aim of the study was to identify novel targets to combine with BRAFi/MEKi, and to compare the results of the screens preformed in vitro and in vivo.

The biggest technical challenge for an in vivo pooled screening approach is achieving adequate library representation after the bottleneck of cell implantation and engraftment in mice. Our in vivo screen had an additional bottleneck due to the dabrafenib/trametinib combination treatment. Therefore, by performing a pilot screen with the BRAF-mutant cell line model HT29 we aimed to address two questions: 1) whether the in vivo screen under treatment pressure would be technically feasible and 2) if novel combination partners to dabrafenib/trametinib would be identified to potentially improve efficacy beyond that observed with the triple combination with EGFR inhibitors.

We were able to achieve comparable intra-group variability and repeatability between in vitro and in vivo conditions, whereby gene level analysis revealed several differential hits between the two conditions, which were both sensitizers and activators to the dabrafenib/trametinib combination treatment. We identified targets specific for the in vivo condition that had not been identified in vitro and vice versa. Thus, in vivo screening may identify powerful hits that would not be realized by in vitro investigations. With success of this pilot effort, the screen is currently being expanded into additional BRAF-mutant CRC models.

#395

Aberrant HER2 signaling is a therapeutic target in a subset of castration-resistant prostate cancer.

Joshua W. Russo,1 Xin Gao,2 David J. Einstein,1 Glenn J. Bubley,1 Steven P. Balk1. 1 _Beth Israel Deaconess Medical Center, Boston, MA;_ 2 _Massachusetts General Hospital Cancer Center, Boston, MA_.

Background: Aberrant HER2 signaling promotes androgen receptor activity and is associated with 18-25% of castration-resistant prostate cancer (CRPC). We present preclinical and clinical data supporting signaling by canonical HER3/HER2 interaction and a constitutively active splice variant of HER2 (d16HER2) as potential oncogenic drivers of CRPC. Further, we provide preclinical evidence that increased HER2 signaling may be targeted with irreversible HER2-targeted tyrosine kinase inhibitors (TKIs) to block tumor growth.

Methods: Human-derived VCaP prostate cancer xenografts were serially biopsied prior to castration, at castration-resistance, and after resistance to abiraterone + enzalutamide (Abi/Enza). Biopsies were submitted for RNA sequencing and RT-PCR, immunohistochemistry (IHC), and reverse-phase protein array (RPPA) for validation of gene/protein expression changes. Castration-resistant VCaP and LuCaP-70CR xenografts were treated with reversible EGFR/HER2 TKI lapatinib (100 mg/kg) or irreversible pan-HER TKIs afatinib (20 mg/kg) or neratinib (20 mg/kg). Transcript levels of total HER2 and d16HER2 variant were determined for TCGA and SU2C prostate cancer (PCa) datasets. IHC for phospho-HER2 (Tyr1221/1222) and phospho-HER3 (Tyr1289) were performed on tumor samples from 49 heavily-treated metastatic CRPC (mCRPC) patients and compared to 18 hormone-naïve prostatectomy samples.

Results: VCaP xenografts exhibit increased activation of HER3/HER2 signaling and increased expression of d16HER2 with progression through castration and Abi/Enza therapy. Increases in pHER2 and pHER3 were observed by IHC and RPPA in serial biopsies of individual tumors, and increases in total HER2, wtHER2, and d16HER2 transcripts were observed by RT-PCR. Castration-resistant VCaP and LuCaP-70CR xenografts rapidly become resistant to lapatinib within 5 days, while the irreversible pan-HER TKIs inhibited tumor growth for at least 15 days. Analysis of TCGA (mostly hormone-naïve PCa) and SU2C (mCRPC) RNAseq datasets shows enrichment of tumors expressing d16HER2 in mCRPC. pHER3/pHER2 IHC in patient tumor specimens showed 9/49 (18%) mCRPC tumors exhibited concurrent pHER3/pHER2 staining compared to 1/18 (6%) of hormone-naïve PCa. An additional 4/49 (8%) mCRPC tumors stained positive for pHER2 alone, possibly representing d16HER2 activity.

Conclusions: HER2 overexpression and d16HER2 expression are associated with PCa progression, castration-resistance, and resistance to second-line hormonal therapy. HER2-targeted therapy with irreversible TKIs is more effective at inhibiting CRPC growth than the reversible TKI lapatinib. Our data indicate a subset of CRPC with aberrant HER2 signaling may be clinically actionable, and further studies to investigate potential treatment with an irreversible pan-HER TKI such as neratinib in biomarker-selected metastatic CRPC patients is warranted.

#396

**Combined inhibition of DDR1with CDK4/6 induced synergistic effect in ER-positive, HER2-negative breast cancer having** PIK3CA/AKT1 **mutations.**

Maryam Shariati, Funda Meric-Bernstam. _Md Anderson Cancer Center, Houston, TX_.

Molecular alterations in the PI3K/AKT/mTOR pathway occur frequently in estrogen receptor-positive (ER-positive) invasive breast tumors. Patients with ER-positive, human epidermal growth factor receptor 2-negative (HER2-negative) advanced or metastatic breast cancer (MBC) are currently treated with palbociclib, CDK4/6 inhibitor, in combination with endocrine therapy. However the contributions of PI3K pathway genomic alterations including PIK3CA and AKT1 to palbociclib, an FDA approved drug, sensitivity remain unclear. In this study we demonstrated that the efficacy of CDK4/6 inhibitor is reduced in the presence of PIK3CA/AKT1 mutations using ER-positive isogenic breast cancer cell line variants. In addition, utilizing a shRNA library screening targeting 530 cancer related human kinases, we identified that genomic suppression of discoidin domain receptor 1 (DDR1), a tyrosine kinase being active in various cancers, induced a significant growth reduction. We also found that DDR1 pharmacological inhibitor, 7rh benzamide with potent DDR1 inhibition in preclinical models, enhanced the sensitivity of PIK3CA/AKT1 mutant cells to palbociclib. Our data indicate that the combined targeting of DDR1 and CDK4/6 could be an effective rational strategy approach for targeted therapy of ER-positive, HER2-negative breast cancer harboring PIK3CA/AKT1 mutations.

#397

SMARCA4 is a selective vulnerability in SMARCA2-deficient esophageal squamous cell carcinoma cell lines.

Katharina Ehrenhöfer-Wölfer, Teresa Puchner, Silvia Blaha-Ostermann, Alexandra Hörmann, Wolfgang Sommergruber, Norbert Schweifer, Thomas Zichner, Andreas Schlattl, Ralph A. Neumüller, Manfred Koegl, Mark P. Petronczki, Mark Pearson, Simon Wöhrle. _Boehringer Ingelheim, Vienna, Austria_.

Esophageal squamous cell carcinoma (ESCC) is a highly prevalent tumor type with poor prognosis and limited treatment options. In this study, we aimed to discover potential novel drug targets using a pooled epigenome sgRNA viability screen in a panel of ESCC cell models. We identify the BAF chromatin remodeling helicase subunit SMARCA4/BRG1 as a novel dependency in a subset of ESCC cell lines. In line with the established synthetic lethal interaction of SMARCA4 and SMARCA2/BRM, the two mutual exclusive catalytic subunits of the BAF complex, SMARCA4-dependent cell lines display low or absent expression of SMARCA2. In rescue studies using SMARCA4 variants, we demonstrate that SMARCA4-dependency is linked to its ATPase/helicase activity, but not to bromodomain function. Similarly, ectopic expression of wild-type and bromodomain-mutant SMARCA2, but not a helicase-dead variant, rescues from loss of SMARCA4 in SMARCA2-low ESCC cells. SMARCA2-proficient ESCC cell models are rendered SMARCA4-dependent upon CRISPR/Cas9-mediated knock-out of SMARCA2. We further identify SMARCA4-dependent cell models from additional tumor types (colon, ovarian and pancreas carcinoma) with low or absent SMARCA2 expression. These findings expand the concept of SMARCA2/SMARCA4 paralog dependency and indicate pharmacological inhibition of SMARCA4 as a novel opportunity for targeted therapy of SMARCA2-deficient cancers.

#398

Overriding drug resistance in Philadelphia chromosome-positive acute B lymphoblastic leukemia by targeting both BCR-ABL and MAP4K signaling pathways.

Ruibao Ren, Xinhua Xiao, Jianming Zhang, Bo Jiao. _Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China_.

The Philadelphia chromosome (Ph) translocation, which leads to creation of a fusion gene product BCR-ABL, is found in chronic myelogenous leukemia (CML) and 20% of adult and 2-5% pediatric acute B-lymphoblastic leukemia (B-ALL). The selective ABL tyrosine kinase inhibitor (TKI) has been shown a remarkable clinical activity in patients with CML. The ABL TKI has also improved the clinical outcome of Ph+ B-ALL dramatically in combination with standard chemotherapy, but the morbidity and mortality of this disease remain high. The existence of ABL inhibitor-resistant leukemia cells in these patients indicates the presence of additional oncogenic pathways in Ph+ B-ALL. Discovery of a new therapeutic strategy to eradicate Ph+ B-ALL cells is, therefore, urgently needed. By using a chemical genetics approach, we identified a novel small molecule, AR4-13, which shows robust ABL kinase inhibition and potent pharmaceutical efficacy in Ph+ leukemias among 1094 cell lines from the Cancer Cell Line Encyclopedia (CCLE) project. Interestingly, AR4-13 possesses preferable potency than Dasatinib, a potent dual SRC/ABL TKI approved to treat CML and Ph+ B-ALL, to kill Ph+ B-ALL cells, but not do so in CML cells, both in vitro and in vivo. In particular, AR4-13 inhibits S6 and p38 phosphorylation much more potently than that of Dasatinib. Furthermore, kinome profiling data indicate that AR4-13 also inhibits Mitogen-activated protein kinase (MAPK) kinase kinase kinases (MAP4Ks), which belong to a kinase family that controls the MAPK signaling in lymphoid cells. Consistently, RNAi-based synthetic lethal screen with TKIs show that suppression of MAP4Ks could significantly enhance the sensitivity to ABL TKIs in Ph+ B-ALL cells, implying that MAP4K family kinases function as a parallel pathway to BCR-ABL signaling and confer ABL TKI resistance in Ph+ B-ALL. Our findings indicate that simultaneous targeting both the ABL and MAP4K signaling pathways might serve as a promising TKI therapeutic strategy for Ph+ B-ALL. 

## CLINICAL RESEARCH

### Circulating and Cell-free Biomarkers for Diagnosis and Monitoring of Cancer 1

#399

ctDNA a promising predictive marker for treatment with PD-1 inhibitors in KRAS mutated NSCLC after platinum based chemotherapy.

T.Jeroen.N. Hiltermann, Lucie B.M. Hijmering-Kappelle, Maria L. Aguirre Azpurua, Anthonie.J. vd Wekken, Birgitta.I. Hiddinga, Wim Timens, Harry.J.M. Groen, Ed Schuuring. _UMCG, Groningen, Netherlands_.

Introduction: Immunotherapy is a breakthrough in non-small cell lung cancer (NSCLC), showing long lasting responses in metastasized disease. However, only a limited number of patients responds to treatment. Selection of patients by known biomarkers (eg PD-L1, TMB) and combining PD-(L)1 inhibitors with other treatments (eg. chemotherapy or CTLA4 inhibitors) enriches the population for responders, however still around 60% do not respond with a durable response. We hypothesized that in KRAS mutant positive advanced NSCLC the detection of changes of ctDNA levels will be a relevant predictive marker in immunotherapy ( and stronger than eg PD-L1 expression in tumor tissue)..

Purpose: To test ctDNA as an early predictive marker for durable response in KRAS mutated lung cancer, in patients treated with PD-1 inhibitors after progression on platinum containing chemotherapy.

Methods: Patients with advanced NSCLC treated with nivolumab with a known KRAS or BRAF mutation from primary biopsy, with disease progression after platinum based chemotherapy are eligible. ctDNA obtained from EDTA plasma at baseline, 1, 2, 4 , 6 weeks and thereafter every 12 weeks compared to durable response as primary outcome (ie. benefit from treatment> 6month). Secondary outcomes were tumor response according to RECISTv1.1, progression-free and overall survival. ctDNA was determined and analyzed using a digital droplet PCR assay (Biorad) that was confirmed by a second technique (Idylla). ctDNA was categorized according to its response pattern in time: increasing, decreasing and undetectable KRAS mutant allele frequencies over time.

Results: In this ongoing study 31 patients with a known KRAS and 2 patients with a BRAF mutation participated. 24 (72%) had detectable levels of KRAS (n=22) or BRAF (n=2) in plasma. Concordance between Idylla and ddPCR was good and correlation was high (R2 =0,52). Of first 27 patients ctDNA increasing n=11, decreasing n=10, no change n=6. Data on all included patients will be presented.

Conclusion: Early detection of decreasing KRAS/BRAF mutant alleles in ctDNA predicted durable tumor responses, and increasing levels were associated with early disease progression.

#400

Digital PCR combined with melting curve analysis for highly multiplexed genotyping.

Junko Tanaka,1 Tatsuo Nakagawa,1 Akiko Shiratori,1 Yuzuru Shimazaki,1 Kunio Harada,1 Chihiro Uematsu,1 Kohara Yoshinobu,1 Erina Takai,2 Shinichi Yachida2. 1 _Hitachi, Ltd., Tokyo, Japan;_ 2 _Osaka University, Tokyo, Japan_.

Background

Digital PCR (dPCR) is a promising method for liquid biopsies and is able to quantify nucleic acids more sensitively than real-time PCR. However, dPCR has a large fluctuation in the fluorescence intensity of the droplets or wells due to insufficient PCR in the small partitions, limiting the multiplexing capability of using the fluorescence intensity. In this study, we propose a new measurement method that combines dPCR with melting curve analysis for highly multiplexed genotyping.

Methods

A sample is digitized into a silicon chip with up to ~104 wells in which asymmetric PCR was performed to obtain more single-stranded amplicons that are complimentary to molecular beacon probes. Fluorescence images were captured while controlling the temperature of the chip, and melting curve was measured for each well. Then, genotyping was performed by using the fluorescence intensity, dye color of the probe, and melting temperature (Tm). Because the Tm of the PCR products does not considerably depend on the amplification efficiency of PCR, genotyping accuracy is improved by using Tm values, enabling highly multiplexed genotyping.

Results

The concept was confirmed by measuring wildtype KRAS and its mutants, G12D, G12R, G12V, and G13D. The results showed that the peaks of the Tm distributions of the DNA groups were 69.2°C and 62.8°C for the wildtype and G13D with the first color dye (HEX), in addition to 66.9°C, 63.5°C, and 59.1°C for G12R, G12D, and G12V, respectively, with the second color dye (FAM). By combining the fluorescence intensities for HEX, FAM and the Tm values, the wildtype and mutant groups were clearly identified and able to be quantified. Furthermore, genotyping of cfDNA of pancreatic cancer patients was successfully performed by this method.

Conclusion

We have proposed a new measurement method that combines dPCR with melting curve analysis, and successfully performed the multiplexed genotyping assay. To our knowledge, this is the first demonstration of genotyping of five DNA groups with a single mutation of cancer-related genes by combining dPCR with melting curve analysis.

#401

Clinical impact of viable circulating tumor cells (v-CTC) detection and PD-L1 expression on v-CTC in the patients with resectable pancreatic cancer.

Masahiro Tanemura,1 Kenta Furukawa,1 Manabu Mikamori,1 Takurou Saito,1 Masahisa Otsuka,1 Yozo Suzuki,1 Kentaro Kishi,1 Hironao Yasuoka,1 Masahiko Tsujimoto,1 Yasuo Urata,2 Hiroki Akamatsu1. 1 _Osaka Police Hospital, Osaka, Japan;_ 2 _Oncolys BioPharma Inc., Tokyo, Japan_.

[Introduction] The capture and analysis of CTCs as "liquid biopsy" provides the possibility to avoid invasive biopsies, obvious implications in cancer diagnosis and staging. We tested novel methods for viable CTCs (v-CTC) isolation in the patients (pts) with pancreatic cancer (PC), and investigated the clinical potential of v-CTCs in prognosis. We analyzed the PD-L1(L1) expression in both PC tumors and v-CTCs.

[Pts and Methods] 7.5 ml of venous blood was collected prospectively from 39 PC pts, either surgery first (S group) or pre-treatment, consisted of Gem:800mg/m2; and S-1:80mg/m2 given concurrently with IMRT to 60Gy (NACRT:N group). To detect v-CTCs, we employed a telomerase-specific replication-selective adenovirous expressing GFP. For S group, samples were obtained before/after resection. For N group, samples were obtained before/after NACRT and after resection. To distinguish between leucocyte and cells with either epithelial or mesenchymal origin, cells were stained by anti-CD45, anti-Cytokeratin and anti-Vimentin Abs. GFP-positive and CD45-negative cells were counted as v-CTC. To assess L1 expression in PC tissues (IHC) and v-CTCs, L1 IHC kit (22C3, for tissues) and anti-human L1 mAb(MIH1,for CTCs) were employed.

[Results]S group: 24 pts aged 53~85 years (male/female=12/12) were enrolled. 24 pts underwent curative resection. No v-CTCs were detected in 6 pts at both before and after resection, and 5 of 6 pts survived without recurrence. V-CTCs were identified in 18 of 24 pts, and 13 of 18 pts developed liver metastasis. Marked decrease of CTC counts were seen after resection in 10 of 18 pts, but 9 pts developed recurrence. N group: 15 PC pts aged 44~77 years (male/female=4/11) were enrolled. 15 pts underwent curative resection. No v-CTCs were detected in 5 pts, and 5 pts survived without recurrence. V-CTCs was identified in 10 of 15 pts, and only 3 out of 10 pts developed disease recurrence. Marked increase in CTC counts was observed after NACRT in 5 of 6 CTC-positive pts before NACRT, and 3 of 5 pts developed liver metastasis and died. NACRT may induce tumor cell dissemination into the blood circulation for CTC-positive pts. PD-L1 expression: L1 expression were assessed for 21 pts (S group:18, N group: 3). No patients with IHC-L1 high expression (≥50%) were observed (<1% [negative]:9, 10%:6, 20%:4, 40%:2). For S group, IHC-L1 low +/CTC-L1 + were detected in 12/18 pts (66%). IHC-L1-/CTC-L1+ were detected in 3/18 pts (17%). IHC-L1 - /no detectable CTCs were detected in 3/18 pts (17%). For N group, IHC-L1 - were detected in 3 pts, but CTCs-L1 + were detected in 2 pts (1 pt had no CTCs). The majority of detectable v-CTCs were positive for L1-expression (89% [92/103 CTCs]), despite L1-negative or -low expression in PC tissues.

[Conclusions] Viable CTC detection appears as a promising prognostic marker. Immunotherapy with anti-PD-1/PD-L1 Abs may effectively target v-CTCs.

#402

RNA-sequencing of plasma exosomes reveals specific transcriptomic profiles in patients with hepatocellular carcinoma.

Johann von Felden, Amanda J. Craig, Mehmet Eren Ahsen, Ismail Labgaa, Delia D'Avola, Gabriela Hernandez Meza, Kimaada Allette, Navneet Dogra, Teresa Garcia Lezana, Parissa Tabrizian, Amon Asgharpour, Douglas Dieterich, Robert Sebra, Gustavo Stolovitzky, Myron Schwartz, Augusto Villanueva. _Icahn School of Medicine at Mount Sinai, New York City, NY_.

Hepatocellular carcinoma (HCC) is the tumor with the highest increase in incidence and mortality in the last 20 years. Exosomes are nano-sized particles loaded with nucleic acids and proteins involved in cell-to-cell communication. The aims of this study are: 1) to establish a method to isolate exosomes from plasma of HCC patients, and 2) to detect de-regulated transcripts in exosomes from HCC patients compared to controls.

In this pilot study, we included 10 HCC patients and 5 controls at high-risk of HCC development (i.e. patients with cirrhosis and/or chronic hepatitis B). Plasma exosomes were isolated by differential ultracentrifugation. Presence of exosomes was confirmed by morphological assessment with electron microscopy, nanoparticle tracking analysis and immune-labeling of EV specific markers (CD9, CD63, CD81, and TSG101) with ExoView and Western Blotting. RNA from exosomes was extracted and quantified with Bioanalyzer. Library preparation was done with SMARTer smRNA-Seq kit and whole transcriptome sequencing was performed on an Illumina HiSeq2500 instrument. Differential gene expression was done with LIMMA and enrichment analyses (i.e., gene ontology) with Enrichr.

HCC patients and controls were matched for age (median 67 vs. 63 yrs.), sex (70% vs. 67% males), and etiology (viral 70% vs. 67%). Sequencing reads obtained from exosomes mapped mRNA, lincRNA and small RNA, including miRNAs and Y-RNAs. Compartment ontology enrichment analysis of the top 250 expressed genes matched to exosomes and membrane-bounded vesicles (FDR<0.01). Genes from hepatic lineage were among the top expressed genes including ALB, BAAT, CRP, C3/5, FGA/B, CYP and APO family. This confirms the presence of liver-derived exosomes in human plasma. We identified 252 differentially expressed genes between HCC and controls (FDR<0.05). Among them, we found genes with known aberrant expression in HCC tissue samples such as ARF1 (Fold-change [FC]: 5 fold up-regulated), CDKN2A (FC:-3.2), H3F3A (FC:8) or TAGLN2 (FC:7). Among the 35 highest differentially expressed genes (FC>8) between HCC and controls, there was an enrichment in previously unreported Y-RNAs (37%, FDR<0.001) and microRNAs (11%, FDR<0.001).

We conclude that whole transcriptomic analysis of plasma exosomes is feasible in patients with HCC. Our pilot study detects significant differences in the transcriptomic profiles of HCC patients compared to controls, including an enrichment of upregulated miRNAs and Y-RNAs.

#403

COX-2 expression on circulating tumor cells in nasopharyngeal carcinoma predicts a worse survival: A prospective analysis.

Yongjiang Li, Xiaoqi Xie, Feng Wang. _West China Hospital, Chengdu, China_.

COX-2 expression on circulating tumor cells in nasopharyngeal carcinoma predicts a worse survival: A prospective analysis

Yong-Jiang Li1, Xiao-Qi Xie2, Feng Wang1*

1Department of Oncology, West China Hospital, Sichuan University, Chengdu, PR China

2Department of Critical Care Medicine, West China Hospital, Sichuan University, Chengdu, PR China

*Corresponding author

Short title: COX-2 Expression on CTCs in NPC patients

Abstract

Background and purpose: The prognostic significance of circulating tumor cells (CTCs) in head and neck cancer is still under debate, as only a few studies have been reported and limited conclusions are reached. Besides, CTCs count alone was utilized as an indicator in the previous researches. As a form of 'liquid biopsy', the further identification of genetic or phenotypic biomarkers on CTCs could possibly provide further clinical significance.

Materials and methods: The prospective study enrolled 131 patients with nasopharyngeal carcinoma (NPC). CTCs were isolated at baseline and at the end of concurrent chemoradiotherapy, and cyclooxygenase-2 (COX-2) expression status of CTCs was detected by RNA-in situ hybridization (ISH) method. Results were correlated with patient's clinicopathological parameters and treatment outcomes. Univariate and multivariate survival analysis were performed to determine the prognostic significance.

Results: COX-2 expression was found in 87/131 (66.4%) patients at baseline and 53/115 (46.1%) patients at the end of concurrent chemoradiotherapy. Patients with post-therapeutic COX-2 expression had significantly poorer treatment response (P=0.011) and higher risk of tumor relapse (P=0.026) and metastasis (P=0.007). Besides, multivariate analysis revealed that post-therapeutic COX-2 expression on CTCs remained an independent prognostic indicator for poorer

progression-free survival (HR 2.17, P=0.019) and overall survival (HR 2.41, P=0.024).

Conclusion: The study demonstrated that post-therapeutic COX-2 expression on CTCs was a novel and promising prognostic indicator for NPC patients. Future studies are needed to validate our findings and further clarify the value of integrating the indicator with current clinical strategies in improving survival of NPC patients.

#404

Prognostic value of soluble and cell surface immune-checkpoint molecules in advanced non-small cell lung cancer (NSCLC) patients receiving anti-PD-1/-L1 immunotherapy.

Imad Tarhoni,1 Mary Jo Fidler,1 Ibtihaj Fughhi,1 Connor Wakefield,1 Revathi Kollipara,1 Maneet Multani,1 Marta Batus,1 Sanjib Basu,1 Wen-Rong Lie,2 Donna Russell,2 Jeffrey Martinson,1 Alan L. Landay,1 Timothy M. Kuzel,1 Philip Bonomi,1 Jeffrey A. Borgia1. 1 _Rush University Medical Center, Chicago, IL;_ 2 _Millipore Sigma, Burlington, MA_.

Current methods to stratify immunotherapy candidates, including PD-L1 expression and tumor mutational burden (TMB) profiling, have limitations that hamper their clinical value. This study explores the prognostic potential of soluble and cell-surface immune-checkpoint (IC) molecules as a means to improve treatment selection for NSCLC patients being considered for PD-1/-L1 blockade.

Pretreatment sera from 111 cases of previously-treated advanced NSCLC receiving PD-1/L-1 targeting checkpoint inhibitors (nivolumab, atezolizumab, or pembrolizumab) were evaluated for 16 soluble IC molecules and immune regulators via the MILLIPLEX® MAP Human Immuno-Oncology Checkpoint Protein Panel (MilliporeSigma) using manufacturer-defined protocols. PBMCs from a subset of this cohort (n=28) were profiled on a LSRFortessa™ for cell-surface IC molecules. T-cell subsets (CD4,CD8) were analyzed for CD27\CD28, iCOS, PD-1, CTLA-4, TIM-3, TIGIT, LAG-3, GITR and PD-L1. NK cells (CD56\CD16) were analyzed for CD96, CD94, CD86, CD80, PD-1, CTLA-4, TIM-3, TIGIT, LAG-3 and PD-L1. Classical (CD14++\CD16-), intermediate (CD14++\CD16+) and non-classical (CD14dim\\-\CD16++) monocyte subsets were analyzed for these identical markers. All statistical relationships were determined using the Log-Rank test in relation to overall survival (OS) and progression-free survival (PFS). Pearson correlations were calculated between the circulating and PBMCs levels of IC molecules.

Sixteen soluble IC molecules were detectable in patient serum, with twelve of these being prognostic for OS, PFS, or both (see Table). In the 28 patients with PBMCs, a set of cell-surface molecules showed prognostic value, but had no apparent correlation to soluble molecules.

These findings suggest that both soluble and cell-surface IC molecules may have promise for identifying advanced NSCLC patients that may benefit from anti-PD-1/-L1 immunotherapy. | PFS | OS

---|---|---

|

p-value | HR | p-value | HR

Soluble molecules

BMI | 0.003 | 0.79 | 0.054 | 0.75

Lymphocyte | 0.004 | 0.70 | 0.002 | 0.60

NLR | 0.005 | 1.69 | <0.001 | 2.12

BTLA | 0.014 | 0.79 | 0.01 | 0.90

CD27 | 0.002 | 0.68 | 0.017 | 0.80

TIM3 | 0.037 | 0.88 | 0.028 | 0.93

HVEM | 0.004 | 0.62 | 0.004 | 0.53

GITR | 0.001 | 0.003 | <0.001 | 0.00

LAG3 | 0.040 | 0.82 | 0.03 | 0.72

GITRL | 0.014 | 0.10 | 0.007 | 0.03

PD1 | 0.002 | 0.49 | 0.003 | 0.34

CTLA4 | <0.001 | 0.18 | 0.002 | 0.27

CD80 | 0.001 | 0.68 | 0.002 | 0.67

CD86 | <0.001 | 0.63 | <0.001 | 0.54

iCOS | 0.014 | 0.84 | 0.047 | 0.90

PBMCs - cell surface molecules

CD3 | 0.052 | 0.66 | 0.11 | 0.65

CD3-CD80 | 0.37 | 0.99 | 0.005 | 1.45

CD4-iCOS | 0.05 | 0.74 | 0.49 | 1.35

CD8-CTLA4 | 0.02 | 0.66 | 0.2 | 1.34

CD8-GITR | 0.023 | 0.49 | 0.3 | 0.70

CD8-TIGT | 0.02 | 0.63 | 0.37 | 0.78

CD8-LAG3 | 0.016 | 0.62 | 0.03 | 1.18

CL-CD86 | 0.36 | 0.90 | 0.05 | 0.56

CL-CD64 | 0.14 | 1.33 | 0.015 | 1.89

NC-CD64 | 0.026 | 1.52 | 0.002 | 1.74

NC-PDL2 | 0.038 | 2.84 | 0.35 | 1.52

NC-TIM3 | 0.003 | 2.07 | 0.04 | 1.19

#405

Using a ctDNA liquid biopsy assay for post-surgical serial monitoring and early detection of disease progression in advanced colorectal cancer patients.

Marek Minarik,1 Tereza Halkova,2 Anastasiya Semyakina,2 Barbora Belsanova,1 Jiri Pudil,3 Filip Pazdirek,4 Miroslav Levy,5 Jaromir Simsa,5 Miroslav Hoch,4 Miroslav Ryska,3 Lucie Benesova1. 1 _Genomac Research Institute Prague and Elphogene, a.s., Prague, Czech Republic;_ 2 _Genomac Research Institute Prague, Prague, Czech Republic;_ 3 _Military University Hospital, Prague, Czech Republic;_ 4 _Motol University Hospital, Prague, Czech Republic;_ 5 _Thomayer hospital, Prague, Czech Republic_.

Introduction: ctDNA liquid biopsy has been recognized as a useful approach to monitor effect of ongoing cancer treatment. The ctDNA dynamics is reflecting the overall tumor burden. Here we present a utility of a simple ctDNA assay for longitudinal monitoring of patients with advanced stages of colorectal cancer in conjunction with surgical treatment.

Patients and Methods: In a prospective setting the disease course of a total of 121 patients in Stage III and IV of colorectal cancer was monitored by ctDNA. A panel of somatic mutations was initially screened in primary and/or metastatic tissue. The found mutations were then traced in ctDNA from plasma acquired before surgery and during the subsequent days and months. In a subgroup of rectal cancers ctDNA was also analyzed prior and during the neoadjuvant chemoradiotherapy. The ctDNA levels were then correlated to the clinical parameters such as surgical radicality, disease relapse or response to anticancer therapy.Results: A brief overview of the ctDNA data is shown in Table I.

TABLE I: ctDNA status in patients monitored in this study

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Time of plasma sampling | ctDNA negative | ctDNA positive | sample not available

prior to surgery | 30 | 91 | 0

2 - 7 days after surgery | 30 (56%) | 24 (44%) | 37

3 months after surgery | 18 (54%) | 15 (46%) | 21

6 - 9 months after surgery | 8 (42%) | 11 (58%) | 14

12 - 14 months after surgery | 4 (31%) | 9 (69%) | 4

18 - 24 months after surgery | 2 (28%) | 5 (72%) | 6

A high correlation between the surgical radicality and appearance or absence of ctDNA after surgery was confirmed with 22 of 24 patients with R0 resection as ctDNA negative and 22 of 30 patients with R1/R2 resection as ctDNA positive. Of the 30 ctDNA-negative patients following surgery, 4 patients had ctDNA-detectable progression during the first 9 months and 12 during the next 14 months. At 4 occasions the ctDNA has outperformed CT imaging in detecting the progression. The anti-angiogenic therapy resulted in disappearance or decrease of ctDNA levels in 6 patients during the first month of treatment. In a subgroup of rectal cancers, neoadjuvant chemoradiotherapy resulted in rapid decrease of ctDNA already in the first week of administration. Conclusion: The ctDNA dynamics closely follows clinical course of disease and may serve as a useful biomarker in post-operative follow-up. (supported by AZV 15-27939A)

#406

Profiling plasma cell-free RNA (cfRNA) with the NanoString low input nCounter assay.

Chung-Ying (Alan) Huang,1 Sarah Warren,1 Megan Wurden,2 Tim Riordan,1 Rich Boykin,1 Mingdong Liu,1 David Henderson,1 Dan Hanson,1 Jay Gerlach,1 Joseph M. Beechem1. 1 _NanoString Technologies, Seattle, WA;_ 2 _California Polytechnic State University, San Luis Obispo, CA_.

Background: Liquid biopsy offers advantages over tissue biopsy for early disease diagnosis, monitoring of disease progression, and evaluation of therapeutic interventions because sample collection is rapid, easy, minimally invasive, and repeatable. In particular, measuring cell-free RNA (cfRNA) in the plasma offers the opportunity for direct monitoring of a tumor cell byproduct without the need for direct access to the tumor. However, robust cfRNA detection has been difficult historically due to weak and highly variable signal detection. The nCounter platform and low input profiling protocol have been adapted to enable robust and reproducible detection of cfRNA from plasma, which is demonstrated here in proof of concept experiments using synthetic targets and patient samples to quantify transcription of standard gene transcripts and fusion products.

Methods: The standard nCounter detection platform which leverages direct hybridization of optical barcodes to RNA transcripts of interest has been adapted to enable detection of rare targets by leveraging an initial targeted amplification step to increase transcript abundance with minimal bias of relative transcript abundance. Feasibility of this process to detect cfRNA was assessed with synthetic DNA oligos and commercially sourced patient-derived plasma samples. Plasma cfRNA was isolated and concentrated using commercial cfRNA kits. Relative expression of targets was benchmarked to universal reference RNA and compared between samples by both nCounter and qPCR.

Results: Using synthetic ultramer DNA oligo targets for 7 genes, the limit of detection of the assay was as low as 24 molecules per target with 100% specificity. Using cfRNA from healthy donors, the nCounter assay was shown to profile relative abundance of 40 targets. 9 genes were selected to benchmark nCounter against qPCR and showed high correlation (R2 = 0.84) with linearity (slope = 0.97). cfRNA from healthy donors or patients with advanced CRC or NSCLC was profiled by the 770-gene PanCancer IO360 panel with a pre-amplification by the adapted nanoString low RNA input protocol. Unsupervised hierarchical clustering revealed that CRC derived cfRNA had different transcriptional profiles from healthy donors, and NSCLC cfRNA had an intermediate profile. Furthermore, a number of therapeutically relevant targets, including EGF, EGFR, PD-1, and PD-L1 could be detected in cfRNA from cancer patients but not in cfRNA from healthy donors.

Conclusion: The nCounter low input protocol can be used for transcriptional profiling of cfRNA from healthy and diseased patients with high sensitivity and specificity. The flexible primer design strategy enables profiling of a variety of transcripts, including fusion gene products, with the possibility of profiling up to 800 transcripts from a single sample. In summary, the nCounter platform (RUO) provides a robust flexible molecular profiling tool for liquid biopsy research.

#407

Gene expression and mutation profiles of single lung circulating tumor cells.

Sarah Owen, Ting-Wen Lo, Shamileh Fouladdel, Ebrahim Azizi, Nithya Ramnath, Sunitha Nagrath. _University of Michigan, Ann Arbor, MI_.

In the era of personalized medicine, many targeted therapies have been developed against cancer-specific antigens, which have greatly improved patient outcome. The efficacy of targeted therapies was first shown in patients with non-small cell lung cancer (NSCLC) containing activating epidermal growth factor receptor (EGFR) mutations who responded to tyrosine kinase inhibitors (TKIs). To determine if a patient qualifies for targeted therapy, the patient must undergo a tumor biopsy. Usually done at the time of diagnosis, this single time-point examination dictates the treatment plan. Despite strong initial response to TKI therapy, nearly all patients develop resistance, most commonly by acquiring a secondary EGFR mutation. At the time of halted response, an additional biopsy may be necessary to screen for the presence of these secondary, resistance-causing mutations.

Circulating tumor cells (CTCs) are cancer cells present in the blood that provide an alternative to an invasive tumor biopsy. Shed from the primary tumor, CTCs circulate throughout the vasculature, and extravasate leading to metastasis. CTCs have been shown to carry tumor-matched characteristics in both genotype and phenotype. Easily accessed through a blood draw, they can be used to serially track patients throughout treatment to detect the early emergence of new tumor clones.

One of the biggest hurdles with CTCs being clinical prognostic markers is their low abundance in the blood. We have previously developed a high-throughput, label-free microfluidic technology, the Labyrinth, to isolate CTCs from the blood of breast, lung, and pancreatic cancer patients. This technology allows for quick and efficient isolation of CTCs from the millions of surrounding blood cells. After isolation, the CTCs are used for enumeration or single cell analysis. The single cell gene expression analysis performs a highly multiplexed RT-qPCR of a pre-designed 96 gene panel.

Here we report a complementary single cell analysis approach to detect the presence of EGFR point mutations, L858R and T790M, implicated in TKI response using digital PCR (dPCR). dPCR is a highly sensitive approach which enables detection and quantification a single-cell level. dPCR relies on small sample loading and partitions the sample into individual PCR reaction droplets. This single molecule resolution provides the ability to distinguish if a cell is homo- or heterozygous for a mutation as well as their relative expression levels. We have validated this system for single cell analysis using lung cancer cell lines with known L858R and T790M mutation status. We present initial findings of EGFR mutation screening of lung CTCs from patients with known EGFR status based on tissue profiling.

By tracking patients over time, comparing the CTC profiles, and correlating it to patient outcomes, we may be able to better identify early treatment resistant clones and adjust their treatment to improve patient outcome.

#408

Expression of CCR5 associated with HER2 in circulating tumor cells (CTCs) is a novel biomarker for patients with metastatic breast cancer (MBC).

Qiang Zhang,1 Lorezo Gerratana,2 Ami N. Shah,3 Andrew A. Davis,3 Lisa Flaum,3 Youbin Zhang,3 Richard G. Pestell,4 Firas Wehbe,3 Amir Behdad,3 Leonidas Platanias,1 William Gradishar,1 Massimo Cristofanilli1. 1 _Northwestern University, Lurie Cancer Center, Chicago, IL;_ 2 _Northwestern University, University of Udine (Italy), Chicago, IL;_ 3 _Northwestern University, Chicago, IL;_ 4 _Pennsylvania Cancer and Regenerative Medicine Research Center, Baruch S. Blumberg Institute, Pennsylvania Biotechnology Center, Wynnewood, IL_.

Introduction: The Chemokine C-C motif ligand 5 (CCL5) and its receptor 5 (CCR5) play a significant role in solid tumors, particularly triple negative breast cancer (TNBC) and HER2+ subtypes with prognostic implications. CCL5-CCR5 axis was reported to govern cancer stem cells expansion and play key role in MBC progression. Our group recently reported that overexpression of HER2 was associated with CTC-clusters which caused poor prognosis of patients with MBC. Herein, we first reported the expression of CCR5 in CTCs of MBC patients, and described the correlation between CCR5 and HER2 expression in CTCs.

Methods: Whole blood samples (7.5ml/each) were collected from stage III/IV MBC patients before systemic therapy. CTCs enumeration was performed in FDA approved CELLTRACKS ANALYZERII® System (Menarini) by using CTC Kit contains antibodies targeting the Epithelial Cell Adhesion Molecule (EpCAM) antigen for capturing CTCs, and immunofluorescent staining reagents including anti-CK-PE (specific for epithelial cells), DAPI (for nucleus), anti-CD45-APC (specific for leukocytes), and anti-HER-2/neu-FLU. The CTCs were classified as CK+, EpCAM+, DAPI+ and CD45-. After confirming CTCs were positive by CellSearch system, CCR5 expression were evaluated by using CXC kit for multiple staining includes anti-CCR5-PE (R&D Systems), Anti-CK-FITC, DAPI and Anti-CD45-APC. The correlation between HER2 expression and CCR5 expression was analyzed by Kruskal-Wallis test was used for statistics.

Results: CTCs were found positive (≥5) in all seven MBC patients with a range of numbers between 124 and 442, and HER expression was identified in 6 out 7 cases (between 57 and 149 total cells). The ratios of HER2+ CTCs/total CTCs were varied between 28.5% and 88.1%. Meanwhile, CCR5 expression were found in 3 out 7 patients (Group 1), with CCR5+ CTCs/ total CTCs ratios were 4.45%, 43.1% and 59.1% respectively. There were 4 patients without CCR5 expression in CTCs (Group 2). The average HER2+ CTCs was 127.6 with HER2\+ CTC/Total CTC as average of 59.4% in Group 1, which were significantly higher in compared with the corresponding numbers as 80.3 and 29.97% respectively in Group 2, which indicated that upregulation of CCR5 was positively associated with high level of HER2 expression in CTCs.

Conclusions: Our data provides the first evidence of strong expression of CCR5 in CTCs of MBC as potential new marker. The significant correlation between overexpression of HER2 in CTCs and high level of CCR5 indicated that CCR5 may contributes to more aggressive MBC subtypes with HER2 expression, which promotes carcinogenesis and metastasis at least partly by maintaining and increasing cancer stem cells leading to increased invasion. We conclude that the further understanding of the molecular interactions of CCR5 and HER2 in CTCs will be important to elucidate the mechanism of metastasis of MBC and predict prognosis.

#409

A novel algorithm to redefine blood-based tumor mutational burden for optimized prediction of clinical benefits from immunotherapy.

Zhijie Wang,1 Guoqiang Wang,2 Jianchun Duan,1 Jing Zhao,2 Zhengyi Zhao,2 Hua Bai,1 Shuhang Wang,1 Shangli Cai,3 Jie Wang1. 1 _State Key Laboratory of Molecular Oncology, Department of Medical Oncology, National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China; _2 _The Medical Department, 3D Medicines Inc., Shanghai, China;_ 3 _The Medical Department, 3D Medicines Inc. Shanghai, P.R. China, Shanghai, China_.

Purpose: Blood-based tumor mutational burden (bTMB) was recently identified as a predictor of progression free survival (PFS) benefit for non-small cell lung cancer (NSCLC) patients treated with atezolizumab over docetaxel. However, the reported bTMB failed to predict overall survival (OS) outcome from atezolizumab over docetaxel, or to predict PFS or OS benefit in the atezolizumab arm upon our analysis. We further revealed that bTMB correlates with maximum somatic allele frequency (MSAF), which is an independently negative prognostic factor and impeded the predictive value of bTMB. Here we aimed to explore a novel algorithm to eliminate the interference from MSAF in bTMB calculation and redefine bTMB to predict clinical benefits in both PFS and OS from immunotherapy.

Patients and Methods: Data from POPLAR (NCT01903993, N = 211) and OAK (NCT02008227, N = 642) trials was first adapted for algorithm development in bTMB redefinition. The derived algorithm was validated with another gene panel (NCC-GP150) in two independent NSCLC cohorts with blood samples (N = 184) and patients treated with mono-immunotherapy (N = 50).

Results: The previous reported bTMB-H (bTMB ≥ 16) was not associated with favorable PFS (P = 0.09, HR 0.81, 95% CI, 0.64-1.03) or OS (P = 0.68, HR 1.06; 95% CI, 0.81-1.38) in patients treated with immunotherapy, instead, it was associated with poor OS outcomes (HR 1.29, 95% CI, 1.02-1.63). Further analysis revealed that bTMB correlates with MSAF (Pearson r = 0.47), which is a negative prognostic biomarker. As a result, the predictive value of bTMB was impeded by the negative prognostic performance contributed by a high MSAF. After we redefined bTMB by filtering out mutations with AF > 5%, the correlation between redefined bTMB and MSAF was no longer observed (Pearson r = 0.09) and the negative prognostic property of bTMB was eliminated. More importantly, the redefined bTMB-H (cutoff=12) serves as an optimal predictor for both PFS and OS from atezolizumab over docetaxel as tested in POPLAR (PFS HR 0.41, 95% CI, 0.21-0.80; OS HR 0.34, 95% CI, 0.16-0.71) and validated in OAK (PFS HR 0.49, 95% CI, 0.33-0.71; OS HR 0.47, 95% CI, 0.29-0.77), respectively. Meanwhile, the redefined bTMB-H can identify patients with favorable PFS (HR 0.62, 95% CI, 0.47-0.80) and OS (HR 0.70, 95% CI, 0.52-0.95) in the immunotherapy arm. Consistent results were obtained when this algorithm was tested in our independent cohort (Pearson r reduced from 0.36 to 0.07; bTMB-H population with immunotherapy, PFS HR 0.28, 95% CI, 0.09-0.94).

Conclusion: MSAF plays a dominant role in previous reported bTMB and should be adjusted in bTMB calculation to remove the prognostic property contributed by MSAF. We have redefined bTMB and demonstrated the feasibility of the redefined bTMB as an actionable predictor of favorable PFS and OS from immunotherapy in future clinical practice.

#410

**Quantification and downstream analysis of circulating tumor cells isolated using CapioCyte** TM **liquid biopsy.**

Michael J. Poellmann,1 Jiyoon Bu,2 Dominic Moon,3 Kyle Wagner,3 Andrew Z. Wang,3 Seungpyo Hong1. 1 _Univ. of Wisconsin Madison, Madison, WI;_ 2 _University of Wisconsin Madison, Madison, WI;_ 3 _Univ. of North Carolina at Chapel Hill, Chapel Hill, NC_.

Background: Circulating tumor cells (CTCs) have great potential as biomarkers for the diagnosis and prognosis of many cancers. The CapioCyteTM chip isolates live CTCs through a unique combination of biomimetic cell rolling and nanoparticle-mediated multivalent immunorecognition. Recently-published work reported the highly sensitive and specific CTC capture from 24 patients in a pilot study [Myung et al., Clinical Cancer Research 2018, 24(11):2439-2547]. Here, we report results from additional cohorts of patients and demonstrate downstream analysis of captured CTCs.

Methods: Peripheral blood samples were collected from patients undergoing radiotherapy (RT) (n=12; oligometastic n = 5, head and neck cancer n = 1, non small cell lung cancer n = 2, prostate cancer n = 3, cervical cancer n = 1) or immunotherapy (n=4). Samples were processed the next day by CapioCyteTM chips designed for the immunoisolation cells expressing epithelial cell adhesion molecule (EpCAM), human epidermal growth factor-2 (HER-2), and epidermal growth factor receptor (EGFR). Captured CTCs were identified by immunocytochemistry as containing round nuclei, positive expression of cytokeratin, and negative expression of CD45. Select samples were recovered from the capture surface and submitted for single cell gene expression assays (RNASeq) using 10X Genomics Chromium barcoding and Illumina next-generation sequencing.

Results: Immunocytochemical staining identified CTCs in all pre-treatment blood samples from patients undergoing RT (mean 95 CTC/ml whole blood, SE 54, range 4-680) and immunotherapy (mean 70 CTC/ml whole blood, SE 16, range 39-104). CTC counts decreased with time points collected during and post-treatment, consistent with other measures of treatment progress. Single cell RNASeq confirmed the presence of tumor-derived cells in select samples and, importantly, demonstrated the ability to conduct downstream analysis of CTCs isolated on the CapioCyteTM chip.

Conclusions: The CapioCyteTM chip effectively captures CTCs for quantification and downstream analysis requiring viable cells such as RNASeq. The liquid biopsy technology has great potential to contribute to diagnosis and personalized treatment of cancer.

#411

Rapid and sensitive detection of EGFR C797S mutations using a blood-based droplet digital PCR assay.

Victoria Edwards, Leisa Jackson, Jordan Reese, Hestia S. Mellert, Gary Pestano. _Biodesix, Inc., Boulder, CO_.

The adoption of third-generation tyrosine kinase inhibitors (TKIs) to treat non-small cell lung cancer (NSCLC) for EGFR positive patient cases has led to the emergence of acquired resistance pathways. Approximately 40% of EGFRT790M-positive NSCLC cases display an acquired mutation of amino acid 797 (C797S) that mediates this resistance, which can develop after a median of 10 months following treatment. The effectiveness of treatment for patients with EGFRC797S may depend on whether the mutation is present in cis or trans with EGFRT790M, the type of sensitizing mutation (EGFRdel19 or EGFRL858R), and previous treatment(s). Patients with EGFRdel19 or EGFRL858R in conjunction with EGFRT790M and EGFRC797S remain resistant to all current targeted therapies. Several fourth-generation inhibitors are in development but have yet to be validated in clinical trials. We have developed a blood-based test that can detect two of the most common C797S nucleotide mutations (T>A and G>C) in cis or trans with EGFRT790M using the droplet digital PCR (ddPCR) technology and assays. Analytic sensitivity and specificity were assessed using synthetic DNA designed to mimic the EGFRC797S variants and their locus detected in the assay. Normal healthy donors as well as reference ddPCR positive and negative NSCLC donor samples were assessed for clinical specificity and sensitivity. Finally, the precision of the assay was evaluated with both clinical and analytical samples. Specifically, we evaluated the assay at high, medium, and low mutation frequencies over three consecutive days, including repeat runs on one day, and with multiple operators. This assay is capable of accurately and precisely detecting multiple EGFRC797S variants and their locus from blood specimens in the clinical laboratory. Consistent with other ddPCR blood-based EGFR variant assays we have developed, the limit of detection was 0.02%for C797S variants detected in both cis and trans with the T790M mutation. Once validated, the EGFR C797S assay described here may be of utility in diagnosing patients who have developed resistance to osimertinib.

#412

Application of a serial liquid biopsy ctDNA assay for monitoring efficacy of anti-angiogenic lung cancer therapy.

Marek Minarik,1 Martin Svaton,2 Barbora Belsanova,1 Anastasiya Semyakina,3 Jan Baxa,2 Ondrej Fiala,2 Milos Pesek,2 Lucie Benesova1. 1 _Genomac Research Institute Prague and Elphogene, a.s., Prague, Czech Republic;_ 2 _Faculty of Medicine in Pilsen, Charles University Hospital in Pilsen, Czech Republic., Pilsen, Czech Republic;_ 3 _Genomac Research Institute Prague, Prague, Czech Republic_.

INTRODUCTION: Patients in advanced stages of non-small cell lung carcinomas (NSCLC), who and unsuited for targeted biological therapy because of lack of actionable molecular predictors are frequently treated by anti-angiogenic therapy. The effectiveness of such therapy is primarily relying on imaging techniques including CT or hybrid PET/CT evaluating a combination of morphological factors (dimensions and volume) and, more recently, also functional parameters including the metabolic activity, tumor vascularization etc. The ability to track the course of the disease at the same time all of these parameters is prerequisite to enable timely monitoring and therapy change in case of an early detection of resistance. This study was aimed at utility of circulating-tumor DNA (ctDNA) as an tool for monitoring of therapy response. and assessment of phenotypic parameters of the tumor.

PATIENTS AND METHODS: A total of 50 patients with confirmed Stage IV lung adenocarcinomas showing negativity on ALK, BRAF, EGFR, RET, ROS1 and MET predictors were prospectively enrolled into the study. Patients were treated under a standard protocol by a combination of paclitaxes/carboplatin/bevacizumab and followed by dual PET/CT. For each patient cytology tissue sample was subjected to test for a panel of somatic mutations. The found mutations were subsequently detected in ctDNA in plasma extracted from peripheral blood collected prior to therapy start and then in 1-month intervals during the therapy. The occurrence and quantity of ctDNA were correlated with the objective therapy response (RECIST) and to the functional imaging parameters of metabolic activity and vascularization.

RESULTS: ctDNA was initially positive in 29 patients. ctDNA levels closely reflected the response to the therapy with complete or partial remission expressed as reduction or absence of ctDNA, while disease stabilization or progression signaled by persisting or increasing ctDNA levels. There was a borderline correlation between ctDNA and vascularization evaluated by dual PET/CT (p=0.055).

CONCLUSION: In a subset of patients ctDNA can readily serve as a surrogate marker for semi-continuous monitoring of the effect of anti-angiogenic therapy. Work supported by Czech Ministry of Health project AZV 17-30748A.

#413

Development of a Highly Customizable Platform for Variant Detection in Circulating Tumor DNA.

Matt Egleston, Paula Roberts, Ian Hoskins, Aaron Garnett, Laura Griffin, Abel Licon, Ryan Walters. _ArcherDX, Inc., Boulder, CO_.

Introduction: Variant detection in circulating tumor DNA (ctDNA) provides an unprecedented opportunity to study the mutational landscape of cancers with minimal patient invasiveness. Meanwhile, new variants providing information about drug response and disease prognosis are being rapidly discovered. Therefore, to fully harness the power of ctDNA variant detection it is critical to be able to rapidly modify the set of variants targeted in an assay. Here we present a platform for next generation sequencing-based ctDNA assays that allows researchers to readily customize their panel targets.

Methods: Anchored Multiplex PCR (AMP™) is a molecular biology technique that allows for near infinite modularity of assay targets while maintaining variant detection sensitivity. In order to ensure assay specificity, AMP utilizes molecular barcoded adapters (MBC) to uniquely label input molecules prior to amplification, enabling post-sequencing error correction. This increases analytical sensitivity by reducing background noise. We also developed bioinformatics methods, which allow the characterization of position-specific error levels. By combining molecular barcode-based error correction and error profile characterization, we can reliably detect low frequency variants with high specificity.

Results: We tested the modularity of the AMP ctDNA platform by creating ctDNA panels targeting either 6 kb of cancer variant hotspots or 34 kb of tumor suppressors plus the same 6kb of cancer hotspots (40 kb total target sequence). We then prepared and sequenced libraries using ctDNA-like inputs containing important cancer variants at low allele fractions (AF). We used these data to characterize the noise inherent in AMP library preparation and Illumina sequencing. We then performed a power analysis to measure the lowest AF at which we could detect variants at each target position given the noise level and sequencing coverage. Our results show that we can confidently detect variants with an AF of less than 0.18% in 50% of bases and 0.47% in 95% of bases covered in the 6kb panel. Variant detection sensitivity remained consistent over the 6kb of hotspot targets in the context of the 40kb panel (50% of bases powered to detect 0.17% AF variants and 95% powered to detect 0.45% variants). Across the full 40kb of the larger panel we had statistical power to call 0.23% AF variants at 50% of target bases and to call 0.56% variants at 95% of target bases. These data demonstrate that ctDNA panels can maintain assay performance upon panel modifications.

Conclusions: The modular nature of AMP ctDNA panels allows for endless combinations of targeted genes to maximize detection sensitivity in relevant regions while also maintaining assay performance. Our experience with AMP assays indicates that the range in the number of targets that can be included in a panel is quite large, allowing broad implementation of the assays developed on the AMP platform.

#414

Detection of AXL-expressing circulating tumor cells (CTCs) in non-small-cell lung cancer (NSCLC) patients using an automated microcavity array (MCA) system.

Mio Ikeda,1 Yasuhiro Koh,1 Shunsuke Teraoka,1 Jun Oyanagi,1 Kuninobu Kanai,1 Atsushi Hayata,1 Nahomi Tokudome,1 Hiroaki Akamatsu,1 Yuichi Ozawa,1 Keiichiro Akamatsu,1 Masayuki Higuchi,2 Masanori Nakanishi,1 Hiroki Ueda,1 Nobuyuki Yamamoto1. 1 _Wakayama medical university, Japan;_ 2 _Hitachi Chemical Co., Ltd., Japan_.

Purpose: Noninvasive diagnostics has been developed over the last decade and we have previously reported that CTCs can be utilized for evaluating molecular features of NSCLC. AXL, a receptor tyrosine kinase is linked to epithelial-to-mesenchymal transition (EMT) leading to cancer progression and regarded as a potential therapeutic target. However, many of current technologies rely on epithelial markers to detect CTCs and that makes it difficult to detect AXL-expressing CTCs. Here, we established the detection of AXL expression on CTCs using MCA system.

Methods: Preclinical experiments were performed using NSCLC cell lines H1299, PC9, and HCC827 and a breast cancer cell line MDA-MB231 with varying cytokeratin (CK) and AXL expression levels. The cells were spiked into 3 ml of peripheral blood from healthy donors, then enriched using MCA system, and detected by staining for CD45, DAPI, CK or vimentin (VM) with the addition staining for AXL. For clinical evaluation, 3ml of peripheral blood was collected from advanced NSCLC patients.

Results: DAPI-positive, CK or VM-positive, and CD45-negative cells were defined as CTCs. In spike-in experiments, when CK was used as a marker, AXL expression was detected in 5 and 17% of high CK-expressing HCC827 and PC9 cells, respectively and detected in 52 and 75% of low CK-expressing H1299 and MDA-MB231 cells, respectively. On the other hand, when VM was used as a marker, AXL expression was detected in 72 and 88% of high VM-expressing MDA-MB231 and H1299 cells, respectively whereas detected in 1 and 7% of PC9 and HCC827 cells with low VM expression, respectively. Twenty-four patients were enrolled in the clinical study. The patient characteristics were as follows: median age 69.5 years (range, 49-84); male 88%; stage III/IV, 25/75%; adenocarcinoma/ squamous cell carcinoma/ other, 63/33/4%. Both CK and VM staining in 17 patients, only CK in 6 patients, and only VM in 1 patient were performed. CK-positive single CTCs were detected in all patients (median, 4; range, 1-50) and AXL-expressing CK-positive single CTCs were detected in 26% of patients (median, 0; range, 0-1). On the other hand, VM-positive single CTCs were detected in 94% of patients (median, 6.5; range, 0-128) and AXL-expressing VM-positive single CTCs were detected in 89% of patients (median, 1; range, 0-106). Significantly more AXL-expressing single CTCs were detected in VM-positive than CK-positive (p < 0.001). Notably, all of identified CTC clusters were positive for only VM, not CK and AXL-expressing CTC clusters were detected in 33% of patients (median, 0; range, 0-22).

Conclusion: Our data indicate that incorporating VM staining is necessary to detect AXL-positive CTCs due to EMT. Further clinical evaluation is warranted for validating the method and the clinical significance of AXL-positive CTCs.

#415

Ultrasensitive analysis of circulating exosomes for liquid biopsy-based cancer diagnosis using 3D nano-engineered microfluidic chips.

Peng Zhang, Xin Zhou, Yong Zeng. _University of Kansas, Lawrence, KS_.

Liquid biopsy is extremely appealing in early diagnosis, prognosis, and precision treatment of cancer, as tissue biopsy is highly invasive, costly, and often infeasible to repeat. Extracellular vesicles (EVs), including exosomes, are emerging as a new paradigm of liquid biopsy for cancer diagnosis and monitoring response to therapy. Current affinity isolation and analysis methods rely on surface capture of EVs which is governed by the fundamental limits in mass transfer, surface binding reaction, and the boundary effects due to the hydrodynamic resistance at the liquid-solid interface. A major conceptual constraint in existing techniques is that they only partially addressed these fundamental factors. Here we report an innovative strategy for 3D nano-engineering of microfluidic chip to address the major limits in one device. This lithography-free engineering strategy exploits microfabricated channels to guide nanoparticle self-assembly, enabling simple, large-scale integration of self-assembled microelements with unique 3D nanostructures for biosensing. Using this method, we devised a nanoporous herringbone (nano-HB) chip that presents distinct advantages to address the aforementioned limits in one device, as it 1) effectively promotes microscale mass transfer; 2) increases the surface area and probe density to enhance binding efficiency and speed; and 3) reduces near-surface hydrodynamic resistance to promote particle-surface interactions for binding. Using the nano-HB chip, we demonstrated highly efficient (>80%) isolation of EVs purified from COLO-1, MCF-7, and SKOV3 cell lines spiked in healthy plasma. The nano-HB chip was further validated for elution and downstream analysis of captured EVs, i.e., targeted mRNA profiling by droplet digital PCR (ddPCR) and protein profiling by microplate ELISA. Assessed with spiked-in EV standards, the nano-HB chip was seen to afford an extremely low limit of detection of 10 μL-1 (200 vesicles per assay) and excellent compatibility with complex blood plasma. Such analytical capabilities enabled ultrasensitive and quantitative detection of low-level folate receptor alpha (FRα) positive exosomes in ovarian cancer patient plasma that are otherwise undetectable to standard assays. We demonstrated sensitive and specific detection of exosome subpopulations expressing CD24, EpCAM, and FRα proteins for non-invasive diagnosis of ovarian cancer using only 2 μL plasma. Comparative evaluation showed that our nano-HB chip greatly improves diagnostic power over the standard microplate ELISA and the combined nano-HB capture and ddPCR analysis of mRNAs. Our findings suggest exosomal FRα as a potential biomarker for early detection and monitoring of progression of ovarian cancer. Our technology would offer enabling exosome isolation and sensing abilities to facilitate liquid biopsy-based cancer diagnosis.

#416

Predictive significance of serum protein levels in advanced non-small-cell lung cancer patients treated with pembrolizumab.

Jun Oyanagi, Yasuhiro Koh, Shunsuke Teraoka, Kuninobu Kanai, Atsuhsi Hayata, Nahomi Tokudome, Hiroaki Akamatsu, Yuichi Ozawa, Keiichiro Akamatsu, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. _Wakayama Medical University, Wakayama, Japan_.

Background: We have previously reported that serum IL-8 and G-CSF were correlated with clinical benefit and immune-related adverse events (irAE), respectively in non-small-cell lung cancer (NSCLC) patients treated with nivolumab. Here, we measured multiple serum proteins serially in NSCLC patients treated with pembrolizumab and explored the potential of predicting clinical response or irAE onset. This study was registered at UMIN (ID: 000024414).

Patients and Methods: Advanced NSCLC patients received pembrolizumab monotherapy (200 mg/body, q3W) until progressive disease (PD) or unacceptable toxicity. Serum samples were collected at baseline and at week 6. Best response was classified into partial response (PR), stable disease (SD), or progressive disease (PD) according to RECIST v1.1. Using LuminexTM xMapTM technology, serum levels of 41 proteins consisting of cytokines, chemokines, growth factors, and angiogenesis factors were measured. All statistical analyses were carried out using JMP Pro software (ver. 13.0) and Mann-Whitney U test were performed accordingly. A p value <0.05 was considered as significant.

Results: Thirty-seven patients were registered in the study between March 2017 and October 2018 at Wakayama Medical University Hospital and 32 were included in the final analysis. Demographics of the patients were as follows: median age 70 (range, 50 to 91); male 75%; smoker 88%; stage III/IV, 22/78%; squamous/non-squamous, 31/69%, previous treatment; 0/1≤, 47/53%. Objective response rate was 28% and disease control rate was 56% in the entire cohort and 40% and 67%, respectively in the first-line subset. Among 41 serum proteins measured, no serum protein at baseline was associated with efficacy or irAE onset in the entire cohort. Levels of serum VEGF-C and sCD40L at baseline, however, in the first-line subset were found significantly lower in the patient who had PR and SD than those who did not. RANTES was also found significantly lower in patients who had PR in the 2nd or later line subset. With regard to irAE prediction, changes of HB-EGF levels between baseline and week 6 were significantly smaller in irAE patients in the entire cohort and changes of MCP-1 levels were significantly smaller in irAE patients in second or later line subset. The serum proteins identified in the current study were not overlapped with those identified in advanced NSCLC patients treated with nivolumab in the previous study.

Conclusions: We identified potential serum protein markers associated with clinical benefit and irAE from pembrolizumab treatment in advanced NSCLC by multi-analyte protein-based assay. Our results suggest that serum proteins associated with efficacy and irAE may vary among different anti-PD-1 antibodies and also between in the first-line setting and later one.

#417

Urine as an alternative to blood for germline genomics and cancer genetics.

Surbhi Jain,1 Tai-Jung Lee,2 Adam Zhang,1 Jonathan Cheng,1 Jamin D. Steffen,1 Chi-Tan Hu,3 James P. Hamilton,4 Harry Luu,4 Hie-Won Hann,5 Fwu-Shan Shieh,1 Selena Y. Lin,1 Wei Song,1 Ying-Hsiu Su2. 1 _JBS Science, Inc, Doylestown, PA;_ 2 _The Baruch S. Blumberg Institute, Doylestown, PA;_ 3 _Hualien Tzu-Chi Hospital, Buddhist Tzu-Chi Medical Foundation, Hualien, Taiwan;_ 4 _The Johns Hopkins University School of Medicine, Baltimore, MD;_ 5 _Jefferson Medical College, Thomas Jefferson University Hospital, Philadelphia, PA_.

Currently, blood is the body fluid of choice to provide comprehensive germline genomics via Peripheral Blood Mononuclear Cells (PBMC) and cancer genetics via the detection of circulating tumor DNA (ctDNA, liquid biopsy) for precision medicine. In this study, we examined if urine can be an alternative to blood for comprehensive germline genomics and for liquid biopsy. We have previously shown that human urine contains DNA derived both from sloughed-off cell debris of the urinary tract (greater than 1 KB/High Molecular weight or HMW) and trans-renal DNA from the circulation (less than 1 KB/Low molecular weight or LMW). We performed whole genome sequencing (WGS) to compare the quality and comprehensiveness of the genomic data between 3 sets of matched PBMC and HMW urine DNA. There was no statistically significant difference between the NGS data obtained from PBMC and urine DNA upon comparing the number of reads, average coverage, percent aligned reads, percent reads passing Phred score Q30 (p value less than 0.05, paired t-test) and coverage of human genome. Next, we compared the detectability of ctDNA in the matching LMW urine and plasma DNA obtained from 44 HCC patients by employing a panel of HCC biomarkers including genetic mutations at TP53 codon 249 [G:C to T:A transversion (TP53 249T)], CTNNB1 exon 3 regions 32-37 (CTNNB1 32-37), and hTERT promoter region position 124 (hTERT 124) and aberrant methylation of RASSF1A (mRASSF1A) promoter. In this study, a systematic analysis of this HCC biomarker panel demonstrated that urine can complement blood for liver cancer screening. A combination of the plasma and urine biomarkers with serum AFP the current most widely used biomarker for HCC can provide higher sensitivity (up to 30% more) for liver cancer detection. In order to characterize and determine if LMW urine DNA is derived from plasma DNA, we performed WGS to compare the size profiles and coverage of these two sources of cell-free DNA in 3 sets of matching HCC urine and plasma samples. Overall shorter read lengths were obtained from urine DNA as compared to the corresponding plasma DNA. A series of peaks occurring at 10 bp periodicity was displayed in both sources of cell-free DNA Further analysis regarding the genome coverage, overlapping fraction and variant detection is ongoing. In conclusion, our data suggests that (i) HMW urine DNA can replace PBMC DNA for providing comprehensive genomic sequencing data and (ii) urine can complement blood for liver cancer liquid biopsy, precision medicine, and potential applications to other cancers.

#418

Ex vivo **isolation and culture of circulating tumor cells using microfluidics chip.**

Huirong Zhang, Pan Zhao, Han Wu, Chang Zou. _Shenzhen People's Hospital, Shenzhen, China_.

Background: Circulating tumor cells (CTCs) are the tumor cells shed from primary tumor, which enter the bloodstream and travel to distant anatomic sites to form metastasis. CTCs have been identified as blood-based biomarkers to guide cancer diagnosis and prognosis in many reports. The huge challenge which limits the clinical utility of CTCs, especially in early stage cancer patients, is the rarity. Specifically, there are only 1-10 CTCs in one milliliter of whole blood.

Methods: A microfluidic CTC capture device was used for capture and analysis of CTCs from gastric cancer patients. The patients' samples were referred to the regulation of ethnic commission of Shenzhen People's Hospital and informed parental consent for the study. CTCs were collected by cells adherent culture using the CCC medium. Immunofluorescence, karyotyping and mude mouse tumorigenesis were used to verify the CTCs.

Results: A epithelial phenotype cell cluster which named S-CTC came out in the 24-well plate after 8 days. S-CTC cells can steadily formed clones and showed stem-cell like properties. Positive rate of CD133 were reached to 4.7% which demonstrated by flow cytometry. Immunofluorescence identification of S-CTC were DAPI-positive, Cytokeratin-positive, and CD45-negative. The karyotyping results indicated that the chromosomes were aneuploidthe, the chromosome number of S-CTC varied from 55 to 63. Also S-CTC presented a larger nuclear/cytoplasmic ratio and limitless replicative potential. Furthermore, the S-CTC had huge sphere formation ability, the tumorigenesis of S-CTC cells can be happened by xenografting in immunodeficient mice.

Conclusion: A cell S-CTC were successfully isolated and cultured by the technology microfluidic chip. S-CTC was identified using a series of analysis and can be confirmed to be the tumor cell, which would be provide more drug sensitivity information of the patient and open up novel avenues towards the understanding of gastric cancer.

#419

Single cell-driven secreted proteins as a surrogate biomarker for Cetuximab therapy in colorectal cancer.

Ye-Lim Park,1 Hwang-Phill Kim,1 Jun Kyu Kang,1 Yoo Joo Lim,2 Sang-Hyun Song,1 Sae-Won Han,2 Tae-You Kim2. 1 _Cancer research institute, Seoul national university, seoul, Republic of Korea;_ 2 _Seoul national university Hospital, seoul, Republic of Korea_.

Epidermal growth factor receptor (EGFR) blockade is efficacious in KRAS wild-type of colorectal cancer, but acquired resistance is inevitably generated. Here, we established the 5 acquired resistant single cell models (CR cell lines) to cetuximab and investigated the mechanism of acquired resistance using single cell-based whole-exome sequencing and transcriptional profiling. Of these, 4 cell lines have KRAS Q61L mutation and the other has BRAF V600E mutation. From gene enrichment analysis (GSEA), we found that epithelial to mesenchymal transition (EMT) occurs and CXCL chemokine family, which are known as secreted proteins, are overexpressed in the CR cell lines compared with parental cells. Interestingly, we found that CXCL chemokines were strongly secreted/expressed in KRAS/BRAF mutant cell lines and plasma/serum samples from colorectal cancer patients who acquired KRAS/BRAF mutation during cetuximab therapy. CXCL chemokines are regulated coordinately by Slug and NF-B transcription factors and trans-activate EGFR through CXCR2/MMP1/sHB-EGF axis. This process activates MAPK pathway via autocrine. To overcome the resistance to cetuximab, combination treatment of cetuximab and MEK/BRAF inhibitor had a larger antitumor effect compared to the single agents, decreasing the expression of MAPK pathway and secretion of CXCL chemokines. This finding highlights that CXCL chemokines, as a secreted proteins circulating in the bloodstream, show correlation with KRAS/BRAF mutation and could be clinical biomarkers for predicting resistance to anti-EGFR therapy in CRC.

#420

Characteristics of circulating tumor DNA in lung cancer patients.

Tomonori Abe, Chiho Nakashima, Akemi Sato, Yohei Harada, Eisaburo Sueoka, Shinya Kimura, Naoko Aragane. _Saga University, Saga, Japan_.

Liquid biopsy using circulating tumor DNA (ctDNA) has been spread world-wide. We established fully automatic sensitive mutation detection system, mutation-biased PCR and quenched probe system (MBP-QP) method, and accomplished multicenter prospective study to investigate the utility of ctDNA in lung cancer patients who acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI). The results of the clinical study showed that ctDNA was frequently detected in lung cancer patients with distant metastasis, and detection of ctDNA was associated with poor prognosis. To investigate the significance of these clinical data, we analyzed biological and clinical characteristics of ctDNA. We examined 130 plasma samples from 92 lung cancer patients in addition to 18 benign pulmonary disease patients and 20 healthy individuals at Saga University Hospital.Circulating free DNA (cfDNA) was extracted from 1000μl plasma by automated DNA extraction system using cellulose magnetic beads. The DNA concentration was quantified by Quantus®, the fluorescent measurement of dsDNA intercalated dye, and the DNA size distribution was analyzed by Bioanalyzer®, capillary electrophoresis system. We found difference of cfDNA size distribution between lung cancer patients and healthy individuals: former showed two peaks of 5kb and 170bp, and latter showed single peak of 170bp. The DNA concentration was higher in lung cancer patients compared to those in benign pulmonary disease patients and healthy individuals. Among lung cancer patients, DNA concentration was increased in those with advanced stages, especially in presence of metastasis. In addition, 5 kb fragments DNA was significantly increased in these cases compared to 170 bp fragments DNA. To investigate which fragment contained tumor-derived DNA, 170 bp and 5 kb fragments were separately isolated, and EGFR mutation, L858R was examined. L858R was detected in both ctDNA fragments, 170 bp and 5 kb, indicating that both sized DNA fragments contain tumor-derived DNA. In order to evaluate DNA resolution, we measured the concentrations of DNase1 and DNase1L3 in plasma, but no significant difference was observed among three groups. Although the 170 bp fragments DNA are well known as an apoptotic product, the origin of 5 kb long fragments DNA has not been clarified. We hypothesized that long fragment DNA has a mechanism to escape from DNase, extracellular vesicles or DNA associated protein. In order to examine that, We analyze whether long fragment DNA is associated with extracellular vesicles or DNA associated protein.

#421

Potential biomarkers for monitoring therapy with Denosumab in patients affected by giant cell tumor of bone.

Amalia Conti,1 Alessandra Luchini,2 Gastone Castellani,3 Enrico Giampieri,4 Laura Pazzaglia,5 Serena Pollino,5 Giovanna Magagnoli,5 Emanuela Palmerini,5 Lance L. Liotta,2 Piero Picci,5 Kerry J Rhoden,1 Maria Serena Benassi5. 1 _Department of Medical and Surgical Sciences, Bologna, Italy;_ 2 _Center for Applied Proteomics and Molecular Medicine, Manassas, VA;_ 3 _Department of Physics and Astronomy, Bologna, Italy;_ 4 _Department of Experimental, Diagnostic and Specialty Medicine, Bologna, Italy;_ 5 _Rizzoli Orthopaedic Institute IRCCS, Bologna, Italy_.

Giant Cell Tumor (GCT) of Bone is an intramedullary tumor histologically composed by spindle-shaped mononuclear stromal cells, that are the neoplastic and proliferative component of the lesion, and osteoclast-like multinucleated giant cells, that express receptor activator of nuclear factor κB (RANK). The mononuclear stromal cells express RANK ligand, a mediator of osteoclast activation. GCT represents 5% of all bone tumors and 20% of all benign tumors. It usually affects young adults and 2-3% is metastatic, generally to the lung. Curettage is the preferred surgery, while current therapeutic treatment is represented by Denosumab, an anti-RANK-L monoclonal antibody that blocks RANKL binding to RANK, thereby stopping the 'vicious cycle' involved in the biological process of GCT. Identification of circulating biomarkers could provide tools for monitoring the effectiveness of therapy and to better understand its biological mechanism. Low molecular weight (LMW) serum proteins or protein fragments, considered to be a rich source of new potential biomarkers and often masked by the presence of abundant proteins, were detected in serum of 25 GCT patients before and after 9 months of treatment with Denosumab, using the hydrogel nanoparticle technique followed by Mass Spectrometry (MS). 25 healthy sera were also analyzed. Hydrogel core-shell nanoparticles selectively entrap LMW proteins by size exclusion and affinity chromatography, protecting them from degradation and amplifying their concentration for subsequent MS detection. Statistical differences in LMW protein abundance between the three groups (GCT pre-treatment, GCT post-treatment, and healthy) were analyzed using the Wilcoxon test or Mann-Whitney U test as appropriate, with the Benjamini-Hochberg False Discovery Rate for multiple test correction. Proteomic analysis revealed 14 differentially expressed analytes in sera from patients pre- and post-treatment, including Complement factor H, Immunoglobulin kappa variable 4-1, Insulin-like growth factor-binding protein 4, Insulin-like growth factor II, Beta-2-glycoprotein 1 and Immunoglobulin lambda constant 6, while 6 proteins were unaltered by the treatment. Furthermore, 52 proteins showed differential abundance in GCT patients before treatment compared to the healthy group, including several (Vitamin D binding protein, Ceruloplasmin, CD5 antigen-like, Serum amyloid A-4 protein, Complement factor D and Apolipoprotein B-100) that we previously identified in a smaller subset of GCT patients and controls, validating our former results. In conclusion, using a noninvasive proteomic technique, we have identified candidate serum biomarkers that could be useful to monitor Denosumab therapy in patients with GCT of bone, for patient management and to better understand its molecular mechanism.

#422

Duplex sequencing for MRD detection in acute myeloid leukemia.

Jacob E. Higgins,1 Lindsey N. Williams,1 Christopher S. Hourigan,2 Jerald P. Radich,3 Jesse J. Salk1. 1 _TwinStrand Biosciences, Seattle, WA;_ 2 _National Institutes of Health, Bethesda, MD;_ 3 _Fred Hutchinson Cancer Research Center, Seattle, WA_.

The purpose of this study is to develop a universally applicable Duplex Sequencing (DS) assay for extremely sensitive detection of minimal residual disease (MRD) in acute myeloid leukemia (AML). The presence of MRD plays a critical role in AML recurrence and mortality, and is an important prognostic marker to guide clinical care and clinical trial risk stratification. Unfortunately, current MRD detection methods, including light microscopy, cytogenetics, qPCR, and flow cytometry have shortcomings in terms of sensitivity, general applicability, and/or inter-laboratory reproducibility. Duplex Sequencing eliminates technical artefacts observed with other sequencing technologies and delivers an unprecedented level of accuracy and sensitivity, on widely available NGS platforms. Here, we report validation of our DS MRD mutation detection assay, with a background mutation level more than 100-fold below single-stranded tag-based error correction (SSCS).

Our complete DS MRD probe panel targets mutation hotspots or full coding sequences in 29 genes recurrently mutated in AML, representing mutations found in approximately 90% of patients. Supplementing with a targeted RNA-seq panel for recurrent gene fusions would approach 100%. To simulate low frequency mutations in MRD, we generated 2 DNA dilution series: either AML patient DNA or cell line DNA with known AML mutations that were serially diluted into normal human DNA. Replicate libraries were prepared with custom targeted probe panels and sequenced to over 50,000x or over 1,000,000x total Duplex depth (original Duplex DNA molecules sequenced per site), respectively. Expected mutations were detected down to MAFs of 1.9x10-5 and 6.0x10-6. Background mutation levels of 5.7x10-7 and 5.8x10-7, were respectively seen in each series, and likely reflect the true age-associated biological background, rather than technical noise. SSCS artefacts in these samples were approximately 100x higher than the frequency of DS mutations, and obscured expected mutations in the lower dilutions.

We are further validating our method in both retrospective and prospective studies. In retrospective samples from a completed clinical trial, we are comparing the sensitivity and accuracy of DS vs. multicolor flow cytometry for MRD with known clinical outcomes. The relative prognostic value of DNA from blood vs. bone marrow will be quantified with DS as well. We will also perform DS on prospective samples to examine the prognostic value of specific genes and MAFs to eliminate uninformative variants, which will also allow accurate quantification of low level mutations in non-leukemic clones that accumulate with aging and can confound detection of MRD. Elimination of sequencing artefacts is critical for the application of NGS assays to MRD detection, especially when expected mutations are unknown, and DS is an important new tool in high sensitivity cancer diagnostics.

#423

The use of the sTRA glycan for predicting responses to neoadjuvant therapy in patients with pancreatic ductal adenocarcinoma.

Ben Staal,1 Ying Liu,1 Peter Hsueh,1 Mohammed Aldakkak,2 Herbert Zeh,3 Douglas Evans,2 Randall E. Brand,3 Susan Tsai,2 Brian B. Haab1. 1 _Van Andel Research Inst., Grand Rapids, MI;_ 2 _Medical College of Wisconsin, Milwaukee, WI;_ 3 _University of Pittsburgh Medical Center, Pittsburgh, PA_.

The sTRA glycan has been validated as a serological and cell-surface biomarker of pancreatic ductal adenocarcinoma (PDAC). In previous work, a biomarker panel using sTRA and CA19-9 performed significantly better than CA19-9 for distinguishing PDAC from benign diseases of the pancreas; here we explored whether the subgroup identified by sTRA has a different prognosis than other subgroups. We examined the plasma levels of sTRA and CA19-9 in two cohorts of patients with resectable or borderline-resectable PDAC. In each cohort, two samples per patient were collected: one prior to any therapy, and another after neoadjuvant therapy but before surgery. In the first cohort (n = 54), the ratio of post-therapy to pre-therapy plasma sTRA was statistically higher (p < 0.05, student's t-test using logged ratios) in patients with poor response, as assessed by surgical pathology. CA19-9 had higher ratios in a subset of the poor-response subjects but without statistical significance. In the second cohort (n = 75), the pre-treatment and post-treatment levels of sTRA were significantly higher (p < 0.01, Wilcoxon Rank-Sum test) in patients with shorter survival (<2 years). In contrast, CA19-9 did not show statistical significance in the pre-treatment or post-treatment levels, but the post:pre ratios were substantially higher in a subset of the short-survival group. In both cohorts, sTRA identified a subset of poor responders that was non-identical to the one identified by CA19-9. As a result, a biomarker panel combining the markers provided better accuracy for identifying poor responders than either marker alone. In an analysis of 24 cell line models of PDAC, the cell lines that secreted sTRA had a statistically higher resistance to chemotherapy than those not secreting sTRA, whereas the cell lines secreting CA19-9 showed no such association. Therefore, pancreatic cancers that produce sTRA may be a distinct subtype with higher resistance to chemotherapy, and sTRA in combination with CA19-9 could be a valuable serological predictor of responsiveness to chemotherapy.

#424

Genomic instability changes of circulating tumor DNA reflect the responses to chemotherapy or targeted therapy in advanced gastric cancer.

Zuhua Chen,1 Cheng Zhang,1 Beifang Li,1 Yunyun Niu,2 Limeng Chen,2 Mengqi Zhang,1 Jing Yang,1 Sijia Lu,2 Jing Gao,1 Lin Shen1. 1 _Peking University Cancer Hospital & Institute, Beijing, China; _2 _Yikon Genomics Co. Ltd., Shanghai, China_.

Background: Gastric cancer was characterized by frequent somatic copy number alterations and high genomic instability, yet the significance of genomic instability and its changes implicated by drug treatment in advanced gastric cancer (AGC) remain unclear. In this study, we investigated genomic instability changes of circulating tumor DNA (ctDNA).

Methods: A total of 101 plasmas and paired blood cells from 26 patients with AGC before (N=26) and after (N=75) treatment were prospectively collected. Plasma ctDNA was separated and assessed for copy number variations (CNV) by whole genome sequencing (WGS), and we assessed the correlation between genomic indexes and therapeutic response.

Results: Genomic instability with diverse patterns was observed in 20 of 26 patients (77%) prior to drug treatment. For patients with high (N=20) level genomic instability prior to drug treatment, the response rate (55%, 11/20) was significantly higher than that (17%, 1/6) in patients with low level (N=6) genomic instability (P=0.17). Notably, genomic instability changed with the administration of drugs, which reflected its correlation with therapeutic response. All ctDNAs (94%, 14/15) collected at the time of partial response (PR) after treatment displayed low-genomic instability. However, 52% ctDNAs (13/25) collected at the time of progressive disease (PD) after treatment showed high-level genomic instability. Furthermore, several patterns of CNV were categorized in ctDNAs. CNV pattern after acquiring drug resistance were largely unchanged with respect to CNV baseline patterns.

Conclusions: Genomic instability based on ctDNA can be used to predict and monitor therapeutic response in gastric cancer, although validation in larger cohort would be necessary.

#425

Development and validation of liquid biopsy assay for copy number loss in cancers.

Simo Zhang, Feng Xie, Amy Chang, Zhixin Zhao, Carlos Montesinos, Xiaohong Wang, Kemin Zhou, Shidong Jia, Jianjun Yu, Pan Du. _Predicine, Hayward, CA_.

Introduction: Copy number loss (CNL) of tumor suppressor genes, such as PTEN, RB1 and BRCA1/2, are common and key characteristics of many cancers. However, non-invasive detection of CNL has represented a strong demand and practical challenge in the clinic. Here we report a high-sensitive liquid biopsy NGS assay coupled with a proprietary bioinformatics pipeline to profile and detect CNL using cell-free circulating DNA (cfDNA).

Method: We developed PrediSeq, a proprietary liquid biopsy NGS assay to detect CNL, in addition to SNV, indels, rearrangement, and copy number gain. We leveraged unique molecular identifier (UMI) and proprietary bioinformatics algorithm to suppress background noise caused by PCR pre-amplification and sequencing steps. In-house developed bioinformatics pipeline, DeepSea, is used for ultra-sensitive detection of CNL and other genomic alterations. Reference materials, matched tissue and plasma samples were used for assay validation.

Results: PrediSeq CNL assay was validated using serially diluted reference materials with known copy number status. The method was evaluated with the orthogonal whole-genome sequencing using a set of clinical plasma samples with matched tissues. About 82% of CNL calls (ATM, BRCA1, BRCA2, CDKN2A, RB1) in were confirmed between plasma PrediSeq and tissue WGS. In alignment with literature, CNL of ATM, CDKN2A, BRCA1, BRCA2, was observed in plasma samples of a cohort of Her2+, HR+, and triple-negative breast cancer patients. Similarly, we observed the dynamic changes of plasma-based CNL of BRCA2, PTEN, RB1, CDKN2A, ATM genes in patients of metastatic castration-resistant prostate cancer (mCRPC) and metastatic hormone sensitive prostate cancer (mHSPC).

Conclusion: PrediSeq CNL assay has been successfully developed and validated to detect copy number loss in circulation, providing valuable molecular biomarkers to guide patient selection and treatment monitoring in precision oncology and clinical drug development.

#426

Autoantibodies: A promising prognostic tool for immunotherapy response in advanced non-small cell lung cancer.

Imad Tarhoni,1 Cristina Fhied,1 Melissa Pergande,2 Revathi Kollipara,1 Connor J. Wakefield,1 Katherine Gallo,1 Apoorva Tangri,1 Marta Batus,1 Mary Jo Fidler,1 Philip Bonomi,1 Jeffrey A. Borgia1. 1 _Rush University Medical Center, Chicago, IL;_ 2 _University of Illinois at Chicago, Chicago, IL_.

Background: Immune-checkpoint blockade has revolutionized cancer therapy in advanced non-small cell lung cancer (NSCLC). Tissue expression of programmed death protein ligand (PD-L1) remains the gold standard for patient stratification, however, the limited performance of this marker encourages investigations for improved molecular diagnostics. The objective of this study is to identify and evaluate the role of neoantigen-associated autoantibodies to predict the clinical response to anti-PD-1/-L1 in advanced stage NSCLC.

Method: Lung adenocarcinoma A549 and H358 cell lysate proteins were resolved via 2-dimensional electrophoresis, electroblotted onto nitrocellulose, and immunoprobed with pooled, pretreatment sera (n= 4/ group) derived from patients with advanced NSCLC who received PD-1/-L1 directed immunotherapy. These patients have documented disease progression within 12 weeks ("rapid progression") or demonstrated radiographical stable disease/progression after the first 180 days of therapy ("late progression"). Immunoreactive spots were detected with an HRP-conjugated, anti-human IgG secondary antibody with digital densitometry. A 4-fold cutoff threshold in expression was used to prioritize spots for identification via tandem mass spectrometry. From A549 cells, recombinant proteins were selected for STIP-1, annexin A2, HSPA8, and GAPDH. These proteins were then analyzed via immunoblotting methods using sera from each indicated group (n=4 per group). In addition, identified proteins from H358 cells include FH, HSP70B, IMPDH2, NY ESO-1, PGAM-1, and vimentin. Recombinant versions of a selection of autoantigens identified in this manner were commercially acquired and used to develop custom Luminex immunobead assays to quantitatively assess autoantibody production in individual patient sera (rapid progressors, n=14; late progressors, n=18). Values were statistically compared via Mann-Whitney test.

Results: Series of differentially expressed autoantigens predictive of clinical response to PD-1/-L1 directed immunotherapy were identified. Western blots of neoantigens identified from A549 cells; STIP-1, annexin A2, HSPA8, and GAPDH were significantly able to distinguish between response groups (p-value < 0.001). Six H358 targets resulting from the custom bead-based immunoassay development; FH, HSP70B, IMPDH2, NY-ESO-1, PGAM-1, and vimentin were also able to distinguish between groups (p-values of 0.01, 0.01, 0.022, 0.005, 0.034, and 0.027, respectively).

Conclusion: Our study demonstrates that serum autoantibodies have great promise to serve as a robust tool to prognosticate response for patients receiving PD-1/-L1 directed immunotherapy and potentially aid current treatment selection methods. Additional targets are currently being developed into multiplexed immunobead assays for evaluation across larger cohorts of patients.

#427

Detection and molecular characterization of circulating tumor cells in advanced head and neck squamous cell carcinoma.

Hiroe Tada,1 Hideyuki Takahashi,1 Hiroki Ishii,2 Yuki Kuwabara-Yokobori,1 Masato Shino,1 Kazuaki Chikamatsu1. 1 _Gunma Univ. Graduate School of Medicine, Maebashi, Japan;_ 2 _National Cancer Institute, Bethesda, MD_.

Malignant properties of tumor cells are genetically and epigenetically altered during tumor development and progression. Moreover, tumor cells rapidly change to treatment resistant phenotype by various therapeutic pressures, and not only become more heterogeneous but also possess metastatic potential. Detection and characterization of circulating tumor cells (CTCs) in the bloodstream of cancer patients have been currently remarked as a valuable tool for real-time evaluation of tumor tissue with phenotypic heterogeneity in the diagnosis and treatment of various cancers, however these have been still challenging. In the present study, we investigated the presence of CTCs and their molecular characterization by the combination of microfiltration system and RT-qPCR method. Peripheral blood samples were collected from 30 patients with advanced head and neck squamous cell carcinoma (HNSCC) and passed through two CellSieveTM microfilters using a low-pressure filtration system. The existence of CTCs collected from the first filter was determined by the expression of epithelial-related genes (KRT19, EPCAM, EGFR, or MET), then the expression of cell proliferation (PIK3CA and CCND1)-, EMT (SNAI1 and VIM)-, cancer stemness (CD44, NANOG, and ALDH1A1)-, and immune regulation (CD47, CD274, and PDCD1LG2)-related genes was analyzed for molecular characterization of CTCs. The gene expression in cells collected from the second filter was used for normalization of target genes. Sixteen (53.3%) of 30 patients were positive for at least one epithelial-related gene. Interestingly, the number of patients with locoregional recurrence or distant metastasis in 16 CTC-positive patients was 7 (43.8%) or 5 (31.3%); whereas that in 14 CTC-negative patients was 2 (14.3%) or 1 (7.1%), respectively. The gene expression profile in CTCs collected from 16 CTC-positive patients has revealed remarkable heterogeneity between HNSCC patients. Of 16 CTC-positive patients, PIK3CA was overexpressed in 56.3%, CCND1 in 43.8%, SNAI1 in 62.5%, VIM in 43.8%, CD44, in 50.0%, NANOG in 31.3%, ALDH1A1 in 56.3%, CD47 in 62.5%, CD274 in 37.5%, and PDCD1LG2 in 31.3%. Our results suggest that CTCs may play an important role in locoregional recurrence as well as distant metastasis in HNSCC. Further molecular profiling at the single-cell level of CTCs is needed to clarify the clinical significance of molecular phenotype of CTCs.

#428

Phenotyping pancreatic cancer CTCs as biomarkers for RX-3117 clinical trials.

Andrew Eisen,1 DJ Kim,1 Jie Yang,2 Weiguo Wu,2 Darren W. Davis2. 1 _Rexahn Pharmaceuticals, Rockville, MD;_ 2 _ApoCell, Houston, TX_.

Background:

RX-3117 is an oral, small molecule nucleoside analogue pro-drug being used in combination with nab-paclitaxel in a Ph2 clinical trial for pancreatic cancer. RX-3117 can be transported intracellularly by SLC29A1 (hENT1) and is converted to an active agent by the cancer-enriched enzyme, uridine-cytidine kinase 2 (UCK2) for incorporation into RNA and DNA. This leads to cancer cell apoptosis. We developed an approach to enumerate and phenotype CTCs from pancreatic cancer subjects by quantitative immunofluorescence (QIF) to assess whether CTC numbers, phenotypic features and/or early apoptotic responses to therapy predict or presage clinical response.

Methods:

QIF staining parameters were developed with cancer cell lines sensitive and resistant to RX-3117 utilizing monoclonal antibodies to hENT1 and UCK2. The staining methods were then applied to CTCs isolated from 10 ml anticoagulated blood by dielectrophoretic (DEP) properties that differentiate cancer cells and normal PBMCs using the ApoStream device (ApoCell, TX). In addition to the drug-related markers, CTCs were stained for a panel of either Epithelial or Mesenchymal markers, CD45 and nuclei (DAPI). Phenotyped CTCs were binned into 6 categories defined by whether they were EPI+/-, EMT+/- or CD45+/-. The percentage of cells in each category also positive for hENT1 or UCK2 was determined as was the mean fluorescence intensity of the marker+ sub-population. In addition, assessment of hENT1 and UCK2 expression was performed by qRT-PCR utilizing mRNA isolated from isolated CTCs, the buffy coat from spun blood and in plasma.

Results:

In a pilot study, blood was obtained from five subjects with Stage IV pancreatic cancer on various regimens. CTCs were isolated, enumerated and phenotyped. CTC numbers ranged from 100 – 20,000 per ml. The percentages of UCK2+ cells were higher in EPI+/EMT+ subsets than EPI-/EMT- subsets. The percentages of hENT1+ cells were generally low in all the subsets. hENT1 and UCK2 transcripts were observed at acceptable levels in the cell populations, and borderline, but consistent levels in plasma samples. CEACAM5 transcripts were observed only in one CTC and PBMC fraction from one subject. After normalization vs. housekeeping transcripts, a greater than 2-fold increase of UCK2 was observed in three CTC samples compared to PBMC samples. A greater than 2-fold increase of hENT1 was observed in two CTC samples.

Conclusion:

CTCs from patients with pancreatic cancer can be obtained in numbers suitable for multiparameter phenotyping to identify features that might serve as selective, predictive or prognostic biomarkers in clinical trials. Multiparameter phenotyping of several relevant markers is being performed by quantitative immunofluorescence on CTCs isolated from pancreatic cancer patients in an on-going Ph2 clinical trial of RX-3117 in combination with nab-paclitaxel.

### Current Developments in Non-invasive Biomarkers for Assessment of Cancer 1

#429

Increased chromatin heterogeneity in circulating tumor cells (CTCs) is associated with high levels of HER2 expression in metastatic breast cancer (MBC).

Qiang Zhang,1 Lorenzo Gerratana,2 Ami N. Shah,3 Andrew Adam Davis,3 Lisa Flaum,3 Xiang Zhou,4 Youbin Zhang,3 Vadim Backman,4 Amir Behdad,3 Firas Wehbe,3 William Gradishar,1 Leonidas Platanias,1 Massimo Cristofanilli1. 1 _Northwestern University, Lurie Cancer Center, Chicago, IL;_ 2 _Northwestern University, University of Udine (Italy), Chicago, IL;_ 3 _Northwestern University, Chicago, IL;_ 4 _Northwestern University, Robert R. McCormick School of Engineering and Applied Science, Evanston, IL_.

Introduction: Molecular and genomic characterization of CTCs may help to understand MBC prognosis and predict treatment benefit. We previously reported that overexpression of HER2 in CTCs is associated with detection of clusters, more aggressive clinical features and prognosis in MBC (2018 AACR #5195). Herein, we report a new finding that a widespread variation on chromatin distribution in CTCs is associated with HER2 expression in MBC which may indicate more aggressive tumor.

Methods: Whole blood sample (7.5ml/each) was collected from stage III/IV BCa patients before therapy. CTC enumeration were performed in FDA approved CELLTRACKS ANALYZERII® System (Menarini) by targeting the Epithelial Cell Adhesion Molecule antigen for capturing CTCs. After confirming CTCs were positive by CellSearch system, live CTCs enrichment was performed by using Parsortix system (ANGLE) utilizes microfluidic based 10μm separation cassettes. The captured CTCs were stained by Anti-CK-PE (specific for epithelial cells), DAPI (for nucleus), anti-CD45-APC (specific for leukocytes), and anti-HER2-FITC. CTCs chromatin packing density was scanned by Partial Wave Spectroscopic (PWS) microscopy which is a label-free spectroscopic microscopy method that resolves structures in cells and quantify the cell nucleus heterogeneity of chromatin packing density scaling between 20-350 nm, or from the kb-pair to 10 Mb-pair range. For each nuclei, the nanoscale heterogeneity of chromatin packing was analyzed as nuclear statistical parameter standard deviation Σ (sigma, RMS). Kruskal-Wallis test was used for statistics.

Results: We identified 400 CTCs by CellSearch system, including 115 HER2+ CTCs and 285 HER2- CTCs. The live CTCs were sorted by Parsortix system and were proceeded with multiple staining and scanning by PWS microscopy. CTCs were classified based on morphology and correct phenotype as CK+, DAPI+ and CD45-. There were 97 acquired nuclei that were scanned and evaluated successfully, including 17 HER2+ CTCs, 12 HER2- CTCs and 80 non-CTC cells. According to the images, stronger nanoscopic variations at each pixel in internal structure represents higher chromatin distribution heterogeneity quantified by RMS. The average RMS was 0.05125 in CTC cells which is significantly higher than the non-CTC population (RMS=0.0381, p<0.01). Moreover, the HER2+ CTCs demonstrated the highest nuclear sigma (RMS=0.05366) among all the subgroups when compared to HER2- CTCs (RMS=0.04546, p<0.01).

Conclusion: This is the first report of chromatin heterogeneity in CTCs, and the association between HER2 expression and high variations of chromatin distribution in MBC patients. This novel property of CTCs describing nanoscale heterogeneity of chromatin packing requires further evaluation and validation but, it may offer a novel dimension in the understanding of the metastatic process.

#430

Prostate specific antigen (PSA) as a diagnostic and prognostic marker of prostate cancer.

Ronald Simon,1 Martina Kluth,1 Kristine Fischer,2 Sarah Bonk,1 Claudia Hube-Magg,1 Doris Höflmayer,1 Maximilian Lennartz,1 Florian Lutz,1 Guido Sauter,1 Franziska Büscheck,1 Ria Uhlig,1 David Dum,1 Andreas M. Luebke,1 Corinna Wittmer,1 Frank Jacobsen,1 Eike Burandt,1 Stefan Steurer,1 Waldemar Wilczak,1 Sarah Minner1. 1 _Univ. Medical Ctr. Hamburg-Eppendorf, Hamburg, Germany;_ 2 _ONCOdianova GmbH, Hamburg, Germany_.

Since several decades, prostate specific antigen (PSA) is regarded as a specific marker for healthy and neoplastic prostate tissues, and PSA level changes in blood and tissue are discussed as prognostic markers of prostate cancer. Using a custom-made mouse monoclonal anti-PSA antibody, we studied expression of PSA in a tissue microarray (TMAs) comprising 17,456 prostate cancers with long-term follow-up data. PSA staining was typically present in the cytoplasm and pronounced at the apical cell pole. PSA expression was often reduced (15%) or even completely lost (1%). Reduction of PSA expression was significantly linked to adverse features of prostate cancer, including advanced stage, high classical and quantitative Gleason grade, presence of lymph node metastasis, a positive surgical margin, increased cell proliferation, and reduced time to recurrence (p<0.0001 each). Complete lack identified a small subset of patients with the worst prognosis. There was also in impact of the subcellular localization of PSA expression: Loss of the apical staining pattern worsened the prognosis in cancers with otherwise identical levels of cytoplasmic staining. In a multivariable analysis including pT stage, Gleason grade, nodal stage, and resection margin status, loss of PSA expression proved to predict outcome independently from these parameters. Of note, PSA even stratified the prognosis of 971 cancers harboring PTEN deletions in our cancer set. In conclusion, gradual reduction of PSA staining, including a loss of the apical staining pattern, is a strong and independent prognostic marker in prostate cancer.

#431

Semi-automated detection, isolation and molecular analysis of single disseminated melanoma cells from lymph nodes.

Barbara Alberter,1 Sebastian Scheitler,1 Giancarlo Feliciello,1 Alberto Ferrarini,2 Melanie Werner-Klein,3 Sebastian Haferkamp,4 Christoph A. Klein,1 Bernhard Polzer1. 1 _Fraunhofer ITEM, Regensburg, Germany;_ 2 _Menarini Silicon Biosystems SpA, Castel Maggiore (BO), Italy;_ 3 _Regensburg Center for Interventional Immunology (RCI), Regensburg, Germany;_ 4 _University Medical Centre, Regensburg, Germany_.

We recently showed that lymph node disaggregation followed by immunocytology enables precise quantification of disseminated cancer cells (DCCs) and demonstrated that this approach has a 20-fold higher sensitivity to detect melanoma DCCs than routine histopathology (Ulmer et al., PLoS Medicine, 2014). Moreover, genetic profiling of single melanoma DCCs identified a colonization signature consisting of specific copy number alterations and point mutations that identify patients with high risk of progression (Werner-Klein et al., Nature Communications 2018). Here, we present the adaptation of this method to a semi-automated workflow for detection, isolation and molecular analysis of single melanoma DCCs. The developed workflow includes a mechanical disaggregation of lymph node tissue and collection of the mononuclear cells, immunofluorescence staining against melanoma-associated markers gp100 and MCSP and depletion of CD45-positive cells. Individual melanoma cells are then detected and isolated by DEPArrayTM technology enabling single cell whole genome amplification (Ampli1TM) for subsequent molecular analysis. In total, we processed 20 lymph nodes of melanoma patients and detected melanoma DCCs in 11/20 samples (55%). The quality of isolated cells was checked by Ampli1TM QC and 174 isolated single cells were further analyzed by Sanger sequencing for specific point mutations (BRAF, NRAS and cKIT) and Ampli1TM low pass kit for Illumina for copy number variation (CNV). Successful molecular analysis was correlated with genome integrity score (GII) as determined by Ampli1TM QC , with more than 95% of cells with GII 3-4 showing good performance in low pass sequencing. In conclusion, a new DepArrayTM based application for marker-dependent single cell isolation from malignant melanoma lymph nodes was successfully established and tested on a cohort of 20 melanoma patients. Molecular analyses of isolated single cells confirmed the tumor origin by CNV profiling and mutational analysis of melanoma-associated mutations. In the future, this approach could help to select individualized therapies for melanoma patients.

#432

Novel method for NGS analysis of actionable mutations in circulating tumor DNA specimens: improved quality control and 20-fold lower sequencing required.

Daniel J. Craig,1 Erin L. Crawford,1 Joshua Xu,2 Thomas M. Blomquist,1 Leihong Wu,2 Thomas Morrison,3 James C. Willey1. 1 _Univ. of Toledo, Toledo, OH;_ 2 _National Center for Toxicological Research, Jefferson, AR;_ 3 _Accugenomics, Inc., Wilmington, NC_.

Background: Identification of actionable mutations in circulating tumor DNA (ctDNA) enables gene-targeted therapy of solid tumors based on a simple blood test. NGS methods that attach a unique molecular identifier (UMI) to each DNA molecule reduce variant allele fraction (VAF) limit of detection (LOD) to <0.01% at a cost of 10-20-fold higher sequencing requirement. Importantly, the VAF lower limit of detection (LOD) for analysis of ctDNA specimens does not typically extend below 0.5% due to limits of ctDNA specimen quantity. For example, the average ctDNA specimen is 32 ng (10,000 genome copies) and at 0.1% VAF, 10 mutant molecules would be added to library prep. At this level, signal loss during library prep (typically 70-90%) will be associated with stochastic sampling variation. For example, in the Sequencing Quality Control Consortium (SEQC) 2 study, we measured inter-site and inter-replicate reproducibility of VAF measurement in 50 ng (15,000 genome copies) of synthetic ctDNA reference material. Thus, even using UMI NGS, measurement, reproducibility at VAF >0.5% was >95%, but reproducibility at 0.1-0.5% VAF was about 70%. Given limited value of UMI NGS when size of ctDNA specimen restricted VAF LOD to 0.5%, we tested the hypothesis that synthetic internal standard (IS) spike-in molecules provide reliable alternative quality control while eliminating UMI-imposed sequencing burden.

Methods: We synthesized IS for 61 actionable mutations, cloned them into pUC vectors, confirmed each IS to be wild-type sequence, linearized, quantified abundance and combined at a 1:1 genome copy mixture. An aliquot of IS mixture was added to the ctDNA reference material at 1:1 genome copy prior to library prep, followed by Illumina HiSeq sequencing, separation of target from IS reads using custom splitter, then pipeline analysis on Qiagen CLC Genomics Workbench.

Results: Analysis of each target relative to IS reliably identified base-substitution sequencing errors at each measured actionable mutation site and determined that VAF for each was <0.2%. Further, although a typical UMI method requires >200,000 reads per target in each sample, with analysis relative to IS spike-ins, 20,000 reads was sufficient (10,000 reads for the specimen and 10,000 reads for the spike-in). Because the IS spike-in molecules were validated to be wild-type, any variants were assumed to be due to technical error. Thus, a z-score comparing sequence count for a) specimen wild-type, b) specimen mutant, c) IS wild-type, and d) IS mutant enabled calculation of confidence level regarding each biological variant call.

Conclusion: Preliminary data indicate that IS spike-in mixtures enable reliable analysis of ctDNA mutation fraction to LOD of 0.5% without need for UMI.

Disclaimer: The views expressed here are those of the authors only and do not necessarily express the views/policies of the FDA.

#433

Genome-wide 5-hydroxymethylcytosine mapping in circulating cell-free DNA Identifies robust diagnostic markers for pancreatic adenocarcinoma.

Zhou Zhang,1 Lei You,2 Xingyu Lu,3 Huanyu Wang,2 Jinshou Yang,2 Taiping Zhang,2 Emily Kunce Stroup,1 Wenhui Lou,4 Chuan He,5 Wei Zhang,1 Yupei Zhao2. 1 _Northwestern University, Chicago, IL;_ 2 _Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China;_ 3 _Shanghai Epican Genetech Co. Ltd., Shanghai, China;_ 4 _Zhongshan Hospital, Fudan Univesity, Shanghai, China;_ 5 _University of Chicago & Howard Hughes Medical Institute, Chicago, IL_.

Pancreatic adenocarcinoma is the most common malignancy of the pancreas, accounting for about 85% of all pancreatic cancer cases. Although it is only the 11th most common cancer globally, pancreatic adenocarcinoma comprises a disproportionate number of total cancer deaths. It ranks 7th worldwide for cancer mortality, responsible for over 400,000 annual deaths globally. Though advances in cancer research and clinical oncology have significantly improved patient outcomes for many human cancers, the prognosis of pancreatic adenocarcinoma remains dismal: ~5% would live for five years after diagnosis. Early detection in high risk individuals or population screening appears to be the only viable option for increased chances of patient survival. In this study, we sought to develop a robust diagnostic model for pancreatic adenocarcinoma based on genome-wide mapping of 5-hydroxymethylcytosines (5hmC), a novel epigenetic marker that has been implicated in cancer pathobiology, in circulating cell-free DNA (cfDNA). Specifically, we prospectively recruited 158 patients with pancreatic adenocarcinoma (age: 60.9 ± 11.5; males: 57.0%), 91 patients with non-cancer pancreatic diseases (age: 48.3 ± 15.6, males: 41.8%), and 159 healthy individuals (age: 47.5 ± 12.0, males: 62.2%) from Peking Union Medical College Hospital and Zhongshan Hospital of Fudan University in China. This cohort was split into a training set and two validation sets. The patient diagnosis was confirmed by a study pathologist. We excluded patients treated with chemotherapy, radiation therapy or immunotherapy and then obtained baseline clinical, laboratory, and treatment data from medical records using a standard protocol. We reviewed pathology and medical records to verify disease progression or recurrence. We then performed genome-wide 5hmC mapping in cfDNA using the 5hmC-Seal technique, a highly sensitive method utilizing only 1-2 ng of cfDNA from <5 mL of plasma. A diagnostic model was then developed using a case-control design and elastic net regularization on a multivariate logistic regression model. Notably, the 5hmC-Seal data in cfDNA were enriched in genomic areas of pancreas-derived cis-regulatory markers (e.g., H3K4me1, a mark for tissue-specific enhancers). A weighted diagnostic score (wd-score) based on 10 genes (e.g., HOXD3, CSF1, and PLEKHG ), some of which have been found to be dysregulated in pancreatic adenocarcinoma (e.g., HOXD3), accurately distinguished cancer patients from healthy individuals in the training (AUC = 91.2%; 95% CI, 86.4-96.0%) and two validation sets (AUC = 91.0%; 95% CI, 83.4-98.5%; and AUC = 85.7%; 95% CI, 77.0-94.5%). This model markedly outperformed conventional cancer biomarkers (e.g., CEA, carcinoembryonic antigen) and shows that 5hmC-Seal mapping in cfDNA can identify a robust diagnostic model for detection of pancreatic adenocarcinoma.

#434

Breast cancer bone metastasis mediated by the exosomal miR-19a and secreted IBSP.

Kerui Wu,1 Jiamei Feng,2 Fei Xing,1 Sambad Sharma,1 Yin Liu,1 Shih-Ying Wu,1 Abhishek Tyagi,1 Ravi Singh,1 Kounosuke Watabe1. 1 _Wake Forest Univ. School of Medicine, Winston-Salem, NC;_ 2 _Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China_.

Bone metastasis (BM) is the one of the most predominant complications in breast cancer (BC) patients. It causes considerable symptoms termed skeletal-related events due to osteoclast-mediated bone resorption. Current treatments targeting BM are proven to reduce the symptoms, but not to extend the patients' survival. Thus, there is an urgent need to develop early predictive markers and preventive or curative therapies for BCBM. Recently, circulating miRNAs have been found to hold great potential for diagnostic and prognostic purposes. Due to the low stability of cell-free RNA in plasma, these circulating miRNAs exist mostly in a form of membrane-wrapped extracellular vesicles (EVs) and they play important roles in cancer progression. In this study, we aim to identify EVs-derived miRNA from serum of BC patients as biomarkers for diagnosis and prognosis of BCBM. We performed miRNA microarray of EVs from BC cell lines (231 and MCF7), as well as their bone metastatic variants (231BoM-1833 and MCF7BoM2d). Using bioinformatics analysis of circulating miRNA in a cohort of BC patients and real-time PCR measurement for blood-derived EVs from BC patients, we found that extracellular miR-19a is up-regulated in BCBM patients. Kaplan-Meier survival analysis indicates that miR-19a is positively correlated with metastases. Interestingly, miR-19a was also found to be highly enriched in exosomes compared to microvesicles and apoptotic bodies. To study the role of miR-19a in BCBM, we applied the CRISPR/Cas9 technology to knockout the miR-19a in 231BoM-1833 and MCF7-BoM2d cells. The knockout of miR-19a didn't alter the proliferation or migration of BC cells. However, it significantly decreased BM in our xenograft mouse models. We also found that the knockout of miR-19a decreased osteolytic lesions in the tumor-bearing bones. We treated osteoclast cells with miR-19a-enriched exosomes or miR-19a-knockout exosomes followed by tartrate-resistant acid phosphatase assay and bone resorption assay. Our results indicated that miR19a-enriched exosomes significantly promoted osteoclast differentiation and activity. In addition to exosomal miR-19a, we also identified IBSP as another necessary factor involved in the BCBM. IBSP is over-expressed in ER+ BC patients with BM, and is negatively correlated with bone metastasis-free survival of ER+ patients. We found IBSP functions as a chemoattractant to create an OC precursor enriched microenvironment to facilitate the delivery of exosomal miR-19a to OC in a concentration-dependent manner. Our animal experiment suggests that both exosomal miR-19a and secreted IBSP are necessary for ER+ BC cells to colonize in the bone microenvironment. In this study, we identified exosome-derived miR-19a and IBSP as important mediators of cell-cell communication between breast cancer and osteoclast cells, which warrants further investigation on both as novel biomarkers and therapeutic targets of BCBM.

#435

Cell-free circulating tumor DNA (ctDNA) detects somatic copy number loss in homologous recombination repair genes.

Catalin Barbacioru, Victoria M. Raymond, Marcin Sikora, Elena Helman, Sante Gnerre, Stephen Fairclough, Darya Chudova, Richard B. Lanman, AmirAli Talasaz. _Guardant Health, San Diego, CA_.

Background: In addition to BRCA1/2 germline and somatic inactivating mutations, loss of heterozygosity (LOH) and biallellic somatic copy number loss are associated with BRCA1/2 loss of function. Patients with cancers harboring these somatic features may benefit from treatment with PARP inhibitors. ctDNA analysis has proven a viable alternative to tumor tissue genotyping, especially in tissue- or time-limited clinical scenarios. However, ctDNA assessment of LOH and biallelic copy number loss is challenging given that ctDNA is diluted by cell-free leukocytic DNA making it difficult to confidently call these genomic events. We developed a method to identify somatic biallelic copy number loss in ctDNA using targeted sequencing of cell-free (cfDNA) across a wide range of cancer types.

Methods: A novel statistical model was developed using coverage profiles and single nucleotide polymorphism (SNP) allelic frequencies estimated from plasma samples to determine the presence of LOH and biallellic copy number loss in BRCA1 or BRCA2. For each gene of interest, the model uses observed coverage and fragment size distribution, together with allele frequency of germline SNPs. The model was applied to plasma samples from 28,000 patients with advanced solid tumors sequenced using a 73-gene next generation sequencing ctDNA panel (Guardant360®, Guardant Health, Redwood City, CA).

Results: The model was analytically validated using in-silico simulations in order to assess both the limit of blank and limit of detection. This method shows 95% sensitivity in detecting LOH and bi-allellic copy number loss for samples with a maximum somatic variant allele frequency as low as 9%. Sensitivity is mainly driven by the panel size and the depth of coverage of the gene of interest. The model was then applied to the 28,199 patient cohort. BRCA1 and BRCA2 LOH was observed in 2.4% (134/5568) and 7.4% (415/5568) of classic homologous recombination deficient (HRD) cancers including breast, ovarian, prostate, and pancreas. BRCA1 and BRCA2 biallelic copy number loss was observed in 0.3% (19/5568) and 0.6% (31/5568) of this same group of HRD cancers.

Discussion: In this cohort of 5,568 patients with classic HRD associated cancers, somatic LOH and biallelic copy number loss was detected in BRCA1 in 2.7% of samples and in BRCA2 in 8.0% of samples, which is aligned with previously reported tissue prevalence. BRCA1/2 somatic LOH and biallelic copy number loss can accurately be detected in ctDNA utilizing likelihood-based scores based on coverage and germline SNPs allele frequencies. The ability to identify this therapeutically targetable genomic alteration through a non-invasive ctDNA assessment has significant clinical implications.

#436

Prediction of aggressive disease in metastatic castration-resistant prostate cancer: The role of very small nuclear circulating tumor cells.

Pin-Jung Chen,1 Yu Jen Jan,2 Pai-Chi Teng,2 Jie-Fu Chen,3 Shirley Cheng,2 Nu Yao,2 Mariana Reis-Sobreiro,2 Amber Lozano,2 Amy Gomez,2 Hsian-Rong Tseng,1 Michael Freeman,2 Edwin Posadas2. 1 _California NanoSystems Institute, University of California, Los Angeles, Los Angeles, CA;_ 2 _Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA;_ 3 _Washington University in St. Louis, Los Angeles, CA_.

Background: Circulating tumor cells (CTCs) have arisen as a contemporary noninvasive prognostic biomarker for prostate cancer (PC). Previously, a subgroup of PC CTCs, with particularly small nuclei (<8.54 μm), were found to be correlated with poor prognosis and the emergence of visceral metastases (VM). This subgroup was named very-small nuclear CTCs (vsnCTCs). We proposed vsnCTCs as a biomarker of aggressive disease in metastatic castration-resistant PC (mCRPC). Previous Studies also showed that the disruption of emerin, a linker of nucleoskeleton and cytoskeleton complex proteins, increases the cancer cells' capacity of migration and invasion. We hypothesize that emerin mislocalization is potentially associated with vsnCTC formation and may be a critical step for metastasis.

Methods: 35 blood samples were obtained from PC patients who failed first-line androgen deprivation therapy (ADT) and starting 2nd line treatment with abiraterone, enzalutamide, or taxane-based chemotherapy. Using our NanoVelcro CTC assay, we captured, subclassified, and enumerated the CTCs from patient samples with high-resolution imaging. Survival analyses were performed to exam the correlation between vsnCTC presence and patients' prognosis. Concurrently, emerin staining was performed and the distribution and expression levels of emerin were analyzed in selected vsnCTC samples.

Results: The presence of one or more vsnCTCs strongly correlated with inferior overall survival (OS), progression free survival (PFS), and time to VM (TTVM). We also observed lower emerin content in vsnCTCs compared to WBC, and more prominent emerin mislocalization in vsnCTCs compared to CTCs with larger nuclei.

Conclusion: Our study strongly demonstrated the importance of morphologic characterization of CTCs and suggested that vsnCTC is a blood-borne biomarker for prediction of aggressive disease. Additionally, emerin mislocalization in vsnCTCs could be a potential biological pathway behind this morphologic phenomenon.

#437

Combining circulating stromal cells with cell free DNA for increased sensitivity in profiling oncogenic mutations and indicates highly aggressive non small cell lung cancer.

Daniel L. Adams,1 Steven H. Lin,2 Ashvathi Raghavakaimal,3 Glenn Weiss,4 Andrew Ford,4 Charmaine Brown,4 Chen-Hsiung Yeh4. 1 _Creatv MicroTech, Inc., Monmouth Junction, NJ;_ 2 _MD Anderson Cancer Center, Houston, TX;_ 3 _Rutgers, the State University of New Jersey, New Brunswick, NJ;_ 4 _Circulogene, Birmingham, AL_.

Background: Circulating cell free DNA (cfDNA) in the plasma of cancer patients may provide oncogenic mutation status for NSCLC. However, the low sensitivity of current cfDNA has clinical utility limited to late stage multifocal or some recurrent diseases. Recently, specific phagocytic stromal cells found in blood, i.e. cancer associated macrophage-like cells (CAMLs), were shown to contain large quantities of tumor DNA. We hypothesized that a single blood sample can provide both cfDNA and parallel CAML DNA, providing more sensitive tumor mutation screening in a broader array of patients. We screened untreated NSCLC patients with a range of stages (stage I=3, stage II=5, stage IIIa=10, stage IIIb=7, & stage IV=5), that had available primary tissue for NGS. Blood was drawn prior to induction of radiotherapy, and plasma was sequenced using a 50 gene oncopanel. Separately, CAMLs were isolated from the same blood sample, lysed and sequenced using the same panel. Our data suggests that CAMLs contain clinically relevant oncogenic variants that correspond to both the primary tumor and the matched plasma.

Methods: Whole peripheral blood was taken from 30 newly diagnosed NSCLC patients with confirmed stage I-IV disease in a preservative collection tube. Plasma was separated and frozen at -80C. CAMLs were purified from the cell pellet using a standard CellSieveTM Assay, which isolates CAMLs based on size separation. Purified CAMLs were enumerated, then removed from the filter and lysed in 150 µL of buffer. The primary tumor biopsies, plasma and lysed CAMLs were blinded and anonymized for sequencing against a standard 50 oncogene NGS panel. Total mutations in tumor/plasma/CAMLs were used to evaluate progression free survival (PFS) by censored univariate analysis.

Results: Forty three mutations were identified from primary tumor DNA (averaging 1.4 variants in 87% of patients) vs 28 mutations from cfDNA (0.9 variants in 47% of patients) with no exon variant matches, although a nonmatching variant was found on the TP53 loci. CAML lysate had 2.8X more mutations than cfDNA, with 78 variants averaging 2.6 variants in 80% of patients. Further, 2 CAML variants exactly matched the primary tumor and 3 more matched at the gene level. cfDNA and CAMLs had the best concordance, with 6 matching exon variants and 6 more matching on the same gene. Notably, patients with cfDNA or CAML mutations had lower PFS, with ≥1 variants in cfDNA (HR=4.0, 95%CI=1.4-11.3, p=0.021) and ≥4 variants in CAMLs (HR=4.3, 95%CI=1.4-12.9, p=0.020).

Conclusions: Our data suggests that a single blood sample to screen for oncogenic tumor mutations from cfDNA and CAMLs provides increased sensitivity and specificity than cfDNA alone. Further, while preliminary, we find that high mutation burden in cfDNA or CAMLs may be indicative of aggressive NSCLC that is more likely to progress within 2 years.

#438

Multicenter evaluation of circulating tumor DNA assays.

Dan Brudzewsky,1 Rita Lampignano,2 Sabrina Weber,3 Alexander Sartori,4 Sumitra Mohan,5 Yves Konigshofer,1 Lorn Davis,1 Dominic Rothwell,5 Ed Schuuring,6 Russell K. Garlick,1 Thomas Schlange,2 Ellen Heitzer3. 1 _SeraCare Life Sciences, Milford, MA;_ 2 _Bayer AG, Wuppertal, Germany;_ 3 _Medical University Graz, Graz, Austria;_ 4 _Agena Biosciences GmbH, Hamburg, Germany;_ 5 _CRUK Manchester, Manchester, United Kingdom;_ 6 _University Medical Center Groningen, Groningen, Netherlands_.

Measurements of circulating tumor DNA (ctDNA) hold great potential to detect residual disease, therapeutic response monitoring and tumor evolution. However, there are many technical challenges including trace levels of circulating cell free DNA 1-10 ng/ml in plasma (300-3,000 genome equivalents/ml), DNA fragment size 170 bps found in the circulation, low variant allelic fractions ~ 0.1% and the need to detect all variant types SNVs, insertions, deletions, CNVs and structural variants. CANCER-ID partners comprise a consortium of experts, companies and institutions for blood -based biomarker validation, assay development, clinical sciences and bioinformatics. This CANCER-ID ring trial was established to help standardize complex ctDNA diagnostic assays by testing a highly complex reference material. The trial looks to improve concordance and to evaluate analytical performance by collecting metrics covering the entire workflow from sample preparation through variant reporting. The common reference material, Seraseq ctDNA Complete, used in the trial is human genomic DNA background material from the Genome-in-a-bottle consortium mixed with synthetic DNA sized to ~170 bp and formulated in artificial plasma. The material contains 25 variants per sample including 12 SNVs (8 genes), 5 deletions (3 genes), 2 insertions (2 genes), 3 CNVs (3 genes) and 3 fusion pairs (3 genes). The variant allelic fractions range from 0.1% to 5% for the SNVs, INDELS and ~ 1 -6 additional copy number for ERBB2, MET and MYC genes. Interlab and intralab testing was done on Roche AVENIO targeted Assay, Agilent SureSelect XT custom assay, QiaSeq Targeted DNA panel, Qiagen GeneRead QIAAct Lung Assay, one clinically validated digital droplet assay and one research digital droplet PCR assays and Agena Bioscience's UltraSEEK Lung Panel. Data analysis, peer comparison and reporting of common and unique QC metrics was accomplished through the cloud based, analytical QC software iQ NGS QC Management. The results demonstrate that a common full process reference material can be used to compare distinctly different assays formats at different levels of input DNA by mutations. For the 1% AF material, the BRAF V600 variant was detected by all assay formats and the cv's ranged from 12%-52% or on average for all variants measured by the assay ranged from 11% to 46%. The study furthermore shows that peer review analytical QC software is useful in establishing standardization of ctDNA assays. This work is supported by IMI JU & EFPIA (grand no. 115749, CANCER-ID).

#439

Targeted single cell DNA sequencing without prior whole genome amplification for mutational analysis of circulating tumor cells.

Nolan G. Ericson,1 Arturo B. Ramirez,1 Alisa C. Clein,2 Celestia S. Higano,2 Daniel E. Sabath,2 Eric P. Kaldjian1. 1 _RareCyte, Inc., Seattle, WA;_ 2 _University of Washington, Seattle, WA_.

Background. RareCyte has developed platform technology for visual identification and single cell retrieval of rare cells in blood, including circulating tumor cells (CTCs). There is increasing interest in mutational analysis of circulating tumor cells (CTCs) as a liquid biopsy application. Because of the minuscule amount of DNA present in a single cell (~6 pg), whole genome amplification (WGA) is typically performed prior to next generation sequencing (NGS) library preparation. Existing WGA methods have inherent amplification biases leading to non-uniform genome coverage that can cause dropout of desired targets, as well as elevated error rates that can lead to false positive mutations. Amplicon-based next generation sequencing (abNGS) is a high-throughput method which enables genetic confirmation of malignancy and discovery of de novo pathogenic mutations. Here we present a method for performance of single cell abNGS on model CTCs without prior WGA using a commercially available pan-cancer hotspot panel.

Methods. A549 lung cancer cells as model CTCs (mCTCs) were spiked into whole blood, which was processed by AccuCyte® separation onto slides. After formalin fixation, multi-parameter immunofluorescence and automated imaging (CyteFinder®) were used to identify CTCs - visualized as nucleated cells expressing epithelial markers (cytokeratin or EpCAM) and not expressing white blood cell markers. mCTCs were mechanically retrieved by CytePicker® into PCR tubes and either amplified by WGA (PicoPLEX®) or lysed in a PCR-compatible lysis buffer. WGA products or cell lysates were used as template for the AmpliSeq™ Cancer HotSpot Panel v2 for Illumina® library preparation; additional PCR cycles were added during target amplification to compensate for low DNA input in the non-WGA samples. Libraries were sequenced on an Illumina MiSeq and analyzed using the BaseSpace bioinformatics suite.

Results. Single mCTCs that underwent amplicon-based NGS library prep direct from cell lysate (non-WGA) displayed increased uniformity of coverage with decreased target dropout when compared to WGA cells. Median read depth increased 7-fold with the non-WGA method. On average, 8 of 15 variants present in bulk A549 genomic DNA were observed in single mCTCs sequenced after WGA, while 12 out of 15 were observed with the non-WGA method. Additionally, the false positive error frequency of non-WGA samples was < 5% of the WGA samples. The non-WGA method was applied to CTCs identified in blood from a prostate cancer patient and confirmed presence of PTEN and TP53 mutations identified by cell-free DNA analysis.

Conclusions. Amplicon-based targeted single-cell sequencing without prior WGA resulted in libraries with more complete and consistent coverage and lower error frequencies, enabling efficient and accurate assessment of somatic mutations in CTCs.

#440

Use of longitudinal samples from the DOD serum repository to identify candidate biomarkers for HNSCC.

Ju Yeon Lee,1 Tujin Shi,1 Vladislav Petyuk,1 Athena Schepmoes,1 Thomas Fillmore,1 Wayne Cardoni,2 George Coppit,2 Joseph Goodman,2 Shiv Srivastava,3 Craig Shriver,2 Tao Liu,1 Karin Rodland1. 1 _Pacific Northwest National Laboratory, Richland, WA;_ 2 _Walter Reed National Military Medical Center, Bethesda, MD;_ 3 _Center for Prostate Disease Research, Richland, WA_.

The Department of Defense Serum Repository (DODSR) represents a unique resource enabling longitudinal studies of cancer risk, progression, and response to therapy. The DODSR was initiated in 1989, initially as a means for HIV surveillance, and is comprised of serum samples from active and reserve military personnel drawn at enlistment and annually or biennially throughout the service members' participation in the Military Health System, accompanied by the service member's electronic health records. This enables the identification of serum samples for all service members with a particular diagnosis, such as Head and Neck Squamous Cell Carcinoma (HNSCC), a cancer significantly represented in the military population with substantial impact on military operations. For HNSCC, 175 cases diagnosed between 2003 and 2013 were identified, as well as 175 corresponding healthy controls matched for age at the time of diagnosis, gender, and ethnicity. Serum samples drawn at the time of diagnosis, 2 and 4 years prior to diagnosis, and 2 years after diagnosis were retrieved and analyzed by targeted mass spectrometry analysis using a panel of 148 qualified selected reaction monitoring (SRM) assays selected from the curated literature and discussions with military oncologists at the Murtha Cancer Center. Initial analyses of 212 samples (38 cases and 38 matched controls, each with 2-4 longitudinal samples) from the total 978 samples available focused on comparisons of pre-diagnosis and at diagnosis serum proteins. Preliminary results identified proteins that were differentially abundant between serum samples taken from HNSCC cases at the time of diagnosis, compared to age-matched controls. Proteins such as A2GL displayed a trend of gradually increasing abundance as a function of time to diagnosis, in HNSC cases, suggesting behavior that may be proven useful for early detection of HNSCC, prior to overt symptoms. These preliminary results suggest that the DODSR can be used to provide serum samples of sufficiently high quality for extensive proteomic analysis, and form cohorts sufficiently large to enable meaningful statistics. Access to longitudinal samples representing years prior to diagnosis enables the identification of serum factors that predict the risk of cancer and may also enable early diagnosis. Access to post-treatment serum samples accompanied with data indicating the clinical response to treatment enables the development of algorithms predicting response to treatment and may eventually contribute to the selection of appropriate therapies.

#441

The stochastic nature of errors in next-generation sequencing of circulating cell-free DNA.

Hunter R. Underhill, Preetida J. Bhetariya, Sabine Hellwig, David A. Nix, Carrie L. Fuertes, Gabor T. Marth, Mary P. Bronner. _University of Utah, Salt Lake City, UT_.

Purpose: Challenges with distinguishing circulating tumor DNA from next-generation sequencing (NGS) artifacts limits variant searches to established solid tumor mutations. Identifying the source(s) of errors associated with the NGS analytics of circulating cell-free DNA (ccfDNA) would enable the determination of an optimal strategy for eliminating noise and broaden ccfDNA clinical applications.

Methods: Buffy coat DNA and ccfDNA were isolated from seven healthy adults. For each participant, a single buffy coat DNA library was generated using duplex adapters (dual unique molecular identifiers [UMIs], dual index), while two ccfDNA libraries were separately produced - one library with singleton adapters (single UMI, single index) and one library with duplex adapters. The assignment of a UMI to each template DNA molecule prior to library formation reduces false positives. A family is a set of DNA amplicons (PCR duplicates) with the same UMI. Representing a family with a single consensus sequence reduces PCR errors and sequencing artifacts. Duplex adapters have been developed to abrogate the early PCR errors that beset singleton adapters.

Results: The error rate using duplex adapters was significantly lower by 26.4±5.9% (P < 0.001) compared to singleton adapters at family size ≥2, where family size is defined as the number of PCR duplicates that yield a single consensus sequence. Due to the persistence of noise in both singleton and duplex adapters even at large family sizes (i.e., ≥10), we explored potential sources of the residual error. Noise in ccfDNA due to effects from clonal hematopoiesis of indeterminate potential (CHIP) accounted for <4% of error. Removing locations with errors present in all seven samples (i.e., highly patterned error likely due to regions difficult to sequence, align, or both) reduced noise in the duplex and singleton adapters by 18.7±5.1% (P < 0.001) and 14.5±3.1% (P < 0.001), respectively. Finally, we explored the effects of stochastic noise as a source of error. A complete replicate with duplex adapters was generated beginning from the source ccfDNA and library preparation. Using replicate data, the error rate was reduced by an additional 59.9±4.3% (P < 0.001) for the duplex adapters at family size ≥2. Using duplex adapters, accounting for CHIP artifacts, removing locations with highly patterned errors, and including replicate data reduced error by 85.4±4.6% (P < 0.001) compared to the error rate for singleton adapters at family size ≥2. Error continued to decline with each family size increment.

Conclusion: Early stochastic PCR errors are a principal source of NGS noise that persist despite duplex molecular barcoding and after removal of patterned errors. Replicates are necessary to eliminate noise and their use in NGS analytics may broaden ccfDNA applications particularly in pre-metastatic and recurrent solid-tumor malignancies by enabling untargeted variant investigations.

#442

Repotrectinib, a next generation TRK inhibitor, overcomes TRK resistance mutations including solvent front, gatekeeper and compound mutations.

Alexander Drilon,1 Dayong Zhai,2 Wei Deng,2 Xin Zhang,2 Dong Lee,2 Evan Rogers,2 Jeffrey Whitten,2 Zhongdong Huang,2 Armin Graber,2 Juliet Liu,2 Shanna Stopatschinskaja,2 J. Jean Cui,2 Dong-Wan Kim,3 Byoung Chul Cho,4 Robert C. Doebele,5 Sai-Hong Ignatius Ou,6 Jeeyun Lee,7 Alice T. Shaw8. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _TP Therapeutics, Inc., San Diego, CA;_ 3 _Seoul National University Hospital, Seoul, Republic of Korea;_ 4 _Yonsei Cancer Center, Seoul, Republic of Korea;_ 5 _University of Colorado Denver, Aurora, CO;_ 6 _University of California Irvine School of Medicine, Orange, CA;_ 7 _Samsung Medical Center, Seoul, Republic of Korea;_ 8 _Massachusetts General Hospital, Boston, MA_.

Oncogenic TRKA/B/C fusions are identified in multiple cancer types in adults and children. TRK inhibitors (TRKis) have demonstrated marked efficacy in patients with these cancers, however, acquired on-target resistance mediated by kinase domain mutations can occur. Next-generation TRKis targeting both wildtype and mutant TRK fusions can address this unmet need. Repotrectinib was designed to potently inhibit wildtype (WT) TRKs and overcome resistance mutations. The anti-proliferative activity of 1st generation (larotrectinib/entrectinib) and next-generation (repotrectinib/LOXO-195) TRKis were compared using engineered Ba/F3 cells expressing WT or mutated TRKs (Table). Repotrectinib was over 10-fold more potent than LOXO-195 against WT TRK fusions and solvent front mutations (SFMs), and more than 100-fold more potent against the gatekeeper mutations TRKA F589L and TRKC F617I. Furthermore, repotrectinib was the only TRKi active against the compound mutation TRKA G595R/F589L in cis in preclinical Ba/F3 cells. In xenograft tumor models, repotrectinib led to significant tumor regression in tumors carrying WT or mutated TRK fusions. In the ongoing TRIDENT-1 phase 1 clinical trial of repotrectinib (NCT03093116), the SFMs TRKA G595R, TRKC G623R and TRKC G623E and the gatekeeper mutation TRKA F589L were detected in plasma cfDNA samples at baseline from three TRKi-resistant patients. Repotrectinib was active against ETV6-TRKC G623E in an entrectinib-resistant patient with a salivary gland tumor (-82%, confirmed partial response, RECIST v1.1). Tumor regression (-33%) was achieved in a larotrectinib-resistant cholangiocarcinoma patient with LMNA-TRKA G595R and F589L mutations in trans. TRIDENT-1 is currently enrolling NTRK fusion-positive patients with advanced solid tumors. | |  | |  | |  | |  | |

|

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

|

Ba/F3 Cell Proliferation Assay IC50 (nM)

|

LMNA-TRKA | ETV6-TRKB | ETV6-TRKC

TRK Inhibitor | WT | G595R | G667C | F589L | G595R/F589L | WT | G639R | WT | G623R | G623E | F617I

Repotrectinib | <0.1 | 0.1 | 9.2 | <0.1 | 17.4 | <0.1 | 3.2 | <0.1 | 0.4 | 0.9 | <0.2

LOXO-195 | 8.6 | 13.1 | 94.9 | 31.6 | 531.1 | 1.0 | 28.4 | 1.7 | 24.6 | 49.1 | 53

Larotrectinib | 15.9 | 1898 | 1863 | 625.3 | 10000 | 13.1 | 3000 | 23.2 | 6999 | 2162 | 5000

Entrectinib | 0.3 | 614 | 186.7 | <0.2 | 2000 | 0.2 | 2000 | 0.4 | 1340 | 1712 | 61.7

#443

Blood-based genotyping and clinical outcomes in patients with advanced breast cancer receiving the CDK4/6 inhibitor palbociclib in real-world settings: Results from POLARIS.

Aditya Bardia,1 Joanne L. Blum,2 Steven L. McCune,3 Kamal Patel,4 Richard C. Frank,5 Kailash Mosalpuria,6 Meghan S. Karuturi,7 Lloyd A. Shabazz,8 Gabrielle B. Rocque,9 Yuan Liu,10 Zhe Zhang,11 Jian Wang,12 Yao Wang,13 Debu Tripathy7. 1 _Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA;_ 2 _Texas Oncology, Baylor-Sammons Cancer Center, US Oncology, Dallas, TX;_ 3 _Northwest Georgia Oncology Centers, Marietta, GA;_ 4 _CARTI Cancer Center, Little Rock, AR;_ 5 _Whittingham Cancer Center at Norwalk Hospital, Norwalk, CT;_ 6 _Nebraska Hematology Oncology, Lincoln, NE;_ 7 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 8 _Delta Oncology Associates, PC, Portsmouth, VA;_ 9 _University of Alabama at Birmingham, Birmingham, AL;_ 10 _Pfizer Inc., San Diego, CA;_ 11 _Pfizer Inc., La Jolla, CA;_ 12 _Cytel Inc., Waltham, MA;_ 13 _Pfizer Inc., New York, NY_.

Background:POLARIS is a prospective, real-world (RW) study of palbociclib (PAL) in patients (pts) with hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2‒) advanced breast cancer (ABC) in the US and Canada. Analyses include evaluating the association between circulating tumor DNA (ctDNA) and clinical response to PAL.

Methods: All pts provided informed consent. Blood samples are collected on day 1 of each 4-wk cycle for 6 cycles, at day 1 of every 2-3 cycles, and end of treatment. ctDNA is sequenced using the Guardant360 platform for somatic single-nucleotide variants in complete or critical exons of 73 genes. Analyses include association between clinical outcomes and ctDNA mutation frequencies at baseline and change from baseline to cycle 2, day 1 (C2D1), stratified by line of prior therapy.

Results: As of July 2018, 127 pts had ≥1 evaluable ctDNA measurement. Of these, 104 had baseline and C2D1 results; 96 had tumor response assessed. The most common mutations at baseline were PIK3CA (41%), TP53 (33%), and ESR1 (20%). PAL was associated with a decline in mutation frequency of PIK3CA, TP53, and ESR1 alleles but not all genes, highlighting differential response based on genomic profile and clonal heterogeneity (Table). Overall, fast progressors (progression <6 mo, n=19) tended to have higher baseline ctDNA values and displayed smaller decreases in ctDNA at C2D1 than slow progressors (progression ≥6 mo, n=4).

Conclusions: This study is among the first to provide RW data on blood-based genotyping. Interim data indicate that genomic architecture and longitudinal changes may be predictive and prognostic in pts with HR+/HER2‒ ABC receiving PAL. Updated data from 239 pts will be presented, including association between individual ctDNA mutation dynamics, clonal heterogeneity, and clinical outcomes (therapeutic response and progression-free survival). Pfizer; NCT03280303

Change from baseline in ctDNA mutation frequency

---

Mutation | Overall (n=104)* | First Line (n=70) | Second Line and Later (n=34)

|

Baseline,

n (%)† | C2D1, n (%)† | Baseline,

n (%)† | C2D1, n (%)† | Baseline,

n (%)† | C2D1, n (%)†

PIK3CA | 43 (41) | 26 (25) | 27 (39) | 14 (20) | 16 (47) | 12 (35)

TP53 | 34 (33) | 27 (26) | 23 (33) | 17 (24) | 11(32) | 10 (29)

ESR1 | 21 (20) | 11 (11) | 9 (13) | 3 (4) | 12 (35) | 8 (24)

ARID1A | 19 (18) | 14 (13) | 13 (19) | 7 (10) | 6 (18) | 7 (21)

NF1 | 11 (11) | 11 (11) | 7 (10) | 6 (9) | 4 (12) | 5 (15)

BRCA2 | 10 (10) | 10 (10) | 8 (11) | 7 (10) | 2 (6) | 3 (9)

GATA3 | 10 (10) | 5 (5) | 8 (11) | 5 (7) | 2 (6) | O (O)

None detected | 13 (13) | 24 (23) | 7 (10) | 20 (29) | 6 (18) | 4 (12)

ctDNA=circulating tumor DNA; C2D1=cycle 2, day 1. *Patients with both baseline and C2D1 ctDNA data available. †Percentages are relative to the corresponding number of patients (n).

#444

Continuous, high throughput microfluidic device to monitor circulating tumor cells in cancer patients.

Kaylee Smith,1 Tae Hyun Kim,1 Costanza Paoletti,2 Douglas H. Thamm,3 Daniel F. Hayes,2 Sunitha Nagrath1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _University of Michigan Rogel Cancer Center, Ann Arbor, MI;_ 3 _Colorado State University, Fort Collins, CO_.

Analysis of circulating tumor biomarkers, designated "liquid biopsies" are less invasive and lower risk to the patient and might replace or complement standard tissue biopsies to determine prognosis, predict benefit from specific therapies, or monitor patients with cancer. Circulating tumor cells (CTCs) are extremely rare, with only 1-10 CTCs per mL of blood. Current technologies typically process 1-10 mL of blood ex vivo. We report an in vivo, indwelling CTC-capture device that is analogous to a Holter monitor used to interrogate cardiac rhythm abnormalities over long periods of time. The system incorporates a dual lumen intravenous catheter that directs blood from the vein into a small (~ the size of a wristwatch) capture carriage by virtue of a peristaltic pump. The carriage contains a CTC capture chip which separates CTCs from whole blood based on flow differences and solid phase anti-EpCAM capture, while the remaining whole blood is returned to the venous system via the dual lumen catheter. The chip is then removed from the carriage for further enumeration and characterization of the CTCs. In a proof of principle set of experiments, the system was placed into investigational canines into which fluorescently labeled human breast cancer cells were injected intravenously. Although the vast majority of these xeno-cancer cells were rapidly removed from circulation, the system captured "CTCs" over a 2 hour time period more successfully than was seen in simultaneous blood draws for ex vivo enumeration over the same time. No adverse effects were observed in the subjects at the time of the experiments or over the succeeding 48 hours after removal of the IV system.Our system permits interrogation of a much larger volume of blood than can be processed with standard blood draws. The larger processed volume will drastically increase the number of cells isolated which in turn increases the number of downstream assays that can be performed. This study is focused on developing a high throughput microfluidic device that can detect CTCs and implementing it in a canine model.

#445

A validated genomic reference material with known content applicable to panel assays requiring low frequency variant allele detection.

Wendell Jones,1 Joshua Xu,2 Binsheng Gong,2 Onco-panel Working Group of the Sequencing QualityControl Phase 2 (SEQC2) Consortium. 1 _Q2 Solutions, Morrisville, NC;_ 2 _Food and Drug Administration, Jefferson, AR_.

Prospective and GCP testing of tumors for important somatic variants in cancer is becoming more commonplace. Currently, there is a lack of a reliable and perpetual publicly available genomic material that can be used across a wide range of genomic testing methods for assessing the potential accuracy of a given test. A great variety of the next-gen sequencing (NGS) methods and processes currently in place now or in the future will have heterogeneous genomic targets, limits of detection, input material, and cost requirements. The variety of these tests extends from small panels (targeting 10 or more genes) to larger panels (targeting > 100 genes) to comprehensive tests (several hundred genes or WES/WGS). There is considerable variability in the sensitivity requirements of various assays with some designed to detect variant allele frequencies (VAF) of 10% or more while others wish to detect VAF < 1%. In addition, the testing itself has several layers of complexity: 1) the size and complex nature of the human genome; 2) the additional complexity of the damaged genome in cancer cells; 3) the amount and origin of the input material; and 4) the complex instruments and software algorithms necessary to proficiently perform the tests. The SEQC2 consortium has extensively evaluated and documented a pooled cell line reference material that has > 40,000 known variants with the great majority having allele frequencies < 10% in the standard pool. This effort evaluated DNA pooled from 11 cell lines, 10 of which are from distinct cancers, used for generating RNA material for the Universal Human Reference RNA (UHRR). This consortium defined ground truth (primarily SNVs, Indels) for each cell line using related and orthogonal genomic testing methods (multiple platforms, enrichment methods, and software algorithms) so that the content of various standardized admixtures and pools of the material will be known. The final results were validated by additional sequencing of the individual pools and through ddPCR testing of a large number of identified variants. The result is a series of reference materials with a large number and variety of validated variants at various VAF, suitable for a wide range of testing, including variants with VAF < 1%. These materials can be used as an ongoing reference material when developing new assays, conducting proficiency and reproducibility tests, or for quality control purposes when running regulated assays (positive and negative control variants).

#446

Plasma genome sequencing identifies prostate cancer patients that are sensitive to platinum-based therapy.

Naveen Ramesh, Emi Sei, Pei Ching Tsai, Christopher Logothetis, Paul Corn, Ana Aparicio, Amado J. Zurita, Nicholas E. Navin. _MD Anderson Cancer Center, Houston, TX_.

Castration-resistant prostate cancer (CRPC) is a lethal disease. A subset of these patients present with atypical clinical characteristics and aggressive disease behavior. These patients, classified as Aggressive Variant Prostate Cancer (AVPC), may derive benefit from platinum-based chemotherapy. However, the identification of AVPC patients is often challenging due to heterogeneity in clinical presentations and potentially in biological drivers, which may not be fully reflected in a biopsy obtained from a single tumor site. To address these challenges, we developed an unbiased genome sequencing method called PEGASUS (Plasma Exome and Genome Analysis by Size-Selection and Unbiased Sequencing) that enables the simultaneous detection of copy number aberrations and exome mutations from circulating-tumor DNA (ctDNA). We applied this method to plasma specimens obtained from 160 CRPC patients with and without AVPC participating in a randomized trial of cabazitaxel alone or in combination with carboplatin. We were able to successfully isolate ctDNA (>2 ng) for genomic profiling using PEGASUS in 91/160 (57%) CRPC patients. Among these 91 CRPC patients, we detected common prostate cancer mutations as well as genomic copy number changes in genes such as RB1, PTEN and TP53 and several other genes associated with DNA repair. One of the most salient features that distinguished the patients' outcomes was whether they had diploid or aneuploid genomes detected by whole genome profiling of the ctDNA. Patients with aneuploid ctDNA had worse progression-free and overall survival. Overall, our data shows the feasibility of performing whole-genome and exome profiling of ctDNA in CRPC patients to characterize the molecular features associated with distinct clinical presentations. Candidate markers associated to benefit from platinum-based chemotherapy are being evaluated. These results pave the way for future clinical applications in biomarker discovery to assist treatment decisions in prostate cancer patients.

#447

Sensitive detection of ctDNA in low burden and 'challenging' cancers.

Christopher G. Smith,1 Jonathan C. Wan,1 Katrin Heider,1 Florent C. Mouliere,2 Richard Mair,1 Matthew Eldridge,1 Kevin Brindle,1 Pippa G. Corrie,3 Grant D. Stewart,4 Nitzan Rosenfeld1. 1 _CRUK Cambridge Institute, Cambridge, United Kingdom;_ 2 _Amsterdam UMC, Amsterdam, Netherlands;_ 3 _Cancer Research UK Major Centre, Cambridge, United Kingdom;_ 4 _Cambridge University Hospitals NHS Foundation Trust, Cambridge, United Kingdom_.

Cell-free tumour derived DNA (ctDNA) analysis offers the potential for minimally-invasive early detection, diagnosis and monitoring of cancer in patients. However, the utility of ctDNA is proving challenging for some cancer types and in earlier stages of disease. We developed an approach for sensitive detection of ctDNA through combined application of Tailored Panel Sequencing (TAPAS) and aggregation of signal across mutant loci by INtegration of VAriant Reads (INVAR). We demonstrated its application to 'challenging' cancers that have been shown to release lower concentrations of ctDNA as compared to other cancers of similar size and stage: renal cancer (RCC) and glioblastoma (GB). We also utilised INVAR-TAPAS for early detection of cancers, as well as detection of residual disease after surgery. We applied INVAR-TAPAS to plasma and urine from 22 patients with RCC of varying subtype and disease stage, and demonstrated ~45% detection in both fluids (10/22 in plasma and 6/14 in urine). Of note, considering both fluids ctDNA was detected in a patient with a benign oncocytoma, and in 2/6 (33.3%) and 10/15 (66.7%) stage I-II and III-IV RCCs respectively. We also applied INVAR-TAPAS to plasma and cerebrospinal fluid (CSF) from 8 GB patients and observed ctDNA detection rates of 75% in both fluids, though levels were higher in CSF. For both diseases, comparison of INVAR-TAPAS and disease burden, as assessed by imaging, demonstrated that ctDNA levels in longitudinal plasma samples track disease course. Furthermore, by basing our analysis on mutations identified from multi-region sampled tissue data, we observed that mutations private to distinct tumour regions were represented in ctDNA (in all fluids tested), in-turn demonstrating that ctDNA overcomes heterogeneity. Finally, INVAR-TAPAS was applied to plasma samples collected from 33 patients with resected stage II-III melanoma. Samples were obtained within the first 6 months after surgery. We detected ctDNA in 50% of patient samples who later recurred, with detection associated with a significantly shorter disease-free interval (4.5 months vs. median not reached with 5 years' follow-up; Hazard ratio (HR) = 3.69; 95% CI 1.44-9.46, P = 0.007) and overall survival (2.6 years vs. median not reached). In summary we demonstrated the ability of INVAR-TAPAS to sensitively detect and quantify ctDNA in various cancer settings. Of note, in these clinical samples we detected ctDNA to a mutant allele fraction of 1x10-5. Despite this sensitivity, detection of certain cancer types and earlier disease stages remains challenging. In these settings, improved isolation and detection methods (e.g. multi-analyte assays) may improve detection and ultimate utility of ctDNA in the future. Nonetheless, our data support liquid biopsy as both a predictive and prognostic biomarker with potential to guide clinical decision making in the future.

#448

Genomic instability as a marker of poor prognosis in advanced prostate cancer: Subclonal insights from whole-genome sequencing of single circulating tumor cells.

Paymaneh D. Malihi,1 Ryon P. Graf,2 Carmen Ruiz Velasco,1 Anand Kolatkar,1 Angel E. Rodriguez,2 Jerry Lee,2 Ramsay Sutton,2 Paul G. Corn,3 Christopher Logothetis,3 Ana Aparicio,3 James Hicks,1 Ryan Dittamore,2 Amado Zurita,3 Peter Kuhn1. 1 _University of Southern California, Los Angeles, CA;_ 2 _EPIC Sciences, San Diego, CA;_ 3 _MD Anderson Cancer Center, Houston, TX_.

A subset of castration-resistant prostate cancer (CRPC) patients present with atypical clinical features and aggressive disease behavior. These patients have a poor prognosis and may derive increased benefit from platinum-based chemotherapy. Heterogeneity in clinical presentations and insufficient understanding of biological drivers of progression underscore the need for more precise molecular stratification methods. We hypothesized that genomic characterization of circulating tumor cells (CTCs) at the single cell level would identify biomarkers relevant to the classification of aggressive CRPC tumors. Peripheral blood samples were collected before initiation of cabazitaxel +/- carboplatin from a cohort of 62 metastatic CRPC patients with and without clinically defined aggressive variant prostate cancer (AVPC) enrolled in NCT01505868. Samples were processed and analyzed for CTCs using the Epic Sciences detection and characterization platform in a CLIA-like environment. After CTC detection, single cells were picked and individually sequenced to assess gene gains and losses and genomic large-scale segment transitions (LST). Of 62 collected blood samples, 49 were positive for CTCs. Patients with more rapidly progressive disease (as assessed by a clinician) had higher CTC/mL, higher AR+CTC/mL, and higher median LST per CTC (p-values: 0.00669, 0.0358, and 0.00623, respectively). AVPC status, as marked by clinical features, was also associated with a higher number of LSTs in CTCs (median 24 per patient CTC vs. 10.5 in non-AVPC, p = 0.02672), whereas no difference in AR+CTC/mL or CTC/mL enumeration was observed. CTC count and LST were additive to prognosticate death by 12 months (AUC = 0.753 combined). The individual copy number was further assessed across 574 genes to identify alterations more commonly associated with poor prognosis. Myc gain, TP53 loss, and other aberrations in genes affecting DNA repair, resistance to anchorage-independent apoptosis, and cell motility showed significant positive association with LST. The presence of high genomic LST, a marker of genomic instability, in individual CTC was associated with more aggressive CRPC behavior (faster rate of progression, shorter survival) and specific gene copy number aberrations. High LST added independent prognostic significance to CTC enumeration. These data suggest that single CTC genomic analysis may provide clinically relevant insights and point to genomic instability in CTCs as a candidate marker of poor prognosis in advanced prostate cancer.

#449

Use of comprehensive cell-free circuating tumor DNA (cfDNA) analysis to identify genomic biomarkers in newly diagnosed advanced non-small cell lung cancer (NSCLC) patients.

Ramon Palmero,1 Alvaro Taus,2 Santiago Viteri,3 Margarita Majem,4 Enric Carcereny,5 Javier Garde,6 Enriqueta Felip,7 Lourdes Gomez,8 Andrea Malfettone,9 Miguel Sampayo,9 Noemí López,9 Rebecca Nagy,10 Matthew Jackson,10 Iris Faull,11 Daniel Dix,10 Niki Karachaliou,12 Rafael Rosell13. 1 _Catalan Institute of Oncology, Barcelona, Spain;_ 2 _Hospital del Mar, Barcelona, Spain;_ 3 _Quirón Salud-Dexeus University Institute, Barcelona, Spain;_ 4 _Hospital de la Santa Creu i Sant Pau, Barcelona, Spain;_ 5 _Hospital Gernmas Trias i Pujol (Can Ruti), Badalona, Spain;_ 6 _Hospital Arnau de Vilanova de Valencia, Valencia, Spain;_ 7 _Hospital Vall d'Hebron, Barcelona, Spain;_ 8 _MedSIR Barcelona, Barcelona, Spain;_ 9 _MedSIR, Barcelona, Spain;_ 10 _Guardant Health, South San Francisco, CA;_ 11 _Guardant Health, Barcelona, Spain;_ 12 _Quirón Salud-Dexeus University Institute, barcelona, Spain;_ 13 _Catalan Inst. of Oncology, Barcelona, Spain_.

Background: In advanced NSCLC pts, current guidelines recommend broad molecular profiling for the following genes EGFR, ALK, ROS1, RET, MET, ERBB2, BRAF and KRAS. Tissue may be used for genomic testing in newly diagnosed advanced NSCLC but comprehensive plasma-based genotyping is less invasive and may provide a quicker and more complete assessment. The present study was conducted to prospectively assess performance of cfDNA compared to standard of care (SOC) tissue-based genomic testing to identify guideline recommended alterations in NSCLC pts.

Methods: In this prospective, multicenter study, blood samples from treatment-naïve stage IIIB-IV NSCLC pts were tested using the Guardant360 next-generation sequencing (NGS) cfDNA panel and compared with SOC tissue testing. The primary objective was to demonstrate the non-inferiority of cfDNA vs tissue analysis for the detection of guideline-recommended biomarkers (not including KRAS) in NSCLC (NI margin = 10%). The rate of incomplete tissue genotyping was defined as the proportion of pts who were biomarker negative in tissue but incompletely genotyped for all 8 guideline-referenced biomarkers. Exploratory analyses included estimation of positive predictive value (PPV) of cfDNA against tissue genotyping and the biomarker discovery rate in tissue testing alone vs tissue plus cfDNA testing.

Results: 199 metastatic NSCLC pts were enrolled between August 2016-June 2017, and 185 were included in this interim analysis. The primary objective of non-inferiority was met, with 47 actionable mutations identified by cfDNA vs. 48 by tissue (p<0.002). Tissue testing was incomplete in 117/185 (63.2%, 95%CI = 55.9%, 70.2%) of pts. The majority (70%) of biomarker negative pts had tissue genotyping for 2 or fewer of the recommended biomarkers (most commonly EGFR and ALK). The PPV of cfDNA for EGFR, ALK, and MET alterations was 96.6% (95%CI = 82.2%, 99.9%). The addition of cfDNA increased the biomarker detection rate by 35.4% (95%CI= 22.2, 50.1), including those who were negative (1), not assessed (13), or quantity not sufficient (QNS) (3) for the biomarker in tissue.

Conclusions: This prospective, multi-center study demonstrates that a comprehensive and highly sensitive cfDNA NGS assay is non-inferior to SOC tissue testing in the detection of guideline-recommended actionable genomic alterations in NSCLC. Moreover, the addition of cfDNA to testing algorithms at the time of diagnosis markedly improved the biomarker detection rate in tissue negative/non-assessable pts. The PPV of the cfDNA assay utilized was high, supporting the use of cfDNA in treatment selection. These results demonstrate the clinical utility of comprehensive cfDNA genotyping to identify all guideline-recommended biomarkers in newly diagnosed advanced NSCLC pts.

#450

Novel genomic differences in cell-free circulating tumor DNA (cfDNA) profiles of early versus older onset colorectal cancer (CRC).

Afsaneh Barzi,1 Carin R. Espenschied,2 Victoria M. Raymond,2 Richard B. Lanman,2 Heinz-Josef Lenz1. 1 _University of Southern California, Los Angeles, CA;_ 2 _Guardant Health, Redwood City, CA_.

Background

The incidence of early onset CRC, defined as a diagnosis under age 50 (<50), is steadily rising without an established cause. Small cohort studies have reported on tumor tissue sequencing results from patients (pts) <50 versus >50 and have included all cancer stages and grouped all mutation types. These studies found significant yet inconsistent genomic differences between cohorts concluding <50 CRC may have higher mutation rates and better survival. This study aimed to compare the cfDNA results in a large cohort of <50 versus >50 advanced CRC pts, which to our knowledge has not been reported.

Methods

Consecutive advanced CRC pts who received clinical cfDNA testing (Guardant360™) between 10/15-10/18 were analyzed. cfDNA analysis included next generation sequencing of 70-73 genes, assessing single nucleotide variants (SNVs), insertion/deletion alterations (indels), fusions, and amplifications (amps). High microsatellite instability (MSI-H) status was available for a subset of cases. Characteristics and mutation frequencies were compared between <50 and >50 groups, excluding variants of uncertain significance and synonymous alterations. Gene specific mutation frequencies were compared with Fisher's exact test.

Results

Of 5341 stage IIIB-IV CRC pts tested, 4706 (88.1%) had alterations detected in cfDNA of which 984 (20.9%) were <50 and 3722 (79.1%) were >50. The <50 cohort was 51.7% male and the >50 cohort was 57.6% male. Both cohorts had a median of five alterations per pt (<50 range 1-207, >50 range 1-112). The median maximum variant allele fraction, including co-occurring amps, was 6.9% for the <50 cohort (range 0.02-94.9%) and 4.4% for the >50 cohort (range 0.03-97.1%). Of 2327 tested pts, MSI-H was detected in 3.3% (17/512) of <50 cases and 3.5% (64/1815) of >50 cases (not significant). In both cohorts SNVs and indels were most frequent in TP53, APC, KRAS, and PIK3CA. However, mutations in APC, KRAS, SMAD4, and ARID1A were more frequent in <50 CRC while >50 CRC had more TP53, ERBB2, and ATM mutations (all p<0.05). The most common amps in both cohorts were EGFR, BRAF, and CDK6 which may reflect aneuploidy, and MYC. BRAF, MYC, CCNE1, and CCND1 amps were more frequently observed in the <50 cohort (all p<0.03). RET, FGFR3, ALK, NTRK1, and FGFR2 fusions were seen in about 1% of both cohorts.

Conclusions

In the first comparison of cfDNA findings between <50 and >50 advanced CRC, significant differences in mutation and amp frequencies of several genes were observed, including genes important for prognosis and therapy selection such as KRAS and ERBB2. Rare but targetable biomarkers such as MSI-H and fusions were present in both age groups. Our previously unreported findings may be due to the strengths of this cfDNA analysis including a larger sample size, more uniform cancer stage, and stratification by mutation type. These results may help improve understanding and treatment of <50 CRC.

#451

Transferrin Receptor identifies a distinct pool of circulating tumor cells from metastatic prostate cancer patients with unique molecular profiles enriched in AR variants.

Ada Gjyrezi *, Giuseppe Galletti *, Jiaren Zhang, Rohan Bareja, Ahmed Halima, Daniel Worroll, David Nanus, Scott Tagawa, Himisha Beltran, Paraskevi Giannakakou, *Contributed equally. _Weill Cornell Medicine, New York, NY_.

Androgen receptor (AR) drives prostate cancer (PC) progression, even in castrate-conditions due to expression of AR variants (AR-V), which are constitutively active in the nucleus. Castration-resistant prostate cancer (CRPC) is currently lethal, due to the development of drug resistance. The molecular basis of clinical drug resistance remains poorly elucidated, due to tumor heterogeneity and limited availability of serial tumor sampling as disease progresses. Circulating tumor cells (CTCs), isolated from patient peripheral blood, have emerged as a reliable, easy to obtain source of tumor cells which can be and used to interrogate the molecular disease evolution. To date, EpCAM-based CTC enrichment is commonly used but has important shortcomings as EpCAM is often lost during EMT, thus, missing the more aggressive mesenchymal CTCs. On the other hand, Transferrin Receptor (TfR) which is overexpressed in cancer cells, is a cell surface protein required for iron uptake and is critical for several biological processes such as cell proliferation and metabolism. Mining large gene expression databases in PC we observed that TfR expression increased with disease stage, from adenocarcinoma (TCGA) to CRPC (SU2C) to the more aggressive neuroendocrine PC (NEPC), while EpCAM expression remained unchanged. We also found that TfR expression was increased in EMT cell models, while EpCAM expression was lost, suggesting that TfR may perform well in CTC isolation across the EMT spectrum. We then tested TfR- and EpCAM-based CTC enrichment in a cohort of 31 CRPC patients, where we observed a significantly higher number of TfR+ CTCs (median, 304) than EpCAM+-CTCs (median, 21) (p<0.01). RNA-sequencing of TfR+ CTCs in comparison to EpCAM+ CTCs, followed by gene-set enrichment analyses revealed that the TfR+ subpopulation was significantly enriched in prostate specific oncogenic pathways including EMT, suggesting distinct molecular profiles between the two subsets.

To investigate the clinical relevance of the two CTC subsets, we quantitated AR-FL and splice variants AR-V7 and AR-v567es expression by digital droplet PCR (ddPCR) in pools of TfR+-CTCs vs EpCAM+-CTCs. Our results from 16 CRPC patients showed that while expression of AR-FL was similar in the two CTC subsets, both AR variants were enriched in TfR+-CTCs. To better map the tumor heterogeneity of AR-V expression we isolated 102 single TfR+\- and 63 single EpCAM+-CTCs from 3 CRPC patients and quantified AR-V expression by ddPCR. The single CTC analysis revealed that AR-V7 and AR-v567es were expressed in 21% and 18% of TfR+-CTCs, respectively, vs 0% in EpCAM+-CTCs. These data identify TfR as a promising marker for CTC identification across the EMT gradient, and provide a unique insight into tumor cell heterogeneity, which was not appreciated before, and which may have significant clinical impact.

#452

Salivary HPV cell free DNA levels predict locoregional disease burden and response in oropharyngeal cancer.

Glenn J. Hanna, Christie J. Lau, Umair Mahmood, Robert I. Haddad, Cloud P. Paweletz, Pasi A. Janne. _Dana-Farber Cancer Institute, Boston, MA_.

Purpose: The human papillomavirus (HPV) is linked to the majority of oropharyngeal squamous cell carcinomas (OPSCC). We have previously shown that tumor-derived HPV cell-free (cf)DNA can be detected and quantified in circulation with high sensitivity and specificity using droplet digital (dd)PCR1. Further, we have shown that plasma cfDNA levels correlate with disease burden and indicate treatment response in advanced OPSCC. In this study we pair plasma with salivary HPV cfDNA to understand its clinical application in monitoring locoregional spread of disease.

Methods: We present a proof-of-concept prospective observational cohort of recurrent, metastatic (R/M) HPV+ OPSCC patients treated with standard systemic therapies or on active surveillance. We utilized ultrasensitive ddPCR to identify and quantify both plasma and salivary HPV cfDNA (subtypes 16, 18, 31, 33 and 45) at multiple time points. Salivary HPV cfDNA was normalized to total DNA concentration as measured by fluorometric quantification. We then compared matched plasma and normalized salivary HPV cfDNA concentrations at various timepoints with clinical parameters, such as tumor burden and therapeutic response.

Results: Clinicopathologic data from 15 R/M patients revealed a male cohort with a median age of 57 at diagnosis. Five (33%) were on immunotherapy and 6 (40%) on standard chemotherapy during the study period (3 months). Salivary HPV cfDNA was detected in 13/15 (87%) samples (range 0-729 copies/mL) in at least one timepoint during the study (plasma cfDNA was detected in 12/15 or 80% of the cohort, range 0-12,949 copies/mL). All patients with measurable locoregional (LR) disease had detectable salivary cfDNA. While matched plasma and salivary cfDNA levels showed no correlation among individual patients, plasma cfDNA levels correlated with site of disease (LR, pulmonary or extra-pulmonary) (p < 0.001), while median salivary cfDNA levels strongly correlated with LR tumor burden (R = 0.81, p < 0.001). Salivary cfDNA levels declined by 80% of their baseline value within a median of 6.5 days among all patients with LR disease experiencing a response to treatment.

Page 1 of 1

Conclusion: Our results suggest that high sensitivity salivary HPV cfDNA levels correlate with locoregional disease burden and can be an early indicator of locoregional treatment response, while our prior work has supported the role of plasma HPV cfDNA levels in monitoring metastatic disease. Evaluating paired plasma and salivary HPV cfDNA levels in a curative HPV OPSCC population is underway to further validate their prognostic and predictive potential.

1Hanna GJ, Supplee JG, Kuang Y, Mahmood U, Lau CJ, Haddad RI, et al. Plasma HPV cell-free DNA monitoring in advanced HPV-associated oropharyngeal cancer. Ann Oncol. 2018;29:1980–6.

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#453

A circulating tumor cell assay for dynamic assessment of drug sensitivity in metastatic castration-resistant prostate cancer.

Pai-Chi Teng,1 Yu Jen Jan,1 Junhee Yoon,1 Jie-Fu Chen,1 Pin-Jung Chen,2 Nu Yao,1 Shirley Cheng,1 Amber Lozano,1 Michael R. Freeman,1 Sungyong You,1 Hsian-Rong Tseng,2 Edwin M. Posadas1. 1 _Cedars-Sinai Medical Center, Los Angeles, CA;_ 2 _University of California, Los Angeles, Los Angeles, CA_.

Background: Tissue-based gene signatures can predict clinical behavior in prostate cancer (PC). Our objective was to extend their application to circulating tumor cells (CTCs) and to show that changes in the signature were associated with changes in clinical behavior.

Methods: Our approach combined the Thermoresponsive(TR)-NanoVelcro CTC purification system with the Nanostring nCounter system for cellular purification and transcriptomic analysis. The Prostate Cancer Classification System (PCS) panel was modified for use in CTCs. We selected 31 blood samples from 23 PC patients receiving androgen receptor signaling inhibitors (ARSI) and measured the PCS1 Z score (probability). These findings were compared with clinical outcome data (responsiveness/resistance).

Results: A modified, 16-gene PCS1 signature was established and validated through a rigorous bioinformatics process. We performed analytical validation of our combined CTC-RNA system to ensure reproducibility and specificity. In patient bloods, ARSI-resistant samples (ARSI-R, n=14) had significantly higher PCS1 Z scores as compared with ARSI-sensitive samples (ARSI-S, n=17) (Rank-sum test, P=0.003). The analyzed bloods contained samples from 8 patients who developed resistance to an ARSI allowing for dynamic measurement of gene expression. Our analysis found that the PCS1 Z score increased at the time that ARSI-resistance emerged (Pairwise T-test, P=0.016).

Conclusions:Using this new methodology, contemporary, clinically-relevant gene signatures such as PCS could be measured non-invasively in CTCs. These findings can be used to relate gene expression to clinical drug response. This approach also allowed for measurement of dynamic variations of gene expression in individual patients over time that correlated to ARSI sensitivity.

#454

Transferrin receptor identifies a novel circulating tumor cell population in patients with pancreatic cancer with a unique metastasis-associated molecular signature.

Ahmed Halima, Jiaren Zhang, Giuseppe Galletti, Allyson J. Ocean, Paraskevi Giannakakou. _Weill Cornell Medical College, New York, NY_.

Pancreatic Ductal Adenocarcinomas (PDAC) is the fourth most common cancer, expected to be the second leading cause of cancer mortality by 2030. Most PDAC patients are diagnosed with metastatic disease, with dismal prognosis of less than 1% 5-year survival. A major challenge in PDAC treatment is the absence of actionable molecular targets and the scarcity of available tumor tissue impeding molecular understanding of PDAC progression. Circulating Tumor Cells (CTCs) provide an easily accessible source for tumor tissue. CellSearchTM is the only FDA-cleared platform for CTC isolation based on positive selection of EpCAM+ cells. However, this method performs poorly in PDAC failing to show any prognostic relevance, likely due to EpCAM down-regulation during epithelial-to-mesenchymal transition (EMT), which precedes metastasis. To overcome this limitation, we tested Transferrin Receptor 1 (TfR) in the identification and isolation of CTCs from PDAC patients. TfR is a cell surface protein that mediates iron uptake, is overexpressed in multiple tumors with sustained expression throughout EMT, making it a suitable candidate for CTC isolation. To test this hypothesis, we first assessed TfR expression in a panel of 9 human PDAC cell lines, and we observed that TfR was expressed in 9/9 (100%) in contrast to 6/9 (66.7%) being EpCAM positive. No TfR expression was detected in healthy donor and PDAC patients' PBMCs. To assess the performance of TfR in CTC identification and isolation from the peripheral blood of PDAC patients, we collected samples from 37 PDAC patients with stage 4 disease, enriched CTCs by negative CD45 depletion (RossetteSepTM) and subsequently stained live CTCs with TfR, EpCAM, and CD45. CTCs were defined as CD45- cells, positive for TfR+ or EpCAM+. We observed that the number of TfR+ CTCs (median: 148, range 2-4182) was significantly higher than EpCAM+ CTCs (median: 68, range 0-1552) within the same patient and across different patients (P-value 0.007). In addition, serial sampling at baseline (chemotherapy-naïve) and at disease progression from 5 PDAC patients revealed that TfR+CTC enumeration correlated with disease progression radiologically and/or CA19-9 (PDAC tumor marker) levels. In contrast, there was no correlation between the number of EpCAM+CTCs and clinical outcomes.

RNA-Sequencing of isolated pools of TfR+ or EpCAM+ CTCs, followed by gene set enrichment analysis, revealed significant enrichment in oncogenic pathways, such as EMT, G2M, MYC and KRAS in TfR+ CTCs as compared with EpCAM+ CTCs. Taken together, our results reveal that TfR identifies a clinically relevant CTC subpopulation which correlates with disease progression and is molecularly distinct. Ongoing studies are focused on the function of TfR in disease progression and the potential use of TfR+ CTC molecular profiles in personalized PDAC treatment.

#455

Innovations in large scale liquid biopsy analysis and the Broad/IBM Cancer Resistance Project.

Carrie Cibulskis,1 Brendan Blumenstiel,1 Matthew DeFelice,1 Mark Fleharty,1 Justin Abreu,1 Viktor Adalsteinsson,2 Laxmi Parida,3 Susanna Hamilton,1 Gad Getz,4 Niall Lennon1. 1 _Broad Institute, Cambridge, MA;_ 2 _Broad Institute, Koch Institute, Massachusetts General Hospital, Cambridge, MA;_ 3 _IBM, Cambridge, MA;_ 4 _Broad Institute, Harvard Medical School, Massachusetts General Hospital, Cambridge, MA_.

Broad Genomics offers a comprehensive liquid biopsy sequencing platform designed to provide the optimal flexibility for conducting research studies in a broad range of applications including: biomarker discovery, treatment resistance monitoring, and clinical grade ctDNA profiling. By utilizing low cost, low coverage whole genome sequencing in conjunction with dual unique molecular indexed (UMI) libraries we can offer a range of analysis that allow researchers to select the most appropriate samples for whole exome profiling or for deeper coverage, higher sensitivity targeted gene panels. To date we have generated over 3000 liquid biopsy whole genome copy number profiles and purity estimates and are supporting driver projects including the Broad/IBM Cancer Resistance Project and Count Me In. The study design for the Broad/IBM effort takes advantage of the discovery potential of tissue-based sequencing combined with serial liquid biopsy analysis to elucidate resistance events by tracking clonal and subclonal populations in patient samples over time. Sourcing samples for this and other similar efforts is a major undertaking and a combination of methods for maximally broad and deep genomic profiling are required to assay patients throughout the course of care, as tumor fraction in blood fluctuates. Responding to this need, and other applications requiring increased sensitivity we have developed a high throughput, automated workflow to efficiently assay cfDNA samples with lower tumor content. Benchmarking data using healthy donor pooled cfDNA samples indicates our assay is capable of detecting > 90% of variants present at ~1% minor allele fraction with less than 1 false positive variant called per megabase. This established laboratory and analytic process forms the basis of our 2Mb, 400 gene CLIA targeted assay currently undergoing validation. Through this suite of products we hope to enable an expansion of cfDNA sequencing efforts in support of clinical and research applications. Early results from emerging studies utilizing this platform to be presented.

#456

Circulating tumor cells and cfDNA changes during radiotherapy in patients with head and neck cancer.

Sweet Ping Ng, Carolyn S. Hall, Salyna Meas, Vanessa N. Sarli, Houda Bahig, Baher Elgohari, Amy Moreno, Heath D. Skinner, Adam S. Garden, David I. Rosenthal, William H. Morrison, Jack Phan, G Brandon Gunn, Steven J. Frank, Clifton D. Fuller, Anthony Lucci. _MD Anderson Cancer Center, Houston, TX_.

Introduction: Head and neck cancer treatment response relies heavily on macroscopic clinical findings. Blood monitoring of circulating markers such as circulating tumor cell (CTC) and cell-free DNA (cfDNA) during treatment may improve earlier detection of responders versus non-responders during definitive radiotherapy. Our work prospectively describes the changes in CTC and cfDNA enumeration during definitive radiotherapy.

Methods: Patients with mucosal head and neck squamous cell carcinoma and intact gross tumor were eligible for the prospective IRB-approved PREDICT-HN study. Blood samples were collected pre-treatment, after first treatment, weekly, and post-treatment (CellSave and EDTA tubes). CTC was enumerated using the FDA-approved CellSearch (Menarini Silicon Biosystems) system. Filtered plasma were collected from the EDTA tubes and stored at -80C. cfNA from pre-, mid- and post-treatment timepoints were isolated from the filtered plasma using the MagMAX Nucleic Acid Isolation Kit (Thermo Fisher Scientific) and cfDNA were quantified with Qubit high sensitivity dsDNA assay (Invitrogen).

Results: 339 blood samples were collected from 45 patients; 137 had detectable CTC. Eight patients had detectable CTCs (1-3/7.5ml blood) in pre-treatment samples. After 1 fraction of radiotherapy, 16 patients had CTCs detected with 12 who had no CTC detected pre-treatment. 19 (42%) patients had detectable CTC in week 6 of treatment. All patients had detectable CTC at some point during radiotherapy except for one patient who had no CTC detected pre, during and post-treatment. There was no correlation between cancer stage, nodal status and gross tumor volume with detection of CTC. Overall circulating cfDNA levels increased during treatment, with their highest level in the final week of treatment similar to when the majority of CTC were detectable in patients, and lowest at the post-treatment time-point.

Conclusions: Our interim results showed, for the 1sttime, to our knowledge, that CTCs can be detected during radiotherapy of head and neck cancer, suggesting mobilization into peripheral circulation during treatment, with as-yet-unknown viability. cfDNA kinetics during treatment correlated with CTC release, may indicate apoptotic change during radiotherapy. Combined cfDNA-CTC as an early marker of treatment response should be investigated.

#457

HER2, AR protein expression and chromosomal instability in circulating tumor cells (CTCs) of metastatic breast cancer (MBC) patients (pts).

Priscilla Ontiveros,1 Connie Landaverde,1 Maren K. Levin,2 Sarah Hippely,2 Mark Landers,1 Yipeng Wang,1 Ryan Dittamore,1 Joyce A. O'Shaughnessy2. 1 _Epic Sciences, San Diego, CA;_ 2 _Baylor Scott & White Research Institute, Dallas, TX_.

Background: Upregulation of HER2 and AR protein expression, and increase in chromosomal instability, are mechanisms of acquired resistance to endocrine therapy, and are being investigated as treatment-guiding biomarkers. However, measurement of these biomarkers requires metastatic biopsies, which are costly, invasive, and prone to under-sampling which precludes their utility to guide treatment in late stage metastatic pts. A CTC-based test could expand the clinical utility. Using the Epic Sciences platform, MBC blood samples were characterized for CTC prevalence, HER2 and AR expression on CTCs, and CTC chromosomal instability at time of disease progression.

Material and methods: Blood samples were acquired from 127 total pts with HR+/HER2-, HR-/HER2+ and TNBC metastatic breast cancer pts. 114 samples were analyzed for HER2 and 110 were analyzed for AR using the Epic CTC Platform. Single-cell whole genome sequencing was performed on 157 CTCs from 19 pts to assess large scale transitions (a surrogate of chromosome instability) and gene copy number alterations.

Results: 87/114 (76.3%) of pts had detectable CTCs, with a median of 1.1 CTC/mL. 17/114 (14.9%) had at least one HER2(+) CTC, 21/110 (19.1%) had at least one AR(+) CTC, and 10/110 (9%) had both AR(+) and HER2(+) CTCs detected in technical replicates. HER2 expression on individual CTCs showed distinctive cytoplasmic membrane staining, and AR expression on individual CTCs showed frequent nuclear localization. The majority of HER2+ and AR+ CTCs had high cytokeratin expression. Most pt samples showed heterogeneous expression of these markers at disease progression indicating subclonal sensitivity to targeted therapies. CTCs with larger nuclear area and higher N/C ratio had higher frequency of chromosomal instability. ERBB2, FGFR1 gain, PTEN, CDH1, BRCA1, BRCA2 loss were frequently observed and the prevalence were associated with CTC phenotypes.

Conclusions: CTCs are detected in most MBC pts upon disease progression. CTC based biomarkers assessing HER2, AR and chromosomal instability status could guide therapy selection. The results warrant prospective evaluation of these biomarkers on MBC pts' CTCs as a resistance marker for targeted therapies.

### Precision Medicine

#458

Precision systems medicine in acute myeloid leukemia: real-time translation of tailored therapeutic opportunities arising from ex-vivo drug sensitivity testing and molecular profiling.

Disha Malani,1 Ashwni Kumar,1 Bhagwan Yadav,2 Mika Kontro,2 Swapnil Potdar,1 Oscar Bruck,2 Säri Kytölä,2 Jani Saarela,1 Samuli Eldfors,1 Riikka Karjalainen,1 Muntasir M. Majumder,1 Imre Västrik,1 Pekka Ellonen,1 Matti Kankainen,1 Minna Suvela,1 Siv Knappila,1 Alun Parson,1 Aino Palva,1 Pirkko Mattila,1 Evgeny Kulesskiy,1 Laura Turunen,1 Karoliina Laamanen,1 Elina Lehtinen1,1 Maria Nurmi,1 Katja Suomi,1 Astrid Muruimägi,1 Bjorn T. Gjertsen,3 Satu Mustjoki,2 Simon Anders,4 Maija Wolf,1 Tero Aittokallio,1 Krister Wennerberg,1 Caroline Heckman,1 Kimmo Porkka,2 Olli Kallioniemi1. 1 _Inst. for Molec. Medicine Finland (FIMM), Helsinki, Finland;_ 2 _Hematology Research Unit Helsinki, Helsinki, Finland;_ 3 _University of Bergen, Bergen, Norway;_ 4 _Center for Molecular Biology of University of Heidelberg (ZMBH), Heidelberg, Germany_.

Acute myeloid leukemia (AML) is an aggressive disease of clonal hematopoietic progenitor cells. Here, we applied ex-vivo drug sensitivity and resistance testing on AML patient cells with 362 emerging and 153 approved cancer drugs together with genomic and transcriptomic profiling to identify and tailor therapies for patients with advanced disease. Ex-vivo testing with freshly isolated patient cells revealed cancer-specific efficacies of approved drugs in 97% of the 164 patient cases, including 47% of the cases with no actionable driver mutations. We identified 142 statistically significant associations between drug responses and somatic mutations, including increased sensitivity to JAK inhibitors in patients with NPM1 mutations. Transcriptomic profiles predicted drug responses better than genomics and helped to identify additional response markers, especially beyond mutations. For example, overexpression of HOX family genes was associated with sensitivity to JAK inhibitors in patients with NPM1 mutation. In a prospective study, we translated the functional drug response and molecular profile data to the clinic and suggested tailored therapy with targeted drugs for 26 relapsed or refractory AML patients. In an observational intervention study, acting on these recommendations resulted in a temporary complete clinical remission or leukemia-free state in 39% of the cases. In summary, we conclude that ex-vivo testing of drugs on patient AML cells i) revealed clinically actionable drug efficacies in almost all AML patients, including patients with no actionable mutations, ii) predicted cases with actionable driver mutations with no pharmacological dependency on the target, and iii) enabled real-time tailoring of therapy with 39% clinical response rate in chemorefractory advanced AML. Taken together, we believe this real-time systems medicine approach could become a powerful strategy for tailoring therapies for individual patients in the future.

#459

Comprehensive molecular characterization of treatment naive muscle invasive bladder cancer.

Ann Taber, Philippe Lamy, Iver Nodentoft, Emil Christensen, Frederik F. Prip, Sia V. Lindskrog, Karin Birkenkamp-Demtröder, Mads Agerbæk, Jørgen B. Jensen, Lars Dyrskjøt. _Aarhus University Hospital, Aarhus, Denmark_.

Background: The interplay between carcinoma cells and their microenvironment plays an important part in cancer development, progression, metastasis and treatment efficacy. In bladder cancer, however, little is currently known about how the tumor microenvironment affects disease outcome and treatment. To study this, we analyzed tumor tissue from a clinically well characterized cohort of 608 patients with muscle invasive bladder cancer (MIBC).

Methods: Multiplex Immunofluorescence (IF) was used to assess the tumor microenvironment in 608 patients with MIBC. Digital pathology was used to quantify the immune context (CD8, CD3, FOXP3) within carcinoma cells and in the adjacent stroma. Immune cell densities were correlated to clinical characteristics, survival outcome and chemotherapy response (n=187). To explore chemotherapy response mechanisms further, a total of 107 patients with metastatic disease are also being characterized by multi-omics analysis (exome-, transcriptome-, and methylome-analysis). Patients receiving chemotherapy were included based on the following criteria: 1) treatment naive primary tumors, 2) received a minimum of 4 cycles of cisplatin based chemotherapy, 3) ECOG Performance Status <2, 4) Response evaluated according to the RECIST criteria (version 1.1).

Results: In total, 384 of 608 patients had metastatic disease. Analysis of the immune cell compositions revealed a immune profile consisting of high levels of CTLs and T helper cells, and a low level of Tregs significantly associated with a better outcome (p<0.0001). For patients receiving chemotherapy for metastatic disease, we observed no significant differences in the treatment naive primary tumors when correlating individual or combined immune cell types (CTLs, T helper cells, Tregs) to treatment response. Preliminary analysis of exome seq data showed a tendency towards a higher mutation burden in complete responders (p=0.047), however response was not associated with DDR mutations nor mutational signatures in this patient cohort. Furthermore, gene expression subtypes did not predict treatment response.

Conclusions: Results from the multiplex IF showed that immune profiles (CTLs, T helper cells, Tregs) correlated with recurrence and survival outcome. However, they did not predict chemotherapy response. A full and integrated molecular characterization of our clinically well annotated metastatic subgroup (n=107) will be presented at the AACR 2019 meeting.

#460

Understanding transcriptomic profiles that might explain discordance of the PAM50 and immunohistochemistry classification methods in luminal tumors from Colombian women.

Silvia J. Serrano-Gomez,1 Maria Carolina Sanabria-Salas,1 Jone Garay,2 Juan C. Mejia,1 Oscar Garcia,1 Jovanny Zabaleta,2 Laura Fejerman3. 1 _Instituto Nacional de Cancerología, Bogota, Colombia;_ 2 _Louisiana State University Health Science Center, New Orleans, LA;_ 3 _University of California San Francisco, San Francisco, CA_.

Background: Breast cancer is the most common cancer in Colombian women. Five intrinsic subtypes have been described based on gene expression profiles and genetic signatures, such as PAM50, have been developed to classified tumors with different prognosis. In Colombia, we have previously reported that the most prevalent breast cancer subtype was luminal B based on immunohistochemistry (IHC) surrogates, which are routinely used in the clinical practice. We analyzed the concordance between IHC surrogates and PAM50 classification in luminal tumors from Colombian women and explored gene expression differences that may account for the observed differences.

Methods: We performed whole-transcriptome RNA-seq analysis in 46 breast cancer samples from Colombian women diagnosed with estrogen receptor positive tumors (27 luminal A and 21 luminal B) at the Colombian National Cancer Institute. Genefu package from R was used to perform PAM50 classification, and the DESeq2 package was used to test differential gene expression between discordant cases between PAM50 and IHC. Genes with adjusted p value less than 0.05 were considered statistical significant.

Results: We found that 23 patients (50%) were discordant between both methodologies (IHC and PAM50). For the 27 luminal A tumors assessed by IHC, 10 (37%) were re-classified as luminal B by PAM50 and 3 (11.11%) as normal-like. On the other hand, for the 21 tumors classified as luminal B by IHC, 4 (19.04%) were re-assigned as HER2-enriched, 4 (19.04%) as luminal A and 2 (9.5%) as normal-like. Next, we tested differential expression in discordant vs. concordant cases with PAM50 within each luminal subtype assessed by IHC. For luminal B IHC-tumors, we found 67 differentially expressed genes. Some of the top differentially expressed genes are part of the ERBB2 signaling pathway (ERBB2, padj < 0.01, log2FC = 2.5; GRB7, padj < 0.01, log2FC = 2.95), which is consistent with the re-assignment of luminal A tumors to luminal B. Moreover, we found overexpression of ARID5B (padj< 0.01, log2FC=1.72) a histone modifier gene that has been described mutated in breast cancer. This result might suggest that an epigenetic mechanism may be involved in defining the luminal profile of these tumors. On the other hand, for luminal A IHC-tumors, we found 12 differentially expressed genes. The gene with the highest fold-change was DSCAM-AS1 (padj = 0.030, log2FC=3.43) which is very interesting as it is a estrogen-regulated long noncoding RNA (lncRNA) that has been recently associated with tumor progression and tamoxifen resistance.

Conclusions: Our results suggest that epigenetic mechanisms such as histone modifications and the lncRNAs might be associated with luminal tumors in Colombian women and might also define the fate of the luminal profile of these tumors.

#461

Distinctive molecular traits of hepatocellular carcinoma in patients with non-alcoholic steatohepatitis.

Sara Torrecilla,1 Roser Pinyol,1 Huan Wang,2 Carla Montironi,1 Carmen Andreu-Oller,1 Wei Qiang Leow,3 Agrin Moeini,1 Claudia Oliveira,4 Venancio Avancini Ferreira Alves,5 Anja Lachenmayer,6 Stephanie Roessler,7 Beatriz Minguez,8 Peter Schirmacher,7 Paolo Boffetta,3 Jean-François Dufour,6 Swan N Thung,3 Andrew Uzilov,2 Flair Jose Carrilho,5 Charissa Chang,3 Daniela Sia,3 Josep M Llovet1. 1 _Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clínic, Universitat de Barcelona, Barcelona, Spain;_ 2 _Sema4, New York, NY;_ 3 _Icahn School of Medicine at Mount Sinai, Tisch Cancer Institute, New York, NY;_ 4 _University of São Paulo - School of Medicine, Sao Paulo, Brazil;_ 5 _University of São Paulo - School of Medicine, São Paulo, Brazil;_ 6 _Bern University Hospital, University of Bern, Bern, Switzerland;_ 7 _University Hospital Heidelberg, Heidelberg, Germany;_ 8 _Hospital Universitari Vall d´Hebron, Vall d´Hebron Institut of Research (VHIR), CIBERehd, Universitat Autònoma de Barcelona, Barcelona, Spain_.

Background and aim: Non-alcoholic steatohepatitis (NASH) is emerging as one of the leading risk factors for hepatocellular carcinoma (HCC), but its molecular pathogenesis is still ill-defined. This study aims to identify unique molecular traits that differentiate NASH-HCC from other aetiologies through an integrative molecular characterization.

Methods: A total of 225 tissue samples were collected, including samples from 125 biopsied/transplanted NASH patients; and 100 resected/transplanted NASH-HCC patients. Molecular characterization of FFPE samples, comprised expression array (n=53 NASH-HCC; n=74 NASH), whole exome sequencing (n=50 NASH-HCC), and SNP array (n=44 NASH-HCC). Publicly available data from HCV/HBV/alcohol-related HCCs were used to identify NASH-HCC distinctive traits.

Results: NASH-HCC patients compared to NASH non-HCC patients were prevalently males (82% vs 42%, p<0.001), older (67 years vs 54, p<0.01), with higher diabetes incidence (72% vs 50%, p=0.004), hypertension (80% vs 52%, p<0.01) and cirrhosis (69% vs 29%, p<0.001). Analysis of the transcriptome showed enrichment of liver metabolism pathways in NASH livers, whereas NASH-liver tissues of HCC cases were characterized by inflammation (TNFα-NFkβ, IL6, STAT3), epithelial-mesenchymal transition (TGFβ1), proliferation (AKT, mTOR), and poor-prognosis liver signatures (p<0.05). Gene expression profiles of NASH-HCC tumours demonstrated that they can be classified in proliferation (50%) and non-proliferation (50%) molecular classes, as in non-NASH HCC. Mutational profiling of NASH-HCC tumours identified 4 genes with mutations in ≥10% of cases: TERT (52%), CTNNB1 (28%), TP53 (18%) and the TGFβ-receptor ACVR2A (10%). Interestingly, mutations in ACVR2A were three times more prevalent in NASH-HCC (n=100) than in viral/alcohol-related HCC (n=624)(2.6%, p<0.05). Functional impact of ACVR2A mutations is currently being investigated. Finally, unsupervised clustering of mutational signatures showed that NASH-HCC tumours are clustered in 2 groups, enriched in liver-cancer signatures #16 (44%) and #5(22%), respectively; and that a third cluster (15%) was enriched in signature #3, which is novel in liver cancer. By comparing these results with the clustering of mutational profiles from viral/alcohol-related HCCs, we identified the signature#3-cluster as specific of NASH-HCC.

Conclusions: Non-tumour liver tissue of NASH-HCC patients is characterized by a cancer-field enriched in inflammatory, epithelial-to-mesenchymal transition and proliferation signalling pathways. NASH-HCC tumours showed a high frequency of ACVR2A mutations (10%), and a novel cancer mutational signature #3 (15%), suggesting genotoxic factors specifically associated to this entity.

#462

Results from multiple datasets including the TCGA reveal limited clinical significance of molecular subtypes in bladder cancer.

Sarrah S. Lahorewala, Daley S. Morera, Jiaojiao Wang, Vinata B. Lokeshwar. _Augusta University, Augusta, GA_.

INTRODUCTION AND OBJECTIVE: Molecular subtypes have been suggested to predict outcome in patients with muscle invasive bladder cancer (MIBC). The major subtypes reported have been "luminal" and "basal"; however, there is little consensus regarding subtype-signatures and the clinical parameters used to evaluate outcome. The objective of this study was to examine using multiple datasets, if molecular subtypes are distinct entities within MIBC and whether they predict clinical outcome.

METHODS: We analyzed transcriptome data from all 402 MIBC samples in The Cancer Genome Atlas (TCGA) and 151 high-grade MIBC samples from three datasets in Oncomine™. Transcript levels were also measured in 52 bladder tumor specimens with clinical follow-up (cohort-1). Samples were subtype-scored using gene panels (GP-11, GP-30, BASQ) consisting only of those genes common among published studies. Correlation of subtypes to recurrence/progression-free survival (R/PFS), metastasis, cancer-specific survival (CSS) and overall-survival (OS) was examined by logistic regression, Cox proportional Hazards model and Kaplan-Meier analyses.

RESULTS: Based on GP-11 subtype-scoring, >75% of the MIBC TCGA-dataset samples were mixed, i.e. neither pure-luminal nor pure-basal. When tumors were categorized as pure-luminal, pure-basal, or mixed, the subtypes could not predict OS or R/PFS. Consistent with published studies, when tumors were categorized as luminal or basal the subtypes predicted OS (P=0.051); sensitivity: 53.9%; specificity: 62%. Subtypes also correlated with tumor-grade (P=0.0005); most low-grade MIBC cases (16/21) in the TCGA-dataset were luminal. However, MIBC is rarely low-grade and subtypes could not predict OS (P=0.131) when only high-grade cases were included. Subtypes were not significant prognosticators in multivariate analyses. GP-30 and BASQ panels validated these results. In the Oncomine-dataset and cohort-1, subtypes could not predict metastasis, CSS, or OS.

CONCULUSIONS: Our study of multiple datasets reveals that molecular subtypes may reflect tumor-heterogeneity but are likely not distinct entities within MIBC. Furthermore, molecular subtypes have limited prognostic capability for MIBC patients. Although there is a need to individualize patient care, further examination into the molecular subtypes of MIBC is needed before their incorporation into clinical practice.

#463

Integrated digital pathology and transcriptome analysis identifies molecular mediators of T cell exclusion in ovarian cancer.

Melanie Desbois,1 Akshata Udyavar,2 Lisa Ryner,1 Cleopatra Kozlowski,1 Yinghui Guan,1 Milena Dürrbaum,1 Shan Lu,1 Jean-Philippe Fortin,1 Hartmut Koeppen,1 James Ziai,1 Ching-Wei Chang,1 Amy Lo,1 Shilpa Keerthivasan,1 Marie Plante,3 Richard Bourgon,1 Carlos Bais,1 Priti Hegde,1 Anneleen Daemen,1 Shannon Turley,1 Yulei Wang1. 1 _Genentech, South San Francisco, CA;_ 2 _Arcus Biosciences, South San Francisco, CA;_ 3 _Laval University Cancer Research Center, Quebec, Quebec, Canada_.

Background:

Close proximity between cytotoxic T lymphocytes and tumor cells is required for effective immunotherapy. Three tumor-immune (TI) phenotypes, infiltrated, excluded and desert, have been previously described based on the infiltration patterns of CD8+ T cells. However, no quantitative methods exist to define these phenotypes robustly in human solid tumors. Importantly, the molecular features and mechanisms determining these phenotypes are not well understood. Here we report a novel integrated approach to classify and functionally dissect TI phenotypes in human ovarian cancer.

Methods:

CD8 IHC and RNAseq analysis were performed on 370 ovarian tumors from the ICON7 phase III clinical trial, a front-line trial testing the addition of bevacizumab to chemotherapies. A digital image analysis algorithm was developed to quantify the quantity and spatial distribution of CD8+ T cells. Coupling digital pathology with transcriptome analysis, a random forest machine learning algorithm was applied to identify genes associated with these two metrics using a training set (n=155). A gene expression-based classifier was developed for classifying TI phenotypes and validated using testing sets from ICON7 trial and a vendor collection. Functional characterization of key mediators promoting T cell exclusion were carried out by integrating in situ, in vitro and ex vivo analyses on ovarian tumor tissues, cancer associated fibroblasts (CAFs) and ovarian cancer cell lines. Anti-tumor activity of TGFβ blockade in combination with anti-PD-L1 was evaluated in the mouse BrKras ovarian cancer model in FVB background.

Results:

Integrating digital pathology and machine learning on large ovarian tumor cohorts, we developed and validated a 157-gene molecular classifier. We show the TI phenotypes are of biological and clinical importance in ovarian cancer. Two hallmarks of T cell exclusion were identified: 1) loss of MHC I on tumor cells and 2) upregulation of TGFβ/stromal activities. We show that MHC I in ovarian cancer cells is likely regulated by epigenetic mechanisms and TGFβ is a key mediator of T cell exclusion. TGFβ reduced MHC I expression in ovarian cancer cells and induced extracellular matrix and immunosuppressive molecules in human primary fibroblasts. Finally, we demonstrated that combining anti-TGFβ and anti-PD-L1 in the BrKras mouse model improved the anti-tumor efficacy and survival.

Conclusion:

This study provided the first systematic and in-depth characterization of the molecular features and mechanisms underlying the tumor-immune phenotypes in human ovarian cancer. We illuminated a multi-faceted role of TGFβ in mediating crosstalk between tumor cells and CAFs to shape the tumor-immune contexture. Our findings support that targeting the TGFβ pathway represents a promising therapeutic strategy to overcome T cell exclusion and optimize response to cancer immunotherapy.

#464

Gene expression signatures for the prediction of endocrine treatment outcome in early-stage luminal breast cancer patients.

Werner Schroth,1 Reiner Hoppe,1 Florian Büttner,1 Stefan Winter,1 Siarhei Kandabarau,1 Jörg Kumbrink,2 Heather A. Brauer,3 Peter Fritz,1 Matthias Schwab,4 Thomas Mürdter,1 Hiltrud Brauch5. 1 _Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology and University of Tuebingen, Stuttgart, Germany;_ 2 _Pathological Institute, Faculty of Medicine, Ludwig Maximilian University, Munich, Germany;_ 3 _NanoString Technologies Inc., Seattle, WA;_ 4 _Dr. Margarete Fischer-Bosch-Institute for Clinical Pharmacology Stuttgart and Department of Clinical Pharmacology, Institute of Experimental and Clinical Pharmacology, University Hospital, Tübingen, Stuttgart, Germany;_ 5 _Dr. Margarete Fischer-Bosch-Institute for Clinical Pharmacology and University of Tübingen, and German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ) Heidelberg, Stuttgart, Germany_.

Estrogen (ER) and/or progesterone (PR) receptor-positive, early breast cancer benefits from targeted therapy via long-term estrogen deprivation. Valid treatment options include the selective ER modulator tamoxifen (TAM) that interferes with estrogen-binding at the ER, and aromatase inhibitors (AI) that block the enzyme aromatase to prevent the conversion of androgens to estrogen. Both treatment principles are in clinical use however fail in about one third of the patients. The choice of endocrine treatment is currently not well supported by predictive tumor markers. Gene expression signatures covering critical breast cancer pathways were tested to predict TAM and AI associated outcomes in a prospectively collected postmenopausal, hormone-receptor positive, early breast cancer cohort (IKP211; 1200 patients, median follow up 5.5 years; DRKS00000605). RNA was extracted from formalin-fixed paraffin-embedded tumor sections of 631 patients and subjected to gene expression profiling with 770 genes across 23 key breast cancer pathways/processes (NanoString®BC360 panel) including the prognostic PAM50 signature for intrinsic subtype classification. Signatures were measured with the nCounter Digital Analyzer system (Nanostring) and revealed 60% Luminal A, 31% Luminal B, 6% HER2 enriched and 3% Basal-like tumor subtypes. Predefined signature scores (Nanostring) or single gene expression scores were analyzed in relation to breast cancer recurrence-free survival (EFS). PAM50 subtype designation and its derived Genomic Risk Score (ROR) were strongly associated with EFS of patients treated with AI (Log Rank P<0.05 all comparisons) but not in patients treated with mono-TAM. Preliminary data show that higher levels of FOXA1 and androgen receptor (AR) gene expression are both associated with longer EFS in patients with AI therapy. Likewise, increased (mutant-like) p53 signaling was prognostic for shorter EFS (Log Rank P<0.05). These relations were not observed in patients treated with TAM or with a switch treatment regimen. A trend for longer EFS linked to an increased anti-tumor immune activity, as deduced by cytotoxic cell abundance and activity, was observed in a subgroup of mainly TAM treated patients. Our preliminary data suggest that gene expression signatures pertaining to FOXA1 and AR signaling, p53 and anti-tumor immune activity may define subgroups of patients with different outcomes and may aid in future personalized treatment concepts for hormone-sensitive postmenopausal early breast cancer.

#465

Molecular classification of endometrial cancer provides complementary information but does not outperform current predictive models: The Karolinska and Bern experience.

Denis Nastic,1 Sara Imboden,2 Mehran Ghaderi,1 Filippa Rydberg,1 Daniel Olsson,1 Michael Mueller,2 Tilman T. Rau,3 Elisabeth Epstein,1 Joseph Carlson1. 1 _Karolinska Institutet, Stockholm, Sweden;_ 2 _University Hospital of Bern and University of Bern, Bern, Switzerland;_ 3 _University of Bern, Bern, Switzerland_.

Purpose: The purpose of this study was to evaluate the performance of a proposed prognostic molecular classification of endometrial cancer in a retrospective cohort of n=604 patients. A secondary goal was to compare the performance to existing prognostic models.

Methods: All cases were analysed using 1) sequencing of the exonuclease domain of the POLE gene, 2) microsatellite instability by immunohistochemistry, and 3) TP53 mutation, as evaluated by immunohistochemistry. Additionally, complete histopathological and clinical data was available. The cases were classified using the molecular markers analyzed.

Results: The assembled cohort largely resembled the typical unselected distribution of FIGO grade and stage. Analysis of clinicopathologic variables showed that FIGO grade and stage, LVSI, tumor histotype, depth of myometrial invasion, ESMO 2013 and 2016 risk group, recurrence and survival all were significantly associated with molecular subgroups. A large number of patients showed multiple molecular aberrations (so called multiple classifiers, n=50, 8.4%). Mutation in P53 was a statistically prognostic marker for overall survival (OS) in the univariable Cox regression (p-value 0.003 for OS, and <0.001 for DSS and PFS, HRs of 1.61, 1.83 and 1.72). The POLE mutated cases showed a trend towards favorable prognosis. By contrast, histotype (endometrioid vs. non-endometrioid) was a highly significant predictor of OS, DSS and PFS (HR=2.23, p=<0.001). Both ESMO 2013 and 2016, which represent an aggregate of the various clinicopathologic factors, were highly significant in predicting outcome and especially so for the high and advanced risk groups with HRs up to 70.1 (CI 23.9-206.8) and all p-values of <0.001. In the multivariable Cox regression analysis, however, none of the molecular markers remained significant. The Harrell C-index for 2016 ESMO risk groups was 0.76. Adding molecular markers did not show improved discriminatory ability.

Conclusions: In this retrospective cohort, molecular markers provided important biological information that may be helpful in therapeutic decision making. However, the molecular markers did not outperform current prognostic models.

#466

A novel DNA methylation-based approach for molecular subtyping and improved prognostication of colorectal cancer using formalin-fixed and paraffin-embedded tissue.

Trine B. Mattesen,1 Mads H. Rasmussen,1 Juan Sandoval,2 Halit Ongen,3 Sigrid S. Árnadóttir,1 Anders H. Madsen,4 Søren Laurberg,5 Emmanouil T. Dermitzakis,3 Manel Esteller,6 Claus L. Andersen,1 Jesper B. Bramsen1. 1 _Department of Molecular Medicine, Aarhus University Hospital, Aarhus 8200, Denmark;_ 2 _[1] Epigenomic Unit, Health Research Institute La Fe, Valencia, Spain [2] Biomarkers and precision medicine Unit, IISLaFe, Valencia, Spain;_ 3 _[1] Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva 1211 [2] Institute for Genetics and Genomics in Geneva (iGE3), University of Geneva, Geneva 1211 [3] Swiss Institute of Bioinformatic, Geneva 1211, Switzerland;_ 4 _Department of Surgery, Hospitalsenheden Vest, Herning 7400, Denmark;_ 5 _Colorectal Surgical Unit, Department of Surgery, Aarhus University Hospital Aarhus 8000, Denmark;_ 6 _Cancer Epigenetics and Biology Program, Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet, Barcelona 08908, Catalonia, Spain_.

Background: Colorectal cancer (CRC) is characterized by marked inter-tumor heterogeneity, both molecularly and clinically. Patient stratification using transcriptional subtyping show promise to resolve the heterogeneity and guide precision medicine. This requires high quality RNA, which can be purified from fresh frozen (FF) tissue, but not from clinically collected formalin-fixed paraffin-embedded (FFPE) tissue. Consequently, transcriptional subtyping is not applicable to most CRC patients.

Aim: To establish a DNA methylation-based approach for molecular characterization and subtyping, which is compatible with both FF and FFPE tissue, and use this to improve patient prognostication as compared to histopathological TNM staging.

Methods: Using paired RNA expression and DNA methylation profiles (450K array) from 394 CRCs, we identified 200 CpG sites genome-wide, whose methylation levels correlated best with expression for each gene. With this information, we 1) devised an approach, methCORR, that can infer RNA expression in both FF and FFPE samples using only DNA methylation profiles, 2) established a methCORR network that clusters genes according to overlap in the CpG sites associated with their expression level and 3) used the network to determine and characterize biological traits associated with tumor aggressiveness.

Results: The methCORR-inferred RNA expression profiles in FF tissue consistently exhibited high correlation to matched RNA-seq. profiles (median Pearson r=0.97; range 0.91-0.98). We found a higher correlation between FF RNA-seq. and FFPE inferred RNA profiles (median r= 0.97; range 0.96-0.98) than between FF and FFPE RNA-seq. profiles (median r= 0.78; range 0.51-0.92). Gene expression profiles were inferred in two FF (n=231, n=203), and two FFPE cohorts (n=113, n=56). Clustering of these profiles identified the same two CRC subtypes independently in all cohorts. Characterization using the methCORR network showed that the two subtypes resembled conventional and serrated CRC. Moreover, methCORR network analysis identified subtype-specific traits that associated strongly with tumor aggressiveness, such as T-cell, fibroblast, and epithelial-mesenchymal transition activity, and thus allowed identification of subtype-specific prognostic biomarkers. These better predicted relapse-free survival (HR=3.22, 95%CI 2.00-5.17) than TNM staging (HR=1.99, 95%CI 1.28-3.08). Finally, we derived four simple and clinically applicable DNA methylation-specific PCR assays for subtyping and prognostication of CRC FFPE samples.

Conclusion: We have developed a novel method for characterization, subtyping, and prognostication of CRC, which is compatible with FF and FFPE samples. We envision that application of the methCORR approach to other cancer types will generate similar fruitful results.

#467

Deep single-cell RNA-seq of the putative cell of origin revealed a novel molecular subtype of high-grade serous ovarian cancer with poor prognosis.

Zhiyuan Hu,1 Abdulkhaliq Alsaadi,1 Nina Wietek,1 Laura Santana González,1 Christopher Yau,2 Ahmed Ashour Ahmed1. 1 _University of Oxford, Oxford, United Kingdom;_ 2 _University of Birmingham, Birmingham, United Kingdom_.

Accurate molecular classification in cancer is essential for rationalized therapy. However, achieving stable subtyping is highly challenging due to the underlying genomic complexity of tumors. An example of the most successful classification is in breast cancer where it is possible to link individual tumor types, basal vs luminal, to individual cell types of origin. However, the generalization of such an approach has not been easy because of the lack of knowledge about subtypes of the putative cells of origin. For instance, it has been difficult to establish a stable molecular classification of high-grade serous ovarian cancer (HGSOC). More recent evidence strongly supports the notion that HGSOC originates from the secretory cells in fallopian tube epithelium (FTE). However, whether or not there are multiple subtypes of secretory cells and how such subtypes are linked to tumor molecular subtypes have remained elusive. To address this issue, we applied single-cell RNA sequencing (Smart-Seq2) on around 4000 FTE cells from patients with ovarian or endometrial cancers. We developed and employed an in-house differential-expression-based clustering method, termed ClinCluster, for clinical samples that takes into account inter-patient variability. Apart from the established secretory and ciliated cell types, we discovered four novel subtypes of secretory cells and a rare intermediate cell type that co-expresses secretory and ciliated markers. We termed these four novel secretory subtypes as cell cycle, progenitor, epithelial-mesenchymal transition (EMT) and immunoreactive subtypes. Based on fifty-three biomarkers of these subtypes, we designed a panel of transcriptomic signatures and exploited them to deconvolute two bulk expression datasets of ovarian cancer: the RNA-seq dataset of TCGA (The Cancer Genome Atlas) and the microarray dataset of AOCS (Australian Ovarian Cancer Study). The deconvolution analysis revealed a poor-prognostic EMThigh subtype of HGSOC, which was not driven by copy number aberrations. Cox regression analysis indicated that the EMThigh tumors were strongly associated with poor overall survival in the two independent datasets tested (hazard ratio = 2.7, 95% confidence interval = 1.4 - 5.1, P = 0.002, n = 307 for TCGA; hazard ratio = 3.2, 95% confidence interval = 1.7 - 6.1, P = 0.0002, n = 253 for AOCS). Importantly, we found that the PI3K-Akt signaling pathway is upregulated in the EMThigh tumors (FDR < 0.002 for the two independent datasets) giving a strong potential for effective rationalized therapies. In summary, our work portrays a new model for the landscape of fallopian tube epithelium, the putative origin of HGSOC, and illustrates that the cellular subtypes in FTE are related to the HGSOC tumor types with important prognostic and therapeutic implications.

#468

Clinical relevance of comprehensive genomic analysis in advanced-stage cancers and rare malignancies: Results from the MASTER trial of the German Cancer Consortium.

Peter Horak,1 Christoph Heining,2 Simon Kreutzfeldt,1 Christoph E. Heilig,1 Lino Möhrmann,2 Laura Gieldon,3 Martina Fröhlich,4 Sebastian Uhrig,4 Daniel Hübschmann,4 Katja Beck,1 Daniela Richter,2 Stephan Wolf,4 Katrin Pfütze,1 Christina Geörg,1 Bettina Meißburger,1 Frederick Klauschen,5 Sebastian Ochsenreither,6 Gunnar Folprecht,3 Jens Siveke,7 Sebastian Bauer,7 Thomas Kindler,8 Christian Brandts,9 Melanie Börries,10 Nikolas von Bubnoff,11 Karsten Spiekermann,12 Philipp J. Jost,13 Klaus Schulze-Osthoff,14 Michael Bitzer,15 Peter Schirmacher,16 Christof von Kalle,1 Richard Schlenk,1 Barbara Klink,3 Barbara Hutter,4 Wilko Weichert,13 Albrecht Stenzinger,16 Evelin Schröck,3 Benedikt Brors,4 Hanno Glimm,2 Stefan Fröhling1. 1 _NCT Heidelberg, Heidelberg, Germany;_ 2 _NCT Dresden, Dresden, Germany;_ 3 _Faculty of Medicine Carl Gustav Carus, Dresden, Germany;_ 4 _DKFZ Heidelberg, Heidelberg, Germany;_ 5 _Charité Universitätsmedizin Berlin, Berlin, Germany;_ 6 _Charité Comprehensive Cancer Center, Berlin, Germany;_ 7 _University Hospital Essen, Essen, Germany;_ 8 _Mainz University Medical Center, Mainz, Germany;_ 9 _UCT Frankfurt, Frankfurt, Germany;_ 10 _DKTK Freiburg, Freiburg, Germany;_ 11 _University Medical Center Freiburg, Freiburg, Germany;_ 12 _University Hospital LMU Munich, Munich, Germany;_ 13 _Technical University of Munich, Munich, Germany;_ 14 _University of Tuebingen, Tübingen, Germany;_ 15 _University Hospital Tuebingen, Tübingen, Germany;_ 16 _Heidelberg University Hospital, Heidelberg, Germany_.

Precision oncology implies the ability to predict which patients will likely respond to specific cancer therapies based on high-resolution molecular diagnostics. The value of comprehensive molecular profiling based on whole-exome/genome sequencing (WES/WGS) and global RNA sequencing to guide therapeutic decisions in individual patients remains to be established. We report the results of MASTER (Molecularly Aided Stratification for Tumor Eradication Research), a multicenter registry trial for prospective, biology-driven stratification of younger adults with advanced-stage cancer across all histologies and patients with rare tumors conducted under the auspices of NCT Heidelberg/Dresden and the German Cancer Consortium (DKTK). Based on a standardized workflow for selection and consenting of patients, sample processing, WES/WGS and RNA sequencing, bioinformatic analysis, and technical validation of potentially actionable findings, we have analyzed more than 1300 poor-prognosis (median overall survival, 12 months) patients representing a broad spectrum of entities. Evaluation of the data by a cross-institutional molecular tumor board has allowed categorization into 7 different intervention baskets and formulation of evidence-based recommendations for clinical management in more than 80% of patients, which were implemented in approximately one third of cases. Overall response and disease control rates on molecularly guided treatment were significantly improved compared to prior systemic therapies, which translated into a progression-free survival (PFS) ratio of greater than 1.3 in more than 40% of cases. In 5% of patients, comprehensive genomic profiling allowed to refine the clinical diagnosis, as exemplified by several soft-tissue sarcomas not otherwise specified and carcinomas of unknown primary site that could be categorized based on their genotypes and subsequent histopathologic re-evaluation. Finally, systematic analysis of germline alterations revealed that 11% of patients had pathogenic (ACMG Class 4 or 5) variants in known tumor predisposition genes, and that 4% were carriers for autosomal recessive disorders. This prospective trial demonstrates that molecular profiling based on WES/WGS and RNA sequencing in a multi-institutional clinical setting is feasible, complements and advances routine molecular diagnostics, and creates clinically meaningful therapeutic opportunities in a significant proportion of patients. To improve clinical translation, the MASTER platform is now linked to a growing portfolio of cross-institutional basket trials. In the intermediate term, genomic profiling within MASTER will be integrated with additional layers of patient characterization and extended to additional treatment modalities (e.g. radiotherapy and surgical interventions).

#469

Patient-derived sarcoma models: First results from the SARQMA study.

Manuela Gaebler,1 Alessandra Silvestri,2 Peter Reichardt,1 Eva Wardelmann,3 Guido Gambara,4 Johannes Haybaeck,5 Philipp Stroebel,6 Maya Niethard,1 Gerrit Erdmann,7 Christian R. Regenbrecht5. 1 _Helios Klinikum Berlin-Buch GmbH, Berlin, Germany;_ 2 _cpo - cellular phenomics & oncology, Berlin, Germany; _3 _University of Muenster, Muenster, Germany;_ 4 _German Consortium for Translational Research (DKTK), Berlin, Germany;_ 5 _University Hospital Magdeburg, Magdeburg, Germany;_ 6 _University Hospital Goettingen, Goettingen, Germany;_ 7 _NMI TT Pharmaservices, Berlin, Germany_.

Objective: The development of new targeted therapeutics has allowed an essential improvement in carcinoma treatment. For sarcomas, however, the main approach is still the combination of surgery, chemotherapy and radiation, which is due to their high heterogeneity, with more than 70 histopathological subtypes, and the limited knowledge of the molecular drivers of tumor development and progression. Here we show that patient-derived 3D (PD3D) cell culture models allow for an in vitro system to systematically test compounds and combos in a semi-automated way, generating a pre-clinical dataset that in combination with clinical data, genomic and proteomics profiles may help to better understand the biology and ultimately identify more potent treatment regimens.

Methods: Fresh surgical specimen underwent several steps of dissociation. Cell aggregates were then seeded into 24w plates in matrix-like scaffolds and grown until >100μm. Organoids were then harvested, transferred to 384w plates and treated with a set of compounds that resemble standard-of-care treatments and novel compounds. Finally, viability was calculated. In parallel, protein extracts were used for DigiWest, a multiplexed protein profiling assay allowing to interrogate up to 800 (phospho)-proteins.

Results: Of 49 cases with a biopsy, 23 had too little material available. Of these 23 samples, 5 were reported as sarcoma tissue afterwards, while 5 of the taken 26 samples were not sarcomas according to the final histopathology. Of all sarcomas, 90% (19/21) were taken from tumors localized at the extremities, the rest was located at the trunk. 52% (11/21) were growing in short term cell culture at least to passage 1 (p1), 28% (6/21) were growing for long term analyses. Two of those six were myxoid liposarcomas, two were undifferentiated pleomorphic sarcomas or classified as not otherwise specified (UPS/NOS), one was a myxoid liposarcoma and one a biphasic synovial sarcoma. Here we show that it is feasible to generate organoids from sarcoma tissues for extensive characterization in order to better understand their biology and mechanisms of treatment. High-throughput drug screening allows for an profiling of pharmacokinetic properties of individual sarcomas. Using the same material for additional (phospho)-proteomics provides multiple layers of understanding.

Conclusion: At present, the structure of clinical trials is not amenable to N of 1 studies, so applying the information garnered from this platform, particularly combination therapy drug screens, remains a significant hurdle. The major limitation to the establishment of organoid cultures was insufficient amounts of fresh tissue with viable tumor cells. Increasing the tumor tissue available for organoid production would lead to a greater success rate. Nevertheless, by their recapitulation of the donor tissue architecture, they provide an interesting and important tool to study the huge variety of soft-tissue tumors.

#470

Chromosomal instability and bad prognosis both connote a multiple myeloma (MM) sub-type carrying 13qCN loss and 1qCN gain.

Carolina Terragna,1 Marina Martello,1 Andrea Poletti,1 Vincenza Solli,1 Enrica Borsi,1 Rosalinda Termini,1 Lucia Pantani,1 Elena Zamagni,1 Giulia Marzocchi,1 Paola Tacchetti,1 Nicoletta Testoni,1 Serena Rocchi,1 Luca Cifarelli,1 Luca Dozza,1 Giovanni Martinelli,2 Michele Cavo1. 1 _Seragnoli Inst. of Hematology & Medical Onc., Bologna, Italy; _2 _Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori, IRST, Meldola, Italy_.

BACKGROUND MM patients (pts) are characterized by a prevalence of aneuploidies and Copy Number Alterations (CNAs) broadly scattered over the whole genome. Along with translocations and/or single nucleotide variants, they account for the MM-distinctive genomic heterogeneity, which characterizes the onset of symptomatic MM and identifies each pts.

AIM. To deeply explore the genomic landscape of newly diagnosed, homogeneously treated MM pts, to assess the prognostic relevance of whole genome aneuploidies, CNAs, and structural aberrations.

PATIENTS AND METHODS 344 highly purified BM CD138+ samples were profiled by SNPs array. R scripts were employed to analyse genomic data. Pts, enrolled in the EMN02 phase III trial, were randomized to receive VMP (121 pts) or high-dose MEL and autologous stem cell transplantation (223 pts), after bortezomib-based induction. At a median follow-up of 46 months (m), the estimated PFS and OS rates were 55% and 83%, respectively.

RESULTS To evaluate the contribution of each genomic variables to the overall pts variability, a Principal Component Analysis of the whole genome's numerical and structural aberrations was performed, highlighting that a "common ground" of at least 4 chromosomal aberrations (hyperdiploidy, H, 13qCN loss, IgH-t and 1qCN gains) was sufficient to describe and resume the MM overall genomic heterogeneity. Therefore, since 13qCN loss and 1qCN gains co-segregate, 3 pts sub-types might be identified, partially overlapping, even though well-defined by the presence of either H, or IgH-t or 13qCN loss and 1qCN gain. Outcomes of the latter MM sub-type was explored. Pts carrying 13qCN loss and 1qCN gain were characterized by the presence of several small deletions in MM-critical genes (YAP1, TRAF3, CYLD), and by the markedly de-regulated expression of CCND2 and CCND1 (as evaluated by the analysis of CoMMpass-derived RNAseq expression dataset). Their 46-m PFS and OS estimates were shorter as compared to that of patients carrying either just one or any of these CNAs (PFS: 39%, 44% and 73%, respectively; p=0.0001. OS: 68%, 83% AND 91%; p=0.0001). PFS and OS hazard ratios (HR) of pts carrying 13qCN loss and 1qCN gain (PFS: 1.76; OS: 2.24) were comparable to that of pts carrying either del(17p) (PFS: 2.1; OS: 2.1) or t(4;14) (PFS: 1.80; OS: 1.7), resulting as an independent factor predicting both PFS and OS in a Cox multivariate analysis. Survival data were confirmed on additional SNPs array data derived from 151 unrelated MM pts not included in the EMN02 trial, as well as on 700 pts, whose CNAs data were extrapolated from the MMRF CoMMpass Study dataset.

CONCLUSIONS We propose a simple way to stratify MM pts, according to the detection of 2 CNAs, which is able to select pts with high (25%) and with low (40%) risk of progression and/or death, allowing to predict the clinical outcome in most MM pts. Supported by AIRC, Fond. del Monte Bo-Ra, Fond. Berlucchi, BO-AIL, Harmony

#471

Tumor clonal status predicts clinical outcomes of non-small-cell lung cancer.

Eun Young Kim, Sanghoon Lee, Arum Kim, Tae Hee Kim, Yoon Soo Chang. _Yonsei Univ. College of Medicine, Seoul, Republic of Korea_.

Background: Intratumoral heterogeneity is a major cause of resistance to therapeutic agents and treatment failure. In this study, we evaluated clonal status, which reflects intratumoral heterogeneity, in non-small-cell lung cancer (NSCLC) and we discussed its clinical implications.

Materials and Methods: Targeted sequencing was performed using a customized panel comprising 70 solid tumor-associated genes on samples obtained from 99 lung adenocarcinoma cases. In parallel, TCGA-LUAD cases with clinical information were extracted from the GDC data portal for validation. Clonal status was identified from the allele frequency of the mutated gene using the Maftools package.

Results: We found no significant relationships with stage between the total number of mutations or the number of variants when classified according to the variant effect predictor. There was a positive correlation between the maximum primary tumor diameter and the number of clones (σ=0.2318, P-value = 0.0339). Additionally, the number of clones increased with stage (p-value= 0.0220, Kruskal-Wallis chi-squared test). Disease-free survival (DFS) was significantly shorter among cases wherein tumors comprised two or more clones than among cases in which tumors were comprised of only one clone (P-value = 0.008, Log-rank test). A multivariate model that controlled for total number of mutations, tumor stage, sex, age, and smoking indicated that the number of clones was an independent determinant of DFS.

Conclusions: Number of clones comprising the primary lesion was positively correlated with tumor size and stage, and was an independent factor affecting clinical outcome, indicating that a description of tumor clonality may be required for next-generation sequencing.

#472

Transcriptome profiling-based identification of prognostic subtypes in glioblastoma: Novel therapeutic strategy targeting invasiveness.

Junseong Park, Jin-Kyoung Shim, Seon-Jin Yoon, Se Hoon Kim, Jong Hee Chang, Seok-Gu Kang. _Yonsei University College of Medicine, Seoul, Republic of Korea_.

Glioblastoma (GBM) is a lethal tumor, but few biomarkers and molecular subtypes predicting prognosis are available. This study was aimed to identify prognostic subtypes and multi-omics signatures for GBM. We identified 80 genes most associated with GBM prognosis using correlations between gene expression levels and overall survival of patients. The prognostic score of each sample was calculated using these genes, followed by assigning three prognostic subtypes according to prognostic scores. This novel classification was validated in several independent datasets including our patient dataset. Functional annotation revealed that invasion- and cell cycle-related gene sets were enriched in the poor and favorable group, respectively. Therefore, the three subtypes were named as invasive (poor), mitotic (favorable), and intermediate. Interestingly, invasive subtype showed increased invasiveness, and MGMT methylation was enriched in mitotic subtype, indicating the need for different therapeutic strategies according to prognostic subtypes. For clinical convenience, we also identified the genes that best distinguished the invasive and mitotic subtypes. Immunohistochemical staining showed that markedly higher expression of PDPN in the invasive subtype and of TMEM100 in the mitotic subtype. We expect that this comprehensive transcriptome-based classification, with several multi-omics signatures and biomarkers, can improve molecular understanding of GBM. Because invasive subtype showed worse prognosis, we next evaluated invasion-modulating transcriptional regulatory networks for novel therapeutic interventions. After classification of 23 GBM patient-derived tumorspheres into low and high invasion groups, we applied single sample gene set enrichment analysis using transcription factor (TF) target gene sets. According to scores, TFs responsible for low (PCBP1) and high (STAT3 and SRF) invasiveness were identified. Consistently with computational prediction, knockdown of PCBP1 significantly increased invasion area, whereas knockdown of STAT3 or SRF significantly suppressed invasive properties in all tested TSs and mice. Notably, MR images showed coherent patterns with invasion of originated TS, and high invasiveness was associated with poor prognosis in both mice and GBM patients, interrelating these transcriptional regulatory networks, invasive phenotype, and prognosis. We suggest that these transcription factors are deterministic molecules for invasion, which can be utilized as novel drug targets for GBM.

#473

Higher levels of genomic complexity correlates with an advanced plasma cell differentiation status in newly diagnosed multiple myeloma patients.

Marina Martello,1 Rosalinda Termini,1 Enrica Borsi,1 Vincenza Solli,1 Andrea Poletti,1 Lucia Pantani,1 Serena Rocchi,1 Katia Mancuso,1 Elena Zamagni,1 Paola Tacchetti,1 Mario Arpinati,1 Gabriella Chirumbolo,1 Nicoletta Testoni,1 Giulia Marzocchi,1 Giovanni Martinelli,2 Michele Cavo,1 Carolina Terragna1. 1 _Univ. of Bologna, Bologna, Italy;_ 2 _IRST - Istituto Tumori, Meldola, Italy_.

Introduction: plasticity is a hallmark of Multiple Myeloma (MM) clone(s), where both quiescent MM cells, acting as tumor-initiating cells, and proliferative MM cells, able to invade and disseminate, might co-exist. Aim of the study is to stratify patients (pts) according to both the level of chromosomal instability (CIN) and their plasma cells (PCs) differentiation stages, and to evaluate the impact of this stratification on the disease outcome.

Patients & Methods: 145 newly diagnosed MM pts were included in the study. Whole-genome copy number alterations (CNAs) were analysed by SNP array both in the CD138+PCs and CD19+B-cells. In each pts, both the CD138+/CD38high PCs and CD19+B-cells compartments were characterized by 6-color multi-parameter flow cytometry analysis, combining CD138-PE, CD38-PE-Cy7, CD20-APC, CD19-APC-Cy7, CD27-FITC, CD45-FITC, CD28-APC, CD44-FITC, CD54-APC, CD81-PerCP-Cy5.5, CD56-APC.

Results: According to the CD138+ PCs' CIN, as described both by the total amount of CNAs and by the portion of genome changed (GC), three major pts subgroups were identified: the most representative, including 21/64 pts, was characterized by a higher CIN (median CNAs: 550 %GC ≥ 25) as compared to the others (intermediate and low CIN, median CNAs: 220 10≤ GC%≥25). Hyperdyploidy, but also high-risk features [i.e. 17p del (TP53)] mainly characterized CD138+ PCs with high CIN. On the contrary, in the same pts, the CD19+B-cells display a quite simple karyotype with very few microalterations (>50kb), mostly involved in the signal transduction pathway (loss on KRAS, chr12p12.1 and on SIRPB1, chr20p13). According to the the co-expression of CD19/CD81, describing the MM clone(s) differentiation status, PCs with a high level of CIN resulted more mature CD19-/CD81-. Both the high expression of CD28 and CD44 and the reduced expression of CD20, CD27 and CD45 confirmed the advanced differentiation status.

Finally, although baseline clinical features of pts with more mature, genomically instable PCs, are associated to bad prognosis (e.g. PET lesions, k/l ratio, ISS III, β2-microglobulin; p<.05), they are more likely to obtain high quality response rates (≥CR) to PI induction therapy.

Conclusion: High level of genomic complexity correlates with advanced PCs differentiation stages, and this is lastly associated with a prevalence of poor prognosis features. Both CIN and phenotypic pliancy represent important, yet poorly defined, mechanisms by which MM clone(s) accelerate their own evolution and survival.

Acknowledgements: AIRC, AIL, HARMONY, Berlucchi

#474

Genome-wide DNA methylation profiling reveals novel candidate epigenetic gatekeepers in hepatocarcinogenesis.

Gabriela Hernandez Meza,1 Johann von Felden,1 Amanda Craig,1 Sergi Sayols,2 Anna Portela,3 Manel Esteller,3 Myron Schwartz,1 Vincenzo Mazzaferro,4 Josep Llovet,1 Augusto Villanueva1. 1 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 2 _Institute of Molecular Biology, Mainz, Germany;_ 3 _IDIBELL Bellvitge Biomedical Research Institute, Spain;_ 4 _Fondazione IRCCS Istituto Nazionale dei tumori, Italy_.

Epigenetic deregulation is a critical event in human malignancies. Besides mutations in TERT promoter, found in 20% of dysplastic nodules, little is known about the key molecular alterations driving early hepatocarcinogenesis. The aims of this study were: 1) to analyze DNA methylation changes during the transition from preneoplastic lesions to early hepatocellular carcinoma (HCC), and 2) to identify candidate epigenetic gatekeepers in the transition between dysplasia and early tumors.

Methylome profiling was done with Illumina HumanMethylation450 (485,000 CpG, 96% of known CpG islands). We evaluated differentially methylated CpG sites between groups using an F-test. To detect novel epigenetic gatekeepers we defined hypermethylation as a B value higher than 0.5. We profiled 144 fresh-frozen tissues from resection or liver transplant specimens and integrated these data with our previously report on DNA methylation in HCC (Villanueva et al. Hepatology 2015) resulting in: 16 normal liver, 139 cirrhosis, 8 dysplastic nodules and 227 HCC samples. Correlation between methylation and RNA expression (n= 361) was quantified with the Pearson's coefficient.

Patients were mostly male (76%), with a median age of 66, and with underlying liver disease mainly due to hepatitis C (43%) and B (23%). All tumors were early stage according to BCLC classification, including 43 cases below 2 cm in diameter. A phylo-epigenetic tree derived from the euclidean distances between differentially DNA methylated sites (n=421,997) reveals a gradient of methylation changes that spans normal, cirrhotic, dysplastic nodules and HCC. Epigenetic analysis confirmed closer proximity of dysplasia to HCC than to cirrhotic tissue. We validated aberrant methylation of previously reported HCC epidrivers within the unpublished dataset (e.g. EFBN2, TBX15) (Villanueva et al. Hepatology 2015). Focusing on CpG sites located in promoter regions (i.e., TSS200, TSS1500, 5'UTR, and 1st exon), we selected candidates hypermethylated in less than 1% of normal and cirrhotic tissue, in all high grade dysplastic nodules, and in more than 50% of small HCC nodules (<2 cm). When we integrated DNA methylation and gene expression we found a significant (all P<0.001) inverse correlation in TSPYL5 (r=-0.31), KCNA3 (r=-0.33), LDBH (r=-0.46) and SPINT2 (r=-0.43).

Whole-genome DNA methylation profiles accurately discriminate between different histological lesions along the human hepatocarcinogenesis spectrum. We report novel epigenetic gatekeepers in the transition between dysplastic nodules and early HCC.

#475

Clinical significance of eif5-mimic protein 1 in pancreatic cancer.

Yushi Motomura,1 Takaaki Masuda,1 Kuniaki Sato,1 Atsushi Fujii,1 Akihiro Kitagawa,1 Hiroaki Wakiyama,1 Kensuke Koike,1 Junichi Takahashi,1 Dai Shimizu,1 Shotaro Kuramitsu,1 Miwa Noda,1 Yusuke Tsuruda,1 Hajime Otsu,1 Yosuke Kuroda,1 Hidetoshi Eguchi,1 Katsumi Sakamoto,1 Masakazu Hirakawa,1 Hiroshi Honda,2 Koshi Mimori1. 1 _Kyushu University Beppu Hospital, Beppu-shi,Oita prefecture, Japan;_ 2 _Kyushu University graduate school of medical sciences, Fukuoka, Japan_.

Background: Pancreatic cancer (PC) is the most lethal cancer in humans, with a 5-year survival rate of less than 10%. Thus, identification of prognostic biomarkers as well as molecular therapeutic targets should be critical.

Translational reprogramming in cancer has been attracting attention. Several studies have reported that genomic alternations of a translational regulatory gene contributing to malignancy. eIF5-mimic protein 1 (5MP1), previously termed BZW2 is a member of the bZIP superfamily of transcription factors. 5MP1 is reported to be upregulated and has a pro-tumor effect via activation of the Akt/mTOR signaling pathway in osteosarcoma. However, the role and mechanisms of 5MP1 in PC remains unknown. The aim of this study is to clarify the clinical significance of 5MP1 expression in PC.

Material and method: Firstly, we analyzed public datasets of PC (178 RNA-sequencing data from The Cancer Genome Atlas, 36 gene expression array data from GSE 15471, 45 gene expression array data from GSE 28735, 69 gene expression array data from GSE 62452, 145 primary PC and 46 normal pancreas gene expression array data from GSE 71729, 45 Copy number variation and expression array data from Broad-Novartis Cancer Cell Line Encyclopedia(CCLE)). Then we performed Gene set enrichment analysis (GSEA) to identify the gene sets which were associated with the expression of 5MP1 in PC. Finally, we investigated the localization of 5MP1 by immunohistochemical staining data of THE HUMAN PROTEIN ATLAS and also confirmed with staining our histological specimens.

Results: In tumor tissues, mRNA expression of 5MP1 was higher than normal tissues (t-test. p<0.05). Survival analysis showed high expression of 5MP1 was significantly associated with poor prognosis (Kaplan-Meier method, Log-rank test. p<0.05). Analysis of pancreas cancer cell line CCLE indicated a significant positive correlation between Copy number variation and expression of 5MP1 ( r = 0.41 ). Moreover, GSEA revealed the relationship between enhanced 5MP1 expression and cycle-related gene set. Immunohistochemistry staining showed prominent expression of 5MP1 in the plasma membranes of PC cells.

Conclusion and Significance: We demonstrated that 5MP1 was overexpressed in PC cells, and the high expression was associated with poor prognosis. These results suggest that 5MP1 may be associated with tumor progression and can be a promising biomarker for a patient with a poor prognosis of PC. Now we are performing a knockdown experiment for 5MP1 to clarify its biological significance in PC.

#476

MEK inhibitors for treatment of oligodendroglioma.

Nazanin Majd,1 Soon Young Park,2 Marisela Martinez de Kraatz,1 Veerakumar Balasubramaniyan,1 Mittal Sandeep,1 Jian Hu,1 Jihye Paik,3 John de Groot1. 1 _MD Anderson, Houston, TX;_ 2 _Oregan Health and Science University, Portland, OR;_ 3 _Weill Cornell Medical College, New York, NY_.

Oligodendroglioma (OD) is defined by the 2016 WHO classification as IDH mutant gliomas with 1p19q co-deletion. Although ODs are sensitive to chemotherapy, they eventually recur leading to functional deficits and ultimately death. CIC gene mutations, located on chromosome 19q are seen in 60% of 1p19q co-deleted OD and have been linked to worse survival. Despite our growing knowledge of the molecular characteristics of OD, all recurrent OD patients are treated similarly mainly using radiation and/or poorly tolerated cytotoxic chemotherapy with modest benefit. There is therefore an urgent unmet medical need to understand the molecular consequence of recurrent mutations in OD in order to improve patient outcomes. CIC gene mutations result in truncated, degraded or non-functional CIC protein, disrupting CIC protein repressor functions. This leads to upregulation of ETS-Pea3 family of transcription factors which are known oncogenes in a variety of solid cancers. MAPK signaling pathway has been shown to directly activate ETS family of proteins in drosophila. However, the link between MAPK signaling, ETS proteins and CIC in OD is elusive. Here, we propose a model of aggressiveness in CIC mutant OD in which these cells are dependent on MAPK axis for activation of their augmented ETS proteins (i.e ETV1, ETV4) and therefore are sensitive to MAPK inhibition. To test our model, we evaluated mRNA and protein expression of ETS family of proteins in CIC mutant and CIC wild-type mouse OD cell lines and show that ETV1 and ETV4 have elevated protein and mRNA expression in CIC mutant OD cells, respectively. Consistent with our hypothesis, two different MEK inhibitors, trametinib and pimasertib, reduce cell viability of OD cell lines with greater reduction in CIC mutant than wild-type cells. As proof of concept, we demonstrate that CIC mutant cells have increased pERK expression when compared with CIC wild-type cells indicating that MAPK pathway is more active in CIC mutant cells. Overall, we were able to show that MEK inhibitors have anti-tumor effects in OD cell lines, with greater sensitivity seen in CIC mutant cells. This work lays the foundation for repurposing the widely clinically used MEK inhibitors for treatment oligodendroglioma patients with CIC mutation.

#477

Targeted deep sequencing contributes to guiding personalized targeted therapy in advanced biliary tract cancer.

Feiling Feng,1 Qingbao Cheng,1 Dadong Zhang,2 Bin Li,1 Hao Qin,2 Chang Xu,1 Miao Han,2 Yong Yu,1 Zhizhen Li,1 Jing-Yu Li,2 Zhiquan Qiu,1 Lei Xiong,2 Chen Liu,1 Fugen Li,2 Bin Yi,1 Xiaoqing Jiang1. 1 _Shanghai Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China, Shanghai, China;_ 2 _3D Medicine Inc., Shanghai, China_.

Purpose: Biliary tract cancer (BTC) is a devastating disease of the digestive system with poor prognosis. Targeted therapy based on specific genetic alterations has been proven to be an effective treatment for some cancer subtypes. However, the effect of this targeted therapy is unclear in BTC. In this study, we aimed to explore the clinical efficacy and safety of personalized targeted therapy guided by targeted deep sequencing for advanced biliary tract cancer (BTC) patients.

Materials and Methods: In this retrospective study, targeted deep sequencing was employed for 49 patients with BTC and the recommendation of biologic agent was offered. Among 32 patients with stage IV and R2 resection, 21 patients underwent conventional chemotherapy (mGEMOX) (chemotherapy group), while the remaining 11 patients received a personalized targeted agent (targeted therapy group). The progression-free survival (PFS), overall survival (OS), disease control rate (DCR) were used to assess the efficacy of treatments, while the grade of treatment-related toxicities was evaluated for safety.

Results: The genomic landscape of 49 patients with BTC was depicted by targeted deep sequencing. Further analysis of all alterations demonstrated that these altered genes were highly enriched in the ERBB family or cell cycle pathway. After a median follow-up of 12 months, the targeted therapy group had a significant prolonged PFS (4.5 months vs. 1.5 months, P = 0.014) and a trend of prolonged OS (12.9 months vs. 4.1 months, P = 0.104) in comparison to the chemotherapy group. The DCR in the targeted therapy group was marginally higher but without statistical significance (63.6% vs. 33.3%, P = 0.142). In addition, there was no statistically significant difference in the percentage of patients that experienced Grade >2 treatment-related toxicities in either treatment group (36.4% vs 19.0%, P = 0.397).

Conclusions: This real-world clinical study suggests that targeted deep sequencing contributes to guiding personalized targeted therapy based on individual actionable mutations, which may benefit advanced BTC patients. Large umbrella trials of personalized precision therapy are needed to further confirm the clinical efficacy and safety of this therapeutic strategy in BTC.

#478

Investigating prognostic value of proinflammatory M1 gene expression in colorectal cancer patients.

Pankaj Ahluwalia,1 Chance Bloomer,1 Chetan Pundkar,1 Kimya Jones,1 Ashis Mondal,1 Gagandeep Gahlay,2 Ravindra Kolhe1. 1 _Medical College of Georgia, Augusta, GA;_ 2 _Guru Nanak Dev University, Amritsar, India_.

Colorectal cancer (CRC) affects nearly 1.4 million individuals every year, making upto 10% of the global burden of cancer. Progress over the last few decades in surgical, chemotherapy and targeted therapy, have led to tremendous improvement in survival rate but the high risk of relapse and variable survival among the patients, calls for the need of better prognostic biomarkers. The tissue microenvironment and the presence of immune cells can be attributed to diverse immune response of individual patients that can lead to variable survival. Macrophage can showcase two polar phenotypes, M1 (inflammatory) and M2 (immunosuppressive). We set out to quantify the M1 oriented gene expression and its correlation with clinic-pathological characteristics of CRC patients. Under IRB approved protocol, a total of 750 CRC patients at Medical College of Georgia with 5 years follow-up were initially selected. The patients were stratified on the basis of overall survival in two groups, with higher (> 5 years) and lower survival (< 1 year) along with AJCC staging (I to IV), grade, gender and age. A total of 88 patients fitted in our inclusion criteria on the basis of survival duration after diagnosis. Total RNA was isolated and quantified through Nanodrop method. Next, the expression of immunological molecules which has the polarization immune potential were quantified through custom plexset using medium through-put Nanostring platform. We quantified expression of M1 macrophage immune gene signature using a custom 21 gene panel: - IRF5, STAT1, STAT6, CCL13, GATA3, CXCL9, CXCL10, CXCL11, IL15RA, IL15, IL1B, CEBPA, PPARG (along with 8 housekeeping genes). We used univariate and multivariate analysis to identify significant associations between gene expression and overall survival. In stage IV patients, STAT1 (p = 0.04*) and STAT6 (p = 0.03*) were expressed at higher levels. IL15RA (p = 0.01*) and IL1B (p = 0.01*) had higher expression in Stage III and IV patients compared to Stage I and II. Upon Kaplan-Meier Curves analysis, lower expression of CXCL9, 10, 11 correlated with poor prognosis with respect to overall survival (log-rank p = 0.038*, 0.009*, 0.034* respectively). In the multivariate analysis, using Cox-proportional hazard model, CCL13 (HR 3.18* p = 0.16*) and CEBPA (HR 14.4 p = 0.00*) were able to determine overall survival of the CRC patients. These initial findings highlight the clinical significance of transcriptome analysis in determining the prognosis for CRC patients. A comprehensive immune-based gene panel would assist in designing new therapies or can complement existing treatment regimes.

#479

BRAF **and** KRAS m **utation define distinct subtypes of the CpG island methylator phenotype in colorectal cancers.**

Vicki L. Whitehall,1 Lochlan Fennell,1 Troy Dumenil,1 Gunter Hartel,1 Katia Nones,1 Catherine Bond,1 Diane McKeone,1 Ann-Marie Patch,2 Stephen Kazakoff,2 John Pearson,2 Nicola Waddell,2 Pratyaksha Wirapati,3 Paul Lochead,4 Shuji Ogino,5 Sabine Tejpar,3 Barbara Leggett6. 1 _QIMR Berghofer Medical Research Institute, Herston, Australia;_ 2 _QIMR Berghofer Medical Research Institute, Australia;_ 3 _Swiss Institute of Bioinformatics, Switzerland;_ 4 _Massachusetts General Hospital, MA;_ 5 _Dana Faber, MA;_ 6 _Royal Brisbane and Women's Hospital, Australia_.

BACKGROUND AND AIMS: Colorectal cancer is an epigenetically heterogeneous disease, however the extent and spectrum of the CpG Island Methylator Phenotype (CIMP) is not clear.METHODS: Genome scale methylation and transcript expression were measured using the Illumina HM450 DNA methylation and HT12 V3 expression microarrays in 216 unselected colorectal cancers. Mutations in epigenetic regulators were assessed using CIMP-classified Cancer Genome Atlas exomes.RESULTS: CIMP-High cancers dichotomised into CIMP-H1 and CIMP-H2 based on methylation profile, which was supported by over-representation of BRAF (74%, P<0.0001) or KRAS (55%, P<0.0001) mutation. Congruent with increasing methylation, there was a stepwise increase in patient age from 62 years in the CIMP-Negative subgroup to 75 years in the CIMP-H1 subgroup (P<0.0001). CIMP-H1 were predominantly comprised of consensus molecular subtype 1 (CMS1) cancers (70%) whilst CMS3 was over-represented in the CIMP-H2 subgroup (55%). PRC2-marked loci were subjected to significant gene body methylation in CIMP cancers (P<1.6x10-78). We identified oncogenes susceptible to gene body methylation and Wnt pathway antagonists resistant to gene body methylation. CIMP cluster specific mutations were observed for in chromatin remodeling genes, such as in the SWI/SNF and NuRD complexes, suggesting synthetic lethality.CONCLUSION: There are five clinically and molecularly distinct subgroups of colorectal cancer. We show a striking association between CIMP and age, gender and tumor location and identify an unidentified role for gene body methylation in progression of serrated neoplasia. These data support our recent findings that CIMP is uncommon in young patients and that BRAF mutant polyps in young patients may have limited potential for malignant progression.

#480

Biospecimen management for the serial measurement of molecular and architectural responses to therapy.

Brett E. Johnson, Swapnil Parmar, Kiara Siex, Anastasiya Olson, Jennifer Laverdure, Jamie Keck, Annette Kolodzie, Alexander R. Guimaraes, Christopher Corless, Joe Gray, Gordon Mills, Raymond Bergan. _OHSU, Portland, OR_.

Rapid advances in molecular diagnostics have led to a growing need for sophisticated and adaptive biospecimen collection methodologies that employ careful acquisition, preservation, and allocation of limited biopsy tissue and blood specimens. To facilitate the comprehensive characterization of serial biospecimens from patients participating in real-time adaptive treatment precision medicine clinical trials, we designed a multi-omic and multi-imaging analytical platform and sought to maximize the quality and suitability of tissue for each downstream analytic. We optimized this biospecimen management system over 38 research-protocol driven biopsies on 33 patients with late stage metastatic breast, prostate, and pancreatic cancer. Importantly, no biopsy-related adverse events occurred. All tissue samples and derivatives therefrom were barcoded and tracked using a custom database built on the LabVantage platform. Here, we report successful strategies for coordination between medical and surgical oncology, interventional radiology, pathology, and clinical and research laboratories. We developed a biopsy prioritization schema that considers patient safety, site selection, procedure type, and amount of tissue required. We evaluated specimens from a variety of biopsy procedures, including radiologically-guided needle cores, as well as laparoscopic, video-assisted thoracoscopic, and excisional biopsies. Radiologically-guided biopsies using 18 gauge needles yielded a median of 5 (1 cm) cores with 70% tumor cellularity, and was the best substrate to rapidly allocate known quantities of tissue into a variety of preservation formats. We also examined enhanced biospecimen preservation methods that improved performance of downstream analytics. For example, optimization of the formalin fixation process included transferring tissue from bedside to formalin within 2 minutes, maintaining low temperature throughout fixation, and standardizing the time spent in formalin. These efforts resulted in recovery of higher quality RNA from FFPE blocks, less tissue required for RNA sequencing, and opened the door to immunofluorescent staining of phosphoproteomic markers. Our protocol uses several preservation methods, including: FFPE for immunohistochemical and cyclic immunofluorescent staining as well as targeted panel, whole exome, and transcriptomic sequencing; OCT freezing for multiple single cell sequencing methods; flash freezing for proteomic profiling; disaggregation of live cells for 2D and 3D culturing; and a specialized fixative for electron microscopy. In conclusion, we demonstrate the feasibility of collecting high quality biospecimens for a variety of downstream analytical platforms with diverse sample requirements. These methods can be used to comprehensively characterize limited amounts of valuable tissue in molecularly driven clinical trials.

#481

Distinct and overlapping patterns of B-cell growth pathway mutations in CD5-negative B-cell lymphoproliferative disorders.

Sophia Shaddy, Weiqiang Zhao, Huolin Tu, Brianna Sisson, Rongqin Ren, Sean Caruthers, Susan Long, Peng Ru, Narendranath Epperla, Lynne Abruzzo, Kami Maddocks, Dan Jones. _Ohio State University Wexner Medical Center, Columbus, OH_.

Low-grade CD5-negative B-cell lymphoproliferative disorders (CD5- LG B-LPD) encompass several entities that often show mutations in different growth regulatory pathways. These include MYD88 (TLR signaling), CD79A/B (BCR complex), CARD11 (CBM signalosome), CXCR4 (chemokine receptor), and NOTCH1/2 and are complemented by KLF2 mutations and BCL6 and MALT1 overexpression. Using targeted, next-generation sequencing (NGS) for 50 B-cell associated growth regulators and epigenetic modifiers (>1000X depth) and cytogenetic/FISH data, we sought to delineate the complementary mutation and gene amplification patterns in 45 consecutive cases of CD5- LG B-LPD. In mutation-negative cases, a 572-gene NGS panel was used to interrogate these pathways more completely. WHO diagnostic criteria were used, without reliance on molecular data, emphasizing morphology features, paraprotein level and type to distinguish lymphoplasmacytic lymphoma (LPL) from marginal zone lymphoma (MZL). As expected, MYD88 L265P mutations were seen in the majority of LPL (11/17, 65%), but also in CD5- chronic lymphocytic leukemia (CLL) (3/9, 33%) and splenic (S) MZL (1/6, 17%) but not in nodal MZL. In 4 LPL cases lacking MYD88 L265P, extra copies of chromosome 3 (BCL6), 6 (IRF4), 12 and/or 18 (MALT1) were seen, with CARD11 mutations in the other 2. NOTCH1/2 mutations were mostly associated with SLL/CLL (3/9, 33%). Mutations in epigenetic regulators (see Table) were seen in the majority of nodal MZL (8/13, 62%) but only few LPL (2/17, 12%) and SMZL (1/6, 17%). CXCR4 inactivating mutations were seen with MYD88 and CD79B mutations and +3/BCL6 alteration. CARD11 mutations were seen with KLF2, MYD88 and NOTCH1/2 mutations. Less common mutations targets included PTPRC and PLCG2 (pre-ibrutinib treatment). The higher rate of mutation in epigenetic regulators (along with loss of KLF2-mediated BCR signaling) supports a distinct pathogenesis for nodal MZL in contrast to other CD5- B-LPD.

Calls per gene in MYD88 negative cases; *TP53, TET2, ASXL1, DNMT3A, BCOR

---

|

CD79B | CARD11 | NOTCH1/2 | CXCR4 | KLF2 | Epigenetic gene set*

LPL | 0 | 2 | 3 | 0 | 0 | 0

0 | 1 | 0 | 0 | 0 | 0

0 | 0 | 0 | 0 | 1 | 0

CD5- CLL | 0 | 0 | 0 | 0 | 0 | 2

0 | 0 | 1 | 0 | 0 | 1

0 | 0 | 1 | 0 | 0 | 0

0 | 0 | 1 | 0 | 0 | 0

0 | 0 | 0 | 0 | 0 | 1

sMZL | 0 | 1 | 1 | 0 | 1 | 0

0 | 1 | 0 | 0 | 0 | 0

0 | 0 | 0 | 0 | 0 | 1

nMZL | 0 | 0 | 0 | 0 | 0 | 2

0 | 0 | 0 | 0 | 2 | 2

0 | 0 | 0 | 0 | 0 | 1

0 | 1 | 0 | 0 | 1 | 1

0 | 0 | 0 | 1 | 0 | 1

0 | 0 | 0 | 0 | 0 | 1

0 | 0 | 0 | 0 | 0 | 1

0 | 0 | 0 | 0 | 0 | 1

0 | 1 | 2 | 0 | 1 | 0

0 | 0 | 0 | 2 | 0 | 0

#482

Recurrent glioma with lineage conversion from oligodendroglioma to astrocytoma: Another facet of glioma evolution during recurrence.

Kyung-Hwa Lee,1 Tae-Young Jung,1 Shin Jung,1 Jae-Hyuk Lee,1 Kyung-Keun Kim,2 Hangun Kim,3 Kyung-Sub Moon1. 1 _Chonnam National Univ. Hwasun Hospital, Jeonnam, Republic of Korea;_ 2 _Chonnam National University Medical School, Jeonnam, Republic of Korea;_ 3 _Sunchon National University, Jeonnam, Republic of Korea_.

Background: Following the introduction of the molecular classification of gliomas by the WHO in 2016, molecularly-proven lineage conversion during glioma recurrence has never been reported.

Methods: We analyzed two cases of recurrent glioma that began as oligodendroglioma, both histologically and molecularly, but converted to astrocytoma over episodes of recurrence.

Results: The cases were initially diagnosed as oligodendroglioma with 1p/19q-codeletion and mutation of isocitrate dehydrogenase 1 (IDH1)-R132H. The recurrent tumors showed loss of alpha-thalassemia/mental retardation X-linked (ATRX) expression, strong P53 positivity, and 1p/19q-nondeletion. Next generation sequencing (NGS) analysis performed on the first case confirmed the transition of molecular traits from oligodendroglioma to astrocytoma. An IDH mutation of R132H was preserved in the episodes of recurrence, but ATRX and TP53 mutations were newly acquired and TERT promoter mutation C228T was lost at the most recent recurrence.

Conclusions: The issue in question for the presented cases is whether the original tumors were pure oligodendrogliomas that then transdifferentiated into astrocytomas, or whether the original tumor was an oligoastrocytoma having oligodendroglioma cells that outnumbered the astrocytoma cells and where the astrocytoma cells becoming more dominant over the episodes of recurrence. With the recognition of the possibility of lineage conversion, our study suggests that molecular examination should be performed to adjust therapeutic strategies in recurrent gliomas. Indeed, our observation of lineage conversion in glioma recurrence calls into question the current distinction drawn between oligodendroglioma, astrocytoma and oligoastrocytoma, rather than simply bidding "farewell to oligoastrocytoma".

#483

Perceived benefits and barriers towards cervical cancer screening among women >15 years in Arsi zone, Southeastern Ethiopia: Application of the Health Belief Model (HBM) in a community- based cross-sectional study.

Gemechu Chemeda Feyissa. _Arsi Zonal Health Department, Addis Ababa, Ethiopia_.

Introduction: The rate of cancer mortality is18.4 per 100,000 in Ethiopia, and cancer of the cervix is the second most common cancer, with an incidence of 16.4 per 100,000. Cancer of the cervix is easily prevented through vaccinations against HPV, regular screening and treatment. Assessing the perception of women with regard to the benefits and barriers of cervical cancer screening among women is important to decrease the barriers and increase the screening rate for early detection. The aim of this study was to describe women's perceived benefits and barriers to cervical cancer screening and their associated factors in Arsi zone, southeastern Ethiopia.

Methods: This community- based cross-sectional study design was conducted in Arsi zone, southeastern Ethiopia among 906 women aged >15 years using a multi-stage sampling technique. After data had been coded and entered into Epi info, it was exported into SPSS for analysis. A binary logistic regression model was used to determine associations between socio-demographic characteristics and reproductive variables on the knowledge of cervical cancer and perception of screening at a p-value of <0.05.

Results: Most of the participants had higher perceptions of the benefits and barriers to screening (567(62.6%) and 487(53.8%), respectively). The women who would like to undergo cervical cancer screening in the future when the service was available were 2.6 times more likely to believe in the benefits of undergoing screening than those who did not wish to undergo screening [AOR=2.3, 95% CI: (1.2-4.6)]. Women living in rural areas were 4 times more likely to perceive the benefits of cervical cancer screening than those living in semi-urban areas [AOR=3.9, 95% CI: (1.9-7.7)]. Women who started sexual intercourse at age <16 years were 2 times more likely to perceive barriers to cervical cancer screening than those who started sexual intercourse at age >16 years [AOR=2.2, 95% CI: (1.5-3.3)]. Moreover, the women who had >3 children were 1.5 times more likely to perceive barriers to cervical cancer screening than those who had <3 children [AOR=1.5, 95% CI: (1.1-2.0)].

Conclusion and recommendation: There were high perceived benefits and barriers to screening in the community. We recommend decreasing the perceived barriers towards screening through education intervention and expanding screening services, as this can increase the uptake of screening in the community.

Key word: Cervical Cancer, Screening, Perceived Benefits, Perceived Barrier, Health Belief Model

1

#484

Molecular profiling identifies a second malignancy in a patient with medulloblastoma.

Kathleen M. Schieffer, Katherine E. Miller, Daniel R. Boue, Daniel C. Koboldt, Patrick Brennan, Benjamin J. Kelly, Gregory Wheeler, Vincent Magrini, Amy Wetzel, Elizabeth Varga, Devon Dishman, Kristen Leraas, Vibhuti Agarwal, Mohamed S. AbdelBaki, Jonathan L. Finlay, Jeffrey R. Leonard, Peter White, Julie M. Gastier-Foster, Catherine E. Cottrell, Elaine R. Mardis, Richard K. Wilson. _Nationwide Children's Hospital, Columbus, OH_.

Medulloblastoma is a pediatric embryonal tumor that can be classified into four molecular subgroups, each derived from a different progenitor cell. It is estimated that about 30-40% of patients will relapse, typically with recurrence at the primary site and of the same molecular subgroup. We present paired tumor/normal genomic analysis of an 18 year-old male who presented with non-Wnt/non-SHH medulloblastoma at age 12 and relapsed with metastatic disease of the falx cerebri 3 years later. Combination surgery, chemotherapy, and radiation were used in treatment of the primary and recurrent tumor. At a timepoint 6 years from original diagnosis, the patient presented with a cerebellar tumor histologically described as "consistent with recurrent medulloblastoma" with comment recommending genomics to confirm. The diagnosis was made based on near identifical morphology and retention of Neu-N and Synaptophysin in the tumor (confirmed by subsequent genetic analysis). The primary tumor and the tumor occurring 6 years after the primary diagnosis were analyzed by whole exome sequencing (blood and tumor tissues) to assess for germline variants, somatic mutation, and copy number variation. We observed no pathogenic germline variants in cancer predisposition genes. The tumor mutational profiles were distinct, with only 6 (1.8%) shared somatic variants between tumors. Specimen provenance was verified by germline variation and SRY coverage. Two targetable mutations within the RAS-MAPK pathway (PTPN11 p.Glu76Lys and PIK3CA p.Gly1007Arg) were present only in the new CNS tumor. Although the primary tumor harbored isochromosome 17q and a gain of chromosome 4, these somatic chromosomal aberrations were not detected in the new CNS tumor. RNA-seq was performed on both tumors and compared to pediatric CNS tumors from the University of California Santa Cruz Treehouse Initiative (n=434). The primary tumor clustered with the medulloblastoma patients by principal component analysis while the new CNS tumor clustered with a group of gliomas and non-medulloblastoma embryonal tumors. The primary tumor displayed evidence of overexpression of Group 4 medulloblastoma genes (e.g. EOMES, RBM24, SNCAIP, and UNC5D). These genes were not overexpressed in the new CNS tumor. Enrichment of genes commonly found in gliomas (e.g. BCAN, CHI3L2, PDGFRA, and SOX2) were noted in the new CNS tumor only. In summary, tumor genomic profiling of a primary medulloblastoma and the new CNS tumor arising 6 years later revealed two distinct sets of somatic mutations suggestive of second malignancy rather than recurrence in this patient. While second malignancy in the setting of medulloblastoma is a rare event, it has been documented, both in a time period consistent with that described in our patient and in the form of glioma. Thus, tumor profiling refined diagnosis in this patient allowing for a more accurate assessment of treatment and management options.

#485

**Exon 9 mutation of** PIK3CA **associated with poor survival in patients with Epstein-Barr virus-associated gastric cancer.**

Dongwon Baek, Jong Gwang Kim, Jin Ho Baek, Byung Woog Kang, Soo Jung Lee, Yee Soo Chae, Hee Jeong Cho. _Kyungpook National University Hospital, Daegu, Republic of Korea_.

PIK3CA would seem to play an important role in the prognosis of gastric cancer (GC), very few studies have reported on the clinical implications and prognostic effects of these mutations in patients with Epstein-Barr virus-associated gastric cancer (EBVaGC). This study attempted to identify the prognostic significance of PIK3CA mutations in a large number of EBVaGC patients and the relationship of these mutations to the clinicopathologic parameters associated with prognosis. After reviewing 1318 consecutive cases of surgically resected at our institution between January 2011 and November 2014, 120 patients were identified as EBV-positive using EBV-encoded RNA in situ hybridization. Among the 120 patients, 8 patients were excluded due to insufficient or unavailable material (6 patients) and technical issues (2 patients). The related medical files and pathologic reports were all reviewed to identify the clinical and demographic characteristics. PIK3CA mutations were identified in formalin-fixed and paraffin-embedded surgical specimens from 112 EBVaGC patients with available tumor tissue sample. A real-time polymerase chain reaction was used to evaluate hot-spot mutations of exons 1, 4, 7, 9, and 20 of PIK3CA. The frequency of PIK3CA mutations was 25.0% (n=28), and among the 28 patients harboring a PIK3CA mutation, most of the mutations were identified in exon 9 (n=21, 75%). The presence of PIK3CA mutation was also correlated with a higher T category (P<0.001) and N category (P<0.001), as well as the presence of perinueral invasion (P<0.001) and venous invasion (P<0.001). In a univariate analysis, PIK3CA mutation showed no association with overall survival (OS) (P=0.184) or disease-free survival (DFS) (P=0.150). The patients harboring exon 9 PIK3CA mutations did exhibit a significantly shorter OS(P=0.023) and DFS(P=0.013) than the patients lacking an exon 9 PIK3CA mutation, yet without statistical significance in the multivariate analysis. Notably, the exon 9 E542K mutation of PIK3CA was associated with the worst DFS (P=0.011). Plus, the PIK3CA-mutant tumors were not related to such immune-related factors as intratumoral (iTu) tumor-infiltrating lymphocytes (TILs), stromal (str) TILs, iTu-PD-L1, and str-PD-L1 (all P > 0.05). The PIK3CA mutations harbored in exon 9 were also strongly related to clinicopathologic parameters associated with an unfavorable prognosis. Although the exon 9 PIK3CA mutations were not identified as an independently robust prognostic factor, they could play an important role in carcinogenesis and tumor aggressiveness in EBVaGC. Consequently, these findings support the theory that exon 9 PIK3CA mutations can be a prognostic indicator for predicting patients' outcomes and a rationale for therapeutic targeting in EBVaGC.

#486

Pathogenic drivers and their comutations in genes frequently altered in cancer.

Vassiliki Kotoula,1 Elena Fountzilas,2 Georgia-Angeliki Koliou,2 Kyriaki Papadopoulou,2 Eleni Giannoulatou,3 Ioannis Tikas,2 Flora Zagouri,2 Christos Christodoulou,2 Georgios Pentheroudakis,2 Angelos Koutras,2 Dimitrios Pectasides,2 George Fountzilas2. 1 _Aristotle Univ. School of Medicine, Thessaloniki, Greece;_ 2 _Hellenic Cooperative Oncology Group, Athens, Greece;_ 3 _Victor Chang Cardiac Research Institute, Darlinghurst / University of New South Wales, Kensington, Australia_.

Background - aim: The as yet publicized results from Precision Oncology trials highlight the need to define parameters that may interfere with the actionability of common pathogenic drivers. One such parameter may be the presence of multiple drivers within the same tumor. Here, we investigated the properties of single and coexisting pathogenic mutations in genes frequently altered and targeted across cancers.

Methods: We examined informative deep sequencing data for TP53, PIK3CA, KRAS and BRCA1 from 2672 paraffin tumors that were obtained at first diagnosis (88% stage I-III; 12% stage IV) from patients with breast (n=1745), colorectal (n=524), nasopharyngeal (n=143), gastric (n=102), biliary tree (n=81), nasopharyngeal (n=143) and ovarian (n=77) cancer. Single mutation corresponds to the presence of a pathogenic mutation in only one of these genes; co-mutation, to the presence of pathogenic mutations in ≥2 genes. We examined the status of single and co-mutations compared to disease stage and patient overall survival (OS) stratified by tumor type.

Results: Pathogenic mutations in any of the 4 genes were observed in 1337 (50% of all tumors) and co-mutation in 347 tumors (28% of tumors with mutations in these genes). Single mutations and co-mutations were observed in 481 (36.0%) and 322 (24.1%) tumors for TP53, respectively; in 350 (26.0%) and 198 (14.8%) for PIK3CA; in 80 (6%) and 198 (14.8%) for KRAS; and, in 51 (3.9%) and 120 (9.0%) for BRCA1. Co-mutations of BRCA1 and KRAS were more frequent than single mutations in the two genes across tumor types (p's<0.010). The same was observed for PIK3CA and TP53 except for breast cancer, where single mutations were more frequent than co-mutations (p's<0.001). Compared to stage I-III, stage IV tumors were more frequently mutated in TP53, KRAS and BRCA1 (p's<0.010) but were significantly less often mutated in PIK3CA (p<0.001). Co-mutation rates for BRCA1, KRAS and TP53 did not differ between stage I-III and IV tumors but these were significantly different for PIK3CA (33.5% in I-III vs. 64.3% in IV; p<0.001). Upon stratification by tumor type, in stage I-III disease, compared to tumors with co-mutations, single BRCA1, TP53 and KRAS mutations conferred similar OS, but single PIK3CA mutations conferred significantly lower risk for death (HR 0.52; 95%CI 0.35-0.77; p<0.001). In stage IV disease, compared to no mutations, single KRAS or TP53 were associated with poor OS (p=0.017 and p=0.025, respectively) but this effect disappeared in patients with KRAS/TP53 co-mutated tumors.

Conclusions: Co-mutation patterns of known cancer drivers vary with respect to the affected gene, tumor type and disease stage. Known drivers have distinct prognostic impact when occurring singly or co-altered in the non-metastatic and metastatic setting, which may need to be considered in Precision Oncology trials.

#487

A high percentage of LCNEC shows DLL3 expression in association with molecular subtypes and neuroendocrine markers.

Ernst-Jan M. Speel,1 Bregtje C. Hermans,1 Jules L. Derks,1 Erik Thunnissen,2 Robert Jan van Suylen,3 Michael A. den Bakker,4 Harry J. Groen,5 Egbert F. Smit,6 Ronald A. Damhuis,7 Esther C. van den Broek,8 Cecile M. Stallinga,1 Guido M. Roemen,1 Anne-Marie C. Dingemans1. 1 _Maastricht UMC, Maastricht, Netherlands;_ 2 _Amsterdam UMC, Amsterdam, Netherlands;_ 3 _Jeroen Bosch Hospital, 's-Hertogenbosch, Netherlands;_ 4 _Maasstad Hospital, Rotterdam, Netherlands;_ 5 _UMC Groningen, Groningen, Netherlands;_ 6 _Netherlands Cancer Institute, Amsterdam, Netherlands;_ 7 _Comprehensive Cancer Association, Utrecht, Netherlands;_ 8 _PALGA Foundation, Houten, Netherlands_.

Background Pulmonary large cell neuroendocrine carcinoma (LCNEC) can be subdivided in two types: the co-mutated TP53 and RB1 subtype, and the TP53 and STK11/KEAP1 mutated subtype (Derks et al. Clin Cancer Res 2018; J Thorac Oncol 2018). DLL3 is a member of the Notch ligand family and a possible treatment target for neuroendocrine carcinoma. We investigated DLL3 and NE marker expression in mutational subtypes of metastatic LCNEC.

Methods Immunohistochemical (IHC) analysis for DLL3 (clone SC16.65) was performed on 94 pathological reviewed pretreatment stage IV LCNEC. Samples were scored positive if ≥1% tumor cells showed cytoplasmic or dotlike DLL3 immunostaining. Also an H-score was calculated by multiplying intensity by percentage of positive cells. Targeted next generation sequencing (TP53, RB1, STK11, KEAP1) could be performed in 66 patients.

Results DLL3 was expressed in 70/94 (75%) LCNEC, 56 of which showed cytoplasmic immunostaining. 37/70 (53%) samples had an H-score>100. DLL3 staining was more often seen in STK11 and/or KEAP1 mutated LCNEC (13/14;93%) than in tumors with wildtype STK11/KEAP1 (36/52;69%) (trend;p=0.093). In addition, DLL3 positivity was associated with expression of ≥2 NE-markers (64/79 LCNEC (81%) vs 3/8 (37%) tumors with <2 NE-markers; p=0.014).

Conclusions A high percentage (75%) of stage IV LCNEC shows cytoplasmatic DLL3 epression in association with NE markers, half of which with H-scores>100. Interestingly, DLL3 positivity was observed in almost all STK11/KEAP1 mutated LCNEC, which is in agreement with recent data (George et al. Nat Commun 2018). It thus might be worthwhile to investigate the efficacy of DLL3 targeted therapy in LCNEC. 

## IMMUNOLOGY

### Imaging the Tumor Microenvironment

#488

Direct real-time visualization and quantification of T cell receptor dynamics.

Jillian N. Rosenberg, Guoshuai Cao, Fernanda Borja-Prieto, Yanran He, Hans Schreiber, Jun Huang. _University of Chicago, Chicago, IL_.

The mechanism by which a T cell utilizes its T-cell receptors (TCRs) to recognize foreign peptide major histocompatibility complexes (pMHCs) has not yet been fully deciphered. Much contention still exists surrounding TCR nanostructures before, during, and after synapse formation. Accurate measurements of single TCR location, diffusion/trafficking, clustering, and signaling at the live T-cell membrane have been hindered by the microscopy techniques available. Recently developed Lattice Light-Sheet Microscopy uses a structured light sheet to excite fluorescence in successive planes of a living T cell such that it can record 4D (x, y, z directions and time) images with exceptionally high temporal resolution (10ms/frame, ~1s/volume), finally allowing for precise single molecule tracking. Here we show that TCRs pre-exist in small microclusters prior to synapse formation in both naïve and stimulated resting CD4+ T cells. These clusters are very fast-moving, explaining the previous difficulties of tracking TCR motion and revealing a fast responding mechanism for antigen recognition. Upon encountering antigens, these smaller TCR nanoclusters traffic globally and quickly to the cell interface, aggregate into larger microclusters, and form the immunological synapse. Finally, we compare and contrast the receptor dynamics of CAR-T cells with those of naturally occurring T cell receptors. These findings precisely quantify TCR and CAR dynamics three-dimensionally, suggesting new mechanistic details of the speed and accuracy of TCR signaling and providing new information upon which to base future immunotherapies.

#489

Multi-parameter tissue section imaging and retrieval of image-defined micro-regions for RNA sequencing using the RareCyte® platform.

Rebecca Podyminogin,1 Nolan Ericson,1 Jia-Ren Lin,2 Yu-An Chen,2 Peter Sorger,2 Eric Kaldjian,1 Tad George,1 Lance U'Ren1. 1 _RareCyte, Inc, Seattle, WA;_ 2 _Harvard Medical School, Boston, MA_.

Background: Tumor tissue imaging allows for a contextual understanding of tumor cells in relation to the immune microenvironment. The ability to interrogate tissues for multiple proteins that define microscopic regions of interest (ROI) and investigate gene expression of cells in those regions is needed for advancing immuno-oncology therapeutic and biomarker discovery. The RareCyte CyteFinder® instrument provides integrated multi-parameter imaging and micro-region retrieval capabilities for sequencing and transcript-level analysis.

Methods: Formalin-fixed, paraffin-embedded (FFPE) sections of tonsil and breast carcinoma sections were stained with a panel of antibodies to CD3, CD8, CD20, cytokeratin, and Ki-67. Frozen sections were stained with antibodies to CD3, CD4, CD8, CD19, and Ki-67. All sections were also stained with SYTOX Orange (nuclear dye). Whole-slide six-color scanning and ROI identification was performed with the CyteFinder instrument. For frozen sections, 40µm diameter micro-regions were retrieved directly from the antibody-stained section using the CytePicker® module. For FFPE sections, ROI were identified on the antibody-stained sample and micro-regions were retrieved from the same location on an adjacent section stained with DRAQ5 only. RNA from retrieved micro-regions was amplified and sequenced and differential gene expression analysis was performed.

Results: Tonsil staining distinctly identified crypt lining (cytokeratin), T cell (CD3, CD4, CD8) and B cell (CD19, CD20) compartments; Ki-67 was prominently expressed in germinal centers. Breast carcinoma staining distinctly identified tumor cells (cytokeratin) and CD8-positive T cells that were prominent at the periphery of the tumor with smaller numbers interspersed. Ki-67 staining was variably present in tumor cell nuclei. Comparative RNA analysis of four frozen tonsil T cell zone micro-regions against four B cell zone micro-regions resulted identified genes with expected differential gene expression pattern, including T cell zone upregulation of CD8a, CCL19, and CCL21 and downregulation of CD38, CR2, and CXCL13. Analysis of FFPE tonsil micro-regions comparing four germinal centers with areas outside the follicle identified genes differentially regulated within the germinal center, including B cell markers CD19, CD22 and CD79a. Out of 20 selected genes preferentially expressed in the germinal center, 16 (80%) were confirmed by interrogation of the Human Protein Atlas to be specific or enriched in germinal centers by immunohistochemical staining.

Conclusions: This study demonstrates high quality six-parameter imaging of tonsil and breast carcinoma tissues. Confirmation of tonsil immune cell type by gene expression analysis supports use of the RareCyte platform for retrieval of defined tissue micro-regions for RNA sequencing in frozen and FFPE samples.

#490

Highly multiplexed single-cell spatial analysis of tissue specimens using CODEX.

Gajalakshmi Dakshinamoorthy, Jaskirat Singh, Joseph Kim, Nadya Nikulina, Roya Bashier, Sejal Mistry, Maria E. Gallina, Atri Choksi, Meenu Perera, Ashley Wilson, Julia Kennedy-Darling. _Akoya Biosciences, Menlo Park, CA_.

Characterizing the complexities of the tumor microenvironment is elemental to understanding disease mechanisms. The spatial relationships between infiltrating immune cells and the remodeling of the cellular matrix is widely recognized as a key component to defining tumor heterogeneity. Current methodologies for analyzing the spatial dimension in tissues, like traditional immunofluorescence (IF) and immunohistochemistry (IHC), are limited to a few parameters at a time, restricting the scope of identifiable cells. Conversely, single-cell technologies like mass cytometry and NGS-based tools provide multiplexing capabilities, but at the expense of the associated spatial information. Here, we present a novel multiplexed imaging technology, termed CODEX, (CO-Detection by indEXing) that combines the high parameter capabilities of single-cell methodologies with the associated spatial dimension. The CODEX technology involves labeling antibodies with oligonucleotide-based Barcodes followed by a single staining step. Over 50 parameters are measured within a single tissue through fully-automated, iterative cycles of adding and removing corresponding dye-conjugated Reporters. Unlike other cyclic IF approaches involving multiple antibody staining and stripping steps, the CODEX platform involves a single initial staining step and subsequent gentle and relatively fast manipulation of the tissue thereafter. This provides a faster workflow and prevents tissue degradation. Other multiplexed imaging technologies, including imaging cytometry and MIBI, require expensive equipment precluding their routine use across various labs. The CODEX technology, developed by Akoya, is comprised of a fluidics instrument that interfaces with existing microscope hardware, as suite of reagents and associated control and analysis software. Over 100 antibody clones have been validated for this platform with more than ten tissue types analyzed, including both FFPE and fresh-frozen from human and mouse samples. The CODEX technology can be used to ascertain complex cellular niches and spatial associations between multiple cell types based on the staining pattern of more than 50 parameters. The CODEX viewer software package enables users to interact with both raw data and cell annotated tissue maps to determine the underlying spatial relationships within each dataset. CODEX data from various normal and cancer tissue types is shown here with corresponding single-cell analysis of key tissue features. Overall, the CODEX platform is an accessible and versatile technology for high parameter, spatial profiling of tissue specimens.

#491

Tumor heterogeneity and its impact on immunoprofiling data: Whole tumor vs regions of interest (ROI) analysis of multiplex immunofluorescence in four carcinoma types.

Jaime Rodriguez-Canales, Michael Surace, Jennifer Cann. _Medimmune, Gaithersburg, MD_.

Background: Multiplex immunofluorescence (mIF) is a key tool for cancer immunoprofiling in tissue samples. These techniques typically require an investigator to select regions of interest (ROI) within the tumor for digital image analysis. However, tumor heterogeneity and sampling bias may result in data that may not be representative of the whole tumor. Our goal was to compare immunoprofiling data from ROI analysis versus whole tumor in lung, colorectal, and bladder carcinomas using mIF and multispectral image analysis.

Methods: 9 lung adenocarcinomas, 11 lung squamous cell carcinomas, 10 colorectal adenocarcinomas (CRC), and 10 bladder urothelial carcinomas (BC) were stained with a 6-marker mIF panel (PDL1, CD8, Ki67, CD68, AE1/AE3, PD1) using the Opal technique, and imaged using a Polaris multispectral scanner. Two pathologists independently selected 5 ROI (0.64 mm2) within the tumor for each case. To address the possibility of human sampling bias, 2 additional sets of 5 ROI were randomly generated in the tumor by a computer. HALO software was used to analyze the ROIs and the whole tumor area. The data was compared using Spearman's ranked correlation coefficients.

Results: 6 cell populations were assessed in the tumor epithelium and stroma: cytotoxic T-lymphocytes (CTL, CD8+), antigen-experienced CTL (CD8+/PD1+), proliferating CTL (CD8+/Ki67+), macrophages (CD68+), PDL1+ tumor cells (AE1AE3+/PDL1+), and proliferating tumor cells (AE1AE3+/Ki67+). These immunophenotypes were assessed in each set of 5 ROI and compared with data from the whole tumor. Spearman's correlation coefficients (r) ranged from -0.25 to 1.00 depending on markers and tumor type. The best correlations across all tumor types were percentage of PDL1+ tumor cells (r=0.82 to 0.89), while cell densities of CTL and PD1+CTL showed the lowest correlations (r=0.35). Among tumor types, lung tumors and BC showed overall good correlation (r=0.82 to 0.83), depending on the marker. CRC showed the highest variability (average r=0.68), with correlation coefficients as low as 0.35 (PD1+CTL) and 0.46 (PDL1+macrophages). Computer selected ROI were not significantly more or less concordant with whole tumor analysis as compared to ROI selected by pathologists.

Conclusion: Our results suggest that for immunoprofiling data, particularly proportion-based endpoints such as percent of PDL1+ tumor cells in lung and BC, ROI analysis has acceptable correlation with data from the whole tumor. However, CRC showed high variability, particularly in PD1+CTL, Ki67+CTL, and PDL1+macrophages, suggesting that CRC has higher tumor immunological heterogeneity compared with lung cancer and BC. These results suggest that investigators should analyze the whole tumor areas, otherwise, special attention should be paid to the image analysis strategy and its validation.

#492

Spatial overlap analysis of myeloid and T cell compartments identifies distinct subgroups in the cohort of selected colorectal primary tumors.

Natalie Zwing, Marta Canamero, Fabien Gaire, Konstanty Korski. _Roche Innovation Center Munich, pRED, Penzberg, Germany_.

The immune contexture of colorectal cancer (CRC) tumors classifies clinically relevant immune subtypes that are extensively studied. Recent developments in digital computational pathology supporting spatial characterization of tumor immune infiltrate allow more comprehensive analysis of relevant cell to cell interactions. Leveraging that methodology in the current study, we tried to address the question how the distribution of myeloid cells in the tumor microenvironment (TME) of CRC affects the effector functions and distribution of cytotoxic T cells.

We applied multicolored immunohistochemistry to identify monocytic (CD11b/CD14) and granulocytic (CD11b/CD15) myeloid cell populations together with proliferating and non-proliferating cytotoxic T cells (Ki67/CD8). Through automated object detection and image registration using HALO software (IndicaLab), we measured the extent of overlap between the areas occupied by myeloid and T cells. Subsequently, we correlated it with other parameters such as CD8 T cell proliferation ratio, amount of necrosis, MMR status, tumor mutational burden and CMS classes. With this approach, we identified distinct categories of CRC based on the myeloid - T cell overlap irrespective of the MMR status. These categories reflect certain functional areas in the TME and correlate with specific gene signatures that mainly include T effector and memory together with myeloid inflammatory and immunosuppressive signatures.

Our results emphasize the importance of analyzing spatial organization of immune infiltrate in TME and shed a new light on myeloid and T cell interactions in CRC. The approach we used has a potential to be applied in immunotherapy related biomarker studies.

#493

The landscape of adenosine signaling in cancer.

Ben Sidders,1 Pei Zhang,1 Kelly Goodwin,2 Greg O'Connor,2 Alexandra Borodovsky,2 Jonathan Dry,2 Richard Woessner,2 Kris Sachsenmeier2. 1 _AstraZeneca, Cambridge, United Kingdom;_ 2 _AstraZeneca, Waltham, MA_.

Adenosine is a key suppressor of immune cell function. CD73 and other nucleotidases/phosphatases dephosphorylate and convert extracellular AMP into adenosine, which binds the A2A adenosine receptor (A2aR). There are several agents in early clinical trials targeting components of the adenosine pathway including A2AR and CD73. The identification of cancers with a significant adenosine drive is therefore critical to our understanding of the potential for these molecules. However, it is challenging to measure tumour adenosine levels on a pan-cancer scale. Here we describe the generation & validation of a gene expression signature that we use to characterise the pan-cancer landscape of adenosine signalling and its role within the tumour microenvironment.

Natural Language Processing (NLP) was used to define a network of regulatory interactions between the A2aR receptor and downstream entities. 14 genes were identified as being positively regulated by A2aR and robustly expressed in tumours and so constitute the signature, which we demonstrate accurately recapitulates adenosine drive in mouse syngeneic models. We therefore applied the signature to a pan-cancer analysis of tumour transcriptomes from TCGA and found a significant negative impact on both overall survival (HR = 0.6, Cox PH p=0) and progression free survival (HR = 0.77, Cox PH p=0.0000006) in adenosine high tumours. An adenosine related survival deficit was also seen in CD8+ T-cell infiltrated tumours (OS HR = 0.47, Cox PH p=0; PFS HR = 0.65, Cox PH p=0.0000002). Studying adenosine signalling in the context of tumour genetics identified highly significant associations with TGFB super-family members including TGFBR2 (p=0.0000000013). These data support the adenosine pathway as a key mediator of a successful anti-tumour immune response, demonstrate the prognostic potential of the gene expression signature and could inform patient selection strategies for adenosine pathway modulators.

#494

Immune cell screen of tumor micro environment of PD-L1 in positive/negative bladder cancer, melanoma, and lung cancer.

Rachel Gonzalez, Wei Fu, Evelin Logis, Casey Chen, Tiffany Cone, Sonia Merritt, Yiran Wang, Guanli Wang, Donghui Ma. _OriGene Technologies Inc., Rockville, MD_.

PD-L1/PD1 immune therapy has been very successful for some patients. Treatment is usually based on PD-L1 tumor expression levels in tumors; however some patients with positive tumors have shown no response to therapy. Recent, clinical trials have shown that treating patients with more than one immune therapy target results in better outcome suggesting the importance of understanding PD-L1 tumor micro environment. In this study, we look at a number of immune cell markers with PD-L1 expression to see if patterns of the immune cells infiltrating the tumor micro environment vary. PD-L1 expression in the tumors was assessed by multiple PD-L1 antibodies since no single PD-L1 antibody has been FDA approved for all tumor types. Immuno-histo chemistry (IHC) screen used 2 recombinant rabbit monoclonal antibodies (clone OR-5E3 and OR-5H8), one mouse anti PD-L1 clone UMAB229, and the FDA approved antibodies clones (SP142 and 28-8). Immune cell markers used in the IHC screen were CD3, CD8A, CD20, CD68, FOXP3, LAG3 and TIM3. The screen was done on sequential sections of bladder, melanoma, and lung tumors. Variation existed between five PD-L1 antibodies in their sensitivity and specificity to detect PD-L1 in the different tumor types; however, the differences were usually associated with the ability to detect low expressing tumors. Immune cell markers CD3, CD8A, CD20, CD68, FOXP3, LAG3, and TIM3 cell markers all produced strong staining if positive cells were present; however, they vary in number and distribution pattern throughout the three types of tumor. For example, when lung or bladder tumors presented strong PD-L1 staining at the edge of the tumor they often had a number of CD3E or CD8A immune positive cells present, this was not the case for CD20 which were foci clusters or scatter through the tumors. FOXP3 and CD68 staining were scarce event in all three tumor types. Although this study was limited to small sample size 10 tumors each it did show differences in the amount and distribution of immune cells in the 3 different types of tumor positive for PD-L1 expression.

#495

All-in-one flow cytometry staining panel for immune-cell profiling in syngeneic tumor models.

Holger Weber,1 Janette Beshay,2 Cynthia Obodozie1. 1 _ProQinase GmbH, Freiburg, Germany;_ 2 _Charles River, Freiburg, Germany_.

Checkpoint inhibitor treatment has become a common therapy of various cancer types; however, clinical data indicate that only few patients respond to this regimen due to attenuated anti-tumor immune response. Thus, it has been recognized as important to consider the immune response already during preclinical drug development to anticipate such clinical drawbacks and investigate the changes of immune cell populations after treatment. Flow cytometry is widely used for this kind of analysis. However, a comprehensive analysis of numerousness immune cell populations in one tumor is a major challenge when only limited material is available due to small tumor size of e.g. a tumor responding to the treatment or analysis at an early tumor developmental stage. A staining panel that allows the analysis of all major immune cell populations in a single staining would be a major advantage.

Here we present an all-in-one flow cytometry panel based on 15 colors which permits the differentiation of the main immune cell populations in the tumor, such as T cells (CD4+, CD8+, Treg), NK cells, macrophages (M1/M2), MDSCs (neutrophils, M-MDSCs) and dendritic cells. To demonstrate the value of the panel, the immune cell content of CT26wt tumors was compared after subcutaneous, orthotopic or intra-mammary fat pad implantation. During tumor growth profiling of the immune population is performed every 4 days, starting at day 7 after implantation when tumor volume is still very small (50 - 100 mm3). A comprehensive overview is given with valuable insight into the immune biology of the tumor over time and with respect to different tumor sizes.

The established all-in-one staining panel allows the direct comparison of immune cell populations with a single staining which improves the development of new immune-modulating drug candidates.

#496

Block age effect on automated quantitative T cell count using multiplex immunfluorescence.

Igor D. Vidal, Jessica Hicks, Christine Davis, Christopher Heaphy, Angelo Michael De Marzo. _Johns Hopkins University, Baltimore, MD_.

Introduction: Formalin fixation and paraffin embedding is the universal mechanism of tissue preservation. While the data showing detrimental effect on slide storage at room temperature1,2, the evidence supports that paraffin blocks archived at room temperature for as little as two years or as long as 25 years exhibited stable staining for the most of antigens evaluated. 3

Description of experimental procedures: In order to evaluate the effect of block age in inflammatory markers with immunofluorescence, two sets of antibodies were assembled and distributed in two distinct panels of five colors each, using the opal technology based on thyramide signal amplification. The first panel comprised foxp3, p63, CD3, CD4, CK8 and the second, p63, CD3, CD8 and CK8. Both panels were applied to a TMA with samples between sixteen to eighteen years old and compared to one with samples with less than one year old, fixed in FFPE. For each TMA construction, carcinoma prostatic tissue and matched normal prostate not inflamed tissue from forty patients were punched multiple times (between four to six) within regions of interest of each case. The slides were scanned and the image analysis performed. Statistic evaluation was performed using STATA13. The immunofluorescent count was validated using chromogenic CD3 stain controls using separate test TMAs.

Unpublished data: The analysis showed an increased number in CD3 positive T-cells in tumor areas, when compared to normal ones. For block age, there was no significant difference between old and new blocks.

Conclusion: The number of positive T cells for CD3 comparing new and old blocks of FFPE tissue shows no significant difference.

#497

**Characterization of TIGIT expression using MultiOmyx** TM **hyperplexed immunofluorescence assay in solid tumors.**

Qingyan Au, Arezoo Hanifi, Erinn Parnell, Judy Kuo, Eric Leones, Flora Sahafi, Kathy Pham, RaghavKrishna Padmanabhan, Nicholas Hoe, Josette William. _Neogenomics Laboratories, Inc, Aliso Viejo, CA_.

TIGIT (T cell immunoreceptor with Ig and ITIM domains) is a recently identified immune-receptor that is expressed on T cells, natural killer (NK) cells and NKT cells. TIGIT has emerged as an important coinhibitory receptor. TIGIT expression on CD8+ tumor infiltrating lymphocytes (TILs) has been shown to be upregulated in solid cancers such as melanoma, colon cancer, and NSCLC and have been associated with a dysfunctional phenotype in TILs. TIGIT can also be activated on a subset of Regulatory T cells (Tregs) and may be critical in driving CD8+ T cell dysfunction. A second mechanism in which TIGIT inhibits immunosurveillance is through both competition and direct inhibition of CD226, impairing its ability to activate immunosurveillance. TIGIT has also been reported to inhibit T cell responses indirectly by triggering CD155 expression in dendritic cells (DCs), thereby preventing DC maturation. These findings render the TIGIT pathway as an attractive candidate for cancer immunotherapy In this study, we seek to use MultiOmyx hyperplexed immunofluorescence (IF) assay to exploit this new pathway and characterize the TIGIT expression in a total of 20 melanoma and NSCLC samples. The cancer FFPE slides will be stained with a 13-marker panel including TIGIT, CD226, CD155, CD3, CD4, CD8, FOXP3, CD56, CD45, CD11b, CD11c, PD1, LAG3, TIM3 and PanCK. This panel will enable the detection of TIGIT, CD226 and CD115 expression in the Melanoma and NSCLC samples. The TIGIT expression will be further characterized on different TILs including CD4+ helper T cells, CD8+ cytotoxic T cells, Tregs and NK cells. Using the MultiOmyx proprietary algorithm, we can quantify different subtypes of TIGIT expressing cells and measure the distance of different TIGIT expressing cells to the tumor. Increased expression of TIGIT and PD1 has been demonstrated in NSCLC and melanoma. Moreover, TIGIT+ Tregs have been reported to upregulate TIM-3 expression in mice tumor models. In this study, we will also evaluate the expression of TIGIT in conjunction with other coinhibitory receptors such as PD1, LAG3 and TIM3. The percentage of TILs that co-express TIGIT/PD1, TIGIT/LAG3 and TIGIT/TIM3 will be quantified and analyzed. Leveraging TIGIT in combination with other immune therapy may achieve more robust clinical outcomes. There are currently multiple phase I clinical trials using TIGIT monoclonal antibody (for instance, BMS-986207 by Bristol-Myers Squibb and MTIG7192A by Genetech) in combination with anti PD1/PDL1 antibodies in solid tumors. Our data can help provide more insight into how TIGIT modulate antitumor immunity in melanoma and NSCLC tumors. And the findings in this study can also be used to understand the synergistic effects between TIGIT and other coinhibitory receptors and help identify the additional opportunity for combination immunotherapy using checkpoint inhibitors.

#498

Integrating bulk and spatial profiling technologies for the discovery of RNA and protein biomarkers in muscle invasive bladder cancer.

Zhaojie Zhang,1 Subhasree Das,1 Manav Korpal,1 Pavan Kumar,1 Jingjing Gong,2 Yan Liang,2 Margaret Hoang,2 Joseph Beecham,2 Victoria Rimkunas1. 1 _H3 Biomedicine, Cambridge, MA;_ 2 _Nanostring Technologies, Seattle, WA_.

Muscle-invasive bladder cancer (MIBC) is an aggressive disease with limited therapeutic options. Although immunotherapies are approved for MIBC, the majority of patients fail to respond, suggesting the existence of complementary immune evasion mechanisms underscoring the need for comprehensive immune profiling of patient tumor samples. The PPARγ pathway constitutes a tumor-intrinsic mechanism underlying immune evasion in MIBC. Immune cell-infiltration is controlled by activated PPARγ, which in turn inhibits expression/secretion of inflammatory cytokines. Clinical data sets indicate that PPARγHigh impairs CD8+ T-cell infiltration and confers partial resistance to immunotherapies.

Here, we report a comprehensive biomarker study aimed at identifying a single, highly multiplexed platform for profiling the tumor microenvironment in clinical-type specimens as formalin-fixed, paraffin-embedded (FFPE) samples. A cohort of PPARγHigh and PPARγLow muscle invasive bladder cancer samples were used for the technology assessments. Gene expression patterns were measured using a combination of research use only genomics and spatial genomic technologies to determine concordance including RNA-seq, spatial in situ hybridization (GeoMx™ Digital Spatial Profiler) and IO360. We determined that multiple RNA and protein-based technologies were able to accurately characterize the immune phenotypes of PPARγHigh tumors. However, the GeoMx™ technology was ideal for precious samples since it digitally quantifies target abundance while maintaining spatial resolution in only two FFPE slides.

GeoMxTM DSP technology is for Research Use Only and not for use in diagnostic procedures.

#499

NanoString®GeoMx®digital spatial profiling further defines the role of B cells in the response to immune checkpoint blockade.

Beth A. Helmink,1 Sangeetha Reddy,1 Jorge Blando,1 Yan Liang,2 Sarah Warren,2 Vancheswaran Gopalakrishnan,1 Hussein A. Tawbi,1 Rodabe N. Amaria,1 Michael Davies,1 Jeffrey E. Gershenwald,1 Elizabeth Burton,1 Rafet Basar,1 Alexander J. Lazar,1 Courtney W. Hudgens,1 Katy Rezvani,1 James P. Allison,1 Padmanee Sharma,1 Joseph M. Beechem,2 Jennifer A. Wargo,1 Michael T. Tetzlaff1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _NanoString Technologies Inc, Seattle, WA_.

Background: While treatment with immune checkpoint blockade (ICB) has markedly improved outcomes in advanced melanoma patients and other malignancies, predicting response remains a challenge. Biomarkers including tumor mutational burden (TMB), T-cell infiltration, and PD-L1 expression, have been identifiedbut remain inadequate. Other components of innate and adaptive immunity, including B-cells and tertiary lymphoid structures (TLS), have been implicated in the response to other cancer therapies, andpreclinical data suggests B cells may contribute in the response to immunotherapy. Here, we use targeted protein expression profiling-via NanoString digital spatial profiling (DSP) technology (research use only)-to demonstrate a role for B cells in the response to ICB in patients with high-risk resectable melanoma; furthermore, we characterize the B cell subsets that enable this function.

Methods: We conducted a phase 2 clinical trial of neoadjuvant ICB therapy in patients with high-risk resectable melanoma (PD-1 blockade monotherapy or combined CTLA-4/PD-1 blockade) (NCT02519322). Longitudinal tumor samples were taken during therapy. Formalin-fixed paraffin embedded tissue sections from tumor samples (n=10 responders [R], n=10 non-responders [NR]) were analyzed by NanoString DSP technology and stained with a cocktail of S100B, CD45, CD19 and a 40-protein cocktail of antibodies conjugated to UV-photocleavable DNA barcodes. Regions of interest (ROI) were delineated using immunofluorescence followed by UV excitation of the defined ROIs, releasing the DNA barcodes for downstream quantitation on the NanoString nCounter®platform. Utilizing masking strategies, we define the unique expression pattern within discrete subsets of immune cells. These same tumors have concurrently been analyzed by RNAseq, immunohistochemistry, CYTOF, and single-cell RNAseq.

Results: Biomarker counts are highly concordant across samples from the same patient's tumor. High concordance between DSP and quantitative fluorescence is seen as a validation for the DSP method. We identify B cells as components of TLS; the B cells are closely integrated with CD4 and CD8 T cells and follicular dendritic cells. There are significantly more TLS identified in R compared to NR patients. Utilizing CYTOF, we concurrently identify specific subsets of B cells present within the TLS associated with response. We characterize the spatial relationship of these same B cell subsets with other components of the TLS and define the protein expression patterns of these cells.

Conclusion: NanoString DSP data complement our deep molecular and immune profiling of tumors from melanoma patients treated with ICB; together, they provide a novel predictive role for B-cells and TLS in the response to ICB and, importantly, provide mechanistic insight into their potential contribution in the response to cancer therapy.

#500

Predicting patient response to immuno-oncology agents in vitro using 3D cultures.

Kathryn M. Appleton, Qi Guo, Ashley Elrod, Alina Lotstein, Lillia Holmes, Teresa M. DesRochers. _KIYATEC, Inc., Greenville, SC_.

Immuno-oncology (I/O) based therapeutics, including cellular therapies and checkpoint inhibitors have surged in the last 2 years, but the technology to accurately test them in a pre-clinical setting is significantly lacking. While animal models have tried to provide accurate testing platforms, the ultimate goal of a matched patient tumor and immune system is not achievable in mice. To overcome this issue, we have developed two 3D tissue systems for in vitro testing that combine a patient's tumor cells and autologous immune cells for accurate testing and prediction. We hypothesize that our 3D cell culture systems can recapitulate the patient's tumor microenvironment to detect I/O response. Our spheroid-based system allows us to monitor how primary T-cells are affected by paired tumor cells and/or the PD-1 inhibitor pembrolizumab using flow cytometry. We have successfully detected pembrolizumab binding to T-cells in a dose dependent manner, clonal expansion of lymphocyte populations, as well as increased expression of activation markers on CD3+ cells following combination with tumor cells and exposure to pembrolizumab. This model also accurately detects CD3+CD8+ T-cell mediated tumor cell death and can be used to track changes in secreted cytokines and chemokines such as Granzyme B and IFN gamma. Our second model, a 3D microtumor platform, allows us to detect immune cell migration and infiltration and therapy related cell death. Our results show pembrolizumab can increase lymphocyte infiltration while simultaneously decreasing microtumor growth in matched patient samples whose tumor cells express PD-L1 and whose lymphocytes are CD8+. Cytokine secretion detected by multiplex technology from our microtumor model supports our observed enhanced T-cell activation in the presence of pembrolizumab. The data generated from our two complex 3D in vitro models can recapitulate in vivo biology in order to derive correlations to I/O drug response. These models can be utilized for preclinical testing of new I/O agents as well as for patient response predictions to I/O therapies.

#501

Early immunological events in the periphery and the TME following a local immunostimulating instillation into the bladder in the MB49 orthotopic model.

Iliana Kerzeli, Andrea Kurtinovic, Aikaterini Nasi, Ivan Stepanek, Martin Lord, Sara M. Mangsbo. _Uppsala Univ., Uppsala, Sweden_.

The mouse bladder 49 (MB49) syngeneic tumors respond to various immunotherapies, both stimulatory immunotherapies and check-point blockers; when tumors are cured it is often an anti-tumor memory response established (previous publications by our group and others). Herein we present data suggesting that bladders of orthotopic MB49 tumor bearing mice hold a reservoir of lymphocytes surrounding the tumor, wherein they migrate rapidly upon immune adjuvant stimulation. The tumors appear to form an accessible immune microenvironment rich in vessels that mediate the homing of leukocytes to the tumor site. This is in contrast to MB49 tumors that grow subcutaneously and do not appear to provide the same rapid immune cell infiltration capacity. After 24 hours post a bladder restricted TLR9 stimulation, we could identify that CpG ODN 1668-treated tumor-bearing bladders displayed increased vascularization around the tumor. Interestingly it appeared as CCL21 expression intratumorally aligned with increased recruitment of CD11c+ DCs in the tumor TME, rather than a migration to lymph nodes. Intratumorally, CCL21 expression correlated with infiltration of both CD11C and CD8 cells. As CD11c+ dendritic cells migrate towards gradients of CCL21 via their CCR7 receptor, enabling antigen presentation, it is plausible that antigen-presentation takes place in the TME in relation to this finding. Currently we are investigating multiplex analyses on bladder tumors, urine and blood from tumor bearing animals to identify cytokines from the TME or systemically, that correlate to the formation of an adaptive immune response. For the liquid biopsy of the urine and serum we have performed a multiplex proteomic analysis using the proximity extension assay (PEA), an assay that greatly reduces the risk for cross-reactivity in the multiplex format. In a pilot analysis with PEA we noted that a single ortothopic instillation of CpG 1668 ODNs led to decreased TGFa and increased CCL3 in serum. This may reflect a change in the TME and a recruitment of cells into the tumor from the periphery, as a response to the pharmacological intervention. By this preliminary data we conclude that in the orthotopic MB49 tumor model, CCL21 appears to play a role in the induction of adaptive immune responses through the CCL21/CCR7 axis and that a single administration of locally acting TLR9 agonist into the bladder, can change the levels of systemic biomarkers. We will now further elucidate the early and late immunological events that lead to an effective immune response in this murine bladder cancer model.

### Metabolic Mechanisms That Modulate the Immune Response to Cancer

#502

Aldo-keto reductase 1B8 (AKR1B8) deficiency causes intestinal immune deficiency.

Xin Wang, Deliang Cao. _Southern Illinois University School of Medicine, Springfield, IL_.

Background: Intestinal epithelial cells (IECs) are crucial mediators of intestinal immunity. IECs defects may disrupt development and maturation of intestinal immunity. Aldo-keto reductase 1B10 (AKR1B10) is a regulator of de novo fatty acids synthesis and lipid synthesis by mediating acetyl-CoA carboxylase-α (ACCA) stability. Aldo-keto reductase 1B8 (AKR1B8) is an ortholog in mice of human AKR1B10, which has similar regulatory function in fatty acid/lipid synthesis. AKR1B8 deficiency causes IECs defects in proliferation and self-renewal, which may lead to dysregulation of intestinal immunity.

Methods: AKR1B8 -/- mice mouse stain was produced by homozygous recombination. Immune cells were isolated from colonic lamina propria (cLP) and mesenteric lymph nodes (MLN). Flow cytometry and immunofluorescence staining were applied for immune cell sorting and measurements. Microbiota composition were quantified by next generation sequencing.

Results: AKR1B8 deficiency causes infiltration of neutrophils, mast cells and basophils in the colon. Also, AKR1B8 deficiency causes impaired γδT cells and T helper cells (Th cells) immunity, such as IFNγ, IL17, IL4 or IL22 production, in colon and mesenteric lymph nodes, while enhances distinct arm of CD8 activity and Treg cells. These alterations may be due to the changes of MHCII\+ DCs and CD103\+ DCs in AKR1B8 deficient mice. Meanwhile, AKR1B8 inhibits the phosphorylation of p-AKT (T308 and S473) and p-ERK (1/2) in IECs, and suppresses the regulation IKKα/NF-κB signaling cascades. Furthermore, AKR1B8 deficiency induces gut microbiota shifts with higher ratio of Firmicutes/Bacteroidetes.

Conclusions: AKR1B8 is a critical mediators for IECs signaling and immune homeostasis. Loss of AKR1B8 in IECs induces Th cells immune deficiency. This study identified a new compensatory mechanism under IECs defects for maintaining intestinal immune homeostasis.

#503

Prostaglandin E2 produced by tumor cells or by the host tumor microenvironment is not completely abolished by aspirin or celecoxib and limits the ability of the host immune system to control tumor growth.

John N. Snouwaert,1 Leigh Jania,1 MyTrang Nguyen,1 Pragnya Dontu,1 Jérôme Besse,2 Barbara Akla,2 Pierre J. Ferré,2 Beverly H. Koller1. 1 _University of North Carolina at Chapel Hill, Chapel Hill, NC;_ 2 _Inst. de Recherche Pierre Fabre, Toulouse Cedex 01, France_.

Prostaglandins are lipid autacoids derived from arachidonic acid (AA). AA released from the cell membrane is metabolized by the sequential actions of prostaglandin G/H synthase or cyclooxygenase (COX) and respective synthases, such as mPGES-1, which forms the most abundant prostaglandin, PGE2. Prostaglandin biosynthesis is blocked by nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit the activity of either one or both cyclooxygenases. Besides their role in homeostasis, prostanoids can modulate immune responses and contribute to chronic inflammation associated with tumor initiation, vascularization, and growth. The prostanoid pathway is activated in many solid tumors, including breast cancer, and high levels of PGE2 have been associated with aggressive tumors and metastatic spread. The objective of this work was to define the pathways and cellular compartments that contribute to tumor PGE2 levels and their role in tumor growth and immune evasion. The anti-tumor impact of classical NSAIDs combined with immune checkpoint antibodies PD1 or CTLA4 (ICPAb) was also evaluated. 4T1 or CT26 murine tumors were grown in immune competent mice to confirm the presence of PGE2 in tumors. Mice were dosed with NSAIDs Aspirin or Celecoxib monotherapy, given at maximal tolerated dose, or combined with ICPAb. While ICPAb reduced tumor growth, nor Aspirin neither celecoxib was effective in monotherapy or combination with ICPAb. Interestingly, PGE2 concentration in tumors was reduced by NSAIDs but not abolished. To examine the production of PGE2 by tumors or host, we have used CRISPR/cas9 mutagenesis to generate tumor lines lacking various enzymes required for PGE2 production. Those lines were combined with mice carrying null mPGES1 alleles. We show that, while the host tumor microenvironment contributes to overall PGE2 levels in the tumor, large amounts of PGE2 are produced by the tumor epithelial cells. Despite high expression of Ptgs1 in these tumor lines, PGE2 production was dependent on COX-2 expression. Similarly, while 4T1 tumors express all three PGE2 synthases at relatively high levels, we found that mPGES1 was solely responsible for all PGE2 released from epithelial cells. Examining several independent Ptgs1/2 null 4T1 tumor lines, we show that loss of Ptgs1/2 results in decreased tumor growth in immune competent mice but not in an allogenic Rag1/2-/- line. Knowledge of tumor PGE2 metabolic and functional pathways is a critical first step in designing the most appropriate therapeutic interventions. Classical NSAIDs given as monotherapy or combined in mice with ICPAb are ineffective against tumors, possibly because they do not abolish PGE2 production, suggesting that better drugs against PGE2 pathway would be required to counter the immune suppression induced by PGE2.

#504

Acarbose enhances intratumoral CD8 T cell responses in a pre-clinical model of kidney cancer.

Rachael M. Orlandella, Daniel L. Smith, Lyse A. Norian. _University of Alabama at Birmingham, Birmingham, AL_.

Metastatic renal cell carcinoma (RCC) exhibits high mortality rates and chemotherapeutic resistance. Treatment options include immune-stimulatory therapies, such as high-dose IL-2 and anti-PD-1; however, these agents only generate responses in 20-50% of patients. This illustrates the need to find ways to improve patient response rates. Long-term caloric restriction (CR) extends longevity and reduces cancer risk in animal studies. Caloric restriction mimetics (CRMs), which replicate the benefits of CR without reductions in calorie intake, were recently found to enhance chemotherapeutic efficacy by improving CD8 T cell mediated anti-tumor immunity. Here we examined acarbose, an FDA-approved anti-diabetic agent with CRM properties, in a preclinical model of kidney cancer. BALB/c mice were orthotopically tumor challenged with syngeneic luciferase-expressing Renca renal carcinoma cells, which spontaneously metastasize to the lungs. Mice were then randomized to either an acarbose-supplemented or control diet. Compared to mice on control diet, acarbose did not alter renal tumor progression at Day 28. Importantly, acarbose did not exacerbate weight loss associated with renal tumor growth. Flow cytometric analysis at Day 28 revealed increased frequencies of activated (CD44+CD62L-) intra-tumoral CD8 T cells in mice on acarbose (p=0.012). Additionally, renal tumors from mice on acarbose displayed increased frequencies of early effector (CD127-KLRG1-) CD8 T cells, in addition to heightened frequencies of PD-1+ CD8s. These findings suggest that acarbose exhibits immune-modulatory properties which could promote anti-tumor immunity. Future experiments will focus on elucidating mechanisms contributing to alterations in anti-tumor immunity. Importantly, these studies warrant investigating acarbose in combination with currently available treatments as a potentially novel therapeutic adjuvant.

#505

Adipocyte-secreted proteins induce DNA damage and apoptosis in T-cell acute lymphoblastic leukemia.

Miyoung Lee, Curtis J. Henry. _Emory University, Atlanta, GA_.

T-cell acute lymphoblastic leukemia (T-ALL) accounts for 10-15% of pediatric and 20-25% of adult cases of ALL. The cure rates for patients with T-ALL is approaching 80% in children while falling to 60% in adult cases. These numbers further decline in patients with primary resistance or who relapse from their initial diseases. Given the poor outcomes for patients with T-ALL, there has been growing interest in identifying risk factors that contribute to the pathogenesis of this disease. Recently, obesity (characterized by a body mass index > 30) has been shown to reduce the survival of patients with ALL. However, a closer examination of the epidemiological data revealed that obese patients with T-ALL exhibit significant improvements in survival outcomes. These data prompt several questions including; 1) what are the specific factors in the obese microenvironment that impact the function of T-ALL, 2) how do T-leukemia cells respond to these factors, and 3) are the responses influenced by mutations commonly found in T-ALL. To address these questions, we have studied how stromal cell and adipocyte-secreted factors impact the function of human T-ALL cells. Furthermore, we sought to determine if Notch1, which is mutated in > 60% of T-ALL cases, contribute to how T-leukemia cells respond to soluble factors in the microenvironment. When human T-ALL cells were grown in vitro with RPMI, stromal cell-conditioned media (SCM), or adipocyte-conditioned media (ACM), only ACM exposure resulted in extensive cell death of T-ALL (30-80% after 3 days of culture) which occurred independently of the mutational status of Notch1 and p53. The ACM secreted factor(s) that induced cell death in T-leukemia cells was revealed to be proteinaceous in nature, given that protease treatment of ACM completely abolished ACM-mediated apoptosis in T-ALL. Cell cycle analysis of human T-ALL exposed to ACM revealed an accumulation in the sub G1 population in human T-ALL, confirming the induction of apoptosis induced by the adipocyte secretome. Although the mutation status of Notch did not impact cell death, Notch1 protein levels significantly decreased in T-leukemia cells after 3 days of culture in ACM which coincided with increased γ-H2AX activation (indicative of DNA damage). This observation was confirmed using immunofluorescent microscopy, which revealed co-localization of γ-H2AX and 53BP1 (a p53 binding protein) in ACM-exposed human T-leukemia cells. Furthermore, extensive nuclear membrane blebbing was observed in human T-ALL exposed to ACM, but not SCM or unconditioned-media. Our studies reveal that adipocyte-secreted proteins are cytotoxic to human T-leukemia cells, which is independent of the mutational status of Notch1 and p53. Given this global effect, our results suggest that the obese microenvironments presents a unique opportunity to develop novel therapeutics for treating standard and high-risk patients with T-ALL.

#506

Wnt/â-Catenin pathway inhibitor RXC004 enhances the immunity of pre-clinical models of cancer.

Caroline Phillips, Inder Bhamra, Catherine Eagle, Alicia Edmenson Cook, Cliff Jones, Simon Woodcock. _Redx Pharma, Macclesfield, United Kingdom_.

Background: RXC004, a potent and selective inhibitor of the Wnt/β-Catenin pathway regulator porcupine, is being investigated in a safety and tolerability study in cancer patients with solid tumors (NCT03447470). We present pre-clinical data confirming the potential for RXC004 in modulating the immune system of the tumor microenvironment.

Materials and Methods: To evaluate RXC004 as an immunomodulatory anti-cancer agent we took two complementary approaches; first utilizing a syngeneic B16F10 melanoma model in C57BL/6 mice. A range of doses and schedules of RXC004 were tested for 28 days and efficacy was measured by tumor volume. To further probe the mechanism of immune modulation by RXC004 in syngeneic models, global gene expression in the tumor microenvironment (TME) was analyzed using the Nanostring IO 360 gene set, and immune cell populations in the blood and spleen were analyzed by flow cytometry. In the second approach, modulation of a range of immune-relevant biomarkers by RXC004 was assessed in a panel of Wnt ligand-dependent human cancer cell lines using flow cytometry and gene expression.

Results: In the syngeneic B16F10 melanoma model, RXC004 treatment significantly reduced tumor volume in a dose-dependent manner at 0.5, 1.5 and 5 mg/kg QD. In addition, 5mg/kg QD of RXC004 scheduled 5 days on, 2 days off also gave significant efficacy. RXC004 treatment in this model is known to decrease myeloid-derived-suppressor-cells (MDSCs) within the TME. Consistent with this, RXC004 treatment (5mg/kg QD) increased the proportion of MDSCs circulating in blood. MDSCs are therefore either retained in the TME through Wnt pathway signaling, or Wnt pathway signaling promotes chemotaxis of these cells to the TME. Analysis of a panel of immune genes using Nanostring IO 360 demonstrated RXC004 treatment (5mg/kg QD) increased expression of chemokine and cytokine signaling components in the TME, such as the T cell chemoattractant CXCL9. CXCL9 is reported to be produced by CD103+ve dendritic cells in the mouse TME and absence of these dendritic cells is linked to a lack of T cell infiltrate (Spranger et al., 2017). In a panel of Wnt ligand-dependent human cancer cells, RXC004 treatment regulated levels of markers associated with tumors evading the immune system e.g. MYC expression levels were reduced significantly. Modulation of such markers in paired clinical sample may indicate an immune-modulatory response to RXC004.

Conclusion: Taken together, Wnt pathway suppression by RXC004 treatment can enhance the immune response against tumors by 1) directly regulating levels of immune-relevant markers on cancer cells, and 2) enhancing the immunity of the TME by decreasing MDSCs and increasing chemokine and cytokine signaling.

Spranger et al Cancer Cell 2017 8 31(5):711-723

#507

**Identification of CD73 and A** 2A **R/CD73 small molecule inhibitors for cancer immunotherapy as single agents or in combination with Immune-checkpoint therapies.**

Pierre Fons,1 Andrew Bell,2 Michael Esquerre,1 Stephanie Versluys,1 Florie Bertrand,1 Adrian Schreyer,2 Iva Hopkins-Navratilova,2 Leonardo-Silvestre Hernani,3 Celia Bergeaud,1 Celine Poussereau-Pomie,1 Ghislaine Marchand,1 Sean Robinson,2 Simone Culurgioni,2 Richard Cox,2 Jeremy Besnard,2 Andrew Payne,2 Peter Ray,2 Emilie Pelissier,1 Michael Paillasse,1 Joanna Lisztwan,1 Craig Johnstone,1 Mark Whittaker,3 Andrew Hopkins2. 1 _Evotec, Toulouse, France;_ 2 _Exscientia, Oxford, United Kingdom;_ 3 _Evotec, Abingdon, United Kingdom_.

Adenosine generated by CD73 is a key driver of immunosuppression in the hypoxic tumour microenvironment (TME) and particularly involved in angiogenic process and immunity. In immune-inflamed tumours, with CD8+ T-cell infiltrates, adenosine signalling is a cause of resistance to immune checkpoint therapies (ICTs) through the inhibition of T-cells, NK cells and more largely of the overall antitumor immunity. Evotec and Exscientia are collaborating to develop an innovative drug discovery platform for accelerating small molecule development in Immuno-Oncology targeting the adenosine pathway. The platform has integrated a unique biophysical screening approach for the adenosine receptors and CD73 to drive automated medicinal chemistry design with a translational-focused screening cascade. We have initiated research of CD73 specific inhibitory molecules based on SPR fragment screening and have identified lead compounds which fully bind in the active site of the CD73 protein in both the open and closed states. In vitro functional potency has been demonstrated for the CD73 inhibitors for inhibiting adenosine production by Rapid Fire technology performed using CD73 recombinant protein. Moreover, we demonstrated that CD73 inhibitors induce recovery of AMP-induced inhibition of T-cells activation as measured through IL-2 production. The series are under lead optimization to improve ADME/DMPK parameters in order to enable evaluation in PK/PD assays and in vivo efficacy studies specifically developed for the project. Finally, from the perspective of developing a dual pharmacological profile for A2AR and CD73 inhibition, we have assessed, using primary human CD3+ T-lymphocytes isolated from healthy donors, the synergy between the inhibition of the two targets in order to enhance the recovery of T-cell activation. We have demonstrated that EVOEXS21546, our pre-development A2AR inhibitor candidate, and our lead compound for CD73 inhibition act in synergy in functional human T-cell assays. Our SPR screening approach has also identified hit compounds with dual bispecific pharmacological activity against both A2AR and CD73.

#508

Combined MEK and PD-1 inhibition in acute myeloid leukemia: Effect on cell proliferation and phenotype.

Kaycee B. Moshofsky, Hyun J. Cho, Yoko Kosaka, Matthew T. Newman, Jeffrey W. Tyner, Evan F. Lind. _Oregon Health & Science University, Portland, OR_.

Although most patients with acute myeloid leukemia (AML) respond to standard of care chemotherapy, the majority of patients will relapse, and the 5-year overall survival rate persists at 25%. The high rates of relapse and poor survival have contributed to increased interest in the use of other therapies, including immunotherapies, in hematological malignancies in order to achieve more durable responses. In particular, immune checkpoint blockade, such as anti-PD-1, has elicited efficacious responses in Hodgkin's lymphoma. In addition, trametinib, a small molecule MEK inhibitor, was shown to have activity in AML patients with a RAS-mutation in a phase 1/2 trial, but demonstrated the need for rational combinational therapies to produce longer lasting responses. Interestingly, in a murine model of colon carcinoma harboring a KRAS mutation, the combination of these two agents resulted in a more durable tumor response compared to either agent alone. We hypothesized that in AML, combined MEK and PD-1 inhibition would result in direct inhibition of blast proliferation while simultaneously promoting proliferation and activation of T cells with a mature/memory phenotype.

Bone marrow and peripheral blood samples isolated from patients with AML were cultured in our laboratory for 5 days with CD3 stimulation to induce T-cell activation. Cultures were treated with trametinib, anti-PD-1, or both. Additional samples were cultured with interferon-gamma (IFN-γ) to induce PD-L1 expression. Mouse spleen cells from a genetically engineered mouse model of AML (FLT3-ITD/TET2+/-) were cultured under the same conditions for 3 days. Samples were evaluated by flow cytometry to measure treatment effects on T-cell and blast proliferation and phenotype.

In the patient samples, trametinib consistently reduced PD-L1 expression in a dose-dependent manner on the myeloid cell population and was further able to attenuate PD-L1 expression induced by IFN-γ. A mean decrease of 36.6% was seen in PD-L1 expression after treatment with trametinib at 100 nm (p=0.0177). In most samples, trametinib decreased blast proliferation and increased blast cell death. Additionally, in a NRAS-mutant sample with high PD-L1 expression, the combination therapy best restored T cell proliferation and decreased the naive T cell population resulting in increased effector and memory T cells. We observed similar changes in our mouse model of AML.

In conclusion, IFN-γ increased PD-L1 expression on cancer cells suggesting a possible mechanism of immunosuppression inhibiting autologous immune responses, and the increased PD-L1 expression could be at least partially reversed by trametinib. Our study suggests that combined MEK and PD-1 inhibition may result in greater therapeutic efficacy over each agent alone and provide a rationale for administering this combination to patients with AML.

#509

Acarbose, but not metformin, reduces tumor burden and improves intra-tumoral immune responses in a pre-clinical breast cancer model.

William J. Turbitt, Rachael M. Orlandella, Justin T. Gibson, Lyse A. Norian. _University of Alabama at Birmingham, Birmingham, AL_.

Finding ways to enhance T cell infiltration into tumors and boost effector function remains a critical knowledge gap that must be addressed to improve responses to immune-stimulatory therapies. Acarbose and metformin are FDA-approved drugs for the treatment of type two diabetes that display potent anti-hyperglycemic activity. A treatment strategy that decreases intra-tumoral glucose could reduce cancer cell viability and render tumors more susceptible to immunotherapy-mediated clearance. The goal of the current study was to determine if acarbose and/or metformin reduce tumor burdens and promote anti-tumor immunity in a preclinical model of breast cancer. Eight-week-old female C57BL/6 mice were orthotopically injected with EO771-Fluc mammary tumor cells then randomized to control (CTRL), CTRL+acarbose (ACA), CTRL+metformin (MET), or acarbose+metformin (ACA+MET) for four weeks. Compared to CTRL, single-agent ACA reduced primary tumor growth (50% decrease; p<0.001) and spontaneous lung metastases (48% decrease; p=0.035) without altering body weight. No effects of single-agent MET or the combination of ACA+MET on EO771 tumor growth or spontaneous lung metastases were observed. nanoString analysis of whole-tumors revealed increased dendritic cell-, T cell-, T helper 1 cell-, CD8+ T cell-, and exhausted CD8+ T cell-based gene signature scores in ACA compared to CTRL-fed mice. Cellular analysis revealed an ACA-induced increase in the percent of tumor-infiltrating PD-1+CD8+ T cells (p=0.042) and a trend toward decreased tumor-infiltrating myeloid-derived suppressor cells, resulting in improved CD8:MDSC ratios. The beneficial effects of ACA on intra-tumoral immunity, specifically promoting T cell immunity within the tumor microenvironment, illustrate the potential for future combined use of ACA with immunotherapies, such as PD-1 or CTLA-4 blockade.

#510

Selective and potent CDK8 inhibitors enhance NK cell activity and promote tumor surveillance.

Marco H. Hofmann,1 Rajeswaran Mani,2 Harald Engelhardt,1 Maria A. Impagnatiello,1 Sebastian Carotta,1 Marc A. Kerenyi,1 Seila Lorenzo-Herrero,3 Jark Boettcher,1 Dirk Scharn,1 Heribert Arnhof,1 Andreas Zoephel,1 Renate Schnitzer,1 Thomas Gerstberger,1 Girish Rajgolikar,2 Swagata Goswami,2 Sumithira Vasu,2 Peter Ettmayer,1 Segundo R. Gonzalez,3 Mark Pearson,1 Darryl B. McConnell,1 Norbert Kraut,1 Natarajan Muthusamy,2 Juergen Moll1. 1 _Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria; _2 _The Ohio State University Comprehensive Cancer Center, Columbus, OH;_ 3 _Universidad de Oviedo, Instituto de Investigación Biosanitaria del Principado de Asturias (IISPA), IUOPA, Oviedo, Spain_.

Natural killer (NK) cells play a pivotal role in controlling cancer. Activation of NK cells is tightly regulated by multiple extracellular receptors and internal signaling nodes. The production of perforin and granzyme B, key NK cytolytic molecules, is regulated by the suppressive signaling intermediate Cyclin Dependent Kinase 8 (CDK8) through phosphorylation and inhibition of STAT1S727.

We identified highly potent and selective CDK8 inhibitors, including BI-9811 and BI-1347, that we previously reported as valuable probe compounds (Hofmann M.H. et al., AACR Annual Meeting 2017, Abstract 4630). CDK8 inhibition promoted activation of NK cells but had no direct cytotoxic activity on the majority of cancer cell lines tested. Single agent treatment with the CDK8 inhibitor BI-1347 increased the survival of mice bearing melanoma and breast cancer allografts. In addition to this, treatment with BI-1347 resulted in enhanced NK-cell mediated lysis of myeloid leukaemia and lymphoma cells.

We evaluated whether increased efficacy could be observed upon activation of both the innate and the adaptive immune system by combining the CDK8 inhibitor with a SMAC mimetic. SMAC mimetic treatment enhanced T-cell activity and increased NK cell number in the murine EMT6 breast cancer model. Increased survival upon combination treatment was dependent on a pulsatile schedule of the CDK8 inhibitor BI-1347, which permitted activation of NK cells but avoided a hypo-responsive steady state. These results demonstrate that CDK8 inhibitors could be used either as a single agent in hematological cancers or be combined with modulators of the adaptive immune system to inhibit the growth of solid tumors.

#511

**An assessment of pre-conditioning regimens for optimal hCD34+ hematopoietic stem cell humanization of NSG** TM **mice.**

Caitlin S. Parello, Benjamin G. Cuiffo, Alexandria Kury, Kasey Reardon, Brett Van Dam, Sallyann Vu, Catarina Costa, Veronica Ritchie, Gavin Gagnon, Timothy Bateson, Samantha Rogers, Gregory D. Lyng, Stephen T. Sonis. _Biomodels, Watertown, MA_.

Humanized immune system (HIS) mice - immunocompromised Nod.Cg-Prkdcscid Il2rgtm1Wjl/SzJ animals in which the immune system has been humanized by the engraftment of CD34+ hematopoietic stem cells (HSC), peripheral blood mononuclear cells (PBMC), or other human cells/tissues - represent an important tool in pre-clinical oncology research. Here we investigated several pre-conditioning regimens (Busulfan pre-treatment or total body irradiation (TBI)) to determine which would yield maximum human leukocyte engraftment and lineage diversity. Animals were pre-treated either one or two doses of Busulfan (25 mg/kg) or varying doses of TBI (1.75, 2.0, or 2.5 Gy). One to two days later, animals received an adoptive transfer of native hCD34+ HSCs. Engraftment was monitored by flow cytometry assessment of peripheral blood four weeks after adoptive-transfer, and every four weeks thereafter through experiment termination at Week 16. Upon termination, spleen and bone-marrow were assessed for various leukocyte populations by flow cytometry. Animals undergoing pre-conditioning displayed increased human CD45+ cell engraftment at all timepoints as compared to animals without pre-conditioning, with animals treated with two doses of Busulfan demonstrating the greatest human CD45+ engraftment at all timepoints and reaching statistical significance at endpoint (vs no pre-conditioning; p<0.05). Diversity of leukocyte cell types (B cells, T cells, NK cells, and other hCD45+) in the bone-marrow was similar across pre-treatment paradigms, however for some lineages, representation in the bone-marrow was significantly altered between pre-conditioning type. The absolute number of T or B cells were increased in TBI and Busulfan treated groups, respectively, whereas NK cells as a percentage of singlets were increased in TBI treated groups. The diversity of leukocyte cell types in the spleen was likewise similar across groups, with B cells dominating. These data provide important insights into the utility of various pre-conditioning regimens for the humanization of NSG mice for pre-clinical oncology research, and demonstrate that the optimum pre-conditioning regimens should be selected based on treatment target.

#512

Adrenergic modulation of inflammatory cytokines in ovarian cancer.

Claudia B. Colon-Echevarria, Tatiana Ortiz, Lizette Maldonado-Laboy, Melanie J. Hidalgo-Vargas, Alexandra N. Aquino, Guillermo N. Armaiz-Pena. _Ponce Health Sciences University, Ponce, PR_.

Work from our group and others suggest that chronic stress accelerates growth of existing tumors by activating the sympathetic nervous system. Specifically, our data suggest that sustained adrenergic signaling can induce tumor growth, secretion of pro-inflammatory cytokines and macrophage infiltration. Moreover, increased macrophage infiltration was associated with decreased survival in ovarian cancer patients. Hence, we investigated role of adrenergic-stimulated macrophages in ovarian cancer biology. To assess the effect of adrenergic stimulation in pro-tumoral functional outputs, we used cytokine arrays and invasion assays. A pre-clinical orthotopic model of ovarian cancer was used to assess the in vivo effect of daily restraint stress on tumor growth and adrenergic-induced macrophages. Cytokine array analyses showed that adrenergic stimulation modulated pro-inflammatory cytokine secretion in a SKOV3ip1 (ovarian cancer cells) /U937 (macrophages) co-culture system. Among these, 23 significantly upregulated cytokines were identified in both, epinephrine and norepinephrine, treated co-culture systems. PDGF-AA, ENA-78, Angiogenin, VEGF, GM-CSF, IL-5, Lipocalin-2, MIF, and TfR were among upregulated cytokines shared between treatment groups. In addition, daily restraint stress resulted in increased ovarian cancer growth and infiltration of CD68+ macrophages in two orthotopic models of ovarian cancer (SKOV3ip1 and HeyA8), while zoledronic acid (a bisphosphonate that has been shown to impair macrophage activity) abrogated this effect. Immunohistochemistry analyses showed restraint stress increased expression of PDGF-AA and ENA-78 inflammatory cytokines. Here, we show that adrenergic regulation of macrophages might play a key role in the progression of ovarian cancer.

#513

Investigating the expression of indolamine deoxygenase I (IDO1) and other immune markers in tissue microarrays.

Ana Cerezo,1 Gloria Martinez-del Hoyo,1 Sandra Peregrina,1 Tamara Mondejar,1 Eduardo Caleiras,1 Patricia Gonzalez,1 Eva Lospitao,1 Sonia Hernandez-Tiedra,1 Laura Diezma,1 Estela Casas,1 Frank Dorsey,2 Karim Benhadji,2 Raymond Gilmour,2 Sandaruwan Geeganage,1 Susana Velasco1. 1 _Centro Nacional de Investigaciones Oncologicas CNIO, Madrid, Spain;_ 2 _Lilly Research Laboratories, Indianapolis, IN_.

Purpose of the study: To gain insight to the proportion of solid tumors that express IDO1, CD73 and PD-L1 in lung cancer, colon adenocarcinoma, renal cell carcinoma (RCC) and melanoma, and the correlation between expression of these immune checkpoints and immune cell infiltration within the tumor.

Experimental procedures: Using a battery of properly validated antibodies, we have performed an in situ analysis of IDO1, CD73 and PD-L1 expression by immunohistochemistry in a panel of colon cancer (n=68), lung cancer (n=36), RCC (n=67) and melanoma (n=48) tissue microarray samples. Tumor infiltrating T cells, CD3+ and CD8+, were also detected by IHC and counted in serial sections for each sample. We search for a possible a correlation between the expression of these immunosuppressor proteins and the number of infiltrating CD3+ and CD8+ T cells as well as their expression in the different tumor components: tumor cells, stroma and endothelium.

Results: Our IHC analysis allowed us to classify the analyzed samples into two main groups: those with expression of IDO1, CD73 and PD-L1, which were also the ones with a high presence of immune cells within the tumors, "hot tumors", and those that showed to be negative for all three proteins, and that correlated with lower infiltration of T cells, "cold tumors". We also analyzed the location of the proteins within the tumors, and found an effect between endothelial expression of Ido1 and reduced infiltration in lung and RCC tumors.

Conclusions: The response to combined immune checkpoint and IDO1 therapy could depend on the proper stratification of patients; therefore, it is of significant interest to determine the proportion of solid tumors that express IDO1, CD73 and PD-L1, and the correlation of the expression of these three immune check points to immune cell infiltration within the tumor. Thus, proper patient stratification based on the expression of these immune checkpoints may improve the efficacy of IDO1 inhibitor based immune therapies.

#514

**DUOX2-related H** 2 **O** 2 **production contributes to pro-inflammatory cytokine-related DNA damage and tumor cell growth in models of pancreatic ductal adenocarcinoma.**

Yongzhong Wu, Jiamo Lu, Smitha Antony, Jennifer L. Meitzler, Agnes Juhasz, Guojian Jiang, Iris Dahan, James H. Doroshow. _National Cancer Inst., Bethesda, MD_.

Chronic inflammation is an important risk factor for the development of pancreatic ductal adenocarcinoma (PDAC). Dual oxidase 2 (DUOX2), one of the seven NADPH oxidase (NOX) family members, by generating extracellular H2O2, is intimately involved in both host defense and chronic inflammation in the gastrointestinal tract. Previously, we demonstrated that short (24 h) exposures of several human pancreatic cancer cell lines to pro-inflammatory cytokines (IFN-γ, IL-4/IL-13, IL-17A) can effectively enhance expression of DUOX2 and its maturation factor DUOXA2. To better mimic chronic inflammation in vivo, a panel of human PDAC cell lines was exposed to the TH2 cytokine IL-4 (50ng/ml) for longer times (7 days); and the effect of cytokine treatment on Stat6 activation and DUOX2 mRNA and protein expression was explored. In BxPC-3 and AsPC-1 cell lines, prolonged IL-4 stimulation results in a sustained activation of Stat6, dramatic induction of DUOX2 mRNA and protein expression, a 3-5-fold increase in extracellular H2O2 production compared to solvent-treated cells, and--most importantly--a robust DNA double strand break (DSB) response indicated by elevated levels of γH2AX. In marked contrast, for two JAK-Stat6 signaling defective PDAC lines, PANC-1 and CFPAC-1, no enhancement of DUOX2 expression with concomitant DSBs was observed under the same conditions. Moreover, when the anti-inflammatory drug dexamethasone (Dex) was co-administered with IL-4 (for 48h), cytokine-related DUOX2 induction and DNA damage in BxPC-3 and AsPC-1 cells was significantly diminished. Furthermore, in the KPC GEMM of PDAC, RT-PCR and immunohistochemistry revealed that DUOX2 mRNA and protein expression in pancreatic tumor tissues were significantly increased compared to adjacent normal tissues and were associated with areas of chronic inflammation. Finally, xenografts established with BxPC-3 cell clones in which DUOX2 mRNA was silenced by stable expression of DUOX2-specific shRNAs demonstrated severely delayed growth in vivo compared to clones stably expressing scrambled shRNAs. These studies, coupled with our previous data demonstrating elevated DUOX protein expression in pancreatitis, pancreatic intra-epithelial neoplasia (PanIN), and frank pancreatic cancers in humans, suggest that ROS- derived from cytokine-mediated DUOX2 up-regulation may initiate and promote pancreatic cancer progression through ROS-induced DNA damage.

#515

Progesterone receptor inhibition of efficient type I interferon signaling in breast cancer.

Katherine Rose Walter, Merit Goodman, Christy Hagan. _University of Kansas Medical Center, Kansas City, MO_.

A vast majority (65-70%) of breast cancers express the progesterone receptor (PR), a nuclear receptor that is activated in response to its ligand progesterone. Compelling clinical trial data have suggested that progestins have a role in breast cancer development, independent of the widely studied estrogen receptor. The mechanism by which this occurs, however, is vastly understudied.

Traditionally, PR exerts its effects following ligand activation by translocating to the nucleus and binding to DNA where it affects the transcription of a variety of genes involved in growth, survival, and proliferation. Our lab has identified a novel subset of genes that have altered expression following PR activation. Interferon Stimulated Genes (ISGs)--primarily activated in response to interferon signaling (viral response pathway) --are repressed when PR is activated by its ligand.

In attempting to elucidate a mechanism by which PR exerts this effect, we have investigated crosstalk of PR with canonical interferon signaling proteins and have found that PR interacts with these proteins and significantly reduces their ability to become activated. STAT1, a crucial protein involved in the interferon signaling cascade, is unable to efficiently be phosphorylated when PR is activated with ligand. ChIP experiments have also shown that PR inhibits the machinery necessary for ISG transcription from efficiently binding ISG promoters, possibly explaining the reduction in ISG expression observed following PR activation. Because of the importance of this particular signaling cascade in normal cell biology, multiple mechanisms most likely exist to fully inhibit interferon signaling and experiments are currently underway to identify other possibilities to better define PR's functionality in this context.

Evasion of the immune system is considered a Hallmark of Cancer and our preliminary data suggest a potential mechanism by which breast cancer is able to accomplish this. Activation of type I interferon signaling is an early step in marking tumors for immune clearance. By repressing ISG protein expression, it is possible that these tumors are able to avoid detection by host immune cells leading to tumor establishment and subsequent progression. This novel role of PR and progestins offers insight that could aid in improving upon established estrogen only-based therapies for prevention and treatment of hormone dependent breast cancers.

#516

Metformin inhibits monocyte differentiation to tumor associated macrophage (TAM) and may restore anti-tumor immunity.

Akira Saito, Hideyuki Ohzawa, Mineyuki Tojo, Yuko Kumagai, Rihito Kanamaru, Hidenori Tsukui, Satomi Shiba, Homare Ito, Naohiro Sata, Joji Kitayama. _Jichi Medical University, Shimotsuke-shi, Japan_.

Background: Tumor-associated macrophages (TAMs) play pivotal roles to regulate tumor cell behavior in tumor microenvironment and a high density of TAMs is associated with poor prognosis in various cancers. Recent studies have demonstrated that metformin, the first-line medication for the treatment of type 2 diabetes, have suppressive effects on tumor growth. Here, in this study, we investigated the effect of metformin on monocyte differentiation to TAMs.

Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers and CD14+ peripheral blood monocytes(PBMo) were purified by positive selection using the magnetic cell separation system. The PBMo were co-cultured with Panc-1, a human pancreas cancer cell, with metformin (10μΜ~10mΜ) using double chamber inserts of 0.4-μm sized pores at 37℃ in 5% CO2 conditions for 5 days. Separately, PBMo were cultured with M-CSF for 5 days and then with IL-10 (20ng/ml)+TGF-β(20ng/ml) for addition 2 days with or without metformin. The differentiation to TAM were evaluated by the expression of M1/M2 markers as well as PD-L1/L2 by flowcytometry. The macrophages were co-cultured with autologous T cells stimulated with anti-CD3 and anti-CD3 and anti-CD28 microbeads and the effect on T cell proliferation was evaluated with CFSE dilution assay.

Results: PBMo co-cultured with Panc-1 showed significantly enhanced expression of CD163, CD206, as well as markedly increased side scatter, suggesting the differentiation to M2-type macrophages. The macrophage also highly expressed PD-L1 and strongly inhibited the proliferation of autologous T cells. However, the expression of these antigens and inhibition of T cell growth were significantly suppressed by the presence of metformin with dose dependent manner. Metformin was effective at 100μM and almost nullified the effects of Panc-1 at 1mM. Metformin did not affect the antigen expression pattern during the treatment by M-CSF, but did downregulate the expression of CD206 and PD-L1 if added together with IL-10 +TGF-β.

Conclusion: During the co-culture with tumor cells, metformin suppresses the monocyte differentiation to TAM at relatively late stage. Metformin can restore T cell mediated-tumor immunity in tumor microenvironment, at least partly, through the effects on TAM, that may result in tumor inhibition.

#517

Supraphysiological androgens activate innate immune signaling in prostate cancer.

Janet Mendonca, Olutosin Owoyemi, Marc Rosen, Michael Carducci, Mark Markowski, Emmanuel Antonarakis, Drew Pardoll, Samuel Denmeade, Sushant K. Kachhap. _Johns Hopkins Sidney Kimmel Comp. Cancer Ctr., Baltimore, MD_.

Castration resistance prostate cancer (CRPC) first manifests as a sustained rise in the androgen-responsive gene, PSA, consistent with reactivation of a functioning androgen receptor (AR) axis. This observation led to the development of "second-line" therapies aimed at further blocking androgen/AR signaling. Unfortunately, resistance to these agents can also develop quickly. Paradoxically, several studies have suggested that the growth of AR-positive human CRPC cell lines may be inhibited by supraphysiologic levels of testosterone (SupT). These studies suggested that the adaptive reliance on AR signaling by CRPC cells becomes a therapeutic liability that can be exploited through the administration of SupT, which we termed as bipolar androgen therapy (BAT). Understanding how BAT works at the molecular and cellular levels might help in rationally combining BAT with other agents to achieve increased efficacy and tumor responses. Our data indicates that SupT induces DNA double strand breaks (DSBs) in prostate cancer (PCa) cells. Unrepaired DSBs induced by SupT are routed for specialized autophagic degradation, termed nucleophagy. We further show that SupT-induced autophagosomal DNA can activate cytoplasmic DNA sensing pathways and downstream innate immune signaling. Based on our findings, we propose that BAT engages the immune system to inhibit tumor growth. Future combination of BAT with existing immunotherapeutics including immune checkpoint blockade may prove beneficial for treatment of CRPC.

#518

Hypoxia and high interstitial pressure present within the tumor microenvironment can promote T cell suppression and exhaustion.

Chiara Rancan,1 Rachel Marusinec,1 James Lim,2 Lawrence Fong1. 1 _UCSF, San Francisco, CA;_ 2 _Xcellbio, San Francisco, CA_.

Introduction: The tumor microenvironment is thought to be hostile to the optimal functionality of immune cells. In addition to hypoxia, tumors have a higher interstitial pressure than normal tissue. We examined the impact of hypoxia and increased pressure on human T cell function in vitro. We hypothesize that hypoxia and high pressure induced a more profound and durable exhaustion of activated T cells.

Methods: We cultivated human healthy PBMCs stimulated with phytohemagglutinin (PHA) or CD3/CD28 beads under tumor hypoxic conditions (1%O2, 2PSI) and compared it to normoxic in vitro culture conditions (20% O2, 0 PSI). We utilized the AVATAR system (Xcellbio), a novel bioreactor with highly customable cultivation conditions that allows for the modulation of oxygen and pressure. Functionality and phenotypic changes induced by different cultivation conditions were assessed by flow cytometry and cytokine release assays.

Results: Upon stimulation of PBMCs for 3 days, we observed that hypoxia markedly reduced IFNγ and TNFα cytokine release. Additionally, hypoxia induced an overall decrease in surface expression of the activation and metabolic markers CD71 (transferrin) and CD98 (neutral amino acids transporter), while promoting an increased expression of the inhibitory receptor PD1. This was true for both CD4+ and CD8+ T cells. Following this initial 3-day "priming" stimulation, cells spent 2-days in a "resting period" and underwent a second stimulation under a different condition (cells under normoxia were cultured under hypoxia and vice versa). We found that cells initially primed under hypoxic conditions maintained higher levels of PD1 expression after the second stimulation, regardless of whether the latter condition was normoxic or hypoxic.

Conclusions: Taken together these observations suggest that T cell priming in hypoxic and high pressure conditions present in tumors induce an irreversible state of T cell exhaustion and point out a dominant effect of hypoxia over normoxia. Our data suggest that environmental culture conditions greatly affect T cell responses and should be taken into account when studying T cell functionality, especially in the investigation of novel therapeutic agents targeting the immune system.

#519

**EVOEXS21546 is a novel non-brain penetrant A** 2A **R inhibitor for cancer immunotherapy with accelerated drug development.**

Pierre Fons,1 Andy Bell,2 Michael Esquerre,1 Stephanie Versluys,1 Florie Bertrand,1 Iva Hopkins-Navratilova,2 Sean Robinson,2 Virgile Visentin,1 Adrian Schreyer,2 Richard Cox,2 Emilie Mirey,1 Emilie Pelissier,1 Celia bergeaud,1 Simone Culurgioni,2 Jeremy Besnard,2 Andrew Payne,2 Jerome Menegotto,1 Frederic Machet,1 Celine Poussereau-Pomie,1 Eric Cogo,1 Michael Paillasse,1 Federica Ferigo,3 Sabrina Pagliarusco,3 Joanna Lisztwan,1 Mark Whittaker,1 Craig Johnstone,1 Andrew Hopkins2. 1 _Evotec, Toulouse, France;_ 2 _Exscientia, Oxford, United Kingdom;_ 3 _Evotec, Verona, Italy_.

Evotec and Exscientia are collaborating to develop an innovative drug discovery platform for accelerating small molecule development in Immuno-Oncology. The primary focus is to target the immunosuppressive adenosine pathway. We have firstly sought to generate a novel and selective, non-brain penetrant A2AR antagonist. EVOEX21546 has been selected as our lead candidate with in vitro potency on primary human CD3+ T-lymphocytes for inducing the recovery of IL-2 production after CADO-mediated inhibition of T-cell activation. In addition, we have demonstrated the compound's effect on other key biological features of T-cell activation including IFN-gamma production and T-cell proliferation. ADME/DMPK data show that EVOEX21546 has a favourable pharmacological profile consistent with its evaluation in syngeneic tumour models. Furthermore, we are pursuing efforts to understand the Mechanism-of-Action of EVOEXS21546 in order to identify the most relevant biomarker of activity for clinical translation. Finally, from the perspective to accelerate the IND submission, EVOEXS21546 entered an INDiGO campaign, an integrated and rapid process to reach IND complemented by high-end integrated CMC. These results, generated in the frame of the A2AR inhibitor pathway, have paved the way to an optimized process for identifying, improving and accelerating the path from drug discovery to the entering into the clinic for Immuno-Oncology drugs.

#520

Alteration of chemokine gene expression in PC-3 cells treated with NSAIDs.

Niradiz Reyes,1 Juan Rebollo,1 Raj Tiwari,2 Jan Geliebter2. 1 _Universidad de Cartagena, Cartagena, Colombia;_ 2 _New York Medical College, Valhalla, NY_.

Background: The role of chronic inflammation for developing prostate cancer and its progression to metastasis is supported by increasing evidence. Among the pro-inflammatory regulators that seem to play a critical function in prostate tumorigenesis, the most implicated have been the members of the CXC chemokines and their receptors, which have been deemed as significant players in several of the critical steps in tumorigenesis and/or metastasis. CXC chemokines have been classified into two subclasses, ELR+ and ELR−. Most of the ELR+ CXC chemokines are angiogenic, while most of the ELR- are anti-angiogenic. Non-steroidal anti-inflammatory drugs (NSAIDs) have been associated with reduced risk of several cancers, including prostate cancer (PCa). The protective effect of NSAIDs has been attributed to inhibition of cancer metastasis. The aim of this work was to determine the expression profiles of the CXC chemokines in the metastatic prostatic cancer cell line PC-3 compared to the non-tumorigenic PWR-1E cell line and to evaluate the effects of NSAIDs in the expression patterns of these molecular components.

Methods: Differences in gene expression were determined between highly metastatic PC-3 cells and the non-tumorigenic PWR-1E by qRT-PCR and ELISA for a panel of 13 genes encoding CXC chemokines: ELR+ (CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8) and ELR- (CXCL4, CXCL9, CXCL10, CXCL11, CXCL12, and CXCL14). Changes in the expression of these genes in response to treatment with NSAIDs (ibuprofen and nimesulide) were also determined.

Results: Highly metastatic PC-3 cells showed high expression levels of six ELR+ CXC chemokines: CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, and CXCL8; there was no detectable expression for CXCL7. In contrast, expression of ELR- CXC chemokines (CXCL9, CXCL10, CXCL11, and CXCL14) was not detected in either PC-3 or PWR-1E cells. The expression levels of CXCL4 and CXCL12 were comparable in the two cell lines. Treatment of PC-3 cells with ibuprofen and nimesulide affected the expression pattern of the genes evaluated in these cells. Ibuprofen treatment caused decreased expression of ELR+ CXCL6 in PC-3 cells; this effect was not seen with nimesulide treatment. Ibuprofen and Nimesulide treatments caused an increased expression of ELR+ CXCL5 and CXCL8 chemokines. The expression levels of the remaining CXC chemokines: CXCL1, CXCL2, CXCL3, CXCL4, and CXCL12 were not affected by treatment with the NSAIDs assayed.

Conclusions: Highly metastatic PC-3 cells had increased expression levels of ELR+ CXC chemokines compared to non-tumorigenic PWR-1E cells. NSAIDs (Nimesulide and ibuprofen) caused differential changes in the pattern of CXC chemokine expression of PC-3 cells. Pharmacological effects of NSAIDs in prostate cancer warrants further investigation at the clinical settings. Additional studies are required to determine the clinical usefulness of NSAIDs in prostate cancer.

#521

Downregulation of type I IFN response in hypoxia as a possible mechanism of immune escape.

Ana Miar, Jan Rehwinkel, Christos Zois, Adrian L. Harris. _University of Oxford, Oxford, United Kingdom_.

Introduction: Hypoxia is a common phenomenon in solid tumors and is considered a hallmark of cancer. Increasing evidence shows that tumor hypoxia promotes local immune suppression and inhibits immune cell killing functions. However, there is little information about the relationship between hypoxia and the type I interferon (IFN) pathway, which comprises the sensing double-stranded RNA and DNA (dsRNA/dsDNA) in the tumor microenvironment, followed by IFNα/β secretion. The aim of this study is to determine the effect of hypoxia on the type I IFN pathway in breast cancer and the consequences for tumor survival.

Methods: A panel of breast cancer cell lines [MCF7, T47D, BT474, SKBR3, MDA231, MDA453, MDA468] were analyzed by Western blot and RT-qPCR in normoxia and acute hypoxia (0.1% O2) for 24 hours, using medium containing 5mM glucose. Cells were stimulated with the dsRNA mimic poly I:C [10ng/ml to 20ug/ml] to activate the IFN type I pathway.

Results: An overall 50% decrease in expression of the dsRNA-sensing branch (sensors: RIG-I, MDA5; adaptor: MAVS; effectors: IRF3, IRF7, STAT1, STAT2; interferon stimulated genes (ISGs): PKR, ADAR1) was observed in all breast cancer cell lines studied when they were treated with 0.1% O2 for 24h both at RNA and protein levels. Moreover, when the pathway was stimulated using poly I:C, hypoxia also showed a decrease in the stimulation and in the activation of pIRF3 and pSTAT1. Different IFN bioassays were performed using HEK293 cells expressing (i) the IFN stimulated response element (ISRE), which were cultured for 24h with supernatant from MCF7 previously treated with normoxia/hypoxia and different concentration of poly I:C, and (ii) the IFNβ promoter, which were transfected with total RNA from normoxic/hypoxic cells. Consistent with reduced expression of factors involved in the IFN pathway, there was lower activation of the ISRE by supernatants from hypoxic cells compared to samples from normoxic cells, indicating lower IFN production in hypoxia. In addition, RNA from hypoxic cells showed much lower activation of the IFNβ promoter pointing to lower IFNβ transcription in hypoxic cells.

HIF1α and/or HIF2α downregulation using siRNAs partially upregulated the expression of some ISGs (i.e. RIG-I, ADAR1), but this treatment did not completely reverse the effect of hypoxia, suggesting that the effect observed in hypoxia does not fully depend on HIF1α and HIF2α. Further studies on how chromatin conformation under hypoxia could affect the expression of genes in the type I IFN pathway are on going.

Conclusions: Hypoxia downregulates the dsRNA-sensing branch of the type I IFN system in breast cancer, thus favoring an immunosuppressive status that could help immune evasiveness.

#522

PI3K/Akt signaling in tumor cells promotes immune evasion by limiting infiltration, recognition and killing by T cells.

Nithya Sivaram,1 Patrick McLaughlin,1 Ya-Ping Jiang,1 Lisa Ballou,1 Kien Pham,2 Chen Liu,2 Adrianus van der Velden,1 Richard Lin1. 1 _Stony Brook University, Stony Brook, NY;_ 2 _Rutgers University, New Brunswick, NJ_.

The presence of tumor-infiltrating T cells is associated with favorable patient outcomes, yet most pancreatic cancers are immunologically silent and resistant to currently available immunotherapy. In this study, a syngeneic orthotopic implantation model of pancreatic cancer was used to show that PI3K/Akt signaling in tumor cells down-regulate immunogenicity to evade T cell surveillance. Genetic silencing of PI3K catalytic isoform Pik3ca in KrasG12D/Trp53R172H-driven pancreatic tumors leads to infiltration of T cells, complete tumor regression, and 100% survival of immunocompetent host mice. By contrast, Pik3ca-null tumors implanted in T cell-deficient mice progress and kill 100% of the animals. Adoptive transfer of tumor antigen-experienced T cells eliminates Pik3ca-null tumors in immunodeficient mice. Pik3ca silencing or inhibition of its effector, Akt, increases the expression of MHC Class I and CD80 on the surface of murine and human pancreatic cancer cells. Down-regulating MHC Class I and CD80 expression, or increasing Akt activity, in Pik3ca-null tumors increases their susceptibility to T cell-mediated regression in vivo. These results indicate that PI3K/Akt signaling in tumor cells limits T cell recognition and clearance of pancreatic cancer. Strategies that selectively target the PI3K/Akt signaling pathway in tumor cells may improve immunotherapy for pancreatic cancer.

#523

Implications of the A2a receptor (A2aR) on tumor microenvironment in non-small cell lung cancer (NSCLC).

Pedro Viveiros,1 Bhoomika Sukhadia,1 Pratyusha Nunna,1 Keon Woo Park,2 Jeffrey Chuang,3 Lihua Zou,1 Young Kwang Chae1. 1 _Northwestern University, Chicago, IL;_ 2 _Dankook University Hospital, Seoul, Republic of Korea;_ 3 _Jackson Laboratory, Farmington, CT_.

Hypoxia-driven adenosine accumulation in tumor microenvironment is believed to play an important role in tumor evasion of immune response. Adenosine via interaction with its A2a receptor, expressed on immune cells, acts as a negative regulator of cytotoxic T-cell immune activity. Increased A2aR expression was observed in tumor infiltrating lymphocytes in freshly resected NSCLC specimens. Higher expression of A2aR was associated with worse prognostic findings in head and neck squamous cell carcinoma. Conversely, it correlated with favorable clinical outcome in non-metastatic lung adenocarcinoma. It has been hypothesized that A2aR antagonism could lead to enhanced efficacy for immune checkpoint blockade. A2aR antagonists have demonstrated favorable in vitro and ex vivo response in NSCLC and ongoing clinical trials are testing these drugs in conjunction with established immunotherapy. However, little is known about the role of A2aR in NSCLC patients. We investigated how A2aR expression affected tumor immune landscape, PD-L1 expression and clinical outcomes in a large pool of NSCLC patients. Methods: We obtained A2aR expression for NSCLC patients from TCGA; adenocarcinoma (ADC), n=586; squamous cell carcinoma (SCC), n=517. The data was arranged into 4 quartiles according to A2aR expression mRNA-seq z-scores, defining the highest quartile as A2aR-high and the lowest quartile as A2aR-low. We evaluated how A2aR expression correlated with a) immune landscape, b) PD-L1 expression, c) tumor mutational burden (TMB) and neoantigen burden, and d) clinical outcome. Results: In ADC, A2aR-high was significantly associated with lower infiltration of activated CD4 and CD8 T cells when compared with A2aR-low patients (12% vs 48% ; 9% vs 47%, all p<0.001). In SCC, similar results were observed (12% vs 61%; 9% vs 52%, all p<0.001). In addition, higher PD-L1 (CD274) expression was observed in A2aR-high ADC (p<0.05). There was no difference on TMB or neoantigen burden between groups. In ADC, A2aR-high was associated with more favorable overall survival (OS) and progression free survival when compared with A2aR-low (median 71 vs 41 months and 84 vs 27 months, respectively, all p<0.05). An inverse trend was noted in SCC, with A2aR-high patients having significantly shorter OS (median 39 vs 71 months, p<0.05). Conclusion: Higher A2aR expression was associated with lower activated CD4 and CD8 T-cells in NSCLC and higher PD-L1 expression in lung ADC, suggesting a potential target for developing new strategies for immunotherapy in NSCLC.

#524

HMGB1-activated IRF3 and NF-κB contributes to UV radiation-induced immune suppression by upregulating PD-L1.

Wei Wang,1 Nicole Chapman,2 Bo Zhang,1 Mingqi Li,1 Meiyun Fan,1 R. Nicholas Laribee,1 M. Raza Zaidi,3 Lawrence Pfeffer,1 Hongbo Chi,2 Zhaohui Wu1. 1 _Univ. of Tennessee Health Science Ctr., Memphis, TN;_ 2 _St Jude Children's Research Hospital, Memphis, TN;_ 3 _Temple University, Philadelphia, PA_.

Solar ultraviolet radiation (UVR) suppresses skin immunity, which facilitates skin lesion initiation and establishment by promoting immune evasion. It is unclear whether immune checkpoints are involved in the modulation of skin immunity by UVR. Here we report that the expression of immune checkpoint molecule PD-L1 was significantly increased in melanocytes and melanoma cells upon UVR exposure. The damage-associated molecular patterns molecule HMGB1 was secreted by melanocytes and keratinocytes upon UVR, which subsequently activated the RAGE (receptor for advanced glycation endproducts) receptor to promote the NF-κB and IRF3-dependent transcription of PD-L1 in melanocytes. We also found that UVR exposure significantly reduced the susceptibility of melanoma cells to CD8+ T cell-dependent cytotoxicity, which was mitigated by inhibiting the HMGB1/TBK1/IRF3/NF-κB cascade or blocking the PD-1/PD-L1 checkpoint. Taken together, our findings demonstrate that UVR-induced PD-L1 upregulation contributes to the immune suppression in the skin microenvironment, which may promote immune evasion of oncogenic cells, and melanoma initiation and progression.

### Radiation-induced Immunomodulation

#525

Immune-oncologic shift in programmed death-ligand 1 expression and microsatellite instability after neoadjuvant chemoradiation therapy in locally advanced rectal cancer.

SungUk Bae, Hye Won Lee, Sang Jun Byun, Woon Kyung Jeong, Seong Kyu Baek. _Keimyung University Dongsan Medical Center, Daegu, Republic of Korea_.

Purpose: We designed this study to evaluate relations between microsatellite instability (MSI) status and programmed death-ligand 1 (PD-L1) and tumor response following neoadjuvant concurrent chemoradiation therapy (CCRT) in locally advanced rectal cancer.

Materials and methods: In the prospective study, we collected 54 rectal adenocarcinoma patients who underwent neoadjuvant concurrent chemoradiation therapy between August 2016 and December 2017. With exclusion criteria, we finally included 24 patients and evaluated the PD-L1 expression using immunohistochemistry (IHC) and tested the MSI status with pre- and post- CCRT samples. Human colorectal cell lines DLD 1, HT-29 and HCT116 were obtained and cell morphology, cell viability, and PD-L1 expressions were evaluated using flow cytometry 24, 48 and 72 hours after irradiation. Using 4-fields box technique, 50.4 to 54 Gy in daily 1.8Gy fractions of radiation was delivered to pelvis including rectal mass and surgery was performed 6 to 8 weeks after completion of neoadjuvant CCRT. Tumor response was estimated by using Mandard grading system.

Results: According to the tumor regression grade, patients were classified into two groups; responder (grade 0, 1 or 2; 9 patients, 37.5%) and non-responder (grade 3 or 4; 15 patients, 62.5%). Between the two groups, there was no statistically significant differences in several tumor characteristics including

histologic type, lymphocytic response, tumor budding, BRAF or K-ras mutations except tumor stage; more patients with advanced T stage in non-responder (p = 0.019). MSI-high status was reported in two responders (25%), but no one with pre-CCRT MSI-high status in non-responder group (relative risk = 3.143, 95% CI = 1.705 - 5.794). High PD-L1 expression on tumor marginally significantly increased from 16.7% before CCRT to 45.0% after CCRT by IHC (P = 0.053), especially in non-responder group. PD-L1 gene expression by irradiation in three human cell lines using flow cytometry analysis was increased, mostly in radio-resistant cell line, DLD 1.

Conclusion: This prospective study verified the chemoradiation-induced immune-oncologic shift toward increases in PD-L1 expression that may be associated with radio-resistance. MSI status was associated with tumor response after chemoradiotherapy for locally advanced rectal cancer.

Key words: Locally advanced rectal cancer, neoadjuvant concurrent chemoradiation therapy, tumor regression, microsatellite instability, programmed death-ligand

#526

**Tumor-draining lymph node irradiation reduces tumor-infiltrating stem-like CD8** + **T-cells and abrogates the abscopal effect.**

Zachary S. Buchwald, Tahseen H. Nasti, Christiane S. Eberhardt, Andreas Wieland, David Lawson, Walter Curran, Rafi Ahmed, Mohammad K. Khan. _Emory University, Atlanta, GA_.

Purpose: PD-1 and PD-L1 antagonists are efficacious because cancer induces T-cell exhaustion via upregulation of PD-L1 and persistent antigen exposure. Our lab, has shown that the proliferative burst following αPD-L1 therapy of exhausted CD8+PD-1+T-cells is restricted to a "stem-like" CD8+T-cell subset in a murine chronic viral infection model. The role of these stem-like CD8+T-cells in malignancies and whether these cells respond to other immuno-stimulation including radiotherapy (RT) is unknown. RT's immuno-stimulation includes acting as an in-situ vaccine by liberating cryptic tumor neo-antigens, generating a potent anti-tumor CD8+T-cell response, synergizing with αPD-1/L1 and leading to control at distant sites of disease outside the radiation field (abscopal effect). In initial studies, we found these stem-like CD8+T-cells in B16F10 tumors and at high frequencies in the tumor-draining lymph nodes (TDLN) of immunocompetent mice. Here, we investigated: (1) the impact of tumor-directed RT on this stem-like CD8+T-cell population in the tumor and TDLN in the context of an abscopal response, and (2) due to the high frequency of stem-like CD8+T-cells in the TDLN, whether depletion of lymphocytes from the TDLN via RT influenced the abscopal effect.

Experimental Design: We developed a preclinical model of oligo-metastatic melanoma to evaluate the role of stem-like CD8+ T-cells and the TDLN in the abscopal effect. This was done with unilateral tumor-directed RT with or without TDLN-directed RT. This was also done in the presence and absence of αPD-L1.

Results: Tumor-directed RT improved local tumor control and induced an abscopal response with a concomitant increase in tumor infiltrating tumor-specific stem-like CD8+T-cells. Tumor-specific stem-like CD8+T-cells were also observed in the TDLN of tumors on both sides. Importantly, the tumor-directed RT increased tumor-specific T-cell proliferation in the TDLNs bilaterally despite only being targeted at one tumor. Given this robust proliferative response and the high frequency of stem-like CD8+T-cells in the TDLN, we next evaluated the role of the TDLN in mediating the abscopal effect. We found that the abscopal effect is impaired if the TDLN is lymphocyte depleted with TDLN-directed RT in the presence or absence of αPD-L1. Additionally, the tumor-directed RT mediated increase in stem-like T-cells in the irradiated and unirradiated tumor was abrogated with TDLN-directed RT.

Conclusion: Our results demonstrate slowed distant tumor growth following irradiation of a local site and that this correlates with an increase in tumor-infiltrating stem-like CD8+T-cells which is reduced with TDLN-directed RT. These data suggest a critical role for both the stem-like CD8+T-cells and the TDLN in mediating the abscopal effect.

#527

High-dimensional profiling of the systemic immune response informs on optimal sequencing of radiotherapy (RT) and immune checkpoint blockade (ICB).

Kevin L. Chua,1 Michael Fehlings,2 Pek Lim Chu,1 Xiao-Tian Lin,1 Eugenia Yeo,1 Kar Perng Low,1 Dennis Poon,1 Enya Ong,1 Wai Yee Woo,1 Joseph Wee,1 Alessandra Nardin,2 Gopalakrishna Iyer,1 Daniel S. Tan,1 Kee Chee Soo,1 Evan Newell,3 Melvin Chua1. 1 _National Cancer Centre Singapore, Singapore, Singapore;_ 2 _ImmunoSCAPE pte ltd, Singapore;_ 3 _Singapore Immunology Network, Singapore, Singapore_.

Background: Combinatorial RT-ICB potentiates anti-tumour reactivity by modulating the immune response. We therefore performed in-depth phenotypic profiling of the systemic T cell compartment following treatment with RT-ICB.

Methods: We recruited 20 patients with biopsy-proven metastatic renal cell and non-small cell lung carcinoma, who were treated with a sandwich regime of ICB-RT-ICB under a prospective observational study protocol, and compared against a RT alone-treated cohort (N=10). All patients received ablative RT (8-50Gy/1-5fr) for oligoprogression and/ or local palliation. Blood samples were longitudinally collected at pre-RT, 14 d post-RT and cycle 2 ICB post-RT. Deep T cell profiling was performed by mass cytometry using a customised 41 parameter panel, together with high dimensional analysis tools.

Results: Median follow-up of the overall cohort was 18mo; median duration of ICB received in the ICB-RT-ICB arm was 15mo. We observed significant diversity of the systemic T cell repertoire between patients at baseline, and this corresponded to significant interpatient heterogeneity in T cell responses specific to the central/ effector memory, EMRA and Treg subsets post-RT. Dramatic local response (complete response at 1 mo post-RT) was significantly higher in the ICB-RT-ICB cohort compared to the RT alone cohort (12/20 vs 1/10, P<0.01). This clinical phenomenon corresponded to an increased %Ki67high CD8 and CD4 T cells post-RT exclusively in the combinatorial treated cohort, which was further enhanced upon resumption of ICB (mean = 10% vs 3% [CD8]; 13% vs 2% [CD4]; P<0.01). Deeper immunophenotyping of the Ki67high subsets revealed associated high expression of GranzymeB and Eomes.

Conclusions: Here, we observed changes in the T cell phenotypes that varied remarkably across all patients following RT. We further highlight a RT-dependent T cell proliferation amongst all RT-ICB-treated patients that was further enhanced by ICB in prior responders. This immune phenomenon may account for the dramatic responses to combinatorial treatment, and informs on optimal sequencing strategies for combining RT and ICB.

#528

Radioembolization followed by durvalumab and tremelimumab does not induce immune responses against liver-metastasized MSS colorectal cancer.

Chongkai Wang, John Park, Ching Ouyang, Raju Pillai, Jeffrey Longmate, Holly Yin, Christian Avalos, Maricel Gozo, Colt Egelston, Peter P. Lee, Marwan G. Fakih. _City of Hope, Duarte, CA_.

Introduction: PD-1 inhibitors have been ineffective in microsatellite stable (MSS) metastatic colorectal cancer (mCRC). Preclinical models suggest that radiation therapy may sensitize MSS CRC to PD-1 blockade. This constitutes the rationale for combining radioembolization to the liver in patients (pts) with MSS CRC with liver metastasis.

Methods: Pts with MSS mCRC with liver predominant disease who progressed following at least 1 prior line of treatment, were eligible for study treatment. Treatment consisted of Y90 radioembolization to the liver (SIRTEX®) followed 2-3 weeks later by the intravenous (IV) combination of durvalumab (D) at 1500 mg and tremelimumab (T) at 75 mg Q4W for 4 months, followed by D 1500 mg Q4W x 8 cycles, or until disease progression (PD). Tumor biopsies were obtained at baseline, 1-2 weeks post SIR-Spheres®, and 2-3 weeks after D+T. A Simon 2-stage design was implemented, with a planned expansion to 18 patients if at least 1 response is noted in the 1st 9 pts. Correlative studies included tumor and peripheral blood flow cytometry, serum cytokine assays, and tumor IHC multiplex assay for CD8, CD4, CD68 and Cytokeratin20 expression. Immune and cancer related gene expression of the tumor microenvironment was analyzed via NanoString.

Results: 9 pts enrolled in the 1st stage of the study, all with PD within or after their first 2 cycles of treatment. Per pre-planned design, the study was closed for futility. Here we report our correlative study for this trial. Based on IHC, intratumoral TILs (CD4 and CD8 T cells) were not detectable on any of the serial tumor biopsies (pre-Y90, post-Y90, and post D+T), while heavy CD68+ macrophage infiltration was consistently observed. Such observations were statistically validated by comparing paired serial samples using NanoString. Increased expression of collagen genes, such as COL1A1, COL1A2, and COL3A1, following Y90 was noted, as reported in literatures for cases with chemo and radiation resistance. MDM2, known to associate with resistance to PD-1/PD-L1 inhibitors, was also upregulated following Y90. Furthermore, flow cytometry results showed no difference in CD4+ T cells, CD8+T cells, CD20+B cells, CD33+HLA-DR-MDSCs, and CD4+Foxp3+ regulatory T cells based on paired serial PBMC samples. However, we observed a significant increase in PD-1+CD4+ and PD-1+CD8+ T cell subpopulations in PBMC following D+T, which agrees with other reports that PD-1/PD-L1 targeting leads to the expansion of PD-1+ T cells. In addition, CD3-CD56+ NK cell population was increased following D+T when compared with Y90.

Conclusion: Y90 radioembolization can be added safely to D+T but did not promote tumor-directed immune responses against liver-metastasized MSS CRC. The associated correlative studies do not support a role for Y90 radioembolization to convert immunologically 'cold' tumors into 'hot' tumors.

#529

A PI3Kαδ inhibitor combined with irradiation enhance the effect of PD-1 blockade in syngenic triple negative breast cancer model.

Ji Min Park, Joo Ho Lee, Danhyo Kim, Eric Eunsik Kim, Jun Hyung Bae, In Ah Kim. _Seoul National University, Seongnam-Si, Republic of Korea_.

Background:Breast cancer has been showing relatively poor response to immune checkpoint blockades potentially due to low immunogenicity of tumor and immunosuppressive tumor microenvironment. Radiation (RT) has been showing immune stimulatory effect and PI3Kδhas immune suppressive effect via modulation of regulatory T cell (Treg). Therefore, we hypothesize that in situ tumor vaccination via RT and suppression of immune tolerance via PI3Kαδ inhibition could enhance the clinical efficacy of immune checkpoint blockade. As PI3Kα controls a key oncogenic signaling pathway, we evaluated whether RT or novel PI3Kαδ inhibitor or both could enhance the efficacy of antitumor effect of a PD-1 blockade in immune competent syngenic triple negative breast cancer (TNBC) model.

Materials & Methods:4T1 murine breast cancer cells were grown subcutaneously in the hind limb of BALB/c mice. The mice were grouped as following seven groups: control, RT, PI3Kαδ inhibitor, PI3Kαδ inhibitor + RT, PD-1 blockade, PD-1 blockade + RT, and triple combination group. Tumors were irradiated using 24 Gy/3 fractions. PD-1 blockade (10 mg/kg) and PI3Kαδ inhibitor (4 mg/kg) were administered every other day for two weeks, respectively. Tumor size was measured periodically using a Vernier caliper and bioluminescence imaging to evaluate efficacy of each modality and combination therapy. Immune-modulatory function was evaluated using FACS and immunohistochemical (IHC) study.

Results:Triple combination of RT, PD-1 blockade, and PI3Kαδ inhibitor significantly delayed tumor regrowth whereas PD-1 inhibitor alone showed only modest effect in 4T1 syngenic TNBC model. FACS and IHC study for immune repertoire using tumor samples showed that RT and PD-1 blockade modestly increased the proportion of cytotoxic CD8+ T cells and PI3Kαδ inhibitor led to decrease the proportion of Treg. Triple combination showed remarkable increase of cytotoxic CD8+ T cells suggesting synergistic immune modulatory effect of RT, PD-1 blockade and PI3Kαδ inhibitor. Triple combination led to significant upregulation of c-GAS/STING pathway in the tumor and increased IFN-γ level in blood as well compared to each modality alone.

Conclusions:Taken together, combination of RT and PI3Kαδ inhibitors maximized immune stimulatory effect in immune competent syngenic TNBC model and enhanced the response of PD-1 blockade via non-redundant synergistic immune modulatory effect. This study provides a preclinical rationale for the combination of PI3Kαδ inhibitor and RT with PD-1 blockade to overcome the immune tolerance of breast cancer. (Work supported by the grants from NRF #2017R1A2B4002710 & #2017M2A2A7A01018438 to In Ah Kim)

#530

Phosphatidylserine-targeting antibody combined with chemoradiation enhances systemtic anti-tumor immunity.

Yifan Wang,1 Zhigang Liu,2 Weiye Deng,1 Betty Y. Kim,3 Steven H. Lin,4 Wen Jiang1. 1 _UT Southwestern Medical Center, Dallas, TX;_ 2 _Hunan Cancer Hospital, The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, China;_ 3 _Mayo Clinic, Jacksonville, FL;_ 4 _UT MD Anderson Cancer Center, Houston, TX_.

The discovery of immune checkpoint blockades have resulted in significant clinical benefits for patients with metastatic cancers including non-small cell lung cancer (NSCLC). Nevertheless, a large proportion of patients do not respond to such therapy. Strategies to enhance the therapeutic efficacy of cancer immunotherapy among a larger group of patients are urgently needed. Increasing the recognition of cancer cells by immune system may enhance response to immunotherapies. Tumor-associated macrophage (TAM) is the most abundant immune cell in most solid tumors, and is critical in bridging innate and adaptive immune system functions. Cancer cells overexpress multiple immune suppressive molecules including phosphatidylserine (PS) to inhibit immunological functions of TAMs. PS expression can be enhanced by cancer therapeutics such as radiation to induce immune suppression. Our results show that radiation increased expression of PS on lung cancer cells. The combination of PS blocking antibody with radiation decreased immune-suppressive phagocytosis and enhanced antigen presentation of macrophages. In murine lung cancer models, combination treatment of PS blockade and chemoradiation more effectively controlled tumor growth and prolonged survival than each treatment used alone. In a bilateral lung cancer tumor model, addition of PS blockade to chemoradiation promoted cytotoxic T cell infiltration and induced tumor growth inhibition of the non-irradiated tumors. Together, our results demonstrate that PS-targeting antibodies may be combined with chemoradiation to enhance intrinsic tumor immunogenicity and activate systemic anti-tumor immune responses. This approach may provide new routes for improving the efficacy of modern immunotherapeutics.

#531

The cGAS-STING pathway as a driver of the immune-reactive microenvironment in small cell carcinoma of the ovary, hypercalcemic type.

Elke Van Oudenhove, Jay R. Patibandla, Selim Misirlioglu, Ernesto Arostegui Fernandez, Petar Jelinic, Douglas A. Levine. _NYU Langone Health, New York, NY_.

Introduction: Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare and aggressive malignancy affecting young women. The disease is characterized by a monogenic mutation of SMARCA4, a key member of the SWI/SNF complex. The treatment options are limited, and survival rates are poor. We have previously reported clinical responses in patients undergoing treatment with anti-PD1 immunotherapy, despite a low mutational burden. The responses were attributed to elevated PD-L1 expression and T-cell infiltration in SCCOHT. To better understand the mechanisms of the immune reactive microenvironment we sought to determine the role of the innate immune response driven by the STING pathway after radiotherapy.

Methods: The cGAS-STING expression of cell lines with SMARCA4 loss (H1299), SMARCA4 knockdown (293T SMARCA4) and SMARCA4 wild type (CAOV3, OVCAR4) were analyzed following 10 Gy of irradiation. We evaluated the expression of the cGAS-STING cytosolic DNA sensing pathway by measuring time-dependent expression of cGAS dependent interferon stimulated genes CCL5 and CXCL10. Finally, we analyzed the number of micronuclei formed in SCCOHT model cell lines BIN67 and SCCOHT-1 following irradiation using multi-parametric immunofluorescence.

Results: In H1299 (SMARCA4 loss), there was an 1847-fold increase (p=0.02) in CCL5 and 3954-fold increase in CXCL10 (p=0.09) at 6 days post-irradiation in comparison to non-irradiated controls. In 293T cells with stable knockdown of SMARCA4, there was a 2.8-fold increase in expression of CCL5 (p < 0.01) and a 1.6-fold increase CXCL10 (p=0.26), 6 days after irradiation compared to irradiated wild type cells. In the SMARCA4 wild type cell lines CAOV3 and OVCAR4, there was no statistically significant upregulation of CCL5 and CXCL10. There were more micronucleated cells per high power field after irradiation in BIN67 (21.2% vs 9.30%, p=0.1) and SCCOHT-1 (9.4% vs 0.0%, p=0.03) compared to non-irradiated controls.

Conclusion: SCCOHT is a low mutational burden cancer with demonstrated response to immune checkpoint blockade. Our research shows that following irradiation, the innate immune response triggered by cGAS-STING may be driving the immune reactive microenvironment of SCCOHT. The loss of SMARCA4 may play a role in activating the innate immune response. Better characterization of the association between the SWI/SNF complex and immune reactivity should lead to a broader use for immune checkpoint inhibitors.

#532

Irradiation alters the tumor microenvironment of orthotropic pancreatic tumor in favoring the infiltration of PD1+CD8 and G-MDSC and the delivery of immune checkpoint blockade therapy agents.

Yu Hung Lee. _National Tsing Hua University, Hsinchu, Taiwan_.

Immunotherapy (IT) is trending on cancer research, but its efficacy heavily depends on the tumor microenvironment (TME). IT is also frequently combined with radiation therapy (RT). Here, we explored the effect of radiation therapy (RT) on the TME and its consequence on the combination of IT using a syngeneic orthotropic UN-KC6141 PDAC tumor model. The result of immunohistochemical staining (IHC) demonstrates that 25Gy of IR could effectively decrease the number of CD31+ vessels, but significantly increase distribution area of Dextran, an indication of vessel normalization. The normalized tumor vessels are more effectively on delivering ICBT (immune checkpoint blockade therapy) drug delivery as shown by the increase of anti-PD-L1 antibody in irradiated tumor. In addition, we also found that the number of PD1+CD8+ Tc cells and Ly6G+G-MDSC is also increased in irradiated tumor. This indicates that RT could alter TME of pancreatic tumor in favoring the combination with IT, not only alters an immune desert tumor to become an immune infiltrating tumor, but also enhances the delivery of ICBT agents. For this tumor model, the combination of RT with anti-PD-L1 or anti-Ly6G therapy could be effective. To test this hypothesis, the combination of RT with ICBT agents was performed. The preliminary results show that anti-PD-L1 ICBT could enhance the efficacy of RT on decreasing tumor burden. The combination of anit-Ly6G therapy with RT is currently under investigation.

#533

The immune landscape in colon cancer and its changes after radiotherapy.

Cristiana Lo Nigro,1 Dario Sangiolo,2 Vincenzo Ricci,1 Ornella Garrone,1 Andrea Abbona,3 Antonella Falletta,3 Anna Merlotti,1 Chiara Varamo,2 Massimo Aglietta,2 Loretta Gammaitoni,2 Oscar Bertetto,4 Marco Merlano1. 1 _S. Croce & Carle Teaching Hospital, Cuneo, Italy; _2 _Candiolo Cancer Institute and University of Torino, Torino, Italy;_ 3 _ARCO Cuneo Foundation, Cuneo, Italy;_ 4 _Piedmont Cancer Network, Italy_.

Background: The TRANSLATE project started in 2016 to test the immune effects on circulating immune cells and cytokines of metronomic Cyclophosphamide (CTX), daily low-dose IL-2 administered every other week, and radiotherapy (RT). The rationale is based on self vaccination induced by RT, T cells expansion by IL-2 treatment and selective Tregs down regulation by low dose cyclophosphamide administration.

Materials/Methods: Treatment consisted of CTX 50/mg tab daily continuously until progression or unacceptable toxicity, 8 Gy single fraction on one metastatic lesion given day 8 from CTX start and IL-2, 500.000 UI twice a day i.v. day 1 to 5 every other week, starting the day after RT. We enrolled patients with end-stage for colon, breast, kidney and prostate cancer. Analysis was performed at baseline, the day after RT, after 28 days from treatment start and at disease progression. Assay focused on Tregs, CD8+, NK, MDSC, CD3-PD1, IL-2, IL-4, IL-5, IL- 6, IL-10, IL-12, IL-13, IL-17a, TNFα, IFNγ, TGFβ. We report baseline and post-RT data separately in patients with colon cancer (CC) and other patients (non-CC).

Results: We report data from 8 pts with left colon, 6 with breast, 2 with kidney and 3 with prostate cancer. At baseline, cytokine expression was similar in CC and non-CC except IFNγ and IL-13, both less represented in CC. After RT, TNFα increased in both groups, but more evidently in CC. A clear increase in IL-10 and IFNγ occurred only in non-CC, while IL-2 increased only in CC. Cumulatively, CC and non-CC reacted differently to RT in terms of positive or negative changes of the analyzed cytokines. On the contrary, we did not observe clear differences in immune cells between CC and non-CC neither at baseline nor after RT.

Discussion: The limited number of patients reduced the interpretation of the study. The differences observed are numerical, but not statistically significant. Colon cancer shows some peculiarities, including the low IL-13 and IFNγ value and a different response to RT. Further analyses investigations are in progress.

#534

Combining stereotactic body radiation and PARP inhibitors to enhance tumor immunogenicity of colorectal cancer cells.

Steven N. Seyedin,1 Vivian N. Pham,2 Michael Petronek,2 Bryan G. Allen,1 Joseph M. Caster1. 1 _University of Iowa Hospital and Clinics, Iowa City, IA;_ 2 _University of Iowa, Iowa City, IA_.

Introduction: Many human cancers, including microsatellite stable (MSS) colorectal cancers (CRC), are resistant to immunotherapies including T-cell checkpoint inhibitors (CPIs). Stereotactic body radiotherapy (SBRT) has shown some promise in improving systemic immune responses to CPIs in preclinical studies although results from clinical trials have been modest. Several classes of radiosensitizers, including PARP and HDAC inhibitors, can enhance responses to CPIs by modulating the tumor microenvironment towards a more "immunogenic" phenotype. We hypothesize that combining immune-modulating radiosensitizers with SBRT will improve the susceptibility of non-immunogenic tumors to immune therapies like CPIs. In the present study, we screened candidate radiosensitizers in syngeneic CRC models and assessed the effects of radiosensitizers and SBRT on surface immunogenic proteins including MHC-1 and PDL-1.

Methods: Murine CT26 and MC38 CRC cells were treated with various doses of ABT-888 (a PARP inhibitor), MS-275 (an HDAC inhibitor), IPI-549 (a PI3Kγ inhibitor), or VE-821 (an ATR inhibitor), and alone and in combination with a single fraction of radiation (0-8 Gy). Radiation survival curves were generated using a linear quadratic model and the IC10 (dose of radiation yielding 10% cell survival) was determined. The sensitizer enhancement ratio (SER) was calculated as the ratio of the IC10 with drug/IC10 radiation alone. Surface localization of MHC-1 and PDL-1 were quantified using FLOW cytometry 24 hours after treatment with radiation +/- ABT-888 pretreatment.

Results: ABT-888 and VE-821 both sensitized CT26 and MC38 cells to ionizing radiation which was more pronounced with ABT-888. MS-275 sensitized CT26 but not MC28 cells and IPI-549 did not sensitize either line to ionizing radiation. Radiation dose-dependently increased MHC-1 and PDL-1 surface localization in both cell lines. Maximal stimulation of both MHC-1 and PDL-1 was observed after 8 Gy which increased both markers by a factor of 1.8-2.0 compared to untreated cells. ABT-888 pretreatment significantly increased surface localization of both markers following 8 Gy (2.5-2.9-fold increase compared to untreated cells) but not lower doses of radiation.

Conclusions: ABT-888 potentiates alterations in surface localization of immunogenic proteins of cancer cells following SBRT-equivalent doses of radiation. These data suggest that radiosensitizers like ABT-888 should be investigated for the potential to improve systemic immune responses.

#535

Preclinical use of focal radiation and immune checkpoint blockade to improve therapeutic response in an immunologically cold tumor.

Maryland Rosenfeld Franklin, David Draper, Sumithra Urs, Scott Wise. _MI Bioresearch, Ann Arbor, MI_.

Radiotherapy is a highly utilized clinical treatment modality. More than 50% of all cancer patients receive some type of radiation therapy during the course of their illness. In mouse models, radiation treatment has been shown to increase the level of tumor antigen presentation and the variety of peptides available for cross-presentation. Current work in the field focuses on using radiation as a tool to bridge the gap from tumor equilibrium to tumor elimination, which could improve the response rate of immuno-oncology agents.

4T1 is a murine breast cancer model known to have a large percentage of myeloid derived suppressor cells (MDSC) making the model resistant to many immunotherapies and is considered an immunologically cold tumor. We hypothesized that treatment with focal radiation (RT, Xstrahl) would sensitize 4T1 tumors to anti-CTLA-4 treatment.

To determine an appropriate dose of RT, mice with 4T1-Lu2 tumors were treated with a single, focal dose of 5, 10 or 20Gy RT. No response was seen at 5Gy, 10Gy resulted in 10 days growth delay, and 20Gy resulted in 16 days growth. In subsequent work, mice were placed into groups and treated with either isotype control (10mg/kg, MPC-11), anti-CTLA-4 (10mg/kg, 9D9), 10Gy RT, or the combination. Mice were monitored over time for changes in primary tumor volume by caliper measurements and for metastatic disease by bioluminescence imaging (BLI) of the thoracic region. Satellite groups were included for immunohistochemistry and the evaluation of changes in tumor infiltrating lymphocytes.

Median tumor growth delay with anti-CTLA-4, RT, or the combination, was 2.4, 3.5 and 9.4 days, respectively. Time to progression, to 1,200mm3 tumors, was increased by 25% in the combination group but not significantly in the monotherapy groups. While all mice showed evidence of metastatic disease, mice in the combination group displayed a lower level of BLI signal on day 30 when compared to other groups.

To examine the effects on immune cell infiltration, 11 subsets were profiled by flow cytometry. B cells and regulatory T cells were significantly reduced (>95% and 90%, respectively) in the combination group when compared to an untreated group. B cell reductions that exceeded 90% were also observed in some animals within the isotype control group. Examination of the CD8+ T cell phenotype demonstrated that combination therapy, but not monotherapies, increased expression of both CD69 and PD-1 on CD8+ T cells suggesting an enhancement of anti-tumor cytotoxicity in these cells. Interestingly, we found that the combination selectively reduced the levels of phosphorylated STAT3 in MDSC subsets. Collectively, this intimates that anti-CTLA-4 and RT selectively disrupt STAT3 signaling in MDSCs and combine to enhance CD8+ T cell activity, which may play a role in the 4T1 tumor growth delay observed.

#536

NBTXR3 potentiate cancer-cell intrinsic interferon beta response to radiotherapy.

Maria E Rodriguez-Ruiz,1 Karsten Pilones,1 Camille Daviaud,1 Jeffrey Kraynak,1 Audrey Darmon,2 Sebastien Paris,2 Sandra Demaria1. 1 _Weill Cornell Medicine, New York, NY;_ 2 _Nanobiotix, Paris, France_.

NBTXR3 represent first in class functionalized radioenhancer optimized for efficient tumor binding and sustained local permanence after intratumoral injection. Recent results of phase III in locally advanced Soft Tissue Sarcoma patients established the significant clinical benefits of NBTXR3 activated by radiation therapy compared to radiotherapy alone. Moreover, preclinical studies using CT26 cells have demonstrated that NBTXR3 activated by RT can induce an anti-tumor immune-response and the abscopal effect. Radiotherapy (RT) can induce T cell-mediated anti-tumor immune responses by multiple mechanisms which include enhancing the recruitment of Batf3-dependent cross-priming dendritic cells (DCs). The latter is contingent on tumor-intrinsic IFN-I induction by cytosolic dsDNA via cGAS/STING pathway, a process that is regulated by the upregulation of Trex1 in a RT dose-dependent fashion (Nat Commun 2017; 8:15618). Here, we utilized the murine TSA breast cancer cell line to test the hypothesis that NBTXR3 strengthens the pro-immunogenic effect of local RT by potentiating tumor cells IFN-I response. For in vitro experiments, TSA cells were exposed to NBTXR3 (800uM) 16 hours prior to RT delivered by a 220-kV source (SARRP) at a dose rate of 283.2 cGy/min. Secreted IFNb in the supernatants was measured by ELISA. Pre-treatment with NBTXR3 enhanced TSA cell radiosensitivity as determined in clonogenic assays. Cell death, as measured by loss of plasma membrane integrity was accelerated by NBTX3 after irradiation of TSA cells with 6 and 8Gy after 24 hours of treatment. NBTXR3 enhanced the secretion of IFNb by irradiated cells. Data suggest that NBTXR3 may enhance the effectiveness of radiation and improve tumor immunogenicity, likely via the induction of IFN-I. The role of the cGAS/STING pathway in the effects of NBTX3 are under investigation in vitro and in vivo. 

### Therapeutic Antibodies 1

#537

Antibody drug conjugate to treat triple negative breast cancer.

Yingnan Si, Nghi Dang, Seulhee Kim, Lufang Zhou, Xiaosi Han, Runhua Liu, Xiaoguang (Margaret) Liu. _The University of Alabama at Birmingham, Birmingham, AL_.

Triple negative breast cancer (TNBC) is characterized by rapid growth, metastasis, and high recurrence. The severe adverse effects and drug resistance associated with standard cytotoxic chemotherapies minimize their clinical benefits. The anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb) had been developed and tested to treat TNBC, but no significant efficiency was demonstrated in clinical trials. The goal of this study was to develop a novel antibody-drug conjugate (ADC) to treat TNBC. Specifically, we established a platform to conjugate anti EGFR mAb with mertansine, which inhibits cell division by blocking tubulin polymerization, and also improved the conjugation efficiency by optimizing the linker and drug-to-antibody ratio. Flow cytometry and live cell confocal laser scanning microscopy analysis confirmed that the anti-EGFR ADC can specifically bind to multiple TNBC subtypes, is effectively internalized, and release potent drug intracellularly within 1 hour after mixing. The IC50 study showed that the anti-EGFR ADC killed >90% TNBC cells with dose of 5 nM. The TNBC xenograft mouse model also showed that the constructed ADC had high anticancer efficacy and successfully inhibited tumor growth. Moreover, the live-animal imaging using In Vivo Imaging System and the tissue microarray samples with immunohistochemical analysis of TNBC and organs indicated that our ADC specifically target TNBC in vivo. Further evaluations, such as maximal tolerated dose (MTD), pharmacokinetics (PK), and biodistribution, are needed. To summarize, we developed an EGFR-targeted ADC, which could provide an effective targeted therapy to treat TNBC and improve the life quality and survival rate of patients with TNBC.

#538

LC-MS based catabolite identification study of an ADC with DM21-C, a novel maytansinoid linker-payload.

Janet Lau, Paulin Salomon, Kerstin Sinkevicius, Juliet Costoplus, Megan Fuller, Raymond Xu, Stuart Hicks, Ravi Chari, Wayne Widdison, Nicholas Yoder, Thomas Keating. _ImmunoGen, Waltham, MA_.

ImmunoGen's newest antibody-drug conjugate (ADC) design uses the novel maytansinoid linker-payload, DM21-C that bears a peptidase/protease cleavable linker. These ADCs show good bystander killing of proximal antigen negative cells, suggesting the generation of cell-permeable catabolites. The goal of this study was to identify the catabolites generated upon incubation in antigen-positive cancer cells (both cell pellet and media), in mouse plasma, as well as in in vitro catabolic systems. Upon incubation with a cysteine-conjugated DM21-C ADC, the small molecule fraction was extracted with an organic solvent and analyzed by high resolution mass spectrometry after chromatographic separation. Through comparison to reference standards, mass spectral signal extraction of possible products, and database search of unknown peaks, the potential in vitro catabolite products of DM21-C conjugate were identified. Information on linker-payload stability in these model systems, including plasma stability, in vitro metabolism of the ADC, and catabolic products that were both retained and effluxed from cancer cells was obtained. As a result, we identified DM51 (the thiol- resulting from self-immolation of the cleaved linker-payload) as a major catabolite of the DM21-C ADC.

#539

A bispecific Fc-silenced IgG1 antibody (MCLA-145) requires PD-L1 binding to activate CD137.

Patrick Mayes,1 Paul Tacken,2 Steve Wang,1 Pieter-Fokko van Loo,2 Thomas Condamine,1 Hans van der Maaden,2 Eric Rovers,2 Steef Engels,2 Floris Fransen,2 Ashwini Kulkarni,1 Yao-bin Liu,1 Arpita Mondal,1 Leslie Hall,1 Soyeon Kim,3 Marina Martinez,3 Shaun O'Brien,3 Edmund Moon,3 Steven Albelda,3 Peggy Scherle,1 Gregory Hollis,1 Reid Huber,1 Mark Throsby,2 Cecile A. Geuijen2. 1 _Incyte Corporation, Wilmington, DE;_ 2 _Merus NV, Utrecht, Netherlands;_ 3 _University of Pennsylvania, Philadelphia, PA_.

CD137 (4-1BB) is a transmembrane costimulatory receptor on T and NK cells that enhances adaptive immune responses and is a critical mediator of antitumor immunity. The development of CD137 targeted agents for cancer therapy has been hampered by on-target off-tumor toxicity in the case of agonist monospecific, bivalent mAbs or limited antitumor activity in the case of crosslinking mAbs. Here we have developed an Fc-silenced bispecific IgG1 antibody to CD137 and PD-L1 with monovalent binding specificity to each target. MCLA-145 drives transactivation of CD137 in the vicinity of cells expressing PD-L1, such as in the immunosuppressive tumor microenvironment. The degree of CD137 agonistic activity in T cells correlated with the expression level of PD-L1 on neighboring cells, as demonstrated in transactivation assays whereby reporter T cells were co-cultured with cells expressing different levels of PD-L1. PD-L1 expression as low as 6000 receptors per cell was sufficient to activate CD137 in neighboring T cells. In contrast, MCLA-145 blocked PD-1 signaling without requirement for CD137 binding in a PD-1/PD-L1 reporter assay. CD137 signaling was induced by MCLA-145 in multiple primary human immune cell assays including the mixed lymphocyte reaction, human PBMC, and whole blood SEB stimulation assays. MCLA-145 reversed T cell suppression mediated by M2 macrophages or Tregs, in vitro. In addition, MCLA-145 enhanced Ag-specific expansion and differentiation of human naïve CD8+ T cells in vitro.

In vivo, MCLA-145 treatment resulted in significant tumor immune activation and antitumor responses in two separate humanized mouse tumor models. In one model, human T cells expressing NY-ESO specific TCR were adoptively transferred to mice bearing A549 tumors which expressed NY-ESO antigen and human PD-L1. MCLA-145 treatment at 5 mg/kg resulted in 54% tumor growth inhibition (TGI) as compared to T cell only treated mice. In the tumors of MCLA-145 treated mice, the percentage of NY-ESO specific CD8+ T cells were significantly increased compared to controls. In a second model, mice engrafted with human CD34\+ cells were implanted with the breast tumor cell line MDA-MB-231. MCLA-145 at 0.5 mg/kg and 5 mg/kg induced significant tumor growth inhibition (55 and 57% respectively) as compared to vehicle control or Fc-silenced huIgG1 controls. Additionally, two out of nine animals in the 5 mg/kg MCLA-145-treated group had complete tumor regression. MCLA-145 increased the number of infiltrating CD8+ T cells, as well as the percentage of central memory CD8+ T cells. The cured animals were then re-challenged with MDA-MB-231 tumor cells, and tumors of previously cured mice were rejected as compared to no growth inhibition in treatment-naïve CD34+ NSG mice. In conclusion, these data support the clinical evaluation of MCLA-145 as a novel, PD-L1 dependent CD137 agonist immune therapy.

#540

AO-176, a normal cell sparing humanized anti-CD47 antibody.

Prabir Chakraborty,1 Myriam N. Bouchlaka,1 Benjamin J. Capoccia,1 Ronald R. Hiebsch,1 Michael J. Donio,1 Robyn J. Puro,1 Vicki Sung,2 Daniel S. Pereira1. 1 _Arch Oncology Inc., St. Louis, MO;_ 2 _Arch Oncology Inc., San Francisco, CA_.

AO-176 is a next generation humanized anti-CD47 IgG2 that binds human and cynomolgus monkey CD47 equivalently. AO-176, like other CD47 antibodies, blocks the interaction of CD47 with SIRPα, inducing phagocytosis of tumor cells by activated macrophages. Unique to AO-176 is its ability to directly kill tumor cells via programmed cell death type III and immunogenic cell death in a cell autonomous (non-ADCC) manner. AO-176 also exhibits preferential binding to tumor vs. normal cells, a notable characteristic, as CD47 is expressed on many normal cells including red blood cells (RBC), platelets, T cells and endothelial cells. While other CD47 antibodies induce hematological toxicities such as anemia and thrombocytopenia in both primate models and in patients, AO-176 negligibly binds RBCs and platelets and even at high doses, minimally impacts hematology in cynomolgus monkey toxicology studies. AO-176 is currently being evaluated in phase 1 clinical trials for the treatment of solid tumors.

In recent head to head experiments conducted with other CD47 antibodies, AO-176 bound all normal cells tested to a significantly lower degree. For example, unlike anti-CD47 antibodies such as Hu-5F9-G4, we observed negligible and minimal ex vivo binding to healthy human RBCs and platelets respectively and significantly lower binding of AO-176 to other hematologic and non-hematologic cells such as T cells and endothelial cells. Although AO-176 binds cynomolgus monkey RBCs to a slightly greater degree than human RBCs ex vivo, AO-176 versus comparative published Hu5F9-G4 findings has demonstrated dramatically reduced receptor occupancy when evaluated in monkey toxicology studies. We have also developed a clinic-ready receptor occupancy assay to measure AO-176 binding to circulating PBMCs in patients treated with AO-176 and to demonstrate minimal binding to CD47 on normal human cells.

We have begun to evaluate mechanisms that may underlie the normal cell sparing effects of AO-176. Published studies have demonstrated that in addition to CD47 protein expression level, clustering and mobility of CD47 at the cell surface may impact binding to ligands, downstream signaling and subsequent cellular responses such as apoptosis. For example, avidity for SIRPα and clearance by macrophages is modulated by CD47 clustering that can be modulated by protein glycosylation, association with lipid rafts, cytoskeleton, integrins or other cis-acting factors. Here, we compare the distribution of CD47 on tumor and RBCs to evaluate receptor association with other cellular membrane components that may impact distribution on the cell surface.

In summary, AO-176 is a next generation anti-CD47 antibody that, aside from induction of phagocytosis, possesses additional attributes that include direct tumor cell killing and preferential binding to tumor versus normal cells. These attributes of AO-176 differentiate it from other CD47 axis targeting agents currently in clinical evaluation.

#541

An unbiased screen identifies a CD137xPD-L1 bispecific IgG1 antibody with unique T cell activation and binding properties.

Cecile A. Geuijen,1 Paul Tacken,1 Rinse Klooster,1 Horacio Nastri,2 Shaun Stewart,2 Jing Zhou,2 Steve Wang,2 Cheng-Yen Huang,2 Arjen Kramer,1 Linda Kaldenberg-Hendriks,1 John de Kruif,1 Renate den Blanken-Smit,1 Vanessa Zondag-van de Zande,1 Abdul Basmeleh,1 Willem Bartelink,1 Patrick Mayes,2 Gregory Hollis,2 Reid Huber,2 Mark Throsby1. 1 _Merus NV, Utrecht, Netherlands;_ 2 _Incyte Corporation, Wilmington, DE_.

CD137 (4-1BB) is a transmembrane costimulatory receptor on T and NK cells that enhances adaptive immune responses and is a critical mediator of antitumor immunity. CD137 signaling requires receptor clustering normally facilitated by the trimeric CD137 ligand (CD137L). Alternatively, CD137 signaling can be triggered either directly by agonistic monoclonal antibodies (mAbs) or indirectly via crosslinking of CD137 binding mAbs by Fcγ receptors on neighboring cells. The development of CD137 targeted agents for cancer therapy has been hampered by on-target off-tumor toxicity in the case of agonist, monospecific, bivalent mAbs or limited antitumor activity in the case of crosslinking mAbs. To address the issues of toxicity and efficacy a highly selective and potent CD137xPD-L1 bispecific antibody (bAb) was identified by applying an unbiased functional screening approach. Collections of common light chain Fabs recognizing CD137 and PD-L1 were produced based on antibody panels from immunized MeMo® mice. A large and diverse panel of CD137xPD-L1 bAbs was then produced by combining different CD137 and PD-L1 Fabs based on epitope and sequence diversity in the IgG1 Biclonics® format. The bAbs were screened for activity in reporter cell lines expressing the receptors. This unbiased combinatorial screening identified a CD137xPD-L1 bAb (MCLA-145) for which CD137 mediated activation is dependent on the presence of PD-L1 on a neighboring cell and, as such, the antibody acts in 'trans'. Flow cytometry experiments demonstrated that MCLA-145 is fully cross-reactive to cynomolgus monkey CD137 and PD-L1. The CD137 Fab arm blocks the interaction of CD137 with CD137L as demonstrated in a competition assay by flow cytometry. The PD-L1 Fab arm blocks the interaction between PD-1 and PD-L1 as demonstrated in ELISA. Binding epitopes were mapped by shotgun mutagenesis using a flow-based screen. In addition, hydrogen-deuterium exchange experiments were performed to map the binding domain on CD137. Data show that MCLA-145 binds the ligand binding domain of CD137 domain (CRDII). The PD-L1 Fab arm binds PD-L1 in the PD-1 binding N-terminal V domain. Both epitope mapping data sets are consistent with the CD137 and PD-L1 ligand blocking activity of MCLA-145. Monovalent binding affinities were measured by surface plasma resonance (SPR) and radioactive iodine labeling and demonstrated affinities in the low nM (CD137) and subnanomolar (PD-L1) range. SPR experiments also confirmed that MCLA-145 was able to bind simultaneously to both CD137 and PD-L1 recombinant proteins. The unique binding properties of MCLA-145 may result in an increased therapeutic window by specifically activating CD137 expressing cells in the tumor niche where PD-L1 is expressed while simultaneously blocking inhibitory input from the PD-1/PD-L1 axis.

#542

Anti-CD19 therapy for precursor B-acute lymphoblastic leukemia: Response and resistance mechanisms.

Firas El Chaer,1 Brandon Carter-Cooper,2 Rena G. Lapidus,2 Maria R. Baer,1 Zeba N. Singh,1 Ashkan Emadi1. 1 _University of Maryland School of Medicine, Baltimore, MD;_ 2 _University of Maryland Greenebaum Comprehensive Cancer Center, Baltimore, MD_.

Introduction: CD19 is a transmembrane protein restricted to B-lineage cells and follicular dendritic cells. In conjunction with CD21, CD81, and CD225, CD19 acts as a B cell co-receptor to decrease the threshold for antigen receptor-dependent stimulation. CD19 trafficking and expression are orchestrated by a multitude of molecules. Resistance to blinatumomab (Blina, anti-CD19 therapy) in acute lymphoblastic leukemia (ALL) is thought to be due in part to the emergence of CD19-negative escape variants. Loss of expression of CD81 and/or CD21, which are required for CD19 trafficking, results in the lack of CD19 expression. In addition, alternative CD19 splicing and other non-CD19-mediated resistance mechanisms have been described. Therefore, we aimed to study CD19 trafficking and response to Blina in ALL patients.

Methods: Peripheral blood was collected at our Center. Mononuclear cells (PBMCs) were isolated and viably frozen. Cells were then thawed and washed with PBS with 10% FBS, and viable cells were counted using trypan blue dye exclusion. 1x10^6 cells/test were stained for CD19, CD81 (BD Biosciences, San Jose, CA) and CD21 (Biolegend, San Diego, CA). After incubation, cells were washed 2x with FACs buffer, resuspended and acquired on a BD Canto II flow cytometer.

Results: Blood was obtained from 10 patients with relapsed or refractory pre-B-ALL with blood blasts prior to Blina treatment. Five patients subsequently achieved complete remission (CR). Cells from four of these patients had CD21 expression on <5% of cells, but CD81 expression on >40% of cells. Of the 5 patients whose disease did not respond to Blina, four also had CD21 expression on <5% of cells and had CD81 expression on an average of 40% of cells. CD19 was expressed on >40% of cells in all samples.

Conclusion: Although CD21 and CD81 are required for CD19 trafficking and expression, loss of either of these antigens measured by flow cytometry did not correlate with the response to Blina. | |  | |  | |

|

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

Patient | Age

(years) | Diagnosis | Karyotype | CD19* | CD21* | CD81* | Response to Blinatumomab

1 | 76 | Pre-B-ALL | 46,XX,t(9;22)(q34.1;q11.2) | 53 | 2 | 41 | Yes (CR)

2 | 55 | Pre-B-ALL | 46, X,inv(Y)(p11.2q11.23), dic(9;12)(p13;p13), del20(q11.2) | 42 | 1 | 46 | Yes (CR)

3 | 47 | Pre-B-ALL | 46, XX,t(4;11)(q21;q23) | 94 | 12 | 56 | Yes (CR)

4 | 84 | Pre-B-ALL | 46,XX,i(9)Iq10) | 65 | 3 | 72 | Yes (CR)

5 | 51 | Pre-B-ALL | 46, XX, t(9;22)(q34.1;q11.2) | 77 | 5 | 78 | Yes (CR)

6 | 33 | Pre-B-ALL | 46, XY, +X, der(17;18)(q10;q10) | 67 | 1 | 85 | No

7 | 25 | Pre-B-ALL | 46, XY, der(20;21)(q10;q10),+21 | 46 | 4 | 32 | No

8 | 25 | Pre-B-ALL | 46, Y, t(X;4)(p11.2;p15), der(9)t(9;14)(p21;q12) del(9)(p21),del(14)q13), +mar[cp14] | 74 | 24 | 29 | No

9 | 79 | Pre-B-ALL | 46, XY | 48 | 1 | 23 | No

10 | 73 | Pre-B-ALL | 46, XY | 59 | 1 | 29 | No

*% cells; CR = complete remission

#543

Dinutuximab binds specifically to disialoganglioside-2.

Shahriar Yaghoubi,1 Travis Harrison,2 Gerald Messerschmidt,2 Suzana Corritori2. 1 _United Therapeutics Corp., Silverspring, MD;_ 2 _Precision for Medicine, Carlsbad, CA_.

Dinutuximab is a human/murine chimeric monoclonal antibody, approved for maintenance treatment of pediatric patients with high-risk neuroblastoma (NB) in the US, and currently under investigation in a phase III trial (NCT03098030) for 2nd line treatment of relapsed/refractory small cell lung cancer (SCLC). Despite four decades of development, the specificity of dinutuximab binding to its intended target, disialoganglioside-2 (GD2), has not been fully elucidated. Although GD2 is a relatively cancer cell specific antigen, other cell surface gangliosides are ubiquitously expressed on the outer membrane of healthy human cells. Therefore, the main goal of this study was to confirm binding specificity of dinutuximab to GD2. We also compared relative affinity of dinutuximab to GD2 with 14G2a, which is the original murine anti-GD2 antibody from which dinutuximab was derived. To measure dinutuximab and 14G2a binding kinetics to various relevant gangliosides (GD1a, GD1b, GM3, GD3, GM4, GM2 and GD2), an in vitro assay was developed and optimized using the Biacore surface plasmon resonance platform. The results showed that GD2 was the only ganglioside that binds to dinutuximab (relative KD=0.000337M). The relative affinity of GD2 to dinutuximab and 14G2a antibody were comparable and within 2-fold of each other (Table). In conclusion, dinutuximab specifically binds to GD2. The absence of binding to GM3, GD3, GD1a, or GD1b suggests that an exposed N-acetylgalactosamine (GalNAc) moiety is necessary for antibody recognition. The absence of binding to GM2 or GT2 (sugar moiety), which differ from GD2 by the number of sialic residues attached to the basal galactose moiety, suggests that the number of sialic acid residues is also critical for antibody recognition.

GD2 binding kinetics of Dinutuximab and 14G2a

---

Ligand | KD (M) | ka (1/Ms) | kd (1/s) | Chi2 (RU2)

Dinutuximab | 3.37E-04 | 2.48E+02 | 8.34E-02 | 0.786

14G2a | 2.00E-04 | 7.11E+02 | 1.42E-01 | 0.0273

#544

Anti-tumor efficacy and potential mechanism of action of a novel therapeutic humanized anti-Globo H antibody, OBI-888.

Yu-Chi Chen, Ming-Chen Yang, Yi-Chien Tsai, Hui-Wen Chang, Chang-Lin Hsieh, Yu-Jung Chen, Kuang-Hsiu Lee, Jiann-Shiun Lai, I-Ju Chen. _OBI Pharma, Inc., Taipei, Taiwan_.

Background: Globo H (GH) is a hexasaccharide expressed on various cancer types. We therefore designed OBI-888, a humanized monoclonal IgG1 antibody targeting GH, as a treatment for GH expressing cancers. This study evaluated OBI-888's mechanisms of action (MOA) and in vivo efficacy.

Methods: Antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) were assessed by bioluminescent cell-based reporter assays. Binding affinity of OBI-888 to various FcγRs and complement-dependent cytotoxicity (CDC) were evaluated by ELISA-based binding assay. We studied the anti-immunosuppressive effect via Jurkat/NFAT-luciferase reporter based assay (Promega). In vivo anti-tumor efficacy of OBI-888 was measured in multiple GH expressing xenograft mouse models. The GH expression levels of tumor cells in xenograft models before tumor inoculation and after tumor excision were determined by flow cytometry and immunohistochemistry (IHC), respectively. The lymphocyte populations in tumor microenvironment were evaluated by IHC.

Results: EC50 values of ADCC and ADCP activity were 4.68 and 29.15 μg/mL, respectively. In addition, OBI-888 demonstrated binding affinity to FcγRs, including FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, FCRN, and a low affinity genotype FcγRIIIA V176F. The EC50 of OBI-888 binding to C1q, the complement component required for CDC initiation, was 0.86 μg/mL. Addition of 5-40 μM GH ceramide (GH-cer) showed dose-dependent immunosuppresive effect on Jurkat/NAF-Luciferase cells. 8.7 μM of OBI-888 can reverse the inactivation of Jurkat/NAF-Luciferase cells induced by GH-cer. In GH positive (GH+) MCF7 (breast), HCC-1428 (breast), HPAC (pancreas), NCI-H526 (lung), and SW480 (colorectal) xenograft cancer models, OBI-888 exhibited tumor growth inhibition of 30-85%. Based on IHC, GH staining was observed on the immune cells at intra- and peri-tumor regions. OBI-888 reduced the population of M2 macrophage, but not that of pan-macrophage.

Conclusions: Study demonstrated OBI-888's ability to trigger ADCC, ADCP, and CDC, mechanisms by which antibody induces tumor lysis. ADCC was further supported by OBI-888's binding affinity to multiple FcγRs, including a low affinity genotype which can potentially benefit those patients who belong to low affinity genotype. GH-cer showed immunosuppresive effect, as manifested by GH staining on the immune cells. OBI-888 has anti-immunosuppresive function to reverse the effect. In vivo tumor growth inhibition with decreased M2 population was observed in various GH+ xenograft models, suggesting the potential MOA through immune modulation. The aforementioned findings suggest that OBI-888 has substantial therapeutic potential to treat various types of cancer that express GH. A first-in-human (FIH) clinical trial of OBI-888 (NCT03573544) has been initiated.

#545

Preclinical development of a bispecific antibody-trap selectively targeting CD47 and CD20 for the treatment of B cell lineage cancer.

Wenzhi Tian, Song Li, Dianze Chen, Guangjun Liang, Li Zhang, Wei Zhang, Xiaoping Tu, Liang Peng, Jie Weng, Gui Zhao, Dandan Liu, Huiqin Guo, Chunmei Yang, Ruliang Zhang. _ImmuneOnco Biopharma, Shanghai, China_.

Tumor cells over expressing CD47 can evade immune surveillance through delivering a "Don't eat me" signal to macrophages. Developing CD47-targeted modalities has been a hot pursue in the pharmaceutical industry. However due to the ubiquitous expression of CD47 in normal tissues, especially in red blood cells (RBC), severe antigenic sink as well as hemotoxicity including anemia set up a higher bar for this particular targeted therapy development. To overcome these hurdles, we have developed a bispecific recombinant antibody-trap, IMM0306, designed to simultaneously target CD47 and CD20 on B cells but avoid binding to human RBCs. Extensive in vitro characterization demonstrated that IMM0306 binds to both CD47 and CD20 with affinity 3-8 folds lower than either single-targeted molecule, but has strong pro-phagocytosis activity over CD47-positive target cells, and even stronger ADCC activity than Rituximab. Intriguingly, IMM0306 has no binding activity at all toward human RBCs, albeit much lower binding activity toward monkey RBCs. Treatment of tumor-implanted SCID mice with IMM0306 significantly inhibited tumor growth and led to eradication of the tumor cells from 5 out of 8 mice, which is much more effective than Rituximab and than co-administration of Rituximab and SIRPα-Fc (IMM01), although profound synergistic effect has been observed for the co-administration. Preclinical study in non-human primates demonstrated a favorable pharmacokinetic profile with no obvious hemotoxicity following single as well as multiple administrations at different dosage. Our study suggests that antibody-trap like IMM0306 might be an ideal approach for CD47-targeted immunotherapy development since selective avoidance of RBC mediated antigen-sink as well as anemia could be achieved along with the robust anti-tumor activity.

#546

Selectively targeting CD47 with bispecific antibody to efficiently eliminate mesothelin-positive solid tumors.

Limin Shang, Vanessa Buatois, Eric Hatterer, Xavier Chauchet, Hasnaà Haddouk, Stefano Majocchi, Krzysztof Masternak, Marie H. Kosco-Vilbois, Nicolas Fischer, Walter G. Ferlin. _Novimmune SA, Plan les Ouates, Switzerland_.

The CD47- signal regulatory protein α (SIRPα) axis, originally discovered as a mechanism of self-recognition, has emerged as a novel innate immune check-point employed by cancer cells to escape immune surveillance. Over-expression of CD47 on a plethora of hematologic and solid cancers has been shown to be associated with poor prognosis. CD47 blockade is thus considered as an attractive strategy to retune the host immune system toward eliminating cancer cells. Clinical efficacy has been achieved in patients with Non-Hodgkin lymphoma (NHL) treated with a combination of the anti-CD20 monoclonal antibody (mAb), rituximab, and the anti-CD47 mAb, Hu5F9-G4. However, in parallel, due to the ubiquitous expression of CD47 on healthy cells, toxicity is observed limiting exposure which impairs clinical development of anti-CD47 mAbs.

To harness the tumoricidal potential and avoid the liabilities of CD47 blockade, we have developed bispecific antibodies (bsAbs), co-targeting CD47 and a tumor associated antigen (TAA), for selective blockade of CD47 on malignant cells. Mesothelin (MSLN) has been selected as one of these TAAs as it is over-expressed on multiple types of solid tumors. An anti-CD47xMSLN bsAb with a fully functional IgG1 Fc domain has been generated and tested for efficacy and safety in vitro and in vivo using cell-based assays and animal models.

With a panel of human MSLN+ target cells, the bsAb kills more efficiently through Ab-dependent cellular phagocytosis (ADCP) and cell-mediated cytotoxicity (ADCC) as compared to the anti-MSLN mAb, amatuximab. Efficacy of the bsAb was minimally affected by soluble MSLN as compared to amatuximab. Mechanistic studies demonstrated that the increased ADCP and ADCC were due to co-engagement-mediated blockade of both target proteins. In vivo using xenograft models, whereas amatuximab was unable, the bsAb inhibited tumor growth of OVCAR3 and MSLN-transfected HepG2 cell lines. Phenotypic analysis showed that treatment with the bsAb induced the accumulation of myeloid cells with increased F4/80 expression in the tumor microenvironment. Importantly, the bsAb was well-tolerated in an exploratory four-week repeated dose study at the highest dose tested (10 mg/kg) in cynomolgus monkeys.

Conclusion: Selective CD47 targeting on tumor cells with an anti-CD47xMSLN bsAb showed efficient killing of MSLN+ tumor cells in vitro and in vivo. A study in non-human primates administered weekly a therapeutically relevant dose over 28 days was well tolerated demonstrating no adverse hematological profiles.

#547

Recombinant immunotoxins targeting B-cell maturation antigen induce complete responses in bone marrow myeloma mouse model.

Zoe Shancer, Xiu-fen Liu, Emily King, Tapan Bera, Ira Pastan. _National Cancer Institute, Bethesda, MD_.

Background: Multiple myeloma (MM) is a B-cell malignancy for which new treatments are urgently needed. B-cell maturation antigen (BCMA) is a lineage-restricted differentiation protein highly expressed on MM and an ideal target for immunotherapy of MM. Recombinant immunotoxins (RITs) are chimeric proteins composed of the Fv or Fab portion of an antibody fused to a fragment of Pseudomonas Exotoxin A (PE). One of these, Lumoxiti, is FDA approved for the treatment of drug-resistant hairy cell leukemia, another B-cell malignancy. LMB-75 is an immunotoxin with an anti-BCMA dsFv of mAb BM306 fused to domain III of PE, and LMB-70 is an anti-BCMA Fab fused to domain III of PE. We previously treated H929 myeloma cells grown as subcutaneous tumors with anti-BCMA immunotoxins that are very effective in killing cultured cells and cells from patients. We observed tumor growth inhibition but not complete tumor responses. Goal: Here we used H929 cells to develop a bone marrow mouse model that is more relevant to human disease and to determine if these immunotoxins are more active against H929 cells growing in bone marrow.

Methods: The NCI-H929 cell line was transfected with luciferase and GFP, recycled through the bone marrow of a mouse and enriched by flow to isolate cells with high GFP expression, which were used to inoculate tumors IV into NSG mice. Mice were treated IV with immunotoxins and tumor burden was assessed using bioluminescence imaging.

Results: H929-luc-GFP cells grow uniformly in the bone marrow of mice, cause hind limb paralysis around day 40, and provide a relevant in vivo model for testing RITs. We achieved complete responses when treating mice with 1.5 mg/kg LMB-75 or LMB-70 given QODX5 beginning on days 4 or day 8. Mice in all treatment groups were disease free at 3 months. An immunotoxin targeting CD22, which is not expressed on H929 cells, or an anti-BCMA immunotoxin with a mutation that inactivates toxin activity did not have any anti-tumor activity.

Conclusions: LMB-75 and LMB-70 induce complete, durable remissions of H929-luc-GFP cells growing in mouse bone marrow without toxicity to mice. These promising results support further pre-clinical development of these agents for MM.

#548

Evaluation of novel SIRP antibodies as potential cancer therapeutics.

Ronald R. Hiebsch, Myriam N. Bouchlaka, Benjamin J. Capoccia, Michael J. Donio, Prabir Chakraborty, W. Casey Wilson, Robyn J. Puro, Daniel S. Pereira. _Arch Oncology Inc., St. Louis, MO_.

Targeting immune checkpoints of adaptive immunity has shown great therapeutic efficacy in oncology, but in a limited fraction of patients. Innate immune cells represent the most abundant immune cell types in many solid tumors and are often linked to a poor prognosis.

SIRPα is expressed by innate immune cells and its interaction with CD47, expressed by most tumor cells, is an important immune checkpoint of the innate response, involved in the regulation of phagocytosis by macrophages, dendritic cells and neutrophils.

Recently, first generation agents targeting CD47 (CD47 antibodies and SIRPα-Fc fusion proteins) have shown promise in clinical trials, but they have also experienced hematological toxicities such as anemia or thrombocytopenia. Consequently, we have previously reported on the development of AO-176, a next generation anti-CD47 antibody that not only blocks the CD47/SIRPα interaction and induces phagocytosis, but also preferentially binds tumor versus normal cells (including RBCs where it binds negligibly) and directly kills tumor cells via a programmed cell death type III and an immunogenic cell death process.

Here we report the discovery of novel anti-SIRP antibodies that recognize either SIRPα selectively or SIRPα/γ. These antibodies are being evaluated for their ability to induce phagocytosis of tumor cells - we have identified antibodies that induce phagocytosis of tumor cells alone and in combination with Rituxan. The ability of our anti-SIRP antibodies to induce immunomodulatory activities in a variety of ex vivo cultured immune cells expressing either SIRPα or SIRPα/γ is also under investigation and will be presented.

#549

**A novel T cell engaging bispecific antibody induces specific and efficacious lysis of small cell lung cancer cells** in vitro **and potent T cell re-directed anti-tumor activity** in vivo **.**

Susanne Hipp,1 Vladimir Voynov,2 Barbara Drobits-Handl,1 Francesca Trapani,1 Craig Giragossian,2 Justin M. Scheer,2 Paul J. Adam1. 1 _Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria; _2 _Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT_.

Small cell lung cancer (SCLC) is the most lethal and aggressive subtype of lung carcinoma characterized by highly chemotherapy resistant recurrence in the majority of patients. To effectively treat SCLC we have developed a unique T cell engaging bi-specific antibody that can potently re-direct T cells to specifically lyse SCLC cells expressing Delta-like Ligand 3 (DLL3). DLL3 is frequently expressed on the cell surface of neuroendocrine tumors, with no to very little expression in normal tissues, thus making DLL3 an ideal targeting antigen with which to safely re-direct cytolytic T cells to tumor cells. The bi-specific monovalent DLL3/CD3 IgG-like T cell Engager (ITE) is designed to have sustained serum exposure and simultaneously bind to CD3 on T cells and DLL3 on SCLC cells, resulting in formation of the cytolytic synapse. In vitro, unstimulated peripheral blood mononuclear cells (PBMCs) were co-cultured with several DLL3 expressing SCLC cell lines and increasing concentrations of the DLL3/CD3 ITE. The DLL3/CD3 ITE induced potent dose-dependent lysis of SCLC cell lines with EC90 values ranging from 15 to 150 ng/mL, whereas viability of DLL3-negative cells was unaffected, demonstrating the specificity of the DLL3/CD3 ITE for the DLL3 antigen. In addition, the DLL3/CD3 ITE induced DLL3-dependent activation and proliferation of T cells, and granzyme B and cytokine secretion. In vivo anti-tumor activity of the DLL3/CD3 ITE was assessed in NOG mice bearing subcutaneous xenografts of the SHP77 SCLC cell line and reconstituted with human T cells. Complete tumor regressions were observed with a dose of 0.25 mg/kg administered once weekly by i.v. administration, with the onset of activity being observed after the first dose. Consistent with the mode-of-action the DLL3/CD3 ITE led to a profound infiltration of both CD8 and CD4 T cell subsets in the tumors, which correlated with cleaved caspase 3 expression on tumor cells and tumor shrinkage. The inflammatory tumor microenvironment that was created by treatment with the DLL3/CD3 ITE also led to an increase of PD-1 on T cells and PD-L1 on the tumor cells. The DLL3/CD3 ITE shows cross-reactivity to both DLL3 and CD3 of human and cynomolgus monkey origin respectively, thus allowing the assessment of pharmacodynamics, pharmacokinetics, and safety in cynomolgus monkeys. In a single dose study in cynomolgus monkeys, the DLL3/CD3 ITE demonstrated antibody-like pharmacokinetic properties. These pre-clinical data demonstrate that the DLL3/CD3 ITE is a highly potent, efficacious, and DLL3-selective T cell redirecting agent, and supports future clinical development.

#550

Tumor-conditional anti-CTLA-4 uncouples anti-tumor efficacy from immunotherapy-related toxicity.

Steven C. Pai,1 Donald M. Simons,2 Xiaoqing Lu,3 Wendy Ritacco,3 Michael Evans,1 Gillian Kingsbury,2 Lawrence Fong1. 1 _UCSF, San Francisco, CA;_ 2 _Agios, Worcester, MA;_ 3 _Abbvie, Worcester, MA_.

While immune checkpoint blockade leads to potent anti-tumor efficacy, it also leads to immune-related adverse events in cancer patients. These toxicities stem from systemic immune activation resulting in multi-organ inflammation including the gastrointestinal tract, lung, and endocrine organs. We developed a dual variable domain immunoglobulin of anti-CTLA-4 antibody (anti-CTLA-4 DVD) possessing an outer tumor-specific antigen-binding site engineered to shield the inner anti-CTLA-4 binding domain. Upon reaching the tumor, the outer domain is cleaved by membrane type-serine protease 1 (MT-SP1) present in the tumor microenvironment, leading to enhanced localization of CTLA-4 blockade. Anti-CTLA-4 DVD significantly reduced multi-organ immune toxicity by preserving tissue-resident regulatory T cells (Tregs) in Rag 1-/- mice that received naïve donor CD4+ T cells from wild-type C57BL/6j mice. Moreover, anti-CTLA-4 DVD induced potent antitumor effects by decreasing tumor-infiltrating Tregs and increasing the infiltration of antigen-specific CD8+ T lymphocytes in TRAMP-C2 bearing C57BL/6j mice. Tregs depletion was mediated through the antibody-dependent cellular cytotoxicity (ADCC) mechanism, as anti-CTLA-4 without the FcγR binding portion (anti-CTLA-4 DANA) spared Tregs, preventing treatment-induced toxicities. In summary, our results demonstrated a new approach to anti-CTLA-4 blockade that depletes tumor-infiltrating, but not tissue-resident Tregs, preserving anti-tumor effects while minimizing toxicity. Thus, our tumor-conditional anti-CTLA-4 DVD provides an avenue to uncouple anti-tumor efficacy from immunotherapy-induced toxicities.

#551

Human recombinant anti BSP antibody fused to cell penetrating peptides has improved cytotoxic properties.

Marineta Kovacheva,1 Michael Zepp,1 Franz-Paul Armbruster,2 Martin R. Berger1. 1 _German Cancer Research Ctr., Heidelberg, Germany;_ 2 _Immundiagnostik Comp., Bensheim, Germany_.

Elevated serum levels of bone sialoprotein (BSP) indicate subsequent bone metastasis and are related to poor survival of respective breast cancer patients. Here we describe the preclinical activity of a human recombinant anti-BSP antibody (AF165) in MDA-MB-231 human breast cancer cells in vitro and in vivo. In addition, this antibody was fused to cell penetrating peptides (CPPs) and the resulting fusion proteins were examined for their cellular uptake and antiproliferative activities.

AF165 was produced in HEK cells and tested for its affinity against BSP by microscale thermophoresis. Its antiproliferative activity was evaluated by MTT assay, the antimetastatic efficacy was determined in soft tissue and skeletal lesions, which had been induced in nude rats by intra-arterial injection of human MDA-MB-231 breast cancer cells. Fusion of AF165 to CPPs (Pep1, TAT) was accomplished by adding the respective DNA sequences with or without a linker (glycine-serine (GS)) to the AF165 gene. The affinity of these fusion proteins to BSP was measured by microscale thermophoresis, as well. In addition, their intracellular uptake and antiproliferative activities were determined by confocal fluorescent microscopy and MTT assay. Finally, changes in protein expression in response to these fusion proteins were detected by western blot.

AF165 showed no uptake into MDA-MB-231 cells and inhibited their growth in cell culture only marginally. However, this antibody induced dose dependent remissions in nude rats harboring MDA-MB-231 induced soft tissue and skeletal lesions. Subcutaneous administration of AF165 caused complete remission in 2 of 9 animals (10mg/kg/week), as well as in 3 of 6 animals (20 mg/kg/week). Interestingly, the affinity of AF165 (Kd = 7.7×10-7 M) was lower than that of fusion proteins, Kd values of which ranged from 3.6×10-8M (AF165-multiGS-Pep1) to 2.2×10-9M (AF165-multiGS-TAT). In contrast to AF165, all fusion proteins were taken up into MDA-MB-231 cells and after 24-48h caused morphologic changes, indicative of apoptosis. MDA-MB-231 cell proliferation was inhibited by 50 % after 48 hours of exposure to the conjugates AF-GS-TAT and AF-multiGS-Pep1 (600nM). The other conjugates caused similar cytotoxicity at higher concentration only (1200nM). The exposure to the fusion proteins led to downregulation of BSP protein levels and upregulation of proteins EGR1, ATF3 and ID2.

In conclusion, the human recombinant anti-BSP antibody AF165 is inactive in vitro, but highly active in a rat model mimicking skeletal metastasis. Fusion proteins of AF165 with CPPs show 10 to 100 fold higher affinity to BSP, as well as uptake into MDA-MB-231 cells, thus causing cytotoxicity in vitro. Therefore, the fusion proteins may have potential for antimetastatic therapy in vivo.

#552

Anti-tumor effects of anti-neoantigen antibodies: Tumor growth retardation and increased survival in a syngeneic model of cancer.

Girja S. Shukla, Yu-Jing Sun, Stephanie C. Pero, David N. Krag. _Department of Surgery & University of Vermont Cancer Center, University of Vermont Larner College of Medicine, Burlington, VT_.

This study was designed to decrease tumor growth by treatment with multiple anti-neoantigen antibodies (Abs) combined with PD1 inhibition. Dramatic reduction in tumor growth was observed. Despite tremendous excitement of checkpoint inhibitors only a minority of patients have durable complete responses. Success of checkpoint inhibitors appears related to the immune response to neoantigens. We investigated the potential enhancement of checkpoint inhibitors by directing a cocktail of antibodies to multiple neoantigens simultaneous with PD1 inhibition. The choice of neoantigens was simply based on the potential rabbit humoral immunogenicity of a mutated epitope present at the tumor cell surface or secretary proteins, without assigning any weightage to the protein function. Rabbits were vaccinated with each of the 9 selected mutated epitopes of B16-F10 melanoma tumor proteins for generating antibodies. Rabbit polyclonal Abs were affinity purified for each antigen with EC50s mostly in picomolar range. All 9 polyclonal antibodies were mixed equally as a cocktail for treatment experiments. The tumor model was C57BL/J strain of mice implanted with 3x105 B16-F10 melanoma tumor cells in their dorsal flank region. The treatment with anti-neoantigen Abs cocktail were administered in two different schedules; 1) a single treatment by implanting tumor cells suspended in 0.2 mg Abs cocktail/mouse and 2) local injection (0.2 mg Abs cocktail/mouse) into the tumor implantation site on 3, 6, 9 and 13 days post-tumor implantation. Except for a completely untreated control group all mice received 4 intraperitoneal injections (0.2 mg/mouse) of anti-mouse PD1 Ab (clone RMP1-14) or its isotype control Ab (IgG2a, k). Normal rabbit polyclonal IgG was used as control against the 9 anti-neoantigen Abs cocktail. No treatment resulted in rapid growth of large tumors and these untreated mice either died or were euthanized by day 20. The treatment with anti-PD1 Ab or its isotype control Ab alone did not affect tumor growth and survival in comparison to untreated mice. Furthermore, PD1 inhibition combined with normal rabbit IgG had no effect on tumor growth or survival. However, the single treatment of tumor cells with the 9 Abs cocktail during implantation combined with PD1 inhibition significantly retarded tumor growth and increased survival. The results were even more dramatic with the group of mice that were implanted with tumor cells in PBS and received 4 treatments of the 9 Abs cocktail in combination to PD1 inhibition. All the animals in this group had substantial retardation of tumor growth and 50% of the mice have shown no tumor whatsoever out to post-implantation day sixty. This is the first study to show anti-tumor effects of multiple anti-neoantigen Abs. These results are important particularly in a poorly immunogenic tumor model which is non-responsive to PD1 checkpoint inhibitor therapy alone.

#553

**Melanoma subtypes that emerge during adaptive resistance to therapy are targets for bispecific T cell engager (BiTE** ® **) antibody constructs directed to CDH19 and DLL3.**

Julie M. Bailis,1 Fei Lee,1 Michael Giffin,2 Paul Hughes,2 Jennifer Tsoi,3 Lidia Robert,3 Thomas G. Graeber,3 Antoni Ribas,3 Angela Coxon2. 1 _Amgen, Inc., South San Francisco, CA;_ 2 _Amgen, Inc., Thousand Oaks, CA;_ 3 _University of California Los Angeles, Los Angeles, CA_.

The treatment landscape for metastatic melanoma has changed considerably over the last several years with the approval of targeted therapy, such as BRAF inhibitors, and immunotherapy, such as immune checkpoint inhibitors that block cytotoxic T lymphocyte associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1). However, approximately 40% of patients do not respond to the current standard of care, and up to one-third of patients who initially respond to therapy can relapse. Novel therapies that act by a distinct mechanism of action may be needed to increase durable response rates in metastatic melanoma. Bispecific T cell engager (BiTE®) antibody constructs directly connect T cells to tumor cells and redirect T cell lysis to achieve tumor cell killing. BiTE®-mediated cytotoxicity is not dependent on a specific T cell receptor or peptide-MHC complex and has the potential to circumvent common immune resistance mechanisms. We have generated a BiTE® with half-life extension (HLE) that targets the atypical cadherin CDH19 on melanoma cells and CD3 on T cells (anti-CDH19/CD3 HLE BiTE®). We have also generated an HLE BiTE® that targets delta-like ligand 3 (DLL3) on tumor cells and CD3 on T cells, anti-DLL3/CD3 HLE BiTE®, or AMG 757. CDH19 and DLL3 are expressed on about half of primary and metastatic melanoma tumors prior to treatment. In this study, we investigated the expression of CDH19 and DLL3 and the activity of the corresponding BiTE® antibody constructs in a panel of 14 melanoma cell lines that were derived from patients that were resistant to either targeted therapy or immune checkpoint therapy and expanded the spectrum of recently defined multistage differentiation melanoma subtypes from melanocytic, transitional, neural crest-like and de-differentiated subtypes (Tsoi et al. Cancer Cell 2017). The cell surface expression of CDH19 and DLL3 was determined by flow cytometry. We then used T cell-dependent cytotoxicity (TDCC) assays to assess the activity of the anti-CDH19/CD3 HLE BiTE® and AMG 757 against the cell lines. 13/14 (93%) of the treatment-resistant melanoma cell lines expressed CDH19 on the cell surface, and 9/14 (64%) of the cell lines expressed cell surface DLL3. The anti-CDH19/CD3 HLE BiTE® and AMG 757 demonstrated potent cell killing of CDH19-positive or DLL3-positive melanoma cells, respectively, in TDCC assays, with EC50 values in the low picomolar range. These data show that melanoma cells that become resistant to targeted therapy and/or immune checkpoint blockade can be effectively targeted with BiTE® antibody constructs and suggest that the anti-CDH19/CD3 HLE BiTE® and AMG 757 may be potential novel therapies for treatment of relapsed and/or refractory metastatic melanoma.

#554

Tumor-antigen 5T4-dependent activation of the CD137 costimulatory pathway by bispecific 5T4 x CD137 x CD137 TRIDENT™ molecules.

Liqin Liu,1 Ling Huang,1 Vatana Long,1 Yinhua Yang,1 Robert Burns,1 Jonathan Li,2 Jennifer DiChiara,1 Qihong Xu,1 Arin Whiddon,1 Chia-Ying K Lam,1 Jim Tamura,1 Valentina Ciccarone,1 Syd Johnson,1 Ezio Bonvini,1 Paul Moore1. 1 _MacroGenics, Rockville, MD;_ 2 _MacroGenics, Brisbane, CA_.

Introduction: Trophoblast glycoprotein 5T4 is expressed on the cell surface of multiple cancers yet sparingly on normal adult tissues. CD137 (4-1BB) is a co-stimulatory molecule expressed by activated T and NK cells that support cell activation, proliferation and survival. Previously we described bispecific tumor associated antigen x CD137 DART® molecules limiting CD137 mediated immunostimulation to the tumor microenvironment (AACR2017) as an approach to reduce the unwanted systemic CD137 effects associated with agonistic anti-CD137 mAbs. We subsequently identified a bispecific TRIDENT format bearing bivalent CD137 and monovalent tumor antigen engagement that provides maximal CD137 activation in a tumor antigen anchored manner. Here we apply that format to generate a 5T4 x CD137 x CD137 TRIDENT molecule that promotes CD137 activation in proximity to 5T4-expressing tumor cells leading to enhanced T-cell co-stimulation, proliferation and tumor cell killing activity when combined with CD3-engaging tumor-targeting bispecific DART molecules.

Methods: TRIDENT molecules were constructed comprising bivalent CD137 binding and monovalent 5T4 binding. Binding properties were evaluated by surface plasmon resonance (SPR) and flow cytometry. Signaling was assessed using a NF-κB luciferase reporter cell line expressing CD137. Co-stimulatory activity was characterized with primary human or cynomolgus monkey T cells. T cells were incubated with or without antigen-expressing cells and submaximal concentrations of either anti-CD3 bead or CD3-engaging tumor-targeting bispecific DART molecules.

Results: SPR and flow cytometry analyses demonstrate that 5T4 x CD137 x CD137 TRIDENT molecule binds human and monkey target antigens. The 5T4 x CD137 x CD137 TRIDENT molecule induces CD137 signaling in a reporter cell line and promotes cytokine release in primary human & cynomolgus monkey T-cell assays in the presence of 5T4+ tumor cells. In the absence of 5T4+ tumor cells, the TRIDENT molecule lacks agonistic activity. Furthermore, 5T4 x CD137 x CD137 TRIDENT molecule enhances T-cell proliferation and tumor cell cytolysis induced by CD3-targeted DART molecules. Consistent with preferential induction of CD137 by CD8 T-cells, 5T4 x CD137 x CD137 TRIDENT increases the fraction of CD8+ central memory and effector memory T cells in the presence 5T4+ tumor cells.

Conclusions: 5T4 x CD137 x CD137 TRIDENT molecule promotes T-cell co-stimulation in a tumor antigen-dependent manner offering an opportunity to target CD137 immunostimulation, while potentially limiting non-specific systemic T-cell activation and related side effects.

#555

A novel human TNFR2 antibody (MM-401) modulates T cell responses in anti-cancer immunity.

James F. Sampson, Vinodh B. Kurella, Violette Paragas, Sandeep Kumar, James E. Lulo, James A. Qiu, Maja Razlog, Ross B. Fulton, Adam J. Camblin, Jennifer M. Richards, Christina S. Wong, Alexander Koshkaryev, James J. Suchy, Stephanie Grabow, Marco Muda, Andreas Raue, Daryl C. Drummond, Eric M. Tam. _Merrimack Pharmaceuticals, Cambridge, MA_.

TNFR2 is an emerging therapeutic target in immuno-oncology. We have previously shown that an agonistic mouse TNFR2 antibody was able to activate CD8+ T cells in vitro and has anti-tumor activity in multiple syngeneic mouse tumor models. This activity required CD8+ T cell and NK cell response and involves Fc γ receptor engagement. Based on this compelling evidence, we have developed MM-401, a human antibody targeting the TNFR2 receptor. MM-401 was humanized from a mouse hybridoma antibody by CDR grafting and includes additional mutations for improved affinity and biophysical properties. Consequently, MM-401 binds with low nanomolar affinity to a region in human TNFR2 that corresponds to the mouse TNFR2 epitope of our mouse surrogate antibody. Although the antibody competes with TNF α in binding the receptor, MM-401 has agonistic activity and induces TNFR2 signaling as observed using a NFκB reporter cell assay. Upon incubation of MM-401 with CD4+ and CD8+ T cells from healthy human blood, we observed upregulation of activation markers and cytokine production comparable to utomilumab (anti-4-1BB), MEDI6469 (anti-OX40), and TRX518 (anti-GITR). We also observed that MM-401 promotes antibody-dependent cellular cytotoxicity (ADCC) in an NK cell-mediated in vitro assay and a reduction in the number regulatory T (Treg) cells in ovarian cancer ascites samples. These data suggest that MM-401 could also promote anti-tumor immunity by mediating ADCC, as well as by direct co-stimulation of T cell responses. Currently, we are evaluating MM-401 in patient derived xenograft (PDX) models in humanized mice generated using NSG-SGM3 mice with cord blood CD34+ hematopoietic stem cells. These results justify the continued development of MM-401 as a modulator of anti-tumor immunity for the treatment of cancer.

#556

Discovery and characterization of next generation monoclonal antibodies targeting the inhibitory Fc gamma receptor CD32b for the treatment of B and plasma cell malignancies.

Haihui Lu, Dongshu Chen, Sunyoung Jang, Babette Wolf, Stefan Ewert, Meghan Flaherty, Fangmin Xu, Bernd Voedisch, Sinan Isim, Yeonju Shim, Meike Scharenberg, Xavier Leber, Eugene Choi, Rebecca Ward, Jennifer Johnson, Thomas Calzascia, Isabelle Isnardi, Juliet Williams, Heather Huet, Emma Lees, Matthew J. Meyer. _Novartis Insts. for BioMedical Research, Cambridge, MA_.

CD32b (FcγR2b), the sole inhibitory Fcγ receptor, negatively regulates immune function and is expressed throughout B cell development and on the malignant counterparts with the highest expression found on multiple myeloma. Additionally, CD32b expression on tumor cells is known to sequester IgG Fc thereby providing a mechanism of resistance to therapeutic monoclonal antibodies (mAb) with Fc dependent activity. Taken together, CD32b represents an attractive tumor antigen for targeting with a mAb. To this end, two anti-CD32b mAbs, NVS32b1 and NVS32b2, were developed. The complementarity-determining regions (CDRs) of these antibodies bind the CD32b Fc binding domain with high specificity and affinity while the Fc region is afucosylated enabling enhanced activation of FcγR on immune effector cells. This specificity and optimized potency is highlighted in whole blood assays where NVS32b2 depletes CD32b positive B cells but spares immune subsets with low CD32b expression or expression of the homologous CD32a. The antibodies mediate potent killing of opsonized cells via antibody dependent cell-mediated cytotoxicity, antibody dependent cellular phagocytosis, and complement dependent cytotoxicity. Additionally, NVS32b mAbs' CDR block the CD32b Fc binding domain, thereby minimizing CD32b mediated resistance to therapeutic mAbs with Fc dependent activity including rituximab, obinutuzumab, and daratumumab. In vivo, NVS32b mAbs demonstrate robust antitumor activity against CD32b positive xenografts and immunomodulatory activity including recruitment of intratumoral macrophages. The NVS32b mAbs' activity against malignant B and plasma cells featuring a range of CD32b expression demonstrates their therapeutic potential, as a single agent or in combination with therapeutics including mAbs with Fc dependent activity.

#557

COBRA: A novel conditionally active bispecific antibody that regresses established solid tumors in mice.

Danielle Dettling, Eilene Kwok, Lucy Quach, Jeremiah D. Degenhardt, Maia Vinogradova, Anand Panchal, Pui Seto, Jessica L. Krakow, Russell Wall, Brian J. Hillier, Ying Zhu, Robert B. DuBridge, Chad May. _Maverick Therapeutics, Brisbane, CA_.

Despite clinical success with bispecific antibodies (bsAbs) targeting hematological malignancies (e.g. blinatumomab, a CD19xCD3 bsAb), efficacy in solid tumor indications remains a significant challenge. Because T cell redirecting bsAbs are so potent, even very low levels of cell surface target antigen expression on normal tissues may quickly become a safety liability and severely restrict the dose levels that can be achieved in patients. This limits the likelihood of reaching efficacious concentrations and reduces the therapeutic potential of these highly active molecules. Additionally, identifying "clean" target antigens that are uniquely expressed on the tumor and not on normal tissues has been very difficult at best.

To overcome these challenges, we have developed a novel recombinant bsAb platform called COBRA™ (Conditional Bispecific Redirected Activation). COBRAs are engineered to enable targeting of more widely expressed and validated tumor cell surface antigens by focusing T cell engagement within the tumor microenvironment. COBRA molecules are designed to bind to target antigen, which may be expressed on both tumor and normal cells, yet not engage T cells unless exposed to a proteolytic microenvironment, which is common in tumors but not in normal healthy tissues. Once bound to the tumor target antigen, protease-dependent linker cleavage allows COBRAs to convert an inactive anti-CD3 scFv to an active anti-CD3 scFv binding domain. Upon conversion, COBRAs are then able to simultaneously co-engage T cells and target antigen, resulting in a potent cytolytic T cell response against the tumor cells. In addition, COBRAs are designed with a half-life extension moiety that is removed from the active molecule upon proteolytic cleavage. This allows for a sustained presence in the circulation of the inactive COBRA prior to tumor target binding, and more rapid clearance of unbound active COBRA molecules, thereby decreasing the potential for cytotoxic activity in normal tissues.

Here we reveal the novel design of the COBRA molecule and demonstrate its ability to engage CD3 and Epidermal Growth Factor Receptor (EGFR) to elicit potent cytotoxic activity in T cell culture and in human T cell implanted tumor-bearing mice. We report low-to-sub-picomolar T cell activation and cytotoxicity in vitro, and COBRA linker cleavage dependent T cell mediated regression of established solid tumor xenografts in NSG mice in vivo.

#558

MERTK-specific antibodies that have therapeutic antitumor activity in mice disrupt the integrity of the retinal pigmented epithelium in cynomolgus monkeys.

Kerry F. White, Matthew Rausch, Jing Hua, Katherine H. Walsh, Christine E. Miller, Christopher C. Wells, Devapregasan Moodley, Benjamin H. Lee, Scott C. Chappel, Pamela M. Holland, Jonathan A. Hill. _Surface Oncology, Cambridge, MA_.

MERTK, a member of the TAM (TYRO3, AXL, MERTK) family of receptor tyrosine kinases, is a pleiotropic immune modulator that controls efferocytosis. Engagement of MERTK with its ligand GAS6, found anchored to phosphatidylserine exposed on the outer membrane of apoptotic cells, triggers MERTK phosphorylation and signaling events that culminate in the removal of apoptotic debris. Recent studies have highlighted the expression of MERTK on tumor-associated macrophages, and Mertk-deficient mice show reduced tumor cell growth accompanied by inflammatory cytokine production and alterations in macrophage activation. Thus, MERTK has emerged as a promising therapeutic target for augmenting innate antitumor immune responses. MERTK is also expressed in retinal pigmented epithelium (RPE) cells of the eye where it mediates phagocytosis of photoreceptor outer segment fragments. Mutations in MERTK that disrupt its expression or kinase activity lead to marked retinal degeneration and blindness in mice, rats, and humans. Due to known differences in blood-retinal permeability, we explored whether therapeutic antibodies targeting MERTK could inhibit macrophage-mediated efferocytosis and promote antitumor activity while sparing RPE toxicity. A diverse panel of high-affinity antibodies was developed to explore MERTK blockade in vitro and in vivo. Multiple antibodies disrupted MERTK-GAS6 binding and blocked human and murine macrophage-mediated efferocytosis. Two antibodies targeting distinct GAS6 binding epitopes were selected for further characterization. Both antibodies demonstrated antitumor activity in murine CT26 and MC38 syngeneic colorectal cancer models and led to alterations in immune cell-related gene expression. To investigate potential effects on RPE biology with MERTK antibodies, a multi-dose, 4-week cynomolgus monkey study with several in-life and post-mortem ophthalmologic endpoints was designed. While no abnormal ophthalmic or electroretinography (ERG) findings were detected, all animals treated with either MERTK antibody at all doses showed histological abnormalities of the retina, including vacuolation of the outer segments of photoreceptors, displacement of RPE cells, and single cell necrosis of the outer nuclear layer. These data suggest that inhibition of efferocytosis by antibody-mediated blockade of MERTK can promote immune activation and inhibit tumor growth in vivo; however, retinal toxicity consistent with histological observations made in Mertk mutant animals is an on-target effect. As several therapeutics that block MERTK function are currently in preclinical development, a thorough evaluation of retinal toxicity is warranted.

#559

Preclinical characterization of the bispecific EGFR/CD16A innate immune cell engager AFM24 for the treatment of EGFR-expressing solid tumors.

Michael Kluge, Uwe Reusch, Stefan Knackmuss, Torsten Haneke, Susanne Wingert, Michael Damrat, Michael Tesar, Martin Treder. _Affimed, Heidelberg, Germany_.

Affimed has engineered AFM24 based on its Redirected Optimized Cell Killing (ROCK®) platform to redirect innate immune cells to EGFR-positive tumor cells with the potential for activation of a broad anti-tumor immune response. This fully human tetravalent bispecific EGFR and CD16A binding antibody was designed for the treatment of EGFR-expressing malignancies with the potential to overcome the resistance to other EGFR-targeting agents and offering a better safety profile. The efficacy of current standard of care (SOC), including EGFR-targeting monoclonal antibodies (mAbs) and tyrosine kinase inhibitors (TKIs), depends on intrinsic or acquired mutations that impact EGFR signal transduction. AFM24 exerts a mutation-independent mechanism of action (MOA), redirecting innate immunity via NK cells or macrophages to EGFR-positive tumor cells.

PK analyses of AFM24 revealed a half-life of ~14 days in mice and ~5 days in cynomolgus monkeys. Using radioactively labeled AFM24 in mice, tumor-specific accumulation in subcutaneous human tumors was observed. While binding to CD16A by classical mAbs has low affinity and is further impaired by competition with serum IgG, no substantial changes in the very high binding affinity of AFM24 to NK cells were observed in the presence of IgG. Anti-tumor efficacy has been shown in in vitro cytotoxicity assays and in in vivo tumor models. Due to its MOA, AFM24 was also active against tumor cell lines expressing mutated Ras in in vitro assays. Antibody-dependent cellular phagocytosis (ADCP) of tumor cell lines expressing high levels of EGFR was comparable between AFM24 and classical or Fc-enhanced mAbs, but ADCP of tumor cells with lower EGFR expression mediated by AFM24 was more pronounced.

Owing to their MOA of blocking EGFR-induced signaling, the therapeutic use of current SOC is associated with severe side effects such as skin toxicity, potentially limiting its use due to dosing interruptions and/or treatment termination. AFM24 is designed to exhibit a reduced potential to inhibit EGFR signaling, aiming for an improved safety profile. In a 4-week toxicology study in cynomolgus monkeys, no skin or organ toxicity was observed at AFM24 doses between 24 and 75 mg/kg i.v. once weekly. In toxicology studies investigating EGFR-targeting mAbs, e.g. cetuximab, with a similar experimental design, severe toxicities have been observed at those dosages.

In summary, AFM24 is a novel, differentiated bispecific innate immune cell engager binding to EGFR and CD16A, thereby redirecting tumor cell killing by NK cells and macrophages to EGFR-expressing tumors. Its preclinical characterization described here, encourages AFM24's development for the treatment of EGFR-expressing tumors with the potential to overcome resistance to current SOC while conferring a favorable safety profile. IND-enabling preclinical development is ongoing.

#560

**Tumor-localized, rather than systemic, PD-L1/PD-1 blockade renders the co-administration of áCTLA-4 IgG more tolerable while exhibiting potent anti-tumoral activity** in vivo **.**

Tea Gunde,1 Alexandre Simonin,1 Stefan Warmuth,1 Christian Hess,1 Matthias Brock,1 Eva Oswald,2 Julia Zeberer,1 Dania Diem,1 Dana Mahler,1 Diego Morenzoni,1 Simone Muntwiler,1 Benjamin Küttner,1 Robin Heiz,1 Sebastian Meyer,1 Timothy Egan,3 David Urech4. 1 _Numab Innovation AG, Wädenswil, Switzerland;_ 2 _Charles River Discovery Service Germany GmbH, Freiburg, Germany;_ 3 _Numab US, LLC, Chevy Chase, MD;_ 4 _Numab Therapeutics AG, Pfäffikon, Switzerland_.

Given the overlapping expression of multiple immune-checkpoint receptors in tumor microenvironments (TMEs), the rapid growth of clinical trials evaluating combinations of therapeutic, immunomodulating mAbs has been a logical result of efforts to improve clinical outcomes for cancer patients. Early validation of the potential of combination immunotherapies in cancer emerged with the market approval of αPD-1 IgG + αCTLA-4 IgG regimens. The pharmacological activities of αPD-1 and αCTLA-4 IgGs synergize to generate impressive response rates among patients. Unfortunately, the fairly tolerable toxicities associated with each component IgG also tend to synergize, frequently necessitating discontinuation of their combined administration. We hypothesized that synergistic toxicities in this setting are a consequence of the extratumoral activity of both IgGs simultaneously, and, therefore, toxicities could be considerably reduced by tumor-localizing the blockade of at least one of these two checkpoint pathways. Because PD-L1 is expressed on the surface of tumor cells that also express tumor-specific antigens, we surmised that multi-specific technology could be leveraged to localize blockade of PD-1 signaling and enable tolerable co-administration of systemically active αCTLA-4 IgGs. To test this hypothesis, we generated a HER2/PD-L1/serum albumin (SA) tri-specific scDb-scFv molecule (three monovalent antibody Fvs in a single chain). Critically, the relative affinity between the molecule's αPD-L1 and αHER2 domain is carefully balanced to ensure that HER2-engagement is a prerequisite for PD-L1 blockade. Meanwhile, the αSA domain extends serum half-life and should also promote tumor-localization of the molecule. In the present study, we demonstrate the successful development of a HER2/PD-L1/SA tri-specific scDb-scFv that potently blocks PD-L1 specifically on HER2+/PD-L1+ cells but not on HER2- cells. In a HCC1954 breast cancer xenograft model, the scDb-scFv molecule exhibited potent antitumoral activity both as a monotherapy and in combination with ipilimumab (αCTLA-4 IgG) in humanized mice. Finally, we demonstrate that the tri-specific molecule is better tolerated than the combination of nivolumab and ipilimumab, as determined by its reduced tendency to exacarbate xenograft versus host disease (xGvHD) in hPBMC-supplemented NOG mice. In conclusion, tumor-localized PD-L1 inhibition, enabled by next-generation multi-specific technology, appears to be a promising strategy to preserve the efficacy and enhance the tolerability of combined immunomodulation of PD-L1/PD1 and CTLA-4.

#561

Discovery of a potential best-in-class anti-CD38 therapeutic utilizing Fc multimerization.

Amit Choudhury, Daniel F. Ortiz, Shannon Argueta, Kevin Garofalo, Jonathan C. Lansing, Utsav Jetley, Danice Wilkins, Carlos Bosques, Edward Cochran, Naveen Bhatnagar, Jay Duffner, Abhinav Gupta, Stan Lee, Karunya Srinivasan, Viraj Parge, Radouane Zouaoui, Jason Wang, Anthony M. Manning. _Momenta Pharmaceuticals, Cambridge, MA_.

CD38 targeting antibodies are at different phases of clinical development, with daratumumab already approved as monotherapy and in combination with standards of care in multiple myeloma (MM). Anti-CD38 monoclonal antibodies (mAbs) induce tumor cell depletion in part by Fc-dependent effector mechanisms such as antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), and complement dependent cytotoxicity (CDC). However, not all MM patients achieve minimal residual disease (MRD)-negativity and similar clinical response. In addition, some patients on daratumumab develop resistance due to reduced cell surface CD38 and high levels of complement inhibitors (CD55 and CD59). We have leveraged Fc multimerization technology (Ortiz et al Sci Transl Med. 2016; 8: 365) to generate an optimized platform (SIF; selective immunomodulator of Fc receptors) that utilizes the valency effect of Fc multimerization to enhance binding to the Fcγ receptors and complement. We combined the Fab-region of CD38 targeting mAb to SIF platform to generate an anti-CD38 SIFbody to enhance immune and complement mediated cytotoxicity against tumor cells. In several human tumor cell line-based cytotoxic assays using primary human effector cells (NK cells and macrophages) and complement, the anti-CD38 SIFbody demonstrates up to 10-fold increase in efficacy and ≥16-fold increase in potency compared to daratumumab and the surrogate therapeutic anti-CD38 mAb (TAK-079). In isolated whole human blood incubated with tumor cells, the anti-CD38 SIFbody demonstrated 40-100 fold increase in potency and 2-3 fold increase in efficacy. In bone marrow cells isolated from MM patients with >80% plasma cells anti-CD38 SIFbody showed better potency and a 3-5 fold increased efficacy (with 100% plasma cell elimination) than daratumumab, suggesting the SIFbody may be more suitable molecule for achieving greater MRD-negativity rates in MM patients. Daratumumab fails to induce CDC against tumor cell lines with low CD38 and high CD55 and CD59, however the SIFbody achieves 100% efficacy in such settings, suggesting this molecule may be effective in patients who are developing resistance to treatment. In single dose pharmacodynamic and tolerability studies in cynomolgus monkeys SIFbody demonstrated up to 5-fold increase in B cell depletion from peripheral blood compared to TAK-079 across all dose ranges (0.3, 1, & 3 mg/kg) tested without any adverse events. Therefore, by leveraging our Fc multimerization technology we have generated a differentiated potential best-in-class anti-CD38 therapeutic.

#562

Antibodies to Sirpα enhance innate and adaptive immune responses to promote anti-tumor activity.

Tracy C. Kuo, Amy Chen, Ons Harrabi, Steven E. Kauder, Emma R. Sangalang, Laura Doyle, Sangeetha Bollini, Bora Han, Janet Sim, Jaume Pons, Hong I. Wan. _ALX Oncology, Burlingame, CA_.

Targeting the CD47-SIRPα myeloid checkpoint pathway represents a novel therapeutic approach to enhance anti-cancer immunity. CD47 is a widely expressed cell surface protein that functions as a marker of self. Signal regulatory protein-α (SIRPα) is an ITIM-containing inhibitory receptor of CD47 on myeloid cells. Tumor cells up-regulate CD47 expression to evade immune surveillance. Clinical trials targeting CD47 to inhibit SIRPα interaction have shown promising results (N Lakhani et al, 2018 SITC #P335). Unlike CD47, which is expressed ubiquitously, SIRPα has limited expression, mainly on myeloid cells including macrophages, dendritic cells, and neutrophils, and neurons. Targeting SIRPα circumvents the need to overcome the CD47 sink that is necessary for CD47 targeting molecules. Antibodies to SIRPα that bind mouse, human and cynomolgus monkey SIRPα with high affinity, were evaluated for their effects on macrophage function, anti-tumor activity and normal blood cells.

Humanized antibody to SIRPα was generated to bind both human SIRPα allele V1 and V2 with high affinity (pM) and cross-react with cynomolgus and mouse SIRPα. Anti-SIRPα antibody in combination with targeted anti-cancer antibodies enhanced antibody-dependent cellular phagocytosis (ADCP) of tumor cell lines by human monocyte derived macrophages. In preclinical xenograft models of SIRPα positive and negative malignant cell lines, administration of anti-SIRPα antibody enhanced anti-tumor activity of rituximab and cetuximab via ADCP.

The impact of SIRPα blockade on anti-tumor immunity was assessed in immunocompetent tumor mouse models. SIRPα antibodies enhanced anti-tumor activity of checkpoint inhibitors in syngeneic tumor models. Combination therapy with SIRPα antibodies and checkpoint inhibitors in tumor models showed eradication of tumors, acquired memory immune response upon tumor rechallenge and decreased lung metastasis. The cellular mechanisms driving efficacy include dendritic cell activation, reduction of myeloid suppression and enhancement of T cell effector profile.

Exploratory safety and PK/PD profiles of anti-SIRPα antibodies were assessed in cynomolgus monkeys. Antibodies to SIRPα demonstrated a typical antibody PK and complete SIRPα target occupancy. There were no adverse clinical observations, no changes in hematological parameters and no impact on serum chemistries.

In summary, anti-SIRPα antibodies combined with targeted anti-cancer antibodies or checkpoint inhibitors integrate innate and adaptive immune responses to promote anti-tumor activity in preclinical models. Together with the favorable safety profile in cynomolgus monkeys, these data support the development of SIRPα antibodies for solid tumors and hematological malignancies.

#563

A biparatopic agonist antibody for OX40 that exhibits superior activity without secondary crosslinking.

Bonnie Hammer,1 Mandar Bawadekar,1 Matthew Bissen,1 John Painter,1 Lauren Lehmann,1 Francis Qufei Li,2 Lucas Bailey,2 Bryan Glaser,1 Roland Green1. 1 _Invenra, Madison, WI;_ 2 _Former Invenra, Madison, WI_.

The development of agonistic antibodies that activate T-cell co-stimulatory pathways represents a therapeutic strategy with significant clinical potential. However, challenges remain for the translation from in vitro efficacy to clinical success. OX40 and other tumor necrosis factor receptor (TNFR) superfamily members are notorious for requiring high-order receptor clustering in order to achieve full activity. For monoclonal antibodies, this high-order clustering is generally achieved through secondary cross-linking strategies. In vivo, this secondary cross-linking is often supplied through internal immune effector cells via Fc engagement. Bispecific and biparatopic antibodies represent an emerging class of drug molecules that enable unique mechanisms of action relative to their monoclonal counterparts. Here, we describe the use of our bispecific platform for the generation of large panels of biparatopic antibodies which enabled high-throughput screening for the discovery of an array of OX40 agonistic molecules. We have optimized these multivalent antibodies that exceed the potency of the OX40 ligand in NFkB activation without the need for secondary cross-linking. These agonist antibodies have additionally been characterized using primary T cell assays to monitor the kinetics of growth proliferation and cytokine secretion, outperforming cross-linked antibodies currently being tested in clinical trials. In co-culture systems, these agonist antibodies were effective in inhibiting immuno-suppressive properties of Tregs. Our lead OX40 agonist antibody has been optimized for activity and developability and has entered stable cell line development to further support pre-clinical activities.

#565

A novel T-cell costimulatory Humabody® VH therapeutic for PSMA-positive tumors.

James W. Legg, Brian McGuinness, Sophie Archer, Phil Bland-Ward, Verena Brucklacher, Jenny Craigen, Carolyn Edwards, Emma Hames, Jay Majithiya, pavel Pisa, Bhindu Revi, Nikki Royale, Angelica Sette, Yumin Teng, Lorraine Thompson, Wembin Wang, Chris Wilson, Chris Wyre, Chris Rossant. _Crescendo Biologics, Cambridge, United Kingdom_.

Agonistic monoclonal antibodies targeting CD137/4-1BB have shown much preclinical promise but their clinical development has been slowed due to a poor therapeutic index, in particular liver toxicity. CB307 is a novel half-life extended bispecific Humabody VH targeting CD137 (4-1BB) and prostate specific membrane antigen (PSMA). The design of CB307 enables agonism of CD137 selectively in the presence of PSMA positive tumour cells and in this way enable tumour selective T cell activation whilst minimising systemic activation. The molecular weight of CB307 is less than 50 kDa (around a third of the size of a standard IgG) and it does not contain an Fc domain, thereby avoiding interaction with Fc receptors. Half life extension is achieved through the inclusion of a VH domain with specificity for human serum albumin (HSA). The identification of CB307 using the Crescendo Mouse™ which develops fully human VH domains in a background devoid of light chains will be described along with characterisation of the key properties of the molecule in in vitro and in vivo models. In dual target binding assays CB307 shows potent co-binding to both PSMA and CD137 targets and mediates CD137 signalling in an NFKB cell reporter assay in the presence of PSMA positive cells but not PSMA negative cells. Co-incubation of primary human T-cells from healthy individuals or cancer patients together with PSMA positive tumour cells and CD3 stimulation induces T-cell activation and cytokine release. In an in vivo model using NSG mice engrafted with human PBMCs the growth of PSMA positive DU145 prostate tumour cells is inhibited by a surrogate bispecific. Together these data support progression of CB307 into clinical development.

### Tumor Antigens and Neoantigens

#566

Neoantigens from translated unannotated open reading frames in cancer.

Tamara Ouspenskaia,1 Travis E. Law,1 Karl R. Clauser,1 Susan Klaeger,1 Derin B. Keskin,2 Bo Li,1 Elena Christian,1 Yuen Ting Chow,3 Phuong M. Le,2 Joshua Gould,1 Zhe Ji,4 Wandi Zhang,2 Pavan Bachireddy,2 Siranush Sarkizova,5 Nir Hacohen,6 Steven A. Carr,1 Catherine J. Wu,2 Aviv Regev1. 1 _Broad Institute of MIT and Harvard, Cambridge, MA;_ 2 _Dana Farber Cancer Institute, Boston, MA;_ 3 _Brookline High School, Brookline, MA;_ 4 _Northwestern University, Chicago, IL;_ 5 _Harvard University, Cambridge, MA;_ 6 _Massachusetts General Hospital, Boston, MA_.

Somatic mutations in cancer cells can generate neoantigens which can be recognized by immune cells and trigger an immune response. Patients vaccinated with neoantigen-based peptides display expanded neoantigen-specific T cells, suggesting that this could be a promising avenue for cancer treatment. Currently, neoantigen predictions are based on mutations detected by whole exome sequencing, which covers a pre-determined set of annotated exons, and often falls short for cancers with few somatic mutations.

Ribosome profiling (Ribo-seq), which allows to monitor mRNA translation, has predicted a plethora of translated novel unannotated ORFs (nuORFs). Here we hypothesized that nuORFs can provide another source of neoantigens in cancer cells. In particular, we focused on nuORFs in the following categories: 1) nuORFs expressed in healthy and cancer cells, that have acquired tumor-specific somatic mutations; 2) nuORFs upregulated in or specific to cancer cells.

To explore this hypothesis, we performed Ribo-seq on primary healthy and cancer cells and cell lines from melanoma, glioblastoma, colon carcinoma and chronic lymphocytic leukemia. Using this extensive dataset, we performed hierarchical ORF prediction analysis to build a database of highest confidence predicted translated nuORFs across healthy and cancer cell types.

To determine if peptides from nuORFs can be a source of antigens, we searched our collection of mono-allelic MHC class I immunopeptidome mass spectrometry (MS) spectra from 94 common HLA alleles against our pan-tissue nuORF database. Additionally, we performed MHC class I immunoprecipitation followed by MS on the same cells used for Ribo-seq. We found HLA-presented unmutated peptides derived from thousands of nuORFs, found within, but out-of-frame with annotated protein-coding ORFs, within 5' and 3' untranslated regions of annotated protein-coding transcripts, long non-coding RNAs (lncRNAs), pseudogenes, and other RNA species. The HLA-binding motifs of peptides from nuORFs correspond to the expected motifs for given HLA types, indicating that 1) nuORFs are translated and 2) nuORF-derived peptides are presented on MHC I.

To identify tumor-specific somatic mutations in nuORFs, we performed whole genome sequencing on patient-matched healthy and cancer cells and mapped somatic mutations to annotated ORFs and nuORFs. Finally, to identify nuORFs upregulated in or specific to cancer cells, we compared translation levels of nuORFs between healthy and cancer cells of the same origin. We found translated nuORFs with cancer-specific somatic mutations and nuORFs highly upregulated in and specific to cancer cells, suggesting that they can give rise to neoantigens.

In conclusion, nuORFs are translated, contribute peptides to MHC I presentation, acquire somatic mutations, are expressed in tissue- and cancer-dependent manner and should be considered in the search for neoantigens in cancer.

#567

Comparative mining of normal and tumor tissue RNAseq gene expression datasets to define expression-based neo-antigens.

Jan O. Kemnade, Maria Cardenas, David Wheeler, Andrew G. Sikora, Mitchell J. Frederick. _Baylor College of Medicine, Houston, TX_.

Background: Mutational neo-antigens (MuNeoAgs) are attractive targets for cancer vaccines because they are unique to tumors. However, limited epitope potential of point mutations, inherent difficulties predicting immunogenicity, and lack of shared mutations across patient tumors has hindered vaccine development. Alternatively, we elected to identify non-mutated, expression-based tumor-specific neo-antigens (EbNeoAgs) defined by their overexpression in tumors and negligible expression in healthy tissue. EbNeoAgs have a low probability of cross-reactivity, while allowing epitope creation across the entire protein. Additionally, they should be present in a higher proportion of patients and could be used to make modular off-the-shelf vaccines.

Methods: RNAseq data from the NIH Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) projects, previously harmonized with a common calling/ mapping algorithm and available from the University of California Santa Cruz TOIL RNAseq recompute project, were used to calculate upper quartile FPKM gene expression values for all samples based on protein coding RNAs. RNAseq data from 27 normal tissue types (excluding testis) from GTEx and TCGA normal tissue was analyzed to identify genes with negligible expression across all healthy tissue. Genes with negligible expression were then analyzed to determine if they were elevated (i.e. expressed at intermediate or higher levels) in samples across 32 different cancer types (excluding testicular tumors). Thresholds for intermediate expression were calculated individually for each tumor type based upon the median FPKM expression values for all protein coding genes with 95% confidence intervals.

Results: 1296 genes were expressed below threshold in all healthy tissues, and 107 of these genes were elevated in 5% or more of samples in at least one cancer. 40/107 EbNeoAgs (37%) are known Cancer Testis Antigens (CTAs), with the remaining 67 representing potentially novel EbNeoAgs. If the threshold for elevated expression is lowered to just 1% of samples for at least one cancer, 58 of the known 276 CTAs are detected. The remaining CTAs are overexpressed in at least one healthy tissue or were not elevated in cancer. In melanoma (cancer with the most EbNeoAgs), 53 (52.0%), 69 (67.6%), and 88 (86.3%) samples expressed at least 5, 3, or 1 EbNeoAg respectively. Comparatively, in lung adenocarcinoma, 81 (15.8%), 97 (18.9%), and 228 (44.4%) samples expressed at least 5, 3, or 1 EbNeoAg respectively.

Conclusion: We leveraged publicly available tumor and normal RNAseq datasets to comprehensively identify EbNeoAgs in an unbiased fashion across 32 tumor types. Many of the EbNeoAgs were known CTAs, but a significant number of EbNeoAgs were novel. Approximately half the cancers exhibited EbNeoAg expression in a large proportion of patients making them broad targets for cancer vaccine development.

#568

Decoding shared antigenic epitopes and their cognate TCR genes in melanoma TILs using a library of paired human cell-based pHLA multimers and artificial APCs.

Kenji Murata, Kayoko Saso, Linh T. Nguyen, Douglas Millar, Marcus O. Butler, Pamela S. Ohashi, Naoto Hirano. _Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada_.

HLA-restricted T cell responses toward immunogenic peptides, whether mutated or non-mutated, can induce antitumor responses in patients with advanced cancer. The fact that potential non-mutated antigens are greater in number than mutated antigens by multiple orders of magnitude and the high polymorphism of HLA genes may have hampered comprehensive analyses of the specificity of antitumor T cell responses toward non-mutated antigens than mutated antigens. Unlike shared antigens, the vast majority of neoantigens are not shared and are unique to each patient. The elucidation of T cell epitopes derived from shared antigens may facilitate the robust development of an efficacious and safe adoptive T cell therapy that is readily available to a larger cohort of cancer patients. It is well established that melanoma tumor-infiltrating T lymphocytes (TILs) contain antitumor T cells that are specific for both non-mutated and mutated antigens. The adoptive transfer of TILs can induce sustained clinical responses in some patients with advanced melanoma. However, a precise and extensive understanding of the shared antigen targets of TILs has been lacking. In this study, TILs were isolated from 8 metastatic melanoma patients, polyclonally expanded in vitro, and their shared antigen specificities for all 45 (25 different) class I alleles were examined. The combination of structure-based analysis using human cell-based peptide/HLA (pHLA) multimers and functional analysis using artificial antigen-presenting cells (APCs) were used to determine antigen-specific T cell responses. We were able to determine the specificity of 12.2 ± 7.2% (mean ± SD, max 25.8%, min 4.6%) of CD8+ T cells in an expanded TIL culture toward 3.0 ± 1.8 (mean ± SD, max 6, min 1) previously known or novel peptides derived from shared antigens. Furthermore, we isolated a number of cognate TCR genes with potent tumor reactivity from the CD8+ T cells. The strategy employed in this study using a library of paired human cell-based pHLA multimers and artificial APCs has enabled us to decipher the antigen specificity of tumor-specific T cells for any given HLA class I allele, regardless of allele frequency and for an infinite number of peptides. It has also allowed us to build a large database of class I-restricted peptides and cognate tumor-reactive TCR genes at an unprecedented scale. This database will be a valuable tool in defining the immune response at the level of each individual cancer patient with precision as well as the identification and validation of biomarkers to aid in patient-based selection of a cancer immunotherapy regimen. Furthermore, this database will help the robust development of novel cancer vaccines and TCR gene therapies for patients with a low mutation burden.

#569

Expression and modulation by IFN of HLA class I APM components in melanoma: Potential biomarkers of clinical response to checkpoint inhibitors.

Lidia Giraudo,1 Loretta Gammaitoni,1 Bortolot Valentina,2 Ilenia Iaia,3 Giulia Cattaneo,3 Paolo Becco,3 Susanna Gallo,1 Alessandro Zaccagna,1 Alberto Pisacane,1 Luca Crotto,1 Soldano Ferrone,4 Massimo Aglietta,3 Fabrizio Carnevale Schianca,1 Dario Sangiolo3. 1 _Candiolo Cancer Institute FPO - IRCCS, Candiolo (TO), Italy;_ 2 _University of Torino, Candiolo (TO), Italy;_ 3 _University of Torino, Candiolo Cancer Institute FPO - IRCCS, Candiolo (TO), Italy;_ 4 _Massachusetts General Hospital, Harvard Medical School, Boston, MA_.

Purpose: The goals of this study are i) to analyze the expression of human leukocyte antigen class I (HLA-I) antigen processing machinery (APM) components in surgically removed melanoma tumors, ii) to assess their modulation by Interferons (IFNs) and iii) to determine their value as predictive biomarkers of the clinical response to immunotherapy with checkpoint inhibitors (CI).

Methods: The expression levels of HLA class I APM components [HLA-I (ABC) chains, beta(2)microglobulin (β2m), Calnexin, Calreticulin, ERp57, Tapasin, TAP1-2, LMP2, LMP7, LMP10] were evaluated by staining with mAbs and flow cytometry in melanoma cells isolated from surgical biopsies from 16 patients with advanced melanoma. In addition the modulation of these molecules by IFN-γ or IFN-α2 (IFNs) was evaluated.

Results: The frequency of cells expressing HLA-I/β2m was >90% in 15/16 Mel tumors. In 1 case extracellular HLA-I/β2m molecules were not detected. The expression levels of extracellular HLA class I APM molecules were quite heterogeneous. Based on Mean Fluorescence Intensity (MFI) of HLA-I/β2m, Mel segregated into 2 groups with high (MFI >200, n=8) and low (MFI<200, n=8) expression levels. In 11 cases (5 from the high and 6 from the low expression groups) we could explore the modulatory activity of IFNs. In vitro exposure to IFNs globally enhanced the expression levels of HLA-I/β2m, allowing the transition from the low to the high expression group in 1 case. The distribution of expression defects in the intracellular APM components resulted intensely heterogeneous. Highly defective (<25%) expressions were detected notably for LMP2 (12/16), TAP1 (13/16), TAP2 (10/16) and Tapasin (14/16). In 10 cases we could explore the beneficial modulatory activity of IFNs on defective APM components. IFNs could restore the expression of LMP2 (4/10), TAP1 (6/10), and TAP2 (7/8), but not that of Tapasin. Seven patients were treated with CI (either anti-PD1 or anti-CTLA4). Four of them had clinical responses (PR or CR); in 3 of them Mel expressed high levels of HLA-I/β2m. Conversely, Mel from all 3 patients that did not respond to CI expressed low HLA-I/β2m levels which could not be upregulated by IFNs. Highly defective expression of APM components, not responsive to IFN restoration, was detected in all 3 Mel from non-responders.

Conclusions: We report a wide heterogeneous distribution of HLA-I/β2m expression levels and deficits of APM components in Melanoma, partially restorable by IFNs. Our data support the possibility that defects in HLA expression, not responsive to IFN restoration, may predict low responsiveness to CI. We provide rationale to incorporate the evaluation of HLA-I/β2m and APM in clinical immunotherapy studies with CI.

#570

A glioblastoma translational pipeline: discovery of novel tumor antigens that drive GBM recurrence.

Parvez Vora,1 Chitra Venugopal,1 Chirayu Chokshi,1 Maleeha Qazi,1 Nazanin Tatari,1 Kevin Brown,2 Nicholas Yelle,1 Jarrett Adams,2 David Tieu,2 Mathieu Seyfrid,1 Mohini Singh,1 Neil Savage,1 Minomi Subapanditha,1 David Bakhshinyan,1 Laura Kuhlmann,2 Thomas Kislinger,2 Sachdev Sidhu,2 Jason Moffat,2 Sheila Kumari Singh1. 1 _McMaster University, Hamilton, Ontario, Canada;_ 2 _University of Toronto, Toronto, Ontario, Canada_.

Background: Glioblastoma (GBM) is the most common malignant primary adult brain tumor, characterized by extensive cellular and genetic heterogeneity. Even with surgery, temozolomide chemotherapy and radiation, tumor re-growth and patient relapse are inevitable, with a median survivorship of just 15 months. Genomic profiling studies have shown that clonal evolution within GBM may be driven by cancer treatment, such that the recurrence may no longer resemble the genetic landscape of the original primary tumor. Furthermore, intratumoral heterogeneity associated with clonal evolution complicates biomarker discovery and treatment personalization and underlies treatment failure. Thus, modeling clonal heterogeneity and evolution to understand cancer progression is critical for the development of effective therapeutic approaches. We aim to identify new therapeutic targets that drive clonal evolution in treatment-refractory GBM and develop novel and empirical therapeutic paradigms targeting recurrent GBM.

Experimental Procedure: We employed a transcriptomic, proteomic and functional genomics approach to discover and validate genes that drive GBM recurrence. Using a therapy-adapted patient-derived xenograft (PDX) model of treatment-refractory GBM, we profiled the transcriptomic and proteomic landscape of treatment-naïve primary GBM through conventional chemotherapy and radiation therapy, and into recurrence. To complement the transcriptomic data, we used an unbiased genome-wide CRISPR-Cas9 screening platform to identify genes essential for self-renewal in recurrent GBM, as well as to identify novel sensitizers and suppressors of conventional therapy. Furthermore, we coupled cellular DNA barcoding technology with our PDX model to profile the clonal evolution of tumor cells through therapy.

Results: Integrative analysis of deep sequencing and surface proteomics of tumor cells harvested at tumor formation, minimal residual disease after chemoradiotherapy, and tumor recurrence from the PDX model resulted in the identification of novel therapeutic targets in treatment-refractory GBM. Using CRISPR, potential targets were knocked out in patient-derived GBMs in order to characterize the effect on self-renewal and tumor formation. We report the successful barcoding of patient-derived primary, treatment-naïve GSCs at a single cell resolution that were expanded into clonal populations, intracranially engrafted in immunodeficient mice and treated with SoC therapy. Conclusion: We have generated a translational pipeline from initial target discovery, through target validation, to building new biotherapeutics against novel targets, and preclinical testing in our PDX model of treatment-resistant GBM. A promising lead panel of biotherapeutic modalities is being translated into early clinical development, generating targeted therapies and hope for future GBM patients.

#571

**The shared mutation and** neo **antigen landscape of MMR-deficient colorectal cancers suggests immunoediting during tumor evolution.**

Matthias Kloor,1 Alexej Ballhausen,1 Moritz Przybilla,1 Michael Jendrusch,1 Elisabeth Pfaffendorf,1 Markus Draxlbauer,1 Florian Seidler,1 Sonja Krausert,1 Aysel Ahadova,1 Simon Kalteis,1 Daniel Heid,1 Johannes Gebert,1 Maria Bonsack,2 Sarah Schott,3 Hendrik Bläker,4 Toni Seppälä,5 Jukka-Pekka Mecklin,6 Sanne Ten Broeke,7 Maartje Nielsen,7 Julia Krzykalla,8 Axel Benner,8 Angelika Riemer,2 Magnus von Knebel Doeberitz1. 1 _Department of Applied Tumor Biology, University Hospital Heidelberg, Collaboration Unit Applied Tumor Biology, German Cancer Research Center, Molecular Medicine Partnership Unit, University Hospital Heidelberg and EMBL, Heidelberg, Germany;_ 2 _Immunotherapy and Immunoprevention, German Cancer Research Center, Molecular Vaccine Design partner site Heidelberg, German Center for Infection Research, Heidelberg, Germany;_ 3 _Department of Obstetrics and Gynecology, University Hospital Heidelberg, Heidelberg, Germany;_ 4 _Department of General Pathology, University Hospital Charité, Berlin, Germany;_ 5 _Department of Gastrointestinal Surgery, Helsinki University Hospital and University of Helsinki, Helsinki, Finland;_ 6 _Department of Education and Research, Central Finland Central Hospital, and Sports and Health Sciences, University of Jyväskylä, Jyväskylä, Finland;_ 7 _Department of Clinical Genetics, Leiden University Medical Center, Leiden, Netherlands;_ 8 _Division of Biostatistics, German Cancer Research Center, Heidelberg, Germany_.

The immune system can recognize and attack cancer cells and their precursors, especially those with a high load of mutation-induced neoantigens. Such neoantigens are particularly abundant in DNA mismatch repair (MMR)-deficient cancers. MMR deficiency results in microsatellite instability (MSI), which leads to multiple insertion/deletion mutations at coding microsatellites and to neoantigen-inducing translational frameshifts. The significance of immune selection and immunoediting potentially shaping the neoantigen landscape during the progression from premalignant MMR-deficient lesions into cancers has not yet been analyzed. We hypothesized that the neoantigen landscape of MSI cancers may reflect the impact of immunoediting. We developed a novel tool for quantitative analysis of microsatellite mutations to explore the neoantigen landscape of MSI colorectal (CRC, n=139) cancers. Frameshift mutations were examined in 41 coding microsatellite (cMS) regions using our new algorithm. We predicted the resulting frameshift neoantigen sequences and used the publicly available prediction tool NetMHCpan 4.0 for prediction of MHC binding sequences. Immunological scores were generated to quantify the likelihood of defined cMS mutations to generate immunogenic neoantigens in different populations with defined HLA allele distributions. Across the 41 cMS analyzed, 77% of all mutations were in the reading frame of 1 nucleotide deletions (m1). The cMS mutation frequency and FSP epitope distribution across HLA genotypes (described by a general epitope likelihood score, GELS) showed a significant negative correlation (Pearson's r=-0.42, p=0.0149). Some cMS presented with high mutation frequencies despite a high GELS (i.e. TGFBR2: pmut = 88%, GELS = 78.9%), suggesting mutation-induced driver effects, which may outweigh the increased immunogenicity. Our results show that MSI cancers share several highly immunogenic neoantigens. Importantly, a negative correlation between the antigenic strength of neoepitopes and their mutation frequency in MMR-deficient cancers points towards continuous immunoediting during their evolution. These findings will have substantial impact on the optimization of vaccines designed to potentially prevent or treat MSI-driven cancers.

#572

Human endogenous retroviruses as a source of shared tumor antigens in triple negative breast cancer.

Paola Bonaventura,1 Laurie Tonon,2 Virginie Mutez,1 Vincent Alcazer,3 Laura Urbini,2 Jenny Valladeau-Guilemond,1 Alain Viari,2 Sebastian Amigorena,4 Christophe Caux,1 Stephane Depil5. 1 _Centre Léon Bérard- Centre de Recherche en Cancerologie de Lyon (CRCL), Lyon, France;_ 2 _Centre Léon Bérard- Synergie Lyon Cancer, Plateforme de Bioinformatique 'Gilles Thomas', Lyon, France;_ 3 _Centre de Recherche en Cancerologie de Lyon (CRCL), Lyon, France;_ 4 _Institut Curie, Paris, France;_ 5 _Centre Léon Bérard- Centre de Recherche en Cancerologie de Lyon (CRCL) et Université de Lyon, Lyon, France_.

Aim: To identify potential tumor antigens derived from Human Endogenous Retroviruses (HERVs) shared within different cancer subtypes, especially here in triple negative breast cancer (TNBC).

Experimental procedures: HERVs expression was analyzed using RNAseq data from 84 breast cancer samples (TNBC: 42) and compared with 56 normal and peri-tumoral tissue samples. The association with different immune signatures (MCPcounter and GSEA) was established. Protein sequences (Gag, Pol and Env from Uniprot) of HERVs overexpressed in TNBC were aligned together (Clustal Omega) and shared peptides were extracted. Common potential HLA-A2 strong binder epitopes (NetMHCI) were identified and the corresponding peptides were synthetized. Peripheral blood mononuclear cells (PBMCs) from HLA-A2+ healthy donors were pulsed with these peptides and T cells expanded in vitro for 12 days, according to a validated protocol of ex vivo priming. T cells were then co-cultured with T2 cells pulsed with the cognate peptide and CD8+ T cell activation was assessed using cytokine intracellular staining. Peptide-specific dextramers were also used to identify and sort specific CD8+ T cells, the functionality of which was evaluated by ELISPOT against peptide-pulsed T2 cells. The presence of HERV-specific CD8+ T cells was evaluated among tumor infiltrating lymphocytes (TILs) isolated from TNBCs using dextramers.

Results: Among the 66 HERV subtypes analyzed, 19 were specifically overexpressed in TNBC, the majority of them belonging to the HERV-K family. HERVs expression significantly correlated with T cell and antigen presentation transcriptomic signatures. This specific set of HERVs overexpressed in TNBC was then used to identify shared HLA-A2 T cell epitopes. Ex vivo priming assay showed that some epitopes were able to generate functional IFN-γ+ TNF-α+ specific CD8+ T cells degranulating (CD107A+) after co-culture with T2 cells pulsed with the cognate peptide. The epitope-specific CD8+ T cells were identified and sorted by dextramer staining and then expanded, showing functionality of these clones against peptide-pulsed T2 cells by ELISPOT (double positive GrzB+ IFN-γ+ producing cells). TILs isolated from TNBCs and directly expanded in vitro (without any specific peptide stimulation) led to the identification by dextramers of HERV-specific T cells spontaneously present inside the tumor.

Conclusions: A specific set of HERVs is overexpressed in TNBC and contains shared epitopes capable of inducing strong T cell responses. The HERV-specific CD8+ T cells are functional and active against target cells presenting the cognate peptides. TILs recognizing HERV epitopes can be identified, showing that these predicted epitopes are naturally processed. Our results suggest that HERV-derived products represent a new class of tumor antigens shared in a specific tumor subtype that could be used in a strategy of innovative cancer vaccine.

#573

Mutations found by targeted next generation sequencing is associated with intra-tumor immune profile and may predict response to anti-PD1 therapy in lung adenocarcinoma.

Audrey Lupo,1 Jerome Biton,2 Marco Alifano,1 Diane Damotte,1 Jennifer Arrondeau,3 Pascaline Boudou,3 François Goldwasser,1 Karen Leroy,1 Marie Wislez,1 Pierre Laurent-Puig,1 Isabelle Cremer,4 Helène Blons,1 Ronald Herbst5. 1 _University Paris Descartes APHP, Paris, France;_ 2 _University Paris 13, Bobigny, France;_ 3 _APHP, Paris, France;_ 4 _UMRS U 1138, Paris, France;_ 5 _Medimmune, Gaithersburg, MD_.

Purpose: the immune environment of cancer is the target of immunotherapies with programmed cell death 1 (PD-1) therapeutic antibodies. However, factors that can robustly predict long-lasting responses are still needed. Previous studies showed a role of cancer cells on immune response setting, but the impact of somatic mutations on the composition of the immune stroma is not fully characterized.

Experimental Design: We characterized the immune profile of 221 lung adenocarcinomas using immunohistochemistry (CD8 T cells, Macrophages, neutrophils, PD-L1) and compare to tumor mutational status using targeted next-generation sequencing. The response to PD-1 blockers was analyzed from a prospective cohort of 31 lung carcinomas according to tumor mutational profiles and to tumor cell PD-L1 expression.

Results: We report that distinct combinations of STK11, EGFR and TP53 mutations, allows the identification of immune cells combination and prediction of PD-L1 expression on tumor cells. Lung adenocarcinomas with the highest CD8 T cell density and PD-L1 expression were those with TP53 mutations without co-occurring STK11 or EGFR alterations (TP53-mut/STK11-EGFR-WT), and independently of KRAS status. Interestingly in TP53-mut/STK11-EGFR-WT tumors, pathways related to T cell chemotaxis, immune cell cytotoxicity, and antigen processing were up-regulated. In accordance with these results we found a prolonged progression-free survival (PFS: HR=0.32; 95% CI, 0.16-0.63, p<0.001) in anti-PD-1 treated patients harboring TP53-mut/STK11-EGFR-WT tumors. This clinical benefit was even more remarkable in patients with associated strong PD-L1 expression.

Conclusions: Our study reveals that mutations in lung adenocarcinoma impact intra-tumor immune profile and predict response to PD-1 blockade.

#574

Overexpression of HER2 in head and neck cancer represents a potential target for T cell immunotherapy.

Emilie A. Warren,1 Hsuan-Chen Liu,1 Caroline E. Porter,2 Kershena S. Liao,3 Meenakshi Hegde,1 Wendong Yu,1 Patricia D. Castro,1 Vlad Sandulache,1 Nabil Ahmed,1 Masataka Suzuki,2 Andrew Sikora1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX;_ 3 _The University of Texas Southwestern Medical Center, Dallas, TX_.

HER2, commonly referred to as ErbB2, is a receptor tyrosine kinase that, along with EGFR, makes up one of the four members of the ErbB family of proteins. These ErbB proteins are expressed in most epithelial cell layers and play a key role in cell differentiation. HER2 has been found to be overexpressed in a number of human cancers, including breast and gastric carcinomas. Overexpression of the HER2 receptor represents a potential target for chimeric antigen receptor (CAR) T cell therapy. CAR T clinical trials in leukemia and lymphoma have demonstrated durable remission of the disease or even cure, but application of CAR T cells to solid tumors such as head and neck squamous cell carcinoma (HNSCC) lags behind. To evaluate HER2 expression in HNSCC, we prepared 4 different tumor microarrays (TMAs) of patient tumors based on their location (larynx, salivary gland, oropharynx or oral cavity) for IHC staining. These TMAs were then stained and scored by two different methods: with an anti-HER2 monoclonal antibody (CB11) and scoring based on breast cancer guidelines, or with the FDA-approved HER2 HercepTest IHC procedure.

Based on the level of HER2 expression determined by TMA staining, we identify HNSCC as a candidate tumor type for preclinical testing of HER2-specific CAR T therapy. We utilize the chick embryo chorioallantoic membrane (CAM) tumor model, a naturally immune-deficient platform for growth of vascularized, three-dimensional tumors, as a rapid and inexpensive system to assess CAR T efficacy against human HNSCC tumors in vivo. To demonstrate tumor killing efficacy of HER2-specific CAR T cells (HER2.CAR Ts) on the CAM, we engrafted tumors derived from the HER2-positive (+) HNSCC cell line FaDu and HER2-negative (–) breast adenocarcinoma cell line MDA-MB-468 onto the CAM of day 7 fertilized chicken eggs. Both cell lines had been previously genetically engineered to express firefly luciferase (ffLuc). On day 10, established tumors were treated with HER2.CAR Ts generated by retroviral transduction of primary activated human T cells with a second-generation CAR T construct incorporating a CD28.ζ signaling domain. Four days after CAR T treatment, tumors were excised from the CAM, homogenized, and lysed; the luminescence of the resulting cell lysates (ffLuc activity) was used to quantify the relative number of viable tumor cells. While CAR T treatment resulted in an average 56% decrease in tumor size in the HER2+ FaDu tumors, there was no significant change in HER2– MDA-MB-468 tumor size. These results suggest that HER2-expressing HNSCC can be effectively targeted by HER2-directed CAR T calls and demonstrate the potential of the CAM tumor model as a cost-effective tool for rapid preclinical assessment of CAR T cell therapy.

#575

Adaptive CD4 Th1 response against telomerase in blood counteracts T-cell exhaustion in non-small cell lung cancer.

Caroline Laheurte,1 Magalie Dosset,1 Dewi Vernerey,1 Elodie Lauret Marie Joseph,1 Laura Boullerot,1 Vincent Kaulec,2 Marion Jacquin,3 Laurie Cuche,2 Guillaume Eberst,2 Pascale Jacoulet,2 Virginie Westeel,2 Olivier Adotevi1. 1 _INSERM UMR 1098, BESANCON, France;_ 2 _University Hospital of Besançon, BESANCON, France;_ 3 _INSERM CIC-1431, BESANCON, France_.

Despite the critical roles played by IFN-γ+ CD4 Th1 response in tumor immunity, the translation of their potential in clinic remains challenging. Here, we evaluate the clinical significance of circulating anti-tumor CD4 Th1 response in non-small cell lung cancer (NSCLC) patients. 170 naïve-treatment patients were enrolled in this immune monitoring study. The antitumor adaptive Th1 response was assessed by IFN-γ ELISPOT assay in blood lymphocytes using a mixture of eight highly promiscuous and Th1-polarized HLA class II-restricted epitopes from telomerase (TERT). The presence of anti-TERT Th1 response was detected in 59/170 patients (35%). We found an opposite link between anti-TERT Th1 and hyper exhausted T cells co-expressing PD-1+ and TIM-3+ in NSCLC patients. In contrast to hyper exhausted CD8 T cells, the presence of anti-TERT Th1 response was associated with a low rate of hyper exhausted CD4 T cells. We showed that NSCLC stage dissemination is associated with a decrease of anti-TERT Th1 response but an increase of circulating hyper exhausted PD-1+/TIM-3+ CD4 T cells. Notably, anti-telomerase Th1 response and hyper exhausted CD4 T-cells displayed opposite prognosis value in NSCLC. While high level of anti-TERT Th1 cells play a protective role, hyper-exhausted PD-1+TIM-3+ CD4 T cells negatively affect patients' survival. By using these two circulating immune factors, we stratified patients in distinct prognostic group. Patients with anti-TERT Th1high/CD4 PD-1 TIM-3low immune profile had better overall survival than anti-TERT Th1low/CD4 PD-1 TIM-3high group (median OS : not reached versus 4 months respectively p<0.0001). In conclusion, high circulating anti-TERT Th1 response plays a strong protective role in NSCLC by counteracting the deleterious effect associated with hyper exhausted T cells in blood. Our study provides a dynamic blood-based immunomonitoring tool allow patients stratification.

#576

A novel intestinal microbiome-derived peptide modulates immune cell activity and the tumor microenvironment.

Helena Kiefel, Dhwani Haria, Yuliya Katlinskaya, Divya Ravichandar, Lily McLaughlin, Sunit Jain, Thomas Weinmaier, Shoko Iwai, Todd DeSantis, Toshi Takeuchi, Karim Dabbagh, Kareem Graham. _Second Genome, South San Francisco, CA_.

The composition of the gut microbiota affects cancer development, progression, and response to therapy. A number of commensal bacteria, including Bifidobacterium, have been associated with increased response to immune checkpoint inhibitors in mouse tumor models and in cancer patients.

We hypothesized that secreted peptides or proteins are driving the Bifidobacterium-mediated effects. Using our unique bioinformatic-driven discovery platform we nominated putatively secreted Bifidobacterium-derived peptides for evaluation in immune cell effector assays. We demonstrate that several of the peptides induce secretion of pro-inflammatory cytokines (e.g., IL-6, TNF) by in vitro-generated mouse and human dendritic cells, as well as effector cytokine secretion (e.g., IFNγ, IL-2) by mouse splenic T lymphocytes in vitro.

To investigate the function of Bifidobacterium-derived peptides in the context of the tumor microenvironment we injected the candidate peptide SG-A directly into the tumor of CT26 tumor-bearing mice and analyzed the immune phenotype using the Nanostring PanCancer Immune Profiling panel and flow cytometry. Both Nanostring as well as flow cytometry analysis showed an increase in CD45+ tumor-infiltrating lymphocytes in SG-A treated tumors. SG-A peptide also induced the upregulation of dendritic cell function genes (CD40, CD83, and CD86) and multiple effector cytokines and chemokines. Re-stimulation of tumor-draining lymph node cells with AH1 peptide (a CT26 tumor-derived antigen) also increased induction of IFNγ in SG-A treated animals (vs. vehicle-treated controls).

Collectively, our results demonstrate the utility of the Second Genome discovery platform for leveraging microbiome science to identify novel immunoregulatory factors. This platform offers a promising approach to identify agents with potential for use as therapeutics in cancer immunotherapy.

#577

Optimization of methods for the analysis of class I MHC peptides by mass spectrometry.

Michael J. Ford,1 Richard C. Jones,1 Ravi Amunugama,1 David Allen,1 Paul Del Rizzo,2 James Mobley,1 Michael Pisano1. 1 _MS Bioworks, Ann Arbor, MI;_ 2 _Cayman Chemical, Ann Arbor, MI_.

Immuno-oncology describes therapeutic approaches exploiting the body's immune system to fight cancer. One approach involves educating the immune-system to recognize and destroy tumor cells by targeting tumor-specific neoantigens. Neoantigens are antigens presented by tumors but not recognized by the immune-system. Identification of Neoantigens is therefore an active area of research and development. The major histocompatibility complex (MHC) plays a crucial role in antigen presentation. Peptides generated by protein degradation in the cytosol are presented, non-covalently bound to MHC Class I molecules, on the surface of cells for inspection by T-lymphocytes. Cytotoxic T lymphocytes (CTL) recognize peptides presented by MHC Class I. The recognition of peptide antigens presented by MHC Class I results in the destruction of the presenting cell by the CTL. Characterization of peptides associated with MHC Class I molecules requires a targeted protein complex enrichment, an unbiased peptide elution and finally a peptide analysis method. Here we present the latest results from our work optimizing and performing a workflow for the analysis of peptides associated with Class I MHC molecules.

#578

Investigation of the effect of hypoxia on presentation of HPV16-derived antigens - implications for therapeutic vaccine design.

Nitya Mohan,1 Maria Bonsack,2 Jonas Förster,2 Alina Steinbach,2 Renata Blatnik,2 Mogjiborahman Salek,2 Angelika B. Riemer2. 1 _German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 2 _German Cancer Research Center (DKFZ) and German Center for Infection Research (DZIF) Partner Site, Heidelberg, Germany_.

Human papillomavirus type 16 (HPV16) is a causal factor of ~50% of cervical cancers, and of ~95% of extra-cervical (other anogenital and oropharyngeal) HPV-mediated cancers. The aim of our group is to develop a therapeutic vaccine against HPV16-induced malignancies. To find optimal vaccine antigens, it is important to assess the effect of the tumor microenvironment on viral antigen presentation. Hypoxia has been reported to regulate HLA class-I expression levels in the context of various tumors. Thus, the aim of the present study was to explore the effect of hypoxia on various components of the antigen processing machinery (APM) in HPV16-positive tumor cells and on the HPV16 E6- and E7-derived HLA class-I epitope repertoire. We are investigating the effect of hypoxia on cervical cancer cells transformed with HPV16 variant lineages (European, Asian-American and African) correlated with differences in oncogenic potential. This is achieved by comparing cells cultured under hypoxia (1.2% O2) or normoxia (21% O2). We investigate E6 protein levels by immunoblotting and HLA-A2 surface expression by flow cytometry. Subsequently, quantitative PCR and immunoblotting are used to assess whether components of the APM are affected by hypoxia. Cervical cancer cell lines transformed by sublineages of the HPV16 European variant (CaSki and SNU17) showed a decrease in E6 protein levels upon hypoxic treatment. HLA-A2 levels were increased in SNU17 cells (HPV16 European Asian prototype), but no change was observed in CaSki cells (European Prototype 2). First qPCR experiments of SNU17 cells showed changes in additional components of the APM at the mRNA level, which still have to be assessed at the protein level. Cell lines transformed by HPV16 variants other than European will be subjected to the same analysis. Potential changes in the HPV16 epitope repertoire will be investigated using a targeted mass spectrometry-based epitope detection strategy established in the lab. These investigations may yield novel insights into HPV16 immune evasion pathways. Furthermore, the identification of epitopes that are presented under both normoxic and hypoxic conditions will provide optimal candidates for therapeutic vaccine design against HPV-induced malignancies.

#579

Immune features and neoantigen recognition in mismatch repair-proficient colorectal cancer liver metastases.

Steven Hébert,1 Mélissa Mathieu,2 David Henault,2 Maud Marques,1 Éric Audemard,3 Mathieu Courcelles,3 Scott D. Brown,4 Pratyaksha Wirapati,5 Sabine Tejpar,6 Michael I. D'Angelica,7 Robert A. Holt,4 Sylvie Mader,3 Pierre Thibault,3 Gerald Batist,1 Claudia Kleinman,1 Simon Turcotte2. 1 _Lady Davis Research Centre, Montreal Jewish General Hospital, Montreal, Quebec, Canada;_ 2 _CHUM - Ctr. Hosp. Université de Montréal, Montreal, Quebec, Canada;_ 3 _Institut de recherche en immunologie et cancérologie, Université de Montréal, Montreal, Quebec, Canada;_ 4 _British Columbia Cancer Genome Sciences Centre, Vancouver, British Columbia, Canada;_ 5 _Swiss Institute of Bioinformatics, Lausanne, Switzerland;_ 6 _University Hospital Leuven, Leuven, Belgium;_ 7 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Immunotherapy for patients with non-highly mutated, mismatch repair-proficient (MMRprof) metastatic colorectal cancer has largely been unsuccessful thus far. Here, we used transcriptomics, genomics and in vitro analyses to study the adaptive immune features of colorectal cancer liver metastases (CRLMs), aiming to provide insights into the development of immunotherapies for this common malignancy.

Analysis of the RNAseq data from 63 core biopsies obtained in patients enrolled in the Q-CROC-01 trial (NCT00984048) revealed 17 (27%) immune-reactive (IR) CRLMs, defined by concurrent relative high expression of antigen processing, immune cell, immune checkpoint, interferon-gamma response, cytokine, and chemokine transcripts. More T-cell receptor (TCR) sequences were found in IR vs. non-IR CRLMs (mean 89.8 ± 13.3 vs. 19.5 ± 4.0 CDR3 counts, p<0.0001), consistent with higher density of tumor-infiltrating T lymphocytes (TILs) confirmed by immunofluorescence. By whole exome analysis, no significant difference was found in non-synonymous somatic mutation (median 76) and neoantigen (neoAg) (median 18) counts comparing IR vs. non-IR CRLMs. High mutation or neoAg loads did not correlate with higher T cell infiltration. TILs were expanded in vitro from five CRLMs, one classified as high IR, two as moderate IR, and two as non-IR by immune gene expression profiling. TIL recognition of predicted neoAgs was tested in co-culture with autologous antigen-presenting cells pulsed with synthetic peptides. Only in the most immune reactive CRLM did we find CD8+ and CD4+ TIL clones (n=8) reactive to neoAg peptides derived from three single nucleotide variant genes, and none reactive to the wild-type peptide counterparts. In this case, by deep sequencing of intratumoral TCRs, the average frequency of neoAg-reactive TIL clones was estimated at 0.95% (range 0.003% to 3.3%), which represented a 3625 fold enrichment compared to the circulating blood. In general, expression of immune checkpoints was more highly correlated in IR vs. non-IR CRLMs. The following T cell-related conceptual pairs of immune checkpoint receptors & ligands were expressed at significantly higher levels in IR compared to non-IR CRLMs: HVEM/LTBR & BTLA/LIGHT, A2A & CD73, TIGIT & PVRL3, FAS & FASL, CD2/2B4 & CD48.

Our results support that naturally occurring neoAg-reactive T cells may be therapeutically harnessed in a subgroup of metastatic MMRprof colorectal cancer patients selected based on the level of intratumoral immune reactivity, and potentially enhanced by targeting immune checkpoints more biologically relevant than PD-1/PD-L1 and CTLA-4/CD80.

#580

**Detailed analysis of immune responses induced against patient-specific neoantigens using the** ex-vivo **induction protocol, NEO-STIM™.**

Divya Lenkala,1 Jessica Kohler,1 Brian McCarthy,1 Michael Nelson,1 Rachel DeBarge,2 Yvonne Ware,1 Yuting Huang,1 Janani Sridar,1 Yusuf Nasrullah,1 Dewi Hartjano,1 Asaf Poran,1 Sejuti Sengupta,1 Joost H. van den Berg,3 Matt Goldstein,4 Richard B. Gaynor,1 Marit M. van Buuren1. 1 _NEON Therapeutics, Cambridge, MA;_ 2 _University of California, San Francisco, San Francisco, CA;_ 3 _Netherlands Cancer Institute, Amsterdam, Netherlands;_ 4 _Tango Therapeutics, Cambridge, MA_.

Background: Neoantigens are tumor-specific antigens that have been shown to be critical in the anti-tumor immune response. These antigens are not subject to central immune tolerance and are therefore potentially more immunogenic than tumor-associated antigens. The goal of our studies is to assess the immunogenic potential of high-quality neoantigen targets, as defined through our bioinformatic engine RECON®, as well as perform detailed characterization of the induced T cell responses towards these neoantigen targets.

Methods: Patient-specific neoantigens were predicted using our bioinformatic engine RECON. The predicted high quality neoantigens were utilized in our proprietary ex-vivo stimulation protocol NEO-STIM to assess immunogenicity. NEO-STIM is used to prime, activate and expand memory and de novo T cell responses from both the CD4+ as well as the CD8+ compartment. In-depth analysis was performed to characterize the specificity, functionality (cytokine production and cytolytic capacity) and diversity of the induced T cell responses.

Results: Here we present the successful induction of memory and de novo CD8+ T cell responses in PBMCs isolated by leukapheresis from three patients using NEO-STIM (2 with melanoma & 1 with NSCLC). We then deeply characterize the induced CD8+ T cell responses of one of the melanoma patients and show that these responses are functional, specific and have cytolytic capacity. Furthermore, we use paired single cell RNA and TCR sequencing to assess the diversity and gene expression profile of these responses.

Conclusions: NEO-STIM is a novel platform to understand the immunogenic potential of high-quality neoantigen-targets in detail. Moreover, this platform can be utilized to generate T cell products for adoptive cell therapy for patients with a variety of solid and liquid tumors.

#581

**Determining the influence of galectin-3 binding properties on its suppression of tumor antigen-specific CD8** \+ **T cells.**

Alexandra B. Pucsek, Todd D. Armstrong, Elizabeth M. Jaffee. _Johns Hopkins University School of Medicine, Baltimore, MD_.

Improving the efficacy of immunotherapeutic cancer treatments requires increased understanding of the factors that contribute to a suppressive tumor microenvironment (TME). Recently, galectin-3 has emerged as an important player in the development and progression of cancer, favoring immune suppression and dysfunction at disease sites. While this protein is consistently identified as a tumor-promoting factor, its unique properties make the mechanisms of its functions difficult to study, and impede the development of therapies against it.

Galectin-3 binds β-galactoside carbohydrate residues through a highly-conserved C-terminal domain (CRD) characteristic of mammalian galectins. It is made unique by its ability to self-associate into homo-oligomers through a disordered N-terminal domain (NTD). We have previously shown that galectin-3 knockout in a mouse model of tumor tolerance improves effector function of antitumor CD8+ T cells and increases duration of tumor-free survival, suggesting a suppressive role for galectin-3 in antitumor immunity. Our current work aims to elucidate how the distinct biochemical properties of galectin-3 contribute to this suppression. To this end, we are developing a new assay to model the suppression of CD8+ T cells by galectin-3 in vitro.

Our assay utilizes TCR transgenic CD8+ T cells specific for rodent Erbb2, the Her-2/neu (Neu) oncogene. These cells are isolated from either normal transgenic mice or those deficient in galectin-3. The T cells are stimulated in culture, then co-cultured with NT2.5 Neu-expressing tumor cells. We use flow cytometry to analyze the levels of CD8+ effector molecules interferon gamma (IFNγ) and Granzyme B, as well as cell surface binding of galectin-3 after co-culture. To further elucidate the roles of carbohydrate binding and self-association in this process, we have developed recombinant mutant galectin-3 proteins deficient in carbohydrate binding through CRD point mutation, lacking the ability to self-associate through NTD truncation, or both. By introducing these recombinant proteins into the co-culture with normal or galectin-3 deficient transgenic T cells, we expect to be able to learn whether galectin-3-mediated suppression is more dependent on carbohydrate binding, self-association, or both properties in synergy.

Current therapies targeting galectin-3 focus only on its carbohydrate binding properties through use of small molecules and glycomimetics, but these have been of limited efficacy. We hypothesize that self-association of galectin-3 through the NTD is an equally important factor in its unique ability among galectins to contribute to an immunosuppressive TME. Learning how these properties each contribute to the mechanism of galectin-3-mediated immunosuppression will allow us to refine strategies for breaking tumor tolerance, contributing to improved outcomes for cancer immunotherapies.

#582

Tumor immunoediting in a lung cancer mouse model harboring EGFR mutations.

Kazuya Nishii, Kadoaki Ohashi, Kiichiro Ninomiya, Go Makimoto, Hiromi Watanabe, Hirohisa Kano, Naofumi Hara, Heiichiro Udono, Katsuyuki Kiura. _Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan_.

Background: Anti-Programed cell death 1 (PD-1) antibodies such as nivolumab or pembrolizumab show promising treatment effects in smoking-related lung cancer. However, anti-PD-1 antibodies have very little effect on epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC). EGFR mutations are the most frequent oncogenic drivers in non-smoking related NSCLC. Therefore, we aimed to explore the mechanism underlying tumor immunoediting in a lung cancer mouse model harboring EGFR mutations.

Methods: We previously established two strains of transgenic lung cancer mouse (C57BL/6) harboring a mouse Egfr exon 19-deletion (mDEL) or a human EGFR L858R (hLR) mutation, driven by a surfactant protein (SP)-C promoter (Ohashi, Cancer Sci. 2008; Ohashi, Cancer Res. 2009). These mice developed EGFR-dependent multifocal lung adenocarcinomas from type II pneumocytes. We modified the two models by transplanting lung tumors subcutaneously into C57BL/6 (Ohashi, AACR. 2018). Evaluation of mice tumor lymphocyte profile was done through flow cytometry or immunohistochemistry (IHC). Effects of T cell depletion or anti-mouse PD-1 antibody (4H2) in vivo were also assessed. Comprehensive analysis of mRNA expression of immuno-checkpoint molecules in the tumors was performed using PanCancer Immune Profiling Panel (NanoString Technologies).

Results: CD4+ T cells and/or CD8+ T cells were depleted in the subcutaneously transplanted tumor model followed by treatment with gefitinib (50 mg/kg/day, 5 days/week) for 2 weeks. As a result, after gefitinib discontinuation, rapid regrowth was seen in the tumors from groups with depletion of CD8+ T cells; depletion of CD4+ T cells had little effect on tumor regrowth, and tumor inhibition continued for 2 weeks after cessation of gefitinib. IHC showed relatively increased numbers of CD4+ T cells in the mice tumors whereas a decrease in the number of CD8+ T cells or Foxp3 positive cells was noted. PD-L1 expression was not observed in the tumors. Consistently, 4H2 showed little effect on tumor growth in vivo. The cancer immune panel showed increased expression of immune mediators such as Ccl6, Clec4n, and Lcn2.

Conclusions: Our results suggest that CD8+ T cells can recognize mice tumors in vivo. New immuno-checkpoint molecules regulating CD8+ T cells from PanCancer Immune Profiling Panel were evaluated. Further, we plan to evaluate the efficacy of various immuno-checkpoint inhibitors in vivo.

#583

DNA demethylating agents enhance susceptibility of lung cancer cells to γδ T cell-based immunotherapy via MHC-independent mechanisms.

Rueyhung Weng,1 Chien-Ting Lin,2 Tai-Chung Huang,1 Hsuan-Hsuan Lu,1 Yi-Chieh Wu,1 Xuan-Hui Lin,2 Rong-Shan Lin,2 Chong-Jen Yu,1 Hsing-Chen Tsai1. 1 _National Taiwan University Hospital, Taipei, Taiwan;_ 2 _National Taiwan University, Taipei, Taiwan_.

Immune-based therapy is revolutionizing lung cancer management in the clinic. In addition to rejuvenating T cells through immune checkpoint blockade, enhancing the immunogenicity of cancer cells and their interactions with immune cells may also be key to boosting the clinical efficacy of immunotherapy. Previous transcriptomic studies have shown that DNA demethylating agents may alter antigen processing/presentation machinery, upregulate cancer-testis antigens, reactivate endogenous retroviruses to facilitate efficacy of immunotherapy. Nevertheless, how DNA demethylating agents modulate surface immune synapse proteins and affect interactions between cancer and immune cells remain unclear. We utilized stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative membrane proteomics to identify surface proteins altered in decitabine-treated human lung cancer cells. The results showed that decitabine upregulated multiple immune synapse proteins. Gene enrichment analyses discovered that these surface proteins are predominantly involved in γδT cell-mediated cytotoxicity. γδT cells are a distinct subgroup of T cells that bridge between the innate and the adaptive immune systems and do not require the major histocompatibility complexes for antigen recognition. Co-culture of decitabine-pretreated human lung cancer cells with allogenic γδT cells in vitro confirmed sensitization effects of decitabine for the killing of γδT cells. A similar effect was also observed in immunocompromised mice bearing xenograft tumors in vivo. Moreover, the comparison of decitabine-induced surface proteomes between sensitizable and unsensitizable lung cancer cells revealed that intercellular adhesion molecule 1 (ICAM1), was among the top differentially expressed proteins. Imaging analyses confirmed that ICAM1 participated in the formation of immune synapses between cancer cells and γδT cells. Increased expression of ICAM1 was also observed in xenograft tumors from mice receiving combination therapy of decitabine and γδT cells. Overall, our data characterized decitabine-induced surface proteomes and uncovered an immunomodulatory effect of decitabine for enhancing MHC-independent killing by γδT cells. These findings have great clinical implications and provide a novel molecular basis for coupling DNA demethylation agents with γδT cell-based immunotherapy in the treatment of lung cancer patients.

#584

Developing a novel engineered T cell to target resistant pancreatic cancer.

Mahboubeh Yazdanifar, Ru Zhou, Shu-ta Wu, Priyanka Grover, Pinku Mukherjee. _Univ. of North Carolina at Charlotte, Charlotte, NC_.

Background: Pancreatic cancer is the 3rd leading cause of cancer mortality in the USA recently surpassing breast cancer. Treatment options are limited to surgery and chemo/radiation, which is normally associated with high toxicity. Targeted immune-based therapies have shown promising results but needs further exploration. Mucin 1 (MUC1), a glycoprotein expressed on the apical surface of epithelial cells, undergoes hypoglycozylation in tumors. This tumor-form of MUC1 (tMUC1) is over-expressed in 80% of pancreatic ductal adenocarcinomas (PDA). We have developed an antibody (TAB004) which specifically detects tMUC1 and not the normal MUC1. Functional fragments of TAB004 antibody (scFv) were incorporated into the chimeric antigen receptor (CAR) construct and used to genetically modify primary human T cells. tMUC1 recognizing domain (TAB004 scFv) is linked to the co-stimulatory molecules of T cells (CD28 and CD3ζ). These CAR T cells are highly activated and reactive to MUC1 expressing tumor cells. Pancreatic cancer is highly resistant to many treatments including immunotherapy. We suggest combining our CAR T cell treatment with low dose chemotherapy drug to improve the treatment efficacy.

Methods: CAR gene was transduced into human T cells using retroviral based technique. Cytotoxicity was evaluated using co-culture method with varying T cell to target cell ratios followed by MTT and LDH assay. Released IFNy, granzyme B and Perforin were measured by flow cytometry and ELISA. To investigate the mechanism involved in resistance of some PDA cells, cancer cells and T cells marker were evaluated by flow cytometry and gene expression profile of sensitive vs resistant cancer cells was assessed by Real-time PCR.

Results: tMUC1-CAR-T cells show increased activation and proliferation compared to normal T cells. Engineered tMUC1-CAR T cells exhibit binding and robust cytotoxicity against a panel of PDA cell lines, associated with high IFNγ, granzyme B and Perforin release. Fortunately, the same CAR T cells display minimum binding and toxicity against normal epithelial cells. CAR T cell function is evaluated in the preclinical mouse model of PDA, as single treatment and in combination with chemotherapy drugs. Several genes expression altered in cancer cells before and after treating with CAR T cells. Combining the CAR T cells with low dose chemotherapy drug (Gemcitabine and 5-FU) improved efficacy of the treatment for resistant PDA cells.

Conclusion: Our results validate the idea of using anti MUC1 CAR T cells to treat pancreatic ductal adenocarcinoma. This therapy has shown to be working synergistically with low dose chemotherapy drug with improved function in treating resistant PDA cells. Hence, tMUC1 CAR T cells have the potential to be further developed for clinical use on resistant PDA.

#585

**Induction of immunogenic cell death and enhancement of dendritic cell function: Development of an** in vitro **,** ex vivo **ICD platform for the identification of novel ICD inducers.**

Akanksha Gangar, Didier Grillot, Raphaelle Guillard-Huet, Jean-Francois Mirjolet, Fabrice Viviani. _Oncodesign S.A., Dijon Cedex, France_.

Immunological cell death (ICD) is a form of cancer cell death induced by radiotherapy, photodynamic therapy and a few chemotherapeutic agents such as Doxorubicin, Mitoxantrone, and Oxaliplatin. Unlike apoptosis or necrosis, ICD can induce an effective immune response directed against the tumor whereby both dendritic cells and T lymphocytes are mediators of this response. Dying cancer cells recruit and activate immune cells by releasing damage-associated molecular patterns (DAMPS) that help and promote the immune response to antigenic tumor neo-epitopes. Three key DAMPS are associated with the ICD process: calreticulin exposition on the cell surface, ATP secretion and high-mobility group box 1 (HMGB1) release. Using our in-house developed screening strategy facilitated by an automated in vitro platform with four assays, we have identified and characterized six cell lines (human breast MDA-MB-436 and MDA-MB-231, human osteosarcoma U-2 OS, murine pancreas Pan02, murine colon CT26, murine liver Hepa 1-6) for the three key DAMPs and cell viability. Furthermore, the screening of Oncodesign's Nanocyclix® library using the ICD bona fide cell lines described above resulted in the identification of ODS2006336, a potential in vitro ICD inducer. As dendritic cells (DCs) play a key role in the recognition of DAMPs associated with ICD and the subsequent uptake and presentation of tumor antigens, we examined the phagocytosis of ICD inducer-treated tumor cells by DCs. ICD inducer-treated CT26 cells when cultured with spleen-derived DCs were efficiently phagocytosed by DCs. An increase in IL-1β secretion in the co-culture supernatant was observed. IL-1β plays an important role in anti-tumor T cell priming. In addition, pro-inflammatory cytokines IL-6 and TNFα that promote T cell differentiation and NK cell activation were also detected. Increased secretion of IL-12 by activated DCs can also enhance NK cell functionality. Thus, ICD activates both innate and adaptive arms of the immune system. With respect to cancer immunotherapy, the ICD process elicits enhanced adjuvanticity and antigenicity from dying cancer cells and consequently, promotes the development of clinically desired antitumor immunity. In essence, we describe a novel strategy for the identification of ICD inducers within large chemical libraries followed by a streamlined ex-vivo co-culture assay to demonstrate enhanced DC function.

#586

**CD4** + **and CD8** + **T cells influence 4T1.2** luc **mammary tumor growth and survival.**

Yitong Xu, Shizhao Duan, William J. Turbitt, Andrea M. Mastro, Connie J. Rogers. _Pennsylvania State University, University Park, PA_.

The 4T1.2 syngeneic murine breast cancer model is an aggressive model of stage IV triple-negative breast cancer. Despite its significant strength, the 4T1.2 model is poorly immunogenic with few defined tumor antigens. The firefly luciferase gene, luc2, has been transfected into various tumor cell lines as a reporter for bioluminescence imaging. However, studies have shown that the luciferase (Luc) may serve as a tumor antigen and induce CD8+ cytotoxic T cell responses. The goal of the current study was to determine if luciferase expression in 4T1.2 tumor cells would enhance the immunogenicity of the tumor, and explore the role of immune effector cells in the parental 4T1.2 and luciferase-transfected 4T1.2luc tumor model. In vitro proliferation of the 4T1.2 and 4T1.2luc cells was assessed using the MTS cell proliferation assay. For in vivo experiments, female BALB/c mice were randomized into 2 groups and orthotopically injected with 5x104 4T1.2 or 4T1.2luc cells, respectively. Tumor growth was assessed until day 35 post tumor implantation. Each group was further randomized into 5 subgroups and received i.p. injections of PBS, rat IgG2b mAb, anti-CD4 mAb, anti-CD8 mAb, or anti-asialo GM1. Mice were sacrificed between day 28-35 post tumor implantation, and lung metastases were analyzed for each group. To further evaluate the immunogenicity of luciferase in the 4T1.2luc model, a separate cohort of mice were orthotopically injected with 5x104 4T1.2luc cells and sacrificed at day 35 post tumor implantation. Splenocytes were harvested and cultured in the presence of 1 or 10 µg/ml peptides representing MHC I-restricted Luc epitopes for 5 days, and cytotoxicity of the effector cells was assessed by the chromium-release assay. In vitro proliferation rates were not significantly different between 4T1.2 and 4T1.2luc tumor cells. However, 4T1.2luc tumor-bearing mice showed a significant reduction in primary tumor growth compared to 4T1.2 tumor-bearing mice. 4T1.2 primary tumor growth was not altered by the depletion of any immune cell type, while 4T1.2luc primary tumor growth was significantly increased by the depletion of CD8\+ T cells, and reduced by the depletion of NK cells. Depletion of CD4+ T cells, CD8+ T cells or NK cells in 4T1.2luc tumor-bearing mice resulted in a significant reduction in median survival. Splenocytes from 4T1.2luc tumor bearing mice demonstrated Luc-specific cytotoxicity upon ex vivo restimulation with Luc peptides, and a stronger response was observed when cells were restimulated with 1 µg/ml compared to 10 µg/ml Luc peptides. These findings suggest that luciferase may serve as a tumor antigen in the 4T1.2luc tumor model, and host immune components (CD4+ and CD8+ T cells) may play a role in controlling 4T1.2luc primary tumor growth and survival. The stronger immunogenicity of 4T1.2luc conferred by luciferase will enable future immunotherapeutic research that ultimately may benefit breast cancer patients. 

## EPIDEMIOLOGY

### Biomarkers in Risk and Outcomes

#587

Highly sensitive and accurate method for measuring serum estradiol in postmenopausal women and other population subgroups.

Lumi Duke,1 Paul H. Kim,2 Julianne Cook Botelho,1 Hui Zhou,1 Candice Z. Ulmer,1 Hubert W. Vesper1. 1 _CDC Atlanta, Atlanta, GA;_ 2 _Battelle Memorial Institute, Atlanta, GA_.

Meta-analyses consistently show that postmenopausal women with elevated levels of estradiol have an increased risk for certain cancers, such as breast cancer. At the same time, estradiol therapy is used to treat menopausal symptoms. Data on blood levels of estradiol are highly variable, especially in postmenopausal women, which prevents the formulation of generally recognized normal ranges and the consistent treatment of postmenopausal women. Similarly, no generally recognized reference ranges for estradiol exist for men and children. One reason for this variability in estradiol blood levels is the lack of appropriate analytical methods. To overcome these challenges, new, highly sensitive, specific, and standardized methods for measuring estradiol are needed. This study describes a new analytical method for measuring estradiol in serum from post and premenopausal women, men and children, which is standardized to the CDC Hormone Standardization Program (HoSt). Data produced by this analytical method can be compared with data produced by other methods standardized to CDC HoSt. The analytical method optimizes the extraction of estradiol from serum using two liquid-liquid extractions with pH and polarity adjustments prior to LC-MS/MS analysis. All sample handling and extraction procedures are automated using 96-well plates. Chromatographic separation is carried out using a phenyl-hexyl HPLC column and a gradient of methanol and methanol:water. E2 and its C13 internal standard were analyzed by selected reaction monitoring (SRM) in the negative ion mode with transitions of m/z 271 to 145 and 274 to 148, respectively.

The mean biases of this method to certified reference materials ranged between −1.3% and −1.7% and were statistically not significant. No significant difference to established metrological reference methods was determined. The limit of detection for estradiol using 200 µL of serum is 11.0 pM (2.99 pg/mL). The method is highly precise with a within-run, among-day (determined over 68 days), and total within-laboratory imprecision ranging between 2.9-5.0%, 1.5-1.8%, and 3.3-5.3% CV, respectively [1].

The sensitivity of the method was found to be suitable for determining estradiol levels in postmenopausal women, men, and children, and is being applied to measure estradiol in postmenopausal women in NHANES 2013-2014. Data generated by this method are comparable to those of other analytical methods that are standardized to the CDC HoSt program. This method allows for the development of generally recognized normal ranges of estradiol in postmenopausal women and other population subgroups.

References:

1. Zhou, Hui et al. "Simultaneous measurement of total estradiol and testosterone in human serum by isotope dilution liquid chromatography tandem mass spectrometry" Analytical and bioanalytical chemistry vol. 409,25 (2017): 5943-5954.

#588

Relationship of serum progesterone and progesterone metabolites with mammographic density.

Manila Hada,1 Hannah Oh,2 Sharon Fan,1 Roni T. Falk,1 Berta Geller,3 Pamela Vacek,3 Donald Weaver,3 John Shepherd,4 Jeff Wang,5 Bo Fan,6 Amir P. Mahmoudzadeh,6 Serghei Malkov,6 Sally Herschorn,3 Louise A. Brinton,1 Xia Xu,7 Mark E. Sherman,8 Britton Trabert*,1 Gretchen L. Gierach*1. 1 _National Cancer Institute, Rockville, MD;_ 2 _Korea University, Seoul, Republic of Korea;_ 3 _University of Vermont and Vermont Cancer Center, Burlington, VT;_ 4 _University of Hawaii Cancer Center, Honolulu, HI;_ 5 _Hokkaido University, Graduate School of Medicine, Sapporo, Japan;_ 6 _University of California, San Francisco, CA;_ 7 _Frederick National Laboratory for Cancer Research, Frederick, MD;_ 8 _Mayo Clinic, Jacksonville, FL_.

Background:

Mammographic breast density (MBD) is a strong breast cancer (BC) risk factor, but its biologic underpinnings are poorly understood. Use of estrogen plus progestin menopausal hormone therapy is linked to increased MBD and BC risk. Experimental data suggest that ratios of tumor promoting (5α-dihydroprogesterone [5αP]) and anti-tumorigenic (3α-dihydroprogesterone [3αP]) progesterone metabolite levels may be related to BC risk. However, relationships of endogenous progesterone and its metabolites with MBD and BC risk have not been established. Accordingly, we assessed levels of circulating progesterone and its metabolites with MBD.

Methods:

In this cross-sectional study, serum progesterone and its metabolites were quantified using a novel liquid chromatography-tandem mass spectrometry assay in 103 postmenopausal and 52 premenopausal (luteal menstrual cycle phase) women, ages 40-65, undergoing diagnostic image-guided ipsilateral breast biopsy. MBD was measured as percent fibroglandular volume (MBD-V) on pre-biopsy digital mammograms using single X-ray absorptiometry. Square-root transformed MBD-V was examined across tertile categories of progesterone/progesterone metabolites using age and body mass index (BMI)-adjusted linear regression models.

Results:

Concentrations of the hormones were as follows among postmenopausal women: progesterone [mean: 12.6 pmol/L (range: 5.2-45.8)], 3αP [5.6 pmol/L (1.4-18.8)], 5αP [100 pmol/L (16.7-388)], and 5αP/3αP ratio [26.1 (2.1-150)]; and among luteal phase premenopausal women: progesterone [2063 pmol/L (13.6-7098), 3αP [12.7 pmol/L (2.4-64.4)], 5αP [243 pmol/L (25.3-774)], 5αP/3αP ratio [25.9 (2.3-73.7)]. Among postmenopausal women, progesterone was positively associated with MBD-V (Tertile 3 vs. 1: β=0.68, p-trend=0.02). A similar borderline positive association was observed among premenopausal women (β=0.74, p-trend=0.10). Additional adjustment for circulating estradiol did not substantively alter observed associations. Levels of 3αP, 5αP and the 5αP/3αP ratio were not associated with MBD-V among pre- or postmenopausal women.

Conclusions:

Concentrations of progesterone and it metabolites show substantial inter-woman variation. We observed a positive association between endogenous progesterone and MBD-V among both postmenopausal and premenopausal luteal phase women. We did not observe an association with the ratio of 5αP to 3αP levels and MBD-V. These findings suggest the need for additional studies to understand the biological basis of the role of progesterone and its metabolites in MBD and BC risk.

#589

Circulating progesterone is associated with increased postmenopausal breast cancer risk: B~FIT cohort.

Britton Trabert,1 Doug C. Bauer,2 Louise A. Brinton,1 Diane S. Buist,3 Jane A. Cauley,4 Cher M. Dallal,5 Gretchen L. Gierach,1 Roni T. Falk,1 Trisha F. Hue,2 James V. Lacey,6 Andrea Z. LaCroix,7 Jeffrey A. Tice,2 Xia Xu8. 1 _National Cancer Institute, Bethesda, MD;_ 2 _University of California San Francisco, San Francisco, CA;_ 3 _Kaiser Permanente Washington Health Research Institute, Seattle, WA;_ 4 _University of Pittsburgh, Pittsburgh, PA;_ 5 _University of Maryland, College Park, MD;_ 6 _City of Hope, Duarte, CA;_ 7 _University of Washington, Seattle, WA;_ 8 _Leidos Biomedical Research, Inc., Frederick, MD_.

BACKGROUND: Epidemiologic data provide compelling evidence of the association between elevated endogenous estrogens and androgens and increased postmenopausal breast cancer risk. However, the role of progesterone remains largely unexplored, primarily due to limitations in assay sensitivity and precision of progesterone measurements at low concentrations in postmenopausal women. Recently identified progesterone metabolites may provide etiologic insights as experimental data suggest that relative changes in concentrations of 5-α dihydroprogesterone (5αP) and 3-α dihydroprogesterone (3αHP) reflect cancer promoting and cancer inhibiting properties, respectively.

METHODS: We developed a sensitive and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay and quantified prediagnostic levels of progesterone/progesterone metabolites in a case-cohort study within the Breast and Bone Follow-up to the Fracture Intervention Trial (B~FIT) including 405 breast cancer cases diagnosed during follow-up and a subcohort of 495 postmenopausal women not using exogenous hormones at blood draw. Multivariable adjusted hazard ratios (HR) and 95% confidence intervals (CIs) were estimated using Cox regression and linearity was assessed using splines.

RESULTS: Hormone concentrations among women in the subcohort were on average 4.6 pg/mL (range 2.3-21.6) for progesterone; all measured values were above the assay detection limit. Women with higher circulating progesterone levels had an elevated postmenopausal breast cancer risk [HR (95% CI) per 10 pg/mL increase in progesterone: 1.18 (0.99-1.41)]. This association was linear in a 5-knot spline and strengthened [1.24 (1.03-1.49)] in models excluding women who reported current use of tamoxifen/raloxifene. Higher levels of 5αP relative to 3αHP were associated with a 4% increased postmenopausal breast cancer risk [per unit increase in ratio: 1.04 (1.00-1.07)]. For the individual metabolites, higher levels of both 5αP [per 10 pg/mL increase: 1.04 (0.93-1.18) and 3αHP [per 3 pg/mL increase: 1.11 (0.999-1.24)] were associated with elevated risk. All associations remained after adjustment for circulating estrogen levels.

CONCLUSIONS: Our prospective data suggest that postmenopausal women with increased serum progesterone concentrations, measured using a highly sensitive LC-MS/MS assay, are at increased risk of breast cancer. Consistent with experimental studies, higher levels of 5αP relative to 3αHP were indicative of increased breast cancer risk. Unlike experimental studies, our data do not suggest that endogenous concentrations of 3αHP are associated with reduced breast cancer risk; instead both metabolites were associated with elevated risk. The identification of these risk-related progesterone metabolites supports the need for additional research regarding their role in the etiology of breast cancer.

#590

Circulating estrogen metabolites and risk of postmenopausal breast cancer in the Nurses' Health study.

Kristen D. Brantley,1 Regina G. Ziegler,2 Susan E. Hankinson,3 A. Heather Eliassen3. 1 _Harvard TH Chan School of Public Health, Boston, MA;_ 2 _Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD;_ 3 _Channing Division of Network Medicine, Brigham and Women's Hospital and Harvard Medical School, MA_.

The positive association between endogenous estrogen levels and breast cancer risk in postmenopausal women has been well established. However, evidence for whether irreversible hydroxylation of the parent estrogens (estrone and estradiol) at the 2- or 16-position plays an important role in breast cancer etiology among postmenopausal women remains limited.

We performed a nested case-control study within the Nurses' Health Study to examine plasma estrogens and estrogen metabolites (jointly referred to as EM) among postmenopausal women not using menopausal hormone therapy at blood draw (in 1989-1990). Breast cancer cases (N=341) were diagnosed between 1996 and 2004. Controls (N=679) were individually matched to cases. Total concentrations of each EM, including conjugated and unconjugated forms, were measured by liquid chromatography-tandem mass spectrometry.

Multivariate conditional logistic regression, adjusting for breast cancer risk factors, was used to estimate the relative risk (RR) and 95% confidence interval (CI) of breast cancer comparing highest v. lowest quintiles of individual EM, summed 2-hydroxylation catechols, summed 16-hydroxylation pathway, and pathway ratios. Secondary analyses included adjustment for unconjugated and total estradiol and estrone. Unconditional logistic regression with adjustment for batch, matching factors, and breast cancer risk factors was performed to estimate the RR by breast tumor subtype, defined by estrogen receptor (ER) and progesterone receptor (PR) status.

Higher concentrations of both total estradiol and total estrone were associated with increased risk of breast cancer overall (both p-trend<0.0001) and among ER+/PR+ tumors [estradiol overall RR=2.7, 95% CI (1.6-4.4); ER+/PR+ RR=3.7, 95% CI (1.9-7.4)]. A positive association was observed between 2-hydroxylation pathway catechols and overall breast cancer [RR=2.7; 95% CI (1.5-4.6); p-trend<0.0001], which remained significant, though attenuated, after adjusting for total estradiol and estrone [RR=2.0; 95% CI (1.1-3.6)]. This association remained consistent among ER+/PR+ tumors [RR=2.4; 95% CI (1.3, 4.5)], with slight attenuation following adjustment for parent EM. Though there was a suggestive positive association between the 16-hydroxylation pathway and overall breast cancer [RR =1.5, 95% CI (0.9-2.5)], it was not maintained after adjustment for total estradiol and estrone. However, one 16-hydroxylation EM, 17-epiestriol, was associated with risk of overall breast cancer even after adjustment for total estradiol and estrone [RR=1.8, 95% CI (1.0-3.0)].

The 2-hydroxylation of parent estrogens to 2-catechols was associated with a higher risk of overall breast cancer, independent of parent estrogens. This pathway may provide etiologic insight and targets for further research into risk assessment and prevention.

#591

Circulating testosterone in modifying the association of BMI change rate with serum PSA in prostate cancer-free men with initial-PSA less than 4 ng/mL.

Kai Wang,1 Mattia Prosperi,2 Peihua Qiu,2 Ting-Yuan David Cheng,2 Victoria Y. Bird,2 Xinguang Chen,2 Mingyang Song1. 1 _Harvard University, Boston, MA;_ 2 _University of Florida, Gainesville, FL_.

Background: Body mass index (BMI)-adjusted prostate-specific antigen (PSA) model has been proposed to improve the predictive accuracy of serum PSA in prostate cancer (PCa) screening. However, how BMI change rate may influence PSA levels in PCa-free men has not been well studied. The current study is to examine the relationship between BMI change rate and serum PSA in PCa-free men and whether this relationship is modified by circulating testosterone.

Methods: We conducted this study at a tertiary hospital in the Southeastern US using the Electronic Medical Records of PCa-free men with initial PSA less than 4 ng/mL (cutoff for prostate biopsy), at least 1 testosterone measurement and at least 2 BMI measurements during the study period. Time when the first BMI measurement was recorded served as the baseline, and the study period was defined from baseline to the most recent hospital visit. The included medical records ranged from Jun 2001 to Oct 2015. BMI change rate was created in two ways depending on the number of data points. For men with only 2 BMI measurements, it was calculated by firstly subtracting baseline BMI from the second BMI, then dividing the difference by time interval (months) between the two BMI measurements. For men with more than 2 BMI measurements, we firstly regressed BMI to time interval (months) between that measurement and baseline, then took the β regression coefficient (slope) as the BMI change rate for that men. Multivariable linear regression was used to assess the association of BMI change rate with three PSA measures, including peak, the most recent, and mean PSA during the study period. Effect modification by testosterone was assessed through stratified analysis by testosterone level of 280 ng/dL as cutoff.

Results: A total of 470 men with a mean study period of 97.6 months were included. Median age at baseline was 62 years. After adjusting for covariates including baseline BMI, no significant association of BMI change rate was observed with peak PSA (β =0.416, P =0.078), the most recent PSA (β =0.360, P =0.139), or mean PSA (β =0.405, P =0.064) in the overall sample. However, testosterone-stratified analyses indicated that BMI change rate was positively associated with peak PSA (β =1.118, P =0.013), the most recent PSA (β =0.932, P =0.044), and mean PSA (β =1.034, P =0.013) in men with testosterone <280 ng/dL, but no significant association was observed in men with testosterone ≥280 ng/dL (for peak PSA, β =0.076, P =0.785; for the most recent PSA, β =0.072, P =0.802; for mean PSA, β =0.099, P =0.700).

Conclusion: Accelerated BMI increase in middle-to-late adulthood might correlate with higher PSA level if a low circulating testosterone occurred concurrently. Further studies are needed to confirm this finding.

#592

Associations of mammographic breast density with breast stem cell marker-defined breast cancer subtypes.

Lusine Yaghjyan,1 Ashwini Esnakula,1 Christopher Scott,2 Akemi Wijayabahu,1 Matthew Jensen,2 Celine Vachon2. 1 _Univ. of Florida, Gainesville, FL;_ 2 _Mayo Clinic, Rochester, MN_.

Background: High mammographic breast density is a strong, well-established breast cancer risk factor. A recent study found increased expression of stem cell markers in the areas of high breast density, but, whether stem cells may explain high breast cancer risk due to high breast density is unknown. We investigated the association between breast density and the risk of breast cancer by the status of stem cell markers CD44, CD24, and ALDH1A1 in the tumor.

Methods: We included 223 postmenopausal women with primary invasive or in-situ breast cancer and 399 age-matched controls from Mayo Clinic Mammography Study. Percent breast density, absolute dense area and non-dense area were assessed using computer-assisted thresholding technique. Immunohistochemical analysis of the markers was performed on tumor tissue microarrays according to a standard protocol with commercial antibodies. Cases with positive staining (≥1% positively stained cells) on one or more cores were considered marker-positive. We used polychotomous logistic regression to quantify the associations of breast density measures with breast cancer risk by the status of each of the markers. Heterogeneity tests were conducted to compare the differences in associations by marker-defined tumor subtypes.

Results: Of the 223 cancers in the study, 182 (82%) were positive for CD44, 83 (37%) for CD24 and 52 (23%) for ALDH1A1. Associations of percent density were not significantly different across any of the marker-defined subtypes (density 51%+ vs. 11-25%: OR=2.83, 95% CI 1.49-5.37 for CD44+ vs. OR=1.87, 95% CI 0.47-7.51 for CD44-, p-heterogeneity=0.66; OR=2.80, 95% CI 1.27-6.18 for CD24+ vs. OR=2.44, 95% CI 1.14-5.22 for CD24-, p-heterogeneity=0.61; OR=3.04, 95% CI 1.14-8.10 for ALDH1A1+ vs. OR=2.57. 95% CI 1.30-5.08 for ALDH1A1-, p-heterogeneity=0.94). Positive associations of absolute dense area and inverse associations of non-dense area with breast cancer risk were similar across marker-defined subtypes. Similar associations of breast density measures with breast cancer were seen when cancer subtypes were defined using combined marker expression status (none, one positive marker, or two and more positive markers).

Conclusions: We found no evidence of differential associations of breast density measures with breast cancer risk by the status of stem cell markers. However, our power was limited and further studies in larger study populations are warranted to confirm these associations in a larger sample.

#593

Mammographic breast density in postmenopausal women is affected by the age of first full term pregnancy.

Magda J. Vandeloo,1 Kristof Y. Neven,2 Liesbeth Bruckers,2 Jose Russo,3 Liesbeth Vancoullie,4 Esmée Bijnens,2 Eliane Kellen,4 Tim S. Nawrot,2 Chantal Van Ongeval4. 1 _University of Leuven, Leuven, Belgium;_ 2 _Hasselt University, Hasselt, Belgium;_ 3 _Fox Chase Cancer Center, Philadelphia, PA;_ 4 _University Hospital Leuven, Leuven, Belgium_.

Mammographic breast density (MBD) is accepted as a risk factor in breast cancer, whereas age of first full term pregnancy (FFTP) has been demonstrated to be a protective one. The objective of this study is to investigate the association between reproductive history and MBD at menopause. Women, age 50-69y, participating in the Flemish (Belgium) governmental organised breast cancer screening program were invited to participate in the study during their visit at the mammography department in Leuven (hospital and the mobile unit). The study was approved by the ethical committee of the hospital. After signing the informed consent, a self-administered questionnaire (SAQ) providing information on lifestyle and environmental exposure was completed by the participants. In the present work we present the analysis of the first 640 women that completed the SAQ and underwent mammography. Questionnaire data were registered via the Qualtrics software. The three parameters of MBD (VolparaDataManager®, USA) included the percentage glandular tissue of the total breast volume (GLAND), the ratio of the glandular tissue volume compared to the whole breast volume or Volumetric Breast Density (VBD) and the BIRADS density classification. We applied linear regression models to investigate the association between the continuous breast density markers (GLAND and VBD, log transformed) and the following explanatory variables: age of woman, ever pregnant, number of pregnancies, age of FFTP, ever breastfed (BF), amount and duration of BF, and breastfeeding in FFTP. An ordinal logistic regression model was used for the breast density marker BIRADS. The average GLAND and VBD changes with a multiplicative factor of 1.073 (p = 0.013) and 1.072 (p = 0.013) for a 5-year increase of age of FFTP. Thus, an increase of 5 years in age of FFTP is associated with about 7% increase in geometric mean of GLAND and VBD. The odds ratio for a higher versus a lower score of the BIRADS classification is 1.24 (p = 0.023) greater for every 5-year increase in age of FFTP. These results are independent for the inverse association between the age of the woman and the MBD markers. For a 5 year increment in age, geometric GLAND (p = 0.019) and VBD (p = 0.024) are approximately 5% lower and the odds ratio for a higher BIRADS score is 12% higher (p = 0.079). There is no association between the MBD markers and breastfeeding, the number and duration BF, and the number of pregnancies after correcting for age and age of FFTP. In conclusion, this study is to our knowledge the first one to show that MBD is significantly reduced when pregnancy takes place at early age.( This study was supported by a Grant of the Breast Cancer Organization Think Pink of Belgium, the Department of Breast Radiology and Mobile Unit of the University Hospital Leuven, the Center of Environmental Sciences and the Center of Biostatistics of the Hasselt University, and NIH Grant CA06927 to FCCC, PA, USA.)

#594

Associations of hemoglobin A1c with risk of diabetes-related cancers in the Cancer Prevention Study-II Nutrition Cohort (CPS-II NC).

Peter T. Campbell,1 Christina Newton,1 Eric J. Jacobs,1 Michael Pollak,2 Susan M. Gapstur1. 1 _American Cancer Society, Atlanta, GA;_ 2 _McGill University, Montreal, Quebec, Canada_.

Self-reported type 2 diabetes mellitus (T2DM) is convincingly associated with higher risks of liver, pancreatic, colon, rectal, female breast, and endometrial cancers, and may also be associated with higher risks of ovarian, bladder and kidney cancers. This evidence largely relies on self-reported T2DM, which does not properly classify the many individuals with pre-diabetes or with undiagnosed T2DM or adequately reflect glucose control among people with T2DM. To clarify these associations, we conducted a case-cohort analysis of hemoglobin A1c (HbA1c), an indicator of circulating glucose over the past 2-to-3 months used to diagnose and monitor T2DM. Participants were identified from the CPS-II NC. From an initial cohort of 32,328 participants who were cancer-free and provided a blood sample at baseline in 1998-2001, we selected a random sub-cohort of 3,000 participants. Further, we selected all participants diagnosed between baseline and June, 2013 with a verified, incident cancer of the colorectum (n=479), liver (n=35), pancreas (n=176), female breast (n=889), endometrium (n=155), ovary (n=93), bladder (n=344), or kidney (n=110). Weighted Cox proportional hazards regression models estimated hazards ratios (HRs) and 95% confidence intervals (CI) for associations of HbA1c with cancer risks combined and stratified by organ site. HRs were adjusted for age, gender, smoking, physical activity, alcohol, and hormone-use (women only). HbA1c levels reflective of clinically-defined T2DM (>=6.5%), compared to HbA1c levels in the non-diabetes range (<5.7%), were associated with statistically significantly higher risks of all 9-T2DM-associated cancers combined (HR: 1.30; 95% CI: 1.05-1.60) and colorectal cancer (HR: 1.58; 95% CI: 1.14-2.19), and associations, although not statistically significant, were in the same, hypothesized directions for risks of liver (HR: 2.30), pancreas (HR: 1.60), endometrial (HR: 1.61), ovarian (HR: 1.85), bladder (HR: 1.23), and kidney (HR: 1.23) cancers. There was no suggestion of association between HbA1c and breast cancer risk (HR: 0.95). Further analyses of colorectal cancer risk combined self-reported T2DM and measured HbA1c levels. Compared to participants who had non-diabetes levels of HbA1c (<=6.5%) and did not report T2DM, participants with high HbA1c levels (>=6.5%) were at higher risk of CRC whether they self-reported T2DM (HR: 1.54), or not (HR:1.56), whereas participants who self-reported T2DM but had good glycemic control (HbA1c =<6.5%) were not at higher risk (HR: 0.96). Results for c-peptide and CRP are forthcoming. This study suggests that HbA1c, a clinically meaningful marker of circulating glucose, is related to the etiology of some cancers.

#595

Circulating levels of coenzyme Q10 and lung cancer risk.

Chris Shidal,1 Hyung-Suk Yoon,1 Wei Zheng,1 Jie Wu,1 Adrian A. Franke,2 William J. Blot,1 Xiao-Ou Shu,1 Qiuyin Cai1. 1 _Vanderbilt University School of Medicine, Nashville, TN;_ 2 _University of Hawaii at Manoa, HI_.

Background: Coenzyme Q10 (CoQ10) is an ubiquitous molecule in living organisms that serves as a cofactor in energy production via the electron-transport chain (ETC). Research has previously shown that deficiencies in CoQ10 can result in a variety of detrimental outcomes including cardiovascular disease and neurological disorders. More recently, epidemiological studies have reported low CoQ10 levels being associated with increased risk of breast, ovarian, and skin cancers. However, studies exploring the link between CoQ10 and lung cancer risk are largely lacking.

Methods: A nested case-control study including 201 lung cancer cases and 395 matched controls from the Southern Community Cohort Study (SCCS) was conducted. Plasma CoQ10 levels were measured using high-performance liquid chromatography (HPLC). A conditional logistic regression model was applied to estimate the odds ratio (OR) and 95% confidence interval (CI) for the association between plasma CoQ10 concentrations and lung cancer risk. CoQ10 level was categorized by tertile distribution among controls.

Results: Plasma CoQ10 concentration was inversely associated with risk of lung cancer. Compared to the first tertile, the ORs (95% CI) were 0.82 (0.52-1.31) and 0.57 (0.34-0.96) for the second and third tertile, respectively, (P for trend=0.038), after adjusting for age, smoking status and pack-years of smoking. Additional adjustment for alcohol consumption, education, income, history of COPD and BMI slightly attenuated the association (OR=0.60, 95% CI=0.34-1.08, comparing third to first tertile). No notable difference was found between African Americans and European Americans. Stratified analysis identified a significant association between CoQ10 and lung cancer in current smokers (OR=0.47, 95% CI=0.26-0.87, third vs. first tertile), but not in former/never smokers (OR=0.84, 95% CI=0.35-2.03). The significant associations between CoQ10 and lung cancer risk were more evident in cases who were diagnosed within one year of blood draw (OR=0.17, 95% CI=0.04-0.73, third vs. first tertile, P for trend=0.023) than in cases diagnosed after one year following blood draw (OR=0.72, 95% CI=0.36-1.41).

Conclusions: Findings from this study suggest that Coenzyme Q10 may be a potential marker for lung cancer, although the stronger effect shortly after diagnosis indicates the association may be related to dietary or other changes associated with the cancer diagnosis rather than CoQ10 being a causal risk biomarker. Further studies exploring the link between CoQ10 and lung cancer risk are necessary to better understand the role of CoQ10 in lung carcinogenesis.

#596

Association between an empirically-derived inflammatory lifestyle score and incident colorectal cancer.

Mark Guinter,1 Susan Gapstur,1 W. Dana Flanders,2 Ying Wang,1 Erika Rees-Punia,1 Marjorie McCullough,1 Kassandra I. Alcaraz,1 Michael Pollak,3 Peter Campbell1. 1 _American Cancer Society, Atlanta, GA;_ 2 _Emory University, Atlanta, GA;_ 3 _McGill University, Montreal, Quebec, Canada_.

Inflammation is often suggested as major pathway by which lifestyle influences the development of colorectal cancer (CRC). In the present analysis, we empirically derived an inflammatory lifestyle score (ILS) based on associations of nine lifestyle factors with serum levels of high-sensitivity C-reactive protein (hsCRP) and subsequently examined its association with incident CRC in the Cancer Prevention Study-II Nutrition Cohort. Lifestyle factors other than smoking were chosen a priori from the American Cancer Society's Guidelines on Nutrition and Physical Activity for Cancer Prevention to include body mass index, physical activity, sedentary time, fruit/vegetable intake, variety of fruits/vegetables consumed, red/processed meat intake, ratio of whole to refined grains consumed, alcohol consumption, and smoking status. Reduced rank regression was used to create sex-specific lifestyle scores that correlate the nine lifestyle factors with hsCRP in a testing subset of 2,707 men and women with no history of cancer at the time of blood draw. In a validation subset (n=540), the derived ILS was positively correlated with serum hsCRP levels (r=0.19, p-value<0.001). Model weights from reduced rank regression in the testing subset were used to calculate the ILS among 114,974 men and women of the cohort for whom hsCRP levels were not available. The association between the ILS and incident CRC was examined using Cox proportional hazards regression to estimate hazard ratios and 95% confidence intervals adjusted for age, sex, education, non-steroidal anti-inflammatory drug use, multivitamin use, postmenopausal hormone therapy, and diabetes status. Starting at baseline in 1999, during a mean follow-up of 12.6 years, 2,082 incident CRC cases were identified (1,649 colon; 433 rectum). Participants in the third and fourth ILS quartiles had 15% (95% CI: 1%, 31%) and 34% (95% CI: 18%, 53%) higher risk of CRC, respectively, than those in the first ILS quartile. A 1-standard deviation increase in the score was associated with 10% higher risk of CRC (95% CI: 1.05, 1.15). The association between the ILS and CRC did not differ across strata of sex or age (<70, ≥70 years). A Lunn-McNeil competing risk model showed no evidence of a differential association of the ILS on CRC organ subtypes (colon vs. rectal). Results support evidence that a lifestyle indicative of high inflammatory potential is associated with an increased risk of developing CRC. To examine other mechanisms by which lifestyle influences CRC risk, additional analyses will use serum levels of C-peptide and hemoglobin A1c. The biomarkers will be examined individually, together (i.e., glucose homeostasis), and in combination with hsCRP (poor metabolic function) to develop lifestyle scores.

#597

Occupational exposure to diesel engine exhaust and alternations in serum microRNAs.

Wei Hu,1 Bryan A. Bassig,1 Yufei Dai,2 Dianzhi Ren,3 Huawei Duan,2 Yong Niu,2 Jun Xu,4 Wei Fu,3 Kees Meliefste,5 Baosen Zhou,6 Jufang Yang,3 Meng Ye,2 Xiaowei Jia,2 Tao Meng,2 Ping Bin,2 Jason YY Wong,1 Dean H. Hosgood,7 Nathaniel Rothman,1 Roel C. Vermeulen,5 Debra T. Silverman,1 Yuxin Zheng,8 Qing Lan1. 1 _National Cancer Institute, Rockville, MD;_ 2 _Chinese Center for Disease Control and Prevention, Beijing, China;_ 3 _Chaoyang Center for Disease Control and Prevention, Chaoyang, China;_ 4 _The University of Hong Kong, Hong Kong, Hong Kong;_ 5 _Utrecht University, Utrecht, Netherlands;_ 6 _China Medical University, Shenyang, China;_ 7 _Albert Einstein College of Medicine, New York, NY;_ 8 _Qingdao University, Qingdao, China_.

Diesel engine exhaust (DEE) is a known lung carcinogen and may be associated with other tumors, however, the mechanisms of action by which DEE causes cancer is not well understood. MicroRNAs (miRNAs), which are small non-coding RNA molecules that play a role in post-transcriptional regulation of gene expression, are altered in multiple tumors and have been observed to be differentially expressed in a variety of biospecimen types in smokers as well as in relation to short-term air pollution exposure. To evaluate whether serum levels of miRNAs are altered in healthy workers occupationally exposed to DEE, we analyzed samples collected in a cross-sectional molecular epidemiology study of diesel engine truck testing facility workers and comparable unexposed controls in China. A panel of 44 miRNAs were measured in 46 workers exposed to relatively high air levels of DEE and 45 controls using the Fireplex circulating miRNA assay (Abcam, Inc.), which profiles miRNAs directly from biofluids. The exposure-response relationship between categorical EC levels and each miRNA was analyzed by linear regression adjusted for age, body mass index, smoking status, current alcohol use and recent infection. We identified two miRNAs that showed a monotonic inverse exposure-response association with DEE: miR-191-5p and miR-93-5p. Levels of miR-191-5p in arbitrary units (A.U.) of fluorescence were 400.7, 333.0, 322.6, and 260.0 in controls and across increasing tertiles of EC, respectively (p for trend = 0.001, FDR = 0.05). Levels of miR-93-5p were 747.1, 713.7, 720.5, and 625.4 A.U. in controls and increasing tertiles of EC, respectively (p for trend = 0.008, FDR = 0.18). Both miRNAs have been reported to influence several biological processes important in carcinogenesis. Our results suggest that occupational exposure to DEE may affect circulating miRNAs in healthy workers.

#598

Genetically predicted concentration of circulating proteins and epithelial ovarian cancer risk.

Guochong Jia,1 Xiang Shu,1 Xiao-Ou Shu,1 Jirong Long,1 Qiuyin Cai,1 Kirsten B. Moysich,2 Ellen L. Goode,3 Thomas A. Sellers,4 Wei Zheng1. 1 _Vanderbilt University, Nashville, TN;_ 2 _RoswellPark Cancer Institute, Buffalo, NY;_ 3 _Mayo Clinic, Rochester, MN;_ 4 _Moffitt Cancer Center, Tampa, FL_.

Epithelial ovarian cancer (EOC) is the most common gynecological cancer, and ranks fifth in cancer deaths among women in the United States. Previous studies have identified several circulating proteins biomarkers for EOC risk, such as CA125 antigen and insulin-like growth factor-1 (IGF-1). However, previous studies on protein biomarkers for EOC were limited with small sample sizes, and only a few proteins investigated. In this study, we integrated genomics and proteomics data to search for novel circulating protein biomarkers for EOC risk. We constructed study instruments using genetic variants for protein quantitative trait loci (pQTL) for over 1,400 circulating proteins. Beta coefficients and standard errors of the selected pQTL variants in relation to EOC risk were obtained from 25,509 EOC cases and 40,941 controls from the Ovarian Cancer Association Consortium (OCAC). An inverse-variance weighted method was used to evaluate the association of these genetically predicted protein levels and risk of overall and serous EOC. We identified 35 protein biomarkers, for which their genetically predicted levels were significantly associated with the risk of either overall or serous EOC (false discovery rate <0.05). There are 25 proteins showing significant associations with both overall and serous EOC; most of the associations were stronger for serous EOC than overall EOC. The association for 31 proteins could be attributed, in part, to variants at ABO 9q34.2, a known ovarian cancer susceptibility locus. Five ABO locus-related proteins are in the PI3K-Akt signaling pathway, including insulin-like growth factor 1 receptor, hepatocyte growth factor receptor, interleukin-3 receptor subunit alpha, insulin receptor, and vascular endothelial growth factor receptor 2. Four of these proteins are also in the Rap1 and Ras signaling pathway. These five proteins are all inversely associated with the risk of overall EOC, with odds ratios ranging from 0.64 to 0.93 per unit increase in genetic risk scores (p values: 7.55×10-7 to 5.66×10-2). Four EOC associated proteins which were not influenced by variants in 9q34.2 are microfibrillar-associated protein 2, protein delta homolog 1, semenogelin-2, and netrin-G1, all of which showed significant associations with overall EOC, but not the serous subtype. Our study identifies potential novel biomarkers for ovarian cancer and suggests that genetic variants at locus 9q34.2 may play a role in the relation between levels of these proteins and risk of ovarian cancer.

#599

**Smoking-associated** AHRR **demethylation in cord blood DNA: Impact of CD235a+ nucleated red blood cells.**

Matthew A. Bergens, Gary S. Pittman, Isabel J. Thompson, Michelle R. Campbell, Xuting Wang, Ma Wan, Cathrine Hoyo, Douglas A. Bell. _NIH, Durham, NC_.

Prenatal tobacco smoke exposure is associated with numerous adverse health outcomes at birth and in later life. Numerous studies have demonstrated that Aryl Hydrocarbon Receptor Repressor gene (AHRR) hypomethylation in umbilical cord blood is significantly associated with prenatal tobacco smoke exposure and is suitable as a fetal exposure biomarker. The mechanism driving this demethylation and whether all cord blood cell types are impacted is unclear. Nucleated red blood cells (nucleated CD235a+ cells, nRBCs) are developmentally immature RBCs that display genome-wide demethylation and are observed at increased frequency in the cord blood of smoking mothers. We assessed if nRBC counts or methylation level affected smoking-associated DNA methylation in the AHRR gene as measured in whole cord blood. We analyzed methylation in DNA from whole cord blood, CD14+ monocytes, and nRBCs across four AHRR CpGs using pyrosequencing and also with Illumina 850K arrays (cg05575921) from smoking (n=34) and nonsmoking (n=19) mothers. nRBCs present in cord blood were determined by conventional CBCs and also estimated with a Houseman deconvolution model. AHRR methylation levels were significantly lower in nRBCs relative to whole blood and CD14+ monocytes. Methylation values at each AHRR CpG averaged 14.6% lower in nRBCs (range 0.4% to 24%, p=3.9E-13) relative to CD14+ monocytes with nonsmokers displaying significantly greater hypomethylation in nRBCs than smokers. Methylation levels at each CpG across the AHRR DMR were strongly associated with either self-reported smoking or cord blood cotinine levels in both CD14+ monocytes (r2=0.45, p=4.6E-08) and nRBCs (r2=0.26, p=1.2E-04). The most significant prenatal smoking effect on methylation occurred in CD14+ monocytes at the CpG located at chr5:373490 (-16.7%, p=6.7E-09), 112nt downstream of cg05575921. For subjects with 850K data, adjusting a regression analysis model with estimated cell-type composition, including nRBC counts or estimates, marginally increased the magnitude and significance of the prenatal smoke exposure effect on AHRR cg05575921 methylation. Prenatal tobacco smoke exposure strongly affects DNA methylation in the AHRR gene and this prenatal smoke exposure biomarker is largely unaffected by cord blood cell type composition.

#600

Clues to understanding the association between B-activation markers and the risk of B-cell non-Hodgkin lymphoma.

Lauren R. Teras,1 Mia M. Gaudet,1 Krishnaveni Subbiah,1 Esmeralda J. Krop,2 Roel Vermeulen,2 Susan M. Gapstur1. 1 _American Cancer Society, Atlanta, GA;_ 2 _Utrecht University, Utrecht, Netherlands_.

Although severe immune dysregulation is an established risk factor for B-cell non-Hodgkin lymphoma (B-NHL), the role of subtle immune perturbations is unclear. However, associations between plasma/serum immune markers and B-NHL in immunocompetent individuals suggest a role for more moderate immune dysregulation in lymphomagenesis. To further understand the associations of immune markers with B-NHL risk, we evaluated associations between these markers and subsequent B-NHL risk; and also examined potential lymphomagens as predictors of the markers. Among the eligible population of 32,704 cancer-free individuals in the Cancer Prevention Study-II Nutrition Cohort who donated a blood sample between 1998 and 2001, 273 B-NHL cases were identified during ~13 years of follow-up. Cases were incidence-density matched to cancer-free controls on age, race, and sex. Plasma level of soluble CD23 (sCD23), sCD27, sCD30, and CXCL13 were measured by ELISA at the Institute for Risk Assessment Sciences, Utrecht University. Rate ratios (RR) and 95% confidence intervals (CI) were estimated using conditional logistic regression. Associations between infectious, lifestyle, and demographic factors and B-activation marker level were examined using multivariable linear regression. We observed strong associations for all four markers with risk of B-NHL (per standard deviation increase in the biomarker: CXCL13: Risk Ratio (RR)=4.18, 95% confidence interval (CI): 2.46-7.11; CD23: RR=3.59, 95% CI: 2.50-5.18; CD27: RR=2.04, 95% CI: 1.48-2.81; CD30: RR=3.12, 95% CI: 2.01-4.84). The associations remained strong regardless of time between biomarker measurement and B-NHL diagnosis with the exception of sCD27 which was attenuated ≥8 years after biomarker measurement. We did not observe differences by major B-NHL subtype with the exception of a notably stronger association for sCD23 with chronic lymphocytic leukemia (CLL). Age was the strongest predictor of CXCL13 (p<0.0001), sCD27 (p=0.0009), and sCD30 (p=0.0043) level, but was not associated with sCD23 (p=0.1018). Body mass index, smoking, and alcohol intake were not associated with any of the markers. However, two Epstein-Barr virus reactivation markers (early antigen and ZEBRA) were associated with sCD27 (p=0.004 and p=0.003 respectively).These varied risk factor associations and time-since-biomarker measurement patterns, suggest that there may be different roles in lymphomagenesis for different B-activation markers. Understanding these associations may help identify high risk individuals—and potential targets for early intervention for B-NHL.

#601

Early pregnancy serum sRANKL, OPG, and TRAIL and maternal breast cancer risk: Results from the Finnish Maternity Cohort.

Danja Sarink,1 Theron Johnson,1 Helja-Marja Surcel,2 Rudolf Kaaks,1 Renée Turzanski Fortner1. 1 _German Cancer Research Ctr.(DKFZ), Heidelberg, Germany;_ 2 _University of Oulu and Biobank Borealis, Oulu, Finland_.

The Receptor Activator of Nuclear Factor kappa-B (RANK)-axis mediates structural changes in the breast during pregnancy in preparation for lactation, but is also implicated in breast tumor development. The RANK-axis includes RANK, its ligand (RANKL) and osteoprotegerin (OPG). OPG is the decoy receptor for RANKL and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL). Based results from experimental and epidemiologic studies, we hypothesized that relatively high serum concentrations of RANK-axis members in early pregnancy would be associated with higher risk of breast cancer in the mother, with differential effects in estrogen (ER)+ and progesterone receptor (PR)+ and ER-/PR- disease. A case-control study including 288 case-control sets (165 ER+/PR+, 79 ER-/PR-) was nested in the Finnish Maternity Cohort. Women eligible for this study donated a blood sample at age ≤40 years and ≤140 days gestation age (GA) in a primiparous term pregnancy; cases were diagnosed within 10 years of this pregnancy. Serum OPG and TRAIL were measured using an electrochemiluminescence assay, and sRANKL (soluble RANKL) using an enzyme-linked immunosorbent assay. Odds ratios (ORs) and 95% confidence intervals [CI] were calculated using conditional logistic regression adjusted for GA at blood collection. Relatively high sRANKL concentrations were associated with higher risk of ER+/PR+ breast cancer (Tertile3vs.1, OR=2.08 [1.25-3.44]) (Table); this association was similar after adjustment for OPG levels. sRANKL concentrations were not associated with ER-/PR- disease, and we observed no associations for OPG and TRAIL. In line with previous studies in non-pregnant women, we provide the first evidence for an association between early pregnancy sRANKL and risk of breast cancer in the decade following pregnancy. Relatively high circulating sRANKL concentrations in early pregnancy may identify women at increased risk of breast cancer subsequent to pregnancy. | |  | |  | |

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

Table. Serum concentrations of sRANKL, OPG and TRAIL in early pregnancy and breast cancer risk by ER/PR subtype: results from the Finnish Maternity Cohort.

|

Tertiles | |

|

|

1 | 2 | 3 | ptrenda | ORlog2 | phetb

sRANKL, cut points (pmol/L) | ≤ .0005 | 0.005 - 0.09 | > 0.09 | |

|

Full population | |  | |  | |

Cases/Controls | 86/181 | 99/181 | 103/180 | |

288/542

|

OR (95% CI) | Ref. | 1.27 (0.88-1.83) | 1.42 (0.99-2.06) | 0.05 | 1.14 (1.00-1.29) | 0.36

ER+/PR+ | |  | |  | |

Cases/Controls | 43/114 | 61/103 | 61/93 | |

165/310

|

OR (95% CI) | Ref. | 1.76 (1.08-2.88) | 2.08 (1.25-3.44) | 0.03 | 1.21 (1.02-1.44)

|

ER-/PR- | |  | |  | |

Cases/Controls | 27/46 | 24/52 | 28/53 | |

79/151

|

OR (95% CI) | Ref. | 0.89 (0.45-1.76) | 1.03 (0.52-2.06) | 0.45 | 1.09 (0.87-1.36)

|

|  | |  | |

|

OPG, cut points (pmol/L) | ≤ 6.94 | 6.94 - 8.62 | > 8.62 | |

|

Full population | |  | |  | |

Cases/Controls c | 83/167 | 73/166 | 104/166 | |

260/499

|

OR (95% CI) | Ref. | 0.85 (0.57-1.26) | 1.21 (0.82-1.79) | 0.64 | 1.10 (0.73-1.65) | 0.99

ER+/PR+ | |  | |  | |

Cases/Controls | 53/98 | 42/97 | 56/94 | |

151/289

|

OR (95% CI) | Ref. | 0.77 (0.46-1.28) | 1.08 (0.65-1.82) | 0.99 | 1.00 (0.58-1.70)

|

ER-/PR- | |  | |  | |

Cases/Controls | 19/45 | 23/41 | 29/51 | |

71/137

|

OR (95% CI) | Ref. | 1.36 (0.61-3.03) | 1.29 (0.60-2.77) | 0.95 | 0.98 (0.45-2.12)

|

|  | |  | |

|

TRAIL, cut points (pmol/L) | ≤ 0.40 | 0.40 - 1.07 | > 1.07 | |

|

Full population | |  | |  | |

Cases/Controls c | 93/166 | 79/166 | 88/166 | |

258/492

|

OR (95% CI) | Ref. | 0.86 (0.59-1.26) | 0.93 (0.65-1.34) | 0.62 | 0.93 (0.71-1.22) | 0.74

ER+/PR+ | |  | |  | |

Cases/Controls | 54/95 | 42/96 | 55/98 | |

150/285

|

OR (95% CI) | Ref. | 0.73 (0.44-1.21) | 0.94 (0.59-1.51) | 0.93 | 0.99 (0.70-1.40)

|

ER-/PR- | |  | |  | |

Cases/Controls | 24/47 | 27/47 | 20/42 | |

70/134

|

OR (95% CI) | Ref. | 1.09 (0.55-2.16) | 0.91 (0.45-1.84) | 0.69 | 0.90 (0.55-1.53)

|

a ptrend based on log2-transformed sRANKL and OPG concentrations; b pheterogeneity comparing ER+/PR+ to ER-/PR- subtypes, based on log2-transformed sRANKL concentrations (244 case-sets); c 28 case sets (OPG) and 30 case sets (TRAIL) missing concentrations due to equipment failure.

#602

Biological monitoring to clarify potential causes of colorectal cancer in Korean diet.

Mihi Yang,1 Myungah Lee,2 Jeongseon Kim Kim,3 Jong Park,4 Aeson Om,5 Jennifer Damonte4. 1 _Sookmyung Women's University College of Pharmacy, Seoul, Republic of Korea;_ 2 _The Catholic University of Korea, Seoul, Republic of Korea;_ 3 _National Cancer Center, Ilsan, Republic of Korea;_ 4 _H. Lee Moffitt Cancer Center, Tampa, FL;_ 5 _Hanyang Univ., Seoul, Republic of Korea_.

We performed a pilot study (N=30) to look for the Korean specific risk factors, which can explain an increased incidence rate of colorectal cancer (CRC) among Korean population. Based on the promising data from the pilot study, we performed a case-control study (N=218, age: 64.4 ±13.4 years; N of men=116, 53.2%) with biological monitoring to analyze various biomarkers including clinical/hematological indices, oxidative or inflammatory biomarkers, e.g., c-reactive protein (CRP), malondialdehyde (MDA) or homocysteine, the metabolites of polycyclic aromatic hydrocarbons (PAHs) and heterocyclic amines (HCAs), e.g., urinary 1-OHP, MeIQx and PhIP, and their DNA adducts, e.g., dG-C8-MeIQx and -PhIP, with UPLC/MS/MS. We also obtained participants' epidemiological, clinical, and behavioral information and developed food frequency questionnaires (FFQ) to collect their food information for one year before cancer diagnoses. Total subjects consumed 53.4±74.0g/day of red meat, and 1.1±3.7g/day of processed meat. After adjustment for sex and age, there were no associations between CRC risk and red or processed meat intake, or urinary MeIQx or PhIP levels. However, there were inverse associations between CRC and milk or calcium intake (ps<0.05). In addition, urinary MeIQx levels were positively associated with intake of lipid or coffee (p=0.06, p<0.01, respectively). LDL-C and CRP levels in the cases were higher than ones from controls (ps<0.05). Among biomarkers, MDA levels were positively correlated to urinary levels of 1-OHP, PhIP and MeIQx (ps<0.05). These relations support the fact that PAHs or HCAs indirectly induce oxidative stress. Finally, fruits and milk intake showed protective effects, however, gender (man), CRP, and lipid intake were risk factors to CRC, based on multivariable regression analyses. Therefore, total lipid intake, oxidative stress, or inflammation may increase risks of CRC in Korean population rather than red or processed meat intake.

#603

**Signatures of chemoprotection from ambient exposure to outdoor air pollution in the human serum albumin Cys** 34 **adductome.**

Joshua W. Smith,1 Robert N. O'Meally,1 Derek Ng,1 Thomas W. Kensler,2 Robert N. Cole,1 John D. Groopman1. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _Fred Hutchinson Cancer Research Center, Seattle, WA_.

Many components of outdoor air pollution, such as benzene, are known to be carcinogenic to humans. However, due to spatial and temporal variation in air pollution, ambient measures of single pollutant exposures may be limited for generalizing across diverse settings. Covalent adducts of air pollutants with the free thiol at the Cys34 residue of human serum albumin represent biomarkers of recent air pollution exposure (<3 months), while adductomic interrogation enables characterization of exposures to complex air pollution mixtures. We have used Cys34 adductomics to further characterize the protective effects of a successful intervention trial in the Yangtze River delta - a region characterized by consistent and substantial levels of airborne pollution, as well as a rising incidence of lung cancer. Residents of Qidong, China were recruited to participate in a 12-week, randomized, placebo-controlled clinical trial. Participants consumed either a placebo (P) or broccoli sprout beverage (B) daily, and urine and serum samples were collected before and throughout the intervention. Previously published results revealed that, relative to P treatment, B significantly increased detoxication and urinary excretion of benzene (+61%, as S-phenylmercapturic acid [SPMA]). For this pilot study, the serum albumin Cys34 adductome was interrogated in n=8 non-smoking women at study day 56 from each of the P and B treatment groups, selecting subjects whose day 56 SPMA excretion was closest to their respective group's geometric mean. Albumin was precipitated from serum, digested with lysyl endopeptidase, separated by nanoflow LC, and Cys34 adducts were identified and quantified on a Thermo Scientific Orbitrap Fusion Tribrid mass spectrometer. We detected four serum albumin Cys34 adducts arising from three metabolites of benzene: benzoquinone (BQ; BQ1 and BQ2), benzene oxide (BO), and benzene diolepoxide (BDE). In accordance with elevated urinary SPMA excretion, BO levels trended lower with B treatment (-39%, p=0.06), while BQ2 was strongly and significantly diminished (-51%, p=0.005); no effect was observed in BQ1 or BDE adduct levels. Incubation of 1,4-BQ with human serum in vitro suggested that the BQ adducts observed in vivo reflect keto-enol tautomerism of a 1,4-BQ-derived adduct, rather than 1,4-BQ and its structural isomer, 1,2-BQ. Along with benzene-derived adducts, we identified several other putative adducts (cyano, ethylene oxide, aldehydes, oxidation, mixed disulfides), revealing a multidimensional signature. To our knowledge, this is the first report to detect benzene-derived adducts, using an adductomics approach, in humans with only ambient exposures. Work is underway to expand upon these results. Cys34 adductomics represents a promising approach for the discovery and quantification of air pollution exposure biomarkers.

#604

Biomarkers of amino acid metabolism and endometrial cancer risk in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO).

Kara A. Michels, Stephanie J. Weinstein, Nicolas Wentzensen, Ruth M. Pfeiffer, Britton Trabert. _National Cancer Institute, NIH, Bethesda, MD_.

Background: Incidence and mortality rates for endometrial cancer have steadily increased during the past decade. Beyond the well-known risk factors of obesity and unopposed estrogen menopausal hormone therapy (MHT), our understanding of the molecular etiology of this disease is limited and few biomarkers for risk are identified. To address this data gap, we explored circulating markers of amino acid metabolism and endometrial cancer risk—with specific interest in assessing whether the associations with branch chain amino acids (BCAAs) differed by known endometrial cancer risk factors.

Methods: We used data from a nested case-control study within the screening arm of the PLCO trial (353 endometrial cancer cases, 353 controls, age 55-74 years at enrollment). We measured absolute concentrations of amino acid metabolism markers in pre-diagnostic serum using targeted mass spectrometry assays. Conditional and unconditional logistic regression adjusted for potential confounders (and matching factors in the latter) was used to estimate associations between these circulating markers and endometrial cancer overall, by body mass index (BMI), and by MHT use at baseline. We evaluated heterogeneity across these groups via likelihood ratio tests (p for interaction [p-int]).

Results: In the full population, associations with endometrial cancer were generally masked/not identified, but we noted substantial effect modification by MHT use and BMI. A doubling in endometrial cancer risk was suggested with the highest concentrations of BCAAs, but only among women not using MHT (odds ratio and 95% confidence interval for the highest quintile [Q5] versus lowest [Q1]: leucine 1.87, 0.91–3.83; isoleucine 2.44, 1.17–5.09; and valine 1.86, 0.89–3.87; p-int <0.15 for all). We similarly noted that high levels of leucine (Q5 v. Q1: 3.26, 0.96–11.08, p-int 0.39) and valine (Q5 v. Q1: 6.40, 1.49–27.47, p-int 0.08) were associated with risk among obese women, but not those with normal BMIs. The highest levels of 3-hydroxyisobutyrate, a bioactive valine precursor, doubled risk among non-MHT users (CI 1.09–4.51, p-int <0.05) and quadrupled it among obese women (CI 1.45–14.86, p-int 0.33). High proline levels were associated with 4- to 7-fold increases in risk among non-MHT users (CI 1.68–10.01) and obese women (CI 2.11–26.81), respectively (p-int<0.05 for both).

Conclusions: Our findings suggest that the regulation of BCAA metabolism may be involved in endometrial carcinogenesis, but this influence depends on other risk factors for the disease. Use of MHT overshadowed potential risks with most markers evaluated, and we identified increased risks associated with a variety of markers only among obese women. Amino acids play crucial roles in cellular energy metabolism and our results highlight the importance of finding ways to maintain and improve metabolic health in postmenopausal women.

#605

Association of blood lipid profile and lung cancer risk: Results from the Southern Community Cohort Study.

Yan Sun, Jie Wu, Hyung-Suk Yoon, Wei Zheng, William J. Blot, Qiuyin Cai. _Vanderbilt University School of Medicine, Nashville, TN_.

Lung cancer is the second most common cancer and the leading cause of cancer death in the U.S. African Americans (AAs) have a higher incidence rate than any other racial group. Lipid metabolism and homeostasis play important roles in the growth of cancer cells and the regulation of membrane proteins. Some previous epidemiological studies have evaluated the association between blood lipid levels and lung cancer risk; however, the results were inconsistent, and few explored patterns among AAs. To investigate the association of blood lipid levels and lung cancer risk, particularly in a low socioeconomic population and AAs, we conducted a nested case-control study using resources from the Southern Community Cohort Study, a well-characterized prospective cohort study of approximately 86,000 adult men and women, two-thirds of whom are AAs. Blood lipid profiles, including total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides (TRIG), and lipoprotein-a (Lp-a), were measured in 372 lung cancer cases and 721 matched controls. AAs had lower median levels of TRIG, and higher levels of HDL and Lp-a than European Americans (EAs). Conditional logistic regression analysis revealed an inverse association between TC and lung cancer risk, with an adjusted OR for the highest vs. lowest tertiles of 0.67 (95% CI: 0.45-0.99). This association was observed in both AAs and EAs, but was only statistically significant in EAs. Further stratified analysis found that this association was only significant in cases that were diagnosed within 2 years of blood collection. No significant associations of other lipid markers with lung cancer risk were observed. The findings from our study suggest that the inverse associations between TC and lung cancer risk more likely resulted from cancerous occurrence and progression and associated lifestyle changes rather than being indicative of causal markers.

#606

Circulating proteins associated with ovarian cancer risk and survival are enriched for immune-related pathways.

Daniel P. Considine. _University of Cambridge, Cambridge, United Kingdom_.

Most women with epithelial ovarian cancer (EOC) are diagnosed after the disease has metastasized and five-year survival rates in this group continue to remain very poor. Circulating proteins may illuminate EOC etiology and have the potential to serve as biomarkers for early detection and diagnosis. We performed a two-sample Mendelian randomization (MR) study with circulating protein levels as exposures and susceptibility to EOC and its histological subtypes as outcomes. We used previously identified quantitative trait loci (QTLs) robustly associated with plasma levels of 1,478 proteins as genetic instruments and corresponding summary genetic association statistics for these instruments derived from 22,406 EOC cases and 40,941 controls. Among proteins that had only a single QTL variant available as a genetic instrument, we identified 12 proteins whose circulating levels were associated with overall EOC risk at a False Discovery Rate (FDR) of < 0.1. For nine of the 12 proteins, the association was driven by variants near ABO, which is both a genome-wide significant high grade serous histotype risk locus and a hub QTL for several proteins. Three of these 12 proteins and their QTLs were, however, not linked to the ABO locus and included the products of the NOTCH signaling genes MFAP2 and DLK1. Among proteins that had multiple QTL variants available for the creation of more robust multi-variant genetic instruments, we identified 33 protein level-EOC risk associations at FDR < 0.1. Once again, we observed that the majority of associations (19/33) were driven by variants at the ABO locus. Nine of the 14 remaining associations were for overall and high grade serous EOC risk and included predominantly immune system proteins: ARTS1 (HLA class I), CPNE1 (involved in innate immunity), Macrophage mannose receptor (HLA class I), DQA2 (HLA class II), RGAP1 (innate immunity), Cathepsin H (innate immunity), and FCG2B (the low affinity receptor for the Fc region of immunoglobulin gamma). We observed one mucinous histotype-specific association (complement C4) and two clear cell histotype-specific associations (KI2S2 and PSG4). KI2S2 is a Killer cell immunoglobulin-like receptor expressed by natural killer cells and PSG4 is a member of the carcinoembryonic antigen gene family that also includes the gene encoding the tumor marker for colorectal cancer. Finally, we also performed two-sample MR analyses using survival as the outcome and identified two proteins whose plasma levels were associated with overall EOC survival at FDR < 0.1: SULT1E, a phenol sulfotransferase that conjugates sulfates to estrogens, and MA2B2. Thus, we identified circulating proteins that are candidate biomarkers for EOC risk and survival. They represent promising starting points for follow-up studies and the measurement of plasma levels of these proteins in EOC cohorts is required to validate these associations.

#607

Clonal hematopoiesis alters blood cell counts in the UK Biobank.

Mitchell J. Machiela, Weiyin Zhou, Erikka Loftfield, Meredith Yeager, Neal D. Freedman, Stephen J. Chanock. _National Cancer Institute, Rockville, MD_.

Clonal hematopoiesis (CH) is an age-related accumulation of detectable somatic mutations in circulating leukocytes that is associated with diverse outcomes including hematologic cancer, solid tumors, cardiovascular disease, neurodegenerative disorders and overall mortality. The biological mechanisms by which CH is associated with disease is poorly understood, with hypotheses suggesting CH is a potential marker of genomic maintenance capacity or a potential marker of immune system imbalance. The most common age-related chromosomal alteration observed in men with CH is mosaic loss of the Y chromosome (mLOY). The relative high frequency of mLOY makes mLOY an attractive marker for investigating disease-related mechanisms associated with CH. In particular, leukocyte counts are associated with hematologic cancer, cardiovascular disease and mortality in men and may, in part, mediate some of the observed associations between CH and disease. We investigated mLOY in a large collection of 223,182 men from the UK Biobank to study potential impacts of mLOY on blood cell count indices. We used existing UK Biobank array intensity data from the male-specific region of the Y chromosome to detect deviations in the median log2 R ratio (mLRR) that signifies the presence of mLOY. In total, 3,789 men had evidence for mLOY, and of these 596 men had evidence for mLOY affecting high cellular proportions (approximately 24% of cells). Age and smoking adjusted models indicated leukocyte count is, on average, elevated by 5.82% (95% CI=4.82-6.83, P-value=5.38×10−30) in men with mLOY and by 11.95% (95% CI=9.47-14.44, P-value=1.99×10−21) in men with high levels of mLOY. Analysis of leukocyte proportions indicated the association between mLOY and leukocyte count was restricted to myeloid progenitor cells, with significant elevations observed in neutrophil and monocyte counts (P-values=1.06×10−25 and 4.89×10−56, respectively). Neutrophil count was most highly altered with average changes in cell count of 8.69% (95% CI=7.60-9.77) in men with mLOY and 14.70% (95% CI=12.04-17.37, P-value=2.32×10−156) in men with high levels of mLOY. Sensitivity analyses in never smokers observed similar elevations in leukocyte, neutrophil and monocyte count in men with mLOY, suggesting our findings were not a result of residual confounding by smoking. Furthermore, elevations in platelet count and reductions in erythrocyte count in men with mLOY provide additional evidence myeloid progenitor cells are impacted by mLOY. We found no evidence suggesting lymphocytes are impacted by mLOY. Together, these observations indicate that men with mLOY have substantial deviations in blood cell counts, particularly in neutrophil count. In this way, alterations in leukocyte counts may be one mechanism by which CH contributes to disease.

#608

Examining H. pylori core genes for epidemiological typing.

Guoqin Yu. _University of Kansas Medical Center, Kansas City, KS_.

H. pylori is the major causative risk factor of a serious of diseases including gastric cancer. It is highly diverse. Conventional method of typing H. pylori has been based on seven MLST loci, which however are not sufficient to distinguish closely-related strains. High diverse core genes that are present in all H. pylori strains are potential good candidates for investigating genomic variation within closely-related strains, therefore improving our understanding of H. pylori -related disease epidemiology. In this study, we investigated the variation of the H. pylori genome, especially its core genes. We downloaded 80 H. pylori whole genome from National Center for Biotechnology Information (NCBI), and identified core genes (99% <= strains <= 100%: 840 genes), Soft core genes (95% <= strains < 99%: 185), Shell genes (15% <= strains < 95%: 933), Cloud genes (0% <= strains < 15%: 2313). Compared with other gene groups, we found that core genes have higher proportion of polymorphic sites, similar level of nucleotide diversity per site, higher recombination events per site, and lower number of gaps per site. In addition, core genes evolve slower than other gene groups with lower Tajima's D value and nonsynonymous to synonymous ratio. Among the core and soft-core genes, we found that majority have low genetic diversity while a few showed higher diversity. We evaluated these high diverse core genes further in terms of their evolution, function and potential to serve as candidates to distinguish closely related H. pylori strains.

#609

Vitamin D binding protein and risk of renal cell carcinoma in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial.

Tyler B. Kratzer,1 Alison M. Mondul,1 Stephanie J. Weinstein,2 Demetrius Albanes2. 1 _University of Michigan, Ann Arbor, MI;_ 2 _National Cancer Institute, Rockville, MD_.

Introduction/Study purpose: Vitamin D binding protein (DBP) is the major carrier of vitamin D in circulation. In addition to its role in vitamin D transport, DBP has several other biologic mechanisms that may be relevant to cancer risk including macrophage activation, apoptosis, and angiogenesis. Although previous studies have demonstrated no association between vitamin D and RCC, our group has conducted two previous studies which found strong inverse associations between DBP and RCC. We undertook the current analysis to replicate our findings in a different study population.

Methods: We conducted a nested case-control study in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO). Cases (n=323) were matched 1:1 to controls on age (+/- 1 year), race, date of blood collection (+/- 30 days) and sex. We performed conditional logistic regression to estimate the odds ratios and 95% confidence intervals for the association between quartiles of circulating DBP and risk of RCC. In addition to conditioning on the matching factors, multivariable models were further adjusted for history of diabetes or hypertension, family history of renal cancer, body mass index (BMI), and smoking status. Additionally, we performed stratified analyses by sex, family history of renal cancer, BMI, smoking status, and tumor stage at diagnosis.

Results: We observed a statistically significant positive association between DBP and RCC that persisted after multivariable adjustment (mv-adj Q4 vs. Q1 OR=3.1, 95% CI=1.8-5.6; p-trend <0.0001). Sensitivity analyses were performed removing matched pairs where the case was diagnosed within 2 years of blood collection or experienced a large weight loss (>40 lbs), with little effect on the overall trend (Q4 vs. Q1 OR=2.5 CI=1.4-4.6 p-trend=<0.0001).

Conclusions: In the present study, those with higher serum concentrations of DBP were at increased risk of RCC, which differs from previously-published findings. Further research is necessary to determine the true

association between DBP and risk of RCC, and whether different DBP phenotypes via GC variation may have different associations with risk of RCC.

#610

A longitudinal investigation of hematologic perturbations among permethrin-exposed pesticide applicators in the Biomarkers of Exposure and Effect in Agriculture Study.

Joe Shearer,1 Laura Beane Freeman,1 Danping Liu,1 Gabriella Andreotti,1 Jennifer Hamilton,2 Julie Happel,3 Charles Lynch,2 Michael Alavanja,1 Jonathan Hofmann1. 1 _National Cancer Institute, Rockville, MD;_ 2 _University of Iowa, Iowa City, IA;_ 3 _University of Iowa Hospital and Clinics, Iowa City, IA_.

Background: In the Agricultural Health Study, a large prospective cohort of pesticide applicators, high lifetime use of the insecticide permethrin was associated with an increased risk of multiple myeloma. However, the biological mechanisms through which this association occurs remain to be fully understood.

Methods: Hematologic perturbations were assessed among 33 permethrin-exposed male pesticide applicators enrolled in the Biomarkers of Exposure and Effect in Agriculture study, a molecular substudy within the Agricultural Health Study. Serial blood samples were collected from each participant coinciding with permethrin use including visits during the offseason, an exposure visit the day after use, and for 27 participants a follow-up visit approximately three weeks after use. Complete blood count with white blood cell differential and lymphocyte subsets were collected at each visit. Linear mixed models were used to assess the relationship between permethrin exposure and hematologic parameters, controlling for potential confounders including season of blood draw, age, body mass index, recent cold/infection, and use of other pesticides.

Results: A statistically significant increase in the adjusted mean immature granulocyte count (37%, 95% CI=6%, 76%) was observed the day after permethrin use compared to an offseason visit. Statistically significant (P<0.05) alterations in several red blood cell (RBC) parameters were also observed on the day after permethrin use, including decreased RBC count and hemoglobin and increased mean corpuscular volume and RBC distribution width-SD. Decreases in RBC count and hemoglobin, and increased levels of RBC distribution width-SD persisted approximately three weeks after permethrin use.

Conclusions: Altered hematologic parameters could be indicative of disrupted hematopoiesis, providing insights into the potential biologic plausibility of the observed association between permethrin use and multiple myeloma risk among pesticide applicators.

Impact/Significance: Given the widespread use of permethrin in both residential and commercial settings, our findings may have public health implications beyond occupationally exposed pesticide applicators.

#611

MYC DNA methylation in prostate tumor tissue is associated with tumor aggressiveness.

Kathryn Hughes Barry,1 Kareshma Mohanty,1 Gary Rose,1 Ashley Cellini,1 Nicholas Ambulos,1 Jing Yin,1 Liying Yan,2 Matthew Poulin,2 Ann Meyer,2 Yuji Zhang,1 Søren Bentzen,1 Allen Burke,1 Arif Hussain,1 Sonja I. Berndt3. 1 _University of Maryland School of Medicine, Baltimore, MD;_ 2 _EpigenDx, Inc., Hopkinton, MA;_ 3 _National Cancer Institute, Rockville, MD_.

We previously reported an increased risk of aggressive, but not non-aggressive, prostate cancer associated with pre-diagnostic blood DNA methylation levels in exon 3 of the MYC oncogene among Caucasian men in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial. In the present study, we aimed to investigate MYC DNA methylation in prostate tissue in relation to tumor aggressiveness (based on Gleason score). We also aimed to investigate whether MYC methylation in prostate tissue is associated with race or tumor/normal status. We accessed formalin-fixed, paraffin-embedded prostate tumor and normal tissue from 50 Caucasian and 50 African American prostate cancer patients that underwent radical prostatectomy at the University of Maryland; for each race group, we selected 25 patients with a Gleason score of 7 and 25 with a Gleason score of 6. We systematically selected the most recent samples, and year of surgery for our patients ranged from 2001-2017. Age at surgery ranged from 42-75 years. We used pyrosequencing to measure DNA methylation at six CpG sites in MYC exon 3 (Chr8:128753145 - Chr8:128753221). We excluded 11 patients for whom the assays failed, resulting in a final sample size of 43 African American and 46 Caucasian men. The six CpG sites demonstrated moderate correlations with one another in tumor tissue (Spearman rho: 0.30-0.60, p-value<0.01 for all pairings). Correlations between the CpG sites were less pronounced in the normal tissue samples (Spearman rho: -0.08-0.57) and five pairs were no longer significantly correlated. We used paired t-tests to evaluate differences in MYC DNA methylation between tumor and normal samples from the same patients, and unpaired t-tests to evaluate differences by Gleason score and race. We observed significantly higher MYC DNA methylation for all six CpG sites in the normal compared to the tumor samples (p<0.05). The difference in mean percent methylation between normal and tumor for the six sites ranged from 2.3 to 16.3 percentage points. In the tumor samples, we also observed significantly higher MYC DNA methylation for patients with a Gleason score of 6 compared to those with a Gleason score of 7 for three of the CpG sites. These included Chr8:128753145 (mean percent methylation for Gleason 6 group=89.0%; mean for Gleason 7=83.6%; t-test p-value=0.002), Chr8:128753154 (mean for Gleason 6=87.7%; mean for Gleason 7=81.7%; t-test p-value=0.004) and Chr8:128753200 (mean for Gleason 6=82.6%; mean for Gleason 7=78.8%; t-test p-value=0.02). Also in tumor tissue, we observed borderline significantly higher DNAm at Chr8:128753151 for African American than Caucasian men (mean for African American men=79.1%; mean for Caucasian men=73.7%; t-test p-value=0.06); no other CpG sites displayed differences by race. Our results provide further evidence for a role of altered MYC DNA methylation in aggressive prostate cancer.

#612

Overexpression of PCDHB9 is correlated with tumor progression in esophageal cancer.

Naohide Oue, Yuji Yamamoto, Ryuichi Asai, Naoya Sakamoto, Kazuhiro Sentani, Wataru Yasui. _Hiroshima University, Hiroshima, Japan_.

Genes encoding transmembrane proteins expressed specifically in cancer cell may be ideal biomarkers for diagnosis. Previously, we showed that overexpression of PCDHB9, which was identified as a transmembrane protein, promotes peritoneal metastasis and correlates with poor prognosis in patients with gastric cancer. However, expression of PCDHB9 in esophageal cancer remains unclear. In the present study, we investigated the expression and function of PCDHB9 in esophageal squamous cell carcinoma (ESCC). Immunohistochemical analysis demonstrated that 51 (40%) of 128 GC cases were positive for PCDHB9. PCDHB9 expression was correlated with T classification, N classification and tumor stage. There was no significant correlation between PCDHB9 expression and patient survival. Cell adhesion to fibronectin and invasion activity were significantly reduced by PCDHB9 knockdown. These results suggest that PCDHB9 may be a novel biomarker and therapeutic target for ESCC.

#613

Demographic and social patterns of average values of inflammatory markers in U.S. adults: 2010-2017 NHANES analysis.

Raisa Haq, Rose Calixte, Marlene Camacho-Rivera. _CUNY School of Medicine, NY, NY_.

Introduction

Inflammation has been found to play a role in disease prognosis and progression of many conditions, such as cancers, thrombolytic, and cardiovascular diseases. However, reference values for inflammatory markers within the U.S. adult population remains an understudied area. As such, this study aims to (1) establish average values of various inflammatory markers within U.S. adults and (2) explore heterogeneity in average values by socio-demographic and behavioral risk factors.

Methods

Aggregated cross-sectional data from participants ages 20 and older within the 2010-2017 National Health and Nutrition Examination Survey (NHANES) were used. Outcomes included validated measures of general inflammation (lymphocyte count, monocyte count, segmented neutrophil count, eosinophil count, basophil count, platelet count, neutrophil/lymphocyte ratio); risk factors included socio-demographic and behavioral variables such as race, age, sex, nativity, BMI, smoking history and alcohol use. The final sample consisted of 16,849 participants across all of the survey waves. Adjusted linear regression models were used to explore the association between various demographic and behavioral characteristics and inflammatory markers.

Results

Average lymphocyte count across all participants was 2.17k cells/microliter, neutrophil was 4.23k cells/microliter, and NLR was 2.12. In the fully adjusted linear regression model, average NLR for non-Hispanic Blacks was 0.44 lower than the average NLR for non-Hispanic Whites (95% CI -0.52 to- 0.37). In the fully adjusted model it was found that age, nativity, and smoking status were also statistically significantly associated with NLR. Participants in the 30-59, 60-79, 80+ age categories had 0.09, 0.28. 0.71 higher average NLR values compared to ages 20-29, respectively. Current smokers had a 0.11 average higher than NLR value compared to non-smokers. Former smokers also have an average NLR that is 0.08 higher than non-smokers.

Conclusions

This study provides population-based average values of inflammatory markers in healthy U.S adults. These values significantly varied by race, age, nativity, and smoking status. Clinical implications indicate the need to consider different cut-off points by race and age for risk assessment as a predictive and prognostic marker for various illnesses. These inflammatory markers can be easily measured in cost-effective simple blood tests, are routinely available in other population-based national surveys, and have the potential to predict the prognosis of cancer treatments, thrombolytic diseases and coronary artery disease.

#614

Cytomegalovirus (CMV) reactivation and patient survival in head and neck cancer.

Heather H. Nelson,1 Jeffrey S. Miller,1 DeVon Hunter-Schlichting,1 Rondi A. Butler,2 Karl T. Kelsey2. 1 _University of Minnesota Masonic Cancer Center, Minneapolis, MN;_ 2 _Brown University, Providence, RI_.

There is building evidence that cytomegalovirus (CMV) may contribute to the development of solid tumors, however to date this has not been investigated in head and neck cancer (HNC). CMV is a highly prevalent, latent herpesvirus infecting over 50% of the population. CMV maintains latency in CD34+ cells, and recent work has demonstrated these latently infected cells preferentially differentiate into immunosuppressive monocytes. However, CMV is not solely immune suppressive as highly cytotoxic adaptive natural killer (NK) cells arise specifically in response to CMV reactivation. Here, we have investigated whether biomarkers of CMV reactivation are associated with patient survival in non-smoking HNC (n=184). CMV DNAemia was assessed using digital PCR, and a threshold of 1000 copies/mL serum was defined as reactivation; 22% of patients met this definition of CMV reactivation, however they did not have significantly worse survival than those without CMV reactivation (HR 1.5 (95% CI 0.7 - 3.0)). Monocytes that mobilize to the cancer site contribute to tumor-associated inflammation. Therefore, we explored whether a biomarker of inflammation, CRP, modified the association of CMV reactivation and HNC patient survival. In the absence of inflammation, there was no association between CMV reactivation and patient survival (HR 0.90 (95% CI 0.30 - 2.71)). However, those with both CMV reactivation and high inflammation had dramatically worse survival (HR 8.86 (95% CI 3.63 - 21.64)). These data are consistent with the recent report that CMV infected monocytes are immunosuppressive and suggest that stimulation of these monocytes to the tumor site may negatively impact patient survival. Future work should address the generalizability of this finding to other cancer types and determine whether CMV infected myeloid cells are present in the tumor microenvironment.

#615

**Roles of** XRCC1 **genetic polymorphism in head and neck cancer patients receiving radiation therapy in Taiwan.**

Shiang-Fu Huang,1 Huei-Tzu Chien,2 Chun-Ta Liao,1 Hung-Ming Wang,1 Yuan-Hung Wang3. 1 _Chang Gung Memorial Hospital, Taoyuan, Taiwan;_ 2 _Chang Gung University, Taoyuan, Taiwan;_ 3 _Taipei Medical University, Taipei, Taiwan_.

In Taiwan, oral cancer (including sub-sites in the oral cavity, oropharynx and hypopharynx) is the fourth most common cancer in men. The incidence of oral cancer still increased in the past decade. Oxidative stress generated from cigarette smoking, AQ chewing and alcohol drinking was shown to contribute to oral cancer development. The oxidative stress associated DNA lesions are repaired via the DNA base excision repair (BER) pathway. In constrast, radiation therapy was an important treatment modality either as primary or adjuvant in these patients. Ionizing radiation induced DNA damage was also repaired by BER related mechanisms. To investigate the role of BER genes XRCC1 in patients receiving radiation therapy in Taiwan, a comprehensive study on evaluation of polymorphisms of XRCC1 genes was carried out in 753 oral cancer male cases received radiation therapy as primary or adjuvant treatment. Patients with XRCC1 194Arg, XRCC1 280Arg, and XRCC1 399Gln homozygous genotypes had worse disease-free survival (p = 0.015, p = 0.587 and p = 0.013, respectively). A linkage disequilibrium exists between XRCC1 194Arg and XRCC1 399Gln genotyping. We further investigated the influence of XRCC1 194Arg and XRCC1 399Gln haplotype in the radiation effects in oral cancer patients. XRCC1 194 Arg/Arg homozygous and its combination with XRCC1 399 Arg/Gln genotyping were selected. We found that the XRCC1 194Arg/Arg and XRCC1 399Gln/Gln haplotype had the worse survival (p = 0.022, ). Multivariate Cox regression adjusting tumor stage, lymph node metastasis and tumor differentiation confirmed that XRCC1 194Arg and XRCC1 399Gln haplotype was an independent prognostic factor for oral cancer receiving radiation therapy. These indicated that XRCC1 194Arg and XRCC1 399Gln genotypes had adverse influence in oral cancer patients receiving radiation therapy. XRCC1 194Arg and XRCC1 399Gln haplotype predicts poor prognosis in these patients.

#616

Literature review and meta-analyses of the prevalence of microsatellite instability high (MSI-H) and deficient mismatch repair (dMMR) for colorectal (CRC), gastric (GC), endometrial (EC) and ovarian cancers (OC) in Chinese population.

Xiaoqiao Liu, Jiaqi Fan, Kai-Li Liaw, Mo Xu, Yu Zhou, Mayur Amonkar, Gefei Zeng. _Merck & Co., Inc., Kenilworth, NJ_.

Purpose Given the high prevalence of MSI-H/dMMR in gastrointestinal and gynecologic cancers we systematically collected published studies and investigated its' prevalence for Chinese CRC, GC, EC, OC subjects to better understand the size of potential target population available for pembrolizumab treatment in China.

Experimental Design Both Chinese and English literature were searched in WanFang and PubMed database respectively. Studies were included if they assessed prevalence of MSI-H by PCR based method investigating 5 markers from NCI or Promega panel, and/or dMMR by immunohistochemistry (IHC) for all 4 MMR proteins. Studies focusing only on hereditary or sporadic cancer were filtered out. Among eligible studies, meta-analyses (DerSimonian Laird random effect model) were performed on the proportion of MSI-H/dMMR for each tumor type by R metafor package. Prevalence for advanced stage (III/IV) were also extracted if the data were available.

Results For CRC, the prevalence of MSI-H estimated from 11 studies (13.7% [10.2-17.6%]) was similar as that of dMMR from 22 studies (14.6% [12.3-17.1%]). Pooled prevalence of MSI-H/dMMR from 32 studies (12,341 subjects) was estimated at 14.1% (12.2-16.1%), and 9.7% (7.2-12.5%) from 17 studies (3,933 subjects) for advanced stage CRC. The prevalence estimation for GC, EC and OC were 18.5%, 26% and 12.9% respectively. Further, the prevalence in advanced stage cancer, regardless of the cancer type, tended to be numerically lower than the all-stage combined prevalence.

Conclusions This was the first study comprehensively integrating published studies to estimate prevalence of MSI-H/dMMR across 4 main tumor types for Chinese subjects. The meta-analysis indicated the prevalence of MSI-H/dMMR for all or advanced stage Chinese cancer patients might be similar as that for western population. 

### Lifestyle Factors and Cancer Risk

#617

Aggressive phenotypes of luminal breast cancer demonstrate associations with body mass index analogous to triple-negative breast cancer.

Mustapha Abubakar,1 Changyuan Guo,2 Hela Koka,1 Bin Zhou,2 Eric Tang,1 Joseph Deng,1 Nan Hu,1 Ning Lu,2 Xiaohong R. Yang1. 1 _National Cancer Institute, Rockville, MD;_ 2 _National Cancer Center, Beijing, China_.

Background

Results from epidemiological studies are in support of heterogeneous relationships between body mass index (BMI) and breast cancer (BC) subtypes. Compared with luminal BC (LBC), pre-menopausal obesity is more frequently associated with triple-negative (TN) BC, while the converse has been reported for post-menopausal obesity. Given the dynamic range of hormone receptor expression in LBC, categorical combinations of immunohistochemical (IHC) markers for subtype definition may mask etiological heterogeneity within this subtype. Herein, we leveraged quantitative measures of tumor clinicopathological and IHC characteristics to define phenotypes of luminal BC and to assess relationships with BMI.

Methods

Participants were 6,412 women with invasive breast cancer who were diagnosed and treated in a tertiary hospital in Beijing, China between 2008-2016. We computed the Nottingham prognostic index (NPI)-score (based on tumor size, grade and lymph nodes) and IHC4-score (based on ER, PR, HER2 and KI67) and stratified LBC patients into four subgroups using quartiles of these scores: Lum-Q1 (least aggressive), Lum-Q2, Lum-Q3, and Lum-Q4 (most aggressive). Multinomial logistic regression models, adjusted for age, parity, menarcheal age, and family history, were used to test the associations between BMI [Kg/m2: underweight (<18.5); normal (18.5-25); overweight (25-30); obesity (>30)] and luminal subgroups, overall and by age categories (as proxy for menopausal status). We also included TNBC as a comparison group.

Results

The most aggressive LBC patients (Lum-Q4), defined by the NPI-score, were significantly more likely to be overweight [OR (95% CI) vs normal = 1.44 (1.12-1.86)] and obese [OR (95% CI) vs normal = 1.55 (0.98-2.46)] than the least aggressive LBC (Lum-Q1) patients, regardless of menopausal status. On the other hand, Lum-Q4 patients, defined by the IHC4-score, were significantly more likely to be underweight [OR (95% CI) vs normal = 2.21 (1.07-4.56)] in all women but less likely to be overweight [OR (95% CI) vs normal = 0.65 (0.50-0.85)] and obese [OR (95% CI) vs normal = 0.53 (0.35-0.82)] among post-menopausal women compared to Lum-Q1 patients (P-interaction (BMI & age) = 0.004). These associations were very similar to those when comparing TNBC to Lum-Q1 patients (defined by either NPI or IHC4 scores).

Conclusion

Aggressive phenotypes of LBC demonstrate associations with BMI that are analogous to TNBC. Discordant BMI associations with NPI and IHC4, and by menopausal status for the IHC4-score only, may be indicative of a positive association between excess adiposity and tumor clinicopathological parameters in the NPI-score, regardless of menopausal status. Conversely, its association with the IHC4-score may be driven by complex hormonal mechanisms that vary by menopausal status. Future analysis will focus on disentangling these relationships.

#618

A prospective evaluation of waist circumference over time and incidence of postmenopausal breast cancer in the Atherosclerosis Risk in Communities (ARIC) Study.

Maneet Kaur, Avonne E. Connor, Elizabeth A. Platz, Kala Visvanathan, Corinne E. Joshu, for ARIC Cancer Investigators. _Johns Hopkins Bloomberg School of Public Health, Baltimore, MD_.

Background: Body mass index (BMI) has been consistently associated with development of postmenopausal breast cancer (BC). There is accumulating evidence that waist circumference (WC) is a measure of visceral fat and thus may more accurately reflect the biological sequelae of adiposity on BC carcinogenesis. WC has been associated with development of BC, but its added value to BMI as a risk factor over time is less clear. This study leverages repeated in-person measurements to examine the association between WC, BMI and BC incidence in a diverse cohort of women.

Methods: We conducted a prospective study of 7626 (BC=564) postmenopausal women (29% Black, 71% white) in the ARIC Study who were 44-65 years old and cancer-free at baseline (1987-89). Women were categorized by time-varying WC (low <88cm, high ≥88cm) and BMI (normal <25, overweight 25-29.9, obese ≥30 kg/m2) measured at 4 clinical visits every 3 years, and followed until diagnosis of BC or another cancer, loss to follow up or 12/31/12, whichever occurred first. Cox proportional hazards models were used to estimate adjusted hazards ratios (HR) and 95% confidence intervals (CI) for BC using age as the time metric, overall and stratified by BMI (WC only). In a subset that had ≥3 visits and attended Visit 1 and 4 (N=4760, BC=244), we also 1) calculated the percent difference in Visit 1 and 4 measures, and 2) used linear regression to estimate the slope of change across all measures. The percent difference and slope coefficients were modeled as average change per year. Models were adjusted for age, race and study center, education, age at menarche, parity, frequency of routine exams, family history of BC, height, and hormone replacement therapy use. Change in WC/BMI analyses were also adjusted for WC/BMI at Visit 1.

Results: Compared to low WC, women with high WC had an increased risk for BC (HR 1.39, 95% CI 1.12, 1.73) that was slightly attenuated after adjustment for BMI (HR 1.27, 95% CI 0.97, 1.68). Stratification by BMI among normal and overweight women showed WC was associated with BC in normal weight (HR 1.47, 95% CI 1.06, 2.03) but not overweight women (HR 0.95, 95% CI 0.60, 1.50) (Pinteraction=0.12). In the subset that we analyzed change over time, the HRs for a 1% annual increase in WC and BMI were 1.12 (95% CI 0.99, 1.28) and 1.07 (95% CI 0.95, 1.21), respectively. For the slope change analyses, the HRs for a 1 cm annual increase in WC and 1 kg/m2 annual increase in BMI were 1.11 (95% CI 0.97, 1.27) and 1.22 (95% CI 0.81, 1.85). Results were similar among Black and white women.

Conclusion: In-person measured WC was associated with postmenopausal BC risk even after adjustment for BMI. Even in normal weight women, having a high WC increased risk of BC by 47%. Independent of baseline WC, increasing WC over time was associated with BC. This study highlights the importance of monitoring WC for risk of postmenopausal BC associated with adiposity.

#619

Dietary advanced glycation end-products and postmenopausal breast cancer risk in the NIH AARP diet and health study.

Lindsay L. Peterson,1 Seho Park,1 Yikyung Park,1 Narges Anbardar,2 David Turner2. 1 _Washington University School of Medicine, St. Louis, MO;_ 2 _Medical University of South Carolina, Charleston, SC_.

Purpose: Advanced glycation end-products (AGE) are hypothesized to be involved in carcinogenesis. We analyzed dietary AGE intake in relation to invasive breast cancer (BC) to advance our knowledge of the impact of diet on BC risk.

Methods: Dietary AGE intake was estimated from the food-frequency questionnaire in postmenopausal women in the NIH AARP Diet and Health study, prospective cohort (n=183,548). Incident BC cases were identified through linkage with state cancer registries. Cox proportional hazards regression models were used to estimate relative risks (RRs) and 95% confidence intervals (CIs) for developing BC according to quintiles of AGE levels. Multivariate regression models were adjusted for established BC risk factors.

Results: During a mean follow-up of 12.8 years, 9,851 BCs (1,978 advanced stage) were identified. Median daily AGE intake was 5,932 KU/1,000 kcal (10th\- 90th percentile, 3,555 - 8,644). Women with higher AGE intake tended to have lower education level, higher BMI, younger age at menarche, menopause, first birth, more children, less physical activity, current smoking and higher total fat and meat intake. AGE intake was associated with increased risk of BC in the multivariate model 1, but the association was attenuated and no longer significant after adjusting for total meat and fat intakes (Table). The association between AGE intake and BC did not differ by BMI, hormone therapy use, or alcohol consumption. AGE intake was not related to BC hormone receptor status, histologic type, or grade. However, AGE intake was associated with increased risks of advanced stage tumors (highest vs. lowest quintile of AGE intake, RR 1.37 95% CI 1.09-1.74) even after controlling for total meat and fat intakes. The risk of advanced stage BC increased by 14% (RR 1.14 95% CI 1.04-1.25 in multivariate model 2) per 2,500 KU/1,000 kcal increase in AGE intake.

Conclusions: Dietary AGEs may play a role in the development of postmenopausal BC, particularly in advanced stage BC.

Relative Risks (95% CI) of breast cancer for quintiles of AGE intake

---

|

Quintiles of AGE intake | |

|

1 | 2 | 3 | 4 | 5 | P value | Continuous AGE

Total invasive breast cancer | |  | |  | |

|

No. of cases | 1,899 | 2,023 | 1,932 | 1,995 | 2,002 | |

Age-adjusted model | 1 reference | 1.07 (1.01-1.14) | 1.03 (0.97-1.10) | 1.08 (1.01-1.15) | 1.10 (1.04-1.17) | .005 | 1.05 (1.02-1.07)

Multivariate model 1 | 1 reference | 1.06 (0.99-1.13) | 1.01 (0.95-1.08) | 1.05 (0.99-1.12) | 1.09 (1.02-1.16) | .03 | 1.04 (1.02-1.07)

Multivariate model 2 | 1 reference | 1.00 (0.93-1.08) | 0.94 (0.86-1.02) | 0.96 (0.88-1.06) | 1.00 (0.90-1.11) | .99 | 1.03 (0.99-1.08)

|  | |  | |  | |

Stage | |  | |  | |

|

Localized | |  | |  | |

|

No. of cases | 1,021 | 1,016 | 974 | 1,001 | 989 | |

Age-adjusted model | 1 reference | 1.00 (0.92-1.09) | 0.97 (0.88-1.05) | 1.00 (0.92-1.09) | 1.01 (0.92-1.10) | .89 | 1.02 (0.99-1.06)

Multivariate model 1 | 1 reference | 0.99 (0.90-1.08) | 0.95 (0.87-1.04) | 0.98 (0.90-1.07) | 1.00 (0.92-1.10) | .92 | 1.02 (0.98-1.06)

Multivariate model 2 | 1 reference | 0.90 (0.81-0.99) | 0.84 (0.75-0.94) | 0.86 (0.76-0.98) | 0.90 (0.78-1.04) | .32 | 1.02 (0.96-1.09)

Distant/Regional | |  | |  | |

|

No. of cases | 341 | 414 | 377 | 408 | 438 | |

Age-adjusted model | 1 reference | 1.22 (1.06-1.41) | 1.12 (0.97-1.29) | 1.22 (1.05-1.41) | 1.33 (1.15-1.53) | <.001 | 1.11 (1.05-1.17)

Multivariate model 1 | 1 reference | 1.21 (1.04-1.39) | 1.10 (0.94-1.27) | 1.18 (1.02-1.37) | 1.28 (1.11-1.48) | .004 | 1.09 (1.03-1.16)

Multivariate model 2 | 1 reference | 1.25 (1.06-1.47) | 1.15 (0.95-1.40) | 1.25 (1.02-1.54) | 1.37 (1.09-1.74) | .02 | 1.14 (1.04-1.25)

#620

Evaluating multiple tumor markers in a novel analysis of reproductive factors and breast cancer risk.

Audrey Y. Jung,1 Thomas U. Ahearn,2 Sabine Behrens,1 Haoyu Zhang,3 Pooja Middha,1 Marjanka Schmidt,4 Nilanjan Chatterjee,3 Montserrat Garcia-Closas,2 Jenny Chang-Claude,1 on behalf of the Breast Cancer Association Consortium. 1 _German Cancer Research Center, Heidelberg, Germany;_ 2 _National Cancer Institute, Rockville, MD;_ 3 _Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD;_ 4 _Netherlands Cancer Institute, Amsterdam, Netherlands_.

Background: Reproductive factors are reported to be differentially associated with risk of breast cancer (BC) subtypes; however, subtype defining markers are highly correlated and clarifying which markers are sources of heterogeneity is challenging. We aimed to define the association of parity, age at first full-term pregnancy (AFFTP), and breastfeeding duration (BFD) with BC subtypes defined by estrogen receptor (ER), progesterone receptor (PR), the human epidermal growth factor receptor 2 (HER2), and grade, and to identify which tumor markers are heterogeneity sources.

Methods: We included 56,545 cases and 65,332 controls from 33 population-based studies in the Breast Cancer Association Consortium. A standard polytomous logistic regression with parity (nulliparous (ref), 1, 2, 3, ≥4), AFFTP (<20 years (ref), 20-<25, 25-<30, ≥30), BFD (0 months (ref), >0-6, >6-12, >12-24, >24), age and study was used to estimate odds ratios (ORs) and 95% confidence intervals (CI) with BC subtypes. Subtypes were defined by in situ and invasive intrinsic-like subtypes: luminal A (HR+, HER2-, grade 1&2), luminal B-HER2- (HR+, HER2-, grade 3), luminal B-HER2+ (HR+, HER2+), HER2-enriched (HR-, HER2+) and triple-negative (TN; HR-, HER2-). We used a two-stage polytomous logistic regression to evaluate heterogeneity sources among ER, PR, HER2, grade, and TN specific associations, adjusting for each other and accounting for missing tumor marker data.

Results: Luminal A and TN risk patterns were most distinct. Luminal A risk was inversely associated with parity (OR (95%CI): 0.81 (0.73-0.91), 0.71 (0.64-0.79), 0.64 (0.58-0.72), and 0.53 (0.47-0.59), for 1, 2, 3, ≥4 livebirths, respectively), positively associated with later AFTTP (1.00 (0.93-1.08), 1.14 (1.05-1.23), and 1.36 (1.25-1.49) for 20-<25, 25-<30, ≥30 years, respectively) in parous women, but not associated with BFD. In contrast, parous women had a higher TN risk attenuating with more livebirths: 1.38 (1.15-1.65), 1.26 (1.06-1.49), 1.16 (0.97-1.38), and 1.02 (0.84-1.22) for 1, 2, 3, ≥4 livebirths, respectively. TN risk was also inversely associated with late AFFTP (0.92 (0.82-1.03), 0.86 (0.76-0.98), 0.81 (0.69-0.94) for 20-<25, 25-<30, ≥30 years, respectively) as well as with longer BFD (0.93 (0.83-1.04), 0.76 (0.66-0.88), 0.8 (0.68-0.93), and 0.79 (0.64-0.96) for >0-6, >6-12, >12-24, >24 months, respectively). Risk patterns for the other intrinsic-like subtypes and in situ were most like luminal A. Notable sources of heterogeneity for parity and BFD was TN vs non-TN (Phet=0.003 and Phet=0.005, respectively) and for AFFTP were ER (Phet<0.001) and HER2 (Phet=0.003), while grade was not an important source of heterogeneity after accounting for ER, PR, and HER2.

Conclusion: We found strong evidence of strong heterogeneity among parity, AFFTP, and BFD with BC subtypes with distinct patterns of associations for TN vs luminal A and other tumor subtypes.

#621

Germline testing in African American women with invasive breast cancer.

Leann A. Lovejoy,1 Seth K. Rummel,1 Craig D. Shriver,2 Rachel E. Ellsworth2. 1 _Windber Research Institute, Windber, PA;_ 2 _Murtha Cancer Center, Windber, PA_.

Background: Although African ancestry is not included in the NCCN guidelines for identifying women with invasive breast cancer who may benefit from genetic testing, African American women (AAW) are at increased risk of being diagnosed at a young age and/or with triple negative breast cancer, both factors that do meet NCCN criteria for genetic testing. Because AAW represent a frequently underserved population, we evaluated the utilization and results of germline testing of cancer predisposition genes in AAW with invasive breast cancer.

Methods: 546 self-described AAW with invasive breast cancer enrolled in the Clinical Breast Care Project from 2001-2017). Eligibility status for genetic testing was assessed using the NCCN guidelines version 1.2019. In patients without clinical panel testing performed and with an available genomic DNA sample, targeted sequencing across 94 cancer predisposition genes was performed. Variants were classified as pathogenic, likely pathogenic, uncertain significance (VUS), likely benign or benign using ClinVar.

Results: 60% of the patients were eligible for genetic testing of which 37% of underwent clinical testing. Within the high-risk women who pursued clinical testing, the mutation frequency was 13% and included mutations in ATM (n=2), BRCA1 (n=7), BRCA2 (n=5) and TP53 (n=1). High-risk women who were tested only in the research setting had a mutation frequency of 9% including BRCA1 (n=2) and BRCA2 (n=2) and single carriers of mutations in BLM, CHEK2, MLH1, MUTYH, PALB2, PMS2 and RECQL4. Within the 111 women who did not meet NCCN criteria, the mutation frequency was 5% with mutations in BRCA2 (n=3), PMS2 (n=1), SBHD (n=1) and TSC2 (n=1). Six women had previously unreported variants in BRCA1, BRCA2, TP53, ALK and RB1 and 29% of the women had VUS in genes including BRCA1 and BRCA2.

Conclusions: Under current NCCN guidelines, the majority of AAW with invasive breast cancer were eligible for genetic testing; however, translating eligibility into pursuit of germline testing remains a challenge. For example, 33% of the high-risk women underwent clinical testing and 40% of the mutation carriers within the high-risk group were detected in the research setting. Furthermore, 5% of low-risk women, not currently eligible for genetic testing, were mutation carriers. In addition, while the majority (66%) of mutations detected were in BRCA1 or BRCA2, multi-gene testing allowed for an additional 50% of mutation carriers to be identified. Because identification of germline mutations may alter patient management and prevent secondary cancers, it is critical to identify and address barriers, such as evolving NCCN guidelines, cost, and patient preference, to testing and optimize the selection of AAW for genetic testing. The views expressed in this abstract are those of the author and do not reflect the official policy of the Department of Army/Navy/Air Force, Department of Defense, or U.S. Government.

#622

Reproductive factors and breast cancer risk to women in Ghana, West Africa.

Jonine D. Figueroa,1 Brittny Davis Lynn,2 Lawrence Edusei,3 Nicolas Titiloye,4 Ernest Adjei,4 Joe Nat Clegg-Lamptey,3 Beatrice Wiafe-Addai,5 Baffour Awuah,4 Montserrat Garcia-Closas,2 Louise A. Brinton2. 1 _University of Edinburgh, Edinburgh, United Kingdom;_ 2 _National Cancer Institute, Bethesda, MD;_ 3 _Korle Bu Teaching Hospital, Accra, Ghana;_ 4 _Komfo Anoyke Teaching Hospital, Kumasi, Ghana;_ 5 _Peace and Love Hospital, Kumasi, Ghana_.

Background: Women of African ancestry have a higher proportion of early onset and estrogen receptor (ER) negative cancers compared to women of European descent. Differences in risk associations by age at onset and ER status for reproductive factors, particularly parity and breastfeeding, have been proposed as possible contributors to this racial disparity. We therefore investigated these relations in the Ghana Breast Health Study.

Methods: The study population included 1,126 women diagnosed with invasive breast cancer and 2,106 population controls aged 18-74 years at recruitment (2013-2015) in three hospitals in Accra and Kumasi, Ghana. Factors evaluated included age at menarche, number of livebirths, age at first livebirth, and median months breastfeeding per pregnancy. Odds ratios (OR) and 95% confidence intervals (CI) were estimated using logistic regression models overall and stratified by age. Associations by ER status were estimated using polytomous logistic regression models.

Results: We observed associations with parity and extended breastfeeding duration per pregnancy that were modified by age at onset (<50 vs. >50 years, P-het <0.02 and 0.01, respectively). For women <50 years, the OR was 0.70 (95% CI 0.42-1.18) for those with >5 v. 0 livebirths, but there was no association with breastfeeding months per pregnancy (>18 vs <12 months: OR (95%CI) = 1.04 (0.75-1.44). For women >50 years, both higher number of livebirths and longer durations of breastfeeding months per pregnancy were associated with lower breast cancer risk: OR (95%CI) = 0.40 (0.20-0.83) for >5 vs 0 livebirths and 0.71 (0.51-0.98) for >18 vs <12 breastfeeding months per pregnancy. Data were consistent with a higher risk of early onset (<50 years) ER-negative breast cancer for parous compared to nulliparous women (1.63 (0.82-3.25), that was attenuated by extended breastfeeding (0.72 (0.45-1.14) for >18 vs <12 breastfeeding months per pregnancy).

Conclusion: In this population of women in West Africa, increased number of live births and breastfeeding months per pregnancy were strong protective factors for later onset breast cancer. Among younger women, these trends were modified by ER status, with opposite associations for parity in ER+ vs. ER- tumors and an inverse association with breastfeeding in the ER- tumors that was not seen in the ER+ tumors. Our data support previous reports in African-American women of differential associations of parity and breastfeeding by ER status and age at onset. Further attention should focus on how reproductive factors contribute to observed racial heterogeneity in breast cancer.

#623

Iron intake, oxidative stress-related genes, and breast cancer risk.

Vicky C. Chang,1 Michelle Cotterchio,2 Susan J. Bondy,1 Joanne Kotsopoulos3. 1 _University of Toronto, Toronto, Ontario, Canada;_ 2 _Cancer Care Ontario, Toronto, Ontario, Canada;_ 3 _Women's College Hospital, Toronto, Ontario, Canada_.

Background: Iron has been shown to enhance breast carcinogenesis in animal models through generation of oxidative stress, although epidemiological evidence of the association between iron intake and breast cancer risk remains inconclusive. This study investigated associations between different types of iron intake and breast cancer risk, and whether these associations were modified by genetic polymorphisms in antioxidant enzymes, including manganese superoxide dismutase (MnSOD) and glutathione S-transferases M1 (GSTM1) and T1 (GSTT1).

Methods: A population-based case-control study in Ontario, Canada recruited 3,030 breast cancer cases identified from the Ontario Cancer Registry and 3,402 controls from random digit dialing. Iron intake from foods and supplements was assessed using a 178-item food frequency questionnaire. Multivariable logistic regression analyses were used to evaluate associations between breast cancer risk and intakes of dietary, supplemental, and total (dietary plus supplemental) iron, among all women and stratified by menopausal status. Associations were also examined by hormone receptor [estrogen receptor (ER)/progesterone receptor (PR)] tumor subtype. Among women providing saliva (DNA) samples (1,696 cases and 1,761 controls), interactions between iron intake and genetic polymorphisms were assessed using the likelihood ratio test.

Results: Among all women, intakes of dietary, supplemental, and total iron were not associated with breast cancer risk, overall or by tumor subtype. Among premenopausal women, there was an increase in breast cancer risk for the ER–PR– subtype associated with higher intakes (highest vs. lowest quintile) of dietary [odds ratio (OR) = 1.4; 95% confidence interval (CI): 0.9–2.4] and total (OR = 1.8; 95% CI: 1.1–2.9) iron (P for trend < 0.05). Among postmenopausal women, supplemental iron intake (>18 vs. 0 mg/day) was associated with reduced breast cancer risk (OR = 0.7; 95% CI: 0.5–0.9), with similar associations across tumor subtypes. Associations of dietary and total iron intake with overall breast cancer risk were modified by GSTT1 and/or GSTM1/T1 combined genotypes (P for interaction < 0.05). For example, among women with deletions in both GSTM1 and GSTT1 loci, higher dietary iron intake was associated with increased breast cancer risk (OR = 2.1; 95% CI: 1.1–4.2), whereas null or inverse associations were found among women with other GSTM1/T1 genotypes.

Conclusions: Results from this study suggest that higher dietary and total iron intake may be associated with increased risk of ER–PR– breast cancer among premenopausal women, whereas higher supplemental iron intake may be associated with reduced postmenopausal breast cancer risk. In addition, associations between iron intake and breast cancer risk may be modified by polymorphisms in oxidative stress-related genes. Our ongoing work will investigate heme and non-heme iron intake in relation to breast cancer risk.

#624

A case control study of benign breast disease and breast cancer among indigenous African women.

BABATUNDE ADEDOKUN,1 Stella Odedina,2 Oladosu Ojengbede,2 Adeyinka Ademola,3 Temidayo Ogundiran,3 Dezheng Huo,1 Olufunmilayo Olopade,1 Imaria Agwai,2 Timothy Makumbi,4 Paul Ndom,5 Antony Gakwaya6. 1 _University of Chicago, CHICAGO, IL;_ 2 _Center for Population and Reproductive Health, Ibadan, Nigeria;_ 3 _University of Ibadan, Ibadan, Nigeria;_ 4 _Mulago Hospital, Kampala, Uganda;_ 5 _Hôpital Général Yaoundé, Yaounde, Cameroon;_ 6 _St. Augustine International University, Kampala, Uganda_.

Purpose: To determine the association between benign breast disease (BBD) and breast cancer and if BBD is a mediator of the association between established risk factors and breast cancer among native African women

Methods: We analysed data from 5154 women who participated in a case control study (2405 cases and 2749 controls) between March 1998 and December 2014 in three sub Saharan African countries: Nigeria (n= 4233), Uganda (n=477) and Cameroon (n=444). Breast cancer cases aged 18 years and older were enrolled in two tertiary hospitals in the two countries, unselected for age at diagnosis and family history. Controls who were free of breast cancer were enrolled in the same hospitals. Interviews were conducted to obtain data on potential epidemiological risk factors for breast cancer. We determined the association between breast cancer, history of BBD, age at diagnosis and types of BBD using logistic regression, while adjusting for socio-demographic characteristics and established risk factors. In addition, we estimated the proportion of the effects of other established risk factors mediated by BBD in the etiology of breast cancer.

Results: Less than a tenth (8.1%, 420) of the study participants reported having a history of BBD, 87 of the 420 reported a biopsy diagnosed BBD. Among breast cancer cases, the proportions of BBD and biopsy-confirmed BBD were 10.4% and 3.8%, respectively, compared to 6.3% and 1.3% among controls (both p<0.001). Fibroadenoma (n=100, 45.2%) was the most commonly reported type of benign breast disease, followed by breast cyst (n=31, 15.2%) and fibrocystic diseases (n=19, 10.1%). Overall, the adjusted odds ratio of having breast cancer for those with benign breast disease relative to those without was 1.42 (95% CI 1.13-1.79). The odds ratios were also significantly higher among premenopausal (OR = 1.45, 95% CI: 1.07-1.95) and postmenopausal women (OR = 1.49, 95% CI 1.02-2.19). The proportion mediated by BBD in the association between age at menarche and breast cancer was 13.7%. Less than 1% of the effects of other risk factors that we examined was mediated by BBD.

Conclusions: Benign breast disease is a risk factor for breast cancer among women in SSA. The mediator role of BBD in the effects of established risk factors on breast cancer risk appears very minimal.

#625

Association between obesity and prostate cancer disease risk and mortality among African American men.

Margaret Saira Pichardo,1 Tsion Minas,2 Wei Tang,2 Tiffany H. Dorsey,2 Stefan Ambs2. 1 _Yale University School of Public Health, New Haven, CT;_ 2 _National Cancer Institute, NIH, Bethesda, MD_.

Prostate cancer (PCa) is a major cause of cancer death in U.S. men. Age-adjusted mortality rates for men of African ancestry (40.8 per 100,000) are more than twice that of men of European ancestry (18.2 per 100,000). Men of African ancestry are more likely to have advanced stage cancer at diagnosis and lower prostate cancer survival rates relative to men of European ancestry of similar age and stage at diagnosis. Risk factors that may explain this survival disparity remain poorly understood. Obesity, a major risk factor for cancer development, aggressiveness and progression, disproportionately affects US men of African ancestry. Age-adjusted prevalence of obesity are higher among men of African ancestry (38.0%) compared to men of European ancestry (34.7%). While it has been previously suggested that obesity may worsen disease-related outcomes among prostate cancer patients, the relationship of obesity and prostate cancer mortality remains unclear, with studies showing mixed or null results in men of African ancestry. Less is known about the underlying biological mechanisms that may contribute to the racial differences observed in the link between obesity and prostate cancer. We previously reported an obesity paradox among African-American men in the NCI-Maryland Prostate Cancer-Case Control Study, with overweight and obese men having a lower risk of being diagnosed with the disease. Here, using Cox proportional hazard regression modeling, we estimated the risk of a PCa mortality for the 976 cases in the study. Our primary analysis suggests an obesity paradox, where obesity may protect against disease mortality among the African-American men. In subsequent analyses, we will examine the role of immune and inflammation markers in mediating or moderating the observed relationship of obesity with PCa in these men.

#626

Association of circulating vitamin D concentrations with colorectal cancer risk differs depending on inherited genotypes in combination with dietary lifestyle factors.

David C. Gibbs,1 Marjorie L. McCullough,2 Mingyang Song,3 Caroline Y. Um,2 Kana Wu,3 Mazda Jenab,4 Edward Giovannucci,3 Roberd M. Bostick,1 Veronika Fedirko1. 1 _Emory University, Atlanta, GA;_ 2 _American Cancer Society, Atlanta, GA;_ 3 _Harvard University, Cambridge, MA;_ 4 _International Agency for Research on Cancer, Lyon, France_.

Higher circulating 25-hydroxyvitamin-D [25(OH)D] concentrations - used clinically to assess vitamin D status - are inversely associated with colorectal cancer (CRC) risk. However, it remains unknown whether this association varies by common, inherited polymorphisms encoding common vitamin D-binding protein (DBP) isoforms linked to differences in vitamin D handling and metabolism. We pooled participant-level data from three prospective cohorts in the United States (Cancer Prevention Study-II [CPS-II], Nurses' Health Study [NHS]) and Western Europe (European Prospective Investigation into Cancer and Nutrition [EPIC]), comprising 1,710 incident CRC cases diagnosed between 1990 and 2011, and 1,649 matched controls selected using incidence density sampling. There were 1,106, 246 and 358 CRC cases in EPIC, CPS-II and NHS, respectively. Pre-diagnostic 25(OH)D measurements were calibrated to the same assay and seasonally-adjusted. Study-specific relative risks (RRs) for 25(OH)D and CRC risk according to DBP2 isoform (determined by the GC rs4588 variant) were estimated using multivariable unconditional logistic regression and pooled using fixed effects models. We also stratified our models by dietary vitamin D intake to account for differences in fortification/supplementation in the US and Europe. Variation in 25(OH)D concentrations across studies reflected differences in vitamin D intake and supplement use. Among individuals with lower vitamin D intakes (≤ 330 IU/day), for each 25 nmol/L increase in 25(OH)D, CRC risk was 30% lower among those with the DBP2 isoform (RR = 0.70, 95% CI = 0.59-0.83), and 5% lower among those without DBP2 (RR = 0.95, 95% CI = 0.82-1.10) (Pheterogeneity by DBP isoform = 0.009). Among individuals with higher vitamin D intakes (>330 IU/day, CPS-II and NHS only), for each 25 nmol/L increase in 25(OH)D, CRC risk was 18% higher among those with DBP2 (RR = 1.18, 95% CI = 0.82-1.10), and 33% lower among those without the DBP2 isoform (RR = 0.67, 95% CI = 0.53-0.85) (Pheterogeneity by DBP isoform = 0.002). These findings suggest that the association of 25(OH)D with CRC risk differs depending on inheritance of the functional DBP2 isoform in combination with levels of dietary vitamin D intake. <!--EndFragment-->

#627

Associations of novel dietary and lifestyle inflammation scores with incident colorectal cancer in the National Institutes of Health-American Association of Retired Persons Diet and Health study.

Doratha Byrd,1 Roberd M. Bostick,1 Suzanne E. Judd,2 William D. Flanders,1 Terryl J. Hartman,1 Veronika Fedirko,1 Jill Reedy,3 Tanya Agurs-Collins3. 1 _Emory University, Atlanta, GA;_ 2 _University of Alabama at Birmingham, AL;_ 3 _National Institute of Health, National Cancer Institute, MD_.

Chronically higher inflammation may play a role in colorectal carcinogenesis. Contributors to inflammation likely include dietary and other lifestyle exposures, such as smoking, physical activity, alcohol intake, and obesity. We developed dietary (DIS) and lifestyle (LIS) inflammation scores to characterize the aggregate contributions of dietary and lifestyle exposures to systemic inflammation, and investigated associations of the scores with incident colorectal cancer (CRC) in the prospective National Institutes of Health-American Association of Retired Persons (NIH-AARP) Diet and Health Study cohort (n = 453,465).

To develop the 19-component DIS, we selected a priori 18 whole food groups, including leafy greens, deep-orange/yellow vegetables, apples/berries, tomatoes/tomato products, other fruits and vegetables, processed meats, red meats, poultry, fish, nuts, legumes, refined grains or starchy vegetables, added sugars, saturated/trans fats, coffee and tea, high- and low-fat dairy, plus a supplemental micronutrient score. Similarly, for the 4-component LIS we selected a priori smoking status, physical activity, alcohol intake, and body mass index. The components were weighted based on the strengths of their associations with a panel of circulating inflammation biomarker concentrations (high sensitivity C-reactive protein and interleukin [IL]-6, IL-8, and IL-10) in a subset of 639 male and female, black and white participants in the Reasons for Geographic and Racial Differences in Stroke cohort study (REGARDS). The sums of the weighted components constitute the scores, such that higher scores reflect a higher balance of more pro-inflammatory exposures. Applying the component weights developed in REGARDS, we calculated a DIS and LIS using baseline data from the NIH-AARP study cohort and investigated their associations with incident CRC using multivariable Cox proportional hazards regression, adjusted for established risk factors for CRC.

Over an average of 13.5 years of follow-up, 10,336 participants were diagnosed with CRC. With increasing levels of the DIS and LIS, there was increasing risk for incident CRC. For men and women in the highest relative to the lowest quintiles of the DIS and LIS, the multivariable-adjusted hazards ratios (HR) and their 95% confidence intervals [CIs]) were: 1.3 (95% CI: 1.2, 1.4; Ptrend: <0.001) and 1.4 (95% CI: 1.3, 1.5; Ptrend: <0.001), respectively. The HR for those in the highest relative to the lowest joint DIS/LIS quintile was 1.8 (95% CI: 1.7, 2.0; Pinteraction: <0.001). The DIS and LIS associations were strongest among men and for cancers of the colon among men and women combined.

These results suggest that pro-inflammatory diet and lifestyle exposures may be associated with higher risk for incident colorectal cancer.

#628

Smoking and risk of colorectal cancer by sex and histological subsites: the Multiethnic Cohort (MEC) study.

Inger T. Gram,1 Song-Yi Park,2 Lynne R. Wilkens,2 Christopher A. Haiman,3 Loïc Le Marchand2. 1 _UiT The Arctic University of Norway, Tromsø, Norway;_ 2 _University of Hawai'i Cancer Center, Honolulu, HI;_ 3 _University of Southern California,, Los Angeles, CA_.

Background. Smoking is an established risk factor for colorectal cancer (CRC). The purpose of this study was to examine if the increased risk of CRC due to cigarette smoking differ by sex and tumor subsite in the Multiethnic Cohort (MEC).

Methods: We analyzed data from 188,052 participants (45% men) who were enrolled in the MEC at 45-75 years of age between 1993 and 1996. We identified CRC cases overall and by anatomic subsite (proximal, distal and rectal) via linkage to the Hawaii and California Surveillance, Epidemiology, and End Results Program cancer registries through December 2013. We used Cox proportional hazards regressions to estimate multivariable-adjusted hazard ratios (HR) with 95% confidence intervals (CI).

Results: During a mean follow-up of 16.7 ± 5.2 years, we identified 4,879 incident cases of invasive colorectal adenocarcinoma. For men and women, the annual age-adjusted incidence rate was 126.4 and 91.8 per 100,000 person-years (truncated to ages 45-85), respectively. Men had a higher incidence rate than women for all three subsites of CRC. Men had a larger proportion of rectal and a lower proportion of proximal colon cancer than women.Altogether, 70% of the men and 44% of women reported to be ever smokers. Men compared with women ever smokers, had smoked for more years, smoked more cigarettes per day and consequently had smoked more pack-years. . For both men and women, age at diagnosis was lower for ever compared with never smokers. Among men, compared with never smokers, ever smokers had a 38% (HR=1.38; 95% CI: 1.15-1.65) higher risk of distal, but not of proximal (HR=1.03; 95% CI: 0.90-1.18) colon cancer. The corresponding figures for women were a 21% (HR=1.21; 95% CI: 1.07-1.37) higher risk of proximal, but not of distal (HR= 0.97; 95% CI: 0.81-1.16) colon cancer. We observed a direct association for the three (duration, number of cigarettes per day and number of pack-years) measures of smoking exposure with proximal (all Ptrend < 0.001) colon cancer for women and with distal (all Ptrend < 0.001) colon cancer for men. Male ever smokers had a 40% (HR=1.40; 95% CI: 1.16-1.69) and female ever smokers a 58% (HR=1.58; 95% CI: 1.28-1.95) higher risk of rectal cancer compared with sex specific never smokers. Heavy smokers i.e. those who had smoked >20 pack-years had a 61% (HR =1.61; 95% CI: 1.29-2.00) higher risk of rectal cancer for men and a doubling in risk for women (HR= 2.09; 95% CI: 1.54-2.83) compared with sex specific never smokers (Pheterogeneity by sex = 0.03).

Conclusions: Our main findings shows that the smoking related colorectal cancer risk varies by sex for the subsites of colon cancer, and possibly also for rectal cancer.

#629

Coffee consumption and risk of colorectal cancer in the Cancer Prevention Study II Nutrition Cohort.

Caroline Y. Um, Marjorie L. McCullough, Mark A. Guinter, Peter T. Campbell, Eric J. Jacobs, Susan M. Gapstur. _American Cancer Society, Atlanta, GA_.

Coffee consumption is inconsistently associated with colorectal cancer (CRC) risk, and further research on potential differences in associations by sex, colorectal subsite, smoking status, and caffeinated/decaffeinated coffee is needed. To investigate associations of coffee consumption with risk of CRC, we analyzed data from the Cancer Prevention Study-II Nutrition prospective cohort study. This analysis included 46,970 men and 60,007 women without a history of cancer at baseline. Those who completed a semiquantitative food frequency questionnaire in 1999 were followed through 2013, and 1,706 verified incident CRC cases were identified. Daily servings of caffeinated and decaffeinated coffee were comparable between men and women. Of the total population, 4.6% were current smokers, 46.1% were never smokers, and 47.3% were former smokers (2% missing smoking status). Cox proportional hazards regression models were used to calculate hazard ratios (HR) and 95% confidence intervals (CI) for the association of coffee consumption with CRC risk, adjusted for age, sex, body mass index, smoking, alcohol intake, total energy, total calcium (energy-adjusted), red and processed meat, and decaffeinated coffee in caffeinated coffee models and vice versa. For those who consumed 2 or more cups per day relative to those who consumed none or less than 1 cup per month, caffeinated coffee was not associated with CRC risk (HR 1.06, 95% CI 0.93-1.20), but decaffeinated coffee was associated with 15% lower risk of CRC (HR 0.85, 95% CI 0.73-0.99). Current smokers tend to consume more coffee in our study population and in other studies, and smoking is an established risk factor for some molecular sub-types of CRC; therefore, we repeated analyses stratified by smoking status (controlling for smoking duration for non-smokers and dose for current smokers). Among non-smokers (includes never and former smokers), the associations for coffee and CRC risk were similar to the overall results (HR 1.03, 95% CI 0.90-1.18 for caffeinated coffee; HR 0.87, 95% CI 0.74-1.02 for decaffeinated coffee). Among current smokers, the adjusted HRs and 95% CIs were 1.60 (0.80-3.22) for caffeinated coffee and 0.45 (0.19-1.03) for decaffeinated coffee. There was no evidence of effect modification by sex or colorectal subsite. Our results suggest that decaffeinated coffee may be associated with lower risk of CRC, but do not suggest potential differences in risk between men and women or by colorectal subsites. Further studies are needed to confirm these findings and to explore potential differences in caffeinated and decaffeinated coffee.

#630

Smoking is associated with risks of molecular subtypes of colorectal cancer.

Xiaoliang Wang,1 Efrat Amitay,2 Barbara L. Banbury,3 Hermann Brenner,2 Daniel D. Buchanan,4 Peter T. Campbell,5 Jenny Chang-Claude,2 Steven J. Gallinger,6 Graham G. Giles,7 Tabitha A. Harrison,3 John L. Hopper,4 Mark A. Jenkins,4 Yi Lin,3 Reiko Nishihara,8 Polly A. Newcomb,3 Shuji Ogino,8 Lori C. Sakoda,9 Robert E. Schoen,10 Martha L. Slattery,11 Steven N. Thibodeau,12 Bethany Van Guelpen,13 Michael O. Woods,14 Li Hsu,3 Michael Hoffmeister,2 Ulrike Peters3. 1 _UNIV. Of Washington, Seattle, WA;_ 2 _German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 3 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 4 _The University of Melbourne, Melbourne, Australia;_ 5 _American Cancer Society, Atlanta, GA;_ 6 _University of Toronto, Toronto, Ontario, Canada;_ 7 _Cancer Council Victoria, Melbourne, Australia;_ 8 _Dana-Farber Cancer Institute, Boston, MA;_ 9 _Kaiser Permanente Northern California, Oakland, CA;_ 10 _University of Pittsburgh Medical Center, Pittsburgh, PA;_ 11 _University of Utah, Salt Lake City, UT;_ 12 _Mayo Clinic, Rochester, MN;_ 13 _Umea University, umea, Sweden;_ 14 _Memorial University of Newfoundland, St. John's, Newfoundland and Labrador, Canada_.

Background: Smoking has been associated with colorectal cancer (CRC) risk; but limited evidence has shown the association between smoking and molecular subtypes of CRC.

Methods: We analyzed 9,422 CRC cases and 9,950 controls from 10 observational studies. Multinomial logistic regression analysis was performed to assess the association between sex-study-specific quartiles of pack years of smoking and risk of CRC molecular subtypes, using non-smokers as reference group, adjusting for age, sex, and study. Known oncogenic mutations in four somatic colorectal tumor markers were assessed individually and in combination, including BRAF mutations, KRAS mutations, CpG island methylator phenotype (CIMP), and microsatellite instability (MSI) status. Case-only analysis was also performed to estimate heterogeneity in risk of molecular subtypes of CRC.

Results: Compared with controls, higher pack years of smoking were statistically significantly associated with increased CRC risk when stratified, individually, by all four markers, and the associations got stronger with higher quartiles pack years (p-trend<0.001). Associations between smoking and CRC risk also differed significantly among molecular subtypes. Compared to nonsmokers, the risk of BRAFmut CRC was 83% higher for smokers within the highest quartile of pack-years (OR=1.83; 95% CI: 1.50, 2.25), and 29% higher for BRAFwt CRC (OR=1.29; 95% CI: 1.17, 1.43; Ratio of ORs (ROR)=1.45; 95% CI: 1.18, 1.17; p=3.45x10-4). Similarly, heavy pack-years of smoking was associated with almost two times higher risk of CIMP-high CRC (OR=1.93; 95% CI: 1.60, 2.31), but only 33% higher risk of CIMP-low/negative CRC (OR=1.33; 95% CI: 1.19, 1.48; ROR=1.49; 95% CI: 1.24, 1.79; p=1.72x10-5). The association between smoking and CRC was also stronger in MSI-high tumors (ORMSI-H=1.65; 95% CI: 1.36, 2.00; ORMSI-L/MSS=1.37; 95% CI: 1.23, 1.52; ROR=1.22; 95% CI: 1.00, 1.48; p=0.046). In contrast, the association between smoking and CRC risk was stronger for KRASwt (OR=1.43; 95% CI: 1.27, 1.60), than KRASmut tumors (OR=1.18; 95% CI: 1.02, 1.37; ROR=0.83; 95% CI: 0.71, 0.97; p =0.016). When combining tumor markers, smoking was found to be significantly associated with higher risk of colorectal tumors from the serrated pathway.

Conclusion: In this largest study with a total of 19,372 subjects, we found that heavier pack years of smoking was associated with increased risk of all CRC molecular subtypes. Smokers with heavier pack-years of smoking had particularly higher risk of CRC subtypes with BRAF mutation and CIMP-high, suggesting smoking may be particularly involved in the development of these subtypes of colorectal tumor.

#631

High BMI in relation to low risk of lung cancer among never smokers.

Hung N. Luu,1 Renwei Wang,1 Jennifer Adams-Haduch,1 Aizhen Jin,2 Woon-Puay Koh,2 Jian-Min Yuan1. 1 _University of Pittsburgh, Pittsburgh, PA;_ 2 _Duke-NUS Medical School Singapore, Singapore, Singapore_.

Background. Lean smokers have increased oxidative DNA damage relative to their non-lean counterparts, indicating their heightened susceptibility to tobacco carcinogens-induced DNA damage. Epidemiological association between body mass index (BMI) and lung cancer is inconclusive. We have reported that BMI is a protective factor for smoking-related lung cancer and so did a recent meta-analysis. Knowledge about the association between BMI and non-smoking related lung cancer is limited as this is rare in Western population. We therefore sought to investigate the association between BMI and the risk of never-smoking related lung cancer in an on-going prospective cohort study, the Singapore Chinese Health Study.

Methods. The current analysis used data from the Singapore Chinese Health Study, a cohort of 63,257 middle-aged and older Chinese men and women enrolled between 1993 and 1998. As of December 31, 2015, 2,001 study participants who were free of lung cancer at baseline developed lung cancer of whom 621 were never smokers. Cox proportional hazard regression method was used to calculate hazard ratio (HR) and the corresponding 95% confidence interval (CI) for lung cancer associated with BMI in never-smokers.

Results. Our results shown that increasing BMI was a protective factor for never-smoking related lung cancer and there is no interaction by gender. The respective HRs and 95% CIs were 0.66 (0.45-0.95) for overall and 0.60 (0.39-0.93) in female for those with BMI>27.5 compared to those with BMI<18.5. Even after eliminating those with exposure to any second-hand smoke in childhood or adulthood, the inverse association between BMI and the risk of never-smoking related lung cancer was stronger and significant (HR=0.31, 95% CI: 0.13-0.75; Ptrend=0.01). There was no difference by histologic subtypes (i.e., adenocarcinomas vs. squamous cell).

Conclusion. There was an inverse association between BMI and the risk of never-smoking related lung cancer and there is no interaction by gender for this association. This association was also not different by histologic subtypes.

#632

Associations of coffee and tea consumption with lung cancer risk: A pooled analysis of 17 cohort studies involving over 1.2 million participants.

Jingjing Zhu,1 Wei Zheng,2 Rashimi Sinha,3 Stephanie A. Smith-Warner,4 Yong-Bing Xiang,5 Yikyung Park,6 Shoichiro Tsugane,7 Emily White,8 Woon-Puay Koh,9 Sue K. Park,10 Norie Sawada,7 Seiki Kanemura,11 Yumi Sugawara,11 Ichiro Tsuji,11 Kim Robien,12 Yasutake Tomata,11 Keun-Young Yoo,10 Jeongseon Kim,13 Jian-Min Yuan,14 Yu-Tang Gao,15 Yumie Takata,16 Eiko Saito,7 William Blot,2 Xiao-Ou Shu2. 1 _Vanderbilt University, Nashville, TN;_ 2 _Vanderbilt University Medical Center, Nashville, TN;_ 3 _National Cancer Institute, Bethesda, MD;_ 4 _Harvard T.H. Chan School of Public Health, Boston, MA;_ 5 _Shanghai Cancer Institute, Shanghai, China;_ 6 _Washington University School in St. Louis, MO;_ 7 _National Cancer Center, Tokyo, Japan;_ 8 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 9 _Duke-NUS Medical School, Singapore;_ 10 _Seoul National University, Seoul, Democratic People's Republic of Korea;_ 11 _Tohoku University, Sendai, Japan;_ 12 _The George Washington University, DC;_ 13 _National Cancer Center of Korea, Goyang, Democratic People's Republic of Korea;_ 14 _University of Pittsburgh, PA;_ 15 _Shanghai Jiaotong University, China;_ 16 _Oregon State University, Corvallis, OR_.

Background

Epidemiological studies investigating the associations of coffee and tea intake with lung cancer risk have yielded inconsistent results. These previous studies included mostly lung cancer patients diagnosed among smokers. Because coffee and tea consumption are closely related to smoking behavior, these previous studies could suffer from biases due to residual confounding of smoking. To better characterize the relationship, a large study with a large number of lung cancer cases diagnosed among never smokers and detailed information on tea and coffee consumption, is needed. Using data from a large-scale pooled analysis that consists of over 1.2 million participants in the U.S. and Asia, we carried out a comprehensive evaluation on the association of coffee and tea intake with lung cancer risk.

Methods

Individual-level data from seven prospective cohort studies conducted in the U.S., and ten studies conducted in Asia, were included. Demographic, lifestyle, coffee and tea intake data were collected at the baseline survey for each study. Multivariable-adjusted Cox proportional hazard models were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). The first 2 years of follow-up time was excluded to minimize potential influence of reverse causality on study results. Subgroup analyses by smoking status, sex, race, histologic subtype and coffee type (caffeinated or decaffeinated) were also conducted to assess potential effect modification, as well as heterogeneity of the association.

Results

After a median follow-up of 8.6 years, 20,519 incident lung cancer cases were identified. Both coffee and tea consumption were associated with an increased risk of lung cancer. Comparing non-coffee and non-tea consumption, HRs for lung cancer associated with exclusive coffee drinkers (≥2 cups/day) among current, former and never smokers were 1.30 (95% CI, 1.15-1.47), 1.49 (1.27-1.74) and 1.41 (95% CI, 1.21-1.63), respectively. Similar positive associations were observed for caffeinated and decaffeinated coffee. The HRs associated with exclusive tea drinkers (>2 cups/day) were 1.16 (95% CI, 1.02-1.32), 1.10 (0.92, 1.32) and 1.37 (95% CI, 1.17-1.60) for current, former and never smokers, respectively. These associations did not differ significantly by sex, race or histologic subtypes.

Conclusion

A high consumption of coffee or tea was both associated with an increased risk of lung cancer regardless of race or smoking status. Our study included a large number of never-smoker lung cancer patients, which minimized potential confounding effects due to smoking. The positive association observed for both caffeinated and decaffeinated coffee suggests that compounds other than caffeine may play a role in the etiology of lung cancer.

#633

Association between saturated fat intakes and lung cancer risk among low-income populations in the Southeastern United States.

Hyung-Suk Yoon, Hui Cai, Jae Jeong Yang, Danxia Yu, Harvey J. Murff, Xiao Ou Shu, Wei Zheng, William J. Blot, Qiuyin Cai. _Vanderbilt University Medical Center, Nashville, TN_.

Background: Lung cancer is the leading cause of cancer death in the United States. Previous studies have indicated that a higher intake of dietary saturated fat was associated with lung cancer risk. However, little is known about the association of specific classes of saturated fat with the risk of lung cancer. We conducted a nested case-control study to investigate the association of different classes of saturated fat with lung cancer risk among low-income African Americans (AAs) and European Americans (EAs) in the Southeastern United States.

Methods: A total of 1,528 incident lung cancer cases and 5,699 matched controls from the Southern Community Cohort Study were included in this study. The controls were matched to the cases on age, sex, race, and recruitment site. Using validated food frequency questionnaires, saturated fat was estimated and classed to short-chain (SCSF, 2-4 carbon atoms), medium-chain (MCSF, 6-12 carbon atoms), and long-chain (LCSF, 14-18 carbon atoms). Multivariate logistic regression models were used to estimate odds ratios (ORs) and corresponding confidence intervals (95% CIs) for lung cancer risk associated with saturated fat after adjusting for age, smoking status, pack-years, alcohol consumption, total energy intake, education, household income, body mass index (BMI), and history of chronic obstructive pulmonary disease (COPD).

Results: Total saturated fat intakes were associated with an increased lung cancer risk among EAs with an OR of 1.66 (95% CI: 1.06-2.60) for the highest vs. lowest quartiles (p-trend=0.03). The positive association was only observed for LCSF (highest vs. lowest quartile: OR=1.68, 95% CI: 1.07-2.64 for palmitic acid; OR=1.60, 95% CI 1.02-2.53 for stearic acid; and OR=1.63, 95% CI 1.04-2.56 for total LCSF; all p-trends<0.05). The association was more evident among heavy smokers, which was defined as smoke ≥ 30 pack-years, and for adenocarcinoma subtype. No association between saturated fat intakes with lung cancer risk were observed among AAs. In addition, no association between lung cancer risk and SCSF or MCSF was observed in our study.

Conclusion: Our findings indicate that a high intake of LCSF was associated with an increased risk of lung cancer among low-income EAs. Further studies are needed to confirm our findings.

#634

Coffee and tea drinking and risk of cancer of the urinary tract in male smokers.

Maryam Hashemian, Rashmi Sinha, Gwen Murphy, Stephanie Weinstein, Linda M. Liao, Neal Freedman, Christian C. Abnet, Demetrius Albanes, Erikka Loftfield. _National Cancer Institute, Rockville, MD_.

Introduction: The association of coffee and tea drinking with risk of the urinary tract cancer is unclear. We evaluated these associations in Finnish men with high coffee consumption, using data from the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study, which included information on coffee preparation method.

Methods: The ATBC trial conducted from 1985 to 1993, enrolled 29,133 male smokers. Among 26,841 men with data on coffee and tea intake and without renal failure or extreme caloric intake, we used multivariable Cox proportional hazards regression models to estimate hazard ratios (HRs) and confidence intervals (CIs) for associations of coffee and tea consumption with incident bladder cancer and renal cell carcinoma (RCC). We used those who drank >0 but <1 cup coffee/day as our referent group for coffee analyses, and non-drinkers of tea for tea analyses.

Results: During 472,402 person-years of follow-up (median 17.6 years), 835 incident cases of bladder cancer and 366 cases of RCC were ascertained. Approximately 98% of participants reported drinking coffee and 36% reported drinking tea at baseline. Mean consumption among drinkers was 2.3 cups/day for coffee and 0.7 cups/day for tea. Participants who reported heavier coffee drinking tended to be younger, smoke more cigarettes daily, drink less alcohol and less tea, be less educated, have a lower prevalence of hypertension, and consume more calories and vegetables but less fruit daily. Participants who reported heavier tea drinking tended to have smoked for fewer years, be more educated, drink less coffee, have a higher prevalence of hypertension, and consume more calories, fruits, and vegetables per day. We observed no statistically significant associations for coffee intake with either bladder cancer (HR ≥4 vs >0 to <1 cups/day = 1.16, 95% CI=0.86-1.56) or RCC (HR ≥4 vs >0 to <1 cups/day = 0.85, 95% CI=0.55-1.32) risk, and a non-significant inverse association between tea consumption and bladder cancer (HR ≥1 vs 0 cup/day=0.77, 95% CI=0.58-1.00), but not RCC risk (HR ≥1 vs 0 cup/day=1.00, 95% CI=0.68-1.46). Smoking status, defined using pack-years, appeared to modify bladder cancer association with both coffee and tea (P interaction=0.005 and <0.001, respectively), but not the coffee- and tea-RCC associations (P interaction=0.50 and =0.08, respectively). However, analyses stratified by pack-years revealed non-significant associations. Among 20,603 men with information on preparation method, 71% drank filtered coffee, 21% drank boiled coffee, and 8% drank instant coffee or were not coffee drinkers. We found no impact of coffee preparation on the coffee-cancer associations.

Conclusion: Coffee drinking was not associated with risk of bladder cancer or RCC. Further research on tea and bladder cancer, in populations with a high prevalence of tea drinking, are warranted.

#635

Selenium intake and thyroid cancer: a population-based case-control study in Connecticut.

Qian Wang,1 Huang Huang,2 Nan Zhao,3 Yawei Zhang4. 1 _Icahn School of Medicine Mount Sinai St Luke's and West, New York, NY;_ 2 _Yale University School of Public Health, New Haven, CT;_ 3 _Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing, China;_ 4 _Yale School of Medicine, New Haven, CT_.

Objective: Selenium (Se) is an important substrate for thyroid hormones. It is essential in protecting thyroid from free radicals' damage and oxidative stress. Prior studies regarding different Se exposure measurement routes and thyroid cancer risk have shown inconsistent results. And among them, one cohort study conducted among elderly population using dietary Se intake found no association. In the light of fast increasing thyroid cancer incidence in the United States over the past few decades and majority of thyroid cancer patients diagnosed before age 50, we examined the association between daily Se dietary intake and thyroid cancer risk using data from a population-based case-control study in Connecticut in 2010-2011.

Methods: A total of 386 histologically confirmed cases and 433 population-based controls were included. Daily Se intake was calculated using Diet*Calc Analysis Software based on dietary information collected through food frequency questionnaire. Multivariate unconditional logistic regression models were used to investigate the associations between Se intake and the risk of thyroid cancer, by histology and tumor size (microcarcinoma (≤1cm)/macrocarcinoma (>1cm)) after controlling for confounders.

Results: Moderate to high dietary intake of Se was associated with decreased risk of well-differentiated thyroid cancer (OR=0.38, 95%CI: 0.18-0.81 for the third quartile; OR=0.24, 95%CI: 0.07-0.81 for the fourth quartile). Similar results were found among females and papillary carcinomas. The observed protective effect of high Se consumption was only observed for macrocarcinomas (OR=0.21, 95%CI: 0.05-0.93 for the forth quartile) but not microcarcinomas.

Conclusions: In conclusion, our study suggested an inverse relationship between dietary Se intake and well-differentiated thyroid cancer. A stronger inverse association was found for macrocarcinomas. These results warrant further investigation.

Table. Daily Se intake and thyroid cancer risk by histology, gender and tumor size.

---

Daily Se intake and thyroid cancer risk by histology, gender and tumor sizea.

|  | Q1 (0.00-81.58)(mcg/day) | Q2 (81.58-112.64)(mcg/day) | Q3 (112.64-147.89)(mcg/day) | Q4 (147.89-)(mcg/day)

Overall | Cases/Controls | 123/109 | 105/107 | 77/108 | 81/109

|

Adjusted OR (95%CI) | 1.00 | 0.78 (0.44-1.36) | 0.42 (0.20-0.88) | 0.30 (0.09-0.99)

Women | Cases/Controls | 114/85 | 87/78 | 62/78 | 53/65

|

Adjusted OR (95%CI) | 1.00 | 0.70 (0.38-1.29) | 0.32 (0.14-0.74) | 0.20 (0.05-0.78)

Women well-differentiated | Cases/Controls | 107/85 | 86/78 | 61/78 | 52/65

|

Adjusted OR (95%CI) | 1.00 | 0.72 (0.39-1.33) | 0.31 (0.13-0.72) | 0.18 (0.05-0.70)

Papillary | Cases/Controls | 103/109 | 92/107 | 64/108 | 68/109

|

Adjusted OR (95%CI) | 1.00 | 0.70 (0.39-1.26) | 0.34 (0.15-0.75) | 0.23 (0.07-0.82)

Well-differentiated | Cases/Controls | 116/109 | 104/107 | 76/108 | 80/109

|

Adjusted OR (95%CI) | 1.00 | 0.77 (0.44-1.35) | 0.38 (0.18-0.81) | 0.24 (0.07-0.81)

Micro-Well-differentiated | Cases/Controls | 66/109 | 48/107 | 28/108 | 38/109

|

Adjusted OR (95%CI) | 1.00 | 0.84 (0.40-1.76) | 0.34 (0.11-1.04) | 0.35 (0.06-2.01)

Macro-Well-differentiated | Cases/Controls | 49/109 | 56/107 | 47/108 | 40/109

|

Adjusted OR (95%CI) | 1.00 | 0.71 (0.34-1.48) | 0.46 (0.18-1.16) | 0.21 (0.05-0.93)

|  | |  | |

a. Model was adjusted for age, gender, BMI, education, family history of any cancer, prior benign thyroid disease, total energy intake and alcohol consumption

#636

Salted food intake and risk of gastric cancer: A pooled analysis of Korean cohorts and a global meta-analysis.

Jin Young Yoo,1 Hyun Jeong Cho,1 Sungji Moon,2 Inah Kim,3 Kwang-Pil Ko,4 Sue Kyung Park,2 Jung Eun Lee1. 1 _Seoul National University, Seoul, Republic of Korea;_ 2 _Seoul National University College of Medicine, Seoul, Republic of Korea;_ 3 _Hanyang University College of Medicine, Seoul, Republic of Korea;_ 4 _Gachon University College of Medicine, Incheon, Republic of Korea_.

Backgrounds: Gastric cancer, the fifth most common cancer, is the third most common cause of death from cancer with more than half of the cases occurring in Eastern Asia. The World Cancer Research Fund proposed "foods preserved by salting" as probable risk factors of gastric cancer, as evidenced from epidemiological studies addressing high salt foods and gastric cancer.

Methods: We conducted a pooled analysis of Korean cohorts, The Korean Genome Epidemiology Study (KoGES) and Korean Multi-center Cancer Cohort Study (KMCC) to investigate the association between salted food intake and gastric cancer mortality among Korean population. We used cox proportional hazard models adjusted for potential confounding factors. We also reviewed global observational studies published until October 2018 and conducted a dose-response and a categorical meta-analysis. We used either a random effect model or a fixed effect model depending on the heterogeneity of the studies. We further categorized salted food into salted vegetable, fish, and soup to examine the relative risk (RR) of gastric cancer for specific salted foods.

Results: The pooled RRs (95% confidence intervals, CIs) of gastric cancer among KoGES and KMCC were 0.99 (0.95-1.04) per 50g increase in salted food intake and 0.98(0.94-1.03), 1.79(1.05-3.06), 1.17(0.95-1.43) per 50g increase in intake of salted vegetable, fish, and soup, respectively. The pooled RR (95% CI) of gastric cancer, compared with the lowest category of salted food intake, was 0.95(0.63-1.27) for the highest category of intake. For the dose-response meta-analysis, we included 35 studies (15 cohort and 20 case-control), and the combined global RRs (95% CIs) of gastric cancer per 50g increase in intake were 1.07 (1.02-1.12) for salted food and 1.11(1.03-1.20), 2.10(1.27-3.46), 1.00(0.99-1.01) for salted vegetable, fish, and soup, respectively. When we analyzed prospective studies only, the RRs (95% CIs) per 50g increase in intake were 1.02(0.99-1.06) for salted food and 1.05(0.98-1.13), 1.26(1.01-1.59), 1.00(0.99-1.01) for salted vegetable, fish, and soup, respectively. For the categorical meta-analysis, 69 studies (23 cohort and 46 case-control) were included; the combined RRs (95% CIs) of the highest intake category, compared to the lowest intake category, were 1.19(1.11-1.27), 1.30(1.19-1.40), 1.06(0.95-1.16), and 1.03(0.92-1.14) for salted food, vegetable, fish, and soup, respectively.

Conclusions: The pooled analysis showed a statistically significant association between risk of gastric cancer and salted fish intake among Korean population. The global meta-analyses also showed a significant association between intake of salted food and risk of gastric cancer.

Acknowledgements: This study was funded by the Korean Foundation for Cancer Research (Grant Number. CB-2017-A-2)

#637

A prospective study of tea and coffee intake and risk of glioma.

David J. Cote,1 Alaina M. Bever,1 Kathryn M. Wilson,1 Timothy R. Smith,2 Stephanie A. Smith-Warner,3 Meir Stampfer1. 1 _Channing Division of Network Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA;_ 2 _Department of Neurosurgery, Brigham and Women's Hospital, Boston, MA, Boston, MA;_ 3 _Department of Epidemiology, Harvard T. H. Chan School of Public Health, Boston, Massachusetts, Boston, MA_.

Background: Tea and coffee have anti-inflammatory, anti-oxidant and neuroprotective effects mediated by a variety of compounds, including polyphenols. Observational studies suggest that tea and coffee intake may reduce risk of certain cancers, although the data on glioma risk are limited and inconclusive.

Methods: We evaluated the association between tea, coffee, and caffeine intake and risk of glioma in the female Nurses' Health Study (NHS, n=92,389, followed 33 years) and Nurses' Health Study II (NHSII, n=95,242, followed 23 years) and the male Health Professionals Follow-up Study (HPFS, n=49,885, followed 29 years). Cumulatively updated tea and coffee intake were derived from validated quadrennial food frequency questionnaires. Glioma cases were confirmed by medical record review. Age-adjusted hazard ratios of glioma by category of beverage intake were estimated using Cox proportional hazards models. Associations across the combined cohorts were calculated using fixed effect meta-analysis.

Results: We documented 554 incident cases of glioma, including 256 in NHS, 87 in NHSII, and 211 in HPFS (159, 52, 151 glioblastomas, respectively). Compared to <1 cup/week, higher tea consumption was inversely associated with glioma risk in women (HR=0.65, 95% CI 0.41-1.03 for >2 cups/day, p-trend=0.03), and was inversely, but not significantly, associated in men (HR=0.70, 95%CI 0.30-1.61 for >2 cups/day, p-trend=0.30). In women and men combined, the corresponding HR was 0.66 (95% CI 0.44-0.99) for >2 cups/day (p-trend=0.02). We observed no significant associations between caffeinated, decaffeinated, or total coffee intake and glioma risk in men or women. We found no material differences in the results when baseline values were used, when responses were lagged by eight years, or when cases were limited to glioblastoma only.

Conclusion: In three large, prospective cohort studies of men and women, tea intake was inversely associated with glioma risk, particularly in women. No significant associations were observed for coffee intake of any type and glioma risk. These results merit further exploration in other prospective studies.

#638

Association between calcium intake and pancreatic cancer risk in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO).

Maggie Hoyt, Jianjun Zhang. _Indiana University, Indianapolis, IN_.

Pancreatic cancer is a leading cause of cancer death in the U.S. and its etiology remains largely unclear. As most patients are diagnosed with an advanced, unresectable disease, primary prevention remains a priority for reducing its incidence and mortality. Although ecological, migrant, and temporal trend studies have shown that diet plays a role in the occurrence of pancreatic cancer, few nutrients that alter its risk have been identified from epidemiologic studies. The present study thus sought to investigate intakes of calcium and fat, adiposity, and their potential interactions in relation to pancreatic cancer risk among participants in the PLCO trial. Usual dietary intake among the participants was assessed with Dietary Questionnaire (DQX) at baseline and Dietary History Questionnaire (DHQ) around their third anniversary of randomization. Of 58,477 participants who completed the DQX, 279 cases of pancreatic cancer were documented during a median follow-up of 12.2 years. A total of 101,721 participants responded to the DHQ and gave risk to 380 cases of pancreatic cancer during a median follow-up of 8.9 years. Cox proportional hazards regression was performed to estimate hazard ratios (HR) and 95% Confidence intervals (CI) for pancreatic cancer in relation to total and dietary intake of calcium, intake of fat, and body mass index (BMI). After adjustment for established and suspected confounders, there was a suggestive inverse association between total calcium intake assessed from both food frequency questionnaires and risk of pancreatic cancer [HR (95% CI) for quartile (Q) 2, Q3, and Q 4 vs. Q1: 0.93 (0.67, 1.30), 0.86 (0.60, 1.24), and 0.71 (0.46,1.09); p-trend, 0.11) for DQX and 0.96 (0.72, 1.26), 0.99 (0.74, 1.33), and 0.72 (0.52, 1.01); p-trend, 0.08) for DHQ]. Overall, this inverse association was stronger or its linear trend in reduced risk across the quartiles of total calcium intake was more apparent among overweight participants (BMI: >25-<30) [e.g. HR (95% CI) for Q2, Q3, and Q4 vs. Q1: 0.91 (0.60,1.38), 0.84 (0.54, 1.30), and 0.72 (0.44, 1.18); p-trend, 0.07) for DHQ]. Among the participants with the highest fat intake (Q4) derived from the DHQ, those with the highest intake of total calcium (Q4) experienced a 65% reduced risk of pancreatic cancer compared with those with the lowest intake (Q1) [HR (95% CI): 0.35 (0.17, 0.68)]. However, the possibility of chance finding for this significant risk reduction could not be ruled out due to multiple comparisons. In addition, no appreciable associations of calcium intake from both dietary and supplemental sources with pancreatic cancer risk was observed. In summary, the present study offers suggestive evidence that total calcium intake was associated with a reduced risk of pancreatic cancer and that this potential benefit may be more pronounced among overweight subjects.

#639

Dietary polyunsaturated fatty acids intake and pancreatic cancer risk in understudied populations.

Lang Wu,1 Wei Zheng,1 William J. Blot,1 Mark D. Steinwandel,1 Wanqing Wen,1 Yong-Bing Xiang,2 Yu-Tang Gao,2 Hong-Lan Li,2 Rachael Stolzenberg-Solomon,3 Harvey J. Murff,1 Xiao-Ou Shu1. 1 _Vanderbilt Univ. School of Medicine, Nashville, TN;_ 2 _Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China;_ 3 _National Cancer Institute, National Institutes of Health, Rockville, MD_.

Background:

Pancreatic cancer (PC) is one of the deadliest malignancies. Lab based research suggests that two types of polyunsaturated fatty acids (PUFA), n-3 and n-6 PUFA, may have opposing effects on PC carcinogenesis. However, human evidence on the role of dietary PUFA intake in PC risk is inconsistent. Previous epidemiological studies on this topic were conducted primarily among Whites and included few individuals from low socioeconomic status (SES). The exposure patterns of n-3 and n-6 PUFA differ across racial groups, with African Americans (AAs) in the high end and Asians in the low end of range. To better characterize the associations of PUFA with PC risk, we conducted a nested case-control study focusing on understudied Asians and low SES AAs and Whites.

Methods:

We performed a nested case-control study within three population-based prospective cohort studies: the Shanghai Women's Health Study (SWHS), Shanghai Men's Health Study (SMHS), and Southern Community Cohort Study (SCCS). Dietary PUFA intakes and other variables were collected from the baseline dietary and other questionnaires. Participants were followed up through annual linkage to cancer registries. Controls were matched to cases on age, sex, and blood sample availability in SWHS/SMHS, and age, sex, race, and enrollment location in SCCS, using incidence density sampling (case-to-control ratio:1:4). Included in the study are 244 cases and 973 controls of Asian females in SWHS, 189 cases and 755 controls of Asian males in SMHS, and 307 cases and 1,228 controls in SCCS (including 219 cases and 876 controls of AAs). The PUFA intake was adjusted for total energy intake using the residual method. Conditional logistic regression analyses were performed to estimate the association of PC risk with dietary PUFA consumption after adjusting for potential covariates.

Results:

The average dietary intake (mean ± SD; g/day) of n-3 and n-6 PUFA were 1.0 ± 0.5 and 6.3 ± 3.1, 1.1 ± 0.5 and 7.2 ± 3.0, and 2.0 ± 1.2 and 19.3 ± 11.7 in the controls of SWHS, SMHS and SCCS, respectively. We observed that in Chinese men, high intakes of certain n-3 PUFA were significantly associated with increased PC risk. The adjusted odds ratio (OR) (95% confidence interval (CI)) comparing the highest versus lowest quartile of docosapentaenoic acid (DPA) was 1.79 (1.10-2.93), with a p for trend across quartiles of 0.02. For docosahexaenoic acid (DHA), the OR 4th vs. 1st quartile (95% CI) = 1.71 (1.06-2.78) (p for trend = 0.04). For marine n-3 PUFA, the OR 4th vs. 1st quartile (95% CI) = 1.69 (1.03-2.76) (p for trend = 0.04). There was no significant association for n-6 PUFA. No significant association was observed for n-3 or n-6 PUFA with PC risk in SWHS or SCCS.

Conclusions:

In a nested case-control study, we observed that in Chinese men, the dietary intake of overall marine n-3 PUFA, DPA, and DHA were positively associated with PC risk. Such associations were not observed in Chinese women or low SES AAs/Whites.

#640

Breastfeeding pattern and ovarian cancer risk: Results from the Ovarian Cancer Association Consortium.

Naoko Sasamoto,1 Ana Babic,2 Susan Jordan,3 Harvey Risch,4 Holly Harris,5 Mary Anne Rossing,5 Jennifer Doherty,6 Marc Goodman,7 Pamela Thompson,7 Susanne K. Kjær,8 Allan Jensen,8 Joellen Schildkraut,9 Linda Titus,10 Daniel Cramer,1 Elisa Bandera,11 Weiva Sieh,12 Valerie McGuire,13 Rebecca Sutphen,14 Celeste L. Pearce,15 Anna H. Wu,16 Malcolm Pike,17 Penelope Webb,3 Francesmary Modugno,18 Kathryn Terry1. 1 _Brigham and Women's Hospital, Boston, MA;_ 2 _Dana-Farber Cancer Institute, Boston, MA;_ 3 _QIMR Berghofer Medical Research Institute, Brisbane, Australia;_ 4 _Yale School of Public Health, New Haven, CT;_ 5 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 6 _Huntsman Cancer Institute, Salt Lake City, UT;_ 7 _Cedars-Sinai Medical Center, Los Angeles, CA;_ 8 _Danish Cancer Society Research Center, Copenhagen, Denmark;_ 9 _University of Virginia, Charlottesville, VA;_ 10 _Geisel School of Medicine at Dartmouth, Hanover, NH;_ 11 _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ;_ 12 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 13 _Stanford University School of Medicine, Stanford, CA;_ 14 _University of South Florida, Tampa, FL;_ 15 _University of Michigan, Ann Arbor, MI;_ 16 _University of Southern California Keck School of Medicine, Los Angeles, CA;_ 17 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 18 _University of Pittsburgh School of Medicine, Pittsburgh, PA_.

Background: Ovarian cancer is the fifth leading cause of cancer deaths in US women with few modifiable risk factors. Breastfeeding has been observed to be associated with reduced risk of ovarian cancer in multiple studies. However, prior studies reporting the association between breastfeeding pattern and ovarian cancer risk by histologic subtypes have reported inconsistent findings, likely due to small sample size and lack of details on breastfeeding patterns. The objective of this study is to evaluate the association between breastfeeding patterns and ovarian cancer risk by histologic subtypes using individual questionnaire data from the Ovarian Cancer Association Consortium (OCAC).

Method: We evaluated breastfeeding patterns in relation to invasive ovarian cancer risk in 9,807 cases and 12, 971 controls from 13 studies in the OCAC. We performed pooled unconditional logistic regression to evaluate the association between breastfeeding and ovarian cancer risk and used multinomial logistic regression to evaluate the association by histologic subtype. We restricted the analysis to parous women and adjusted for study site, age, body mass index, race, education, oral contraceptive use, number of full-term births, family history of ovarian cancer, and birth decade (to account for change in breastfeeding practice over time). We additionally adjusted for age at first and last births simultaneously when examining the association between first and last age at breastfeeding and ovarian cancer risk.

Results: Women who reported ever breastfeeding had a 24% decreased risk of overall invasive ovarian cancer compared to women who never breastfed (OR=0.76, 95%CI: 0.71-0.81) and this association did not differ significantly by histologic subtypes (p-heterogeneity=0.09). Considering only episodes when women breastfed, we observed lower risk of ovarian cancer in women who had longer average duration per episode compared to women who breastfed < 6 months on average per episode (OR=0.85, 95%CI: 0.77-0.93 for 6-12 months; OR=0.77, 95%CI: 0.66-0.89 for > 12 months; p-trend <0.0001). Older age at first and last breastfeeding was significantly associated with lower risk of ovarian cancer compared to women who never breastfed (OR=0.65, 95%CI: 0.58-0.73 for age >30 years at first breastfeeding; OR=0.60, 95%CI: 0.53-0.69 for age >35 years at last breastfeeding). Compared to women who never breastfed, women who last breastfed < 10 years ago had 54% reduction of risk (OR= 0.46, 95%CI: 0.39-0.56) while women who last breastfed ≥ 30 years ago had 19% reduction of risk (OR=0.81, 95%CI: 0.74-0.88).

Conclusion: Breastfeeding regardless of age or duration was associated with reduced risk of ovarian cancer. Our findings support the benefit of breastfeeding as modifiable risk factor for ovarian cancer regardless of histologic subtypes, which has the potential for reducing the incidence this deadly disease.

#641

Oral contraceptive use and risk of highly fatal ovarian cancer: Evidence from the Ovarian Cancer Association Consortium.

Jennifer M. Mongiovi,1 Janine M. Joseph,2 Albina N. Minlikeeva,2 Ahmad AlSulimani,2 Hani Almohanna,2 Kirsten B. Moysich2. 1 _University at Buffalo, Buffalo, NY;_ 2 _Roswell Park Comprehensive Care Center, Buffalo, NY_.

Objectives: While oral contraceptives (OC) use is associated with lower risk of ovarian cancer compared to non-users, its role in the etiology of aggressive and highly fatal disease has not been determined. Thus, we utilized data from the Ovarian Cancer Association Consortium (OCAC) to evaluate the association between pre-diagnostic OC use and highly fatal ovarian cancer.

Methods: Data were pooled from 20 case-control studies participating in OCAC to examine the association between oral contraceptive use and risk of highly fatal ovarian cancer; defined as dying within 12 or 18-months of diagnosis. Controls were frequency matched to cases (4:1) on five-year age categories, race, and study site. The study samples included 579 patients who died within 12 months of their diagnosis, matched to 2279 controls as well as 1294 patients who died within 18-months, matched to 5095 controls. Analyses were performed using unconditional logistic regression, and separate models were run for any use of OC and duration of OC use per five year increase. Race, education, prior hysterectomy or tubal ligation, parity, age at menarche, menopausal status, and smoking status were assessed as potential confounders of the association.

Results: After adjusting for age, site, and parity, any OC use was associated with a 46% reduction in the odds of death within 12-months after adjusting for age, site, and parity (OR=0.54, 95% CI: 0.43-0.68). There was also a significant trend of decreased odds with increased duration of OC use. Among those who used OC for over 10 years, a 66% reduction in the odds of death within 12-months was observed (OR=0.34, 95% CI: 0.23-0.49) while there was no significant benefit from use of OC for 1 year or less (OR=0.83, 95% CI: 0.60-1.14). Similar trends were observed among those who died within 18-months, although OC use appeared to be more protective among highly fatal cases. Use of OC was most protective for endometrioid subtypes among both those who died within 12-months (OR=0.43, 95% CI: 0.20-0.93) and 18-months (OR=0.39, 95% CI: 0.23-0.65). Conclusions: To our knowledge, this is the first large, multi-center study to investigate the association between OC use and risk of highly fatal ovarian cancer. These results are in line with current evidence that has demonstrated a substantial decrease in risk of ovarian cancer with OC use for five or more years. Future research should aim at understanding the mechanisms behind this association and identifying specific subpopulations that may benefit most from this chemopreventive strategy.

#642

Increasing BMI is associated with tumor volume and uterine weight in endometrial cancer.

Yingao Zhang, Nerlyne Desravines, Dominic T. Moore, Paola A. Gehrig, Leslie H. Clark, Victoria L. Bae-Jump. _University of North Carolina at Chapel Hill, Chapel Hill, NC_.

Obesity and diabetes is associated with increased risk and worse outcomes in endometrial cancer (EC). Our previous work has found that obesity leads to more aggressive tumor behavior and a doubling of tumor size in EC mouse models. Thus, we explored the association between endometrial tumor volume (TV) and uterine weight (UW) as a function of BMI in EC patients. We performed a retrospective review of EC patients diagnosed and treated from 2005-2010 at a single tertiary care center. Tumor histology, dimensions, stage, grade, and UW were extracted from surgical pathology reports. Patient age, BMI, and diabetic status were also collected. TV was determined using the formula TV=l*d*w*pi/6. Independent variables include obesity classification, BMI, and diabetic status. Univariable and multivariable regression models were used to assess strength of association. A total of 564 EC patients were evaluated, with either endometrioid (n=522) or serous (n=42) histologies (Table 1). Within the endometrioid subgroup, associations were noted for both increased TV and UW with obesity (TV p=0.02; UW p<0.001) and BMI (TV p=0.007; UW p<0.001). Within the serous subgroup, increased TV was associated with obesity and BMI (p<0.001; p=0.007), but UW was only associated with obesity (p=0.005) and not BMI (p=0.07). Controlling for stage and grade, multivariable models demonstrated similar patterns with obesity status and BMI within both subgroups. Multivariable analyses also demonstrated that having diabetes (n=160) was associated with both higher TV and UW in the endometrioid subgroup (TV p=0.004; UW p=0.02), but not in the serous subgroup (TV p=0.71; UW p=0.12). Obesity and increasing BMI appear to be associated with both increased TV and UW in EC patients, consistent with our previously described findings in murine models. Diabetes was also associated with higher TV and UW, but was only significant in patients with endometrioid ECs. Our results may partially account for the worse outcomes seen in obese EC patients.

Table 1

---

EC Histology | |  | Median | IQR

Endometrioid (n=522) | Uterine Weight (g) | Non-obese | 118.1 | 83.0-157.5

|  | Obese | 140.0 | 103.8-200.0

|

Tumor Volume (cm3) | Non-obese | 3.695 | 0.927-9.664

|  | Obese | 4.359 | 1.419-14.216

Serous (n=42) | Uterine Weight (g) | Non-obese | 116.6 | 67.4-166.6

|  | Obese | 189.8 | 127.6-252.6

|

Tumor Volume (cm3) | Non-obese | 3.249 | 0.601-9.382

|  | Obese | 23.824 | 7.356-59.494

#643

Poor oral hygiene is associated with HPV-negative and not HPV-positive oral cancer.

Jitesh B. Shewale,1 Robert Pickard,2 Maura L. Gillison1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _The Ohio State University Comprehensive Cancer Center, Columbus, OH_.

Background Oral health and hygiene have been implicated as risk factors for oral squamous cell carcinoma (i.e. oral cavity and oropharyngeal cancers, OSCC), and we recently reported that these risk factors also dramatically alter the oral microbiome. Here we performed a more detailed analysis of associations between oral health behaviors and OSCC in a matched case-control study. We stratified our analysis to compare and contrast associations with Human Papillomavirus (HPV)-positive versus HPV-negative OSCC.

Methods Cases were patients with newly diagnosed OSCC at The Ohio State University Comprehensive Cancer Center between 2011 and 2014. Controls were patients without cancer from the same outpatient clinic who were matched (2:1) to each case on age, sex, and race. Cases were stratified by tumor high-risk HPV status (HPV-positive, n=117; HPV-negative, n=114) based on E6 or E7 mRNA expression. Data on demographics; tobacco, alcohol, and sexual history; and oral health behaviors were collected using an audio computer-assisted self-interview. Conditional logistic regression was used to calculate odds ratios (OR) and 95% confidence intervals (CI), and oral health behaviors significant in univariable analysis were evaluated in separate multivariable models.

Results In univariable analysis, oral health behaviors such as low frequency of dental clinic visit, tooth brushing, and dental flossing; gum bleeding; tooth loss; and denture use significantly increased risk of HPV-negative OSCC, whereas none of these behaviors increased risk of HPV-positive OSCC. In univariable analysis, covariates associated with increased risk of HPV-negative OSCC were low education and income level, high lifetime tobacco pack-years (0, >0-<20, ≥20-<40, >40) and high lifetime heavy (≥14 drinks/week) drinking years (0, >0-<10, ≥10-<20, ≥20). In contrast, covariates associated with increased risk of HPV-positive OSCC were low education level, high lifetime marijuana use years (0, >0-<10, ≥10-<20, ≥20) and high no. of oral sex partners (0-2, 3-6, ≥7). After adjusting for covariates in multivariable analysis, a frequency of less than once per year dental clinic visits significantly increased risk of HPV-negative OSCC (OR = 2.28 [95% CI = 1.14-4.57]). Dental flossing less than once per day also increased risk of HPV-negative OSCC, overall (OR = 3.56 [95% CI = 1.04-12.14]) and in those without denture (OR = 4.34 [95% CI = 1.07-17.63]). In multivariable analysis, frequency of tooth brushing and gum bleeding, denture use, and tooth loss were not associated with HPV-negative OSCC.

Conclusion Poor oral health behaviors such as infrequent dental visits and dental flossing were independently associated with HPV-negative OSCC, but not with HPV-positive OSCC. These findings are consistent with a hypothesis that poor oral health behaviors alter oral microbial composition to promote chronic inflammation and the development of HPV-negative OSCC.

## PREVENTION RESEARCH

### Experimental Models and Biomarkers in Cancer Prevention Research

#644

Ancestral paternal obesity: Systemic versus local effects on offspring mammary tissue development and tumorigenesis.

Camile C. Fontelles, Anni Wärri, Raquel S. Da Cruz, Maria I. Cruz, Ersilia Barin, Sonia De Assis. _Georgetown University, Washington, DC_.

Background: Obesity and overweight are risk factors for breast cancer, particularly in the post-menopausal years. We recently reported that having a history of ancestral overweight from the paternal lineage is enough to increase breast cancer susceptibility in daughters. Using a mouse model, we demonstrated that paternal consumption of an obesity inducing diet (OID) altered mammary gland development, increased mammary carcinogenesis and disrupted metabolic parameters in the female offspring, compared to the female offspring of fathers who consumed only control (CO) diet. Given both the systemic and local the mammary tissue alterations observed, we aimed to investigate in more details why daughters of overweight fathers are at increased risk for breast cancer. More specifically, we used mammary gland transplantation experiments to study whether this ancestrally-induced breast cancer predisposition is linked to systemic factors and/or mammary tissue confined changes in daughters.

Material and methods: Male mice were exposed either to a control (CO) or to high-fat (OID) diet. CO and OID male mice were then mated with female mice fed CO diet exclusively. Female offspring of both CO and OID male mice underwent a mammary gland transplantation surgery. Briefly, female offspring had their mammary fat pad area between the nipple and the proximal lymph node excised. Afterwards, mammary tissue fragments (1 mm3) of a donor mouse, either CO or OID female offspring, were implanted into a pocket made in the cleared fat pad. The transplantations were performed from CO female offspring donors to both CO [CO(CO)] and OID [OID(CO)] female offspring hosts, as well as from OID female offspring donors to both CO [CO(OID)] and OID [OID(OID)] female offspring hosts. Approximately 10 weeks post-surgery, the mammary glands were collected, photographed and analyzed using ImageJ software to determine branching density, epithelial elongation and number of Terminal End Buds. Cell proliferation was assessed by ki-67 and cell apoptosis was measured on ImageJ software.

Results: Our preliminary data shows that CO(OID) had more (p≤0.05) articulated mammary branching morphogenesis than CO(CO). OID(CO) displayed marginal increase (p≤0.09) in the mammary gland area, as well as in the mammary ductal elongation, compared to CO(OID). Additionally, the number of apoptotic cells within mammary ducts and lobules is higher in CO(OID) compared to OID(CO). In contrast, the number of proliferating cells is higher in OID(CO) mammary gland compared to CO(OID).

Conclusion: Altogether, our findings suggest that daughters of overweight fathers have both systemic and mammary gland confined factors alterations that ultimately lead to higher breast cancer risk in adulthood. However, further data is needed to corroborate this hypothesis. Our ongoing mammary tumor transplantation studies should provide further insight.

#645

Pregnancy inhibits mammary carcinogenesis by altering the mammary epithelial and stromal interactions.

Ramadevi Subramani Reddy,1 Adriana Galvez,1 Diego A. Pedroza,1 Animesh Chatterjee,1 Courtney Perry,2 Elizabeth Penner,1 Rajkumar Lakshmanaswamy1. 1 _Texas Tech Univ. Health Sciences Ctr. El Paso, El Paso, TX;_ 2 _Texas Tech Univ. Health Sciences Ctr., El Paso, TX_.

A full-term pregnancy before the age of 20 is the only known natural phenomenon that can drastically reduce the risk of breast cancer in women of all ethnic backgrounds worldwide. This universal protective effect of early pregnancy is clearly of major consideration in devising prevention strategies against breast cancer. It is well known that parous subjects have an altered systemic hormonal milieu with a significant decrease in circulating GH and PRL. Determining how these hormonal alterations impact the mammary gland of early parous rats is expected to improve our understanding of the dynamics of mammary carcinogenesis. The inguinal mammary fat pad was cleared of the host mammary gland in 3-4-week-old rats. One group of rats were exposed to N-Methyl-N-Nitrosourea (MNU) at 7 weeks of age, and another group of rats were not exposed to MNU. A subset of rats from each of these experimental groups were mated at 9 weeks and allowed to go through a full-term pregnancy. All animals were terminated 6 weeks post-weaning. Mammary epithelial cells (MECs) were isolated using the collagenase cell dissociation procedure. The isolated MECs (5 × 105) were transplanted into the gland-free fat pads of respective hosts. In brief, we studied the following groups to understand the influence of early parity on epithelial-stromal interactions involved in mammary carcinogenesis: 1) carcinogen treated MECs from Age-matched nulliparous (AMNP) rats were transplanted into untreated parous rats; 2) carcinogen treated MECs from parous rats were transplanted into untreated AMNP rats; 3) untreated MECs from AMNP rats were transplanted into carcinogen treated parous rats and 4) untreated MECs from parous rats were transplanted into carcinogen treated AMNP rats. All experimental animals were palpated weekly for the presence of mammary cancers. The cancerous nature of the palpable tumors was confirmed by histopathological analysis. Digital images of immunocytochemistry result was analyzed using Image Pro Plus software. Carcinogenesis experiments were terminated nine months after carcinogen administration. Incidence, multiplicity, and latency of mammary cancers was determined for each group and analyzed for significance by the appropriate statistical test. Our data demonstrated that transplantation of MECs from AMNP rats into parous hosts did not develop into palpable tumors while MECs from parous rats when transplanted to AMNP hosts developed mammary tumors. Analysis of the mammary gland wholemounts and molecular markers clearly indicated that parity alters key signaling mechanisms and inhibits progression of mammary lesions. These findings suggests that the mammary epithelial-stromal interactions in the parous rats is altered making it resistant to mammary carcinogenesis.

#646

Pre conception paternal DDT exposure and programming of metabolic dysfunction and breast cancer risk in offspring.

Raquel Santana Da Cruz, Hong Cao, Camille Castilho Fontelles, Apsra Nasir, M Idalia Cruz, Sonia de Assis. _Georgetown Univ., Washington, DC_.

Background: Breast cancer is the most common cancer in American women, being the second leading cause of cancer-related death in this group. Exposure to environmental chemicals, such as the pesticide DDT (dichlorodiphenyltrichloroethane), has been long suspected to be a contributing factor in the development of metabolic disease and breast cancer. For instance, early life (in utero and early childhood) exposure to DDT has been associated with increased breast cancer risk in women, and these findings are supported by animal studies. While maternal DDT exposure in pregnancy has been linked to metabolic syndrome and breast cancer risk in offspring, the effects of paternal DDT exposure in male germ-line reprogramming and phenotypes in their progeny has not been investigated. Here, we evaluated the effects of pre-conception paternal exposure to DDT on offspring's metabolism and susceptibility of breast cancer, using a mouse model.

Methods: Male mice were exposed to DDT (1.7mg/kg body weight) by oral gavage for two weeks. At the end of this period, DDT and control-vehicle (CO) were housed with female mice, with free access to a standard chow diet, for three days. Pregnancy onset was assessed by the presence of a vaginal plug. The weight and number of pups per litter were determined two days after birth. Pups were weaned from mothers at 21 days of age, fed a standard chow diet for the extent of the study and weighed weekly. Mammary tumors were induced by subcutaneous administration of 15mg of medroxyprogesterone to six-week-old female offspring, followed by oral administration of 1mg 7,12-dimethylbenz[a]anthracene once-a-week for 3 weeks.

Results: Paternal DDT exposure reprogramed the sperm small non-coding RNA content. In line with that, DDT offspring had decreased birthweight (p=0.04) and weaning weight (p=0.01) compared to CO. Further, ancestral DDT exposure caused metabolic dysfunction in offspring (p<0.05) at 7 and 22 weeks of age. Further, female offspring of DDT exposed fathers showed alterations in mammary gland development with a non-significant increase in numbers of terminal end buds and lower rates of apoptosis. In line with that, our preliminary data suggest that ancestral paternal exposures to DDT increases breast cancer risk in offspring: Compared to CO, the DDT offspring presented a non-significant increase in mammary tumor incidence and shorter latency to first-tumor onset. In addition, tumor growth (volume) was significantly increased in DDT offspring compared to CO (p<0.01). In conclusion, our findings support a role for DDT exposure from the paternal lineage in metabolic dysfunction and increased breast cancer risk in their offspring.

#647

Immunoprevention of triple negative breast cancer by a TOP2A multi-peptide vaccine.

Sang Beom Lee,1 Jing Pan,1 Yueqiang Jiang,1 Katie Palen,1 Bryon Johnson,1 Romaine Ingrid Fernando,2 Shizuko Sei,2 Ronald Lubet,2 Ming You,1 Yian Wang1. 1 _Medical College of Wisconsin, Milwaukee, WI;_ 2 _National Cancer Institute, Division of Cancer Prevention, Bethesda, MD_.

Triple negative breast cancer (TNBC) comprises approximately 20% of all breast cancers, and it has a poor prognosis due to aggressive growth characteristics and lack of effective targeted therapies. Emerging data suggest vaccination as a promising approach to prevent TNBC as well as other cancer types, but appropriate vaccine targets need to be identified. In the present study, we employed in silico analyses to select genes that are uniquely expressed in TNBC. Topoisomerase 2 alpha (Top2A) was found to be highly expressed in human TNBC. Top2A is a key enzyme in DNA replication and has been identified as a target of anti-cancer therapy. Overexpression of Top2A was confirmed in human breast cancer tissue microarrays and in mouse TNBCs derived from C3(1)/TAg mice. Top2A-specific MHC class II epitopes with optimal binding affinity were identified using the "combined scoring system", based on their potential to elicit a Th1 immune response. Furthermore, all peptides were selected to have 100% amino acid sequence homology between humans and mice for potential clinical translation. Four Top2A peptides with strong immunogenic responses were selected by immunization of tumor naïve mice followed by IFN-γ-based ELISPOT assays. These peptides were combined to form a multi-peptide Top2A vaccine. The anti-tumor efficacy of the Top2A vaccine was tested in a preventive setting using a syngeneic TNBC model in C3(1)/TAg-REAR mice. Vaccinated animals demonstrated a significant reduction in tumor growth when compared to the adjuvant control with tumor size decreasing from 278.1 mm3 in adjuvant control to 150.4 mm3 in vaccinated group (p < 0.05). Taken together, our data clearly demonstrate that the Top2A multi-peptide vaccine is highly immunogenic and provides immunopreventive efficacy against TNBC in mice.

#648

**Effects of bazedoxifene and lapatinib in the prevention of estrogen receptor positive (ER** + **) and negative (ER** - **) mammary cancers.**

Clinton J. Grubbs,1 Altaf Mohammed,2 Shizuko Sei,2 Robert H. Shoemaker2. 1 _Univ. of Alabama Comp. Cancer Ctr., Birmingham, AL;_ 2 _National Cancer Institute, Rockville, MD_.

Bazedoxifene is a third-generation selective estrogen receptor modulator (SERM) that has been clinically approved in Europe for the prevention and treatment of postmenopausal osteoporosis. Our research previously showed that this agent (at a dose of 5 mg/kg diet) would decrease ER+ mammary cancers in rats by 73%. Lapatinib, an epidermal growth factor receptor (EGFR) inhibitor, is commonly used to treat patients with HER2+ breast cancer. We have also shown that the compound inhibits the development of ER+ mammary cancers in rats and ER- mammary cancers in mice at a dose of 75 mg/kg BW/day. The present studies were done to determine if lower doses and different treatment regimens designed to reduce possible toxicity would also be effective in preventing mammary cancers. Bazeodxifene was added directly to the diet (Teklad, 4% fat), while lapatinib was given by oral gavage. When evaluated in rats receiving methylnitrosourea (MNU), lapatinib given daily at doses of 75, 50 and 25 mg/kg BW/day reduced mammary cancer multiplicity by 93, 84 and 67%, respectively. Equivalent doses given 1x/week caused reductions of 87, 56, and 40%. When lapatinib was given to dimethylbenzanthracene (DMBA) treated MMTV/Neu mice at dose levels of 250, 125, and 63 mg/kg BW, 5x/week, mammary cancer multiplicity was reduced by 89, 60 and 80%, respectively. When equivalent doses were given 1x/week, the number of cancers were reduced by 82, 61, and 70%. Bazedoxifene given at 5.0 and 2.5 mg/kg diet to female MMTV/Neu mice also receiving DMBA caused reduction of mammary cancers by 73 and 51%. In a biomarker study lasting two weeks, bazedoxifene at the 5.0 mg/kg BW/day dose decreased the proliferation rate of normal mammary epithelial cells in MMTV/Neu mice by 60%. Additional studies to determine why this SERM is having a preventive effect against ER- mammary cancers are warranted. Studies to evaluate the efficacy of the combination of low doses of bazedoxifene and lapatinib in prevention of ER+ and ER- mammary cancers are in progress and will be presented.

Supported by the NCI contract number HHSN261201500036I, Task Order HHSN26100005.

#649

Alpha -Santalol, a component of sandalwood oil inhibits migration of breast cancer cells by targeting beta-catenin pathway.

Ajay Bommareddy,1 Kacey Knapp,1 Abigail Nemeth,1 Chandradhar Dwivedi2. 1 _Wilkes Univ., Wilkes Barre, PA;_ 2 _College of Pharmacy, SDSU, Brookings, SD_.

Alpha-santalol, a terpenoid found in sandalwood oil has been shown to inhibit breast cancer cell growth in vitro by inducing apoptosis but the mechanisms underlying the growth inhibitory effects of alpha-santalol are not fully understood. In this study, we demonstrate that alpha-santalol treatment targets Wnt/beta-catenin pathway to inhibit migration of cultured breast cancer cells. Exposure of MDA-MB 231 and MCF-7 cells to alpha-santalol resulted in a significant reduction in their migratory potential and wound healing ability. In addition alpha-santalol affected the localization of beta-catenin from cytosol to nucleus in MDA-MB 231 cells. In conclusion, the present study indicates that alpha-santalol inhibited migration of breast cancer cells may be mediated in part by targeting Wnt//β-catenin pathway, and that beta-catenin represents an important target of alpha-santalol's response for future pre-clinical studies.

#650

Gut microbiota population may be used to predict chemotherapeutic responsiveness in triple negative breast cancer.

Alaa Bawaneh, Adam S. Wilson, Kenysha YJ Clear, Akiko Chiba, Katherine L. Cook. _Wake Forest University, Winston Salem, NC_.

Triple negative breast cancer (TNBC) accounts for 15-20% of all breast cancers and predominately affects young and minority women. TNBC is characterized by a high recurrence rate, with about 34% of TNBC patients relapsing around 2.6 years. This type of breast cancer lacks targeted therapy options and is limited to chemotherapy treatment options. The purpose of our study was to determine whether gut microbiota populations correlate with chemotherapeutic responsiveness and may be a predictive biomarker of outcome. Moreover, we want to determine the impact of targeting the microbiome on chemotherapy efficacy. In this study, 8-week old female BALB/c mice were injected with TNBC 4T1-luciferase cells into their L4/5 mammary fat pad. Once tumors developed, mice were either untreated (control group), treated with 1 x weekly 2.5 mg/kg IV doxorubicin (DOX), or treated with doxorubicin + antibiotics (mixture of streptomycin, ampicillin, and colistin in the drinking water to ablate host bacterial populations). Tumors were collected at the end of the study. Fecal samples were collected at time point 0 weeks (T0; before therapy when tumors were approximately 100 mm3) and time point 4 weeks (T4; after treatment). We subdivided the doxorubicin treated group into DOX-responders (tumors stopped growing or shrank) or DOX-nonresponders (tumors continued to grow on treatment). Tumors from the DOX-responders and DOX + antibiotics groups were significantly smaller and mice from these groups displayed reduced lung weight, suggesting decreased lung metastatic burden. 16S-bacterial sequencing analysis was performed on breast tumor tissue and feces. We demonstrate that at T0, elevated fecal Ruminococcus correlates with DOX-nonresponsiveness. Furthermore, at T4, we show increased fecal abundance of Oscillospira and Bacteroidales are associated with better therapeutic outcome. Protein analysis of primary 4T1 breast tumor tissue indicate a significant elevation of apoptosis (cleaved caspase-3 protein levels) in DOX-responders and DOX + antibiotic treated mice. We also stained breast tumor tissue and lung tissue for bacteria to confirm presence of microbiota by immunohistochemistry. Taken together our data demonstrates that chemotherapy efficacy is modulated by the microbiome. Moreover, fecal microbiota populations could be used as a predictive biomarker of chemotherapeutic responsiveness and suggests that modulation of the gut microbiome through dietary or probiotic interventions may affect therapeutic outcomes.

#651

Analysis of the immune microenvironment to advance breast cancer risk prediction and prevention.

Doug Hinerfeld,1 Keith Knutson,2 Derek Radisky,2 E. Aubrey Thompson,2 Yan Asmann,2 Kim McCoy,2 Amy Degnim,3 Jodi Carter,3 Stacey Winham,3 Michele Cote,4 Jeroen van der Laak,5 Mark Sherman2. 1 _NanoString Technologies, Seattle, WA;_ 2 _Mayo Clinic, Jacksonville, FL;_ 3 _Mayo Clinic, Rochester, MN;_ 4 _Karmanos Cancer Institute, Detroit, MI;_ 5 _Radboud University Medical Center, Nijmegen, Netherlands_.

Existing risk models underperform among women who have undergone a benign breast disease (BBD) biopsy (> one million performed annually in the US) and with respect to estimating the risk of aggressive BCs. We've shown that molecular pathologic analysis of BBD biopsies can improve individual risk prediction, compared with standard risk models relying on self-reported factors, and provide insights into mechanisms mediating BC risk. BC risk factors such as obesity and ethanol use are proposed to increase production of cytokines and chemokines, resulting in chronic inflammation, and leading to production of DNA damaging free radicals and growth factors (e.g., VEGF, IGFs) that activate pro-carcinogenic pathways (e.g. NF-KB and JAK/STAT). The effects of most BC risk factors on immunity are poorly understood. Further, whereas experimental models implicate immunity throughout carcinogenesis, knowledge of immune markers and mechanisms related to BC development among women is limited. Therefore, we aim to define tissue immune cell content throughout BC development, and particularly at the earliest stages, to improve risk assessment and discover immune-based prevention strategies.

Our study combines novel resources and technologies to define the immune landscape in: 1) normal breast tissues in relation to BC risk factors, 2) BBD with respect to BC risk, and 3) invasive BC in relation to neoantigen expression and molecular subtype. We will perform NanoString GeoMxTM Digital Spatial Profiling (DSP) to quantitatively map expression of key immune proteins in healthy breast tissues donated to the Komen Tissue Bank (KTB) in relation to BC risk factors and in BBD biopsies from two cohorts to predict BC risk. We will re-analyze top prognostic markers in BBD biopsies using Vectra multiplex IF staining with machine learning algorithms to refine how immune cell content and organization impacts prognosis, and assess critical immune pathways related to BBD progression with the NanoString PanCancer IO 360 panel, which provides RNA expression of 770 immune genes (13 signatures). Finally, we will evaluate immune responses in BCs categorized for predicted neoantigen load and underlying mutation type with our novel bioinformatics pipeline that enables accurate prediction of MHC class I and class II missense, fusion and frameshift mutation neoantigens generated through faulty DNA repair, aberrant DNA and RNA splicing, insertions, and deletions.

Our project demonstrates a novel approach to defining the immune processes in early stages of BC development which may improve BC risk prediction and prevention. We present a plan to comprehensively analyze immunity throughout BC development and define changes that accompany transitions from normal to BBD and BBD to BC.

#652

Racial/ethnic differences in BRCA testing and test results among adult women.

Fangjian Guo, Erika L. Fuchs, Abbey B. Berenson, Yong-Fang Kuo. _University of Texas Medical Branch, Galveston, TX_.

Introduction: BRCA mutation testing has been recommended for targeted, individualized cancer prevention and treatment among women whose family histories or ethnic backgrounds are associated with increased risks for BRCA mutations. This study was to assess BRCA testing in different racial/ethnic groups in the US.

Methods: We used data from ClinformaticsTM Data Mart (CDM) Electronic Health Records. We included 134,827 women 18-65 years old with BRCA testing results from 1/1/2015-9/1/2017. BRCA testing rates and test result positive rates were calculated by race/ethnicity. Women with positive test results had a high risk for developing susceptible cancers - breast or ovarian cancer. As we only had data through 9/1/2017, we calculated annual test rates based on data in 2016.

Results: The BRCA mutation test rate in 2016 was highest among non-Hispanic Whites (582 per 100,000), while it was 313 per 100,000 in non-Hispanic Black women, 310 per 100,000 in Hispanic Women, and 281 per 100,000 in women of other races/ethnicities. Percentage of positive results was lowest among non-Hispanic White women tested (75.7%), and highest among non-Hispanic Black test takes (85.1%). After adjusting for age, region of residence, education, income, and family history of breast or ovarian cancer, adjusted odds ratio for having a positive test among non-Hispanic Black women tested vs. non-Hispanic White women tested was 1.71, 95% confidence interval 1.61-1.82, and adjusted odds ratio for Hispanics vs non-Hispanic Whites was 1.21, 95% confidence interval 1.13-1.31.

Conclusions: There are significant differences in test utilization and test results. Test utilization was highest in non-Hispanic Whites, who were also less likely to have a positive test result. Non-Hispanic Black women tested were more likely to have a positive test result, suggesting a more stringent test selection criteria for this underserved population.

#653

Ovarian cancer persister cells: 2D and 3D in-depth characterization and analysis.

Kathrin Boepple,1 Meng Dong,1 Andrea Gaissler,1 Bernd Winkler,2 Markus Kleih,1 Frank Essmann,1 Walter E. Aulitzky2. 1 _Dr.Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany;_ 2 _Robert Bosch Hospital, Stuttgart, Germany_.

Ovarian cancer is one of the five cancer types with highest incidence of death in women. Due to a lack of diagnostic options, timely detection of ovarian cancer is difficult and 75% of patients are diagnosed at a late stage. The standard therapy for ovarian cancer is resection of the affected tissue, followed by chemotherapy. However, patients frequently suffer from relapse of the disease, as some tumor cells persist chemotherapy and grow into a new, more aggressive tumor. To offer therapeutic approaches that prevent relapse by eradicating persister cells we aimed to identify the molecular mechanisms and determinants that are characteristic for persister cells. As initial cellular model system OVCAR-3 cells were used to select ovarian cancer persister cells. Selection was performed analogously to serum levels of chemotherapy patients, i.e. OVCAR-3 cells were incubated twice in the presence of 13 µM Cisplatin for 4 hours. Cisplatin-selection identified clones were collected and in further analyses compared to randomly collected untreated control clones. In order to account the native tumor microenvironment and heterogeneity within the tumor and among patients we cultured precision-cut tissue slices from primary ovarian tumor tissue with 280 µm thickness. Tumor tissue slices were cultured either under control conditions or exposed to 13 µM Cisplatin. Slices were further processed and cultured in Matrigel producing tumor-derived organoids, i.e. tumoroids. In contrast to OVCAR-3 control clones, the Cisplatin-selected OVCAR-3 persister cells showed filopodia-like structures. A scratch assay demonstrated enhanced motility of OVCAR-3-persister cells. Interestingly, cell death analysis (Annexin V) of Cisplatin exposed control and persister clonal cell lines showed the presence of surviving cells in both, persister and control groups. To identify the molecular determinants specific for persister cells we performed RNA sequencing. Data showed that, in line with initial results, the motility associated genes Snail and Vimentin are up regulated in persister clones. We successfully generated tumoroids from patient-derived ovarian cancer tissue slices. Tumoroids were cultured for more than two months and displayed a high viability. After exposure to Cisplatin less tumoroids generated from tumor slices were found. OVCAR-3-derived persister clones show morphological alterations (filopodia-like structures), enhanced motility and expression of motility associated genes. We expect the identification of genetic determinants for persister cell characteristics from comparison of RNAseq results of OVCAR-3 persister clones and tumor-derived organoids. The identification of genetic determinants will direct the development of therapeutics approaches targeting persister cells in ovarian cancer to prevent recurrence of tumors.

#654

Dietary interventions ameliorate infectious colitis through differential regulation of Lgr5.

Ishfaq Ahmed,1 Badal C. Roy,1 Rita-Marie T. Raach,2 Sharon J. Manley,1 Pugazhendhi Srinivasan,1 Prasad Dandawate,1 Afreen Sayed,1 Danny R. Welch,1 Shrikant Anant,1 Venkatesh Sampath,3 Shahid Umar1. 1 _Kansas University Medical Ctr., Kansas City, KS;_ 2 _University of Kansas, Kansas City, KS;_ 3 _Children's Mercy Hospital, Kansas City, KS_.

Background: During inflammatory bowel disease (IBD), the disruption of the epithelial barrier and translocation of bacteria drive inappropriate immune responses and unresolved inflammation. Rapid wound healing responses orchestrated by intestinal stem cells (ISCs) are central to inflammatory resolution and normalization of the mucosal barrier. Commensal microbiota ferment fibers to produce short-chain-fatty-acids (SCFAs) such as butyrate and its decrease has been linked to IBD. However, the mechanism through which SCFAs promote wound healing is poorly understood.

Aims: The present study was designed to test the hypothesis that dietary fibers/butyrate ameliorate infectious colitis by differentially regulating Lgr5-dependent crypt regeneration and wound healing.

Methods: Lgr5CreERT2/+; Rosa26LacZ reporter (Lgr5-R) mice and wild-type littermates were infected with Citrobacter rodentium (CR; 108 CFUs) and fed with either 6% Pectin (Pec) or 6% Tributyrin (Tbt) diets followed by euthanasia at 12 days post-infection. To partially deplete microbiota, mice were also given a cocktail of vancomycin (500mg/L), metronidazole (1g/L) and Ciprofloxacin (0.2g/L) for 10 days starting 3-days post-CR infection. Fresh feces pre- and post-infection/treatments were taken for 16S rDNA sequencing. Lineage tracing post-tamoxifen and Lgr5 promoter reporter activity assays were performed.

Results: Both Pec and Tbt reduced the severity of CR-induced colitis as was evidenced by increased body weight and colon length, reduced immune cell infiltration and increased mucus production compared with CR-infected but untreated mice. 16s rDNA sequencing revealed significant dysbiosis during CR infection with the dominance of Proteobacteria and loss of Firmicutes and Bacteroidetes. Both Pec and Tbt diets reduced the levels of Proteobacteria and restored Firmicutes and Bacteroidetes phyla to pre-infection levels. X-gal staining revealed that there was an expansion of LacZ-labeled Lgr5(+) stem cells in the colons of CR infected Lgr5-R mice when subjected to dietary intervention via Pectin and Tributyrin as sources of butyrate compared with controls. Interestingly, Pec-induced Lgr5 regulation was dependent upon the presence of gut microbiota as antibiotics treatment reduced Pec-induced Lgr5 expansion and the extent of crypt regeneration. Tbt-treatment, on the other hand, regulated Lgr5 independently of the microbiota. Butyrate, in a dose-dependent (1-10mM) manner, increased Lgr5 promoter reporter activity. Docking studies further revealed butyrate's ability to efficiently bind Lgr5 with a -4.0 Kcal/mol binding energy. The cellular thermal shift assay showed that butyrate was indeed able to bind Lgr5.

Conclusions: Thus, dietary interventions, by altering the gut microbiota, can differentially regulate Lgr5's ability to orchestrate crypt regeneration and wound healing to ameliorate colitis.

#655

Clinicopathological and genetic features of colorectal cancer in Algerian patients.

Farid Cherbal,1 Asma Lamia Boumehdi,1 Feriel Khider,1 Fatma Narimane Nouredine,1 Karim Layaida,2 Hassen Mahfouf,3 Mustapha Maaoui3. 1 _Univ. of Sciences and Technology 'HB', Algiers, Algeria;_ 2 _University Hospital Mustapha Bacha, Algiers, Algeria;_ 3 _School of Medicine, University of Algiers, Algiers, Algeria_.

Background Colorectal carcinoma is the third most common form of cancer worldwide. It is the second most common cancer in men and women in Algeria.The aim of this study was to determine clinicopathological and genetic features of colorectal cancer in Algerian patients. For the genetic study, we focused on the two major forms of hereditary colorectal cancer, Familial Adenomatous Polyposis (FAP) and Lynch Syndrome (LS), respectively.

Materials and Methods The study population included 366 patients diagnosed with colorectal cancer (CRC). Colorectal carcinoma was diagnosed between January 2017 to March 2018. Clinical and tumor data: age at diagnosis, site of tumor and TNM stage were collected from the medical records of the patients. We screened by PCR-direct sequencing exon 15 of APC gene in 30 patients with a strong family history of classic FAP. 30 patients with Lynch syndrome who fulfilled Amsterdam criteria were screened by PCR- direct sequencing for germline mutations in the MMR genes: MLH1 (exons 9,10,13,16), MSH2 (exons 5,6,7,12) and MSH6 (exon 4). Two FAP patients and two LS patients were screened by NGS using a cancer panel of 30 hereditary cancer genes (Color Genomics, California, USA).

Results In this retrospective study, there were 183 women (50%) and 183 men (50%).The median age at diagnosis cancer was 58 years old. The highest proportions of CRC were observed for the age groups 60-69, 50-59 and 40-49 years, 25.4%, 23.49%, 20.49% respectively. We noticed that colorectal tumors were not uniformly distributed through the large bowel. Rectum was the most common site (26.8%) followed by right colon (20.5%), sigmoid colon (18.9%) left colon (13.7%) and caecum (7.4%). Our results showed that 75.95% of the CRC patients were diagnosed at TNM stage II (9.02%), stage III (48.63%) and stage IV (18.31%), respectively. The analysis of DNA samples of FAP patients revealed that 6 patients carried pathogenic variants in APC gene. 3 distinct pathogenic variants: c.1605dupT ( one patient), c.3784delT (2 related patients), and c.3927_3931delAAAGA (3 unrelated patients) were identified in APC gene. Moreover, three distinct germline mutations: c.55_63delATCGCGGCGinsT in exon 1 of MLH1 (one patient), c.1165C>T in exon 7 of MSH2 ( one patient) and the Newfoundland mutation c.942 +3A>T in exon 5 of MSH2 (3 unrelated patients) were detected in 5 Lynch syndrome families.The APC c.1605dupT and c.3784delT and the MLH1 c.55_63delATCGCGGCGinsT new variants have never been reported in LOVD, UMD and ClinVar databases and could be specific of Algerian FAP patients and Lynch syndrome patients, respectively. Haplotype analysis of the APC c.3927_3931delAAAGA and the c.942+3A>T MSH2 variants will establish their genetic origin in Algeria.

Conclusions The accumulative knowledge about clinicopathological and genetic characteristics of colorectal cancer in Algerian patients will impact on clinical management in the areas of both prevention and treatment.

#656

High resolution metabolomics of aspirin treatment in colon tissue and adenoma risk: Results from a randomized clinical trial.

Elizabeth L. Barry,1 Karan Uppal,2 Chunyu Ma,2 Dean P. Jones,2 John A. Baron,1 Veronika Fedirko2. 1 _Dartmouth Geisel School of Medicine, Lebanon, NH;_ 2 _Emory University, Atlanta, GA_.

Although substantial evidence from cell and animal studies, epidemiological studies, and clinical trials supports the chemopreventive effects of aspirin, especially for colorectal cancer, the molecular mechanisms are uncertain. Aspirin incorporates two bioactive components in one molecule, a reactive acetyl moiety and a salicylate group, and is well known for its pleotropic effects. Its best-characterized pharmacologic activity is the irreversible acetylation of the cyclooxygenases (COX-1 and COX-2). However, there is evidence for numerous COX-independent mechanisms that could also modify colorectal carcinogenesis. This study applied an untargeted, discovery-based approach (metabolomics) to elucidate the effects of aspirin on low molecular weight molecules in human colon tissue and assess their impact on colorectal carcinogenesis. We utilized normal mucosal tissue biopsies collected at colonoscopy after about 3 years of treatment from a sub-set of N=325 participants in the Aspirin/Folate Polyp Prevention Study, a randomized, placebo-controlled trial of aspirin (81 or 325 mg/day) for the prevention of colorectal adenomas. The global metabolic effects of aspirin were assessed using a high-resolution Thermo Fusion mass spectrometer coupled with dual chromatography and dual ionization (HILIC positive and C18 negative electrospray ionization). Multivariable linear regression was used to identify metabolic features associated with aspirin treatment adjusting for age, sex and race. Multivariable Poisson regression was used to assess associations with adenoma outcomes of metabolic features associated with aspirin treatment. Products of aspirin metabolism (salicylate, salicyluric acid) were identified and statistically significantly increased in post treatment compared to baseline plasma samples confirming aspirin treatment status. After quality control exclusions, N=4,879 and N=5,390 features were included in analyses from the C18 and HILIC columns, respectively, of which N=244 and N=222 were associated with aspirin treatment at a raw P<0.05. At the pathway level, aspirin treatment was most strongly associated with perturbations in prostaglandin/arachidonic acid metabolism and the carnitine shuttle, which is involved in energy metabolism from fatty acids. At the metabolite level, only one feature (tentatively identified as a pterosin compound) was statistically significantly associated with aspirin treatment after FDR adjustment, but it was not associated with adenoma risk. Top features associated with aspirin treatment that were associated with adenoma outcomes included creatinine but are mostly of unknown identity at this time. In conclusion, a non-targeted high-resolution metabolomics approach has the potential to identify key downstream effects of aspirin involved in colorectal chemoprevention.

#657

The comparison of calcium-induced differentiation in colonoid cultures derived from the normal human colon tissue and colonic adenomas.

Muhammad N. Aslam, Shannon D. McClintock, Areeba H. Rizvi, Durga Attili, Shailja Pandya, Humza Rehman, Daniyal M. Nadeem, D. Kim Turgeon, James Varani. _University of Michigan, Ann Arbor, MI_.

Introduction: Previous ex vivo studies with colon cancer cell lines and human colon adenoma derived colonoid cultures have demonstrated that calcium and Aquamin (a calcium-rich, multi-mineral natural product) suppressed proliferation and induced differentiation. Aquamin was effective at a lower dose as compared to calcium.

Methods: Colonoid cultures were established from histologically-normal human colon tissue and maintained in a low-calcium (0.25 mM) medium (control) or supplemented with an amount of calcium or Aquamin (1.5 mM), that was shown previously to induce differentiation in human adenoma colonoids. Quantitative immunohistochemistry and differential proteomic expression were assessed after 4 weeks of in vitro intervention with calcium or Aquamin, as performed in the past with adenoma-derived cultures.

Results: The normal colonoids underwent differentiation as indicated by morphological features, a low proliferative index and high-level expression of cytokeratin 20 (CK20) under control conditions. Only modest additional changes were seen in these parameters with either calcium alone or Aquamin (providing up to 3.0 mM calcium). This was an altered response as compared to adenoma-derived colonoids which differentiated in response to calcium or Aquamin at 1.5mM calcium. In spite of this, proteomic analysis and immunohistochemistry of normal colonoids revealed that both interventions induced strong up-regulation of several proteins involved in cell-cell and cell-matrix interaction and in barrier formation. Among these were cadherins, keratins, claudins, CAECAMS, laminin subunits, and desmosomal proteins. Transmission electron microscopy showed an increase in desmosomes in response to intervention.

Conclusion: These findings suggest that normal colonoids were inherently more calcium-sensitive than adenoma colonoids and were readily differentiated at a lower calcium concentration as present in the control.

Table: Quantitative expression of immunohistochemical markers in colonoid cultures

---

|

Human Colonic Adenoma (n=3) | Normal Colon (n=3)

IHC Markers | Control | Calcium 1.5mM | Aquamin 1.5mM | Control | Calcium 1.5mM | Aquamin 1.5mM

Ki67 | 84.8±9.2 | 50.1±14.8* | 41.8±23.6* | 33.2±26.1 | 27.5±26.8 | 27.1±24.3

CK20 | 0.23±0.13 | 0.54±0.13* | 0.53±0.10* | 0.78±0.19 | 0.73±0.22 | 0.76±0.20

E-Cadherin | 0.44±0.06 | 0.70±0.09* | 0.70±0.07* | 0.49±0.10 | 0.61±0.09* | 0.58±0.08*

*reflects significance as compared to the corresponding control (p<0.05).

Each condition has 20-30 crypts in colonoid culture per adenoma or normal colon.

#658

Antiproliferative effects of type II diabetes agents in Fanconi derived oral squamous carcinoma.

Kim Miller, Beverly Wuertz, David Potter, Frank G. Ondrey. _Univ. of Minnesota, Minneapolis, MN_.

Fanconi anemia is a rare disease characterized by bone marrow failure requiring transplantation at a young age. Patients with Fanconi Anemia are at risk for the development of squamous cell carcinomas of the skin and mucosal surfaces at risks several hundred fold higher than control populations and these malignancies can pursue an aggressive, often fatal, course. Our group has been interested in repurposing Type 2 diabetes agents for oral cancer prevention and treatment. Presently, we examined the effects of the thiazolidinedione drug, pioglitazone, and an experimental biguanide, N1-hexyl-N5-benzyl-biguanide (HBB), on the proliferation of Fanconi-derived oral carcinoma cells. We incubated MOP FA1,a, VU-1131, and VU-1365 b, and C squamous cell carcinoma cell lines with vehicle controls or the thiazolidinedione pioglitazone or biguanide (HBB). We found dose-dependent decreases in cellular proliferation in all three Fanconi-derived cell lines at physiologically relevant concentrations (10 - 50 µM) after 1 to 3 days in several experiments. The effects were not due to simple cytotoxicity as judged by trypan blue staining. In other non-squamous cell lines of cervical and oral cavity origins we have previously discovered similar effects. These agents affect PPAR gamma associated pathways (pioglitazone) or mTOR and mitochondrial glucose metaboliscm pathways (HBB). Since Fanconi anemia patients are highly susceptible to DNA damaging effects of cytotoxic chemotherapy, these classes of agents might be attractive in Fanconi Anemia oral cancer treatment or prevention. Metformin, another biguanide, is currently postulated to delay bone marrow failure in the Fanconi population and is currently in use in an early phase clinical trial. Our results may, therefore, be attratciveattractive for oral cancer prevention and treatment in this population, as they are well tolerated. Further examination of molecular mechanisms will likely generate putative pharmacodynamics markers for future clinical trials.

#659

Expression patterns of NF-κB in inflammatory oral potentially malignant lesions.

Iris Lin,1 Lewei Zhang,1 Miriam Rosin,2 Leigha Rock,1 Denise Laronde1. 1 _University of British Columbia, Vancouver, British Columbia, Canada;_ 2 _Simon Fraser University, Burnaby, British Columbia, Canada_.

Introduction: Oral lichen planus (OLP), a common chronic autoimmune inflammatory condition, is recognized as a potentially malignant condition by the World Health Organization. However, some argue that only OLP with epithelial dysplasia - termed lichenoid dysplasia (LD) - have malignant potential. As research continues to characterize the immune microenvironment of OLP, there is a need to elucidate factors favorable for malignant change. Recent research has demonstrated that activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is key to cancer development. This transcription factor has been extensively explored in inflammation-associated cancers, such as colon and gastric cancers. There is a need to investigate these factors in oral potentially malignant lesions (OPML), including OPL. In this study, we aim to compare clinical and risk habit differences between OLP and LD, and determine if NF-κB expression is associated with histological and clinical features of OPML indicative of cancer risk.

Methods: Clinical, demographic, and histological data have been collected from the Oral Cancer Prediction Longitudinal (OCPL) study and the CoPath Vancouver Coastal Health Database. Patients with a primary diagnosis of OLP or low-grade LD were eligible to participate. Patients with previous history of head and neck cancer, or who have less than one year of follow-up are excluded from enrollment. Demographic, risk habit and clinical information was collected.For completed cases, immunohistochemistry (IHC) has been performed on formalin-fixed and paraffin-embedded tissue. Nuclear reactivity of NF-κB in the epithelium was counted in 10 high-power fields, and cytoplasmic positivity classified into 4 categories. Chi-squared tests were performed on categorical demographic and risk habit data.

Results: To date, 51 participants have been recruited into this ongoing study: 37 with OLP and 14 cases of LD. There is no significant difference in gender and age between groups (p=0.297, p=0.120, respectively). Ever smokers and lesion location at a high-risk site were significantly associated with a diagnosis of LD compared to OLP (p=0.002, p<0.001, respectively). Preliminary IHC results show strong NF-κB positivity in the sub-epithelial lymphocytic infiltrate and strong to moderate cytoplasmic reactivity in the epithelium. Nuclear reactivity appears to increase from lichen planus to dysplasia.

Conclusion: Patients with LD were more apt to be smokers, and more often presented with lesions at a high-risk site compared to those with OLP. Strong NF‐κB cytoplasmic positivity in OLP, especially adjacent to areas with intense cytotoxic inflammatory infiltrate, reinforces the prominent role of NF-κB in inflammation.

#660

The molecular characterization of a murine lung premalignancy model for lung cancer interception.

Sarah A. Mazzilli,1 Kahkeshan Hijazi,1 Riley Pihl,1 Anna Belkina,1 Xiaohui Zhang,1 Gang Liu,1 Marc Lenburg,1 Christopher Stevenson,2 Avrum Spira,1 Jennifer Beane3. 1 _Boston University, Boston, MA;_ 2 _Janssen Pharmaceuticals LLC, PA;_ 3 _Jennifer Beane, Boston, MA_.

There is a critical need to identify strategies for lung cancer prevention, as it remains the leading cause of cancer-related death in the US. A critical barrier to developing new approaches to prevent lung cancer is the lack of understanding of key molecular and cellular changes that lead to cancer initiation and progression. Our group has initiated a Pre-Cancer Genome Atlas (PCGA) to define the early molecular alterations that drive premalignant progression of lung cancer. This work has identified transcriptomic alterations in immune pathways associated with progression of bronchial dysplasia (BD) that werefurther validated by immunofluorescent staining showing shifts in immune populations in adjacent bronchial biopsy sections. A challenge to further explore interactions between epithelial and immune cells driving lesion progression is the lack of in vivo models of premalignant transitions ofBD that progress into lung squamous cell carcinoma (LUSC). However, in the NTCU mouse model of LUSC, mice treated with carcinogen develop lesions histologically comparable to human BD. The NTCU-induced murine model serves to act as a robust model for LUSC premalignancy, however, the molecular and cellular alterations driving disease in this model have not been well characterized. This study seeks to investigate the molecular pathogenesis of NTCU-induced BD progression to carcinoma to that of human BD progression to determine the pre-clinical utility of the model for intervention studies.

RNA was isolated from normal, dysplastic and tumor tissue from NTCU-treated mice and sequenced (n=22). Gene expression alterations associated with histological grade were identified using regression models. Concordance between the murine and human data was investigated using GSEA (Gene Set Enrichment Analysis). We identified 503 genes differentially expressed with increasing histological severity in the mouse dysplasia (FDR<0.05) and involved in cancer signaling and immune regulation. We also identified significant concordance of gene expression differences associated with histology between mouse and human dysplastic lesions (FDR<0.05). Furthermore, fluorescent cytometry analysis of lung leucocytes detected immune cell infiltration associated with increasing dose anddisease progression (n=30), and identified a dose-dependent suppression of several lymphoid populations and increase in myeloid populations frequencies associated with disease progression. These observations were concordant with transcriptional and histologic findings in both mouse and human samples. In summary, our data suggest molecular and cellular concordance between human and murine lesions that precede the development of LUSC, and suggest NTCU-induced BD progression to LUSC be a relevant pre-clinical model for testing targeted immunoprevention strategies.

#661

Adipose specific monocyte chemotactic protein-1 knockdown reduces pulmonary metastasis of Lewis lung carcinoma in mice.

Sneha Sundaram, Lin Yan. _Grand Forks Human Nutrition Research Center, ARS, USDA, Grand Forks, ND_.

Adipose accumulation is a strong indicator of detrimental health outcomes because adipose tissue produces pro-inflammatory adipokines that participate in many pathological processes including cancer. Monocyte chemotactic protein-1 (MCP-1) is a potent adipokine and body adiposity is positively correlated with adipose MCP-1 expression. This study tested the hypothesis that adipose-produced MCP-1 contributes to metastasis. In a metastasis model of Lewis lung carcinoma (LLC), male adipose MCP-1 knockdown (MCP-1-/-) and wild-type (WT) mice were fed the standard AIN93G diet or a high-fat diet (HFD) containing 16% or 45% of energy from soybean oil. The duration of experimental feeding was 11 weeks. The expression of MCP-1 in epididymal adipose tissue was analyzed by quantitative real-time PCR and ELISA. Pulmonary metastases from a primary tumor, established by subcutaneous injection of LLC cells, were the primary endpoints. There was no significant difference in fat body mass between MCP-1-/- and WT mice fed the same diet. There were no differences in energy intake among the four groups. The HFD increased and adipose MCP-1 knockdown decreased MCP-1 mRNA and protein in adipose tissue compared to their respective controls. The HFD increased the number of metastases formed in the lungs in WT mice. The number of metastasis was lower in MCP-1-/- mice fed the HFD than in WT mice fed the HFD but was higher compared to WT mice fed the AIN93G diet. The volume of metastases was smaller in MCP-1-/- mice than in WT mice, regardless of diet. Adipose MCP-1-/- mice, compared to WT mice, exhibited lower concentrations of insulin, pro-inflammatory adipokines (leptin, plasminogen activator inhibitor-1, and resistin), and angiogenic markers (vascular endothelial growth factor, hepatic growth factor, and angiopoietin-2) in plasma. These results indicate that adipose MCP-1 knockdown may lead to the down-regulation of inflammatory and angiogenic pathways during malignant spread. We conclude that adipose MCP-1 deficiency attenuates HFD-enhanced pulmonary metastasis of LLC and supports our hypothesis that adipose-produced MCP-1 contributes to malignant spread.

#662

Impact of chemotherapy on the intestinal microbiome and epithelial barrier in PDX models of lung cancer.

Cindy Pensec,1 Dominique Guenot,2 Loreley Calvet,3 Caroline Mignard,4 Olivier Duchamp,4 Thomas Carton,1 Sébastien Leuillet,1 Hervé M. Blottière,5 Françoise Le Vacon1. 1 _Biofortis Mérieux NutriSciences, Saint Herblain, France;_ 2 _Université de Strasbourg (Unistra), EA 3430 U1123, Progression tumorale et microenvironnement, Strasbourg, France;_ 3 _Sanofi, Paris, France;_ 4 _OncoDesign, Dijon, France;_ 5 _MetaGenoPolis, INRA, Université Paris-Saclay, Jouy-en-Josas, France_.

Introduction In the field of lung cancer treatments, significant progresses have been realized during last decade, such as targeted therapies and immunotherapies. Nevertheless, chemotherapy remains the gold standard for cancer. Pemetrexed is a chemotherapeutic agent commonly used in advanced lung cancer. This drug has a broad-spectrum effect that can induce significant side effects in patients. However, the impact of pemetrexed on gut microbiota and gastrointestinal inflammation in PDX mice remains unknown, although the role of the microbiota in carcinogenesis and modulation of efficacy or toxicity of chemotherapy agents has been demonstrated. The aim of this new study was to explore the impact of pemetrexed on the gut microbiota and the integrity of intestinal epithelial barrier and inflammation markers of PDX models following treatment.

Methods Upon establishment of the PDX model, mice were treated with pemetrexed for 2 weeks. Stool specimens were collected at 3 time-points: before, after and one week after treatment. Gut microbiota composition was studied by 16S rRNA gene sequencing. The colon integrity of the epithelial barrier was evaluated by a histological examination, a permeability measurement and a selected cytokines expression. In parallel, body weight was recorded and tissues were sampled for assessment of toxicity and inflammation.

Results Pemetrexed induced a significant body weight loss after each treatment cycle reflecting toxicity as known in clinical results. We have found that pemetrexed and tumor induced several modifications on microbiota composition, and the more important perturbation was the significant increase of the relative abundance of Enterobacteriaceae. A significant alteration of epithelial barrier integrity associated with early inflammation and infiltration of leukocytes into mucosal tissues was observed following treatment. Moreover, we have shown that pemetrexed effect on the microbiota was reproducible on several models of lung PDX models of lung carcinoma, and that dysbiosis seem proportional to the effectiveness of chemotherapy.

Conclusion This work is a preliminary approach, that confirms the relationship between microbiota and chemotherapy. A better understanding of gut microbiota alterations induced by chemotherapy could help reduce side effects. It is essential to expand our knowledge about the chemotherapy impact on microbiota in order to minimize the side effects, avoid infection complications, and improve therapy efficiency.

#663

Diagnostic screen for lung cancer using high throughput metabolomics screen.

Robert S. Plumb, Lee Gethings, Andrew Peck. _Waters Corporation - Milford, MA, Milford, MA_.

Lung cancer is one of the most common cancers in the world. It is a leading cause of cancer death in men and women in the United States, cigarette smoking being leading cause of lung cancer. There are two main types of lung cancer small cell and non-small cell lung cancer (NSCLC). The most common type being NSCLC which accounts for 80-85% of all reported cases. The ability to rapidly detect / diagnose lung cancer at an early stage is critical to successful treatment selection and patient survival.

The use of metabolic profiling in biomedical applications metabolic profiling is being deployed as a method for finding novel, mechanistic, biomarkers of disease with obvious potential for improving diagnosis, and patient stratification. Here we present a rapid, simple and reliable high throughput targeted LC/MS single platform, for the quantification/monitoring of small molecule metabolites, lipids and peptides. The methodology employs a single set of LC/MS conditions which facilitate TCA cycle, bile acids, biogenic amine, free fatty acids, acyl carnitines, lipids and 100 protein panel without need for user intervention.

Plasma from pilot cohort of lung cancer a health control samples were evaluated using this new methodology. The methodology showed excellent reproducibility and accuracy. The peptide analysis showed that 10 peptides were shown to be markers of lung cancer, these are listed in Table 1. Acylcarnitines were quantified over a range of 5 - 625ng/mL data showed that the C14:2 tetradecadienoyl carnitine and C16:1 palmitoleoyl carnitine were elevated in lung cancer samples whereas the C8:1 octenoyl carnitine level was reduced in the lung cancer samples . Although deoxycholic acid and chendeoxycholic acid appeared to be reduced in the lung cancer samples this was determined to be not statistically relevant. A total of 29 amino acids were measured during the analysis. Using t-test with p-value FDR cutoff adjusted to 0.01 the data clearly showed that sarcosine was highly over-expressed in lung cancer samples.

Table 1

|

---|---

P02749 | Apolipoprotein H

P19652 | Alpha-1-acid glycoprotein 2

P68871 | Hemoglobin subunit beta

P02671 | Fibrogen alpha chain

P02656 | Apolipoprotein C-III

P02750 | Leucine-rich alpha 2-glycoprotein (LRG)

P00738 | Haptoglobin

P02748 | Complement component C9

P02763 | Alpha-1-acid glycoprotein 1

P01011 | Alpha-1-antichymotrypsin

#664

Microbial and inflammatory response to electronic cigarette and cigarette use.

Kevin L. Ying,1 Min-Ae Song,1 Daniel Y. Weng,1 Quentin A. Nickerson,1 Joseph P. McElroy,1 Theodore M. Brasky,1 Mark D. Wewers,1 Ewy Mathé,1 Jo L. Freudenheim,2 Peter G. Shields1. 1 _Ohio State Univ., Columbus, OH;_ 2 _University at Buffalo, Buffalo, NY_.

Background: As electronic cigarette (e-cig) use increases in the US, investigation of their effects are critical. Alteration of the lung microbiome, oral microbiome, and inflammation are well established effects of cigarettes; however the effects of e-cigs are yet unknown. Individuals with smoking-related lung disease have alterations in their lung microbiome compared to healthy individuals and their lung microbiomes appear more similar to their oral microbiomes when compared to healthy individuals. To our knowledge, only one study has examined smoking tobacco's concurrent effects in the oral and lung microbiome and none have examined e-cig use. Further, none have studied e-cigs' effect on the lung microbiome and inflammation. We hypothesized that e-cig use would affect the lung microbiome, and that the effects are different from smokers and never-smokers; alteration of the lung microbiome will also affect inflammatory gene expression in the lungs.

Methods: A cross-sectional study of bronchoscopy with bronchoalveolar lavage (BAL) of 10 never-smokers, 8 cigarette smokers, and 10 e-cig users was conducted. RNA was extracted from BAL samples for total transcriptome RNA-seq analysis, allowing measurement of the microbiome and human gene expression. Differences in the microbiome by smoking status were determined by the Kruskal-Wallis test. Pairwise Wilcoxon rank sum tests with Holm correction was used. Effect size (fold change >1.5) and adjusted P-value cutoffs (<0.05) were used to identify microbes of potential interest. The limma-voom package in R was used to determine associations with human gene expression.

Results: We identified 53 differentially-abundant bacterial species in BAL samples by smoking group. Among them, the majority were less abundant in the lung of smokers and ~20 are normally found in the oral microbiome. While there were significant differences in differentially-abundant microbes between e-cig users and smokers and between smokers and never-smokers, the microbiome of e-cig users did not differ from that of never-smokers. In preliminary analyses of gene expression, there were 2,400 differentially-expressed human genes among the three groups, of which 58 are inflammatory pathway genes.

Conclusion: The majority of differentially-abundant microbes observed by smoking group are largely due to smokers. The microbiome of e-cig users is more similar to that of never-smokers. Interestingly, nearly half of microbes that are altered in the lung microbiome due to smoking use are bacterial species normally found in the oral microbiome. These findings suggest that the alterations in the oral microbiome associated with smoking cigarettes may also be reflected in the lung microbiome.

#665

Cancer neoantigen and autoantibody discovery with PEP functional proteomics platform.

Xing Wang, Trevor Landon. _Array Bridge Inc, St. Louis, MO_.

Proteins play essential roles in numerous biological processes and being able to pinpoint functional differences between cell or tissue samples can greatly aid in understanding disease processes and metabolic changes. The PEP technology allows systematic analysis of protein functions within a proteome. Hundreds of functional proteins can be separated and functionally assayed to generate a comprehensive three-dimensional landscape of protein families such as protein kinases, phosphatases, proteinases and oxido-reductases. This information can then be integrated into other genomic and proteomic knowledge bases to provide further insight of important biological processes such as cancer development and aging. Array Bridge has developed a 2-Dimensional separation technology called PEP (Protein Elution Plate) that can separate proteins into two-dimensional display and efficiently recover all the separated proteins into 384-microplates that can be probed in a standard ELISA format with high throughput. Our initial studies using breast cancer cell line proteins indicated that the PEP technology can be used to separate the proteins efficiently and probed with plasma from cancer patient for autoantibody discovery. Using the autoantibodies exist in the cancer patient, strong signals can be detected from a subset of proteins from the breast cancer cell line. We also use this platform to separate proteins from the cancer cell lines and probed with the plasma from the same cancer patient to identify the neoantigen candidates with mass spectrometry identification, the identified neoantigens can be used for the development of personalized immunotherapy such as CAR-T and other treatments.

#666

Determination of biomarkers for neuronal damage in bortezomib (BTZ) induced peripheral neuropathy (BIPN).

Paramjot Kaur, Theodore L. Mathuram, Vineela Nagamalla, Jay Patel, Thiagarajan Venkatesan, Alicia Fernandez-Fernandez, M. Samuel Cheng, Appu Rathinavelu. _Nova Southeastern Univ., Davie, FL_.

Multiple myeloma (MM) is one of the most commonly seen hematological cancers involving plasma cells and bone marrow. Anti-myeloma chemotherapeutic agent BTZ, a proteasome inhibitor, has been established as the foremost cause of BIPN among the MM patients. Chemo-induced peripheral neuropathy is a disabling side effect that causes many cancer patients to discontinue or limit their treatment regimen. Therefore, our initial goal was to determine the molecular mechanisms involved in the onset and progression of BIPN so that we can come up with preventive measures or treatment strategies that would allow for the continuation of therapy with BTZ or similar drugs. Therefore, we conducted in vitro studies to determine the molecular events that may precede and coincide with the onset of BIPN. We also tested some of the novel strategies for preventing and treating the BIPN related cellular events in our study. We used differentiated PC12 cells, as the neuronal model for testing the neurotoxic effects of BTZ. The cell differentiation was achieved using nerve growth factor (NGF) at 100 ng/ml concentration for 9 days. On the 10th day, the differentiated PC12 cells were treated with BTZ for 24 hours along with untreated controls. After treatment with BTZ, the extent of damage to the neurite outgrowth was assessed by immunocytochemistry technique. The BTZ treatment was able to reduce more than 50% of the neurite outgrowth compared to the untreated controls. In addition to measuring the neurite outgrowth, the levels of some of the cellular biomarkers such as Mitogen-activated protein (MAPK), epidermal growth factor receptor (EGFR), cyclic AMP response element binding (CREB) and Neuron-specific enolase (NSE) were assessed using Western blot and ELISA techniques. Our results showed increased expression of MAPK, EGFR, CREB, and NSE indicating the onset and progression of neuronal damage in BTZ treated PC12 cells. Between the two neuroprotective agents, Gamma Linoleic Acid (GLA) and Vitamin B12, tested in our experimental system, GLA appears to block the neuronal damage more noticeably by suppressing the levels of MAPK by 25%. Hence, our study will continue with the assessment of additional neuroprotective agents such as calcium channel blockers and neuronal nitric oxide (nNOS) inhibitors in the in vitro models in comparison with vitamin B12 and GLA. (This project is supported by the President's Faculty Research and Development Grant from NSU and the generous financial support from The Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida).

#667

A mouse model of APOE to define effects of doxorubicin on cognition.

Tamar C. Demby, Yichien Lee, Olga Rodriguez, Christopher Albanese, Jeanne Mandelblatt, G. William Rebeck. _Georgetown University, Washington, DC_.

The purpose of this study was to establish the effects of Apolipoprotein E (APOE) genotype on cognitive function following doxorubicin treatment in a mouse model. Chemotherapy treatment can lead to cognitive impairment in cancer survivors, and, in older breast cancer survivors, APOE4 carriers have been shown to have higher risk for these impairments. Though APOE4 genotype is known as the strongest genetic risk factor for late-onset Alzheimer's Disease compared to the APOE2 and APOE3 alleles, its effects on cognitive outcomes following treatment with common breast cancer chemotherapeutics are not yet well characterized. One year old female mice with targeted replacement of human APOE3 or APOE4 under control of the endogenous murine promoter were treated with 10 mg/kg doxorubicin or saline. Five weeks following treatment, spatial learning and memory was tested using the Barnes Maze. In APOE3 mice, doxorubicin treatment left spatial learning and memory intact. In APOE4 mice, doxorubicin treatment lead to impairment in spatial learning over the four days of training. This represents a promising model for studying the mechanisms behind cognitive impairment following doxorubicin treatment in a genetically vulnerable population.

## BIOINFORMATICS AND SYSTEMS BIOLOGY

### Convergence Science for Therapeutics and Precision Medicine

#668

A recommender system to enhance drug development in oncology from phase I trials: The RESOLVED2 project.

Guillaume Beinse, Virgile Tellier, Valentin Charvet, Isabelle Borget, Christophe Massard, Antoine Hollebecque, Loic Verlingue. _Gustave Roussy Cancer Campus, Paris, France_.

The industry and investigators' communities are currently facing a conjunction of increasing number of anti-neoplastic agents (ANA) candidate for phase 1 clinical trials (P1CT) and an important attrition rate for final approval. Improvement of the drug development strategies in oncology could be achieved by machine learning supporting go/no-go decisions after phase I trial.

PubMed abstracts of P1CT reporting ANA were used together with pharmacological classification and molecular targets of ANA from DrugBank5.0 database. To account for heterogeneous follow-up we computed a new metric entitled Food & Drug Administration approval-free-survival (FDA-aFS). FDA-aFS is a right-censored variable defined by the time between first P1CT publication and the FDA approval date or censoring for compounds not approved up to 2018, July. A training set and a test set were randomly generated from the whole dataset with a 70%/30% ratio. A regression and a classification version of RESOLVED2 have been developed in the training set and evaluated independently on the test set. The RESOLVED2 regression model has been optimized using a Cox logistic regression on FDA-aFS, penalized by Lasso with cross validation on the training set. RESOLVED2 predictions have been compared to a model including common variables used for go/no-go decisions (toxicity, clinical activity, observation of complete tumor response). The RESOLVED2 classification model (predicted approved ANA vs predicted non-approved ANA) was computed in the training set using predicted probabilities derived from the regression model, by the identification of the cut-off reaching minimal log-rank derived p-value when comparing FDA-aFS of predicted approved versus predicted non-approved ANA.

We identified 462 ANA matching with DrugBank entries (P1CT publication interval: 1972-2017). Among 1411 variables, 28 remained in the model after Lasso penalization. The relation of the model to the FDA-aFS had a weighted concordance index of 0.89 on the independent test set, versus 0.69 for a classical model based on efficacy/toxicity. Classified approval predictions achieved a p<10e-10 for association with observed FDA-aFS in the test set: at 6 years of follow-up, 73% (95%CI [49%; 86%]) of predicted approved drugs were indeed approved and 92% (95%CI [87%; 98%]) of predicted non-approved drugs were still not approved. A predicted approved drug was 16 times more likely to be approved compared to a predicted non-approved drug (Hazard ratio=16.4; 95%CI [8.40; 32.2]; p<10e-10).

RESOLVED2 is an applicable machine learning model that could support early go/no-go decision as soon as P1CT completion based on accurate prediction of FDA approval for drugs in oncology.

#669

Dynamic modeling of responses to PDL-1 inhibitors in non-small cell lung cancer: implications for precision combination therapy.

Iman Tavassoly. _Mt. Sinai Icahn School of Medicine, New York, NY_.

Immunotherapy by immune checkpoint blockade has had a promising impact on the treatment of Non-Small Cell Lung Cancer (NSCLC). Although single and combination therapy with checkpoint inhibitors such as PDL-1 inhibitors have been proved to be effective in making the survival of patients with NSCLC longer, but there is always a population of patients which shows resistance to these therapeutic modalities. The source of resistance emerges from signaling pathways and regulatory systems in tumors and the immune system, but the dynamics among tumor components, T cells, and tumor microenvironment contribute in this process as well. To understand the complex system of these interactions, a mathematical model of these pathways and interactions are presented using Ordinary Differential Equations (ODEs). The specific characteristics of NSCLC tumors including metabolic reprogramming have been considered in this model. The model is capable of decoding patient-to-patient variabilities regarding responses to PDL-1 inhibitors, and parameter-sensitivity analysis shows the vulnerable biomarkers to be targeted by combination therapy to give better results in killing the tumor cells. The model can be integrated with PK/PD models to provide an in-silico platform for investigating the therapeutic responses in NSCLC for precision oncology.

#670

Molecular basis of the idling state uncovers vulnerabilities suitable to sequential targeting in melanoma.

Corey E. Hayford, Bishal B. Paudel, Leonard A. Harris, Keisha N. Hardeman, Darren R. Tyson, Vito Quaranta. _Vanderbilt University, Nashville, TN_.

Recent published work from the lab identified a novel "idling" population state in the response of BRAF-mutant melanoma cancer cell lines to BRAF inhibition. The idling state is marked by a long-term net-zero proliferation rate in drug treatment, resulting from a balance of division and death events within isogenic clonal populations. We hypothesize that the idling state is equivalent to stable disease in patients that experience short-term tumor reduction on targeted therapy (e.g. tumor stops growing or shrinking but is still capable of rebound). Specifically, the idling state may be a prelude to resistance as a reservoir for genetic modifications that lead to tumor rebound. It is therefore critical to understand the molecular mechanisms underlying the idling state, since they may point to exploitable treatment vulnerabilities. Here, we have characterized the idling state in several isogenic clonal lineages isolated from the melanoma SK-MEL-5 cell line across several biological scales, including genomics, transcriptomics, metabolism, and cell morphology. Consistently across these measurements, these lineages converge to a molecularly homogeneous phenotype, which we consider to be an idling state molecular signature. Prominent in this signature are genes involved in metabolism redox balance and neuronal cell differentiation. This convergence appears to be a property of many cell lineages in the SK-MEL-5 parental population, since they were observed in barcoded lineages at approximately the same abundance pre- and post-treatment. When treating cells in the idling state with drugs that target the convergent molecular signature, we observe remarkable cell death and a significant drug effect compared to untreated cells. Together, we propose that these data reflect a non-selective convergence mechanism to tolerate drug treatment. We expect idling state drug vulnerabilities may inform sequential drug treatment strategies that exploit convergence mechanisms and reduce the number of cells that could acquire genetic resistance modifications. It may also shed light on the mechanism to achieve complete remission for melanoma patients in the clinic.

#671

Evaluation of machine learning tools for cancer drug response prediction with genomic profile data and drug response data from PDX model studies.

Henry Gu, Jingjing Jiang. _GenenDesign Co, Ltd., Menlo Park, CA_.

Cancer are diseases caused by genomic aberrations which lead to abnormal cell proliferation and metastasis. Cancer treatment based on cancer mutation profiles are the focus of research and development of cancer precision medicine. Currently only a small percentage of cancer patients' genomic information are associated with known drug responses, while others lack known cancer drivers or have cancer drivers that are not druggable. Machine learning (ML) tools have been developed recently for cancer biomarker discovery and cancer drug response prediction. However, due to a lack of sufficient amount of high quality drug response data, especially data for new drugs, the application of ML tools in this area is still in its early stage. In this study, we compared the performance of about a dozen ML methods, such as random forest classifiers, logistic regression classifiers, and support vector machines (SVM), trained on data of about 2300 treatment responses to average 12 different cancer drugs in 98 NSCLC, 55 gastric and 38 HCC PDX models with WES, CNV and RNA-seq information. Data preparation including genomic profile data selection, dimensionality feature reduction and processing was performed with considerations of known cancer gene functions, signal pathways, and pharmacodynamics information. Each dataset was randomly divided into training dataset and test dataset for ML algorithm training and evaluation. Performance of ML algorithms in testing was measured by their drug response prediction accuracy. Different regularization methods, such as LASSO and Elastic Net, were also evaluated in order to improve classification accuracy and identify relevant biomarker features. From comparing results from different algorithms or from the same algorithms but with differently prepared datasets, it seems that selection of functionally unknown gene aberrations for data preparation plays a significant role in prediction accuracy. Increasing dataset number or different data selection in different cancer types can improve prediction accuracy.

#672

**Establishment of a drug-tumor interaction database using Lantern Pharma's Response Algorithm for Drug Positioning and Rescue (RADR** TM **).**

Yuvanesh Vedaraju, Umesh Kathad, Aditya Kulkarni, Barry Henderson, Gregory Tobin, Panna Sharma, Arun Asaithambi. _Lantern Pharma Inc, New York, NY_.

The Response Algorithm for Drug positioning and Rescue (RADRTM) technology is Lantern Pharma's proprietary Artificial Intelligence (Al)-based machine learning approach for biomarker panel identification and patient stratification. RADRTM is a combination of three automated modules working sequentially to generate drug- and tumor-specific gene signatures predictive of response. RADRTM integrates biological knowledge, data-driven feature selection, and robust Al algorithms to facilitate hypothesis-free, drug- and cancer-specific biomarker development. RADRTM uses transcriptomic, drug sensitivity datasets and systems biology inputs and generates gene expression-based responder/non-responder profiles for specific tumor indications with high accuracy. RADRTM uses a unique process flow and a combination of machine learning algorithms to extract drug-specific biomarkers from whole transcriptome level input (~18000 genes). RADRTM comprises three main modules: data pre-processing, feature selection, and response prediction. Data pre-processing includes data cleaning, transformation and normalization. For dimensionality reduction and feature selection, RADRTM performs gene filtering via biological and statistical feature selection methods. In the final component, an AI-driven program reduces the intermediate number (approximately 500) of genes to a more manageable number (10 - 50) of candidate biomarkers capable of predicting drug sensitivity or insensitivity. Lantern's RADRTM AI application incorporates automated supervised machine learning strategies along with big data analytics, statistics and systems biology to enable identification of new correlations of genetic biomarkers with drug activity. Using RADRTM we have created a database of drug response prediction models for more than 120 drug-tumor type combinations in a preclinical setting that is expected to keep growing. These drug- and cancer-specific RADRTM models have further enabled the classification of clinical records into distinct response groups, as well as generated gene expression signatures as features predictive of response. The average response prediction accuracy lies above 80%. We also demonstrate the utility of such a database in clinical translation through various performance metrics including but not limited to true positive rate (TPR), true negative rate (TNR), positive predictive value (PPV), negative predictive value (NPV) and area under the curve (AUC). This database links the majority of FDA approved and selected investigational drugs with appropriate cancer indications and the associated RADRTM-derived responder/non-responder profiles in terms of gene expression signatures. This database could directly inform the drug-companion diagnostic co-developmental pathways for new drugs and cancer indications.

#673

Impact of PD-L1 feedback modulation on the sensitivity of anti-PD-1/PD-L1.

Jian Zhu,1 Rebecca L. Zhu2. 1 _OmicsHealth LLC, Potomac, MD;_ 2 _Winston Churchill High School, Potomac, MD_.

The long-term benefits of anti-PD-1/PD-L1 are limited in most cancer patients. Resistance and insensitivity are major challenges of this therapeutic class. Feedback regulation of PD-L1 could serve as a mechanism of drug resistant, especially negative feedback loops where inhibition of PD-L1 may lead to up-regulation of its own expression and function. In this study, computational models were established to investigate PD-L1 feedback modulations to facilitate discovery of biomarkers for drug sensitivity and to inform design of drug combination studies. As of October 2018, over 10,000 publications on cancer and immunosuppressive factors (e.g. PD-1, PD-L1, CTLA4, IDO1, TIM-3, LAG3, TIGIT) were found in PubMed. About 2500 abstracts of these articles were downloaded and systematically analyzed through natural language processing and text mining. Information on gene/drug, pathway/disease along with results from cell/animal/clinical studies were extracted, standardized and integrated into a directed graph-based database. Algorithms were designed to construct networks and identify PD-L1 feedback loops (FBLs). Fisher's Exact tests were performed to evaluate the significance of factors involved in (+) and (-)FBLs. The computationally generated PD-L1 feedback network comprises 102 nodes, 216 interactions with 1080 possible FBLs (502 positive vs 578 negative). In particular, up-regulation of IL10, pEGFR, SRC, HIF1A, ISX, NF-kappaB, pSMAD2/SMAD2, IFN-gamma were strongly associated with up-regulation of PD-L1 upon inhibition of PD-1/PD-L1 (p<0.0001, (-)FBLs). Interestingly, TIM-3, a potential IO target and commonly up-regulated by anti-PD-1/PD-L1, is involved in both (+) and (-)FBLs. Inhibition of TIM-3 (e.g. anti-TIM-3) could decrease IL-10 in Tregs and enhance efficacy of anti-PD-1/PD-L1, but meanwhile, anti-TIM-3 could activate NF-kappaB, IRF7 and STAT1, leading to up-regulation of PD-L1 expression and potential resistance to anti-PD-1/PD-L1. In conclusion, the literature-based network analysis proved to be effective to identify potential regulatory pathways driving resistance to immunotherapies, to prioritize the selection of biomarkers, and to assess risk of drug combinations. Particularly, through the analysis of feedback regulations of PD-L1, novel therapeutic approaches to boost efficacy of anti-PD-1/PD-L1 could be postulated. Targeting genes involved in both (+) and (-)FBLs of PD-L1, such as TIM-3, needs to be carefully evaluated.

#674

Predicting cellular response to therapy in breast cancer using mathematical modeling.

Wei He,1 Diane M. Demas,2 Isabel Conde,2 Yassi Fallah,2 William T. Baumann,1 Ayesha N. Shajahan-Haq2. 1 _Virginia Tech, Blacksburg, VA;_ 2 _Georgetown Lombardi Comp. Cancer Ctr., Washington, DC_.

About 70% of all breast cancer tumors are estrogen receptor positive (ER+) and are treated with antiestrogen therapies. While the inevitability of developing resistance to these therapies remains uncontested, little is known about the mechanism and prevention of resistance. Our ultimate goal is to use mathematical modeling to optimize dynamic therapies that decrease proliferation and stave off resistance. In this initial study, we used MCF7 cells as a model of ER+ breast cancer and estrogen deprivation as a surrogate for aromatase inhibitors. We developed long-term estrogen deprived MCF7s (LTEDs) that proliferate similarly to untreated MCF7s but are resistant to antiestrogens. We collected time-course data for simultaneous gene expression and protein levels (NanoString Pan Cancer panel, a non-amplification based digital method) over 6 weeks to capture early molecular adaptations of deprived MCF7s and compare them to those present in LTEDs. PCA analysis of the mRNA and protein data shows a dramatic change in the first component due to estrogen deprivation, and a dramatic change in the second component associated with long-term resistance. Correlation analysis shows a large number of cell cycle genes that are similarly regulated, decreasing with estrogen deprivation and increasing with the onset of resistance. There is also a highly-correlated group of genes associated with resistance. This knowledge allowed us to hypothesize a molecular mechanism for resistance that can be tested experimentally. To begin building a dynamic model, we measured a 7-day time course of estrogen related proteins. The model is built around ER signaling and the cell cycle, and simulates protein and proliferation changes in response to deprivation and antiestrogen (ICI182,780; ICI) treatment. To determine which treatments to model in addition to estrogen deprivation and ICI, we looked for a promising sequential therapy to limit proliferation and, hopefully, stave off resistance compared to a single non-stop therapy. We found that alternating the targeting of CDK4/6 using Palbociclib (clinical anti-cancer therapy) with targeting of RUNX1 (gene that is increased at the onset of resistance in our model), could be a plausible strategy to inhibit endocrine resistance. Future work will involve extending the model to longer time scales and using it for treatment optimization.

#675

Network modeling of drug resistance mechanisms and drug combinations in breast cancer.

Jorge Gómez Tejeda Zañudo,1 Pingping Mao,2 Joan Montero,3 Guotai Xu,4 Kailey J. Kowalski,2 Gabriela N. Johnson,2 José Baselga,5 Maurizio Scaltriti,4 Anthony G. Letai,2 Nikhil Wagle,2 Reka Albert6. 1 _Broad Institute, Cambridge, MA;_ 2 _Dana-Farber Cancer Institute, Boston, MA;_ 3 _Institute for Bioengineering of Catalonia, Barcelona, Spain;_ 4 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 5 _Vall d'Hebron Institute of Oncology, Barcelona, Spain;_ 6 _Pennsylvania State University, University Park, PA_.

Durable control of invasive solid tumors is thwarted by the lack of knowledge of effective drug combinations and of the acquired and intrinsic resistance mechanisms of drugs. In an effort to tackle this problem, the SU2C-NSF-TVF Drug Combination Convergence Team is using mechanistic models of cancer cell signaling based on therapeutic and cell line data in order to identify elements within cancer cells that might eventually be exploited through therapeutic combinations.

Here we present a comprehensive mechanistic network model of signal transduction in ER+ PIK3CA-mutant breast cancer. Focusing on PI3K inhibitors, the model recapitulates known resistance mechanisms and predicts other possibilities for resistance: loss of RB1, FOXO3, P27, or PRAS40. To test these predictions, we analyzed genome-wide CRISPR screens of two breast cell lines in the presence of PI3K inhibitors (BYL719 and GDC0032) and found that the predicted genes (RB1, FOXO3, P27, and PRAS40) were significantly enriched in the screens. Some of these resistance genes (e.g. loss of RB1) were found to be cell-line specific and follow-up experiments in RB1-KO cells confirmed the cell-line-specific nature of PI3K-inhibitor resistance. The model also reveals known and novel combinatorial interventions that are more effective than PI3K inhibition alone. For example, the model predicts that the combination of PI3K inhibitors with inhibitors of anti-apoptotic protein MCL1 would be effective. Follow up experiments in cell lines using cell viability assays, cell death analyses, and dynamic BH3 profiling experiments to determine increases in apoptotic priming upon treatment confirmed that MCL1 inhibitors (S63845) enhance the effect of PI3K inhibitors (BYL719) and that this combinatorial effect is cell-line-specific, similarly to what was found in the resistance genes case.

In conclusion, the model predicted drug resistance mechanisms and effective drug combinations, some of which were verified experimentally and found to be cell-line-specific. Next iterations of the model will incorporate the identified discrepancies and newly identified resistance mechanisms to drugs of clinical interest.

#676

Leveraging TCGA gene expression data to build predictive models for cancer drug response.

Evan Clayton. _Georgia Institute of Technology, Atlanta, GA_.

Personalized oncology promises to increase the success rate of cancer drug therapy by using molecular tumor profiles to determine the optimal therapeutic for an individual. 5-Fluorouracil and Gemcitabine are two Food and Drug Administration approved chemotherapeutics commonly prescribed to cancer patients. Here, we build machine learning models using gene expression data from patients' primary tumors to predict their response, positive or negative, to these drugs. We compared several clustering and classification methods for predicting response. Of those tested, Clara was found to be the best clustering algorithm while random forest was the best classification method, with prediction accuracy up to 86%. We determined that models trained across several cancers outperform single cancer models. We also found that genes most informative for predicting drug response were enriched in well-known cancer signaling pathways, highlighting their potential significance in chemotherapy prognosis. Ultimately, such predictive models may aid oncologists with making critical treatment decisions.

#677

A precision dosing application for prostate cancer chemotherapy.

Claire Villettte, Hitesh Mistry, Fernando G. Ortega, David Orrell, Frances A. Brightman, Jim Millen, Christophe Chassagnole. _Physiomics plc, Oxford, United Kingdom_.

The therapeutic window of chemotherapy drugs is commonly established at a population level and patient dose selection is often simply scaled with Body Surface Area. Due to large inter-individual physiological variability, this leads to a significant number of patients being under or over-dosed. While a limited number of precision dosing techniques exist to tailor patient-specific treatment, they typically require costly additonal tests which severly restrict their use in clinical practice. Focusing on the use of docetaxel in metastatic castrate-resistant prostate cancer, we have developed a demonstrator for precision dosing which relies only on routine blood tests that are already universally available in most health systems and could thus fit within the current clinical practice to improve patient outcome at a low cost. This tool was developed using publicly available data from Project Datasphere for the comparator arm of a phase III clinical trial for metastatic hormone-resistant prostate cancer (NCT00617669). This cohort includes 412 patients who were treated with Docetaxel between 2008 and 2011 in cycles of 21 days with weekly blood tests in the first cycle. A population PK/PD model for Docetaxel and leukocyte population was assembled based on literature [1,2]. Individual patient parameters including Docetaxel AUC were estimated by calibrating this model with weekly measured blood count using a Bayesian approach. Survival and toxicity Cox models were developed using estimated PK/PD parameters as well as biomarker levels. They allowed estimations of gains in prognostic as well as toxicity risks associated with modifying patient dose after the first chemotherapy cycle. These models will be made available through a web application to support clinicians in their decision to refine patient doses. Concordance levels of the order of 0.7 were obtained for the survival and toxicity models, with significant predictive variables including Docetaxel AUC, patient liver markers as well as PSA. Patients with low estimated hematologic toxicity (neutrophil count not dropping under 1 billion/L for more than 5 days) presented a median overall survival time of 461 days, against 563 days for patients with higher hematologic toxicity. Patients with low estimated Docetaxel AUC (<1.5 µg.h/mL) presented a median overall survival time of 455 days, against 587 days for patients with higher estimated Docetaxel AUC. For some patients, the models predicted gains in prognosis without severe toxicity when increasing the dose. We have developed a precision dosing app for Docetaxel in prostate cancer which requires a single weekly classical blood test in the first chemotherapy cycle. This app has the potential to significantly improve patient outcome at low cost without disrupting current clinical practice. References [1]: McLeod, H.L. et al (1998). Cancer chemotherapy and pharmacology [2]: Friberg, L.E. et al (2002). Journal of clinical oncology

#678

Drug-induced TMDD: a novel class of PK models relevant to Immune-stimulating therapies.

Fei Hua,1 David Flowers,1 Daniel C. Kirouac,2 John M. Burke,1 Joshua F. Apgar1. 1 _Applied BioMath, Concord, MA;_ 2 _Applied BioMath, Oakland, CA_.

Purpose: Many biologics exhibit target-mediated drug disposition (TMDD). That is, given the high affinity and low doses typical for antibody-based therapeutics, drug clearance is often mediated through target binding and endocytosis, in addition to 1st-order elimination processes. Accounting for the process of TMDD in pharmacokinetic (PK) models is thus necessary to describe and predict drug exposure. TMDD is typically characterized by dose-dependent clearance, wherein drug elimination is faster at lower doses. In standard TMDD-models, target concentration and turnover are assumed to be constant. While this is generally a good approximation, the assumption does not always hold.

Immune-stimulating drugs are the largest class of agents currently being developed in oncology, including checkpoint inhibitors (PD-1/PD-L1, CTLA4), cytokines (IL-2, IL-15), and immune agonists (GITR, OX40, ICOS). For immune-stimulating agents, drug treatment may induce lymphocyte proliferation, expanding the pool of target along with the target-expressing cells. TMDD in such cases is thus induced by drug treatment, and thereby target burden is dynamic; both dose- and time- dependent. PK dose response profiles in such cases could exhibit very different characteristics from standard (static) TMDD.

Methods: The goal of this work is to invest the role of target dynamics on drug pharmacokinetics (PK) and receptor occupancy (RO). To do so, we have developed a mathematical modelling construct termed 'Drug-induced TMDD', wherein drug-target binding induces increase of the target synthesis rate, subsequently target concentration, leading to enhanced TMDD. We incorporate this construct into a 2-compartment model typical of monoclonal antibodies. Simulations were then used to characterize how dose, drug-target affinity, and the amplitude and the delay in target up-regulation affect the PK and RO profiles, and compared static vs. drug-induced TMDD behavior.

Results: For Drug-induced TMDD, target expression increases with increased target engagement. This creates a pseudo-negative-feedback circuit, wherein the rate of TMDD-mediated clearance is dependent upon drug concentration. As a result, drug clearance does not appear dose-proportional at low doses, as is typical of static-TMDD. The AUC of drug concentration for drug-induced TMDD can be higher than static TMDD at low doses, but lower at higher doses. Similar results are predicted for the AUC of RO as well with the nominal parameter set. Increasing target affinity (decreasing KD) however results in faster clearance, typical of both cases of TMDD.

Conclusions: Dynamic-TMDD could lead to different dose-dependent PK and RO behaviors from a static TMDD. Based on these characteristic profiles presented, one can determine whether a static vs. drug-induced TMDD model may be required for PK modelling, particularly important consideration for immune-stimulating biologics.

#679

Quantitative systems pharmacology modeling of immunotherapies in B-cell acute lymphoblastic leukemia.

Oleg Demin Jr, Antonina Nikitich, Oleg Demin. _InSysBio, Moscow, Russian Federation_.

Introduction: Multiple T-cell engaging antibody-based molecules and CAR-T therapies are in development for the treatment of B-cell acute lymphoblastic leukemia (B-ALL). Moreover, two therapies have already been approved by FDA: blinatumomab (CD19/CD3 bispecific T-cell engager) and tisagenlecleucel (anti-CD19 CAR-T therapy). This work employs quantitative systems pharmacology (QSP) modeling approach to investigate factors affecting outcomes of administration of such types of therapy.

Methods: Model was developed on the basis of Immune Response Template (QSP model of immune system). It describes leukemic blasts, hematopoiesis of normal B cells, CD4+ and CD8+ T-cell subsets, and cytokines (IL-6, TNFα, IFNγ, TGFβ, etc.) in bone marrow and blood. The model takes into account the different level of expression of CD19 on various subsets of normal B cells and leukemic cells. Model parameters were estimated and fitted against in vitro, in vivo human data. Small part of clinical data on blinatumomab and anti-CD19 CAR-T therapies was used for model calibration, other part was used for model validation.

Results: Our model reproduced clinically relevant blinatumomab and anti-CD19 CAR-T hallmark data including depletion of CD19+ cells, T-cell subset dynamics and cytokine release post infusion. The model successfully described the clinically observed phenomenon on progressive cytokine release attenuation with subsequent blinatumomab treatment cycles. It confirmed the dose step strategy for decreasing initial cytokine release. Model showed that treatment outcome is very sensitive to target expression, level of immune cells, disease progression rate and expression of PD-L1 on leukemic cells. The proportion of CD19+ and CD19- leukemic blasts before treatment can be used as a biomarker to predict probability of relapse in patients to stratify patients.

Conclusions: This work provides a quantitative modeling framework to predict responses to immunotherapies in B-ALL on the basis of realistic baseline conditions and reveal the factors affecting treatment outcome.

#680

Predicting the effect of radiotherapy on tumor growth inhibition and time to progression in head and neck cancer.

Fernando G. Ortega, Frances A. Brightman, David Orell, Hitesh Mistry, Jim Millen, Christophe Chassagnole. _Physiomics plc, Oxford, United Kingdom_.

We have previously developed a tumor model that replicates and predicts the effect that irradiation (IR) has on tumor growth inhibition in several preclinical studies. These studies utilize different IR doses and regimes as well as combination with therapeutic agents with disparate mechanisms of action. The primary outcome measure of these models was tumor size over time up to 1-2 months post-treatment.Encouraging results in the preclinical space led us to develop an enhanced strategy for modelling RT treatments using a tumor model that has enabled us to predict tumor shrinkage and longer term regrowth in human studies of squamous cell carcinoma head and neck tumors. To achieve this goal we analysed historical clinical data sourced from different clinical trials (NCT00094081; NCT00415194) in head and neck cancers which suggested that i) the initial rate sum of longest diameter (SLD) shrinkage depends on the SLD before treatment where the larger the initial SLD the faster the tumor shrinkage rate; ii) the magnitude of the tumor shrinkage can not only be explained by depletion of the peripheral growing layer of the tumor; and iii) a significant proportion of tumors remained suppressed for years following treatment. We used these findings to update and adapt the mathematical model to the clinical setting as well as to calibrate the model to describe the behavior of head and neck tumors treated with RT alone. We assumed that the mechanism of action of RT at the cell cycle level is unaltered between preclinical and clinical model, i.e. only the growing cell layer is directly depleted by radiation induced DNA damage. However, we hypothesized that the integrity of the growing layer plays a role protecting the necrotic core from degradation by biological or physical processes. Therefore, reducing the width of the growing layer width by depleting the number of viable cells indirectly contributes to overall tumor size shrinkage through erosion or leakage from the necrotic core. The other major challenge was to develop a mechanism to reflect the wide variation in time to tumor regrowth post-treatment. Literature evidence suggested that this regrowth can be explained by variation in cellular doubling times and we were able to calibrate our modeled cell population using a distribution of doubling times that enabled to the model to accurately predict regrowth profiles from literature. We were able to extent the mathematical model from a tool that explained and predicted the effect of RT on tumor growth inhibition in the preclinical space to one that describes both tumor inhibition and regrowth in the clinical space. We have used this model as the basis to predict the effects of different RT regimes as well as combinations with other therapeutic agents in a clinical setting.

#681

Methylation biomarkers of cellular response to demethylating drug.

Xuewei Wang, Jean-Pierre Kocher, Zhifu Sun. _Mayo Clinic, Rochester, MN_.

DNA methylation plays an import role in both tumorigenesis and therapy resistance. Demethylating drug regimens have shown efficacy in hematological malignancies, and are actively studied for other cancer types. High variability in patients' responses to these drugs has been observed and highlights the demand to identify predictive biomarkers for these demethylating drugs. In particular, decitabine (latest FDA-approved demethylating drug) demonstrated as the most potent agent and has the best response on hematological cell lines. Two public datasets were collected to identify methylation biomarkers of decitabine. The first one is from Genomics of Drug Sensitivity in Cancer, with methylation data from Illumina 450K array and drug response from cell lines. The other one is a publicly available patient dataset (methylations from RRBS sequencing and decitabine response from clinic). 64900 CpG sites common to the two methylation platforms were used to train machine learning model (Elastic Net), based on the methylation data and decitabine response (AUC) from 108 hematological cell lines in GDSC. 73 CpGs were identified as predictive biomarkers by Elastic Net. The trained model was then used to make predictions on 39 patients (20 responders and 19 non-responders) with their methylation profiles of the same set of CpGs. The predicted response of responder group is indeed better than non-responder group (p-value = 0.0501), indicating the validity of identified methylation biomarkers. The top one CpG identified by Elastic Net resides in the promoter region of gene C20orf85, and is significantly hypermethylated in non-responder group (p-value = 0.0458), suggesting its potential involvement in the resistance to decitabine.

#682

Drug repurposing engine fueled by diverse drug similarity data.

Jamal A. Elkhader,1 Coryandar M. Gilvary,1 Neel S. Madhukar,1 Olivier Elemento,1 David Solit2. 1 _Weill Cornell Medical College, New York, NY;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Despite recent advances in life sciences and technology, bringing a single drug to market has stayed drastically expensive, leading to a decline in the number of new drugs being clinically approved. Drug repurposing, identifying novel indications for approved drugs, presents an opportunity to avoid many common pitfalls, such as toxicity, and bring treatments to market faster. Additionally, drug repurposing, which aims to identify indications for unapproved, shelved drugs, can also be a short-cut to the clinic due to the previously completed preclinical work. Current computational efforts for indication prediction focus on leveraging the similarities between drugs' approved indications, however these approaches are unsuitable for drug repurposing. Here we introduce a novel big data approach that integrates only compound similarity metrics to identify novel indications for drugs, regardless of the existence of a primary indication. We introduce a computational approach that integrates multiple data types to create a drug-indication network, which can be used to recommend indications a drug may treat. Using this network, we can accurately elucidate novel indications for drugs currently being used as treatments in clinical trials. Additionally, we present multiple promising candidates for future clinical work. This drug-indication network is built using a confidence score that two drugs share an indication, calculated using a gradient boosting classifier. Utilizing only publicly available data, we built this classifier based on drug similarity features and identified previously undocumented associations between approved drugs which share indications such as significantly high side effect (D = 0.26, p < 0.001) and target similarity (D = 0.21, p < 0.001). When trained on FDA approved drugs, this classifier achieved significant predictive performance, with an area under the ROC curve of 0.82.We then used our method to determine potential repurposing opportunities. We predicted numerous statins, including Lovastatin and Cerivastatin, known cholesterol-lowering agents, as potential treatments for nasopharyngeal cancer. Recent work has shown statins to be promising anti-tumor agents, specifically in nasopharyngeal cancer, highlighting the specificity of our approach. Furthermore, we predicted Cinnarizine, a calcium channel blocker with potential antipsychotic effects, to treat schizophrenia. In addition to identifying these and a variety of other novel indications with our drug-indication network, our complete method incorporates large-scale biological/chemical data to help understand the underlying mechanisms behind new indication predictions. Altogether, our method provides a distinctive strategy that can identify novel and diverse indications, allowing for an expedited and more efficient method for future drug development and repurposing efforts.

#683

A machine learning based clinical decision support system (CDSS) for anti-PD-1 therapy using non-invasive blood marker and clinical information for lung cancer patients.

Kyoung-Ho Pyo,1 Beung-Chul Ahn,2 Chun-Feng Xin,1 Dongmin Jung,1 Chang Gon Kim,3 Min Hee Hong,2 Byoung Chul Cho,2 Hye Ryun Kim2. 1 _Yonsei University College of Medicine, Seoul, Republic of Korea;_ 2 _Yonsei Cancer Center, Division of Medical Oncology, Seoul, Republic of Korea;_ 3 _KAIST, Daejeon, Republic of Korea_.

Cancer immunotherapy with anti-PD-1 therapy (αPD-1) has greatly improved the survival outcome of patients with non-small cell lung cancer (NSCLC). However, the response rates of αPD-1 are around 20-30% based on clinical trials. The presence of PD-L1, tumor infiltrating lymphocyte or tumor mutation burden may be used as indicators of response, but are still limited to predict αPD-1 response. In this study, we established the machine learning based clinical decision support system (CDSS) to predict the αPD-1 response by comprehensively combining clinical information and blood-based data which are easily assessable in routine practice.We enrolled 126 patients with NSCLC treated with the αPD-1 at Yonsei cancer center. Clinical data including patient characteristics, mutation, treatment outcomes, and adverse events were collected and analyzed. Forty patients additionally had blood-based immune data by flow cytometry. There were two data sets; clinical data set (n=126) with 15 variables, and immune data set (n=40) with 37 variables. We found that 27 variables out of 52 variables are selected by recursive feature elimination. The responders are defined as PR or SD ≥6 months and the non-responders are defined as the others. Supervised learning algorithms such as the LASSO, Ridge, Elastic Net, SVM, ANN, and RF were applied to each data set for predicting the αPD-1 response. The performances of each model were evaluated according to the ROC curve or cross-validation errors. Variable importance was measured by using the random forest and gradient boosting.Patient characteristics included male (69.8%), age ≥ 60 years (66.2%), ECOG 0/1 (77.7%), adenocarcinoma (69.8%), EGFR mutations (15.1%), and PD-L1 positive (61.2%). We classified the patients into responders (38%) and non-responders (62%) in total 126 patients. A result from the clinical data set of 126 patients demonstrated that the Ridge regression model (AUC: 0.78) can more accurately predict the αPD-1 response than others. Of 15 clinical variables, some are considered to be important in the following order; tumor burden,age, PD-L1, ECOG PS, and irAE based on the random forest. When we performed the machine learning process with clinical and immune data, The Ridge regression model (AUC:0.82) showed the good performance to predict αPD-1 response, compared to the single clinical model. The machine learning based CDSS for aPD-1 to NSCLC patients has benefit for predicting aPD-1 responses. Our prediction model could be easily accessible and fast processed in routine practice. The supervised machine learning based non-invasive predictive score (NIPS) demonstrates the rate of aPD-1 response on NSCLC patients. We will validate NIPS in independent patient cohort and currently are establishing the NIPS as web-based software.

#684

A semi-mechanistic platform model to capture individual animal responses to checkpoint inhibitors in a syngeneic mouse model.

Alison M. Betts,1 Lin Lin,2 Carissa L. Young,2 Wenlian Qiao,1 Jatin Narula,1 Peter O'Brien,3 Derek Bartlett,3 Andrea Hooper,4 Jason Williams,3 John M. Burke,2 Joshua Apgar,2 Fei Hua,2 Lore Gruenbaum2. 1 _Pfizer Inc, Cambridge, MA;_ 2 _Applied Biomath, LLC, Concord, MA;_ 3 _Pfizer Inc, La Jolla, CA;_ 4 _Pfizer Inc, Pearl River, NY_.

Syngeneic mouse models have been widely employed in preclinical discovery of checkpoint inhibitors as they enable study of drug impact on the intact immune system (Lechner, 2013; Murphy, 2015). However, the interpretation of such studies remains challenging partly due to the large variability in individual animal responses to drug treatment. In this work, we describe the generation of a model platform that captures essential aspects of the pharmacokinetics, cellular and tumor growth effects of murine surrogates of two checkpoint therapeutic antibodies, anti-PD1 and anti-CTLA4, in the CT26 syngeneic tumor model. The model describes individual animal responses with regard to drug exposure, key intra-tumoral cell kinetics and tumor volume changes and provides biologically plausible explanations for the observed differences between good and poor responders to treatment with anti-PD1 or anti-CTLA4. We used the model to predict the antibody dose-response relationships for individual animals and to identify dose thresholds above which complete tumor elimination can be achieved in good responders. In contrast, our models predict that poor responders would not achieve complete response even with much higher drug doses. The parameters in our model that impact the response in poor responders are not drug-related. This finding suggests that immune-cell related barriers have to be crossed in order to achieve a therapeutic response in these animals - possibly via combination therapy. In addition, we identified the net tumor cell doubling rate, one potential parameter that contributes to individual variability in response to treatment, as the most sensitive biological parameter determining tumor volume changes upon treatment with anti-PD1 or anti-CTLA4. Measuring individual animal tumor cell growth characteristics may help with the experimental design and qualification of animals for studies (in addition to absolute tumor volume), and thereby reduce inter-animal variability and enhance the interpretability of study results, especially in combination with a model such as the one presented here. This model platform can be adapted to capture and compare checkpoint drug effects in different syngeneic tumor models. Moreover, it can be expanded to add additional drug mechanisms and can serve as a tool to inform the experimental design of mouse studies.

#685

Development of a multiscale QSP model to characterize the tumor suppressive effects of a cytokine mRNA immunotherapy in a preclinical melanoma mouse model.

Kamau Pierre,1 Lin Lin,2 Lore Gruenbaum,2 Carissa Young,2 Joshua Apgar,2 Christian Hotz,3 Timothy Wagenaar,4 Spyros K. Stamatelos1. 1 _Sanofi US Services Inc., Bridgewater, NJ;_ 2 _Applied BioMath, Concord, MA;_ 3 _Biopharmaceutical New Technologies (BioNTech) Corporation, Mainz, Germany;_ 4 _Sanofi US Services Inc., Cambridge, MA_.

Despite the success of checkpoint blockade immunotherapy, most patients do not respond adequately to treatment and tumors effectively evade T lymphocyte and Natural Killer (NK) cell immune surveillance. Within the tumor microenvironment, the activities of immune cells are regulated by a multitude of signals including the local cytokine milieu. Intratumoral administration of our cytokine mRNA cocktail therapy, consisting of transcripts encoding for IL-12sc, IFNα2b, IL-15sushi and GM-CSF, has induced regression in tumor-bearing mice. Leveraging in vitro, in vivo and in silico data, we have developed a QSP platform that captures the observed preclinical tumor responses and allows for systemic predictions under a variety of conditions. A 5-compartment model was employed to account for the pharmacokinetics, cellular interactions and signaling within the tumor, lymph node, peritoneum, blood and periphery. We model the intratumoral (i.t.) injection of the mRNA mixture and its subsequent uptake, translation and secretion of activated cytokines that induce an immune response. Specifically, when released into the tumor, these cytokines promote tumor killing and trigger the activation and proliferation of CD4+/CD8+ T lymphocytes and NK cells. The model's simulation results are well aligned with our preclinical data; indicating increased tumor regression with escalating doses of the mRNA monotherapy. The wealth of data and the intertwined information derived from the interaction of receptors and ligands defining the T cell phenotype necessitate a systems approach in order to identify suitable targets and mechanisms of drug action. Our murine immuno-oncology QSP model could be translated across species and be used as part of a computational platform to test various assets with similar MoA.

#686

Quantitative analysis of the role of homologous recombination in response to radiotherapy for prostate cancer.

Mengdi Qian,1 Alexandru Almasan,2 Evren Gürkan-Çavusoglu1. 1 _Case Western Reserve Univ., Cleveland, OH;_ 2 _Cleveland Clinic, Cleveland, OH_.

The purpose of this study is to computationally analyze how the interaction between androgen receptor (AR) and homologous recombination (HR) affects the treatment outcome for ionizing radiation (IR) combined with androgen deprivation therapy (ADT) for prostate cancer (PCa). Combination treatment with radiation and ADT is currently a standard treatment for PCa. The effectiveness of radiation treatment alone depends on the cells' capacity to repair the damage that is mostly in the form of double-strand breaks (DSBs). Two major pathways that repair DSBs are non-homologous end joining (NHEJ) and HR. The experimental data in the literature show that AR promotes both NHEJ and HR following IR, and inhibition of AR by ADT impairs both of these pathways in PCa cells leading to either increased radiosensitivity or sensitization to PARP inhibitors. In our previous work, we developed mathematical models of the NHEJ pathway and in this current work we have developed such models for HR to comprehensively analyze the response of PCa cells to IR combined with ADT. We have modeled HR using a series of ordinary differential equations. The parameters that needed to be estimated are the kinetic rate constants, and we have used least square estimation to obtain these model parameters. The data sets used in the parameter estimation were obtained from the literature and they were from both in vitro experiments as well as clinical data from PCa patients. The experimental data show that AR influences the DNA end resection that is required for proficient HR and we have computationally included this effect in the kinetics of the models. We have developed models for IR treatment alone or in combination with ADT. We have carried out sensitivity analysis and identifiability tests on all the parameters from both models in order to determine the parameters that are reliably estimated. The outputs from both NHEJ and HR models show that the suppression of AR activity through ADT has the potential to enhance the IR treatment outcomes for PCa patients. The impairment of HR by ADT also opens up an avenue for achieving synthetic lethality using PARP inhibitors as shown in the literature; in the future we are planning to extend our modeling efforts to include the effect of PARP inhibitors on the treatment outcome.

#687

Implementation of inter-patient variability in semi-mechanistic model of melanoma treatment with vemurafenib.

Dmitry Shchelokov, Oleg Demin Jr, Oleg Demin. _InSysBio, Moscow, Russian Federation_.

Introduction: The number of parameters in quantitative systems pharmacology (QSP), mechanistic and semi-mechanistic models is greater than in conventional PK/PD models. That is why it is not possible to implement inter-patient variability using only clinical data. The objective of this study was to describe inter-patient variability in the semi-mechanistic model of melanoma treatment with vemurafenib, a BRAF inhibitor, on the basis of published human in vitro, in vivo and clinical data, and to predict overall response from phase 2 clinical trial.

Methods: Model described melanoma progression and its treatment with vemurafenib. It includes (i) proliferation, apoptosis, and transition between states (c-MET negative/positive and sensitive/resistant to BRAF inhibitor) of cancer cells; (ii) HGF effect on proliferation of melanoma cells; (iii) PK/PD of vemurafenib. Various types of data were used to extract inter-patient variability, for example: (a) in vitro data on effect of vemurafenib on melanoma cell lines [1]; (b) in vivo data on tumor growth in melanoma patients without treatment [2]; (c) in vivo data on HGF level in blood plasma of melanoma patients [3].

Results: Different virtual patients developed by the model reproduce all types of tumor response to vemurafenib monotherapy according to RECIST criteria: complete response, partial response, stable disease and progressive disease. To evaluate the predictive ability of the model in a quantitative manner we compared results of simulation of virtual patients population with clinical outcomes. An overall response rate was 70% in the model versus 53% (95% CI, 44 to 62) observed in phase 2 clinical trial [4]. Overestimation can be explained by intermittent administration and dose reduction in case of adverse events in clinical trials which were not taken into account in the model.

Conclusions: Our work establishes the benefit of using of in vitro and in vivo data in addition to clinical data to predict the variability in treatment outcomes of cancer patients.

[1] Stones et al. Front Genet. 2013 May 8;4:66

[2] Hartung et al. PLoS One. 2017 May 4;12(5):e0176080

[3] Hügel et al. Melanoma Res. 2016 Aug;26(4):354-60

[4] Sosman et al. N Engl J Med. 2012 Feb 23;366(8):707-14

#688

Safety and efficacy of TTFields delivery to the lungs: A computational study.

Hadas Sara Hershkovich, Noa Urman, Ariel Naveh, Zeev Bomzon. _Novocure, Haifa, Israel_.

Objective: This study investigates the efficacy and safety of Tumor Treating Fields (TTFields) delivery to the lungs utilizing computational simulations.

Introduction: Recent results of a phase 2 clinical trial STELLAR (NCT02397928) demonstrated a significant extension in median overall survival among patients with mesothelioma treated with TTFields plus standard of care chemotherapy compared to historical control data of patients who received standard of care chemotherapy alone. A phase 3 clinical trial (NCT02973789) is currently investigating the efficacy of TTFields therapy for the treatment of Non-Small-Cell lung cancer (NSCLC). The efficacy of TTFields therapy in disrupting tumor cells depends on the frequency of the field (150kHz optimal frequency for NSCLC and mesothelioma) and the field intensity. The higher the field intensity, the larger the therapeutic effect, with a therapeutic threshold of 0.7 V/cm above which TTFields begin to exert a significant anti-mitotic effect on cells.

Delivery of an electric field to the body unavoidably leads to deposition of heat in the tissue through Joule heating. The rate at which electrical energy is absorbed and converted to heat, depends on field's frequency and tissue's properties. Since Joule heating may cause thermal damage, it is important to ensure that TTFields intensity in the body is below the thermal damage threshold. In this study we performed numerical simulations to evaluate the thermal safety and the efficacy of the NovoTTF-100L when delivering Tumor Treating Fields to the torso. Thermal safety threshold levels were determined by current density and specific absorption rate (SAR) and TTFields therapeutic threshold was determined by field intensity.

Methods: We investigated field intensity, current density and SAR values that developed within 3 different realistic human computational models in which a virtual representation of the NovoTTF-100L (150kHz) was used to deliver TTFields to the lungs. The models used in this study include: a female model, male model and an obese male model (Virtual Population, IT'IS foundation) with a range of BMI values from normal to obese. Numerical simulations were performed using Sim4life (v3.0, ZMT Zurich).

Results: The simulations show that for all models, the NovoTTF-100L delivers therapeutic intensities of over 0.7 V/cm RMS to over at least 76% of the lungs. Current density within the models is well below the safety threshold of 100 mA/cm^2. SAR values within the internal organs are below the levels at which thermal damage occurs. In the superficial body layers, higher SAR values are observed. However, the NovoTTF-100L incorporates temperature control that prevents the skin from heating to levels at which thermal damage can occur.

Conclusions: Results of this study support the observations that the NovoTTF-100L delivers TTFields to the lungs at therapeutic levels and that the device is safe for use in the entire patient population.

#689

Heating of head tissues during TTFields therapy: A computational study.

Nichal Gentilal,1 Ricardo Salvador,2 Pedro C. Miranda1. 1 _Institute of Biophysics and Biomedical Engineering, Lisbon, Portugal;_ 2 _Neuroelectrics, Barcelona, Spain_.

Tumor Treating Fields (TTFields) is a non-invasive technique that can be applied jointly with chemotherapy to treat glioblastoma (GBM). It consists on delivering an alternating current (900 mA) with a frequency of 200 kHz in two perpendicular directions alternately: Anterior-Posterior (AP) and Left-Right (LR) via capacitively coupled transducer arrays placed on the shaved scalp. This technique showed an anti-mitotic effect when the electric field magnitude in the tumor is at least 1 V/cm and the results were enhanced for values up to 3 V/cm. Optune is the FDA-approved device that was created by Novocure to deliver TTFields in humans. Due to the high daily compliance that is necessary for this technique (at least 18 hours, every day) biological tissue heating can occur. To prevent an excessive temperature increase, Optune shuts down the applied field in both directions every time an electrode reaches 41ºC. Our aim is to estimate Optune's duty cycle, suggest ways to improve it and appraise possible thermal damage mechanisms. The simulations were performed in a realistic head model created from MR images and the software used to run these simulations was COMSOL Multiphysics. The results obtained showed that Optune is only effectively delivering the fields around 40% of the time and that the AP configuration is applied during more time than the LR. However, we show that it is possible to increase Optune's duty cycle if 1) the current is shutdown only for the configuration that has the electrode that reached 41ºC while maintaining the other configuration active; 2) the current is injected homogeneously across all the electrodes of the same array; 3) the room temperature is low. We also calculated the Cumulative number of Equivalent Minutes at 43ºC (CEM 43ºC) for 18 hours to evaluate the possible thermal damage mechanisms. Our results showed that thermal damage might occur for the scalp and for the brain. For the scalp, these findings are corroborated with what is seen experimentally because most of the patients treated with this technique suffer from dermatitis. Regarding the brain, the impact of the temperature increase is still unclear as no related effects have been reported to date in clinical trials. However, according to the literature, an increase in temperature may lead to a higher Blood-Brain Barrier (BBB) permeability, a variation of the brain's blood flux and a change in the concentration of some neurotransmitters such as GABA, glycine and glutamate. An increased BBB permeability can be helpful if there is a more suitable drug that can be used for GBM that under normal conditions would not pass to the brain. An increased blood flux can also be helpful to reduce brain's temperature, while a variation in the concentration of the neurotransmitters is unlikely to affect TTFields efficacy. These results can serve as a starting point for future studies to evaluate the thermal impact of this therapy.

#690

Optimization of biopsy scheduling in clinical studies of T cell bispecifics through an integrated modeling and simulation, digital pathology and machine learning approach.

Lucy G. Hutchinson,1 Antoine Soubret,1 Benjamin Ribba,1 Jean-Eric Charoin,1 Alex Phipps,2 Richard Peck,1 Oliver Grimm3. 1 _Roche Innovation Center, Basel, Switzerland;_ 2 _Roche Innovation Center, Welwyn, United Kingdom;_ 3 _Roche Innovation Center, Munich, Germany_.

In the context of cancer immunotherapy clinical trials, baseline and on-treatment tumor biopsies may provide important insight into whether a treatment is working as expected, and furthermore whether efficacy is anticipated. For tumor-retained antibodies that perturb the behaviour of immune cells, such as T cell bispecific antibodies (TCBs), spatial information derived from biopsy images may be particularly insightful. On-treatment biopsies in clinical trials are usually scheduled at a time point that is considered convenient for the study design and when therapeutic effects, such as T cell infiltration, are expected to be distinguishable in tumor tissue. To our knowledge, there have been no attempts to investigate optimal scheduling of on-treatment biopsy sample collection using quantitative approaches due to lack of clinical data at a sufficiently diverse range of time points.

Our integrated tissue pathology, disease modeling and machine learning workflow is designed to select the time point at which on-treatment biopsies could be most informative for making reliable predictions of response to treatment. Leveraging around 20 baseline and on-treatment digitized biopsy images from patients undergoing treatment with immune stimulating TCBs, we train a mathematical model to simulate tumor cell/T cell interactions in the tumor microenvironment. The mathematical model produces an enriched dataset of "virtual" biopsy images corresponding to predictions at intermediate time points. The virtual biopsies are evaluated based on their ability to predict treatment response.

Specific mechanisms of action of bispecific antibodies, such as upregulation of T cell activation and/or proliferation, are taken into account in the structure of the mathematical model. The model is tuned and validated using machine learning techniques, and a reserved "test" dataset comprising images that were not used to estimate model parameters is used to evaluate model performance. Our workflow has the potential to inform clinical study design by promoting a scientific basis for the selection of an on-treatment biopsy schedule. Future applications of this workflow include identification of tissue properties that may contribute to inter-individual variability, and simulations of novel doses and schedules for combinations of immune-modulating cancer therapies.

#691

Simulating TTFields-induced temperature changes within a patient's brain - a proof of concept study.

Ariel Naveh, Ze'ev Bomzon. _Novocure, Haifa, Israel_.

Introduction: TTFields is an antimitotic cancer treatment that utilizes low intensity (1-3 V/cm) alternating electric fields in the intermediate frequency (100-300 kHz) that are delivered in two orthogonal directions using 2 pairs of transducer arrays. TTFields are currently approved for Glioblastoma Multiforme (GBM). Preclinical studies show that the effect of TTFields is intensity-dependent with a therapeutic threshold of 1 V/cm, and that as the intensity rises so does the efficacy. However, delivery of high field intensities could result in temperature increases within the body that lead to thermal damage to tissue. In practice, during treatment with TTFields, tissue heating is limited by a closed loop that adjusts the delivery of TTFields to ensure that skin temperature remains within the safety limits. Since heating limits the intensity at which TTFields is delivered, accurate models of tissue heating during TTFields therapy could provide insights into new approaches for the delivery of TTFields. Since measuring the temperature spatial distribution within a patient's brain is extremely challenging, simulating the heat flow within the head during treatment is a more practical approach. Here, we present preliminary results from a study aimed at developing a valid computational model of heat transfer in the head during TTFields therapy.

Method: A realistic computational head model of a healthy male was used in this study. Virtual transducer arrays were placed on the head. To simulate the delivery of TTFields to the brain, we used ZMT's Sim4Life v4.0 electro-quasi-static solver. The results of the electric field simulation were then used as heat source for the heat simulation. The heat simulation solves the Pennes bio-heat model, taking into account tissue heat capacity and conductivity, and also accounting for heat convection through blood perfusion. Sensitivity analysis was performed to establish the sensitivity of the temperature distribution within the brain to the thermal properties of the various tissue types.

Results: Average temperatures within the brain tissue were well below 38° C for all simulations. Temperature distributions within the brain were highly sensitive to the perfusion coefficient assigned to the cerebrospinal fluid as well as other tissues.Conclusions: We have established a computational framework for investigating heat transfer in the brain during TTFields therapy. Further work is needed to correctly establish the thermal properties of the various tissue types. This may require experimental measurements in which the heat transfer model is fine-tuned and validated.

#692

Treating spinal cord metastases with tumor treating fields.

Ofir Yesharim, Ariel Naveh, Ze'ev Bomzon. _Novocure, Haifa, Israel_.

Introduction: Tumor Treating Fields (TTFields) is an anti-mitotic technology utilizing low-intensity (1-3 V/cm) electric fields within the medium frequency range (100-300 kHz) to treat solid tumors. TTFields are currently approved for the treatment of Glioblastoma (GBM). Some cancer types, such as breast cancer, lung cancer, and melanoma, frequently result in metastases to the spinal cord. Transducer array positioning used to treat abdominopelvic diseases (e.g. pancreatic cancer), in which one pair of arrays are placed on the anterior and posterior of the patient, and another pair of arrays placed on the lateral aspects of the patient have been shown to be inefficient for treating the spinal cord because the clinical threshold for field intensity of 1V/cm is not reached in the spine when these layouts. The structure of the spine, in which a relatively resistive bone structure encapsulates a highly conductive layer of fluid (cerebrospinal fluid), shunts the current delivered across the body by the arrays away from the spine, reducing the field within the spine to below the therapeutic threshold. To overcome this limitation, a new approach of array placement on the body is needed. Here we present a simulation-based study designed to identify array layouts that effectively deliver TTFields to the spine.

Methods: To simulate delivery of TTFields to the spine, we used a human male 34 years old realistic computerized model (DUKE v3.1 by ITI'S, Zurich). In order to generate TTFields, an alternating current with current density of 200mA/disk with a frequency of 150 kHz was imposed on the outer surfaces of the disks of each pair of arrays. The simulations were performed using ZMT's Sim4Life v4.0 electro-quasi-static solver.

Results: One of the array layouts tested was placement of a pair of arrays on the back of the patient. One array is placed above the target region in the spine to which treatment would be delivered, and the other array is placed below the region. With this configuration, a high electric field is induced within the spinal cord and surrounding CSF. Mean field intensity within the spine and nerves in between vertebrae T8-T9 at the top, and L3-L4 at the bottom were calculated at a value of 1.77 V/cm. It is important to note that the electric field is oriented along the spine in this instance (a vertical field). In general, the whole spine could be treated by placing one array on the neck, and one at the bottom of the spine.

Conclusions: Placing two transducer arrays on the patient's back allows single-direction treatment of the spinal cord and nerves. Further work should be conducted to find an effective treatment in the perpendicular direction.

#693

Deep learning analysis of circulating tumor DNA identified a pan-tumor molecular subtype with enhanced response to durvalumab (anti-PDL1).

Song Wu,1 Sriram Sridhar,1 Han Si,1 Michael Kuziora,1 Brandon W. Higgs,1 Koustubh Ranade,1 Judson Englert,1 Shaad E. Abdullah,1 Philip Dennis,2 Feng Xiao,2 Guozhi Gao1. 1 _MedImmune, Gaithersburg, MD;_ 2 _Astrazeneca, Gaithersburg, MD_.

Tumor mutation burden (TMB) has emerged as a promising predictive biomarker for anti-PD1/L1; however, whether somatic mutations in specific genes can sensitize to immunotherapy is poorly understood. We used deep learning to identify molecular subtypes from mutations detectable in circulating tumor DNA (ctDNA) and associated these with outcomes with anti-PDL1 (durvalumab) and durvalumab + anti-CTLA4 (tremelimumab) therapy.

Patient blood samples from 4 nonrandomized phase 2 trials evaluating durvalumab (NCT01693562, 1L+ solid tumors; NCT02087423, 3L+ NSCLC) or durvalumab+ tremelimumab (NCT02000947, 2L+ NSCLC; NCT02261220, 2L+ solid tumors) were sequenced with the Guardant 360 assay of 73 genes. CtDNA mutations were examined pre-treatment in a discovery cohort of 818 patients composed of >15 tumor types and validated in a cohort of 94 patients composed of >5 tumor types. Deep learning neuron network was computed using Tensorflow to identify 4 unique molecular subgroups. Kaplan-Meier analysis was calculated and objective response rates (ORR) were assessed with RECISIST v1.1.

The subgroups consisted of: DS1 (N = 215/818, 26%) enriched in RB1 mutations (17%) and low TMB, DS2 (N = 237/818, 29%) enriched in ARID1A (33%), NFE2L2 (8%), and cKIT (13%) mutations, DS3 (N = 251/818, 31%) enriched in EGFR, ERBB2, BRAF and STK11 mutations, and DS4 (N = 115/818, 14%) enriched in TERT, FGFR3, and PIK3CA mutations. In the discovery cohort, the DS2 subgroup had most improved outcomes (mOS=24.6 months, mPFS=3.7 months, ORR=27%) compared to the other 3 subgroups. DS1 subgroup had the worst outcomes (mOS=6.3 months, mPFS=1.6 months, ORR=10%), while the biomarker evaluable population (BEP, n=818) had ORR=17%. In the validation cohort, DS2 also had the most improved outcomes (mOS=not reached, mPFS=13.2 months, and ORR=64%), with the BEP (n=94) having an ORR=37%. PD-L1 was not correlated with any specific molecular subgroup, while an IFNg mRNA signature trended with correlation with subgroup DS2.

Deep learning analysis of ctDNA identified a molecular subgroup with enhanced response to anti-PDL1 treatment in multiple tumor types.

#694

Mathematical modeling of stromal fibroblast-mediated adaptive resistance to bortezomib in multiple myeloma.

Elliot Eton,1 Kamran Kaveh,2 Sachin Khiste,1 Andrea Fernandes,1 Ashima Agarwal,1 Shiladitya Sengupta,1 Aaron Goldman1. 1 _Harvard Medical School, Boston, MA;_ 2 _Program for Evolutionary Dynamics at Harvard University, Cambridge, MA_.

Understanding intrinsic and adaptive resistance is crucial to overcoming multiple myeloma chemotherapy failure. We recently demonstrated that standard chemotherapy can induce cancer cells to phenotypically transition to a transient state of dormancy and drug tolerance. Further, targeting cells precisely during this transition can thwart the emergence of adaptive resistance (Goldman, 2015). However, it is not yet understood how the stromal contexture may contribute to this chemotherapy-induced drug tolerance.

We developed a deterministic mathematical model containing a system of differential equations to examine how stromal fibroblasts temporally affect phenotypic switching between active and dormant, bortezomib-tolerant cancer cell states. The equations describe the total growth rate of each cell state population, which is influenced by that population's count and baseline growth rate, the frequencies of transitions to and from the other cell state, the drug treatment concentration, and the population carrying capacity. The model also establishes that the presence of fibroblasts affects the frequency of transitions between the cell states. This framework is calibrated to in vitro data, and model simulations predict that bortezomib-tolerant myeloma cells may escape more quickly from dormancy and proliferate when they are incubated in the presence of fibroblasts than in their absence. This prediction is supported by preliminary experimental observations. The mathematical model could be adapted to predict how stromal fibroblasts, or the tumor microenvironment generally, may influence phenotypic switching in other cancers.

#695

Using PSA dynamics to predict patient-specific responses to intermittent androgen deprivation.

Renee Brady, Heiko Enderling. _H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL_.

Intermittent androgen deprivation therapy (IADT) is an attractive treatment approach for biochemically recurrent prostate cancer, whereby cycling treatment on and off has been shown to limit toxicities and reduce cumulative dose. While IADT has been shown to delay the development of treatment resistance, underlying mechanisms and actionable biomarkers are required to accurately predict when resistance will emerge and how to maximally delay time to progression. We developed a quantitative framework to simulate the enrichment of prostate cancer stem cell dynamics during treatment as a plausible mechanism of resistance evolution. Using simulated dynamics of cancer stem cells and non-stem cancer cells we derive longitudinal blood serum prostate-specific antigen (PSA) concentrations that were calibrated to and validated with data of 86 patients undergoing multiple cycles of IADT. Model analysis suggests that cancer stem cell proliferation patterns correlate with PSA dynamics and patient outcomes. Learning these dynamics adaptively from the earlier treatment cycles in individual patients can predict the evolution of resistance in subsequent IADT cycles with a predictive power of 80%, which warrants prospective evaluation in a clinical setting.

#696

High-throughput screening of therapeutic combinations against ovarian cancer.

Peng Zhou, Alan S. Wong. _The University of Hong Kong, Hong Kong, China_.

Applying synergistic drug combinations can increase efficacy of cancer treatment. However, conventional methods for identifying those combinations involve testing of every possible combination including different dosages for each agent, are difficult to scale due to the huge amount of efforts and costs needed. To tackle this problem, we have developed CombiGEM-CRISPR (combinatorial genetics en masse-CRISPR) to enable high-throughput assembly and screening of pair-wise gene combinations. By applying CombiGEM-CRISPR,phenotypes of combinatorial gene knockouts could be used to predict the effects of drug inhibitor combinations targeting the matching gene products. We designed a pair-wise sgRNA library comprising 25,281 combinations for interrogating 52 genes that are overexpressed in ovarian cancers. By pooled evaluation of their effects on cell proliferation, our screening identified new combinations that inhibit ovarian cancer cell growth. Therefore, our work accelerates the discovery of new therapeutic options against ovarian cancer through applying CombiGEM-CRISPR.

## MOLECULAR AND CELLULAR BIOLOGY / GENETICS

### Apoptosis

#697

Degradation of cancer mitochondria by the mitochondria-eating protein induces iron-dependent cancer cell death.

Yasuyuki Nakamura, Naoki Ikari, Hirofumi Arakawa. _National Cancer Ctr. Research Inst., Tokyo, Japan_.

Parkin/Pink1-mediated mitophagy plays a critical role in mitochondrial quality control, in which the unhealthy mitochondria are sequestered by autophagosomes, and degraded by fusion between autophagosomes and lysosomes. Here, we report non-canonical mitophagy as a new function of tumor suppressor p53. The mitochondria-eating protein (Mieap) was originally identified as a p53-target gene. Mieap induced large vacuole-like structures (Mieap-induced vacuoles: MIVs) in cancer cells. MIVs were generated from around the mitochondria, and directly ate and degraded cancer mitochondria without autophagosomes. Therefore, we defined the function as the Mieap-regulated non-canonical mitophagy. UVRAG was found to be a Mieap-interacting protein that mediates the MIVs formation. Interestingly, degradation of cancer mitochondria by the MIVs induced cell death via iron-dependent production of reactive oxygen species. Introduction of exogenous Mieap strikingly suppressed in vivo tumor growth of Mieap-deficient colorectal cancer cells. Deficiency of endogenous Mieap dramatically promoted cancer development, malignancy, and aggressiveness in colorectal and gastric cancer model mice. The Mieap-induced cell death was found to occur in in vivo hypoxic tumor microenvironment of colorectal cancer tissues. These results suggest that degradation of cancer mitochondria by Mieap-regulated non-canonical mitophagy plays a critical role in p53 tumor suppression via iron-dependent cancer cell death.

#698

**Enhancing apoptosis to improve the therapy of** EGFR **-mutant lung cancer.**

Kosuke Tanaka,1 Song Han,1 Yogesh T. Ganesan,1 James J. Hsieh,2 Emily H. Cheng1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Washington University, St. Louis, MO_.

EGFR-TKIs have shown clinical efficacy in EGFR-mutant lung cancer patients, but most of the cases eventually develop resistance to the drugs, including the third-generation EGFR-TKI. Here, we develop a cell death mechanism-based combination strategy to enhance the therapeutic effect of EGFR-TKIs as well as to overcome the acquired resistance to EGFT-TKI in EGFR-mutant lung cancer. Through high-throughput screening of a custom pathway inhibitor library (-1000 drugs), we discovered Aurora kinase inhibitors that synergized with a third-generation EGFR-TKI (AZD9291) to induce apoptosis in EGFR-mutant lung cancer cells. Aurora kinase inhibitors enhanced AZD9291-mediated induction of BIM and PUMA, two important sentinels interconnecting the EGFR signal transduction pathway and the mitochondrial apoptotic machinery BAX and BAK. Combined inhibition of Aurora kinases and EGFR prevented the emergence of EGFR-TKI resistant clones in clonogenic assays. Interestingly, AZD9291 resistant lung cancer cells exhibited greater sensitivity to Aurora kinase inhibitors compared with parental cells. In conclusion, we found that the combination of AZD9291 and Aurora kinase inhibitors synergistically induced BIM and PUMA-mediated apoptosis, which may render a promising strategy to improve the therapy of EGFR-mutant lung cancer. <!--EndFragment-->

#699

KMT2A regulates cervical cancer cell growth andapoptosisthrough cooperation with VDAC1.

Changlin Zhang,1 Yizhuo Li,1 Qian Long,1 Tianze Liu,2 Ge Qin,1 Dingbo Shi,1 Miao Chen,1 Kefang Zhang,3 Shuihan Shi,3 Wuguo Deng1. 1 _Sun Yat-sen University Cancer Center, Key State Laboratory of Oncology in South China, Guangzhou, China, Guangzhou, China;_ 2 _Radiological Department of Oncology, The Fifth Affiliated Hospital, Guangzhou, China;_ 3 _Global Life Care Institute, Guangzhou, China, Guangzhou, China_.

Cervical cancer is an aggressive cutaneous malignancy, illuminating the exact mechanisms of tumorigenesis and discovering novel therapeutic targets are urgently needed. Lysine methyltransferase 2A (KMT2A) is a transcriptional co-activator regulating gene expression during early development and hematopoiesis, but the role of KMT2A in cervical cancer remains largely unknown. Here, we showed that KMT2A regulated cervical cancer cell growth and apoptosis through cooperation with VDAC1 (voltage dependent anion channel 1). Knockdown of KMT2A significantly inhibited cell viability and cell migration and induced apoptosis in cervical cancer cells. Overexpression of VADC1 reversed the KMT2A knockdown-mediated suppression of cell viability and cell migration and induction of apoptosis. The results from a xenograft mouse model confirmed that KMT2A regulated cervical cancer growth via the VDAC1 signaling. Moreover, analyses of clinical samples demonstrated that the expression of KMT2A and VDAC1 were positively correlated in cervical cancer tissues, especially in cervical intraepithelial neoplasia (CIN) samples. Collectively, our results indicated that KMT2A promoted cervical cancer growth by cooperating with the VDAC1, suggesting that the KMT2A/VDAC1 signaling pathway may be a potential therapeutic target for cervical cancer.

#700

Development of a second-generation TRAIL agonist and predictive biomarker profile for colorectal cancer.

Sara Ghassemifar,1 Yasmin Hashambhoy-Ramsay,1 Haluk Yuzugullu,1 Tamara Utermark,1 Violette Paragas,1 Tim Maiwald,1 Lia Luus,1 Maja Razlog,1 Hannah Hudson,1 Diane Chai,1 Birgit Schoeberl,1 C. Patrick Reynolds,2 Peter Sorger,3 Andrew J. Sawyer,1 Daryl C. Drummond,1 Eric Tam1. 1 _Merrimack, Cambridge, MA;_ 2 _Texas Tech University, Lubbock, TX;_ 3 _Harvard University, Cambridge, MA_.

In colorectal cancer (CRC), RAS mutated cancers comprise 30-50%, for which there are limited treatment options. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has been found to selectively induce cell death in 50% of the CRC cell lines tested in vitro and this appears to be independent of RAS mutation status. Minimal clinical success was observed for first generation TRAIL receptor agonists primarily due to poor agonist activity and poor bioavailability. In addition, a biomarker to identify responsive patients was lacking. Here we present the therapeutic development of MM-201, a next generation TRAIL receptor agonist, and its companion biomarker strategy. MM-201 is composed of an IgG1 Fc fused to a single chain TRAIL trimer (Fc-scTRAIL) engineered for improved stability and activity. Using TRAIL surface display on yeast, we identified three mutations (R130G/N228S/I247V) from a random mutagenesis library that lead to enhanced surface expression and DR5 binding. Incorporation of these three mutations into each TRAIL protomer of MM-201 resulted in an improvement of greater than 10°C in TM and an extended serum half-life of 33 hours in mice compared to wild-type Fc-scTRAIL and comparable activity in a panel of colorectal cancer (CRC) cell lines. To uncover predictive biomarkers for MM-201, we used continuous logic gate analysis on a publicly available dataset of cell survival for 27 CRC cell lines treated with cross-linked TRAIL cytokine. Using this dataset and gene expression data from Cancer Cell Line Encyclopedia, we explored combinations of 33 differentially expressed genes in the apoptosis pathway and identified a two gene biomarker signature that positively correlated with TRAIL response in CRC cell lines. The combined signature of high DR4 and low cIAP-1 was identified with a correlation coefficient of 0.99 with MM-201 response across 15 CRC cell lines both in vitro and in vivo. Subsequently we evaluated MM-201 and our biomarker approach in vivo, in a group of five CRC patient-derived xenograft (PDX) models. Four of the five models were sensitive to MM-201, with an average tumor growth inhibition (TGI) of 91%. The relative expression of DR4 and cIAP-1 in each of the PDX models was measured by RT-qPCR and the biomarker scores were observed to be significantly higher for all responding models. Given the responses observed in preclinical models, we used TCGA data to assess biomarker prevalence in patient tumor tissues. This analysis predicted that 59% of samples would have a biomarker score greater than 0.5, which correlates to a 75% reduction in cell viability in vitro. In contrast, only 12% of normal tissues had a score in this range. Our biomarker signature identifies a potentially responsive patient population and when combined with our next generation TRAIL agonist, MM-201, has the potential for development in CRC, especially for RAS mutated cancers.

#701

**Mitochondrial mass is a critical determinant of** cis **Pt-induced cell death in ovarian cancer.**

Markus Kleih,1 Kathrin Böpple,1 Andrea Gaißler,1 Meng Dong,1 Walter E. Aulitzky,2 Frank Essmann1. 1 _Dr Margarete-Fischer-Bosch Institute, Stuttgart, Germany;_ 2 _Robert-Bosch Hospital, Stuttgart, Germany_.

Platinum compounds are an inherent part of chemotherapy for patients with high grade serous ovarian cancer (HGSC). Platinum based therapeutics, such as cisPt, induce DNA-damage by directly binding to nuclear DNA. Interestingly, cisPt also critically affects mitochondrial DNA. Elucidating the role of mitochondria in cisplatin induced cell death of HGSC cells likely identifies new efficient therapy approaches to overcome resistance. Flow cytometric analysis of cisPt sensitive and resistant HGSC cell lines from the NCI60 panel revealed higher mitochondrial mass and higher levels of mitochondrial ROS (mtROS) in cells that are sensitive to cisPt-induced cell death. In clonal sub-lines derived from OVCAR3 the mitochondrial mass correlates with basal oxygen consumption rate and survival after cisPt exposure. The correlation of mitochondrial mass and sensitivity to cisPt-induced apoptosis is corroborated by data from the human protein atlas (www.proteinatlas.org) that indicates high expression of the mitochondrial transcription factor TFAM and the mitochondrial inner membrane protein TIMM23 as favorable prognostic factors for the survival of ovarian cancer patients. Together, this shows the importance of mitochondrial mass in cellular response to cisPt treatment. Furthermore, exposure to cisPt enhances TFAM expression and increases mitochondrial mass as well as mitochondrial ROS. Incubation with cisPt in the presence of the ROS scavenger glutathione (GSH) or pan-caspase inhibitor zVAD-fmk reduces cisPt induced apoptosis while mitochondrial mass is not affected. However, the oxygen consumption rate in cells exposed to cisPt and GSH shows that increased ROS levels induce mitochondrial dysfunction. In line with a sensitivity-determining role of mitochondrial mass, knock-down of key-regulators of mitochondrial biogenesis, i.e. PGC-1α or TFAM, blocks mitochondrial ROS and protects cells from cisPt-induced apoptosis. Mitochondrial ROS is also reduced in an autoregulatory feedback loop by so-called uncoupling proteins (UCPs). Inhibition of UCP2 by Genipin results in increased induction of mitochondrial ROS by cisPt and consequently results in enhanced apoptosis. A comparable sensitization to cisPt-induced apoptosis and ROS production is evident in cells incubated with the iron chelator VLX600.Mitochondria are a critical element in cell response to cisPt since mitochondrial mass correlates with induction of ROS and apoptosis. We show the relevance of cellular mitochondrial content by manipulation of mitochondrial mass and function. We propose the relative mitochondrial content as a biomarker that indicates the response of ovarian cancer cells to cisPt-induced apoptosis. Furthermore, our data provides evidence that increasing mitochondrial mass or induction of mtROS enhances sensitivity to cisPt-induced apoptosis and therefore is a reliable strategy to overcome resistance of ovarian cancer.

#702

Kinetic analysis identifies determinants of sensitivity to MEK inhibitor-induced cell death.

Zintis Inde, Kyuho Han, Michael C. Bassik, Scott J. Dixon. _Stanford University, Stanford, CA_.

Successful identification of the genetic determinants of drug sensitivity has been a long-standing goal of cancer research and, more recently, of precision medicine. In cancer cells, drug treatment can induce both growth arrest and death. The distinction between these outcomes is important but can be overlooked in large-scale analyses that rely on traditional metabolism-based methods for quantifying cell viability. We recently demonstrated that Scalable Time-Lapse Analysis of Cell Death Kinetics (STACK) effectively quantifies cell death in response to diverse lethal stimuli. We investigated cases in which this approach captured drug-induced lethality more effectively than traditional assays. We found that inhibitors of mitogen activated protein kinase 1 and 2 (MEK1/2) arrested proliferation in diverse cancer cell lines, but induced cell death in only a subset of these lines. We sought to identify the factors that determine whether a cancer cell will undergo growth arrest or death in response to MEK inhibition. MAP kinase pathway activation has previously been reported as a determinant of MEK inhibitor sensitivity; we determined that the dynamics of Bcl-2 family proteins in response to MEK inhibitor treatment correlate with death sensitivity independent of pathway activation. In a genome-wide CRISPR screen, we identified a lipid metabolic enzyme that is a novel modulator of MEKi sensitivity. Our results suggest new approaches for predicting and enhancing sensitivity to cancer therapeutics in tumors and demonstrate that direct measurement of cell death provides clear advantages over proliferation-based metrics.

#703

An anthelmintic drug, flubendazole, exerts antitumor effects in triple-negative breast cancer via targeting cancer stem-like properties.

Yoon-Jae Kim, Eunhye Oh, Tae-Min Cho, Seojin Jang, Jung Min Park, Soeun Park, Minsu Park, Ji Young Kim, Jae Hong Seo. _Korea University, Seoul, Republic of Korea_.

Triple-negative breast cancer (TNBC) remains challenging due to the absence of established molecular targets for the phenotype. These tumors tend to be more aggressive with a higher risk of recurrence and metastasis, leading to poorer clinical outcomes. Flubendazole (FLU) is a widely used anthelmintic agent that also exhibits anticancer activity in several cancer types. The objective of the present study was to investigate the effects of FLU on cell proliferation, apoptosis and breast cancer stem cell (BCSC)-like properties in TNBC. The effect of FLU on TNBC cell lines in vitro was evaluated in terms of apoptosis, cell cycle distribution, caspase-3/-7 activity and BCSC-like characteristics. An orthotopic allograft model with 4T1 mammospheres was used to examine the effect of FLU on tumor growth and angiogenesis in vivo. FLU efficiently induced apoptosis with accompanied by activation of caspase-3 and -7, and PARP cleavage in TNBC. FLU-induced apoptosis is associated with the accumulation of G2/M phase cells by tubulin degradation, leading to mitotic catastrophe. Interestingly, FLU significantly suppressed BCSC-like properties in TNBC, as evidenced by a marked inhibition of ALDH1 activity, the CD44high/CD24low and CD49fhigh/CD24high subpopulations, and the subsequent impairment of mammosphere formation in vitro. This appears to contribute toward the suppression of BCSC-enriched tumor burden and intra-tumoral angiogenesis in TNBC allografts, together with a significant downregulation of ALDH1A1 and CD49f expression in vivo. Our findings suggest that FLU kills not only rapid proliferating tumor cells but also effectively eradicates BCSC-like cells in vitro and in vivo. These findings warrant further investigation of flubendazole as a potential treatment for triple-negative breast cancer.

#704

**RALB GTPase: A critical regulator of DR5 cell surface expression and TRAIL sensitivity in** RAS **mutant colorectal cancer.**

Hajrah Khawaja,1 Jamie Z. Roberts,1 Arman Javadi,1 Paul O'Reilly,1 Darragh McArt,1 Wendy L. Allen,1 Markus Morrison,2 Richard Kennedy,1 Nicolas Vitale,3 Patrick G. Johnston,1 Tim Harrison,1 Daniel B. Longley,1 Emma Evergren,1 Sandra Van Schaeybroeck1. 1 _Queen's University Belfast, Belfast, United Kingdom;_ 2 _University of Stuttgart, Stuttgart, Germany;_ 3 _University of Strasbourg, Strasbourg, France_.

RAS mutant (MT) metastatic colorectal cancer (mCRC) remains a difficult-to-treat group. RALA and RALB GTPases function downstream of RAS and have been found to be key regulators of several cell functions and implicated in KRAS-driven tumourigenesis. However, their role as regulators of the apoptotic machinery remains to be elucidated. Here, we found that inhibition of RALB expression, but not RALA, resulted in caspase 8-dependent cell death in KRASMT cells, that was not further increased following MEK1/2 inhibition. Proteomics analysis and mechanistic studies revealed that RALB inhibition induces marked upregulation of the pro-apoptotic cell-surface TRAIL Death Receptor 5 (DR5) receptor in KRASMT CRC. Moreover, DR5 knockout also attenuated siRALB-induced apoptosis, confirming the role of the extrinsic apoptotic pathway as regulator of siRALB-induced cell death. Importantly, TRAIL treatment resulted in acute association of RALB with the death-inducing signalling complex (DISC) and targeting RALB using pharmacologic inhibition or RNAi approaches, resulted in a potent increase in TRAIL-induced cell death in RASMT CRC. Significantly, high RALB mRNA levels were found in the poor prognostic CRIS-B CRC subgroup. Collectively, this study provides the first evidence for a role of RALB in apoptotic priming and that RALB inhibition may be a promising strategy to improve response to TRAIL treatment in poor prognostic RASMT CRC.

#705

**Tumor intrinsic PD-L1 is mediated by** BRAFV600E **and can regulate drug-induced apoptosis in human colon cancer.**

Daofu Feng, Krishnendu Pal, Debabrata Mukhopadhyay, Xin Liu, Haidong Dong, Shengbing Huang, Frank A. Sinicrope. _Mayo Clinic College of Medicine, Rochester, MN_.

Resistance to chemotherapy remains a major obstacle to improving outcomes of cancer treatment. Recent data suggest intrinsic functions of immune checkpoint proteins, including PD-L1, that may be distinct from their immunoregulatory roles. We found that colorectal cancer (CRC) cells with mutant BRAF(V600E) or KRAS alleles show increased PD-L1 transcripts and expression of cell surface PD-L1 protein compared to those with wild-type copies. Furthermore, ectopic expression of BRAFV600E or mutant KRAS induced PD-L1 whose upregulation was attenuated by inhibition of RAS-RAF-MEK-ERK signaling. Combined knockdown of MEK/ERK effectors c-JUN and YAP markedly reduced PD-L1. Knockout of PD-L1 in RKO cells resulted in a reduction in chemotherapy-induced DNA double strand breaks (pH2AX) and caspase-3 cleavage compared to parental cells. Results were confirmed in PD-L1 knockout MC38 murine CRC cells where re-expression of PD-L1, but not its deletion mutants, was shown to induced pH2AX and apoptosis. In cells with knockout of PD-L1, reductions in p-AKT and the BH3-only proteins BIM and BIK were observed that could be restored by re-expression of PD-L1. Treatment of cells with an anti-PD-L1 antibody reduced p-AKT, BIM and BIK, and also attenuated chemotherapy-induced apoptosis. Re-expression of BIM in PD-L1 knockout cells restored apoptosis. Murine tumor xenografts generated from cells with knockout of PD-L1 displayed resistance to oxaliplatin-induced tumor regression compared to tumors derived from parental cells. Using TCGA datasets, human CRCs with high vs low PD-L1 mRNA levels were significantly associated with better survival. In summary, we identified a non-immune function of tumor cell-intrinsic PD-L1 that upregulates BH3-only proteins BIM and BIK to promote apoptosis, thereby suggesting its potential as a predictive biomarker.

#706

A natural inhibitor of c-Met that sensitizes head and neck cancer cells resistant to EGFR and PI3K co-targeting.

Asm Anisuzzaman,1 Abedul Haque,2 Brian Butler,3 ARM R. AMIN3. 1 _Emory University, Atlanta, GA;_ 2 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _Marshall University, Huntington, WV_.

Purpose: Molecularly targeted agents will play a major role in the next generation of personalized cancer therapies. However, intrinsic and acquired resistance to targeted agents poses challenges to the success of such treatments. The Cancer Genome Atlas data suggest that both EGFR and PI3K pathways are reasonable targets in head and neck cancers (HNC). However, single agent treatment with EGFR inhibitor erlotinib or pan-PI3K inhibitor BKM120 fails to induce apoptosis of most HNC cell lines. We have previously reported that EGFR and PI3K co-targeting has synergistic antitumor effects both in vitro and in vivo and induces variable apoptosis. The purpose of the current study was to investigate the mechanism of intrinsic resistance to apoptosis induced by the combination of EGFR inhibitor erlotinib and PI3K inhibitor BKM120 and to sensitize the resistant cells with natural extracts.

Methods: In a panel of 10 malignant and 1 premalignant HNC cell lines, we evaluated apoptosis by Annexin-V staining. Protein expression levels were measured by Western blotting. Small molecule chemical inhibitors and siRNA-mediated knockdown strategies were used to inactivate and shut down the expression of the relevant proteins, respectively.

Results: The combination of erlotinib and BKM120 induced variable apoptosis. Some cell lines were very sensitive (Tu686, 686LN, 93-VU-147T), some were moderately sensitive (Fadu, SqCCy1, 1483, UMSSC90), and others were resistant (UD-SCC2, MSK-LEUK1, JHU022) to apoptosis induction. JHU022 and 1483 cell lines expressed very high levels of phosphorylated c-Met. Targeting c-Met with the small molecule inhibitor crizotinib or siRNA rendered these cell lines highly sensitive to erlotinib and BKM120 induced apoptosis. Moreover, inhibition of Src family kinases (SFK) with the small molecule inhibitor dasatinib or with c-Src-specific siRNA inhibited phosphorylated c-Met. In addition, inhibition of SFK in these cell lines conferred sensitivity to erlotinib and BKM120 induced apoptosis. We also screened several natural extracts isolated from microorganisms. We found that ACM, one of these extracts, strongly induced apoptosis of the resistant cell lines. Moreover, the extract inhibited constitutively active p-Met in the resistant cell line.

Conclusions: Our results strongly suggest that some of the resistant cell lines expressed high levels of phosphorylated c-Met downstream of SFK. This confers resistance of these cells to the combination of erlotinib and BKM120 as inhibition of this pathway sensitizes the cells. Moreover, the bacterial extract ACM is a natural inhibitor of c-Met which sensitizes these resistant cell lines. It may be an excellent direction for further drug development.

#707

Emerging role of mitochondrial pathway in crosstalk between ferroptosis and apoptosis.

Yong J. Lee. _Univ. of Pittsburgh Cancer Inst., Pittsburgh, PA_.

Introduction: Several processes lead to cell death; ferroptosis, apoptosis, necrosis, and autophagy are the primary mechanisms. Each type of biological death has been believed to have a distinct biochemical and morphological fingerprint. However, emerging evidence suggests that these different types of cell death often share common pathways. In this study, we investigated whether mitochondrial signaling pathways play an important role in the intracellular communication between ferroptosis and apoptosis.

Methods: The human colorectal carcinoma HCT116 cell line was used to evaluate the interplay between cell death pathways. Cells were treated with the apoptotic agent TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) and ferroptotic agents such as artesunate and erastin. Cell death was determined by trypan blue exclusion assay. Apoptosis was measured by western blot. Ferroptosis was assessed using MDA assay. Bax oligomerization was performed by mitochondrial fractionation followed by SDS-PAGE under nondenaturing conditions.

Results: We observed that the combinatorial treatment induced synergistic apoptosis, which was mediated through an increase in caspase activation. We also observed an increase in pro-apoptotic Bax oligomerization in the mitochondrial fraction. The combined treatment-induced apoptosis was suppressed in Bax-deficient HCT116 cells. Ferroptotic agents enhanced the intracellular level of anti-apoptotic Mcl-1. However, the combined treatment decreased the level of Mcl-1. Interestingly, the combined treatment-induced apoptosis was abolished in HCT116 cells with knockin of Mcl-1 phosphorylation site mutant (S121A/E125A/S159A/T163A).

Conclusions: These data suggest a novel mechanism for the combined treatment of the apoptotic agent TRAIL and ferroptotic agent erastin/artesunate-induced synergistic apoptosis through the mitochondrial pathway.

#708

Targeting fatty acid synthase induces apoptosis and senescence.

Travis Van der Steen,1 George Kemble,2 Ruth Lupu1. 1 _Mayo Clinic, Rochester, MN;_ 2 _3-V Biosciences, Menlo Park, CA_.

Fatty acid synthase (FASN) is a novel therapeutic target for cancer as it has increased expression in many different types of cancers. There has been significant understanding that cancer cells can hijack and modify tightly regulated metabolic pathway for its own survival. FASN is the enzyme that synthesizes palmitate from malonyl-CoA for energy storage, structure and biomolecules. FASN expression is low to undetectable in normal cells with the exception of liver and adipose tissue. In this study, we investigated the role of FASN in breast cancer cells. We observed that in highly expressing FASN breast cancer cells, TVB-3166, FASN inhibitor, results in a decrease in cellular proliferation and an increase in cellular death. FASN inhibition increased reactive oxygen species (ROS) and altered NADPH/NADP+ ratio. Increase in ROS and NADPH have been linked to the intrinsic apoptosis pathway. Additionally, there is an increase in expression of the BH3-only proteins Bim, Puma and Noxa, leading to the permeabilization of the mitochondria and the release of cytochrome c. Furthermore, after prolong exposure to TVB-3166 a number of cells enter into a senescent state. This was observed the in detection of β-galactosidase and confirmed by the expression of senescent markers including ARF, INK4A, and CIP. Additionally, when the cells were treated with a CDK4/6 inhibitor in combination with the FASN inhibitor TVB-3166, an increase in stress induced premature senescence was observed. Our data demonstrates that inhibition of FASN induces intrinsic apoptosis while it stimulates cellular senescence to the apoptotic resistant cells. Therefore, the ability for TVB-3166 to both eradicate and overturn cell growth makes it potential therapeutic modality.

#709

Cordycepin induces apoptosis of human ovarian cancer cells by inhibiting CCL5-mediated Akt/NF-κB signaling pathway.

Ik-Soon Jang. _Korea Basic Science Inst., Daejon, Republic of Korea_.

The chemokine, CCL5, is a key mediator for the recruitment of immune cells into tumors and tissues. Akt/NF-κB signaling is significantly activated by CCL5. However, the role of NF-κB inactivation in apoptosis induced by negative regulation of CCL5 remains unclear. Here, we analyzed the effect of cordycepin on NF-κB activity in SKOV-3 cells and found that cordycepin-mediated inhibition of NF-κB signaling induced apoptosis in SKOV-3 cells via the serial activation of caspases. In addition, immune-blotting analysis showed that CCL5 is highly expressed in SKOV-3 cells. In addition to activating caspases, we show that, cordycepin prevents TNF-α-induced increase in CCL5, Akt, NF-κB, and c-FLIPLactivation and that CCL5 siRNA could inhibit Akt/NF-κB signaling. Moreover, cordycepin negatively regulated the TNF-α-mediated IκB/NF-κB pathway and c-FLIPL activation to promote JNK phosphorylation, resulting in caspase-3 activation and apoptosis. Also, we show that c-FLIPL is rapidly lost in NF-κB activation-deficient. siRNA mediated c-FLIP inhibition increased JNK. SP600125, a selective JNK inhibitor, downregulated p-JNK expression in cordycepin-treated SKOV-3 cells, leading to suppression of cordycepin-induced apoptosis. Thus, these results indicate that cordycepin inhibits CCL5-mediated Akt/NF-κB signaling, which upregulates caspase-3 activation in SKOV-3 cells, supporting the potential of cordycepin as a therapeutic agent for ovarian cancer.

#710

Real-time apoptosis and necrosis detection in 3D spheroid cellmodels.

Andrew L. Niles, Kevin R. Kupcho, Terry L. Riss, Dan F. Lazar, James J. Cali. _Promega Corp., Madison, WI_.

The structural and biological complexity of cancer spheroids make them uniquely suited to address or approximate the pharmacodynamics of new chemical entities directed at chemotherapeutic reduction of tumor masses. Unfortunately, this same complexity represents a substantial technical challenge for measuring apoptosis and necrosis using conventional methods in real time. To address this challenge, we explored the use of a multiplexed bioluminescent and fluorescent, real-time assay reagent which utilizes annexin V fusion proteins containing binary elements of a complementing luciferase, a time-released luciferase substrate and an excludable DNA membrane integrity dye. The assay reagent was added to HCT-116 and HepG2 spheroids dosed with serial dilutions of panobinostat, bortezomib, or paclitaxel to examine the magnitude, potency and kinetics of the responses. Luminescent and fluorescent data were continuously collected throughout the 48h exposure using a conventional multimode plate reader equipped with atmospheric control. After the exposure period, the treated spheroids were imaged by fluorescence microscopy to assess necrotic burden as well as analyzed for remaining cellular viability using ATP content compared to untreated control. Consistent with their known modes of cytotoxic action, bortezomib produced the first measurable phoshatidylserine (PS) exposure (~8h), followed by panobinostat (~12h) and paclitaxel (18-22h). The apoptotic potencies and magnitudes of response matured thereafter as a function of time until 48h. Secondary necrosis resulting from completion of the apoptotic program lagged PS exposure in both time and magnitude. Imaging results for necrosis and ATP determinations provided orthogonal and inversely complementary verification of data values obtained with the apoptosis assay reagent. Taken together, this workflow may help define the in vitro pharmacological disposition of new chemical entities with respect to their capability to penetrate and induce apoptosis in spheroids and/or other more complex multicellular models.

#711

p53 causes butein-mediated apoptosis of chronic myeloid leukemia cells.

SeoYeon Lee,1 Yu-Jeong Choi,1 Myeong-sun Kim,1 Sooyeon Kang,1 Ji Hye Kim,1 Sang Mi Woo,1 Seong-Gyu Ko,1 Sukjoong Oh2. 1 _Kyung Hee University, Seoul, Republic of Korea;_ 2 _Sungkyunkwan University School of Medicine, Seoul, Republic of Korea_.

Progression of chronic myeloid leukemia, marked by the oncogenic Bcr-Abl mutation, is tightly associated with an alteration of the p53 pathway. It is known that butein extracted from various plants represses cancer growth. Although the anticancer effects of butein are widely accepted, the mechanisms by which butein induces apoptosis of chronic myeloid leukemia cells remains to be elucidated. The present study demonstrated that butein-induced apoptosis was mediated by p53. KBM5 chronic myeloid leukemia (CML) cells expressing wild-type p53 were more sensitive to butein compared with p53-null K562 CML cells in terms of apoptotic cell death. In addition, butein arrested KBM5 cells at S-phase and altered the expression levels of certain cyclins and the p53-downstream targets, MDM2 and p21. In addition, while butein reduced the protein expression of MDM2 in the KBM5 and K562 cells, it resulted in proteasome-independent MDM2 degradation in p53-expressing KBM5 cells, however, not in p53-null K562 cells. Therefore, the present study suggested that p53 causes the butein-mediated apoptosis of leukemic cells.

#712

Down-regulation and nuclear localization of survivin by sodium butyrate induces caspase-dependent apoptosis in human oral mucoepidermoid carcinoma.

Chi Hyun Ahn, Sung-Dae Cho. _Seoul National University, School of Dentistry, Seoul, Republic of Korea_.

Sodium butyrate (NaBu) is a histone deacetylase inhibitor that possess an apoptotic ability. However, the molecular mechanism by which NaBu induces apoptosis in human oral mucoepidermoid carcinoma (MEC), a type of salivary gland tumor, remains unclear. The anticancer effects of NaBu and its related molecular mechanisms were determined by trypan blue exclusion assay, 4'-6-diamidino-2-phenylindole staining, live/dead assay, human apoptosis array, RT-PCR, western blotting, immunocytochemistry, preparation of nuclear fractions, and nude mice tumor xenograft. In this study, we found that NaBu inhibited growth and induced apoptosis in the human oral MEC cell lines MC3 and YD15 with the acetylation of histone proteins H2A and H3. NaBu apparently down-regulated survivin protein, as evidenced by the results of the human apoptosis antibody array, and modulated it at the post-translational process. Interestingly, NaBu caused nuclear translocation of survivin protein in both cell lines. NaBu also resulted in decreased expression levels of Bcl-xL mRNA and protein, leading to induction of caspase-dependent apoptosis in human oral MEC cell lines. In addition, NaBu administration inhibited tumor growth in vivo at a dosage of 500 mg/kg/day, but it did not cause any hepatic or renal toxicity. This study provides new insights into the molecular mechanism of apoptotic actions by NaBu in human oral MEC and the basis of its clinical application for the treatment of human oral MEC.

#713

BBI-608 modulates DNA repair pathways, induces apoptosis and impacts response to 5-Fluorouracil and ionizing radiation in pancreatic cancer cells.

Batoul Farran, Ganji P. Nagaraju, Sneha G. Shipra, Shipra R. Bethi, Gregory B. Lesinski, Bassel El-Rayes. _Emory, Atlanta, GA_.

Background: BBI-608 has been shown to modulate multiple oncogenic cellular pathways which are often constitutively active in pancreatic ductal adenocarcinoma (PDAC). Among these are key mediators of DNA repair and cellular survival pathways. We hypothesized that BBI-608 would sensitize PDAC cells to chemoradiotherapy.

Methods: The combined effects of BBI-608 and chemoradiotherapy (5-FU + IR) were evaluated in human (MIA PaCa-2) and murine (PANC-02) PDAC cell lines using a Br-dU cell proliferation assay. To elucidate the mechanism of action, effects on the expression of DNA damage and repair (pATM, γ-H2AX, p53; pATR, and Rad51) molecules were examined by Western blot. Apoptosis was assessed via Annexin V staining and flow cytometric analysis to evaluate mechanism of cell death following treatment of MIA PaCa-2 and PANC-02 cell lines. The in vivo activity of BBI-608, 5-FU + IR, alone and in combination, were evaluated in C57BL/6 mice bearing subcutaneous PANC-02 tumors.

Results: The combination of BBI-608 and chemoradiotherapy significantly decreased (p<0.001) cell proliferation and induced Annexin V positive cells in both MIA PaCa-2 and PANC-02 cell lines. BBI-608 and chemoradiotherapy also decreased the activation of pATR, MDM2, and Rad51 and increased pATM, γ-H2AX and p53. Administration of BBI-608 potentiated the effects of chemoradiotherapy in vivo in mice bearing subcutaneous PANC-02 tumors as measured by tumor volume.

Conclusion: These data identify a role for BBI-608 as a modulator of DNA damage and apoptosis. These effects enhanced the efficacy of chemoradiotherapy in cell lines and in vivo models of PDAC and provide rationale for continued investigation in the pre-clinical and clinical setting.

#714

Cannabidiol-induced apoptosis is mediated by activation of Noxa in human colorectal cancer cells.

Hye Kyeong Yun, Soyeon Jeong, Yoon A Jeong, Min Jee Jo, Jung lim Kim, Seong Hye Park, Bo Ram Kim, Yoo Jin Na, Dae Young Kim, Bu Gyeom Kim, Dae Hee Lee, Sang Cheul Oh. _Korea University Guro Hospital, Seoul, Republic of Korea_.

Cannabidiol (CBD), one of the compounds present in the marijuana plant, has anti-tumor properties, but its mechanism is not well known. The authors aimed to evaluate the apoptotic action of CBD in colorectal cancer cells (CRC), and focused on its effects on the novel pro-apoptotic Noxa-reactive oxygen species (ROS) signaling pathway. CBD experiments were performed using the colorectal cancer cell lines HCT116 and DLD-1. CBD induced an apoptosis by regulating many pro- and anti-apoptotic proteins, of which Noxa showed a significantly higher expression. To understand the relationship between Noxa and CBD-induced apoptosis, authors downregulated Noxa levels using siRNA and confirmed that the expression of apoptosis markers was decreased. After ROS production was blocked, the level of Noxa also decreased, suggesting that ROS is involved in the regulation of Noxa. The Noxa and ROS are well-known pro-apoptotic signaling agents. As a result, CBD was found to induce apoptosis in a Noxa-and-ROS-dependent manner. The effects of CBD treatment were re-demonstrated in vivo, thus confirming its role as a novel, reliable anticancer drug

#715

Mechanism-based evaluation of apoptosis in live tumor cells by triple co-fluorescence staining: Testing algorithmic effectiveness of targeted drugs.

Nandini Dey, Jennifer C. Aske, Casey Williams, Pradip K. De, Brian Leyland-Jones. _Avera Research Inst., Sioux Falls, SD_.

Background: Suppression of apoptosis, a morphologically distinct form of cell death is a trait commonly shared by tumor cells which provide them survival advantages and enables them to evolve to drug-resistant states, higher stages, and/or metastasize. Recently we have reported a procedure of Triple co-Fluorescence Staining (FTS) to visualize apoptosis following chemotherapeutic and targeted anti-cancer drugs in live tumor cells (De et al., 2018).

Aim: Using triple-co-fluorescence to simultaneously identify 3 crucial mechanisms of apoptosis, (1) enzyme activity of executioner-caspase3, (2) caspase-dependent cell surface exposure of phosphatidylserine and (3) functional mitochondria, we studied apoptosis following combinations of (a) paclitaxel plus BKM120, (b) trametinib plus paclitaxel or CDK4/6 inhibitor in ovarian models, and (c) p110beta inhibitor plus BNM673 in TNBC models.

Methods: Ovarian cell lines OVK18, A2780Cis, and SUM149 (TNBC) were used for the study. For the standardization of MitoViewBlue (B) staining, a final concentration of 50 nM was used. Treated live cells on cover-slips were incubated under cultured conditions in the dark for 15-30 minutes with the staining cocktail containing MitoViewBlue (B), NucView® 488 (G) and CF®594 Annexin V (R) in the 1X binding buffer. At the end of the incubation period, pictures were taken (Olympus DP72 digital camera) within a span of 10 minutes. The FTS was corroborated with (1) AnnexinV by flow cytometry, (2) live/dead cell assay, (3) WB expression of apoptotic markers, and (4) mitochondrial depolarization.

Results: Using a standardized protocol for FTS we tested apoptosis in ovarian and TNBC models following the combination of chemotherapeutic and targeted anti-cancer drugs. Using B, we stained active mitochondria while using G we stained active caspase3 and R was used to localize plasma membrane asymmetry, flipped phosphatidylserine, a characteristic feature of apoptotic cells. A merged TFS image of live and non-treated (NT) OVK18, A2780Cis and SUM149 cells showed functional mitochondrial stained with bright blue which corroborated with TMRE-based mitochondrial potential. Fewer cells showed membrane fluorescence in red for R and even fewer cells with green fluorescence for G which stained active caspase3. R and G were mutually exclusive. Paclitaxel-treated A2780 cells were significantly devoid of B stains. Membrane blebbing and annexin-V stained cell membrane was identified in cells either with or without G stains but definitely without B stains which indicated the sequence of events of apoptotic in cells. The mutual exclusivity property of B cell to R and/or G cells are preserved as observed with the help of transmission overlay images.

Conclusion: TFS can be used to evaluate mechanism-based apoptosis following chemotherapeutic and targeted anti-cancer drugs in live tumor cells.

#716

**Chakasaponin I from tea (** Camellia sinensis **) flower induce anti-proliferative effect and apoptosis of cisplatin-resistant ovarian cancer cells.**

Yi C. Chen. _Alderson Broaddus Univ., Philippi, WV_.

Ovarian cancer has the highest rate of death among gynecological malignancy and ranks fifth in cancer deaths among women. Adverse side effects and acquired resistance to conventional chemotherapy based on platinum drive the exploration of other anticancer therapies,thus natural products have drawn greater attention for cancer treatment in recent years. Tea (Camellia sinensis) flower being an abundant agricultural by-product in China and Japan, has been studied for its bio-functional effects. A recent research showed that tea flower saponins (TFS) could induce anti-proliferative effect and cell cycle arrest in human ovarian cancer cells. In the present study, Chakasaponin I, one of the major constituents in TFS, whose main structure is oleanane-type triterpene, showed strong growth inhibitory effects on cisplatin-resistant OVCAR-3 cells at 3 μM by MTS assay. Such inhibitory effects might result from the induction of apoptosis. Western blot assay, Hoechst 33342 staining assay, and flow cytometry analysis were used to examine the effects of Chakasaponin I on apoptosis. The results showed that Chakasaponin I treatment dose-dependently activated the pro-apoptotic proteins (Bad and Bax) and therefore induced apoptosis might by the intrinsic apoptotic pathway.

#717

Erianin induces autophagy-dependent apoptosis in oral cancer cells.

Chiao-Wen Lin,1 Shun-Fa Yang2. 1 _Institute of Oral Sciences, Chung Shan Medical University, Taichung, Taiwan;_ 2 _Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan_.

Oral squamous cell carcinoma (OSCC) is a leading cause of cancer-related deaths worldwide. It has a very poor prognosis with 5-year over survival rate of only 50%. Thus, it is important to identify effective therapeutic interventions against oral cancer. The present study was designed to evaluate the effect of erianin, a natural bibenzyl compound found in a traditional Chinese orchid Dendrobium chrysotoxum, on human oral cancer cell proliferation. Results of the study revealed that treatment with erianin significantly reduced the viability of different OSCC cell lines. Erianin exerted its cytotoxic effect by inducing cell cycle arrest at G2/M phase and caspase-dependent apoptotic pathways. Both intrinsic and extrinsic pathways were found to be involved in erianin-mediated cell death. In addition, treatment with erianin also increased autophagy in OSCC cells. With further analysis, it was found that erianin induced both apoptosis and autophagy by regulating MAPK signaling pathways. Taken together, our study indicates that erianin plays an important role in reducing oral cancer cell viability, and thus, can be considered as a potential anticancer agent.

#718

A laboratory friendly visualization method of triple-co-fluorescence staining to identify cytomorphological features of apoptosis simultaneously with biochemical events in live cells.

Brian R. Leyland-Jones, Jennifer H. Aske, Pradip De, Nandini Dey. _Avera Cancer Institute, Sioux Falls, SD_.

Background: During apoptosis, its cytomorphological features are functionally coupled to its tightly regulated and ATP-dependent biochemical events. Traditionally, identification and quantification of apoptosis in the laboratory are based on one of these features at a time from either lysed or fixed cells.

Aim: We present a protocol for staining live cells in a culture undergoing apoptosis in real-time. Live cell-imaging by Triple-co-Fluorescent Staining (TFS) is standardized to test 3 crucial events of apoptosis simultaneously in a single cell; the enzymatic activity of executioner caspase3, caspase-dependent phosphatidylserine presentation on cell-surface and mitochondrial depolarization.

Methods: Live cells were stained simultaneously with the NucView488-Casp3 substrate (G), CF594 AnnexinV (R), and MitoViewBlue (B). Specificity of the staining was tested by co-localization & caspase3 inhibitor assay. We used (1) real-time confluency (Incucyte), (2) Annexin V (Accuri C6), (3) WB expression of cl-caspase3, cl-PARP, and BIM, and (4) TMRE-A based mitochondrial depolarization on validating our method. CalceinAM green and EthD-1red were used as internal staining controls. This method was verified to test combinations of chemotherapy and targeted therapy drugs in tumor cells.

Results: Drug combinations blocked cytoplasmic B staining while increased both G & R staining compared to controls. Merged images showed 100% mutual exclusivity between B & G stains in control and treated cells as determined by overlap and co-localization coefficients. Pixel co-localization between FITC (G and CalceinAM), DAPI (B) and TRITC (R and EthD-1) channels from 2.5 nM paclitaxel + 500 nM BKM120 or paclitaxel only treated OVK18 cells confirmed above results. Caspase3 and AnnexinV staining in treated cells, however, were both separate and overlapped (yellow fluorescence) indicating the sequence of apoptotic-events. Co-localization between FITC, DAPI, and TRITC channels was determined in the scatterplot of merged channels. No co-localization between EthD-1 red and CalceinAM green cells was observed. Ac-DEVD-CHO, a tetrapeptide (amino acid sequence of PARP cleavage site) that acts as a competitive inhibitor for caspase-3/7 was used to test the specificity of the assay.

Significance: The strength of our method lies in our (a) choice of TFS, (b) independent validations, and (c) specificity of staining. To the best our knowledge this is the most stringent method to visualize 3 critical features of apoptosis simultaneously and conclusively deciphering mechanistic involvement of different stages/features of apoptosis in live tumor cells in real-time which can be applied in live tumor cells to test chemotherapy and targeted drugs' efficacy in everyday laboratory practice with the help of a minimalistic laboratory setup.

#719

Combination of atypical protein kinase-C inhibitor and 5-fluorouracil retards the proliferation of colorectal cancer cells.

S M Anisul Islam, Avijit Dey, Mildred Acevedo-Duncan. _Univ. of South Florida, Tampa, FL_.

Colorectal cancer (CRC) is the third most common malignancy and considered as the fourth most common cause of cancer-related death worldwide. Although the number of CRC survivors with stage II and III are increasing, more than 50% of patients are diagnosed with stage III or beyond where the distant disease progression has already occurred. Surgical removal of cancerous tissue coupled with chemotherapeutic intervention is the main treatment of metastatic CRC and only hope of enhanced survival. The treatment of metastatic CRC considered palliative for many years aiming for an improved life, with little hope of a cure, highlighting the need for developing novel targeted therapy for CRC. Dysregulation of kinases has been shown to be pivotal of the various pathological process including cancer. Human protein kinases constitute a complicated system with intricate internal and external interaction which stimulates various cellular processes such as cell growth, metabolism, survival, and apoptosis. In this study, a combination of atypical protein kinase c (aPKC) inhibitor (ICA-I, PKC-ι inhibitor or ζ-Stat, PKC-ζ inhibitor) and 5-FU was used to examine the effect of aPKC and thymidylate synthase on CRC cells viability. The cell lines tested were LoVo and RKO CRC cells. Our findings showed that the combination of aPKC inhibitor and 5-FU significantly reduced the viability and induce apoptosis of CRC cells. These data suggest that the simultaneous knockdown of upstream aPKC protein and downstream DNA replication would be a useful approach to combat CRC and to improve overall patients' survival rate. These results indicate the possibility of utilizing aPKC as the potential therapeutic targets in addition to blocking DNA synthesis in metastatic CRC.

#720

Aspirin plus FOLFOX for colorectal cancer management: A promising alternative for clinical trial in Africans with the disease.

Faruk Mohammed,1 Sani Ibrahim,1 Sirajo Mohammed Aminu,1 Ahmed Adamu,1 Adamu Abdullahi,1 Abdulmumini Hassan Rafindadi,1 Yawale Iliyasu,1 John Idoko,1 Abdullahi Jubril Randawa,1 Mohammed Sani Shehu,1 Atara Ntekim,2 Abdullahi Mohammed,1 Aishatu Maude Suleiman,1 Yahaya Ukwenya,1 Khalid Zahir Shah,3 Ahmad Bello,1 Sani Abubakar,4 Kasimu Umar Adoke,5 Cheh Augustin Awasum,1 Ahmad Mai,1 Hussaini Yusuf Maitama,1 Dauda Maigatari1. 1 _Ahmadu Bello University, Zaria, Nigeria;_ 2 _University of Ibadan, Ibadan, Nigeria;_ 3 _University of Wolverhampton, Wolverhampton, United Kingdom;_ 4 _Bayero University, Kano, Nigeria;_ 5 _Federal Medical Center, Birnin Kebbi, Nigeria_.

Colorectal cancer (CRC) has disproportionately overburden Africans with advanced stage at diagnosis and higher mortality rate due in part to issues that include grossly inadequate resources for management and poor understanding of the pathobiology of the disease. Accumulating evidences support the effectiveness of Aspirin in prevention and inhibition of CRC occurrence and cellular growth respectively. The utilisation of 5-fluorouracil (5-FU)-based regimens which include folinic acid, 5-FU and oxaliplatin (FOLFOX) combination in CRC management have not completely halt the distant recurrences and poor prognosis of CRC. In this study, we highlighted evidences that prove Aspirin plus FOLFOX to be promising alternative for clinical trial in Black men with CRC. Our study aims in this research were: to identify the expression pattern of BIRC7 in human CRC cells, SW480 cell line and N-methyl-N-nitrosourea (NMU)-induced CRC in rat; to determine the relationship between BIRC7 and BCL2, p53, PDL-1, MSH1, PMS2, DARC and Annexin V expression in CRC. to examine the efficacy of Aspirin plus neoadjuvant FOLFOX on the BIRC7 in CRC cells in-vivo in rat. Results show significant (P = 0.0001) expression of the BIRC7 in the CRC tissues compared to normal sections. When comparing this expression with Data from TCGA data bank, we observed a significant increase in BIRC7 expression in Caucasian than the Black and Indian and The expression pattern of the BIRC7 was significantly higher (P = 0.0001) in CRC not otherwise specified compared to mucinous CRC histology. The SW480 cell line show increased expression of the BIRC7 protein in-vitro. There was significant expression of the BIRC7 in the NMU-induced CRC cells compared the normal colonic lesion. There was increased expression of the BCL-2 and reduced expression of p53 in the human CRC cases and in the NMU-induced CRC cells model. There was increased expression of the Annexin V, absence of the MSH1, PMS2, PDL1 and DARC protiens in the CRC sections. Aspirin plus FOLFOX may serve as an important adjuvant treatment component for colorectal cancer metastasis and local recurrence in Blacks, Caucasian and Asians. There is need to explore the potential of Aspirin plus FOLFOX for clinical trial in colorectal cancer management in Blacks and other population using approach from our findings.

#721

CHD4 mediates genotoxic sensitivity and tumor initiation of AML cells through both MBD2-NuRD and MBD3-NuRD co-repressor complexes.

Javeria Aijaz, Justin Sperlazza, Gordon D. Ginder. _Virginia Commonwealth University, Richmond, VA_.

Despite several advances in therapy, the 5-year survival of AML, estimated at 40-45% for the young and 10% for the elderly, remains dismal. New modalities of therapy are thus clearly needed. Research in our laboratory has identified CHD4, an ATPase chromatin re-modeling component of the NuRD co-repressor complex, as an important mediator of genotoxic sensitivity to standard chemotherapy in AML cell lines and primary AML samples. Knockdown of CHD4 also markedly reduced colonies in soft agar. Interestingly, normal hematopoietic progenitor cells did not show these phenotypic alterations. Classical NuRD complex contains either MBD2 or MBD3, and at least four other proteins. We sought to determine if the previously observed CHD4 effects are through MBD2-NuRD or MBD3-NuRD. MBD2 and MBD3 are thought to link DNA to the GATAD2/CHD4 NuRD sub-components through coiled-coil domains of both proteins interacting with those of GATAD2A/B. It was thus hypothesized that if the observed effects of CHD4 are mediated through canonical NuRD, knockdown of either MBD2 or MBD3 should be able to replicate these. In order pursue this aim, we first knocked down MBD2 in U937 cell lines and performed colony forming assays. Persistent efforts to knockdown MBD3, however, were unsuccessful. Therefore, MBD3 was knocked out using CRISPR-Cas9. Single cell cloning to generate a completely knocked out cell line, however, introduces a potential intrinsic bias in colony forming assays by selecting for cells with an inherently increased colony forming potential. A guide RNA with an ~80% overall knockout efficiency was thus selected and the assay performed on the bulk population of U937 cells prior to limiting dilution and plating. U937 cells with CHD4 knocked down were used as positive controls. Neither MBD2, nor MBD3, when knocked down by 90% or knocked out, respectively, showed any effect on colony formation in vitro using the soft agar colony forming assay. However, knocking down MBD2 in U937 cells with MBD3 knocked out, resulted a significant reduction in the colony forming potential. A similar result was obtained in the genotoxic sensitivity assay, whereby cells were treated with cytosine arabinoside (AraC) and the percentage apoptosis determined by flow cytometry through 7-AAD staining as well as caspase cleavage assays. MBD2 knockdown did not increase the genotoxic sensitivity of U937 cells, while MBD3 knockout increased it only marginally. MBD2 knockdown in MBD3 knockout U937 cell lines, however, did markedly increase the sensitivity of these cells to AraC. We conclude that in terms of mediation of AML colony forming potential and sensitivity to standard chemotherapy agents, MBD2-NuRD and MBD3-NuRD may be redundant. Future therapeutic strategies aimed at exploiting the potent effects of disrupting the NuRD complex on AML cell sensitivity to chemotherapy and tumor initiating activity need to take this into account.

#722

**Epigenetic regulation of the** Birc5 **promoter explains mechanism of action of YM-155 in synovial sarcoma.**

Aleksander Mika,1 Sarah Luelling,2 Adriene Pavek,2 Neil Parkinson,2 Alexandra Heyneman,2 Jared J. Barrott2. 1 _University of Rochester, Rochester, NY;_ 2 _Idaho State University, Pocatello, ID_.

YM-155 is an anti-cancer therapy that has advanced into 11 different human clinical trials to treat various cancers. This apoptosis-inducing therapy indirectly affects the protein levels of Survivin (Gene: Birc5), but the molecular underpinnings of the mechanism remain largely unknown. Synovial sarcoma is another cancer with high protein expression of Survivin. We investigated whether YM-155 would be a viable therapeutic option to treat synovial sarcoma. We, therefore, applied YM-155 therapy to human synovial sarcoma cell lines and a genetically engineered mouse model of synovial sarcoma. We discovered that YM-155 exhibited nanomolar potency against human synovial sarcoma cell lines and the treated mice with synovial sarcoma demonstrated a 50% reduction in tumor volume compared to control treated mice. We further investigated the mechanism of action of YM-155 by looking at the change of modifications of the histone tails that were near the Birc5 promoter. Using chomatin immunoprecipitation (ChIP) we discovered that the histone epigenetic marks of H3K27 for the Birc5 promoter changed upon YM-155 treatment. H3K27me3 increased whereas the H3K27ac decreased, highlighting the decrease of the protein Survivin occurs through epigenetic silencing of the gene's promoter. The treatment of YM-155 was accompanied by an increase in NFkB protein expression, which indicates an attempt of the cell to initiate a positive feedback due to the decrease in Survivin expression. This combination of molecular events eventually resulted in Caspace 3/7/8 upregulation and death of the sarcoma cells.

#723

MX69 induces apoptosis by inhibiting XIAP in both rituximab sensitive and resistant lymphomas.

Sumera Khan, Cory Mavis, Ahmad Hanif, Juan Gu, Pallawi Torka, Francisco Hernandez-Ilizaliturri. _Roswell Park Comprehensive Cancer Center, Buffalo, NY_.

Background: X-linked inhibitor of apoptosis protein (XIAP) has been implicated in development of resistance to chemotherapy via its direct inhibition of caspases 3 ,7 and 9. It is overexpressed in several lymphoma cell lines including rituximab-resistant cell lines (RRCL), Raji4RH and RL4RH, produced by our group. We have previously demonstrated that XIAP is critical for chemotherapy-resistance and survival in RRCL by knocking down XIAP using siRNA interference which demonstrated that XIAP is critical for chemotherapy-sensitivity and survival in RRCL. MDM2 inhibition activates p53 transcription and inhibits translation of XIAP, thus promoting apoptosis of cancer cells. Here, we targeted XIAP at a translational level by inhibiting it with MX69, a dual inhibitor of MDM2 and XIAP.

Materials and Methods: Cell lines representing Burkitt's lymphoma (BL), activated B-cell like diffuse large B-cell lymphoma (ABC-DLBCL, germinal center B-cell like DLBCL (GCB-DLBCL), mantle cell lymphoma (MCL) and B-cell lymphoblastic leukemia were exposed to MX69 as a single agent (0-80 uM) over 24, 48 and 72 hrs and IC50 concentrations were calculated. Subsequently, Raji, Raji4RH, RL, RL4RH, HBL-2, TMD-8, Daudi and Reh cell lines were exposed to MX69 (0-80 uM), in combination with doxorubicin (0-1 uM), cytarabine (0-50 uM), vincristine (0-10 nM), etoposide (0-50 uM), carboplatin (0-10 uM), ixazomib (0-1.5 uM), ibrutinib (0-10 uM) and venetoclax (0-10 uM) for 48 hours. Cell viability was determined by Cell Titer-Glo. Coefficient of synergy was calculated using CalcuSyn. Induction of apoptosis was evaluated by flow cytometry (Annexin V/PI stain). MDM2, p53, XIAP and PARP protein expression was determined by Western blotting.

Results: MX69 induced cell death in a dose- and time-dependent manner in all cell lines. Annexin/PI staining showed caspase-dependent apoptosis with MX69 in all cell lines. Western blotting confirmed significant inhibition of MDM2, XIAP and changes in p53 and PARP following exposure to MX69. MX69 demonstrated significant synergistic activity when combined with doxorubicin, ixazomib, ibrutinib or venetoclax; synergy was strongest for the MX69- venetoclax combination.

Conclusion: Our data suggests that in vitro exposure to MX69 resulted in anti-tumor activity in a wide variety of B-cell lymphoma cells lines (including BL, DLBCL, MCL). Perhaps related to its anti-tumor effects, MX69 inhibited XIAP levels. These findings are similar to prior siRNA XIAP knockdown experiments. Strong synergistic activity was observed when XIAP was combined with various chemotherapy agents and small molecules inhibitors (such as venetoclax, ixazomib or ibrutinib). Ex vivo experiments using primary tumor cells isolated from lymphoma patients and lymphoma mouse models have been planned. Targeting MDM2 and XIAP can be an attractive therapeutic strategy in patients with rituximab-sensitive or -resistant B-cell lymphoma.

#724

Preclinical evaluation of TQB3728, a potent orally active IAP antagonist.

Yingchun Liu,1 Xiquan Zhang,2 Ling Yang,2 Xin Tian,2 Tiantian Dong,2 Charles Z. Ding,3 Lihong Hu,1 Zhaobing Xu,1 Yuanfeng Xia,3 Haijuan Tong,3 Wenxi Li,3 Lijuan Hou,3 Chichung Chan,3 Shuhui Chen3. 1 _WuXi AppTec, Wuhan, China;_ 2 _Chia Tai Tianqing Pharmaceutical Group Co., Ltd., Nanjing, China;_ 3 _WuXi AppTec Headquarters, Shanghai, China_.

Objectives: Evasion of apoptosis and avoidance of immune surveillance represent two key hallmarks of cancer. Members of the inhibitor of apoptosis (IAP) gene play important interconnecting roles in both of these characteristic pathways of tumorigenesis, providing a critical nexus in targeting of cancer. Small-molecule antagonists of the IAPs, known as Smac mimetic compounds (SMCs), are in quick clinical development for cancer therapy. We disclose here a novel IAP antagonist TQB3728 and its preclinical evaluation on tumor therapy.

Methods: The biochemical activities of TQB3728 for cIAP1 BIR3, cIAP2 BIR3 and XIAP BIR3 were measured by the corresponding biochemical assays. The in vitro anti-proliferative activity was evaluated on MDA-MB-231 and EMT6 cell lines. The in vivo antitumor activity of TQB3728 was evaluated in two cell-derived xenograft (CDX) models of TNBC (MDA-MB-231) and lymphoma (Karpas 422), and one syngeneic model of TNBC (EMT6). PK/PD was assessed by western blot analysis of cIAP1 expression and cleaved caspase 3 level in the treated tumor tissues.

Results: TQB3728 displayed potent biochemical activities for cIAP1 BIR3, cIAP2 BIR3 and XIAP BIR3 with IC50 of 0.7, 11.8 and 22.5 nM, respectively. The expected anti-proliferative activities were also observed in both tumor cell lines, with a IC50 of 14.3 Nm for MDA-MB-231, and IC50 of 149.5 nM for EMT6 in the presence of exogenous TNFα (1 ng/ml). TQB3728 showed in vivo antitumor activity in the MDA-MB-231 CDX model with 89.8% TGI @30 mpk QD, which is better than LCL-161 (71.9% TGI @30 mpk, QD). TQB3728 also showed good antitumor activity in vivo in the Karpas 422 lymphoma CDX model with 112.2% TGI @100 mpk Q3D. In EMT6 tumor-bearing mice, the combination of TQB3728 and anti-PD1 antibody significantly regressed the tumor growth (TGI = 106.3%), whereas monotherapies only displayed moderate activities ( 60.6% and 0.4% TGI for anti-PD1 and TQB3728, respectively). Western blot analysis of the tumor samples showed that TQB3728 promoted caspase 3 cleavage, indicating that the tumor growth inhibition could be mediated by cell apoptosis signaling.

Conclusions: We have discovered a potent orally active IAP antagonist, TQB3728, which shows activity of cIAP1 inhibition and strong antitumor activity in both in vitro and in vivo preclinical tumor models. These results are highly promising and warrant moving the compound forward to clinical investigation.

#725

Anticancer effect and mechanism of exosomes mediated microRNA let-7b in breast cancer cells.

Xu Biantiao. _Department of Pharmacology, School of Pharmacy, Southwest Medical University, LuzhouSichuan, China_.

Exosome is referred as membrane-bound nano-vesicles secreted by various mammalian cells. It can be found in different body fluids, such as plasma, saliva, urine, latex, cerebrospinal fluid, ascites, and cell supernatant, which contains a large number of proteins, miRNAs, mRNAs, lipids, nucleic acids, peptides and other substances. Exosome plays a pivotal role in the information transmission signals between cells and the diagnosis and prognosis of diseases as well as drug evaluation. It has become an emerging field in cancer research recently. Therefore, we separated exosomes from human kidney embryo cell (HEK-293) by ultra high speed centrifugation. To identify exosomes from their surface specific proteins by Western blotting analysis. We found enrichment of exosome markers CD63 and TSG101 without Calnexin protein in the exosoms so confirmed that exosome was successfully isolated. Then, we transfered mRNA let-7b into the exosomes by electric transfection further evaluated the effects of cytotoxicity and apoptosis induction on human breast cancer MCF-7 cells by CCK 8 assay Hochest 33342-PI and AnnexinV-PI dual staining analysis, respectively. The results showed that exosome mediated microRNA let-7b exhibited significant anticancer activity and apoptosis induction in a concentration-dependent manner in MCF-7 cells. Moreover, exosome mediated let-7b significantly up-regulated the expression levels of apoptosis related proteins casepase 3, casepase 9, and PARP determined by Western blotting analysis. Our results indicate that the exosomes mediated microRNA such as let-7b can effectively inhibit cell growth and induce apoptosis against breast cancer cells and it may be developed as a novel anticancer agent in the treatment of cancer clinically.

#726

HNSCC-associated caspase-8 mutations mediate resistance to apoptosis and up-regulation of immunosuppressive cytokines.

Zhibin Cui, Hadas Tal, Jennifer R. Grandis, Daniel E. Johnson. _University of California, San Francisco, San Francisco, CA_.

Background: The gene encoding caspase-8 is mutated in 10% of the head and neck squamous cell carcinoma (HNSCC) tumors analyzed by The Cancer Genome Atlas (TCGA). HNSCC patients with caspase-8 mutations tend to have worse prognosis. While the role of wild-type (WT) caspase-8 in mediating extrinsic apoptosis is well known, recent studies indicate that it also acts in a noncatalytic fashion to mediate death ligand induction of immunosuppressive cytokines. To determine the potential impact of HNSCC-associated caspase-8 mutations on anti-tumor immunity and the development of HNSCC, we investigated the functional capacity of caspase-8 mutants to mediate death ligand induction of apoptosis and cytokine production.

Methods: Ininitial studies, we knocked out the endogenous CASP8 gene in HeLa cells using CRISPR-Cas9 technology. The HeLa-CASP8 KO cells were then engineered for doxycycline (DOX)-inducible expression of WT or representative caspase-8 mutants (MT; L105H, D303G, S386* and Q465*). The engineered cells were then stimulated with death ligand (eg. TRAIL) and analyzed for induction of cell death using MTT assays or annexin V staining. Induction of immunosuppressive cytokines was assessed by qPCR and ELISA assays.

Results: HeLa-CASP8 KO cells engineered to express WT caspase-8 underwent rapid apoptosis following TRAIL treatment, as indicated by processing of the caspase-8, PARP cleavage, and cell death assays. By contrast, cells engineered to express the MT caspase-8 proteins failed to undergo apoptosis even when treated for 48 hours with very high concentrations of TRAIL. Treatment of parental HeLa cells, but not HeLa-CASP8 KO cells, with TRAIL led to upregulation of mRNAs for the immunosuppressive cytokines IL-6, IL-8, and CXCL1. Exogenous expression of WT caspase-8 in the HeLa-CASP8 KO cells restored TRAIL induction of the immunosuppressive cytokine mRNAs. Interestingly, exogenous expression of the caspase-8 D303G MT, but not the other MTs (L105H, S386*, Q465*), also restored TRAIL induction of the immunosuppressive cytokines, following treatment for as brief as 6 hours.

Conclusion: Our findings demonstrate that the four representative HNSCC-associated caspase-8 mutations have lost the capacity to mediate TRAIL-induced apoptosis. Hence, HNSCC cells harboring these mutations are likely more resistant to killing by CTLs and NK cells which utilize death receptor-mediated apoptosis to kill target cells. Notably, the D303G caspase-8 MT retained the capacity to mediate TRAIL induction of immunosuppressive cytokines. Expression of the D303G MT, which has been observed in more than one patient, is likely to enhance the immunosuppressive tumor microenvironment, further contributing to HNSCC development.

### Cancer Genomics 1

#727

Comprehensive genomic profiling of >1000 plasma and tumor tissue samples from metastatic castration-resistant prostate cancer (mCRPC) patients gives insight into targeted treatment strategies.

Foad Green,1 Jeremy D. Shapiro,2 Ray McDermott,3 Josep Maria Piulats,4 Alison Reid,5 Peter Ostler,6 Jingsong Zhang,7 David Campbell,8 Dominique Spaeth,9 Ivor Percent,10 Arif Hussain,11 Andrew D. Simmons,1 Tony Golsorkhi,1 Simon P. Watkins,1 Andrea Loehr,1 Simon Chowdhury,12 Wassim Abida13. 1 _Clovis Oncology, Inc., Boulder, CO;_ 2 _Cabrini Hospital, Malvern, Australia;_ 3 _Adelaide and Meath Hospital (Incorporating the National Children's Hospital), Dublin, Ireland;_ 4 _Instituto Catalan de Oncologia, Barcelona, Spain;_ 5 _Royal Marsden Hospital, London, United Kingdom;_ 6 _Mount Vernon Cancer Centre, Northwood, United Kingdom;_ 7 _H. Lee Moffitt Cancer Center, Tampa, FL;_ 8 _University Hospital Geelong (Barwon Health), Geelong, Australia;_ 9 _Centre d'Oncologie de Gentilly, Nancy, France;_ 10 _Florida Cancer Specialists, Port Charlotte, FL;_ 11 _University of Maryland Greenebaum Cancer Center, Baltimore, MD;_ 12 _Guy's Hospital and Sarah Cannon Research Institute, London, United Kingdom;_ 13 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Background: The phase 2 TRITON2 (NCT02952534) study is evaluating the poly(ADP-ribose) polymerase inhibitor rucaparib in mCRPC patients with a deleterious germline or somatic mutation in BRCA1, BRCA2, ATM, or 1 of 12 other DNA damage repair (DDR) genes who have progressed on prior androgen receptor-directed therapy and 1 taxane-based therapy. Here we present results from central genomic screening of plasma and tissue samples for TRITON2.

Methods: Plasma samples were profiled for genomic alterations (GAs) in 70 genes and tumor tissue FFPE samples for GAs in 395 genes by Foundation Medicine, Inc., using next-generation sequencing (NGS) assays. Deleterious alterations included frameshift, nonsense, and deleterious missense mutations, protein-truncating rearrangements, and (for tissue samples) homozygous loss. Tissue samples included both archival and more recent specimens, whereas plasma samples were collected at the time of disease progression on prior therapy.

Results: As of July 2, 2018, 359 plasma samples from patients with mCRPC were screened for deleterious GAs in a DDR gene. Of the plasma samples, 96% (343/359) were successfully sequenced for a comprehensive genomic profile. The most commonly altered genes were AR (49%) and TP53 (48%). Deleterious GAs were detected in BRCA1 (2%), BRCA2 (10%), ATM (15%), CDK12 (6%), or other DDR genes (6%). GAs in the MAPK pathway were observed in 5% of plasma samples. Additionally, 738 tissue samples from primary prostate cancer tumors (78%) or metastases (22%) were profiled. Only 66% of tissue samples (487/738) were sequenced successfully, highlighting the challenges associated with NGS of predominantly archival prostate tissues (median sample age, 2.5 years [range, 4 days to 21 years]). The most commonly altered genes were TP53 (38%), PTEN (33%), ERG-TEMPRSS2 fusions (28%), AR (15%), and MYC (9%). Deleterious GAs were observed in BRCA1 (2%), BRCA2 (9%), ATM (7%), CDK12 (7%), or other DDR genes (5%), as well as in the PI3K/AKT (41%), Wnt (11%), MAPK (5%), and mismatch repair (3%) pathways. Further, GAs in the DDR, PI3K/AKT, and Wnt pathways were observed at a higher rate in metastatic (42%, 40%, and 15%, respectively) than in primary prostate tissues (32%, 34%, and 12%, respectively). Updated and expanded genomic analyses and plasma-tissue concordance analyses will be presented.

Conclusions: Genomic NGS profiling of plasma and FFPE tumor tissue samples successfully identified patients with GAs in a DDR gene for the evaluation of rucaparib in mCRPC. The plasma assay is convenient for patients and has a low NGS failure rate, whereas the tumor tissue assay has a higher failure rate but can detect more alteration types. Taken together, these data highlight the strengths and limitations of tissue and plasma testing to identify GAs for targeted therapies in mCRPC.

#728

Genomic alterations in lung adenocarcinoma precursor lesions.

Michael K. Asiedu, Nanette R. Reed, Marie Christine Aubry, Anja C. Roden, Dennis A. Wigle. _Mayo Clinic College of Medicine, Rochester, MN_.

Background: Adenocarcinoma of the lung is the most common type of non-small cell lung cancer and is the subtype found in approximately 50% of all lung cancer cases. Adenocarcinoma in situ (AIS) and minimally invasive adenocarcinoma (MIA) are thought to be precursor lesions of invasive disease. It has been hypothesized that there is a step-wise progression to lung adenocarcinoma, but little is known about genomic alterations in these precursor lesions.

Methods: Genomic analysis including whole genome and exome sequencing, and SNP array analysis were performed on 9 AIS and 18 MIA pathologically confirmed samples to identify single nucleotide variants (SNVs), structural variations and copy number variations. Mutation significance and signature analysis were determined by MutSig and NMF analyses. Altered pathways were determined by Ingenuity variant effect pathway analysis.

Results: The tumor mutation burden (TMB) for AIS and MIA ranged from 0.8 to 23.1 mut/Mb with a median of 3.0mut/Mb and a mean of 5.1mut/Mb. Highly mutated genes identified in AIS and MIA were RPL10, NBPF1, KRAS, GPR52, CAMK2A, ATOX1, STK11, TP53, MET and EGFR. Genes with recurrent mutations included SPTA1, USH2A, CACNA1E, EGFR, KRAS, MUC16, MUC17, OTOG, RYR2, COL20A1, CSMD3, RFX6 and XIRP2, which implicated pathways involving the EGFR-KRAS-TP53-MAPK axis. Somatic mutations were characterized by a C>T, T>C transitions and C>A transversion signature. There were comparable structural variations in the AIS cases compared to MIA.

Conclusion: In contrast to hypothesized models of tumor progression, AIS and MIA can harbor significant genomic alterations and tumor mutation burden, which suggests an intriguing concept of immunotherapeutic options for either treatment or chemoprevention. Deregulation of EGFR-KRAS-TP53-MAPK pathway indicates a role in early events of carcinogenesis.

#729

Zygosity, lineage, and penetrance dictate the role of germline pathogenicity in tumorigenesis.

Chaitanya Bandlamudi, Preethi Srinivasan, Philip Jonsson, Yelena M. Kemel, Shweta S. Chavan, Allison L. Richards, Alex Penson, Craig M. Bielski, Chris Fong, Aijazuddin Syed, Gowtham Jayakumaran, Meera Prasad, Jason Hwee, Nikolaus Schultz, Semanti Mukherjee, Vijai Joseph, Diana Mandelkar, Ozge Birsoy, Liying Zhang, Jinru Shia, Ahmet Zehir, Marc Ladanyi, David M. Hyman, Kenneth Offit, Mark E. Robson, David B. Solit, Zsofia K. Stadler, Michael F. Berger, Barry S. Taylor. _Memorial Sloan Kettering Cancer Center, New York, NY_.

Human cancers arise from environmental, heritable, and somatic factors. While each has been studied extensively, how these factors interact to drive tumorigenesis and disease phenotypes is poorly understood. Here, we integrated germline pathogenicity with somatic alterations inferred from the prospective sequencing of 17,152 advanced cancer patients across 55 cancer types to explore their interplay in tumorigenesis. Leveraging expert curated germline variant calls from 5,358 patients, we developed a combined machine learning and risk stratification-based approach for pathogenicity assessment. Overall, 10.3% of patients harbored a pathogenic allele with penetrance levels ranging from high to low, and an additional 6.3% with a variant of uncertain penetrance. Integrating high-precision zygosity inference in the concomitant cancer diagnoses, we found that only carriers of high penetrance germline pathogenic alleles that are biallelically inactivated in their tumors had an earlier age of onset (51y in carriers vs. 58y in germline WT, p-value=3.5e-12), and an elevated rate of multiple independent cancer diagnoses (23% in carriers vs 13% in germline WT, p-value=1.6e-7). Broadly, penetrance dictated somatic dependence, with only those higher penetrant germline pathogenic alleles associated with lineage-dependent selective pressure for somatic biallelic inactivation (p-value<2e-16). Tumor lineage dictated the substantial variability in the rate of tumor-specific biallelic inactivation of germline alleles, even among high penetrance genes. Among only high and moderate penetrance genes, high-risk cancers for which these alleles predispose and are therefore strongly associated by prevalence had the highest biallelic inactivation rates as compared to those for which no association exists (85 versus 30%, respectively; p-value<2e-16). Consequently, nearly 30% of all tumors diagnosed in carriers of high penetrance germline alleles are likely sporadic cancers whose tumorigenesis is unrelated to the germline dysfunction. Moreover, tumorigenesis in only those carriers of high penetrance germline alleles biallelically inactivated in their tumors required fewer somatic oncogenic driver mutations overall to confer a selective growth advantage, emphasizing their distinct role as a founding event in disease pathogenesis. Collectively, the role of germline pathogenicity in tumorigenesis is determined by penetrance and zygosity in a manner that is lineage-dependent, thereby facilitating the discovery of true germline drivers and multiple distinct routes to tumorigenesis in affected patients, with implications for disease pathogenesis, screening, and ultimately therapy.

#730

Genomic characterization of hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a rare and aggressive kidney cancer.

Pankaj Vats,1 Yuping Zhang,1 Sarvana Mohan Dhanashekaran,1 Narayanan Sathiya Pandi,1 Xuhong Cao,1 Fengyun Su,1 Sudhanshu Shukla,2 Rui Wang,1 Marcin Cieslik,1 Jincheng Pan,3 Christopher J. Ricketts,4 Prem Kumar Kumpati,5 Ajjai Alva,1 Marston W. Linehan,4 Rohit Mehra,1 Arul M. Chinnaiyan1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _Indian Institute of Technology Dharwad, Dharwad, India;_ 3 _Sun Yat-Sen University, China;_ 4 _National Institutes of Health, MD;_ 5 _Bharathidan University, Tiruchirappalli, India_.

Background

Hereditary Leiomyomatosis and Renal Cell Carcinoma (HLRCC) is a rare autosomal-dominant syndromic disorder associated with germline FH mutations. Affected individuals have a predisposition to cutaneous and uterine leiomyoma's, and are at increased risk of developing a renal cell carcinoma (HLRCC- associated RCC) with aggressive clinical outcome. However, aside from FH bi-allelic loss, additional genomic features of HLRCC are currently poorly understood.

Method

Five cases of HLRCC-associated RCC were characterized using integrated next generation sequencing (NGS) technology. This included an index case where multiple tumor sites (kidney, lung, liver, lymph node, skin, and uterine) from a single patient were characterized. For integrative analysis, in-house data were combined with five RCC cases with FH bi-allelic loss from The Cancer Genome Atlas Study. Single cell sequencing was carried out on normal human kidney to study the cell of origin for HLRCC- associated RCC.

Results: Integrative NGS profiling and analysis of HLRCC-associated RCC tumors (n=10) revealed several important observations such as 1) a high frequency loss of whole chromosomes, which is reminiscent of tumors arising from the distal nephron, 2) low mutational burden similar to that observed in other kidney tumors and, 3) a HLRCC specific gene expression signature and discovery of a novel therapeutic candidate (HLRCC-TC1). Importantly, among renal tumors, HLRCC-TC1 was specifically expressed in HLRCC and other RCCs with poor clinical outcome. Application of siRNAs and neutralizing antibodies to HLRCC-TC1 reduced cell proliferation of the HLRCC cell line UOK262, but did not for that of control cell lines.

Conclusion: The observed FH bi-allelic mutation along with recurrent chromosome-wide losses and integrative analysis with human kidney single cell sequencing data support a distal nephron origin for HLRCC- associated RCC. In addition, our study provides evidence for nominating HLRCC-TC1 as a novel cancer-specific therapeutic target. Further studies are needed to conduct an in-depth characterization of HLRCC-TC1.

#731

Clinical genome sequencing uncovers potentially targetable truncations and fusions of MAP3K8 in spitzoid and other melanomas.

Scott Newman,1 Liying Fan,1 Allison Pribnow,1 Antonina Silkov,1 Stephen V. Rice,1 Seungjae Lee,1 Ying Shao,1 Bridget Shaner,1 Heather Mulder,1 Joy Nakitandwe,1 Sheila Shurtleff,1 Elizabeth Azzato,1 Gang Wu,1 Xin Zhou,1 Raymond Barnhill,2 John Easton,1 Kim E. Nichols,1 David W. Ellison,1 Downing R. James,1 Alberto Pappo,1 Philip M. Potter,1 Jinghui Zhang,1 Armita Bahrami1. 1 _St Jude Children's Research Hospital, Memphis, TN;_ 2 _Institut Curie, Paris, France_.

Most melanomas arising in children and adolescents are of the spitzoid subtype. Unlike conventional melanomas in adults, spitzoid tumors are driven by fusions of kinase genes such as ALK, NTRK1/3, MET, RET and ROS1. However, in approximately 50% of cases, no oncogenic driver has been established. This raises the possibility of an, as yet, undescribed oncogene in such tumors.

Clinical whole genome and transcriptome (RNA-Seq) sequencing detected a novel fusion of MAP3K8 in a spitzoid melanoma from an adolescent patient. The fusion preserved the kinase domain of MAP3K8 - a serine threonine kinase that activates MEK/ERK downstream - but replaced the autoinhibitory final exon with an unrelated gene, GNG2. MAP3K8 is a proposed oncogene in breast, ovarian, squamous cell carcinoma and lung cancer and its high expression has been shown to cause resistance to BRAF inhibitors through a MEK-dependent mechanism in melanoma cells (Johannessen et al. 2010 Nature 468:968-72). As the patient had exhausted other therapeutic options, we treated him with the MEK inhibitor, trametinib, and observed a transient response. He later relapsed, and further clinical sequencing showed the MAP3K8-GNG2 fusion had increased in genomic copy number and expression - potentially explaining the acquired resistance to trametinib.

We subsequently screened a cohort of 49 pediatric melanomas with spitzoid features by RNA-Seq, fluorescence in situ hybridization and immunohistochemistry and found that MAP3K8 fusions and truncations were the most common genetic event, supplying the missing kinase driver for 33% of samples. All rearrangements preserved MAP3K8 exons 1-8 but replaced the autoinhibitory final exon with unrelated genetic or intergenic sequence. Strikingly, MAP3K8 rearrangements were mutually exclusive of other known driver mutations such as ALK fusions, further implicating MAP3K8 as a driver oncogene. Transformation assays using NIH 3T3 cells confirmed that the truncated form of MAP3K8 was oncogenic.

Finally, we screened over 11,000 TCGA RNA-Seq samples, and identified seven adult melanomas with analogous MAP3K8 disruptions (1.5% of SKCM samples). Similar to their pediatric counterparts, all seven tumors lacked any other kinase driver mutation. Thus, MAP3K8 rearrangements drive a subset of adult melanomas and these tumors may also be amenable to MEK inhibition. Our experience highlights the need for genome-wide clinical sequencing as MAP3K8 is not covered by popular targeted gene panels.

#732

Clinically significant subgroups of Wilms tumors are defined by genome-wide DNA methylation and RNA expression patterns.

Jack Brzezinski,1 Sanaa Choufani,1 Rodrigo Romao,2 Haiying Chen,1 Cheryl Shuman,1 Ronald Grant,1 Armando Lorenzo,1 Rosanna Weksberg1. 1 _Hospital for Sick Children, Toronto, Ontario, Canada;_ 2 _IWK Women and Childrens Hospital, Halifax, Nova Scotia, Canada_.

Introduction: Wilms tumors (WT) are the most common renal tumor of childhood. Although cure rates are high, the burden of treatment-related late effects is significant. It is evident from clinical experience that there is a high degree of heterogeneity in the natural history of WT. However, currently known molecular features inadequately explain this heterogeneity. Substratification of WT by genome-wide analysis of features such as DNA methylation and RNA expression may identify patients who would benefit from a reduction in therapy.

Methods: WT-kidney pairs (n=28) and unpaired tumours (n=18) were collected at Sick Kids at the time of nephrectomy. An additional 40 tumor-kidney pairs were obtained from the Childrens' Oncology Group (COG). DNA methylation was assessed using Illumina 450K and EPIC methylation arrays. Whole exome sequencing and poly-A RNA sequencing were performed on the Illumina HiSeq 2500.

Results: Unsupervised clustering of DNA methylation and RNA expression data revealed three subgroups of WT – the DIFF group has a DNA methylation profile very similar to differentiated mature kidney, the PRO group has genome-wide dysregulation of DNA methylation, and the INT group has an intermediate profile and most closely resembles pre-neoplastic nephrogenic rests. DIFF and INT tumors share a pattern of RNA expression while PRO tumors have a unique pattern. PRO tumors overexpress genes associated with proliferation and genes involved in early renal development including several HOXA and HOXB genes. The expression of the early renal developmental gene set, as opposed to the proliferation gene set, is driven by dysregulation of DNA methylation.

Exome sequencing of the tumors showed that previously known recurrent variants in microRNA processing genes occurred almost exclusively in the PRO group. As well, the overall rate of non-synonymous missense mutations was higher in PRO tumors suggesting a higher degree of genome instability – supported by the increased incidence of segmental chromosomal aberrations in this group. Recurrent pathogenic variants in histone-modifier SETD2 were identified in WT for the first time.

WT subgroups are clinically important as no cases of relapsed disease nor high-risk histology occurred in PRO or INT. Conversely, bilateral WT were enriched in the PRO group.

Conclusions: Through an analysis of DNA methylation and RNA expression patterns we have separated WT into three subgroups suggesting a new model of tumorigenesis in which early Wilms tumors face an inflection point where they can either undergo differentiation or increase their proliferative capacity. The universally favorable outcomes in DIFF tumors imply that some children with WT may be future candidates for a reduction in therapy. Future work will identify the drivers of increased proliferation in PRO tumors as candidates for future targeted therapy.

#733

Characterizing the genetic profile of endometrioid ovarian carcinoma using exome sequencing.

William E. Pierson, Pamela Peters, David A. Quigley, Lee-may Chen, Jocelyn S. Chapman. _University of California, San Francisco, San Francisco, CA_.

Objective: Endometrioid ovarian carcinoma is poorly understood at the genomic level. Prior sequencing investigations have been limited to small gene panels. In order to characterize the etiology of this disease and identify potential therapeutic targets, we sought to build an unbiased genetic profile of coding gene alterations in endometriod ovarian tumors.

Methods: We sequenced the exomes of primary endometrioid ovarian tumors obtained from our institutional gynecologic oncology tissue bank from 1996 - 2011. We analyzed somatic point mutations, copy number alterations, structural variations and mutational signatures and integrated these data to determine genetic drivers. Demographic and clinical data were collected by retrospective chart review.

Results: Seventeen primary endometrioid ovarian tumors were sequenced. Median age at surgical resection was 51 years (range 33-77 years). The majority of patients presented with early stage disease (71%), many had a history of endometriosis (47%) and 19% had a synchronous primary uterine carcinoma. Microsatellite instability (MSI) was present in 18% (3/17) tumors. All three of these tumors with MSI exhibited a high tumor mutational burden with an average of 174 mutations per megabase of DNA, and two of them had confirmed loss or inactivation of mismatch repair proteins. Approximately half of all cases (47%) had inactivation or truncating mutations in PTEN. 59% of cases (10/17) had alterations in WNT pathway genes, including 6/17 (35%) with missense mutations in the regulatory domain of beta catenin (CTNNB1). 2/17 (12%) had TP53 mutations. We identified one case with a mutation in the exonuclease domain of POLE (P286R), which also exhibited a characteristic POLE-mutant mutation signature.

Conclusion: Using whole exome sequencing, we identified that endometrioid ovarian carcinomas are commonly hypermutated and often harbor microsatellite instability. These findings highlight the importance of genetic sequencing to determine which patients may benefit from targeted immunotherapies.

#734

Large-scale radiogenomic profiling of patient derived xenografts (PDX).

Priyanka Gopal, Titas Bera, Craig Peacock, Mohamed Abazeed. _Cleveland Clinic, Cleveland, OH_.

The determinants of clinical failure after radiotherapy remains poorly understood. Although cancer derived cells grown in vitro have served as a valuable resource to identify tumor cell intrinsic determinants of radiation sensitivity, their propensity to undergo genetic selection due to non-native culture conditions reduces intratumoral heterogeneity and alters transcriptomic fidelity. We show that patient-derived xenografts (PDX) models recapitulate the genetic and transcriptomic features of the primary tumors from which they are derived. We extend this characterization to an extensive collection of PDXs derived from multiple cancer types. We propose our "10,000 mice" PDX effort, which seeks to profile up to 500 PDX across 20 cancer types with multiple single fraction doses of radiation (2, 4, 6, 8, 10 Gy). We establish the feasibility of our approach using a 3x1x1 experimental design (3 mice per PDX per dose of radiation). Initial treatment responses following single doses radiation revealed that tumors exhibit significant variation in their response to radiation. Responses in PDX correlated with clinical outcomes, mainly in patients treated with radiation or chemoradiation alone. We compare pre- and post-irradiation PDX and deoncstruct the phylogenetic architecture of tumors before and after treatment selection. We show that in a substantial proportion of the tumors profiled to date, treatment resistance is regulated by subclonal outgrowths. Taken together, our results demonstrate the feasibility of high-content profiling of PDX to identify determinants of radiation sensitivity.

#735

Squamous cell lung cancer with interstitial lung disease has low tumor mutation burden.

Haruki Kobayashi, Masakuni Serizawa, Tateaki Naito, Hayato Konno, Hideaki Kojima, Tetsuya Mizuno, Mitsuhiro Isaka, Masahiro Endo, Takeshi Nagashima, Masatoshi Kusuhara, Kenichi Urakami, Keiichi Ohshima, Ken Yamaguchi, Yasuhisa Ohde, Toshiaki Takahashi. _Shizuoka Cancer Center, Japan_.

Introduction: Smoking history is considered to be related to high tumor mutation burden (TMB) in patients with non-small cell lung cancer (NSCLC). Although smoking history is also a risk factor for interstitial lung disease (ILD), characterization of TMB in NSCLC with ILD is unclear. Therefore, the present study aimed to evaluate TMB in samples of NSCLC with ILD and clarify factors that influence numbers of TMB.

Methods: Between January 2014 and December 2015, NSCLC samples from whom underwent thoracic surgery at the Shizuoka Cancer Center were collected with informed consent and subjected to whole-exome sequencing (WES) with an Ion Proton system, and clinical factors were retrospectively reviewed using the medical records. ILD was diagnosed by high-resolution computed tomography (HRCT) or by pathological findings in resected specimens. In multivariate analyses, the parameters assessed with regard to the TMB were age, sex, histology, pathological stage, Brinkman index (BI) (400) and existence of ILD.

Results: A total of 238 samples were collected from primary tumors with a median TMB (mTMB) of approximately 2.1 mutation/Mb (M) with the range from 0.06 to 64.38. In adenocarcinoma (Ad), mTMB were approximately 1.7 M with the range from 0.06 to 64.38, and in squamous cell carcinoma (Sq), it was approximately 5.3 M with the range from 0.60 to 18.42. This study included 47 NSCLC samples with ILD. There was no significant difference in the number of TMB between tumors located outside the radiological finding of ILD and those located inside (p = 0.1085). Among 184 Ad of this study, 25 samples were with ILD. In univariate analyses, samples with male, heavy smoker and concomitant ILD showed higher TMB. Multivariate analysis showed that BI was significantly associated with the high TMB (p = 0.0009). On the other hand, among 54 Sq of this study, 22 samples were with ILD. In univariate analyses, samples with ILD showed lower TMB than those with non-ILD. Multivariate analysis showed that concomitant ILD was significantly associated with the low TMB (p = 0.0388).

Conclusions: Concomitant ILD could be associated with low TMB in Sq. Oncogenic pathway other than that activated by smoking might promote carcinogenesis of Sq with ILD. Precise molecular character of each group will be presented at the meeting.

#736

Development of an allele-specific assay for detecting circulating tumor DNA in prostate cancer.

Nicholas T. Terrigino,1 S. Thomas Hennigan,1 Shana Trostel,1 Scott C. Wilkinson,1 Huihui Ye,2 Adam G. Sowalsky1. 1 _NIH, BETHESDA, MD;_ 2 _Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA_.

Background and Hypothesis

Cancer cells release circulating tumor DNA (ctDNA) into the bloodstream, which has the potential to be clinically relevant as a marker of tumor clonality and a marker of clinical response. This "liquid biopsy" may provide a non-invasive approach to cancer care diagnosis and monitoring. We have optimized a PCR assay for rapid and inexpensive selection and detection of a priori sequenced targets, serving as clonal and subclonal markers of cancer evolution. Our hypothesis is that this assay will allow us to detect circulating free DNA and ctDNA while modeling tumor clonality and measuring clinical response in patients undergoing treatment for metastatic prostate cancer.

Methods

From a given panel of somatic point mutations previously identified via unbiased sequencing of matched tumor and normal samples, multiplex PCR primer mixes were generated using the IonTorrent AmpliSeq Design tool, creating locus-specific primers flanking each point mutation by approximately 60bp on each side. To the 5' end of each forward primer, a 7-base degenerate unique molecular identifier (UMI) was synthesized, to which an additional 32-base sequence complementary to the forward Illumina Nextera adaptor was further added. To the 3' end of the reverse primer, a 34-base sequence complementary to the reverse Illumina Nextera adaptor was added. Forward primers for each patient-specific set were pooled and hybridized to aliquots of ctDNA extracted from patient plasma. The tagged product was amplified in exponential PCR with the reverse primer pool and a full-length Nextera i7 Indexing primer. Following purification and size selection, the tagged and partial-adaptor-ligated library was amplified in additional exponential cycles of PCR with full length Nextera i5 and i7 indexing primers. Libraries were then quantified and sequenced via MiSeq.

Results and Conclusions

We have previously shown that for a series of seven patients, primers designed against clonal and subclonal mutations successfully amplified their genomic targets in 269 out of 280 amplicons, averaging 10 amplicons per library and 100,000x target coverage per amplicon. Following duplicate reduction, approximately 1,000 unique molecules were sequenced. Spike-in experiments were utilized to establish the lower limit of reliable detection. This assay is currently being applied to patients with metastatic prostate cancer who had blood drawn across multiple timepoints. We are currently analyzing these samples to model tumor clonality and treatment response across multiple patients.

Relevance and Importance

We have developed a robust, patient-specific assay for detection of ctDNA and monitoring patients over time. This assay has the potential to provide a patient-specific and non-invasive approach for monitoring treatment and remission of metastatic prostate cancer.

#737

NF-κB pathway activation by clonal mutations contributes to the recurrence of nasopharyngeal carcinoma.

Rui You, You-Ping Liu, Ming-Yuan Chen. _Sun Yat-sen University Cancer Center, Guangzhou, China_.

Objectives: Identification and characterization of the genomic features that potentially drive NPC recurrence.

Methods: We performed whole-genome/whole-exome sequencing of fresh tumors and corresponding blood samples from 55 rNPC and 44 pNPC patients. Additionally, previously published pNPC exome data were integrated for analysis. Immunohistochemistry and histologic analyses were performed to identify novel biomarkers among the differential mutational events between pNPC and rNPC in independent cohorts of 148 rNPC and 237 pNPC samples with complete clinical follow-up.

Results: rNPC and pNPC tissues had similar mutational burdens; however, the number of clonal mutations was increased in rNPC samples. TP53 and three NF-κB pathway components (TRAF3, CYLD and NFKBIA) were significantly mutated in both pNPC and rNPC. Notably, mutations in TRAF3, CYLD and NFKBIA were all clonal in rNPC; however, 55.6-57.9% of them were clonal in pNPC (P < 0.05). In general, the number of clonal mutations in NF-κB pathway-associated genes was significantly higher in rNPC than in pNPC (P < 0.05). Moreover, the nuclear abundance of NF-κB protein was significantly greater in pNPC samples with local regional relapse than in those without relapse (P = 0.006). Further, in both the pNPC training and validation cohorts, high nuclear NF-κB levels were an independent negative prognostic marker for locoregional relapse-free survival (LRRFS) in pNPC. Accordingly, concurrent chemotherapy for patients with high nuclear NF-κB reduced the rate of locoregional relapse. Lastly, we functionally validated that inhibition of NF-κB enhanced both radiosensitivity and chemosensitivity in NPC cells, strongly supporting our in vivo results.

Conclusion: NF-κB pathway activation by clonal mutations plays an important role in promoting the recurrence of NPC. Moreover, nuclear accumulation of NF-κB is a prominent biomarker for predicting LRRFS and suggesting treatment for pNPC patients. Finally, blocking NF-κB activity may be a novel and effective therapeutic modality for treating NPC.

#738

Myxofibrosarcoma is characterized by frequent abnormalities in TP53 and increased genetic instability.

Yasuhide Takeuchi,1 Annegret Kunitz,2 Hiromichi Suzuki,1 Kenichi Yoshida,1 Yuichi Shiraishi,3 Kenichi Chiba,3 Hiroko Tanaka,3 Teppei Shimamura,4 Nobuyuki Kakiuchi,1 Yusuke Shiozawa,3 Akira Yokoyama,1 Tetsuichi Yoshizato,1 Kosuke Aoki,1 Yoichi Fujii,1 Yasuhito Nannya,1 Hideki Makishima,1 Satoru Miyano,3 Hironori Haga,5 Frederik Damm,2 Seishi Ogawa1. 1 _Kyoto University, Kyoto, Japan;_ 2 _Charite University Hospital, Berlin, Germany;_ 3 _The University of Tokyo, Tokyo, Japan;_ 4 _Nagoya University, Nagoya, Japan;_ 5 _Kyoto University Hospital, Kyoto, Japan_.

Myxofibrosarcoma (MFS) is a rare subtype of soft tissue sarcomas (STSs) preferentially affecting the elderly. Histologically, MFS is distinct from other STSs, in that it is characterized by the proliferation of pleomorphic spindle cells with varying degrees of myxoid components. However, the molecular pathogenesis of MSF is poorly understood.

We conducted an integrated molecular study involving 70 samples from primary MFS patients, in which samples were analyzed by whole-genome sequencing (WGS) (n=5), whole-exome sequencing (WES) (n=44), targeted-capture sequencing (n=65), RNA sequencing (n=3), and immunohistochemistry (IHC, n=50). Copy number (CN) alterations were detected by sequence-based CN analysis. The obtained genomic data were combined with those from STS cases from the Cancer Genome Atlas (TCGA) cohort, including 17 MSF samples and compared to the data from other STS samples (n=189). To further investigate the genetic basis of mixed histological components and chronological changes in MFS, we performed WES of multi-regional and/or multi-time-point samples from 8 MSF cases.

A total of 4,613 mutations were identified by WES in 61 primary MFS samples with a median of 44.0 mutations/sample. Mutations were dominated by age-related C to T transitions at CpG sites. Among 84 primary MFS samples, most frequently mutated and/or CN-altered genes included TP53 (n=64, 76.2%), RB1 (n=29, 34.5%), CDKN2A (n=25, 30.0%), and ATRX (n=18, 21.4%). A fusion gene involving TRIO was newly identified by RNA sequencing in a single case. A similar mutational profile was observed in undifferentiated pleomorphic sarcoma (UPS), in which TP53 (59.1%), ATRX (34.0%), RB1 (22.7%), and CDKN2A (20.5%)) were the major mutational targets, suggesting the common molecular pathogenesis between these two subtypes. Combined with frequent positive staining in IHC (n=22, 44.0%), TP53 was affected in as many as 83.3% of the MFS cases (n=70). Five MFS cases evaluated by WGS showed complex structural abnormalities suggestive of increased genetic instability, where a median of 179 structural variations were detected per sample. WES with multi-regional sampling (n=5) disclosed a high level of intratumor heterogeneity, in which less than 29.0% of mutations were shared by different samples taken from the same tumor. Finally, an analysis of longitudinal samples (n=6) revealed significantly higher numbers of mutations in relapse samples (1.6 times on average, p = 0.03). In all cases, TP53 lesions were present from at the time of initial diagnosis, while most of the other lesions were subclonal and acquired during the clinical course.

In summary, the genetic profile of MFS is characterized by frequent abnormalities in TP53, RB1, CDKN2A and ATRX, and closely related to other STSs, particularly to UPS. Clonal TP53 abnormalities resulted in complex chromosomal structure and a high degree of

intratumor heterogeneity.

#739

Whole-exome sequencing of neuroendocrine neoplasm of the liver reveals recurrent mutations in components of RTK-RAS pathways.

Lingli Chen,1 Yuan Ji,1 Jing Zhao,2 Lingtong Hao,3 Yixue Li,4 Xi Zhang,3 Huichuan Sun,1 Rongkui Luo,1 Jing Han,1 Jie Huang,1 Yingyong Hou,1 Jia Liu1. 1 _Zhongshan Hospital of Fudan University, Shanghai, China;_ 2 _Obstetrics and Gynecology hospital of Fudan university,Shanghai,China, Shanghai, China;_ 3 _Department of Precision Medicine, Research Institute of Genowis, beijing, China, beijing, China;_ 4 _CAS Key Laboratory of Computational Biology, CAS-MPG Partner Institute for Computational Biology, Shanghai Institute of Nutrition and Health, Shanghai Institutes for Biological Sciences, University of Chinese Academy of Sciences, Chinese Academy of Scienc, Shanghai, China_.

Aims: Primary hepatic neuroendocrine neoplasms (phNENs) is a rare and poorly understood type of primary liver cancer. We aimed to perform a comprehensive molecular characterization of this malignancy.

Methods: Twenty-one cases of phNENs were graded into stepwise increasing malignant potential groups: neuroendocrine tumor grade 1 (NET G1, 3 cases), grade 2 (NET G2, 9 cases), neuroendocrine carcinoma (NEC, 9 cases) based on mitotic activity and proliferative index, according to WHO Classification of Tumors of the Digestive System (2010). The expression of PD-L1(clone 28-8), p53(DO7), NCAM, RB, and synaptophysin were detected. Gene expression profiling, DNA copy number detection, and exome sequencing using formalin-fixed samples from 16 patients with phNENs (9 NETs, 7 NECs) were performed. Comparative genomic analysis was carried out, using independent datasets of pancreas NET (n = 64) and intrahepatic cholangiocarcinoma (ICC) (n = 49).

Results: Integrative genomic analysis of phNENs revealed that NEC represents a distinct biliary- derived entity compared with the NET types. NET were, chromosomally stable (mean chromosomal aberrations 4.7 vs. 14.1, p = 0.008), showed significant upregulation of MAPK and PI3K signaling and enrichment of inflammation-related and immune response signatures (p <0.001). NEC tumors were characterized by the mutation signature associated with failure double-strand DNA break repair homologous recombination on its high mutation burdern in samples harbouring BRCA1 or BRCA2 mutation (6/7 vs. 0/9, p <0.001), neural cell adhesion molecule (NCAM) positive (6/7 vs. 1/9, p <0.001), enrichment of progenitor-like signatures, activation of specific oncogenic pathways (i.e., P53-Rb and Wnt), and signatures related to poor clinical outcome. In the NET type, there was a significant correlation in the copy number variation of the ICC, suggesting a clonal origin. Exome sequencing revealed an average of 104 non-synonymous mutations per tumor (2 mean driver mutations per tumor). Among those, TP53 was the most frequently mutated gene (4/7, 57.1%) in phNECs. Intratumor expression of PD-L1 was significantly associated with poorly-differentiated NEC, p53-positive patients. Overall survival rate was significantly correlated to tumor differentiation, growth pattern, necrosis, TP53 mutation (P<0.05), but not correlated to PD-L1expression and vascular invasion(P>0.05).

Conclusions: phNEN represents a heterogeneous group of tumors, with the NEC type characterized by features of poor prognosis, and the NET type with common lineage for iCCA. PhNET stands alone as a distinct biliary-derived entity associated with chromosomal stability and active RTK-RAS signaling. The elevated expression of immune related genes and pathways in NENs suggests a therapeutic opportunity for immunotherapy.

#740

Mutational landscape of early-onset colorectal cancer tumors in Hispanics.

Maria Gonzalez-Pons,1 Lenis Rovira,1 Kelvin Carrasquillo,2 Ingrid Montes,1 Julyann Perez-Mayoral,1 Abiel Roche,2 Anna M. Napoles,3 Jung S. Byun,3 Eliseo Pérez-Stable,3 Kevin L. Gardner,4 Marcia Cruz-Correa1. 1 _Univ. of Puerto Rico Comprehensive Cancer Center, San Juan, PR;_ 2 _Univ. of Puerto Rico Medical Sciences Campus, San Juan, PR;_ 3 _National Institutes of Health, Bethesda, MD;_ 4 _Columbia University, New York City, NY_.

Background: Colorectal cancer (CRC) is the 3rd and 1st leading cause of cancer-related deaths in men and women in the US and Puerto Rico, respectively. Although the incidence of CRC has been decreasing, annual CRC incidence rates have increased by more than 1.5% per year for more than a decade among patients 20 to 49 years old. Early-onset CRC represents a clinically distinct form of CRC often associated with a poor prognosis, which currently comprises 10% to 18% of newly diagnosed cases. Moreover, a marked increase in early-onset CRC incidence has been reported among Hispanics, compared to other racial/ethnic groups. The aim of this study was to perform whole exome sequencing analysis on sporadic, early-onset colorectal tumors and concordant healthy colonic mucosa in order to identify genetic variations that could contribute to the development of colorectal tumors at a younger age among Hispanics.

Methods: Whole exome sequencing analyses were performed on 42 concordant colorectal adenocarcinoma and colonic mucosa tissue samples from individuals diagnosed with colorectal cancer at <50 years old to identify somatic and germinal mutations associated with early-onset colorectal cancer. All participants were recruited by PuertoRico Familial Colorectal Cancer Registry (PURIFICAR); only participants without known mutations in genes associated with hereditary cancer syndromes were included.

Results: A total of 14 shared somatic tumor-specific point mutations were detected in 16 of the 42 early-onset colorectal tumors analyzed. Six of these variants were located in introns and one was located in an intergenic region. When analyzing normal colonic mucosa, 26,716 shared point mutations were detected in all of the participants in our study. Of these, genetic variations were located in introns (16,443), exons (4,008), non-coding RNAs (3,516), promoters (3,467), intergenic regions (2,774), 3' UTR (1,741), 5' UTR (717), and splice sites (79).

Conclusion: This is the first description of the mutational landscape of early-onset colorectal tumors among Puerto Rican Hispanics, a Hispanic subgroup with a high burden of early-onset CRC. The majority of the mutations detected were not located in exons, suggesting that variations in genomic regions that regulate gene expression may contribute to early-onset carcinogenesis. Additional research is warranted to gain a more comprehensive understanding of the biology of sporadic, early-onset CRC in order to directly address this emerging public health problem and to subsequently shift current clinical practices for young patients across the cancer continuum, from prevention strategies to screening and/or treatment.

#741

Clonal evolution of non-malignant proliferative lesions into breast cancers.

Tomomi Nishimura,1 Kenichi Yoshida,1 Yukiko Kawata,1 Yasuhide Takeuchi,1 Nobuyuki Kakiuchi,1 Yusuke Shiozawa,1 Kosuke Aoki,1 Masahiro Hirata,2 Tatsuki R. Kataoka,2 Takaki Sakurai,2 Satoko Baba,3 Yuichi Shiraishi,4 Kenichi Chiba,4 Kengo Takeuchi,3 Hironori Haga,2 Satoru Miyano,4 Masakazu Toi,1 Seishi Ogawa1. 1 _Kyoto University, Kyoto, Japan;_ 2 _Kyoto University Hospital, Kyoto, Japan;_ 3 _Japanese Foundation for Cancer Research, Tokyo, Japan;_ 4 _The University of Tokyo, Tokyo, Japan_.

[Introduction] Non-malignant proliferative lesions in the breast have been implicated in the development of invasive breast cancer. Previous studies showed that adjacent atypical proliferative lesions and breast cancers shared common genetic alterations, suggesting that these evolved from the same ancestral cell. However, the clonal structure of atypical proliferative lesions and their clonal dynamics during progression to cancer are poorly understood. In this study, we compared genetic profiles of normal ducts, non-malignant proliferative lesions and cancers in the same patients to illustrate the clonal evolution of cancer from a non-malignant epithelial cell.

[Methods] Multiple samples were collected from different proliferative lesions within the cancer-borne breast, including invasive cancers, using micro-dissection from formalin-fixed, paraffin-embedded surgical specimens. Somatic mutations and copy number alterations (CNAs) were then evaluated by whole exome sequencing.

[Results] A total of 39 samples from 6 premenopausal females carrying estrogen receptor-positive cancers were analyzed, where the samples were obtained from normal ducts (N = 5), non-malignant proliferative lesions (N = 9), and non-invasive (N = 21) and invasive (N = 4) cancers. The number of somatic mutations per sample was ranging from 1 to 311 and increased with pathological disease progression. Two cases with bilateral cancers had a pathogenic germline mutation of either BRCA2 or TP53, where no somatic mutations or CNAs were shared by individual proliferative lesions, suggesting multifocal independent cancerous evolutions. By contrast, in the remaining four unilateral cases, no pathogenic germline mutations were detected, but all proliferative lesions, which were separated by a distance of 7-33 mm, shared one or more driver alterations, such as an AKT1 mutation (UID: KU01), a GATA3 mutation (UID: KU03 and KU06), a CBFB mutation (UID: KU06) and concurrent 1q gain and 16q loss (UID: KU02, KU03 and KU06), while harboring private mutations/CNAs of their own. The analysis of phylogenic trees based on the number of shared mutations predicted an early origin of these founder mutations, which frequently predated decades before the onset of cancer.

[Conclusions] Early breast cancer development is thought to be shaped by a simultaneous evolution of multiple precancerous clones. It may be multi-focally initiated by a germline mutation, frequently terminated in bilateral cancers. By contrast, in unilateral cases, cancer clones might be derived from a common ancestral clone, which has acquired a driver founder mutation long before the onset of cancer, and undergo independent evolution, giving rise to multiple proliferative lesions, from which invasive cancer finally evolves. Our findings provide unique insight into the early development of breast cancer.

#742

Genomic alteration, tumor mutation burden and PD-L1 status of Chinese hepatic angiosarcoma patients.

Rongkui Luo,1 Lili Zhang,1 Zhengzeng Jiang,1 Lingli Chen,1 Na Zhu,1 Yingyong Hou,1 Qiang Cui,2 Yueting Qu,2 Hong Li,3 Yuan Ji1. 1 _Department of Pathology, Zhongshan Hospital of Fudan University, Shanghai, China;_ 2 _OrigiMed, Shanghai, China;_ 3 _CAS Key Laboratory of Computational Biology, CAS-MPG Partner Institute for Computational Biology, Shanghai Institute of Nutrition and Health, Shanghai Institutes for Biological Sciences, University of Chinese Academy of Sciences, Chinese Academy of Sci., Shanghai, China_.

Background:

Hepatic angiosarcoma is a rare and aggressive tumor which originating from endothelial cells in the liver. Known etiological factors include exposure to vinyl chloride, arsenic, androgenic-anabolic steroids and thorotrast, which was reported associated with mutation of TP53 and KRAS in angiosarcoma. Currently, there is a lack of data on the genomic profiling of hepatic angiosarcoma in Chinese population. We tried to mapping the mutation tendency of Chinese patients with hepatic angiosarcoma and provide the reference of targeted therapy or immunotherapy for these patients.

Methods:

Formalin Fixed Paraffin Embedded (FFPE) samples of 7 Chinese hepatic angiosarcoma patients were collected for next-generation sequencing (NGS)-based 458 genes sequence assay, and the medical history was recounted. Genomic alterations including single base substitution, copy number variations, gene fusions and rearrangement were assessed. Tumor mutational burden (TMB) and microsatellite instability (MSI) status were also analyzed by NGS algorithm. PD-L1 expression was detected by immunohistochemistry (IHC) assay (SP142).

Results:

None of these 7 patients, including 6 males and 1 female with median age of 61, had any contact with vinyl chloride, arsenic, androgenic-anabolic steroids or thorotrast. The most commonly altered genes were TP53 (6/7),ATRX (5/7),IGF1R amplification (2/7),LRP1B (2/7),NOTCH1 (2/7) and SETD2 (2/7). One patient was found contain substitution and amplification of KRAS. Several actionable genomic alterations were found in this study, including PIK3CA (1/7), CDKN2A/B (1/7), KDR (1/7), KIT (1/7) and PDGFRA (1/7). The frequency of ATRX alteration is relatively high, 5 out of 7 patients showed ATRX mutation. These mutations lead to ATRX inactivation which can induce telomerase instability. Meanwhile, the level of TMB was analyzed by 458 gene-related NGS targeted panel sequencing and it was found that 4 patients (57%) presented high TMB (>10 muts/Mb), which is consistent with angiosarcoma in general. Interestingly, 3 TMB-H patients carried ATRX mutation at the same time. All of the samples were identified as microsatellite stability (MSS). IHC results showed that positive expressions of PD-L1 were found in 2 patients (TC 15%, IC 1%-3%).

Conclusions:

Our study firstly revealed the genomic profiling of Chinese hepatic angiosarcoma patients with no history of exposure with known etiological factors. Three patients harbored at least one actionable genomic alteration. Moreover, 3 of the 5 patients with ATRX mutation presented high TMB value, which shows that inactivation of ATRX may relevant to TMB-H. Overall, our results provide evidences for therapy strategy of hepatic angiosarcoma in China, especially patients with high TMB may potentially benefitted from immunotherapy.

#743

Fusion gene identification from common cancer cell lines and comparison to primary tumors.

Neetha Nanoth Vellichirammal, Abrar Albahrani, Jasjit K. Banwait, You Li, Babu Guda. _University of Nebraska Medical Center, Omaha, NE_.

Fusion transcripts, frequently observed in cancer, contribute to oncogenicity by either altering the expression of tumor suppressors/proto-oncogenes or modifying the original function of a protein resulting in an abnormal chimeric protein that stimulates tumorigenesis. Cancer cell lines, extensively used as in-vitro molecular models to study biological processes related to cancer, often do not represent the genomic profiles of primary tumors. To examine the true representation of the cancer cell lines, we examined fusions in tumor- derived cancer cell lines from 20 cancer types in the Cancer Cell Line Encyclopedia (CCLE) and compared them to fusions present in the primary tumors from the TCGA (The Cancer Genome Atlas). ChimeRScope, an alignment-free algorithm developed by our group, was used to screen fusion genes using level 1 paired-end RNA sequencing read data from CCLE and TCGA (GDC data portal). A total of 8,883 primary tumor samples, 730 normal samples spanning 33 cancer types from TCGA, and 802 common laboratory cell lines from CCLE were analyzed in this study. The pool of recurrent fusions identified from CCLE was vastly different from those identified in primary tumors with merely 2% of an overlap. Canonical fusions were found to be more common than non-canonical fusions (fusions containing genes in antisense strand) between TCGA and CCLE. Hierarchical clustering analysis of both canonical and non-canonical recurrent fusions in CCLE and TCGA clustered the primary tumors and cell lines into separate clusters, revealing that fusions in cell lines are vastly different from primary tumors. However, canonical and non-canonical fusion profiles of endometrial cancer cell lines were very similar to female primary tumors (ovarian and breast) and thyroid cancer cell lines were similar to bladder cancer in TCGA. The results from this study indicate that the recurrent fusions in common cancer cell lines harbor fusions that are significantly different from primary tumors.

#744

Clinically targetable genomic alterations in acral melanoma.

Natasa Broit,1 Ken Dutton-Regester,1 Peter Johansson,1 Antonia L. Pritchard,2 Glen M. Boyle,1 Nicholas K. Hayward1. 1 _QIMR Berghofer Medical Research Institute, Herston, Australia;_ 2 _University of the Highlands and Islands, Inverness, United Kingdom_.

Background: Acral melanoma is a rare subtype of melanoma, which has a distinct genomic profile from cutaneous melanoma. While survival outcomes for late-stage cutaneous melanoma have significantly improved in the last decade, treatment options for acral melanoma remain limited. The purpose of this study was to assess therapeutically targetable genomic alterations in acral melanoma.

Methods: The AACR project GENIE database was accessed via cBioPortal (http://cbioportal.org/genie/; version 3) to identify recurrent gene alterations in acral melanomas analyzed using the MSK-IMPACT oncopanel. These alterations were assessed through Cancer Genome Interpreter (http://cancergenomeinterpreter.org) to identify clinically actionable alterations.

Results: Thirty-six patients with acral melanoma were identified from the cBioPortal-GENIE database. These patients were sequenced using three different versions of oncopanel, which included 341 (n = 6), 410 (n = 22) and 468 (n = 8) genes. Seventy-six genes were found to be somatically mutated in this cohort in at least one sample. The most commonly mutated genes were NF1 (n = 5), BRAF (n = 5), PTPRT (n = 4), NOTCH3, HRAS and KRAS (n = 3). Copy number variations greatly contributed to aberration burden, with 132 unique genes found to either carry an amplification or deletion event. The most commonly amplified genes were CDK4 (n = 9), CCND1 (n = 9), FGF19/FGF4 (n = 8), PAK1 (n = 8), MDM2 (n = 7) and FGF3 (n = 7). The most recurrently deleted genes included CDKN2A (n = 9) and CDKN2B (n = 8), and less commonly JAK2 and PTEN (n = 2). Some genes were altered by various mechanisms; for example, some tumors had KIT activated by amplification while other tumors carried missense mutations. BRAF exhibited two fusion events: BRAF-METTL2B (with concomitant amplification) and BRAF-KIAA1549. Cancer Genome Interpreter identified several therapeutic targets, some of which have been the focus of clinical trials in cutaneous melanoma and other solid tumors, e.g. CDK4/6 inhibitors to target amplified CDK4 and CCND1, or mTOR inhibitors for tumors with NF1 mutations.

Conclusion: Several inhibitors are available which could show efficacy in a subset of acral melanomas with particular genomic alterations. Many of these inhibitors have yet to be tested in the context of acral melanoma and should be explored.

#745

Development of a comprehensive gene fusion NGS panel using an integrated microfluidic circuit enabling highly efficient, multiplex PCR enrichment.

Michael L. Gonzales,1 Jian Qin,1 Xiaohui Wang,1 Sangpen Chamnongpol,1 Jeff Jasper,2 Thomas Goralski,1 Christopher Kubu1. 1 _Fluidigm Corporation, South San Francisco, CA;_ 2 _Q2 solutions, Morrisville, NC_.

Introduction: RNA fusion transcripts result from genomic rearrangements where two distinct genes become juxtaposed and fused. Fusion transcripts are an important class of somatic alterations because they have the potential to create chimeric proteins with altered function, contributing to oncogene activation. These aberrant proteins are expressed in a tumor-specific manner and are thus excellent targets for therapeutic intervention. We have designed an amplicon-based library preparation (LP) kit that targets over 350 fusion gene pairs, representing over 1,000 unique fusion breakpoint events from both solid tumor and hematologic cancers. This panel is specifically designed to run on a newly developed microfluidic integrated fluidic circuit (IFC) that supports flexible amplicon library preparation of 1-6 unique LP panels on a single IFC. Samples can be run against the LP panels in groups of 8, allowing for up to 48 samples per IFC. In this poster we present the results of our analytical validation tests, demonstrating the performance of our comprehensive gene fusion panel on synthetic targets, reference FFPE samples, and subject FFPE samples for use in research studies.

Methods: Primers for amplicon library generation targeting known fusion breakpoints were designed in collaboration with Q2 Solutions®. The panel is comprised of over 1,000 amplicons with an average insert size of 170 bp. Primers were divided into 8 assay pools using informatic parameters that minimize generation of off-target products. Assay pools and cDNA from an off-IFC reverse transcription reaction, along with sample barcodes, were dispensed into designated inlets on an IFC and placed in a Juno™ targeted DNA sequencing library preparation system for mixing the nanoscale reactions, thermal cycling, and amplicon harvesting. Harvested amplicons were collected from the IFC, pooled, and prepared for sequencing on an Illumina® NextSeq™.

Results: The panel was tested against synthetic targets representing all targeted amplicons to demonstrate that each assay reliably detects the intended target of interest. Reference standards and subject FFPE samples were used to assess performance, including positive predictive agreement (PPA), positive predictive value (PPV), and limit of detection. Using these samples at inputs as low as 10 ng of total RNA, PPA and PPV were greater than 99% for the fusion events tested. In addition, fusion events could be reliably detected with as little as 250 copies of target fusion material loaded onto the IFC.

Conclusions: A comprehensive fusion gene panel for targeted next-generation sequencing has been developed using nanoliter-scale PCR based enrichment on a newly developed IFC. Generation of high-quality libraries from a minimum of 10 ng total RNA from FFPE samples for use in research studies has been demonstrated.

#746

Genomic alteration of Chinese dMMR/MSI-H gastric cancer.

Jia Wei,1 Jian Wang,2 Xiangshan Fan,3 Yue Wang,1 Yao Fu,3 Kai Wang,2 Nandie Wu,1 Qin Liu,1 Yang Yang,1 Weifeng Wang,2 Baorui Liu1. 1 _The Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University & Clinical Cancer Institute of Nanjing University, Nanjing, China; _2 _OrigiMed, Shanghai, China;_ 3 _Pathology Department, Affiliated Drum Tower Hospital to Medical School of Nanjing University, Nanjing, China_.

Background: Gastric cancer is a high incidence malignancy often diagnosed at an advanced stage with limited therapeutic options and poor prognosis. A small fraction of gastric cancer with dMMR/MSI-H has extraordinary response to the immune checkpoint inhibitor treatment attributing to the immunogenicity generated by the elevated tumor mutation burden. At the meantime, the extensive genomic variants also cause profound impact on many other cancer related genes. Thus the understanding of dMMR/MSI-H gastric cancer genomic profiling is urgent for exploring clinical strategy to this cohort.

Methods: Formalin Fixed Paraffin Embedded (FFPE) samples of 30 Chinese gastric cancer patients were collected for 450 gene panel based next-generation sequencing (NGS) assay. Genomic alterations including single base substitution, short and long insertions/deletions, copy number variations, gene fusions and rearrangement were assessed. Microsatellite instability (MSI) status and tumor mutational burden (TMB) were also calculated by NGS algorithm.

Results: There were 15 males (50%) and 15 females (50%) diagnosed as gastric cancer with a median age of 67 years old. The TMB value of this cohort ranged from 29 to 118 muts/Mb, with the median value of 58 muts/Mb. The most frequent genomic alterations in Chinese MSI-H gastric cancer patients were revealed as KMT2D (50%), RNF43 (50%), CIC (50%), PIK3CA (50%), KMT2C (43%), ACVR2A (40%), TP53 (30%), ATM (33%), and ERBB3 (33%). Truncation has been found the most frequent gene alteration among these top mutations due to the deficiency of mismatch repair system caused reading frame shift. Epigenetic modifier genes with functions of chromatin remodeling and histone methylation have been found extensively mutated in MSI-H gastric cancer. Among eighteen (60%=18/30) patients carrying truncations in ARID1A whose coding protein participates in chromatin remodeling, seven of them possess loss-of-function biallelic truncations. Moderate frequency of mutation also occur at other genes in chromatin remodeling such as ARID1B (23%), PBRM1 (17%) and SMARCA4 (26%). About 77% patients in this cohort carry at least one truncation in genes belonging to the histone lysine methyltransferase 2 (KMT2) family such as KMT2A, KMT2C and KMT2D. Mutations of B2M gene which are commonly associated with resistance to immune checkpoint inhibitor have been found in 23% MSI-H gastric cancer. About 66% of the patients in this cohort carrying mutations in FBXW7, PIK3CA, PTEN, STK11 and TSC may benefit from the mTOR inhibitors.

Conclusions: In general, dMMR/MSI-H gastric cancers possess elevated and dispersive TMB value. Epigenetic modifier genes involving in chromatin remodeling and histone methylation mutate frequently in MSI-H gastric cancer. About 66% of the patients in this cohort may benefit from the mTOR inhibitors.

#747

Characteristic molecular signature of pericardial effusion identifies malignant cancer in pericardial disorder patients.

Suk Woo Nam, Sang Yean Kim, Hyung Seok Kim, Hee Doo Yang, Min Jeong Na. _Catholic Univ. of Korea College of Medicine, Seoul, Republic of Korea_.

Pericardial effusion (PE) can develop in patients with virtually any condition that affects the pericardium, including acute pericarditis and a variety of systemic disorders. PE is also common in cancer patients, and that may develop by several different mechanisms by direct or metastatic spread of the primary process or as a complication of antineoplastic therapies. Thus, definite differentiation of malignant PE and rapid diagnosis are of particular therapeutic and prognostic importance. We aim to determine etiology-based characteristic molecular changes of PE in pericardial disorder patients, and to identify novel molecular signature for early detection of cancer patients with pericardial disorder. We obtained pericardial fluids from the pericardial disorders including cancer patients. Whole genomic transcriptome scan resulted in 1,639 outlier genes as cancer-specific PE molecular signature. Further analysis showed that a cancer-specific molecular signature exists and discerns cancer patients with non-cancer patients, and that also suggested 191 gene elements as stringent classifier between cancer vs non-cancer (Welch's t test, p < 0.05 and Ven diagrammatic analysis). Gene set enrichment analysis (GSEA) with additional computational analysis of 1,639 cancer-specific PE molecular signature identified CD24, SDC1, and ST14 as strong molecular markers for identifying cancer in PE patients. In conclusion, whole transcriptomic analysis of pericardial fluids of pericardial disorder patients suggest that etiology-specific molecular signatures can discriminate different patterns of PE, and that they provide useful information for mechanistic studies that may lead to a better understanding of the molecular basis of PE pathology.

Keywords: pericardial fluid, differential gene expression, diagnostic markers, CD24, SDC1, ST14

#748

Genomic characterization of recurrent small cell lung cancer through research autopsy reveals clonal diversity and candidate driver of chemoresistance.

Hui-Zi Chen,1 Russell Bonneville,1 Melanie A. Krook,1 Michele R. Wing,1 Julie W. Reeser,1 Jharna Miya,1 Anoosha Paruchuri,1 Eric Samorodnitsky,1 Lianbo Yu,1 Amy M. Smith,1 Thuy Dao,1 Dorrelyn Martin,1 Qishan Guo,1 Hailey Magenheim,1 Aharon G. Freud,1 Sharon Cole,2 Gregory Otterson,1 Peter Shields,1 David P. Carbone,1 Patricia Allenby,1 Sameek Roychowdhury1. 1 _The Ohio State University, Columbus, OH;_ 2 _Blanchard Valley Health System, Findlay, OH_.

Genomic characterization of recurrent small cell lung cancer (SCLC) is needed to dissect the molecular mechanisms of relapse, metastatic dissemination, and chemoresistance. SCLC is a highly aggressive, lethal neuroendocrine cancer arising from the bronchial epithelium that develops in heavy smokers. Although many SCLC patients demonstrate sensitivity to first-line platinum-doublet chemotherapy, the response is short-lived and nearly all patients progress during therapy or relapse within several months of completing treatment. Treatment options beyond first-line chemotherapy are limited and ineffective. Metastatic SCLC has a poor prognosis and is associated with a 5-year overall survival rate of 5-10%. A well-characterized genetic hallmark of SCLC is the concurrent inactivation of tumor suppressor genes TP53 and RB1. Additional SCLC candidate driver genes have been reported and include CREBBP, MLL, NOTCH1-4, SOX2 and MYC. However, most SCLC tumors profiled in these studies were obtained from treatment-naïve patients. Therefore, genomic alterations that are acquired during therapy and drive the development of chemoresistance in recurrent SCLC remain undefined. We present results from whole exome sequencing (WES) of multiple metastatic tumors procured from five treatment-refractory SCLC patients who underwent research autopsy. WES was also performed on pre-treatment samples from three of these patients. Our results showed universal TP53 alterations and to a lesser extent RB1 mutations in our SCLC cohort. Next, we utilized bioinformatics methods to analyze clonal heterogeneity and evolution in these SCLC patients. This analysis demonstrated the existence of multiple clones of tumor cells in both the pre-treatment and treatment-resistant autopsy SCLC samples, suggesting that tumor heterogeneity occurs early in SCLC development and is subsequently maintained. Furthermore, allele-specific analysis of copy number variations in our SCLC cohort demonstrated near universal deletion of a region on chromosome 5q containing the tumor suppressor APC, consistent with a recently reported role for deregulated WNT signaling in chemoresistance development in relapsed SCLC. Finally, in one SCLC patient, we identified PTEN deletion (loss of heterozygosity) as a potential mechanism of metastasis specifically to the brain. In conclusion, we have performed genomic characterization of recurrent SCLC, defined by significant clonal diversity, through research autopsy and identified candidate genetic drivers of treatment resistance and organ-specific metastasis.

#749

Multi-layer molecular characterization of high grade serous ovarian carcinomas.

Robert L. Hollis,1 Alison M. Meynert,2 Michael Churchman,1 Tzyvia Rye,1 Patricia Roxburgh,3 Daniel Stetson,4 Athena Matakidou,5 Brian Dougherty,4 J. Carl Barrett,4 Ruth E. March,5 Colin A. Semple,2 C. Simon Herrington,1 Charlie Gourley1. 1 _Nicola Murray Centre for Ovarian Cancer Research, Edinburgh CRUK Centre, MRC IGMM, University of Edinburgh, Edinburgh, United Kingdom;_ 2 _MRC Human Genetics Unit, MRC IGMM, University of Edinburgh, Edinburgh, United Kingdom;_ 3 _Cancer Research UK Beatson Institute and The Institute of Cancer Sciences, University of Glasgow, Glasgow, United Kingdom;_ 4 _Translational Sciences, Oncology, IMED Biotech Unit, AstraZeneca, Boston, MA;_ 5 _Precision Medicine and Genomics, IMED Biotech Unit, AstraZeneca, Cambridge, United Kingdom_.

High grade serous ovarian carcinomas (HGSOCs) are molecularly and clinically heterogeneous malignancies. Currently, molecular stratification of patient care is limited to tumors rendered homologous recombination deficient (HRD) by BRCA1/2 mutation (BRCAm). Here we explore the overlap, interplay and clinical impact of molecular subgrouping layers in a cohort of 362 FFPE HGSOCs. We overlay genomic subgrouping and tumor-infiltrating lymphocyte (TIL) burden with the three transcriptionally-defined subtypes (Immune, Angio and AngioImmune) we have previously reported as associated with survival and bevacizumab sensitivity.

The BRCAm group, the Immune subtype and those with high CD8+ TIL burden demonstrated prolonged overall survival (OS) as previously reported. The OS benefit of CD8+ TILs was abrogated in the context of gross residual disease following debulking [multivariable hazard ratio (mHR)=0.51 (0.34-0.78) vs 0.93 (0.76-1.52)]. BRCA wild-type patients demonstrating high expression of EMSY, encoding the BRCA2-binding protein EMSY, demonstrated prolonged OS [mHR=0.48 (0.29-0.79)]. BRCA2m and high-EMSY patients displayed greater chemosensitivity (first-line CA125-CR 94.4% and 81.3% vs 48.2%, P<0.001 and P=0.016; CA125-CR at relapse 58.3% and 50.0% vs 14.3%, P=0.002 and P=0.015).

There was significant enrichment and depletion of BRCAm and CCNE1-gain (CCNE1g) in the Immune subtype (28.9% vs 6.3%, P=0.001 and 9.1% vs 17.4%, P=0.050). The Angio subtype harboured far fewer CD8+ TILs compared to the Immune and AngioImmune subtypes (P<0.0001 for both). The frequency of RB1 loss and nonsense TP53 mutation was significantly higher in BRCAm versus BRCA wild-type cases (33.3% vs 13.5%, P=0.020 and 21.4% vs 10.3%, P=0.019).

The clinical impact of CCNE1g was modulated by transcriptomic subtype: in the Immune group, CCNE1g was associated with poor OS [mHR=3.32 (1.49-7.41)], while in the Angio group CCNE1g cases had favourable outcome [PFS mHR=0.26 (0.10-0.68)]. CD8+ TIL burden was not associated with outcome in CCNE1g cases. Missense TP53 mutation was associated with better outcome versus TP53-null mutations in BRCAm patients [PFS mHR=0.43 (0.14-0.82)], but not in the context of HR proficiency [mHR=0.90 (0.67-1.22)].

Integrated classification using the consensus of favourable and unfavourable HR-centric (HRD favourable, HR-proficient unfavourable) and transcriptomic (Immune favourable, Angio/AngioImmune unfavourable) subgrouping yielded three groups with distinct OS [favourable vs unfavourable mHR=0.49 (0.34-0.74); favourable vs no-consensus mHR=0.64 (0.42-0.98); unfavourable vs no-consensus mHR=1.31 (1.00-1.70)].

Together, these data paint a more granular picture of the clinical impact of HGSOC subgroups and demonstrate novel candidate interactions between subgrouping layers. The poorest outcome groups represent those with most to gain from trials of novel treatment regimens.

#750

Ancestry-related differentially spliced and expressed genes in prostate cancer.

Muthana Al Abo,1 Daniel J. George,2 Jennifer A. Freedman,2 Steven R. Patierno2. 1 _Duke Cancer Institute, Durham, NC;_ 2 _Duke Cancer Institute, Duke University Medical Center, Durham, NC_.

Prostate cancer (PCa) affects disproportionally men from different population groups. The Surveillance, Epidemiology, and End Results Program's (SEER's) 2018 report reveals that PCa incidence and mortality rates are ~2 times higher among African American (AA) men in comparison with white men. In addition to differences in social, lifestyle and structural determinants of health, there is accumulating evidence for a biological contribution to racial disparity in PCa. To date, most work focused on understanding further the molecular mechanisms underlying racial disparity in PCa has analyzed differential aggregate gene expression and mutation among PCa from patients of different population groups. Our recently published work reported alternative RNA splicing (ARS) as a mechanism promoting tumor aggressiveness and drug resistance in PCa from AA patients. Here, we analyzed The Cancer Genome Atlas (TCGA) data using the Genomic Data Commons to analyze differential aggregate gene expression (2-fold mean change, p < 0.001, Wilcoxon rank sum test) and TCGASpliceSeq to analyze ARS (20% median change, percent spliced in) between PCa from AA and white patients. From our analysis of the 307 PCa specimens from white patients and 49 PCa specimens from AA patients, we identified 71 differentially expressed genes (DEGs) and 73 differential RNA splicing events (DRSEs) between PCa from AA and white patients. 51 of the DEGs (~72%) exhibit increased expression levels in PCa from AA patients compared with white patients. Among the DRSEs, the majority involve exon skipping (35 events, ~48%). Notably, the genes that exhibit differential aggregate gene expression and the genes that undergo differential ARS do not overlap, indicating that ancestry-related differences in aggregate gene expression and ARS can be independent events. However, a significant number of the Gene Ontology terms corresponding to the genes exhibiting ancestry-related differential aggregate gene expression or differential ARS do overlap, indicating that, despite these two distinct mechanisms of regulation (transcription and RNA splicing), both differentially regulate common pathways in PCa between AA and white patients. In addition, we identified 10 trans-acting splicing factors (SFs), whose aggregate gene expression significantly differed between PCa from AA and white patients, suggesting a potential mechanistic relationship between these SFs and the identified DRSEs. These findings increase understanding of molecular mechanisms underlying racial disparity in PCa. Upon further study, such DEGs and DRSEs have the potential to be candidates for novel precision medicine interventions.

#751

**Ductal carcinoma** in situ **of the breast: Cancer precursor or not.**

Lindy Visser, Marlous Hoogstraat, Tycho Bismeijer, Lotte Elshof, Koen van de Vijver, Emilie Groen, Mathilde Almekinders, Joyce Sanders, Carolien Bierman, Dennis Peters, Ingrid Hofland, Frank Nieboer, Michiel de Maaker, Petra Kristel, Lennart Mulder, Annegien Broeks, Michael Schaapveld, Marjanka Schmidt, Lodewyk Wessels, Esther Lips, Jelle Wesseling, On behalf of the PRECISION team. _Netherlands Cancer Institute, Amsterdam, Netherlands_.

Background. Ductal carcinoma in situ (DCIS) is a potential precursor of invasive breast cancer (IBC). However, the natural course of a particular DCIS lesion is unknown, because almost all women with DCIS are treated. Furthermore, most studies are biased because these comprise DCIS adjacent to IBC, also known as synchronous DCIS and IBC, indicating that such DCIS lesions already have the capacity to progress to IBC. It is still unknown which proportion and type of subsequent ipsilateral IBCs (iIBC) are related to the initial primary "pure" DCIS lesion. Therefore, we performed an extensive molecular characterization of DCIS and matched subsequent iIBC, to better understand the natural course of DCIS.

Patients and methods. We used a unique series of 78 women diagnosed with DCIS and treated by breast conserving surgery (BCS) alone, which subsequently developed iIBC. Mean time to iIBC event was 6.3 years (range 0.5-17.0). These 78 women are a representative sample of a case-control series, nested in a nation-wide, population-based cohort including all patients diagnosed with DCIS between 1989 and 2005 in the Netherlands (Visser, et al Clin Can Res 2018). Data on tumor location (ICD-10) was available for all lesions. DNA and RNA was simultaneously extracted for 78 DCIS lesions and 78 matched subsequent iIBC (DNA >20ng; RNA >100ng), and RNA sequencing (RNAseq) and low coverage whole genome sequencing (CNVseq) was performed. Panel sequencing (PanelSeq), using a custom panel of 53 breast cancer driver genes, was performed with the remaining DNA of 42 DCIS and iIBC matched pairs. We determined if the iIBC lesion and DCIS lesion were related, by comparing tumor location and genomic features.

Results. Based on tumor location and histological grade, >95% of the subsequent iIBC reflected outgrowth of residual disease. Based on RNAseq data, 77% of all DCIS and IBC lesions classified into the same PAM50 subtypes. The CNVseq data showed that the DCIS lesions contained copy number aberrations on typical breast cancer-associated loci, such as 1q gain, 8q gain, 16q loss, 20q gain. However, when we compared DCIS with their matched iIBC, we observed in 41% of the cases very distinct copy number profiles, indicating either outgrow of a different tumor (minority) subclone or a second primary tumor. Analysis of PanelSeq mutation data supported this clonal or independent origin of the subsequent iIBC.

Conclusion. To our knowledge, our study is the first to investigate if subsequent iIBC is likely to originate from the initial primary "pure" DCIS in a large series with long-term follow-up.

Surprisingly, our CNVseq and PanelSeq results indicate that more than one third of the subsequent iIBCs after BCS alone treated primary DCIS are likely to be second primary tumors or represent selective outgrowth of a minority DCIS subclone.

#752

Rapid acquired resistance to alectinib in ALK-positive lung cancers with high tumor mutation burden.

Kadoaki Ohashi, Go Makimoto, Shuta Tomida, Kazuya Nishii, Shinichi Toyooka, Katsuyuki Kiura. _Okayama University, Okayama, Japan_.

Background: Alectinib, a highly selective anaplastic lymphoma kinase (ALK) inhibitor, demonstrates an unprecedented effect in ALK-positive lung cancers. However, some tumors quickly develop resistance to alectinib. In this study, we aimed to investigate the mechanism of the rapidly acquired resistance to alectinib using clinical samples.

Methods: Autopsied specimens of lung, liver, and renal tumors were obtained from a 51-year-old male patient with advanced ALK-positive lung cancer, who had acquired resistance to alectinib in only three months. Two alectinib-resistant cell lines were established from pleural effusion (ABC-14) or liver tumors (ABC-17). A patient-derived xenograft (PDX) model was also developed from liver tumors. Next-generation sequencing (NGS), direct DNA sequencing, and quantitative real time reverse transcription-PCR were performed.

Results: ABC-14 harbored no ALK mutations and showed sensitivity to crizotinib, an ALK/MET inhibitor. It also showed mesenchymal-epithelial transition factor (MET) gene amplification and amphiregulin overexpression. The combination of crizotinib and erlotinib showed beneficial inhibitory effects on cell growth. ABC-17 and PDX tumors harbored ALK G1202R. PDX tumors metastasized to multiple organs in vivo, whereas lorlatinib, the third-generation ALK inhibitor, diminished tumor growth both in vitro and in vivo. NGS demonstrated high tumor mutation burden (TMB) and heterogeneous evolution of the patient tissues. The autopsied lung tumors harbored ALK G1202R (c. 3604 G>A) and the right renal metastasis harbored ALK G1202R (c. 3604 G>C); the mutation thus altered codon sequences.

Conclusions: High TMB and heterogeneous tumor evolution may have conferred the rapid resistance to alectinib. Timely administration of lorlatinib or combination therapies with an ALK inhibitor and other RTK inhibitors may constitute a potent therapeutic strategy.

Financial support: This research was supported by a Grant for Lung Cancer Research (K.O.), provided by the Japan Lung Cancer Society.

#753

In situ RNA expression profiling of 1600+ immuno-oncology targets in FFPE tissue using NanoString GeoMx™Digital Spatial Profiler.

Margaret Hoang, Zoey Zhou, Michelle Kriner, Kristina Sorg, Zach Norgaard, Erin Piazza, Chris Merritt, Dae Kim, Joseph Beechem. _NanoString Technologies, Seattle, WA_.

Clinical specimens including formalin-fixed, paraffin embedded (FFPE) tumor sections preserve spatial and molecular information of tumor cells and their surrounding microenvironment. This valuable spatial information is loss with bulk RNA-seq, the most prevalent method for gene expression profiling of archival FFPE samples. In contrast, the NanoString GeoMx™ Digital Spatial Profiler (DSP) is a high multiplexing assay that can profile thousands of RNAs from user selectable regions of interest (ROIs) in FFPE sections. Here we introduce a 1600+ gene panel of tumor, stroma, and immune cell-specific content derived from NanoString nCounter® PanCancer series. We compare RNA profiling using our 1600+ immune-oncology panel to the RNA-seq methodology in FFPE samples. DSP RNA in situ probes are photocleavable oligonucleotides tags coupled to hybridization sequences that bind to mRNA transcripts in the FFPE tissue section. We gridded 96 ROIs upon the FFPE section, photocleaved oligonucleotide tags from each ROI were collected and prepared into sequencing libraries with our NGS readout workflow. After sequencing, reads were charted back to each ROI in the tissue section, generating a map of transcript activity within the tissue. We found high concordance between "bulk" DSP RNA (counts from all 96 gridded ROIs) and RNA-seq from the same FFPE block. Individually, however, each ROI showed different expression patterns than bulk and ROI expression patterns clustered based on similar tissue morphology. We further profiled the tumor and microenvironment compartments from multiple FFPE cancer tissues, comparing our 1600+ RNA expression profile to RNA-seq. DSP was able to detect a higher number of genes with low expression within each spatial compartment compared to bulk RNA-seq. These data demonstrate that DSP offers unparalleled sensitivity for large-scale gene expression while preserving critical information about tissue architecture. GeoMx™ DSP technology is for Research Use Only and not for use in diagnostic procedures.

#754

A comprehensive immunogenomics profiling platform enables the exploration of the intricate relationship between TMB, MSI, Neoantigen and HLA status among multiple cancers.

Jie Wang, Ao Li, Jiawei Wang, Zhaoze Cheng. _WuXi NextCODE, Shanghai, China_.

With the rapid advancement of immune therapy on cancer treatment, there is an increasing demand for accurate and comprehensive profile of tumor immunogenomics landscape in order to broaden our knowledge on predicting patient response as well as to facilitate drug development process. We established a cloud based analytical platform to comprehensively characterize genomic features that are potentially related to patients' responses to immunotherapy. We implemented a neoantigen prediction pipeline based on whole exome sequencing (WES) and transcriptome sequencing data that integrates accurate HLA typing based on WES, somatic variant detection/phasing, RNA expression, class I MHC binding prediction as well as peptide epitope ranking. Among that, the HLA typing pipeline was validated using 73 Chinese and Japanese subjects covering >60 known class I HLA alleles with an accuracy above 95%. In addition, we also developed and validated methods to compute microsatellite instability (MSI) and tumor mutation burden (TMB) based on either a 600 gene panel or WES. Thirdly, this platform also enables the detection of HLA somatic variants and loss of heterozygosity events based on WES data. Next, we applied this analysis platform to process 127 samples across four cancer types (colon, rectal, endometrial and gastric) from TCGA as well as two Chinese cohorts of 100 non-small cell lung cancers and 30 esophageal squamous cell carcinomas sequenced at WuXi NextCODE. Within the TCGA cohort, the data reveals that patients with MSI High status produces significantly more neoantigens than patients with MS Stable status (7.06±4.60 vs 0.71±0.64, p = 7.65e-16, neo-peptides per Mb). Number of neopeptides predicted from each frameshift event is significantly higher than that from each missense mutation (1.30±1.56 vs 0.58±0.54, p = 2.86e-11, neo-peptides per Mb). We also observed strong correlation between TMB and neoantigen burden across multiple cancer types. In conclusion, we have developed a immunogenomics solution that captures tumors' TMB, MSI, neoantigen as well as deleterious events associated with their HLAs. This platform will allow more comprehensive molecular features assessed to potentially improve the prediction of patients' response to immune therapy.

#755

AVENIO Tumor Tissue Analysis Kits performance across Illumina sequencing platforms.

Isabel Soto, Lewyn Li, Cindy Choi, Garima Kushwaha, Jingchuan Li, Bosun Min, Laura Qin, Seng Salee, Liang Feng, Gregory Turenchalk, Jing Li. _Roche Sequencing Solutions, Pleasanton, CA_.

Introduction: Genomic profiling of tumors is an important clinical tool for biomarker discovery and therapy selection. Tumor profiling often relies on formalin-fixed, paraffin-embedded (FFPE) tissue which is challenging because of fragmentation, cross-linking, and nucleotide modifications that arise from the fixation process. AVENIO Tumor Tissue Analysis Kits (For Research Use Only. Not for use in diagnostic procedures) provide an end-to-end solution for FFPE tumor tissue genomic profiling using hybrid-capture next-generation sequencing (NGS). Three panel options (Targeted, Expanded, and Surveillance Kits) provide flexibility in gene coverage with accurate detection of four major types of cancer mutations: single nucleotide variants (SNVs), insertions/deletions (indels), fusions and copy number variations (CNVs). AVENIO Tumor Tissue Analysis Kits were verified and validated on the Illumina NextSeq 500/550 sequencers but there is a need to show comparable performance on alternative NGS platforms. Confirmation of equivalent performance on the HiSeq 4000 allows for sequencing of up to sixteen multiplexed library pools, and simultaneous sequencing of samples analyzed using the AVENIO Tumor Tissue Analysis Kits and the AVENIO ctDNA Analysis Kits.

Methods: In this study, we compared the performance of the three panel options for the AVENIO Tumor Tissue Analysis Kits (For Research Use Only. Not for use in diagnostic procedures) across three Illumina NGS platforms: Illumina NextSeq 500, Illumina HiSeq 4000, and Illumina HiSeq 2500. Ninety libraries containing variants at targeted limit of detection (5% allele frequency for SNV, Fusions, and Indels, and 4-5 copies for CNV) were prepared, sequenced, and analyzed using an internal analysis pipeline equivalent to the commercially available AVENIO Oncology Analysis Software. The total number of reads per sample were normalized across sequencers, and sequencing metrics and variant calls were compared.

Results: The libraries that were prepared using the AVENIO Tumor Tissue Analysis Kits (For Research Use Only. Not for use in diagnostic procedures) and sequenced on the Nextseq 500 demonstrate sensitivities of >99% for SNVs, >99% for Indels, >99% for CNVs, and >99% for Fusions and >99% specificity for all variant types. We found that overall sensitivity and specificity performance were comparable when the samples were subsampled at the same level across the three Illumina platforms. Key sequencing metrics such as sequencing depth, on-target rate, error rate, and uniformity were also similar across the platforms. This study demonstrates that the high assay performance of the AVENIO Tumor Tissue Analysis Kits is consistent across multiple Illumina sequencing platforms.

#756

Whole exome sequencing identifies frequent mutations of PTPRB and KDR in secondary angiosarcoma.

Tenzin Gayden,1 Brendan C. Dickson,2 Hamid Nikbakht,1 Pierre-Olivier Fiset,1 Nicolas De Jay,1 Javad Nadaf,1 David L. Burk,1 Albert Berghuis,1 Rebecca Gladdy,2 Jay Wunder,2 Robert Turcotte,1 Jacek Majewski,1 Nada Jabado1. 1 _McGill University, Montreal, Quebec, Canada;_ 2 _Mount Sinai Hospital, Toronto, Ontario, Canada_.

Angiosarcoma is an uncommon malignant vascular tumor that can occur anywhere on the body, with a predilection for the skin or soft tissues of the scalp and face.

By whole exome sequencing, we identified recurrent somatic mutations in PTPRB, which encodes an endothelial-specific phosphatase that negatively regulates angiogenesis, in three secondary angiosarcoma tumors (3/10). In addition, four tumors harbored missense variants in the KDR gene, including three germline substitutions. Neither the PTPRB nor the KDR alterations were found in the 5 primary tumors studied. Copy number analysis derived from the exome and methylation data revealed frequent MYC amplification in the majority of secondary angiosarcoma tumors (9/10).

Altogether, our data provide a comprehensive overview of genetic and epigenetic landscapes of angiosarcoma, and demonstrate that aberrant angiogenesis associated with PTPRB and KDR mutations may contribute to the pathogenesis of angiosarcoma. 

### miRNA Regulation of Cancer Biology

#757

MiR-141-3p is upregulated in esophageal squamous cell carcinoma and targets pleckstrin homology domain leucine-rich repeat protein phosphatase-2, a negative regulator of the PI3K/AKT pathway.

Ichiro Akagi,1 Osamu Ishibashi,2 Yota Ogawa,2 Toshihiro Takizawa3. 1 _Tokyo Midtown Clinic, Tokyo, Japan;_ 2 _Osaka Prefecture University, Osaka, Japan;_ 3 _Nippon Medical School, Tokyo, Japan_.

The phosphatidylinositol-3-kinase (PI3K)/AKT pathway is frequently activated in various human cancers and plays essential roles in their development and progression. Accumulating evidence suggests thatdysregulated expression of microRNAs (miRNAs) is closely associated with cancer progression and metastasis. Here, we focused on miRNAs that could regulate genes related to the PI3K/AKT pathway inesophageal squamous cell carcinoma (ESCC).To identify upregulated miRNAs and their possible target genes in ESCC, we performed microarraybased integrative analyses of miRNA and mRNA expression levels in three human ESCC cell lines and a normal esophageal epithelial cell line. The miRNA microarray analysis revealed that miR-31-5p, miR-141-3p, miR-200b-3p, miR-200c-3p, and miR-205-5p were expressed at higher levels in the ESCC cell linesthan the normal esophageal epithelial cell line. Bioinformatical analyses of mRNA microarray data identified several AKT/PI3K pathway-related genes as candidate targets of these miRNAs, which include tumor suppressors such as DNA-damage-inducible transcript 4 and pleckstrin homology domain leucine-rich repeat protein phosphatase-2 (PHLPP2). To validate the targets of relevant miRNAs experimentally, synthetic mimics of the miRNAs were transfected into the esophageal epithelial cell line. Here, we report that miR-141-3p suppress the expression of PHLPP2, a negative regulators of the AKT/PI3K pathway, as a target in ESCC.

#758

Long non-coding RNAH19 as a competing endogenous RNA of KIT in the progression of GIST.

Peter Hohenberger,1 Lin Tu,2 Wenyi Zhao,3 Ming Wang Wang,3 Zizheng Zhang,3 Heike Allgayer,4 Hui Cao3. 1 _Div.of Surgical Oncology & Thoracic Surgery, Mannheim University Medical Center, Mannheim, Germany; _2 _Department of Experimental Surgery-Cancer Metastasis, Medical Faculty Mannheim, University of Heidelberg, Germany, Mannheim, Germany;_ 3 _Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China;_ 4 _Department of Experimental Surgery - Cancer Metastasis, Mannheim, Germany_.

Background: Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor with a wide range of biological behaviors, and activating mutations of KIT are particularly crucial to its development. Long non-coding RNAs (lncRNAs) have been identified as an oncogene in multiple cancer types, but data in GIST are missing. The aim of the study was to evaluate the contribution of lncRNA H19 to the malignant course of GISTs.

Materials and Methods: RNA-seq analysis of lncRNA expression in five paired GIST and ANTs were analyzed to screen for differentially expressed lncRNAs. 274 human lncRNAs analyzed, exhibited significantly differential expressions with 186 genes being up-regulated and 88 genes being down-regulated compared to ANT (fold change>5, p<0.05). Unsupervised hierarchical clustering identified 48 lncRNAs and lncRNA H19 was the most overexpressed novel one. To assess the clinical importance of lncRNA H19 upregulation in GISTs, based on the expression levels 92 GIST patients were categorized into two groups: high (n=46) and low (n=46) lncRNA H19 expression.

Results: After identifying lncRNA H19 as the most important one, validation in the expanded cohort confirmed the frequent upregulation of lncRNA H19 in GIST. Functional assays revealed that lncRNA H19 promotes GIST cell proliferation and metastasis. We could demonstrate that lncRNA H19 was predicted to sponge 19 miRNAs with six potential binding sites shared with KIT 3'-UTR using bio-informative database analysis. It was confirmed that lncRNA H19 was positively correlated with the expression of KIT, and the knockdown of lncRNA H19 led to the depression of KIT and its downstream pathway. Using luciferase reporter assays, lncRNA H19 was observed to sponge several miRNAs including miR-18a/b-5p, miR-19a/b-3p, miR-193a/b-3p, miR-216b-5p and mIR-3666. The combination of lncRNA H19 and KIT inhibition showed increased growth inhibition in GIST cell lines. Taken together, the data indicate that lncRNA H19 functions as a competing endogenous RNA (ceRNA) of KIT and promotes GIST progression. It could be shown that the expression levels of lncRNA H19 were significantly correlated with the progression markers of GIST, tumor size (P<0.001), mitosis count (P<0.001), and the modified NIH risk criteria (P<0.001)

Conclusion: Our results indicate that lncRNA H19 can affect imatinib-induced apoptotic cell death and play a role in facilitating GIST progression by modulating the expression of KIT through the ceRNA network. High lncRNA H19 expression is associated with worse prognosis in patients with GIST and lncRNA H19 may serve as a new predictor of prognosis. Furthermore, targeting lncRNA H19/KIT ceRNA network appears to be a promising treatment for GIST.

#759

microRNA 26a-5p inhibited stemness and enhanced the chemosensitivity of non-small cell lung cancer.

Jiwoong Son, Sun Jung Kwon, In Beom Jeong, Ji Hye Kim. _Konyang Univ Hosp, Seo-gu Daejeon, Republic of Korea_.

Only a subpopulation of cells which have the ability to regulate self-renewal and differentiation of cancer cells, are able to tumorigenic capacity. This subpopulation of cells is called cancer stem cells (CSC). Cancer stem cells are also identified in lung cancer and exhibits resistance to chemotherapy, radiotherapy and targeted therapy. Therefore, the technology to control CSCs is a good strategy to overcome resistance to cancer therapy. Epithelial-mesenchymal transition (EMT) has been linked with the generation of cancer stem cells (CSCs). Recent evidence proposes that EMT-related transcriptomic alterations correlate with the acquisition of CSC phenotype. In previous study, we investigated the miRNA profiles of mesenchymal-like lung cancer cell lines to evaluate the relationship beween EMT and miRNA in non-small cell lung cancer (NSCLC). miR-26a-5p is down-regulated in mesenchymal like lung cancer cell lines. These findings suggested miR-26a-5p may be involved in cancer stemness of lung cancer cells. To test that miR-26a-5p has a role in stemness of lung cancer, we performed colony formation assay, sphere formation assay and fluorescence activated cell sorting. RNA polymerase III subunit G (POLR3G) was selected as a candidate target of miR-26a-5p related to cancer stemness. To verify that miR-26a-5p regulates POLR3G, we performed qRT-PCR, western blot and luciferase assays. Cell proliferation assay was performed to evaluate miR-26a-5p's role of chemosensitizer. The overexpression of miR-26a-5p induced a marked reduction of the colony formation and sphere formation in non-small lung cancer cell lines. Overexpression of miR-26a-5p lead to reduction of CD133+ cells. Co-treatment with miR-26-5p and Paclitaxel treatment showed a significant reduction of growth. miR-26a-5p regulated the expression of POLR3G at the mRNA and protein levels. These data demonstrate for the first time that miR-26a-5p suppresses lung cancer stemness by repressing POLR3G expression. This provides new insight into a potential approach for the treatment of lung cancer by regulating cancer stemness.

#760

miR-760 reduced chemotherapeutic resistance of breast cancer cells through downregulating ABCA1.

Xiaowei Wu,1 Lei Huang,2 Ziyi Fu,3 Shui Wang,1 Hui Xie1. 1 _First Affiliated Hospital of Nanjing Medical University, Nanjing, China;_ 2 _Gulou Hospital, Nanjing, China;_ 3 _Women's Hospital of Nanjing Medical University, Nanjing, China_.

Background: It has been confirmed that chemotherapeutic resistance of breast cancer is associated with dys-regulated miRNAs expression. miR-760 expression pattern was confirmed to be related with chemotherapeutic resistance both in breast cancer tissues and cells. However, it's unclear whether miR-760 itself or specific miR-760 regulated genes play brief roles in this property. In this study, we aimed to elucidate the function and undergoing mechanism of miR-760 in chemotherapeutic resistance of breast cancer.

Methods:Drug sensitivity was validated by using Cell Counting Kit-8 in ADR-sensitive breast cancer cells (MCF-7) and ADR-resistant cells (MCF-7/ADR). The expression levels of miR-760 were detected by quantitative real-time polymerase chain reaction (qRT-PCR) in ADR-sensitive/-resistant cells,patient tissues, and mouse models. High-throughput gene microarray and bio-informatic analysis were performed to identify target genes of miR-760. Dual-luciferase assay was used to identify the binding of miR-760 and potential target gene ABCA1. The expression level of ABCA1 was determined by qRT-PCR, western blotting and immunohistochemistry (IHC). The growth and sensitivity of breast tumor to Adriamycin were assessed by micro-CT in vivo.

Results: miR-760, which was downregulated in chemotherapeutic resistance breast cancer tissues and cells, could induce the sensitivity of breast cancer cells to Adriamycin both in vivo and in vitro. Gene microarray and bio-informatic analysis results consistently showed that ABCA1 expression was correlated inversely with miR-760 and ABCA1 might be one of the target genes. Dual-luciferase assay results confirmed that miR-760 could regulate ABCA1 expression through binding to the 3'-UTR region of ABCA1 mRNA. Additionally, overexpression of ABCA1 could reverse the function of miR-760 induced sensitivity of breast cancer cells to Adriamycin.

#761

Up-regulation of miR-10a affect on prostate cancer racial disparity.

Yutaka Hashimoto, Marisa Shiina, Yuichiro Tanaka, Pritha Dasgupta, Priyanka Kulkarni, Taku Kato, Ryan K. Wong, Varahram Shahryari, Shigekatsu Maekawa, Soichiro Yamamura, Divya Bhagiratha, Sharanjot Saini, Guoren Deng, Laura Tabatabai, Shahana Majid, Rajvir Dahiya, Rajvir Dahiya. _UCSF, San Francisco, CA_.

Purpose: Prostate cancer (PC) is the second most diagnosed cancer in men. African-American (AfA) men have higher incidence and twice the PC mortality rates compared to Caucasian-American (CaA) men. In this study, we investigated the biochemical role of miR-10a in prostate cancer racial disparity. Methods: We used PCa cell lines from CaA (DU145, LNCaP, PC3), AfA (MDA-PCa-2b, E006AA-hT) and a normal epithelial cell line (PWR1E) to examine miR-10a expression levels. Total RNA was extracted from total 138 human prostate cancer clinical samples (AfA: n=75, CaA: n=63) and cell lines. Gene expression levels were determined by quantitative real-time PCR (qPCR). A miRNA target database (miRWalk that integrates mainly three databases, miRDB, TargetScan and miRTarBase) was used for miR-10a target prediction. Cell proliferation and apoptosis were monitored after miR-10a knockdown by using anti-miR-10a. Results: miR-10a was significantly up-regulated in AfA prostate cancer cell line, E006AA-hT compared to CaA cells. Utilizing SFVAMC and NDRI patient cohorts, we confirmed that miR-10a expression was linked to a racial difference between AfA/CaA PCa patients. Knockdown of miR-10a showed decreased growth and apoptosis, though the effect was less in CaA compared to AfA cells. We found NCOR2 which is frequently deleted in castration- resistant prostate cancer patient specimens and tumor-suppressor PTEN as miR-10a targets that overlapped in multiple miRNA target prediction databases. Conclusion: These results demonstrate that miR-10a may be a central regulator of crucial events that contribute to racial differences in prostate cancer. Regulation of miR-10a expression may be a new therapeutic strategy for AfA prostate cancer patients.

#762

A non-invasive miRNA signature for the identification of pancreatic ductal adenocarcinoma (PDAC) patients with poor molecular subtypes and worse prognosis.

Raju Kandimalla,1 Tadanobu Shimura,1 Saurav Mallik,2 Susan Tsai,3 Douglas B. Evans,3 Hideo Baba,4 Yasuhiro Kodera,5 Xi Chen,2 Ajay Goel1. 1 _Baylor Scott & White Research Institute and Charles A. Sammons Cancer Center, Dallas, TX; _2 _Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL;_ 3 _Division of Surgical Oncology, Medical College of Wisconsin, Milwaukee, WI;_ 4 _Graduate School of Medical Science, Kumamoto University, Kumamoto, Japan;_ 5 _Nagoya University Graduate School of Medicine, Nagoya, Japan_.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with dismal prognosis. Recent advances in treatment paradigms have been encouraging; however, identification of appropriate patient populations and a better understanding of the underlying tumor heterogeneity could further improve patient outcomes. Recently, four distinct molecular subtypes of PDAC were established, and in particular, squamous subtype was attributed with worse prognosis. Nonetheless, the clinical application of genome-wide transcriptome-based subtyping is cumbersome. Therefore, in this study, we identified miRNA networks regulating the expression of various genes in each PDAC subtype, with a goal to develop tissue and liquid biopsy-based miRNA biomarkers that can be translated into the clinic.

Experimental Design: We used a mRNA-miRNA network-based approach to unravel miRNA networks regulating various PDAC subtypes. Using logistic regression models and area under the curve (AUC) analysis, we evaluated the performance of a miRNA-signature for identifying poor molecular subtypes of PDAC. Using qRT-PCR assays, we also analyzed the prognostic significance of the miRNA panel in 433 PDAC patients, including TCGA (n=183), multiple in-house retrospective, tissue cohorts (n=199) and a prospective, preoperative serum cohort (n=51). Univariate and multivariate Cox-proportional hazard models were used for data analysis, and Youden's index derived cut-off thresholds were used to plot the Kaplan Meier (KM) curves for overall survival.

Results: We identified a panel of 9-miRNAs that were significantly dysregulated (upregulated: miR-205-5p and -934; downregulated: miR-192-5p, 194-5p, 194-3p, 215-5p, 375-3p, 552-3p and 1251-5p) in squamous subtype; and was highly accurate in identifying all poor molecular PDAC subtypes proposed previously (Squamous AUC=0.90, Basal AUC=0.89 and Quasimesenchymal AUC=0.83). Intriguingly, expression level of all these 9-miRNAs clearly stratified overall survival (OS) in patient subgroups, and patients with high-risk scores exhibited significantly worse OS (Hazard ratio (HR): 2.48, p<0.0001). Intriguingly, our miRNA signature was equally robust even in pre-operative serum specimens for identifying patients with poor OS (HR: 2.85, p=0.02), highlighting its significance for treatment decision-making in the clinic.

Conclusions: We for the first time report a unique miRNA signature for identifying patients with poor molecular subtype PDAC who are at risk for worse overall survival. Our 9-miRNA signature can be easily adapted into the clinical setting using simple qRT-PCR based assays. The discrimination of high-risk patients even in the preoperative setting could improve the selection of PDAC patients for neoadjuvant treatment, especially in those with unresectable disease.

#763

**The regulation of miR-125b1-** peroxiredoxin like 2A **anti-oxidative activity in oral squamous cell carcinoma.**

Yi-Fen Chen, Yun-Yen Wei, Cheng-Chieh Yang, Kuo-Wei Chang, Shu-Chun Lin. _National Yang-Ming Univ. School of Dentistry, Taipei, Taiwan_.

Head and neck cancer, including oral squamous cell carcinoma (OSCC), is the sixth most common cancer worldwide. MicroRNAs are small non-coding RNAs that are involved in the modulation of biological/pathological properties. Peroxiredoxin like 2A (PRXL2A) has been reported to be an antioxidant protein that protects cells from oxidative stress. Our previous study identified an association between PRXL2A up-regulation in OSCC and a worse patient prognosis. The miR-125 family of genes drive pluripotent regulation across a wide variety of cancers. In this study, we identify the oncogene eligibility of PRXL2A and clarify miR-125b as its upstream regulator. Down-regulation of miR-125b can be observed in OSCC tumors. Lower miR-125b expression in tumors results in a worse patient prognosis at the relatively early stage. Reporter assays were able to validate that PRXL2A is a direct target of miR-125b. Exogenous miR-125b expression in OSCC cells results in increased oxidative stress and drug sensitivity, and suppressor activity that is paralleled by the knockout of PRXL2A gene. The suppressor activity of miR-125b is able to be rescued by PRXL2A, which suggests the existence of a miR-125b-PRXL2A regulatory axis that is part of OSCC pathogenesis. Nuclear factor erythroid-2-related factor was found to be a downstream effector of the miR-125b-PRXL2A cascade. As a whole, this study has pinpointed novel clues demonstrating that down-regulation of miR-125b suppressor underlies up-regulation of PRXL2A in OSCC, and this then protects the affected tumor cells from oxidative stress resulting in a worse prognosis.

#764

MicroRNA-1205 contributes to the risk of castration-resistant prostate cancer.

Wen Wen, Yicun Wang, Yue Zhang, Xuelian Cui, Xiaoguang M. Liu, Runhua Liu. _Univ. of Alabama at Birmingham, Birmingham, AL_.

Although death rates for prostate cancer are down about 50% as a result of improvements in early detection and treatment, it is still the second leading cause of death (26,120 cases) for American men. However, the genetic mechanism underlying the development and progression of prostate cancer remains poorly understood. It is therefore important to identify the genetic risk factors for prostate cancer. Genome-wide association studies (GWAS) showed evidence for an association of prostate cancer risk with independent genetic variants on chromosome 8q24, conferring prostate cancer susceptibility at this locus. Nevertheless, at the locus with these genetic variants, no specific gene has been identified as responsible for the risk of prostate cancer. At 8q24.21, there is a long-range interaction of these genetic variants with c-MYC or long non-coding RNAs (lncRNAs) in a tissue-specific manner, including prostate, suggesting master genetic factors at 8q24.21 that contribute to this genetic risk. The chromosome 8q24.21 locus, which contains the proto-oncogene c-MYC, long non-coding RNA PVT1, and microRNAs (miRs), is the most commonly amplified region in human prostate cancer. Here, the copy numbers and expressions of miRs-1204~1208 were investigated using quantitative PCR for prostate cancer cell lines and primary tumors. The data revealed that copy numbers and expression of miR-1205 were increased in both castration-resistant prostate cancer cell lines and primary tumors. In castration-resistant prostate cancer specimens, the copy number at the miR-1205 locus correlated with expression of miR-1205. Furthermore, functional analysis with a miR-1205 mimic, a miR-1205 inhibitor, and CRISPR/Cas9 knockout revealed that, in human prostate cancer cells, miR-1205 promoted cell proliferation and cell cycle progression but not apoptosis. In these cells, miR-1205 downregulated expression of the Egl-9 family hypoxia inducible factor 3 (EGLN3) gene and targeted a site in its 3'-untranslated region to downregulate its transcriptional activity. Thus, by targeting EGLN3, miR-1205 has an oncogenic role and may contribute to the genetic risk of castration-resistant prostate cancer.

#765

The PAX3-FOXO1 oncogene alters exosome miRNA content and leads to paracrine effects mediated by miR-486.

Farah Ghamloush,1 Sandra Ghayad,2 Ghina Rammal,2 Assil Fahs,2 Mohamad Harajly,1 Hasan Zalzali,1 Raya Saab1. 1 _American University of Beirut, Beirut, Lebanon;_ 2 _Lebanese University, Beirut, Lebanon_.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. The alveolar histologic subtype (ARMS) accounts for 20% of tumors, and is characterized by a more aggressive course and tendency for metastasis, and a poorer clinical outcome. The majority of ARMS are driven by an oncogenic fusion protein PAX3-FOXO1 (or less commonly PAX7-FOXO1) which plays a major role in driving oncogenic cellular properties. Exosomes are small, secreted vesicles that play an important role in paracrine signaling, in both normal and cancer cells. Using C2C12 murine myoblasts, we now show that the PAX3-FOXO1 protein alters exosome content of myoblasts, leading to pro-tumorigenic paracrine effects in recipient cells, including increased proliferation, migration, invasion, and colony formation. Analysis of miRNA content of these exosomes revealed alterations in miRNA involved in cell metabolism, growth signaling, tumor suppression, and cellular invasion. We identified miR-486-5p as a downstream effector of PAX3-FOXO1 in this setting, where overexpression and knockdown studies identified it as a mediator of the paracrine effects of PAX3-FOXO1-modulated exosomes, including recipient cell migration, invasion, and colony formation. Importantly, miR-486-5p was also enriched in exosomes of human RMS cell lines, and to a higher extent in ARMS cell lines. In addition, miR-486-5p was increased in exosomes derived from serum of patients with RMS, with the highest level noted in ARMS, and follow-up after chemotherapy showed decrease in levels to control values. Our findings identify a novel role of both PAX3-FOXO1 and its downstream effector miR-486-5p in exosome-mediated oncogenic paracrine effects of RMS, and suggest its possible use as a biomarker in RMS.

#766

Mutual negative regulation of EZH2 and miR-4448 for tumor progression via epithelial mesenchymal transition in small cell lung cancer.

Nobuyuki Koyama,1 Yuichi Ishikawa,2 Yuki Iwai,3 Kazutetsu Aoshiba,3 Hiroyuki Nakamura,3 Koichi Hagiwara4. 1 _Tokyo Medical University Hachioji Medical Center, Tokyo, Japan;_ 2 _Japanese Foundation for Cancer Research, Tokyo, Japan;_ 3 _Tokyo Medical University Ibaraki Medical Center, Ibaraki, Japan;_ 4 _Jichi Medical University, Tochigi, Japan_.

Enhancer of zeste homolog 2 (EZH2) is a catalytic component of polycomb repressive complexes, promotes tumor progression and metastasis. Expression levels of EZH2 are associated with clinical outcomes in a variety of tumors. High expression levels of EZH2 have been reported in cell lines and tissues of small cell lung cancer (SCLC), which is characterized by its invasiveness and metastatic potentials. Epithelial-mesenchymal transition (EMT) is well recognized to contribute to aggressiveness of SCLC, and microRNA (miRNA) is known to be associated with both EMT and the biological characteristics of SCLC. The aim of this study was to identify a novel miRNA defining EZH2-assciated EMT and tumor aggressiveness in SCLC. Immunohistochemistry and quantitative PCR showed that EZH2 expressions levels were decreased in three types of SCLC cell lines and tumor tissues from 34 patients with SCLC. Small interfering RNA (siRNA) against EZH2 suppressed its expression and EMT in SBC3 and SBC5 cell lines, both of which had high EZH2 expression levels as SCLC cell lines. Comprehensive miRNA expression analysis was used to compare changes of miRNA expression between EZH2 siRNA-transfected cells and mock-transfected cells. As a result, miR-4448 expression was significantly decreased in

SBC3 and SBC5 cells transfected with EZH2 siRNA. Cell growth assay and cell invasion assay revealed that overexpression of miR-4448 inhibited cellular proliferation and invasion potential. Furthermore, we confirmed that overexpression of miR-4448 abrogated EMT and decreased EZH2 expression. In conclusion, EZH2 and miR-4448 mutually suppressed these expressions and functions, which was associated with tumor progression via EMT in SCLC. The findings of this study suggest that miR-4448 may play a crucial role in the biological characteristics of SCLC.

#767

Deciphering a signature of MYB-regulated miRNAs and novel target/pathways in Ph+ ALL cells: Implications for BCR-ABL targeted therapies.

Manuela Spagnuolo,1 Giulia Regazzo,1 Marco De Dominici,2 Andrea Sacconi,1 Ana Belèn Dìaz Mèndez,1 Etleva Korita,1 Francesco Marchesi,1 Andrea Mengarelli,1 Sabrina Strano,1 Giovanni Blandino,1 Bruno Calabretta,2 Maria Giulia Rizzo1. 1 _Regina Elena National Cancer Institute, Rome, Italy;_ 2 _Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA_.

Background

The Philadelphia (Ph) chromosome generates BCR-ABL constitutively active chimeric proteins in chronic myeloid leukemia (CML) and in a subset of acute lymphoblastic leukemia (ALL). Ph-positive cells proliferation/survival are dependent on the activity of certain transcription factors (TFs), such as c-MYB, which is required for transformation of Ph+ cells. However, the molecular mechanisms responsible for this "MYB addiction" are not completely understood. MicroRNAs (miRNAs), gene expression regulators, may function as downstream effectors of MYB. To investigate the role of MYB-regulated miRNAs in leukemia, we evaluated the global changes in miRNA expression induced by MYB silencing in Ph+ cells. We found that several miRNAs are modulated, including the oncogenic and direct MYB target miR-17-92 cluster. The biological effects of miR-17-92 in CML cells were modest while restoring its expression in ALL cells rescued only in part the reduced proliferation and enhanced apoptosis of these cells. Our aim is to investigate the functional role, individually or in combination, of MYB-regulated miRNAs and to validate novel potential targets amenable to pharmacologic inhibition for Ph+ leukemia

Experimental Procedures

Ph+ ALL cell lines, stably transduced with the doxy-inducible lentiviral vector pLVTSH-MYB ShRNA, and Primary Ph+ ALL cells, were used through this ongoing study. To investigate the molecular pathways involved in leukemia pathogenesis, the miRNA signature, and the target genes, screened by bioinformatic tools, were evaluated by qRT-PCR

Results

We reassessed our unsupervised cluster analysis only in Ph+ ALL-ShMYB cellular context. We found 152 significantly downregulated miRNAs and potentially good candidate to investigate as involved in the "MYB addiction" of Ph+ ALL cells. Among these, MYB-regulated miR-17, -19a, -425, -191, -324 emerged as the most interesting miRNAs and the screening, by informatics tools, of their potential targets, revealed interesting putative miRNAs target genes with oncosuppressor function and related molecular pathways. Moreover, ongoing experiments show that high expression of the miRNA signature in Ph+ ALL patients correlated with that of MYB, is more abundant than in peripheral blood mononuclear cells from healthy donors. By contrast, expression levels of the selected putative target genes result much higher in cells from healthy donors than in blast cells from the Ph+ ALL leukemia patients.

Conclusions

These studies, although preliminary, support the proof of concept that miRNome modulation has an important role in "MYB addiction" of Ph+ cells and targeting MYB-regulated miRNAs and/or pathways under their control may lead to identification of novel druggable targets.

#768

miR-378a-5p acts as a positive regulator of melanoma progression.

Maria Grazia Tupone, Marta Di Martile, Simona D'Aguanno, Elisabetta Valentini, Marianna Desideri, Sara Donzelli, Andrea Sacconi, Daniela Trisciuoglio, Giovanni Blandino, Donatella Del Bufalo. _Regina Elena National Cancer Institute, Rome, Italy_.

Melanoma, the most aggressive form of skin cancer, is frequently associated with alterations in many genes, among which the Bcl-2 oncogene plays an important role in survival, progression, chemosensitivity and angiogenesis. Also microRNAs play an important role in melanoma development and progression affecting tumor proliferation, migration and invasion.

HUVEC and a panel of human melanoma cells were used. Western Blot, qRT-PCR, miRNA Microarray, ELISA, vasculogenic mimicry, cell proliferation, clonogenic and invasion assays have been performed. MiR-378a-5p mimic and inhibitor have been used to study the biological and functional role of miR-378a-5p. In silico analysis have been carried out to find putative miRs targets using miRWalk, Diana Tools and Target Scan.

To identify putative miRNAs, whose expression could be modulated by Bcl-2, M14 have been transfected with a smart-pool RNA targeting human Bcl-2 mRNA, and a miRNA Microarray has been performed. Four independent experiments indicate that, after Bcl-2 silencing, 13 miRs were significantly downregulated. Among these, miR-378a-5p, has been identified as significantly deregulated in different human melanoma cell lines. Based on the target prediction analysis, we identified and then confirmed, through qRT-PCR, several miR-378a-5p target genes, such as SUFU, STAMBP and KLF9. By performing in vitro experiments, we found that ectopic expression of miR-378a-5p does not significantly affect cell proliferation and clonogenic ability of melanoma cells, while it significantly increases invasion and the expression of MMP2 at protein level. It also facilitates the ability of tumor cells to form de novo vasculogenic structures. We have also evidences that conditioned medium from miR-378a-5p overexpressing melanoma increases the formation of capillary like structures in endothelial cells. To assess the molecular mechanism through which miR-378a-5p induce angiogenesis, ELISA showed that ectopic expression of miR-378a-5p significantly increases VEGF protein secretion, but not IL-8 mRNA expression. Experiments using an antiVEGF neutralizing antibody indicate VEGF implication on miR-378a-5p-induced vasculogenic mimicry. To identify other factors that may be implicated in miR-378a-5p proangiogenic/proinvasive functions, we focused our attention on Sp1/uPAR axis, a pathway we previously demonstrated to be regulated in cancer cells by Bcl-2 under hypoxic conditions. Our experiments indicate that ectopic expression of miR-378a-5p increases uPAR mRNA expression. Surprisingly, this treatment results in a Sp1 reduction, indicating that other transcription factor, other than Sp1, can be responsible for uPAR induction by miR-378a-5p.

Based on these evidences, although a deeper understanding of the molecular mechanism involved in Bcl-2 modulation of miR-378a-5p is needed, our findings strongly suggest a proangiogenic and proinvasive role of miR-378a-5p in melanoma cells.

#769

Mechanistic role of miR-29 in pancreatic ductal adenocarcinoma progression.

Shatovisha Dey, Jason J. Kwon, Sheng Liu, Tricia Factoria, Gabriel A. Hodge, Jun Wan, Janaiah Kota. _Indiana Univ. School of Medicine, Indianapolis, IN_.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers with a 5-year survival rate of 8% (Kota et al., 2017, Cancer Lett.). Lack of curative treatment for PDAC warrants better understanding of its mechanisms. Our previous work demonstrated that miR-29 is commonly downregulated in PDAC, while its restoration exhibits tumor-suppressive effects (Kwon et al., 2016, Oncotarget; Kwon et al., 2015, Sci Rep.). To understand these mechanisms, first we performed siRNA mediated knockdown of several miR-29 promoter binding transcription factors, including MYC, which play a role in PDAC. Our results indicate MYC represses miR-29 expression, and MYC nuclear localization negatively correlates with miR-29 in pancreatic cancer cells (PCCs). Next, to identify global miR-29 targets associated with PDAC, we conducted RNAseq on two miR-29 overexpressing PCC lines. This identified 41 overlapping miR-29 targets downregulated in both datasets. Gene ontology and survival correlation analyses identified seven most prominent targets (LOXL2, MYBL2, TRIB2, HGK, NRAS, CD276 and CLDN1). Ectopic expression of miR-29 significantly altered protein levels of these genes, confirming a translational suppression mechanism mediated by miR-29. RNAi mediated silencing of these targets significantly reduced the migratory ability of the PCCs. Our top target LOXL2 is involved in regulation of EMT/migration and extracellular matrix (ECM) remodeling. Luciferase reporter assay verified LOXL2 as a direct miR-29 target in PDAC. Immunohistochemical analyses in clinical specimens from PDAC patients and KPC mice pancreatic tissues showed higher LOXL2 expression in regions of pancreatic intraepithelial neoplasia (PanIN) lesions as compared to non-cancerous areas. This increased LOXL2 level was accompanied by concomitant reduction in miR-29 expression. Thus, our current study provides insight into new miR-29 mediated regulatory pathways in PDAC and reveals the association of miR-29-LOXL2 axis in the disease progression. Together, our data suggest miR-29 to play a critical role in mechanisms of PDAC progression and could serve as a potential therapeutic target for the disease.

#770

MiR628-5p targets Jagged 1 and inhibits growth, stemness and invasiveness of advanced prostate cancer cells.

Leslimar Rios-Colon,1 Juliet Chijioke,1 Anvesha Srivastava,1 Malathi Ramalinga,1 Habib Kedir,1 Elena Arthur,1 Patrice Cagle,1 Gagan Deep,2 Deepak Kumar1. 1 _North Carolina Central University, Durham, NC;_ 2 _Wake Forest University Health Sciences, Winston-Salem, NC_.

Prostate cancer (PCa) is the most common non-cutaneous cancer in men, and according to the American Cancer Society reports, 164,690 new cases and 29,430 deaths from PCa are estimated in the United States in 2018. To reduce PCa mortality, we need a better understanding of molecular regulators of prostate carcinogenesis. MicroRNAs (MiRs) are small, non-coding RNA molecules that have been implicated in various cancers, and offer a novel opportunity to diagnose and treat cancer. We have previously identified MiR628-5p as a potential biomarker in PCa patients. This miRNA was found to be downregulated in the serum of both African American and Caucasian men with PCa compared to healthy individuals. In the present study, we further characterized the anti-cancer role of MiR628-5p in various PCa cell culture models. Results showed that MiR628-5p mimic treatment (100 nM) for 5 days significantly decreased the cell proliferation (~43% inhibition, p<0.001) in human prostate carcinoma PC3 cells compared to random mimic control. Similarly, MiR628-5p mimic treatment decreased the number of clones (clone size ≥50 cells) formed by PC3 cells by 40% (p<0.001) compared to random mimic control. Interestingly, miRNA 628-5p mimic treatment also reduced the stemness of PC3 cells as reflected in significant decrease in prostasphere size and CD44 expression. In mechanistic studies, we identified Jagged-1 as a molecular target regulated by MiR628-5p. MiR628-5p mimic treatment strongly decreased the Jagged-1 expression in several PCa cell lines (PC3, DU145, LNCaP and C4-2) as well as reduced the Jagged-1 luciferase activity. Jagged 1 inhibition by MiR628-5p was associated with reduced Notch signaling as well as decreased expression of transcription factors (snail and slug) regulating EMT. Moreover, MiR628-5p mimic treatment increased the expression of epithelial biomarkers (E-cadherin and ZO1), reduced the expression of mesenchymal marker (vimentin) and strongly inhibited the invasiveness of PC3 cells. In conclusion, we present evidence that MiR628-5p is a key regulator of prostate carcinogenesis, and offers a novel therapeutic opportunity to inhibit the growth of advanced PCa.

#771

CMTM6-mediated maintenance of sarcoma malignancy.

Yuko Nishiyama, Naoto Tsuchiya. _National Cancer Center Research Institute, Tokyo, Japan_.

Sarcomas are malignant bone and soft tissue tumors that have highly aggressive and metastatic features. To reveal molecular networks associated with sarcomas, comprehensive expression profiles were generated using clinical samples. We identified miR-451a, showing different levels according to the prognosis of Ewing's sarcoma (EWS) patients. High expression of miR-451a was also associated with good prognoses of patients with other types of sarcomas, suggesting repression of malignant properties across histological subtypes. Although miR-451a did not affect cell proliferation, it strongly repressed migration of EWS cells. Using gene expression analysis, we identified that CMTM6, encoding a chemokine-like membrane protein, was a target of miR-451a. Depletion of CMTM6 suppressed cell migration and induced changes in the expression of genes encoding plasma membrane and secretory proteins. Interestingly, we found high levels of CMTM6 mRNA under low-glucose culture conditions. Unexpectedly, under the same conditions, high extracellular miR-451a levels were observed; this suggested that the glucose level might be a modulator of the miR-451a-CMTM6 axis. Our data suggest that the malignant properties of sarcoma are influenced by the nutrient status of the tumor microenvironment.

#772

**MicroRNA regulatory networks: Concomitant** in situ **detection of microRNA, mRNA and protein in cancer samples.**

Boye Schnack Nielsen. _Bioneer A/S, Horsholm, Denmark_.

The canonical mode of action of microRNAs involves the binding to the 3'UTR of mRNAs thereby mediating repression of protein synthesis. MicroRNAs comprise an interesting new group of cancer biomarkers and may also be therapeutic targets in cancer and other diseases using antisense oligos (ASO). The visualization of potential molecular dynamics between a microRNA, its target mRNA and the effect of protein expression can be facilitated by combined detection in tissue sections. MicroRNAs, as well as ASOs, can be detected in tissue sections or cultured cells by in situ hybridization (ISH) using LNA probes. We have developed a novel ISH assay for microRNA detection with improved sensitivity and signal-to-noise that can be used with any LNA probe on both FFPE and frozen sections. The assay is based on double-labeled LNA™ probes and detection can be either fluorescence or chromogenic staining. We have combined the fluorescence assay with immunofluorescence, for double and triple staining. We also combined the microRNA ISH assay with mRNA in situ hybridization using the highly specific RNAscope probes. We show a variety of combinations, including examples of combined staining of microRNA-17, IL-1beta mRNA or TNF-alpha mRNA, and cytokeratin proteins in automated triple staining procedures of colon cancer samples. The focal expression of IL-1beta mRNA or TNF-alpha mRNA and the loss of microRNA-17 at the invasive front involving inflammatory cells and budding cancer cells is discussed.

#773

A global increase in miRNAs and a concomitant decrease in tumor suppressor pathways define preneoplastic transition in TNBC.

Anjana Bhardwaj, Zhenlin Ju, Harpreet Singh, Matthew Embury, Jing Wang, Isabelle Bedrosian. _UT MD Anderson Cancer Ctr., Houston, TX_.

Introduction: The molecular changes that accompany the step wise progression to breast cancer are not well characterized. Recent studies have shown that histologically normal cells adjacent to cancer cells possess a large number of mutations suggesting that molecular aberrations often precede the development of histologic change. The goal of this study was to characterize the timing and extent of genomic changes across several well-defined histologic stages of progression to breast cancer and to identify downstream functional target pathways.

Methods: We performed next generation small RNA & RNA seq using a cell line panel previously shown to replicate the multi-step progression from normal (MCF10A) to preneoplastic stage (hyperplastic/ atypia, MCF10.AT1), ductal carcinoma in situ (MCF10.DCIS), and invasive (MCF10.Ca1d) TNBC. Sequencing outputs were analyzed to identify aberrant miRNAs and their functional target pathways. Biological pathways from MSigDB were downloaded and parametric analysis of gene set enrichment were applied to RNA seq data to calculate pathway scores. K-means clustering algorithm was used to group miRNAs, mRNAs and pathways. Interesting groups were selected based on predefined tumor progression profiles. Target Scan was used to identify miRNA target genes, which were used to determine target pathways. The biological networks between miRNAs, target mRNAs and pathways were then created based on Pearson's correlation. Highly correlated and robust target pathways were identified through Pathway Co-expression Network analysis. miRNA and mRNA expression were compared by ANOVA, and a FDR of <0.05 was considered to be statistically significant.

Results: Approximately 70% of miRNA alterations occur during the initial progression from normal to preneoplastic stage. Interestingly, most of the early changes reflect a global upregulation of miRNAs. This was consistent with a global increase in miRNA processing enzyme-DICER and upregulation in XOP5 transcription, a gene involved in miRNA export. DICER upregulation is a direct result of loss of Let-7b-5p, which regulates DICER during TNBC progression and eventually leads to a global increase in miRNA expression. Consistent with early genome wide-increase in miRNA expression, we found a global (>60%) decrease in gene expression. Lastly, we noticed that compared to a relatively small number of miRNAs that change, a larger number of genes and pathways change during TNBC progression. Several oncogenic and tumor suppressor pathways including PI3K cascade, DNA damage check point & DNA damage response are found to be among these miRNAs mediated networks that change early.

Conclusions: These data suggest that most of the genomic changes in the progression to TNBC occur in the very earliest stages of histologic progression, providing significant opportunities to develop targeted strategies for prevention.

#774

Regulation of autophagy and epigenetic modulator micro RNA by transient receptor potential melastatin 7 (TRPM7) channel in glioma.

Mingli Liu. _Morehouse School of Medicine, ATLANTA, GA_.

High grade malignant gliomas are devastating, uniformly fatal cancers for which no effective therapies currently exist. Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. We have reported that TRPM7 channels, a subfamily member of the transient receptor potential (TRP), regulate glioma stem cell (GSC) growth/proliferation through STAT3 and Notch signaling pathways. Autophagy is a catabolic process that occurs during both physiological and pathological process which has been reported to have dual roles, functioning either as a tumor-suppressor or tumor-promoter in cancer, including glioma. In addition, because of the limited success of the traditional treatment in prolonging the overall survival in GBM patients, epigenetics and epigenetic modulators have been attracting an increasing amount of attention for their influence in many aspects of cancer. microRNAs (miRNA), which can be considered as epigenetic effectors, reversibly regulate transcription through binding to complementary sequences of mRNA and silencing its translation into proteins. Therefore, we first determined the role of autophagy process in regulating glioma by TRPM7 using real time RT2 Profile PCR arrays (SABioscience, PAHS-084Z). We found that ATG16L1, ATG9A, CDKN1B, CLN3, CXCR4, and RGS19 were upregulated in response to TRPM7 silencing with the fold change greater than 1.6 using the A172 cell line of human glioma. The gene changes were further validated in other three glioma cell lines U87MG, U373MG and SNB19, which demonstrated different glioma tumorigenicity. miroRNA microarray analysis were then were performed upon TRPM7 silencing in A172 cells (www.LCsciences.com). In-depth data analysis from miRNA microarray data showed a list of 16 downregulated and 10 upregulated miRNA whose transcripts are statistically significant with fold change greater than 2 by TRPM7 knock-down. Among these, the micro RNA of hsa-miR-26b-5p, has-miR4530, and has-miR-28-5p have shown to exert crucial influence on tumor growth and migration by modulating AKT, ERK and IGF-1 signaling pathways. Stem-loop RT-qPCR were then used to detect and quantify miRNAs identified in miRNA microarray data from U87MG, U373MG and SNB19 cell lines. Our data indicate that autophagy effects serve as tumor suppressors in gliomagenesis and are reversely regulated by TRPM7; while hsa-miR-26b-5p, has-miR4530, and has-miR-28-5p are regulated by TRPM7 and participate in glioma progression.

#775

MicroRNA-1205 regulation of FRYL in neuroendocrine prostate cancer.

Michelle K. Naidoo, Olorunseun Ogunwobi. _Hunter College, New York, NY_.

High mortality rates of prostate cancer (PCa) are associated with metastatic castration-resistant prostate cancer (mCRPC) due to the maintenance of androgen receptor (AR) signaling despite androgen deprivation therapies (ADTs). Resistance to second generation ADTs further leads to progression to neuroendocrine PCa (NEPC), which is observed in nearly 1 in 5 men with mCRPC and is associated with low survival rates. The molecular mechanisms of progression to NEPC are, however, still unclear. Further understanding of molecular mechanisms of NEPC is critical to improving patient outcomes. The 8q24 chromosomal locus is a highly susceptible PCa region that carries high risk genetic variants associated with PCa incidence. This region also carries frequent amplifications of the PVT1 gene, a non-protein coding gene that encodes microRNA-1205 (miR-1205). We have previously reported that miR-1205 is underexpressed in PCa tissues in comparison to normal prostate tissue, and is also underexpressed in vitro in CRPC cells in comparison to non-CRPC cells. We also demonstrated that exogenous miR-1205 significantly inhibited tumor volume in CRPC tumor xenografts in mice, and that miR-1205 directly targets the putative oncogene, Fry-like (FRYL). FRYL is predicted to regulate dendritic branching leading to the hypothesis that FRYL plays a role in NEPC. To test this hypothesis, we first examined miR-1205 expression levels in PCa tissues with Gleason scores ≥8 and <8, using a cohort of histologically confirmed tissues. We observed that miR-1205 was underexpressed in tumors with Gleason scores ≥8 when compared to those with Gleason scores <8, suggesting that miR-1205 underexpression is characteristic of high grade PCa. To examine miR-1205 regulation of FRYL specifically in NEPC, we analyzed RNA sequencing data from a cohort of histologically confirmed NEPC tissues and prostate adenocarcinoma tissues obtained from TCGA via cBioPortal. We observed increased FRYL mRNA expression in NEPC tissues when compared to prostate adenocarcinoma tissues, indicating a putative oncogenic role of FRYL in NEPC. In vitro data revealed that FRYL mRNA was overexpressed and miR-1205 was significantly underexpressed after fourteen days of induced NEPC differentiation using LNCaP cells when compared to undifferentiated LNCaP cells. In conclusion, these data suggest that miR-1205 regulation of FRYL may be a critical mechanism in NEPC. Further understanding this mechanism may provide novel insights into effective therapeutic strategies for NEPC.

#776

Posttransriptional control of steroid biosynthesis pathway in prostate cancer.

Asmita Bhattarai, Girish C. Shukla. _Cleveland State University, Cleveland, OH_.

Prostate cancer (PCa) depends on androgens for growth and maintenance. Despite curative therapeutics of the androgen-sensitive PCa, one-third of patients relapse to a more aggressive form of the disease known as castration-resistant PCa (CRPC). Androgen signaling mediated via Androgen receptor (AR) continues to promote CRPC and therapeutic resistance. The synthesis of testosterone (T) and dihydrotestosterone (DHT) via Intratumoral steroidogenesis appears to play a crucial role in CRPC. It appears that PCa cells synthesize de-novo androgen from cholesterol from acetyl-coA via Terpenoid backbone and Steroid biosynthesis pathways. We found that several enzymes of the intracellular cholesterol synthesis pathways including HMGS1, HMGCR, SREBP1, FASN, SCARB1 are potential targets of hsa-miR-149 5p. Currently, we are characterizing miR-149-5p mediated posttranscriptional regulation of these genes and determining its potential therapeutic application in CRPC.

#777

Down-regulation of miR-506 contributes to EGFR-TKI resistance through inducing sonic hedgehog signaling in non-small cell lung cancer.

Inamul Haque,1 Mukut Sharma,2 Andrew K. Godwin,1 Chao H. Huang1. 1 _University of Kansas Medical Center, Kansas City, KS;_ 2 _Kansas City VA Medical Center, Kansas City, MO_.

Background: Lung cancer is the leading cause of cancer related death in the United States. Epidermal Growth Factor Receptor (EGFR) mutation predicts response to a tyrosine kinase inhibitor (TKI) of EGFR in approximately 25% of patients. Non-small cell lung cancer (NSCLC) patients with EGFR mutation eventually develop resistance to EGFR-TKI. The mechanism of resistance is not fully elucidated but majority of the cases is related to emergence of clones with T790M mutation in EGFR, amplification of cMET and Epidermal Mesenchymal Transition (EMT). Sonic Hedgehog (SHh) signaling activation is involved in EMT through FGF, notch, TGFβ signaling and microRNA (miRs) networks. Several miRs have been shown to correlate with TKI resistance indicating that miRs may serve as novel targets and/or biomarkers for anti-EGFR therapy. MiR-506 is involved in Hedgehog signaling and it is abnormally expressed in several cancers. However, the role of miR-506 in modulating TKI resistance in NSCLC remains unexplored. In this study, we investigated the role of miR-506 in the regulation of SHh in EGFR-TKI-resistant lung cancer cell lines.

Methods: To generate resistant cell lines, two EGFR-TKI sensitive NSCLC cells (HCC4006 and HCC827) were exposed to increasing concentrations of erlotinib up to 20 µM over 6 months. The resultant clones were verified for sensitivity to erlotinib using a crystal violet staining assay. The expression of miR-506 in parental and resistant clones was measured using Taqman microRNA assay. Invasive/migratory ability of parental and resistant clones was measured by Boyden chamber assays. EMT and stemness markers were evaluated by Western blotting.

Results: The studies demonstrated that miR-506 levels were ~90% and 75% lower in most resistant clones of HCC4006 (HCC4006ER4) and HCC827 (HCC827ER3), respectively. These clones showed overexpression of SHh (5-fold in HCC4006ER4 and 2.5-fold in HCC827ER3 as compared to their respective parental cells). Ectopic expression of miR-506 in these resistant cells inhibits the expression of SHh by ~80% as well as its downstream mediator, GLI1 by 55%. Our results showed that the IC50 of erlotinib was 22.4 μM for ER cells transfected with ctrl-miR and 4.7 μM for cells with miR-506 mimics. Moreover, an epithelial marker, E-cadherin is upregulated (~2.7-fold) and mesenchymal/cancer stem cell markers (e.g., N-cadherin, vimentin, Sox-2, Notch1, CD44 and ALDH1A1) were significantly downregulated (75-90%, p-value<0.001) in miR-506 overexpressing cells.

Conclusions: Our data showed that miR-506 downregulation and induction of SHh are associated with EGFR-TKI resistance in EGFR mutated NSCLC cells. MiR-506 interference and inhibition of SHh pathway may be potential therapeutic strategies to reverse resistance to EGFR-TKI in NSCLC with EGFR mutation. (This study was funded by the support from IRG‐16‐194‐07(IH) and Godwin's lab).

#778

EphB2 stem-related and EphA2 progression-related miRNA-based networks in progressive stages of CRC evolution: Clinical significance and potential miRNA drivers.

Mariangela De Robertis,1 Tommaso Mazza,2 Caterina Fusilli,2 Luisa Loiacono,3 Maria Luana Poeta,4 Massimo Sanchez,5 Emanuela Massi,1 Giuseppe Lamorte,2 Maria Grazia Diodoro,6 Edoardo Pescarmona,6 Emanuela Signori,7 Graziano Pesole,8 Angelo Luigi Vescovi,2 Jesus Garcia-Foncillas,9 Vito Michele Fazio1. 1 _University Campus Bio-Medico of Rome, Rome, Italy;_ 2 _Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG), Italy;_ 3 _AstraZeneca iMED Biotech Unit, Cambridge, United Kingdom;_ 4 _University of Bari "A. Moro", Bari, Italy;_ 5 _Istituto Superiore di Sanità, Rome, Italy;_ 6 _IRCCS "Regina Elena" National Cancer Institute, Rome, Italy;_ 7 _Consiglio Nazionale delle Ricerche (CNR), Rome, Italy;_ 8 _Consiglio Nazionale delle Ricerche (CNR), Bari, Italy;_ 9 _Cancer Institute, University Hospital "Fundacion Jimenez Diaz", Autonomous University, Madrid, Spain_.

Background: EphA2 and EphB2 are tyrosine kinase receptors that provide a fine positioning of epithelial cells with distinct stemness or differentiation properties in the intestinal mucosa. At the onset of colorectal cancer (CRC), early events trigger continuous stem-like self-renewing state giving rise to adenoma, while additional driver pathway alterations confer invasive behavior in advanced carcinoma. Since EphA2 and EphB2 undergo progressive dysregulation in the intestinal carcinogenesis that may resemble these different stages, we assumed that these receptors may affect alternative cell behaviors on initial and advanced CRC stages, thus representing attractive druggable targets. MicroRNA (miRNA) capacity to simultaneously regulate large cohorts of genes have shaped by evolution combinatorial miRNA-target networks that profoundly affect cell properties, including promotion of tumorigenesis. Through a translational approach and a miRNAome-guided pathway analysis, we explored EphA2/EphB2-related transcriptional signatures with clinical significance in CRC.

Methods: TaqMan miRNA Low-Density Arrays were performed on EphA2high/low and EphB2high/low FAC-Sorted cells and on laser-microdissected tissues of sequential CRC phases obtained from the AOM/DSS murine model. MiRNA expression was used to identify enriched pathways by correlation with validated mRNA targets through Ingenuity Pathway Analysis. Validation of miRNA expression was carried out in public databases (GEO, TCGA) covering 1,663 CRC patients and on 49 hCRC tissue samples. The EphA2/EphB2-related miRNAs and their enriched pathway targets were analyzed for their clinical value by the use of GEO and TCGA.

Results: Through the characterization of progressive miRNAomes centered on EphA2/EphB2 signaling during CRC development, we showed a miRNA-dependent regulation of EphB2-specific stem-like properties in earlier CRC phases and the EphA2-specific control of tumor progression in the latest CRC phases. Two transcriptional signatures that specifically depend on the EphA2/EphB2 signaling pathways were identified, namely EphA2, miR-423-5p, CREB1, ADAMTS14, and EphB2, miR-31-5p, mir-31-3p, CRK, CXCL12, ARPC5, SRC. EphA2- and EphB2-related signatures were validated for their expression in CRC patients. In multivariate analysis, both signatures were predictive of survival and tumor progression. Interestingly, the early dysregulation of miRs-31 found in mice was confirmed on human preneoplastic lesions and tumors. In light of this, miRs-31 emerged as novel potential drivers of CRC initiation.

Conclusions: Our study adds information to the role of a miRNA-dependent orchestration of EphA2/EphB2 networks at different stages of CRC evolution and proposes EphA2/EphB2-related signatures as new predictive biomarkers and therapeutic targets for CRC.

#779

Unraveling exosome-enabled cancer signaling: An integrated genomic approach.

Juan Cui, Jiang Shu, Tian Gao, Haluk Dogan. _Univ. of Nebraska, Lincoln, NE_.

Exosomes play important roles in mediating intercellular communication by transferring various cargos (lipids, RNAs and proteins) into recipients. Emerging evidence has indicated strong implication of exosomes and their RNA cargos in human and animal health including cancers, however, little is known about the mechanisms. In this study, we focus on a specific class of exosomal cargo, microRNAs, to explore new machineries underlying exosome-controlled microRNA secretion and trafficking and subsequently, to assess the regulatory impacts of cancerous noncoding RNAs to surrounding cells or remote tissues in terms of signaling transduction. Particularly, we developed a series of novel bioinformatics techniques that address important problems related to sequence motif finding (primarily based on short RNAs) and gene regulation network analysis (modeling complex structures that reflect multifaceted RNA cooperation and competition). Applying public exosomal microRNA sequence data and TCGA cancer transcriptomic profiles on various types of cancer, we have identified several candidate RNA motifs that might be responsible for guiding the transporter-mediated microRNA loading into exosomes, along with corresponding gene networks that are controlled by those circulating intruders. For example, in human colon cancer SW620 case, three 4-mer sequence motif patterns including [AU][CUG][UG]G, [CGU][UA][GU]G, [AUG][CG]U[UG] are highly enriched among exosomal microRNA sequences (with > 85% coverage, adj.p-value <=1.83E-07). Cell transfection experiments were performed on mutants of the constituent motifs versus their wild-type sequences, and have confirmed motifs, e.g., GGUG, were associated with microRNA loading into exosomes, mostly specific to a certain type of cancer. Results from network analysis further shows such exosomal microRNAs can regulate key signaling processes related to TGF-beta, MAPK, Neurotrophin, and Ras, in the recipient cells through interacting with significant numbers of target genes participating in those pathways. Moreover, the dynamic and conditional aspect of the network analysis enables the identification of microRNA regulatory modules as well as the characterization of regulatory transition across stages along cancer progression. Overall, we demonstrate here that exosome-mediated microRNAs regulation could be an effective channel for cancer cells to communicate with surroundings; and the packaging process is selective depending on unique sequence properties. More importantly, we present an integrative approach using computational modeling and genomic data mining to facilitate the discovery of disease-related exosome and microRNA functions, which can be generally useful for generating new insights and novel intervention tools.

#780

High expression of miR-155 promotes prostate cancer aggressiveness in African-Americans compared to Caucasians.

Marisa Shiina, Yutaka Hashimoto, Priyanka Kulkarni, Pritha Dasgupta, Varahram Shahryari, Guoren Deng, Divya Bhagirath, Laura Tabatabai, Sharanjot Saini, Shahana Majid, Soichiro Yamamura, Yuichiro Tanaka, Rajvir Dahiya. _UCSF/VA Medical Center, San Francisco, CA_.

Background: African-Americans have higher risk for developing prostate cancer and when diagnosed, the cancer is more aggressive and African-Americans are more than twice as likely to die of the disease compared to Caucasians. To clarify the mechanisms involved in this disparity, we analyzed the role of miR-155 in African-American and Caucasian prostate cancer tissues and cell lines.

Materials and Methods: Human tissue samples from African-Americans and Caucasians clinical FFPE (Formaldehyde Fixed Paraffin Embedded) were used to analyzed miR-155 expression by Real Time PCR. We selected prostate cancer cell lines from Caucasians (DU-145, LNCaP, PC3), African-American (E006AA-hT) and normal epithelial cell line (PWR-1E) which express different levels of miR-155, to mimic the tissue samples and help identify the mechanisms related to racial disparity. We knocked down miR-155 in cells lines and performed cell viability assays using a CellTiter-Glo luminescent assay. For migration analysis, miR-155 knockdown cells were seeded in chambers and we measured the capacity of cell motility towards a chemo-attractant. To identify miR-155 mediated downstream regulators, three target prediction algorithms (miRDB, Targetscan and miRecords) were used.

Results: We found miR-155 expression to be higher in African-American prostate cancer tissues compared to Caucasian patients with localized disease. Also, expression of miR-155 in African-American cell line, E006AA-hT, was significantly higher compared to Caucasian cell lines, DU-145, LNCaP and PC3. To determine the role of miR-155 in the differences between African-Americans and Caucasians, miR-155 was knocked down in E006AA-hT and DU-145 cells. miR-155 knockdown in E006AA-hT cells caused a decrease in cell viability compared with negative control, while there was no difference in DU-145 cells. We observed inhibition of migration E006AA-hT miR-155 knockdown cells but not on DU-145 miR-155 knockdown cells, suggesting that miR-155 knockdown have the more potent effect on African-American cell line. We analyzed three target prediction

algorithms and found that four genes overlapped, SPI1, JARID2, ETS1 and CEBPB that may play a pivotal role in the treatment of aggressive African-American prostate cancers.

Conclusion: We conclude that high levels of miR-155 is associated with prostate cancer aggressiveness in African-Americans.

#781

The tumor suppressive microRNA-15 family targets MYCN in neuroblastoma.

Kishore B. Challagundla,1 Patrick Reynolds,2 Santhi Gorantla,1 Larisa Poluektova,1 Don Coulter,1 Surinder K. Batra,1 Srinivas Chava1. 1 _University of Nebraska Medical Center, Omaha, NE;_ 2 _Texas Tech University Health Sciences Center, Lubbock, TX_.

Background: Neuroblastoma is the most common extracranial solid malignancy in children. Amplification/stabilization of the MYCN oncogene defines a group of neuroblastoma with a high risk of recurrence. Despite current aggressive treatment regimens, the prognosis for high-risk neuroblastoma patients remains poor with survival less than 40%. Most of the affected children ultimately die of the disease, which highlights the urgent need for novel therapeutic approaches. MicroRNAs (miRNAs) are small non-coding RNAs with gene expression regulatory functions whose expression is dysregulated in all types of human cancers, including neuroblastoma. However, the role of miRNAs in the regulation of MYCN and the development of drug resistance is currently unknown.

Hypothesis: We hypothesize that miRNA-15 family act as a tumor suppressor in neuroblastoma.

Methods: Neuroblastoma patient tumors were expanded in vivo. miRNAs were profiled by Nanostring and validated by qRT-PCR using TaqMan assays. miRNAs or their inhibitors were overexpressed by lipid reagent mediated transfection or lentiviral infection. Western blotting and qRT-PCR assays detected MYCN expression. Luciferase reporter and Ago-2 immunoprecipitation assays were performed to test miRNA-15 directly targeting of MYCN through interaction with 3'UTR of MYCN mRNA. Cell proliferation assay was carried out through MTT; cell migration by wound-healing; whereas invasion by examining the ability of cells to pass through a Matrigel-coated membrane matrix in neuroblastoma cells. Tumor growth was measured in neuroblastoma xenografts treated with neuroblastoma cells alone or with miRNA-15. MYCN (mRNA and protein), and miRNA-15 levers were assessed in xenograft tumors. Student's t-test was used to evaluate the differences between treatment groups. All statistical tests were two-sided.

Results: Based on the MYCN expression, neuroblastoma patient-derived xenografts (PDX) were divided into MYCN higher and lower expression. miRNA-15 family such as miRNA-15a-5p, miRNA-15b-5p, and miRNA-16-5p were downregulated in PDX with higher MYCN expression. By luciferase and Ago-IP assays, we found miRNA-15a-5p, miRNA-15b-5p, and miRNA-16-5p can directly bind with 3'UTR region of the MYCN mRNA. R2 database involving 105 neuroblastoma patients show an inverse correlation between MYCN mRNA and miRNA-15 host gene. The forced expression of the miRNA-15a-5p, miRNA-15b-5p, and miRNA-16-5p in neuroblastoma cells resulted in a significant reduction in the levels of MYCN and suppressed cell proliferation, migration, and invasion. Conversely, the inhibition of miRNA-15 family expression dramatically accelerated MYCN expression. Injection of neuroblastoma cells with miRNAs inhibits the tumor formation in neuroblastoma xenografts.

Conclusion: These data suggest that miRNA-15 family may function as a tumor suppressor by targeting MYCN in neuroblastoma.

#782

Epigenetic modulation of SEC23/SEC24 and circulating miR-605 by histone deacetylase inhibitors in renal cell carcinoma.

Eri Banno, Nur P. Damayanti, Justin A. Budka, May F. Elbanna, Roberto Pili. _Indiana University, Indianapolis, IN_.

Background: Recent studies have shown that circulating microRNAs (miRNAs) are associated with prognosis in clear cell renal cell carcinoma (ccRCC), and we previously reported the alterations of miR-605 in serum of ccRCC patients receiving treatment with a histone deacetylase inhibitor (HDACi) vorinostat in combination with a VEGF inhibitor bevacizumab. In patients who achieved an objective response (Responders), significant upregulation of miR-605 at baseline and downregulation of miR-605 at post-treatment was observed compared to patients who had progression disease. These alterations of circulating miR-605 suggests its availability inside the cells. SEC23 and SEC24 are components of coat protein complex II (COPII) responsible for vesicle budding including exosomes which contain miRNAs, and alterations of SEC23A or SEC24D are linked to poor prognosis in RCC. Additionally, miR-605 is transcriptionally activated by p53. In this study, we examined the effect of HDAC inhibition on SEC23A/SEC24D and p53 in ccRCC cells. We also evaluated intracellular and extracellular miR-605 expressions in ccRCC cells and circulating miR-605 levels in plasma from patients on our clinical trial with vorinostat and a PD-1 inhibitor pembrolizumab.

Method: ccRCC cell lines 768-0 (p53 mutant) and UMRC-2 (p53 wild type) were treated with the HDACi vorinostat or entinostat, and endogenous protein expression and mRNA expression of SEC23A/SEC24D were evaluated in Western blot analysis and quantitative real-time PCR (qRT-PCR) respectively. Exosomes were isolated from culture medium treated with these HDACis and plasma from the clinical trial participants using ExoQuick Exosome Precipitaion Solution and exosomal RNAs were extracted by SeraMir RNA amplification Kit. MiR-605 expression in cells and in exosomes were assessed in qRT-PCR using TaqMan microRNA Assay.

Results: SEC24D protein expression decreased in both 786-0 and UMRC-2 cells after treatment with HDACis. In contrast, HDACis inhibited p53 more strongly in p53 mutant RCCs (786-0) than in p53 wild-type RCCs (UMRC-2) in Western blotting, showing that modulation of SEC23A/24D may affects mutant p53 protein expression. Upregulation of intracellular levels of miR-605 and downregulation of circulating miR-605 levels was observed in 786-0 cells. Interestingly, our preliminary data showed high circulating miR-605 expression at baseline and downregulation of circulating miR-605 in Responders. These results implicated that high levels of circulating miR-605 levels are regulated by SEC23A/24D and this regulation could be different depending on p53 status.

Conclusions: Our results suggest the therapeutic biomarker potential of circulating miR-605 in ccRCC treated with HDACis in combination therapies. The association between miR-605 and p53 indicates circulating miR-605 may be a novel non-invasive method to detect p53 status.

#783

miR-375 downregulates VEGFA via YAP1-EBRR2 pathway in esophageal cancer.

Shuchang Ren,1 Xiaohui Tan,1 Xiaoling Wu,2 Tao Chen,2 Patricia Latham,1 Sidney W. Fu1. 1 _George Washington Univ., Washington, DC;_ 2 _Chengdu Military General Hospital, Chengdu, China_.

Esophageal cancer (EC), including esophageal squamous cell carcinoma (ESCC) and adenocarcinoma (EAC), is one of the deadliest cancers with extremely aggressive nature and poor survival rate. Diagnostic methods for EC mainly include endoscopy, barium swallow and ultrasound, which are either invasive or lack of sensitivity and specificity at early stages. Therefore, the development of a non-invasive and reliable diagnostic method with high sensitivity and specificity is crucial in improving early detection, diagnosis and treatment. microRNAs (miRNAs) are small RNA molecules that regulate the expression of protein-coding genes by directly binding to target mRNAs in a sequence-specific manner. The aim of this study is to identify novel miRNA biomarkers for EC diagnosis. By cross-referencing our data with others' from NCBI, we identified a list of dysregulated miRNAs in EC, including miR-375. We assessed the expression of miR-375 in EC cell lines and tissues using real-time reverse transcriptase-polymerase chain reaction (qRT-PCR). We found that miR-375 expression is significantly decreased in human EC cell lines (KEYS-70, KEYS-180, FLO-1 and JHU-ad1) compared to the normal esophageal squamous cell line, Het-1A. Using microdissection technique, we isolated normal epithelium, dysplasia, and invasive EC components from formalin-fixed, paraffin-embedded (FFPE) tissue followed by qRT-PCR analysis. Decreased expression of miR-375 was detected in 12 of 14 EC tissue samples (86%) compared to the matched normal tissues. MTT and Matrigel invasion assay demonstrated that forced expression of miR-375 suppressed the proliferation and invasion in EC cell lines. TargetScanS and miRanda were used to identify potential target genes of miR-375, which includes epidermal growth factor receptor 2 (HER2) and YES-associated protein (YAP1), both of them act as oncogenes in EC development. Interestingly, forced expression of miR-375 resulted in significantly reduced expression of human vascular endothelial growth factor-A (VEGF-A) gene, which is a downstream target gene of YAP1/ERBB2 pathway involving in tumor angiogenesis in EC cell lines. More functional analysis is under way. These findings suggest that miR-375 may act as a tumor suppressor, and could serve as a marker for EC diagnosis and management.

#784

**Epigenetic silencing of miR490-3p by** H. pylori **activates DARPP-32 and induces resistance to gefitinib in gastric cancer cells.**

Shoumin Zhu, Zheng Chen, Dunfa Peng, Tian-ling Hu, Heng Lu, Mohammed Soutto, Wael El-Rifai. _University of Miami, Miami, FL_.

Background: H. pylori causes epigenetic dysregulation to promote gastric carcinogenesis, but the roles and functions of microRNAs (miRNAs) in this multistage cascade are not fully explored. In this study, we proposed to investigate the expression and biological functions of miR490-3p in gastric cancer, as well as to identify its downstream target genes and pathways.

Methods: miRNAs expression were profiled by RNAseq. Western blot analyses, Quantitative real-time PCR, ATP-Glo, clonogenic survival, Annexin V, and bisulfite PCR amplification were used. The survival rate was calculated using the Kaplan-Meier method and log-rank tests.

Results: DARPP-32 was markedly upregulated in gastric cancer (P < 0.01), whereas, significant downregulation of miR-490-3p was confirmed in mice and human gastric cancer tissues (P < 0.05). Importantly, our clinical data demonstrated negative correlation of DARPP-32 and miR-490-3p in human gastric primary tumors, strongly supporting a role for miR-490-3p regulating DARPP-32 expression in gastric cancer (R = - 0.58, P < 0.01). miR490-3p host gene CHRM2 is hypermethylated and downregulated in mice and human gastric tissues (P < 0.05). Infection with H. pylori decreased the expression of miR490-3p in the in vitro and in vivo models (P < 0.05). qRT-PCR and Western blot analysis data showed higher protein and mRNA levels of DARPP-32 in H. Pylori infected mice than in control mice (P < 0.05). Reconstitution of miR490-3p in MKN45 cells sensitized cancer cells to gefitinib treatment by directly targeting DARPP-32, and inactivating AKT and STAT3 signaling pathways. Conversely, miR490-3p inhibitor increased DARPP-32 protein expression and reversed these signaling effects leading to decreased sensitivity to gefitinib, as measured by ATP-GLO Cell Viability and Clonogenic Survival Assay (P < 0.01). Knockdown of DARPP-32 significantly attenuated the pro-tumorigenic effects of miR490-3p inhibitor, whereas, enforced expression of DARPP-32 promoted gefitinib resistance in gastric cancer cells (P < 0.01). In survival analysis, gastric cancer patients with low levels of miR490-3p and high expression level of DARPP-32 had poor clinical outcome with decreased overall survival (P < 0.05).

Conclusion: Hypermethylation-mediated silencing of CHRM2 and miR-490-3p by H. pylori increased DARPP-32 expression. Downregulation of miR-490-3p in gastric cancer plays a role in gefitinib response by inducing DARPP-32 and PI3K/AKT, STAT3 signaling pathways.

#785

The inhibitory effect of compound ChlA-F onhuman bladder cancer cell invasion can be attributed to its blockage of SOX2 protein.

Xiaohui Hua,1 Jiheng Xu,1 Chuanshu Huang,1 Jingxia Li,1 Zhongxian Tian,1 Haishan Huang2. 1 _New York Univ., Manhattan, NY;_ 2 _Wenzhou Medical University, Wenzhou, China_.

Sex-determining region Y-box 2 (SOX2), a well-known stemness biomarker, is highly expressed in a variety of cancers, including human highly invasive bladder cancer (BC). However, the role of SOX2 may vary in different kinds of malignancy. In the present study, we discovered that ChlA-F, a novel conformation derivative of isolate Cheliensisin A (Chel A), remarkably inhibits the invasive ability of human invasive BC cells through downregulation of SOX2 protein expression. We found that ChlA-F treatment dramatically decreases SOX2 protein expression in human high-grade invasive BC cells. Ectopic expression of SOX2 reversed ChlA-F inhibition of cell invasion ability in human bladder cancer cells, suggesting that SOX2 is a major target of ChlA-F during its inhibition of human BC invasion. Mechanistic studies revealed that SOX2 is downregulated by ChlA-F at both the protein degradation and protein translation levels. Further studies revealed that ChlA-F treatment induces HuR protein expression and that the increased HuR interacts with USP8 mRNA, resulting in elevation of USP8 mRNA stability and protein expression. Elevated USP8 subsequently acts as an E3 ligase to promote SOX2 ubiquitination and protein degradation. We also found that ChlA-F treatment substantially increases c-Jun phosphorylation at Ser63 and Ser73, initiating miR-200c transcription. The increased miR-200c directly binds to the 3'-UTR of SOX2 mRNA to suppress SOX2 protein translation. These results present novel mechanistic insight into understanding SOX2 inhibition upon ChlA-F treatment and provide important information for further exploration of ChlA-F as a new therapeutic compound for the treatment of highly invasive/metastatic human BC patients.

### Mitochondrial Function and Metabolism in Cancer

#786

Metastasis is driven by mitochondrial protein A via mitochondrial reprogramming in colorectal cancer.

Dayeon Kim, Sang-Mo Kwon. _Pusan National University, Yangsan, Republic of Korea_.

Colorectal cancer (CRC) is the third most common cancer worldwide, and CRC metastasis is the main cause of cancer-associated mortality. The hypoxia signaling pathway controls mediators to facilitate cancer progression and metastasis. Recently, mitochondrial reprogramming has emerged as a one of the mechanisms of hypoxia-induced metastasis; however, the link between hypoxia-induced metastasis and mitochondrial reprogramming remains to be elucidated. Here, we have focused on the novel mitochondrial protein A, to determine its role in the molecular mechanism of hypoxia-induced metastasis. Our results indicate that mitochondrial protein A is a downstream target of hypoxia-inducible factor 1 (HIF-1α) and plays an essential role in HIF-1-induced migration and invasion. In addition, we found a clinical correlation between hypoxia and mitochondrial protein A. To identify the role of mitochondrial protein A, we generated mitochondrial protein A deficient or silenced cells, which showed impaired migration, invasion, and colony-forming ability even under hypoxic condition. In contrast, ectopic expression of mitochondrial protein A elevated metastatic potential, both in vitro and in vivo. Mechanistically, mitochondrial protein A directly interacted with dynamin-related protein (DRP1) to retain it in mitochondria, thereby promoting mitochondrial fission. To confirm the effect of mitochondrial fission on mitochondrial protein A-induced metastasis, we blocked DRP1 in mitochondrial protein A overexpressing cells by pharmacological inhibition, which led to impairment of mitochondrial protein A-induced metastatic potential. Furthermore, mitochondrial protein A-silenced cells showed decreased metabolism and lactate production, suggesting that mitochondrial protein A regulates the mitochondrial reprogramming. In summary, we demonstrate that mitochondrial protein A is a novel DRP1 adaptor protein, facilitating mitochondrial fission and mitochondrial reprogramming, and plays a critical role in hypoxia-induced metastasis. We suggest that targeting of mitochondrial protein A maybe a strategy for cancer therapy.

#787

**Aberrantly increased expression of** ZIC2 **is correlated with altered cellular metabolism in prostate cancer.**

Nathan J. Bowen, Alira Danaher. _Clark Atlanta University, Atlanta, GA_.

Prostate cancer incidence and mortality rates are consistently reported to be higher in American men of recent African ancestry when compared to American men of recent European or Asian ancestry. However, actionable factors contributing to these different rates remain undefined. In order to identify common molecular events to assist in the design of interventions aimed at prevention and treatment of lethal prostate cancer, we analyzed existing public RNA-Seq data and report that Zic family member 2 (ZIC2) RNA levels are significantly higher in prostate and other cancers. ZIC2 encodes a C2H2 zinc finger domain containing protein that is expressed during embryonic development in metazoans. Altered ZIC2 expression results in holoprosencephaly in humans. The ZIC2 locus also encodes several RNA species that have been co-purified with members of the polycomb repressive complex 2 (PRC2). In prostate cancer, we found that ZIC2 levels correlated significantly with primary cancer grade, i.e., the highest ZIC2 expression was found in lethal, metastatic prostate cancer. Therefore, we hypothesize that increased ZIC2 contributes to cancer initiation and progression. To establish models for identifying functional roles for ZIC2 in cancer, we targeted the first exon in ZIC2 by CRISPR/Cas9, with the intent of abrogating ZIC2 expression. We established clonal cell lines from PC-3, E006AA-Par and 293T cells with mutant ZIC2 loci. ZIC2 mutant cells displayed significant decreases in proliferation and altered phenotypes. RNA-Seq revealed expression patterns consistent with reduced glycolysis and increased oxidative phosphorylation, as well as many altered signaling pathways, in cells with ZIC2 mutations. Metabolism was monitored by extracellular flux analysis and revealed increased oxygen consumption and significantly higher respiration rates in ZIC2 mutant cells. ZIC2 localization to mitochondria was predicted using computational methods and confirmed by sub cellular fractionation and immunoblot analysis as well as by GFP-tagged microscopy. JC-1 fluorescence indicated increased mitochondrial membrane potential in ZIC2 mutant cells. We present these results and suggest that ZIC2 promotes anabolic metabolism consistent with embryonic development and cancer cell proliferation. We found that altering ZIC2 expression in PC-3 cells increased oxidative phosphorylation and produced a 5X increase in the number of apoptotic cells. Our results suggest that ZIC2 expression contributes to the cancer phenotype, given that altered cellular energy metabolism and evasion of apoptosis are defining characteristics of nearly all cancers. We further propose that the targeted inhibition of ZIC2 in cancer cells will decrease mortality rates in men diagnosed with lethal prostate cancer.

#788

Identification and optimization of pro-apoptotic molecules targeting adenine nucleotide translocator 2 (hANT2) in tumor mitochondria.

Nelly Buron, Claire Pertuiset, Roxane Loyant, Cécile Martel, Mathieu Porceddu, Annie Borgne-Sanchez. _MITOLOGICS SAS, Paris, France_.

The mitochondrial protein ANT2 is one of the four isoforms of the ADP/ATP translocase and is expressed in highly proliferating cells. ANT2 plays a crucial role in the maintenance of transmembrane potential and mitochondrial integrity in tumor cells by importing glycolytic ATP into the mitochondrial matrix. Thus, this protein is required for tumor cell survival and displays anti-apoptotic function. Recently, ANT2 upregulation was shown to be involved in drug resistance process in various cancer types. Considering ANT2 role in tumor cell metabolism, we searched for ANT2-ligand small molecules. Ligands were first identified by virtual screening of chemical library on 3D model of human ANT2 and validated as ADP/ATP translocase inhibitors using our screening platform on isolated mitochondria. Compound specificity for ANT2 isoform was validated by cellular knock-down and pull-down experiments. ANT2-ligands optimization lead to the selection of MTL105 compound that induce characteristic intrinsic apoptotic cell death, specifically in tumor cell lines at sub-µM concentrations with no effect on healthy cells, suggesting a strong safety margin. According to the NCI60 compare analysis, this compound constitutes a first-in-class product in cancer therapy with this MoA and would be particularly interesting to overcome multidrug-resistant cancers.

#789

**Multi-targeted novel 5-substituted pyrrolo[3,2-** d **]pyrimidines with** **tumor-selective targeting and inhibition of** **cytosolic** de novo **purine biosynthesis and mitochondrial one-carbon metabolism.**

Nian Tong,1 Khushbu Shah,1 Aleem Gangjee,1 Carrie O'Connor,2 Adrianne W. Porvirk,2 Aamod Dekhne,2 Zhanjun Hou,2 Larry H. Matherly2. 1 _Duquesne Univ., Pittsburgh, PA;_ 2 _Wayne State University School of Medicine, Pittsburgh, PA_.

One-carbon (C1) metabolism supports a number of physiological and pathophysiological processes ranging from stem cell renewal to cancer progression. Clinically used antifolates are transported into both tumor and normal cells by the ubiquitously expressed reduced folate carrier (RFC). Uptake of targeted agents via tumor-specific folate receptors (FRs) over RFC would permit tumor-selectivity, while limiting dose-limiting toxicities associated with standard chemotherapy. Serine catabolism in mitochondria is the major source of glycine and C1 units for cytosolic biosynthesis, preserves redox balance and minimizes reactive oxygen species, and is an important source of ATP. Among the mitochondrial C1 enzymes, serine hydroxymethyltransferase 2 (SHMT2) and 5,10-methylene tetrahydrofolate (me-THF) dehydrogenase 2 (MTHFD2) are highly expressed in tumors versus normal tissues. SHMT2 has been suggested to be an important oncodriver. However, there are no clinically relevant inhibitors of these enzymes. To generate potential inhibitors of these enzymes, we synthesized 5-substituted pyrrolo[3,2-d]pyrimidine analogs as structural hybrids of cytotoxic 5-substituted pyrrolo[2,3-d]pyrimidines and me-THF. The 5-substituted pyrrolo[3,2-d] pyrimidine with a four carbon bridged phenyl side chain AGF300 afforded selective uptake via FRα over RFC, with inhibition of mitochondrial C1 metabolism and de novo purine biosynthesis, resulting in inhibition of KB human tumor cell proliferation. Inhibition of KB cells by AGF300 was reversed by glycine and adenosine. As previous studies of related 5-substituted pyrrolo[2,3-d]pyrimidines established that the nature and length of the bridge plays an important role in determining tumor cell potency and transport selectivity, we replaced the carbon adjacent to the phenyl ring in AGF300 with heteroatoms, including O (AGF323), S (AGF346) or NH (AGF350). These compounds were tested as growth inhibitors against engineered Chinese hamster ovary (CHO) cells singly expressing human FRα (RT16) or RFC (PC43-10). Incorporation of the O, S and NH in the pyrrolo[3,2-d]pyrimidine analogs preserved excellent inhibition of FRα-containing CHO cells (IC50s of 57 nM, 77 nM and 50 nM, respectively); there was no inhibition of cells with RFC uptake up to 1000 nM. AGF323, AGF346 and AGF350 inhibited proliferation of KB cells which was reversed by excess glycine and adenosine. This establishes that for AGF323, AGF346 and AGF350, both mitochondrial and cytosolic C1 metabolism were inhibited. The development of novel compounds targeting mitochondrial and cytosolic C1 pathways with tumor-selective uptake is highly significant in that this would overcome the drawbacks of currently used cytotoxic agents for cancer.

#790

STAT3 control of mitochondrial RNA stability and translation is a vulnerability in tumors.

Chamira D. Fernando, Daniel J. Garama, Daniel J. Gough. _Monash University, CLAYTON, Australia_.

Signal Transducer and Activator of Transcription (STAT)3 is a critical signalling molecule and transcription factor activated downstream of cytokines and growth factors to regulate immune responses, metabolism, proliferation and cell death. Whilst the activity of STAT3 is crucial for sustenance of life, aberrant activity is observed in around 50% of all human cancers. However, recent evidence has shown non-transcriptional activities of STAT3 that are critical for tumor growth. Mitochondria are the central powerhouse of the cell and provide the biomolecules required for rapid cell proliferation. Recently we showed that a pool of STAT3 resides in mitochondria and enhances the activity of complex I, II and V of the electron transport chain (ETC) driving Ras-dependent malignant transformation. Whilst STAT3 can physically interact with complex I, its low abundance compared to complex I suggests that a direct protein-protein interaction is unlikely to dictate metabolic activity of the ETC.To elucidate mitochondrial activity of STAT3, we isolated STAT3 from mitochondrial fractions of Ras-driven lung adenocarcinoma (A549 cell line) and identified its interactome using mass spectrometry. We show that STAT3 forms a stable 400 kDa complex with the LRPPRC/SLIRP proteins which are key regulators of mitochondrial mRNA stability and translation. STAT3 loss significantly reduced steady state levels of polyadenylated mitochondrial RNA which is due to instability of the polyadenylated transcripts but not due to changes in mitochondrial DNA copy number, differences in mitochondrial transcription kinetics or mitochondrial RNA processing. Furthermore, in the absence of STAT3 there is defective delivery of these transcripts to the mitochondrial ribosomes resulting in delayed mitochondrial translational kinetics. We propose that the ability of STAT3 to regulate mitochondrial transcription and translation is the fundamental mechanism by which a small pool of STAT3 can regulate highly abundant key component proteins of the ETC. Importantly, this activity is required for tumor formation, but is dispensable in normal tissues which makes this activity of STAT3 an attractive therapeutic target that will diminish tumor growth whilst preserving the critical nuclear functions of STAT3.

#791

V-ATPase in glioma stem cells: V1G1 subunit expression correlates with metabolic behavior and mitochondria activity.

Alessandra M. Storaci,1 Irene Bertolini,2 Manuela Caroli,3 Stefano Ferrero,3 Valentina Vaira1. 1 _Università degli Studi di Milano, Milan, Italy;_ 2 _The Wistar Institute, Philadelphia, PA;_ 3 _IRCCS Ca' Granda Foundation, Milan, Italy_.

Background The vacuolar H+-ATPase (V-ATPase) is a multisubunit proton pump with a role in the acidification of the extra- and intra-cellular environments. Recent evidence connected V-ATPase deregulation and human diseases, including cancer. We previously demonstrated that the overexpression of the catalytic subunit V1G1 is correlated with glioma stem cells (GSC) maintenance. According to V1G1 expression, we sorted glioblastoma patients-derived neurospheres cultures (NS) into two categories: V1G1HIGH and V1G1LOW NS. These NS differ in terms of stem cell genes expression, sphere formation, invasion ability, lysosomal acidity and bioenergetics.

Methods V1G1HIGH, V1G1LOW NS treated with 10nM of BafilomycinA1 (BafA1) or vehicle for 24h (n=3 per group) were used for the functional experiments. Lactate, glucose, ATP levels were measured by commercial kits. Metabolic proteins expression was assessed by western blot. ROS production and mitochondria function were measured by flow cytometry using MitoSox and TMRE assays, respectively.

Results At basal conditions, V1G1LOW NS produce higher levels of lactate (p=0.0379) whereas V1G1HIGH NS show higher levels of extracellular glucose and intracellular ATP. Accordingly, the two NS cultures show an opposite trend in LDH subunits expression with LDHA, which pushes lactate production, being more expressed in V1G1LOW (p=0.0027). Conversely, LDHB that facilities the production of pyruvate from lactate, and the transporter MCT1 are more expressed in V1G1HIGH NS. Interestingly, using the V-ATPase specific inhibitor BafA1, metabolic characteristics of V1G1HIGH are redirected to the V1G1LOW phenotype. Moreover, in V1G1HIGH NS BafA1 induces ROS production and affects mitochondria activity, increasing their depolarization.

Conclusions Taken together these data suggest two distinct metabolic behaviors of NS according to V-ATPase expression. Indeed for energy production V1G1LOW NS could rely on a glycolytic metabolism whereas V1G1HIGH NS on oxidative phosphorylation. Moreover, when overexpressed the proton pump could also have a role in preserving mitochondria polarity. Although preliminary, this is the first evidence reporting a role for V-ATPase in the regulation of cancer stem cell metabolism.

#792

Acetate production from glucose and coupling to mitochondrial metabolism in mammals.

Xiaojing Liu,1 Daniel E. Cooper,2 Ahmad A. Cluntun,2 Marc O. Warmoes,2 Steven Zhao,3 Michael A. Reid,2 Juan Liu,2 Peder J. Lund,3 Mariana Lopes,3 Benjamin A. Garcia,3 Kathryn E. Wellen,3 David G. Kirsch,2 Jason W. Locasale2. 1 _North Carolina State University, Raleigh, NC;_ 2 _Duke University, Durham, NC;_ 3 _University of Pennsylvania, Philadelphia, PA_.

Background: In conditions of hyperactive cellular metabolism, excessive cellular nutrient uptake results in incomplete metabolism and excretion of intermediates. These intermediates may serve as unconventional fuel sources satisfy metabolic demands during nutrient scarcity. Interestingly, acetate metabolism provides a parallel pathway for acetyl-CoA production and allows for protein acetylation and lipogenesis independent of citrate conversion to acetyl-CoA. This pathway is important in tumorigenesis, immune alertness, neural plasticity, and other diverse contexts but the origin of acetate has been unclear. Thus, we have conducted a re-evaluation of endogenous acetate generation and the biological relevance.

Method: Cancer cells were cultured in RPMI medium with 13C labelled nutrients in the presence of 18O2. Mouse models of soft tissue sarcoma were generated in a mixed 129/SVJae and C57BL/6 background. A jugular vein catheter was surgically implanted and exteriorized via a vascular access port, which allows infusion of [13C6]-glucose via the venous catheter. Acetate in medium and blood is quantified using [2H3] labelled acetate as the standard after 2-hydrazinoquinoline (HQ) derivatization, and other polar metabolites were directly analyzed after cold methanol extraction. All metabolites were measured using liquid chromatography coupled with high resolution mass spectrometer.

Results: By employing multiple-isotope tracing technology, quantitative proteomics, and mouse genetics tools, we demonstrated that acetate is quantitatively generated from pyruvate, the end product of glycolysis and key node in central carbon metabolism in cancer cells and tumor. One reaction mechanism found to generate acetate occur through altered enzyme activity of thiamine-dependent keto acid dehydrogenases, which transforms their activity to keto acid decarboxylases. The other reaction mechanism to generate acetate occur by reaction with reactive oxygen species (ROS), a finding which potentially links this pathway to numerous physiological and pathophysiological processes. Thiamine starvation and the addition of exogeneous ROS greatly stimulated the ROS contribution to acetate production, which can be used to replenish intracellular acetyl groups. Thus, increased acetate production and release could potentially favor the neighboring cells deficient in cytosolic acetyl-CoA, as demonstrated by co-culturing ACLY KO cells with HCT116 cells.

Conclusion: We have not only provided direct evidence that acetate arises from endogenous metabolism of glucose in mammalian cells, but also identified the regulatory mechanisms, which involve ROS and mitochondrial functions.

#793

The prometastatic effects of subcytotoxic doses of doxorubicin are countered by agents inactivating mitochondrial reactive oxygen species.

Paolo E. Porporato,1 Pierre Sonveaux2. 1 _University of Torino, Torino, Italy;_ 2 _University of Louvain (UCL) Medical School, Brussels, Belgium_.

Mitochondrial reactive oxygen species (mtROS) possess both tumor-promoting and tumor-suppressive activities according to their net concentration resulting for production levels and antioxidant detoxification. mtROS promote cancer cell fitness at low doses (mitohormesis), are cytotoxic at high doses and promote cancer migration, invasion and metastasis at intermediate doses. These intracellular doses vary from a model to another. Yet, we recently identified that mitochondria-targeted ROS scavengers mitoTEMPO and mitoQ prevent cancer metastasis in several in vivo models (Porporato et al. Cell Reports 2014;8:754-66). Interestingly, several anticancer drugs, among which doxorubicin, are well known to trigger mtROS production in cancer cells, but whether this response participates in cell killing or in resistance is still debated. We therefore tested whether combining mitochondria-targeted antioxidants with doxorubicin could help controlling cancer cell proliferation in vitro and tumor growth in mice. Using breast cancer cells lines MCF7 and 4T1 as models, we first found that subcytotoxic doses of doxorubicin increased cell respiration and, thereby, mitochondrial superoxide production. Downstream, mtROS activated the TGFβ pathway and induced cancer cell migration, invasion, epithelial-mesenchymal transition (EMT) and metastatic take in lungs. MitoTEMPO and mitoQ almost totally blocked these phenotypical changes. Together, our data provide a strong rationale for combining doxorubicin chemotherapy with drugs lowering mtROS production in breast cancer.

#794

Inhibition of mitochondrial metabolism by a novel agent targeting RUNX2-DNA binding in breast cancer.

Myoung Sook Kim,1 Ramkishore Gernapudi,1 Yessenia C. Cedeno,1 Brian M. Polster,1 Geoffrey D. Girnun,2 Antonino Passaniti1. 1 _University of Maryland, Baltimore, MD;_ 2 _Stony Brook University, Stony Brook, NY_.

The RUNX2 transcription factor promotes breast cancer growth and metastasis through interactions with a variety of cofactors that activate or repress target genes. RUNX2 knockdown decreases expression levels of glycolytic genes such as LDHA, HK2 and GLUT-1, but upregulates PDHA1 mRNA level and enzymatic activity, which is consistent with lower glycolytic potential. Using a direct computer-assisted drug discovery (CADD) approach, we identified CADD522 as a small molecule that inhibits the RUNX2-DNA binding, transcriptional activity of RUNX2, and expression of RUNX2 target genes. We also observed that CADD522 inhibits breast cancer growth and metastasis using in vitro and in vivo tumor models. RUNX2-activated glucose utilization and GLUT-1 levels were also reduced by CADD522. New data also define CADD522 as a potential antitumor agent that downregulates the levels of metabolites in the TCA cycle and inhibits mitochondrial oxygen consumption rates. Our results support the observations that CADD522 could regulate cellular metabolism through modulation of both glycolytic metabolism and mitochondrial oxidative phosphorylation. To address the mechanisms by which CADD522 exerts its inhibitory effects on mitochondrial metabolism, we performed RNA-Sequencing analysis in breast cancer cells and identified several specific molecular signatures that determine cellular response to RUNX2 and CADD522. Further pathway changes in mitochondrial function as well as validation of differentially expressed genes that play critical roles in mitochondrial metabolism are under investigation.

#795

GDF15, the putative mitokine factor, promotes tumor progression in thyroid cancer via STAT3 regulation.

Yea Eun Kang,1 Jae Won Chang,2 Lihua Liu,3 Kyoungmin Lee,2 Mi Ae Lim,2 Seung-Nam Jung,2 Chan Oh,3 Ho-Ryun Won,2 Minho Shong,1 Bon Seok Koo2. 1 _Division of Endocrinology and Metabolism, Department of Internal Medicine, Chungnam National University, College of Medicine, Daejeon, Republic of Korea;_ 2 _Department of Otolaryngology-Head and Neck Surgery, Chungnam National University, College of Medicine, Daejeon, Republic of Korea;_ 3 _Department of Medical Science, College of Medicine, Chungnam National University, Daejeon, Republic of Korea_.

Purpose: As mitochondrial dysfunction is an integral component during the initial stages of tumorigenesis, mechanistic insight on the interaction between these two pathways may lead to novel therapeutic approaches. The aim of this study is to investigate the role of GDF15, the putative mitokine factor in thyroid tumorigenesis.

Methods: Expression levels of GDF15 was investigated using The Cancer Genome Atlas database. The role of GDF15 in tumor aggressiveness was investigated by analyzing the effects of knock-down of GDF15 on proliferation, migration, and invasion of thyroid cancer cells. Serum levels of GDF15 were measured in healthy subjects and patients with papillary thyroid cancer (PTC), and expression of GDF15 by IHC was investigated in PTC samples. To investigate the exact mechanism of GDF15 in thyroid cancer, both IPA analysis and GSEA analysis were performed using TCGA database and RNA seq data from Chungnam national university hospital.

Results: TCGA data revealed the higher expression of GDF15 in tumor samples compared to in paired non-tumor samples. Thyroid cancer cells with knock-down of GDF15 revealed the decrease of proliferation, migration, and invasion compared to control cells via regulation of STAT3. Higher serum levels of GDF15 were found in patients with thyroid cancer than in control participants, and were significantly associated with tumor stage, lymphovascular invasion, and recurrence. In addition, patients with high GDF15 expression by IHC revealed the aggressive phenotype compared to patients with low GDF15 expression.

Conclusions: GDF15 is a new biomarker for predicting tumor progression, and therapies targeting GDF15 may be effective for treating thyroid cancer.

#796

Progestins target mitochondrial respiration to shift metabolic phenotype in luminal breast cancer.

Shawna B. Matthews, Lynsey M. Fettig, Ashley V. Ward, Matthew R. Jackman, Paul S. MacLean, Carol A. Sartorius. _University of Colorado Anschutz Medical Campus, Aurora, CO_.

The dichotomous nature of progesterone receptor (PR) in estrogen receptor (ER)-positive breast cancers has confounded its use as an endocrine target. Historically, progestins were used to treat some advanced breast cancers and ongoing clinical trials are testing several PR ligands. PR generally opposes ER activity through multiple mechanisms, most notably by altering global ER transcriptional activity to suppress breast tumor growth. However, progestins can also stimulate expansion of endocrine-resistant cancer stem cells (CSCs). Thus, better understanding of potential consequences of progestins in breast cancer is necessary to gauge therapeutic efficacy. Given that ER-driven tumor growth relies on mitochondrial respiration, we evaluated whether PR modulates cell metabolism machinery as a mechanism of ER antagonism. We used functional and morphometric assays in an ER+ breast cancer cell line and patient-derived tumor models to show that progestin treatment dramatically alters mitochondrial function. Multivariate metabolomics analysis highlights a progestin-associated metabolic signature involving accumulation of multiple TCA cycle intermediates, including fumarate, malate, and succinate. Functional assays indicate that progestins impair mitochondrial oxidative reserve capacity and oxygen consumption while ablating estradiol-induced accumulation of ATP in PR+ but not PR− cells. Morphometric assessment of mitochondria via laser-scanning confocal and transmission electron microscopy indicate a shift away from reticular filamentous mitochondria towards smaller, rounder, more perinuclear mitochondria with altered membrane potential. These results suggest that PR targets mitochondrial respiration as part of a broader strategic shift from ER-driven tumor growth to PR-driven thrifty metabolic dormancy. Whether the latter is indicative of an acquired propensity for glycolytic metabolism in indolent tumor cells, CSCs, or perhaps both, is addressed in ongoing studies.

#797

Obesity-associated, but not obesity-independent, tumors respond to insulin by increasing mitochondrial glucose oxidation.

Aviva Rabin-Court, Gerald I. Shulman, Rachel J. Perry. _Yale University School of Medicine, New Haven, CT_.

Obesity is associated with increased incidence and worse prognosis of more than one dozen tumor types; however, the molecular mechanisms for this association are unknown. We hypothesized that insulin, which is elevated in obesity-driven insulin resistance, would increase tumor glucose oxidation in obesity-associated tumors. To test this hypothesis, we developed a stable isotope method in which tumor cells are incubated in physiological concentrations (5 mM) of [13C6] glucose and the ratio of pyruvate dehydrogenase flux to citrate synthase flux (VPDH/VCS, i.e. the percent of total mitochondrial oxidation fueled by glucose oxidation) is given as [4,5-13C2]glutamate/[13C3]alanine, measured by gas chromatography/mass spectrometry and liquid chromatography-tandem mass spectrometry respectively. Using this method, we found that both breast cancer (4T1) and colon cancer (MC38) cells, tumors that are associated with obesity, exhibit greater relative rates of glucose oxidation to total mitochondrial oxidation (VPDH/VCS 61±3% and 60±2%, respectively) than melanoma (YUMM) and Renca (renal cell carcinoma) cells (41±4% and 37±2%, respectively; P<0.05 for all comparisons between obesity-associated and non-obesity-associated tumors). In addition, both 4T1 and MC38 cells increased VPDH/VCS upon incubation in 100 nM insulin (75±3% and 81±2%, respectively, P<0.01 vs. the same tumor type without insulin), whereas there was no change in glucose oxidation with insulin in YUMM or Renca cells (35±3% and 36±2%, respectively). These data reveal that a shift in substrate preference may comprise a metabolic signature of obesity-associated tumors that differs from that of those not associated with obesity. In addition, contrary to the conventionally held view that glucose oxidation in tumor cells is constitutively high, these data reveal that obesity-driven tumors respond acutely to insulin by increasing mitochondrial glucose oxidation, whereas obesity-independent tumor cells do not. CONCLUSION: These data demonstrate that obesity associated tumors (breast and colon), in contrast to tumors that are not associated with obesity (melanoma, renal cell), are insulin-responsive and have higher rates of basal and insulin-stimulated mitochondrial glucose oxidation. Therefore assessment of in vitro tumor mitochondrial substrate preference may predict whether interventions to reverse hyperinsulinemia or reduce insulin-responsive glucose oxidation in tumors may be attractive therapeutic modalities in cancer types associated with obesity.

#798

Epithelial-mesenchymal transition directs stem cell polarity via regulation of mitofusin.

Meng-Ju Wu, Mi Ran Kim, Silpa Gampala, Yingsheng Zhang, Yueyang Wang, Jer-Yen Yang, Chun-Ju Chang. _Purdue University, West Lafayette, IN_.

Mitochondria are dynamic organelles that have been linked to stem cell homeostasis. However, the mechanism involved in mitochondrial regulation of stem cell fate determination remain elusive. Here we discover that epithelial-mesenchymal transition (EMT), a key process in cancer progression, induces mitochondrial fusion through regulation of miR200c-PGC1a-MFN1 pathway. EMT-activated MFN1 forms a complex with PKCz and is required for PKCz-mediated NUMB phosphorylation and dissociation from the cortical membrane to direct asymmetric division of mammary stem cells, where fused mitochondria are tethered by MFN1-PKCz to the cortical membrane and asymmetrically segregated to the stem cell-like progeny with enhanced glutathione synthesis and reactive oxygen species scavenging capacities, allowing sustaining of a self-renewing stem cell pool. Suppression of MFN1 expression leads to equal distribution of the fragmented mitochondria in both progenies that undergo symmetric luminal cell differentiation. Together, this study elucidates a novel role of mitofusin in stem cell fate determination to mediate EMT-associated stemness and provides therapeutic implications for targeting EMT-induced tumor stem cell populations.

#799

Aberrant mitochondrial protein involvement through early PDAC initiation and progression using multiplexed DNA-PAINT and correlative histology.

Matthew J. Rames, Fehmi Civitci, Ting Zheng, Josiah Wagner, Jason Link, Xiaolin Nan. _Oregon Health & Science University, Portland, OR_.

Prior to pancreatic ductal adenocarcinoma (PDAC), pancreatic acini cells change their morphology through pancreatic intraepithelial neoplasia (PanIN) stages, becoming increasingly dysplastic as stroma and tissue hypoxia increase. This increasing hypoxia forms a cancer promoting microenvironment, wherein we propose metabolic changes trigger aberrant mitochondrial networks form healthy tissue. We developed tissue superresolution imaging to directly quantify structural mitochondrial response through patient histology, whereby DNA-PAINT can provide super-resolution detail decoupled from photo bleaching to visualize mitochondria through tissue layers. Expanding from preliminary data, target mitochondrial dynamics proteins' organization will also be correlated to PanIN stages.

Introduction: Early oncogene involvement in PDAC progression links to metabolic and mitochondrial regulatory changes. Typically nutrient deprivation and hypoxia trigger cell death from increased reactive oxygen species production and organelle damage which trigger apoptosis, yet these prolonged effects can be cancer promoting when less severe. Mitochondrial dynamics and proteins related to mitochondrial fission and fusion can reduce apoptotic signaling, enhance aerobic glycolysis, and increase ROS to allow cancer progression.

Materials and Methods: Adapting from previous proof of concept, mitochondria (TOM20) within formalin-fixed paraffin embedded (FFPE) tissue sections were imaged with stochastic optical reconstruction microscopy (STORM). In brief: cadaver healthy pancreas FFPE tissue samples underwent deparafinization, antigen retrieval, indirect immuno-labeling with AlexaFluor647, TIRF illumination with a 60x objective was used for data collection, whereby data processing was conducted using the open-source FIJI and custom MATLAB software.

Results and Discussion: Preliminary data shows proof of principle that both STORM and DNA-PAINT can be correlated to histological staining of human pancreas.

Conclusions: Superresolution imaging reveals ultrastructural details of mitochondria in FFPE patient samples not resolved via conventional fluorescence imaging. Through quantitative feature analysis, we would be able to correlate aberrant mitochondria structure and abundance to PDAC progression.

Acknowledgements: Funding provided by the Cancer Early Detection Advanced Research (CEDAR) Center of OHSU Knight Cancer Institute.

#800

ESI-09 and HJC0197, known EPAC inhibitors, sensitize lung cancer cells to glucose starvation by uncoupling mitochondrial electron transport, leading to bioenergetic crisis.

Yuki Iwai,1 Ryota Kikuchi,1 Hiroyuki Nakamura,1 Nobuyuki Koyama,2 Koichi Hagiwara,3 Kazutetsu Aoshiba1. 1 _Tokyo Medical University Ibaraki Medical Center, Inasiki-gun, Japan;_ 2 _Tokyo Medical University Hachioji Medical Center, Inasiki-gun, Japan;_ 3 _Jichi Medical University, Japan_.

Cancer cells utilize aerobic glycolysis and proliferate continuously beyond the capacity of their blood supply, leading to microenvironmental stresses such as hypoxia, nutrient (glucose) deprivation and extracellular acidosis. We have previously showed that extracellular acidosis prevents glucose starvation-induced death of lung cancer cells by reducing glycolytic energy production and de novo protein/RNA synthesis that consume ATP (AACR 2018). This acidosis-dependent, glucose starvation-resistant, and ATP-saving phenotype is thought to serve as an adaptive response to glucose deprivation in cancer cells to survive an energy-restricted tumor microenvironment. In the present study, we screened small-molecule inhibitors to explore therapeutic reagents that can exert cytotoxicity against the glucose starvation-resistant phenotype in the lung cancer cell lines, A549 and H1299 cells. Among the various inhibitors tested, we found that ESI-09 and HJC0197, known as inhibitors of the exchanger protein directly activated by cAMP (EPAC), reduced the cellular ATP levels and survival under glucose deprivation stress under both acidic (pH 6.8) and neutral (pH 7.4) conditions. Interestingly, the ATP-reducing effect of ESI-09 and HJC0197 was not due to inhibition of the EPAC function, because the effect cannot be reproduced with other EPAC inhibitors, such as CE3F4, EPAC5376753 and ESI-05. Rather, the effect of ESI-09 and HJC0197 was attributed to their previously unknown action of uncoupling the mitochondrial electron transport chain (ETC). We found that ESI-09 and HJC0197 induced mitochondrial proton leak, causing ATP deficiency and cell death via the following three mechanisms: 1) decreased mitochondrial ATP production due to ETC uncoupling, 2) increased ATP consumption resulting from reversal of F0/F1-ATPsynthase/ATPase to maintain the mitochondrial membrane potential, and 3) glucose deficiency as a result of increased glucose consumption due to a compensatory stimulation of glycolytic ATP production. The energy-saving phenotype of cancer cells is thought to serve as an adaptive strategy that allows cell survival in an energy-restricted, acidic tumor microenvironment. Our results suggest that ESI-09 and HJC0197 disrupt bioenergetic tumor metabolism for energy homeostasis in both acidic and neutral microenvironment, and may exert a therapeutic effect against the starvation-resistant phenotype of cancer cells.

The complete chemical structures of ESI-09 and HJC0197 will be divulged at the time of the presentation at the meeting.

#801

Dual inhibition of distinct metabolic features targets osteosarcoma stem cells.

Shingo Kishi, Kanya Honoki, Shinji Tsukamoto, Hiromasa Fujii, Yumiko Kondo, Yasuhito Tanaka, Hiroki Kuniyasu. _Nara Medical Univ., Kashihara, Japan_.

[Objective] Mitochondria are the places for the energy production of the cells, while reactive oxygen species (ROS) are also produced alongside. In recent years, it has been reported that cancer stem cells metabolize predominantly through oxidative phosphorylation (OXPHOS) rather than glycolysis in certain cancer cells. Targeting OXPHOS achieved by suppression of ATP synthesis through mitochondrial ATP synthase could be a potential therapeutic option against cancer stem cells. In the current study, we have identified the mitochondria metabolism as the potential therapeutic target in osteosarcoma (OS) stem cells, presenting the synergistic effects of combination of OXPHOS inhibition by pterostilebene (PTE) with c-Myc inhibitor, which target both OXPHOS-dominant cancer stem cells and glycolysis-dominant non-cancer stem cells as a 'two hit' or 'dual inhibition' of metabolic pathways, OXPHOS and glycolysis.

[Materials & Methods] Using human OS cell lines of SaOS2, U2OS and MG63 cells, cell survival and the ability of sphere formation was assessed with or without PTE, and the expression of stem cell markers mRNA such as Oct3, NS, CD44 was examined by RT-PCR. Next, the activity of mitochondrial ATP synthase, mitochondrial respiration capacity of oxygen consumption rate, and the amount of ATP as well as ROS production were measured under the treatment of PTE. Furthermore, we examined the synergistic effect of PTE with cMyc transcription inhibitors of JQ1 or Honokiol (HNK).

[Results] PTE treatment on human OS cell lines reduced the viabilities of all cell lines in dose-dependent manner and expression of stem cell marker and the ability of sphere formation were also decrease in terms of sphere number and size. PTE reduced the activity of F0F1-ATP synthase; Complex V predominantly, and the mitochondrial oxygen consumption rates and synthetic amount of ATP were also decreased in spheroid condition. These results suggest that PTE possibly targets stem cell population which preferably relies on OXPHOS, suppressing ATP synthesis via F0F1-ATP synthase inhibition as well as increased ROS production in OS cells and changes metabolic flax to glycolysis dependent feature. The dual inhibition of OXPHOS by PTE and c-Myc inhibition by HNK or JQ1 showed the synergistically inhibition of OS cell growth in a dose-dependent manner.

[Discussion] Prognosis of the patients with osteosarcoma has reached to plateau without any breakthroughs over the last quarter century, and nearly 30% of patients still have to face very severe poor prognosis, especially with metastatic disease. Current study suggests that modulation of metabolic flux by c-Myc and OXPHOS inhibitors showed a greater synergistic effect with 'two metabolic hit' or 'dual metabolic inhibition' of distinct metabolic features and it could be a novel therapeutic strategy against osteosarcoma, possibly targeting both stem-like cell population and general tumor cell population.

#802

Effective breast cancer combination therapy targeting BACH1 and mitochondrial metabolism.

Jiyoung Lee. _The University of Chicago, Chicago, IL_.

Mitochondrial metabolism is an attractive target for cancer therapy. Reprogramming metabolic pathways could improve the ability of metabolic inhibitors to suppress cancers with limited treatment options like triple negative breast cancer (TNBC). Here we show that BACH1 (BTB and CNC homology1), a heme-binding transcription factor whose expression is enriched in TNBC patients, targets mitochondrial metabolism. BACH1 decreases glucose utilization in the TCA cycle and negatively regulates transcription of electron transport chain (ETC) genes. BACH1 depletion by shRNA or degradation by hemin sensitizes cells to ETC inhibitors such as metformin, suppressing growth of both cell line and patient-derived tumor xenografts. Expression of a heme-resistant BACH1 mutant in shBACH1 cells rescues the BACH1 phenotype and restores metformin resistance in hemin-treated cells and tumors. Finally, BACH1 gene expression inversely correlates with ETC gene expression in breast cancer patients as well as other tumor types, highlighting the clinical relevance. This study demonstrates that mitochondrial metabolism can be exploited through targeting BACH1 to sensitize breast cancer and potentially other tumor tissues to mitochondrial inhibitors.

#803

Glucolipotoxicity & altered oxidative stress responses in cancer cells.

Arwa MT Al-Nahdi, Annie John, Haider Raza. _UAE Univ., Al Ain, United Arab Emirates_.

Alterations in energy metabolism, such as aerobic glycolysis and lactic acid production, and mitochondrial bioenergetics are the key metabolic changes associated with increased glucose uptake in cancer cells (Warburg effect). Metabolic adaptation, inflammatory responses and oxidative stress have been implicated in cancer etiology and pathology. Both experimental and epidemiological studies have suggested that caloric restriction is beneficial in reducing the complications of cancer cells. Studies have also suggested that increased availability of energy metabolites, such as glucose and fatty acids, as also seen in obesity and diabetes, which induces inflammatory and oxidative stress responses, may also be key factors in cancer development. In the present study, we have used human hepatoma, HepG2 and rodent pancreatic cancer derived Rin-5F cells to investigate the effects of high glucose (25mM) and high palmitic acid (up to 0.3mM) on cell survival and cell death along with the metabolic, inflammatory and oxidative stress associated alterations in these cells. Our results have shown that both increased glucose and fatty acids induce DNA damage and caspase-dependent apoptosis and mitochondrial respiratory dysfunction. Glucolipotoxicity in these cells, due to increased ROS production and oxidative stress has been suggested to be involved in altering the mitochondrial membrane potential and bioenergetics. Altered Nrf-2/NFk-B/AMPK/mTOR-dependent cell signaling and inflammatory (IL6/PGE2) responses have also been observed when cells were treated with high glucose and palmitic acid. Interestingly, pretreatment with N-acetyl cysteine (NAC, 10mM), a GSH precursor, resulted in partial recovery from the oxidative stress-induced glucolipotoxicity. Our results suggest that the presence of high energy metabolites enhance inflammatory and oxidative stress responses that may be responsible for alterations in cancer cell signaling and metabolism.

#804

Environmental regulation of mitochondrial dynamics in cancer.

Rayees A. Padder, Mohammad Husain. _Jamia Millia Islamia -A Central University, New Delhi, India_.

Mitochondria are the metabolic organelles found in eukaryotic cells, with a chief function to carry on the Oxidative phase of respiration- OXPHOS. The organelle remains in a continuous state of fission-fusion dynamics and its movement along cytoskeleton to meet the varying cellular energy requirements. Deregulated metabolism being an emerging cancer hallmark has sought a keen attention towards mitochondrial architecture and how it governs cellular transformation. Recent studies have shown that metabolic differences between transformed vs. normal cells are due to the result of interplay of the genes regulating mitochondrial fission-fusion dynamics, with higher fission rates associated to high proliferation and invasiveness and vice-versa. However, the underlying mechanism(s) governing this phenotype are largely elusive. To this end, here we present that how metabolic environment governs mitochondrial dynamics and hence tumorigenesis in colorectal cancer cells. The cells were grown under different environmental conditions- High glucose/ normoxia; high glucose/ hypoxia; low glucose/ hypoxia for 24 hrs. (to mimic different solid tumor environments), followed by re-oxygenation of the corresponding replicates of same sets to next 24 hrs. (to mimic tumor re-oxygenation). It was found that hypoxia (1% O2/ 24 hrs.) gave rise to a fragmented mitochondrial phenotype with higher glycolytic rates compared to the corresponding normoxic groups, as observed through mitochondrial morphology analysis, using mitotracker CMX Ros and elevated expression of fission mediators- DRP 1/ PDRP1s616. However, hypoxia could not induce mitochondrial fragmentation under low glucose conditions (5mM), where the cells showed an elongated mitochondrial network as observed through mitochondrial imaging and a decrease in DRP 1/PDRP1s616. Surprisingly, the corresponding re-oxygenation of low glucose/hypoxia treatments gave rise to a more fragmented mitochondrial phenotype. Further, this hypoxia and/or reoxygenation mediated mitochondrial fragmentation seemed to enhance cellular migration as observed through wound healing and/or expressional analysis of N/E cadherins besides bestowing a glycolytic phenotype. This hypoxia mediated mitochondrial fragmentation and hence metabolic deregulation was found to be regulated through HIF 1α axis, however how this phenotype is governed under reoxygenation conditions seems obviously through a HIF 1α independent manner.

Taken together, the results show that how mitochondrial morphology changes in response to changing environmental conditions to fuel colorectal carcinogenesis.

#805

Adaptation of mitochondrial organization to aggregation in serous ovarian cancer.

Joseph P. Grieco,1 Stephanie Edwards,1 Mitchell Allen,1 Yao Wang,2 Justin Perry,2 Yipei Song,3 Nathan Swami,3 David Brown,1 Eva Schmelz1. 1 _Virginia Tech, Blacksburg, VA;_ 2 _Virginia Tech, VA;_ 3 _University of Virginia, VA_.

During metastasis, ovarian cancer cells exfoliate from the primary tumor and disseminate throughout the peritoneal cavity mostly as aggregates with a higher survival rate and metastatic potential. To determine how the mitochondrial morphology and dynamic expression of fusion and fission-regulating proteins contribute to spheroid survival, in this study we investigated changes in mitochondrial morphology in order to identify phenotypic alterations that are associated with changes in mitochondrial function. We compared monolayers and spheroids of murine and human benign, late-stage, and tumor-initiating cells in hypoxia using fluorescent microscopy and western blot analysis. In addition, we investigated how the mitochondrial membrane potential (Δψm) and ROS production contributed to this phenotype using the InCell analyzer with TMRM and Amplex Red dyes respectively. The data from this study suggest that the morphology and dynamic protein expression are highly altered during tumorigenesis, aggregation, and in hypoxic conditions, indicating a direct correlation of mitochondrial morphology in regulating survival, apoptosis and proliferation. The identification of these molecular events and changes that allow for the survival of metastatic cancer cells in an environment low in oxygen and nutrients may provide specific targets for interventions that would suppress successful metastatic outgrowth of disseminating cells and could potentially be used as a biomarker for treatment efficacy.

#806

Mitochondrial oncobioenergetic Index (MOBI) mediated by oxidative stress as a marker for progression from indolent to aggressive cancer.

Praveen Kumar Vayalil. _Univ. of Alabama at Birmingham, Birmingham, AL_.

Cellular Metabolic profile is governed by the way which the mitochondria functions in a cell. Any change in mitochondrial function would alter the cellular metabolic profile. During the evolution of tumor from onset to aggressive cancer, the metabolic needs also differs as alterations in oncogenes, tumor suppressor genes, and changes in the epigenetics and microenvironment occurs at each stage of the tumorigenesis process. Therefore, we hypothesized that mitochondrial function will be stably and dynamically altered at each stage of the tumor development. Moreover, the ability mitochondria to withstand stress will also be different. We recently defined MOBI, which is a mathematical representation of oncobioenergetic profile of a cancer cell. MOBI increases significantly upon transformation into localized premalignant form and rapidly falls below the normal as they become aggressive. We also show that the MOBI of cancer cells at various stages of tumorigenesis after oxidative stress depend on their initial MOBI. Aggressive cancer cells completely lost their MOBI upon stress, even though some of the cancer cells had high initial reserve capacity. However, cells from pre-malignant cells had significantly higher MOBI than the normal epithelial cells. This study thus reveals that MOBI could be a potential biomarker to distinguish aggressive cancer from that of indolent disease and screening of compounds that specifically target the bioenergetics of cancer cells.

#807

HDAC1 -dependent epigenetic loss of ATP5H confers a stem cell-like phenotype with features associated with metastatic capacity through mitochondrial reprogramming.

Suyeon Kim, Kwon-Ho Song, Tae-Woo Kim. _Korea university, Seoul, Republic of Korea_.

The failure of most cancer therapies is the emergence of tumor cells that are refractory to available agent. Here we report that single-modality cancer therapy triggers broad-spectrum cross-resistance to multiple clinical interventions. This multi-modality resistance arises through a core metabolic reprogramming pathway instigated by loss of the ATP synthase subunit ATP5H, which sequentially leads to accumulation of reactive oxygen species and stabilization of HIF-1α under normoxia. Furthermore, we found that NANOG/HDAC1 axis regulates epigenetic loss of ATP5H, this pathway confers tumor cells a stem cell-like phenotype with features associated with metastatic capacity. ATP5H loss in the tumor is strongly linked to failure of therapy, and poor survival in cancer patients. Integration of these biological features provides the best basis for understanding patient outcome in this setting and suggests that loss of ATP5H shapes the tumor resistance to multiple anti-tumor therapy.

#808

Aggregation alters the metabolism of serous ovarian cancer cells.

Stephanie Edwards, Emily Pyne, Lu Lui, Jack Guinan, Madlyn Frisard, Eva Schmelz. _Virginia Tech, Blacksburg, VA_.

Ovarian cancer is typically diagnosed at late stages with a poor prognosis due to high recurrence. Exfoliation from the primary tumor results in aggregation of ovarian cancer cells into spheroids in the peritoneal cavity, which may increase survival of cancer and metastasis to other tissues. We have previously reported metabolic changes in adherent ovarian cancer cells to rely more heavily on glycolysis. However, metabolic changes in cancer cell aggregates are poorly understood. In this study, we investigated differences in spheroid metabolism in normoxic and hypoxic conditions as compared to adherent cells. We examined late-stage and tumor-initiating murine ovarian cancer monolayers and spheroids. Using a Seahorse XF extracellular flux analyzer, we investigated changes in metabolism over time in the presence of mitochondrial inhibitors (oligomycin and rotenone) or uncouplers (FCCP). Data show a reduction in basal respiration, maximal oxygen consumption rate, and spare respiratory capacity of spheroids compared to adherent monolayers. This suggests a reduction in energetic demand and metabolic flexibility in spheroids, which may confer increased survival capabilities. The identification of these metabolic changes that may increase survival and metastasis of spheroids provide targets for treatment to control the ability of spheroids to survive in the peritoneal cavity.

#809

Response of metabolic phenotype to chemotherapy agents in a simulated tumor microenvironment.

David L. Hoffman, Julie M. Rumble. _Cayman Chemical Co., Ann Arbor, MI_.

Chemotherapeutic agents inhibiting fatty acid oxidation can sensitize cells to the tumor microenvironment.

Chemotherapeutic agents (CA), while standard-of-care for treating numerous types of cancer are, by nature, poisonous. Studies have shown that treatment with certain CA, such as Irinotecan, leads to chemotherapy associated steatohepatitis (CASH) resulting from the inhibition of mitochondrial fatty acid oxidation (FAO). While inhibition of FAO may have harmful effects on the liver, it may have a different effect in the tumor microenvironment (TME). Within the TME exists a unique metabolic environment, consisting of low [O2], low pH and high concentrations of fatty acids. Occupying the TME are immunosuppressive myeloid derived suppressor cells (MDSCs), which aid in tumor growth by suppressing T cells, which would normally attack the tumor. It is thought that the MDSCs localized to the TME undergo an alteration in metabolic phenotype, allowing them to utilize the high concentrations of fatty acids found in the TME as their primary carbon source, becoming reliant on FAO. It is therefore hypothesized that normoxic MDSCs will alter their metabolic phenotype in response to a simulated TME to favor FAO. This altered metabolic phenotype will sensitize MDSCs to inhibition of FAO by SN-38, the active metabolite of Irinotecan. The response of metabolic phenotype to hypoxic conditions will also be examine in HCT116 colorectal carcinoma cells.

#810

Differential use of lactate for mitochondria respiration by NSCLC cells.

Pamela Swain, Natalia Romero, Yoonseok Kam, Brian P. Dranka. _Agilent Technologies, Lexington, MA_.

Metabolic liabilities in cancer cells provide the opportunity for novel therapeutic approaches. This is especially true as the dogma that all cancer cells are glycolytic is being challenged. Phenotypic screening of cancer cell lines can demonstrate what these liabilities are and suggest approaches for drugs that target these vulnerabilities. As an example, non-small cell lung cancer (NSCLC) is typically driven by oncogenic mutations in either KRAS or EGFR. However, the impact of these mutations on cellular metabolic phenotype is not well-studied. In this study, we observed that the two KRAS-mutated NSCLC cell lines (A549 and H460) primarily relied on glycolysis for ATP production while EGFR-mutated cell lines (H1975 and PC9) were more reliant on mitochondrial respiration to meet energy demands. There is emerging evidence to suggest that lactate is a major fuel for cancer cell energy metabolism especially in the glucose-limited tumor microenvironment. Thus, we next compared the use of lactate with other carbon sources for ATP production in NSCLC cells. We found there was differential usage of lactate for mitochondrial respiration between KRAS and EGFR-mutated NSCLC cells. EGFR-mutated NSCLC cells used lactate and increased mitochondrial respiration when lactate was acutely administrated regardless of glucose availability. In contrast, the use of lactate for the mitochondrial respiration by KRAS-mutated cells was limited. In A549 cells, no significant change in mitochondrial respiration rate was observed at all by acute injection of lactate. Together, these results imply that NSCLC variants have adopted different metabolic phenotypes depending on the oncogenic background and more oxidative phenotype may be correlated to higher lactate use for TCA cycle in production of ATP.

#811

**Multi-targeted inhibitors of mitochondrial one-carbon metabolism and cytosolic** de novo **purine synthesis enzymes as anti-tumor agents.**

Md Junayed Nayeen. _Duquesne University, Pittsburgh, PA_.

Targeting cellular one-carbon (1C) metabolism in cancer cells with pyrrolo[3,2-d]pyrimidine analogs provides in vivo antitumor efficacy. We discovered a first-in-class series of 5-substituted pyrrolo[3,2-d]pyrimidine analogs (AGF291, AGF320, AGF347) with inhibition of serine hydroxymethyltransferase 2 (SHMT2), a key enzyme in mitochondrial 1C metabolism and a reported oncodriver. In addition, AGF291, AGF320 and AGF347 inhibited de novo purine nucleotide biosynthesis at glycinamide ribonucleotide formyltransferase (GARFTase) and 5-aminoimidazole-4-carboxamide (AICA) ribonucleotide formyltransferase (AICARFTase), and serine hydroxymethyltransferase 1 (SHMT1). In vivo studies demonstrated excellent antitumor efficacy for AGF347 against MIA PaCa-2 pancreatic adenocarcinoma xenografts in SCID mice with a median tumor growth delay (T-C) of >38 days for 4 mice, and 1 of 5 mice tumor-free 122 days post-treatment. AGF359, a new analog in this series, was a potent inhibitor of KB human tumor cell proliferation in vitro. AGF359 inhibition of KB human tumor cells was reversed with glycine and adenosine, establishing mitochondrial 1C metabolism and de novo purine biosynthesis as the targeted pathways; AICA plus glycine was incompletely protective, implicating AICARFTase as a direct cellular target. AGF359, like AGF291, AGF320, and AGF347, inhibited purified human SHMT2 (Ki = 0.399 ± 0.174 µM), SHMT1 (Ki = 0.70 ± 0.088 µM) and AICARFTase (Ki = 8.86 ± 2.83 µM). There was no inhibition of 5,10-methylene tetrahydrofolate dehydrogenase 2 (MTHFD2). Further structural modifications of these multi-targeted agents afforded pyrrolo[3,2-d]pyrimidine analogs AGF307 and AGF312. Cell-based glycine/nucleotide rescue experiments in KB tumor cells established that, as with AGF359, AGF307 and AGF312 were dual inhibitors of the mitochondrial 1C metabolism (AGF307) and de novo purine biosynthesis (AGF307, AGF312). These compounds are currently in further preclinical evaluation as a prelude to possible clinical development as antitumor agents.

#812

γ-tocotrienol induces mitophagy in breast cancer cells through activation of LKB1-AMPK pathway.

Venkatesh Dronamraju, Paul W. Sylvester. _University of Louisiana at Monroe, Monroe, LA_.

The dysregulation of glucose metabolism in cancer cells manifests as a characteristic phenomenon, referred to as the Warburg effect. Warburg effect is an over-reliance on aerobic glycolysis by cancer cells to meet the energy demand of proliferation. Previous studies have shown that γ-tocotrienol, a natural vitamin E derivative, inhibits aerobic glycolysis in human breast cancer cells through its modulation of key regulatory enzymes protein kinase B (Akt) and 5'-AMP activated kinase (AMPK). In the present study, we investigated the effects of γ-tocotrienol-induced activation of AMPK on mitochondrial dynamics in human breast cancer cells. Western blotting and immunocytochemistry analysis revealed that in human breast cancer cells MDA-MB-231 and T-47D, γ-tocotrienol removed the negative regulation of Akt on liver kinase B1 (LKB1), an upstream promoter of AMPK by increasing active complex formation and cytosolic localization of LKB1. In addition, γ-tocotrienol-induced activation of AMPK improved mitochondrial membrane potential through inhibition of uncoupling protein 1 (UCP1) as revealed by western blot analysis and mitochondrial membrane potential assay. High performance liquid chromatography (HPLC) analysis of NAD+/NADH ratio showed that γ-tocotrienol-induced AMPK activation improved the NAD+ ratio in human breast cancer cells, which indicates improving mitochondrial oxidative phosphorylation. In addition, γ-tocotrienol induced AMPK activation in human breast cancer cells causes increased mitochondrial fission, an indicator of mitophagy, as revealed by Western blot analysis of mitochondrial fission factor (MFF). In summary, these results show that γ-tocotrienol-induced AMPK activation modulates metabolic reprogramming in part by influencing the mitochondrial dynamics in human breast cancer cells. This work was supported in part by funds provided by the Louisiana Cancer Foundation.

#813

PERIOD2, a core circadian clock protein, initiates adaptive radioprotection via PER2-WNT controlled mitochondrial bioenergetics.

Aris T. Alexandrou,1 Ming Fan,2 Andrew Vaughan,2 Clifford Tepper,2 Loning Fu,3 Gayle Woloschak,4 David Grdina,5 Jian Jian Li2. 1 _Holy Cross College at Notre Dame, IN, Notre Dame, IN;_ 2 _University of California at Davis, Sacramento, CA;_ 3 _Baylor College of Medicine, Houston, TX;_ 4 _Northwestern University, Chicago, IL;_ 5 _University of Chicago, Chicago, IL_.

Circadian clocks are intimately involved in the homeostatic maintenance of metabolic and physiological processes that may have increasingly important roles to enhance bone marrow transplantation success in cancer patients; to protect normal tissue surrounding tumors from radio/chemotherapy; and to enhance the quality of diet and sleep for astronauts during space exploration. Herein, we report that expression of PERIOD2 (PER2; a core circadian clock component and "the inherent driver" of radioprotection) is required for adaptive protection against environmental radiation stress. PER2 expression is induced by exposure to low-dose radiation (LDR; 10cGy) in human epithelial cells, mouse epidermal cells, primary mammary epithelial cells, and mice liver and brain. siRNA blockade of PER2 ablates LDR-induced adaptive radioprotection in MCF10A human mammary epithelial cells. LDR-induced PER2 transcription is regulated by NF-κB and β-catenin and is released from phospho-glycogen synthase kinase-3ß (p-GSK3ß), as a serine-threonine kinase, in the WNT/β-catenin pathway. Unbound β-catenin interacts with the TCF/LEF domain on the Per2 promoter to promote feed-forward PER2-pGSK3β complex formation. Mitochondrial metabolism (bioenergetics) measured by oxygen consumption and ATP generation was attenuated in bone marrow isolated from PER2 deficient (Per2def) mice. Furthermore, RNA-seq profiling of bone marrow-derive progenitor hematopoietic stem cells (BM-pHSCs; Lin-/Sca1+/cKit+; LSK) cells isolated from LDR-treated wild-type (WT; C57BL/6) versus Per2def mice showed a cluster of genes involved in mitochondrial metabolism and DNA repair capacity. In addition, melatonin (N-acetyl-5-methoxytryptamine), a natural anti-oxidant and hypothalamic circadian synchronizer, stimulates PER2 expression and confers mice radioprotection through weight maintenance and increased survival. These results demonstrate that a core circadian regulator plays an indispensable role in defending mammalian cells against environmental genotoxic stress through the PER2/WNT pathway, a potential therapeutic target to enhance cell survival under radiation.

#814

Involvement of unique endosomes in the Mieap-induced vacuoles formation during Mieap-mediated cancer cell death.

Naoki Ikari, Yasuyuki Nakamura, Hirofumi Arakawa. _National Cancer Center Research Institute, Tokyo, Japan_.

Background: Mitochondria-eating protein (Mieap), a p53-inducible protein is known to have tumor suppressive functions by repairing or degrading unhealthy mitochondria. When Mieap degrades mitochondria, Mieap-induced vacuoles (MIVs) are formed to eat mitochondria, which subsequently induce iron-dependent cell death. Previously, we elucidated that the MIVs contain lysosomal proteins and degraded mitochondria, suggesting the MIVs represent non-canonical mitophagy. However, the process of MIV formation is still unclear.

Methods: The process of MIV formation was visualized by high spatial and temporal resolution confocal live cell imaging with the EGFP-tagged Mieap protein (EGFP-Mieap) that was ectopically expressed in A549 cells. To interpret the imaging data, we performed the Mieap-interactome analysis, and consulted the list of proteins immunoprecipitated by anti-Mieap antibody. Further, we investigated the mechanism for MIV formation using various deletion-mutant forms of EGFP-Mieap.

Results: The enforced expression of EGFP-Mieap generated characteristic endosomes whose membrane contained Mieap. The unique endosomes showed directed movement toward clusters of mitochondria, where MIVs were generated, and enlarged by eating mitochondria and fusing to each other. When we reviewed the list of Mieap-interacting proteins considering the directed movement, alpha tubulins were found to be included. The endosomes consisting of C-terminus deficient Mieap showed nondirected movement and accumulated at the edge of the cells.

Conclusions: Besides lysosomes and mitochondria, MIVs are derived from the Mieap-related endosomes via the endocytosis/pinocytosis. Possibly, the interaction between the C-terminus region of Mieap and alpha tubulins is critical for the endosome delivery to the mitochondria in MIV formation. 

### New Trends in DNA Methylation

#815

Genome-wide DNA methylation signatures to predict pathologic complete response from combined neoadjuvant chemotherapy with bevacizumab in breast cancer.

Ping-Ching Hsu, Susan Kadlubar, Eric Siegel, Lora J. Rogers, Valentina K. Todorova, Joseph Su, Issam Makhoul. _University of Arkansas for Medical Sciences, Little Rock, AR_.

Neoadjuvant chemotherapy is given before surgery to patients with primarily unresectable, advanced-stage cancers to render the tumor resectable, and to facilitate breast conservation. Previously, we have reported a prospective phase II trial for women with stage IIA-B/IIIA-B-C breast cancer with improved pathologic complete response (pCR) when using bevacizumab in the neoadjuvant setting. Chemotherapy agents are given orally or intravenously during multiple cycles of systemic treatments. However, the effect of the treatment can only be evaluated upon the completion of therapy. Here, we aim to identify epigenetic signatures in blood for patients at baseline as non-invasive markers to predict their pCR from the treatment, and to determine if treatment-related changes in epigenetic profiles reflect responsiveness to therapy. We performed genome-wide DNA methylation profiling using blood-derived DNA, and found that pre-treatment methylation status of BRD9 was predictive of responsiveness to therapy. Post-treatment global methylation differences were also observed between responders and non-responders. Most differentially methylated CpGs were located in promoter CpG island regions for responders, but in the open sea region for non-responders. DNMT3B was hypomethylated while most of the other genes were hypermethylated after 4 cycles of treatment. Hypomethylation of DNMT3B could potentially lead to the increased methylation of oncogenes and genes responsible for cell growth and proliferation, facilitating responsiveness to the therapy. These results support the development of BRD9 as a biomarker for treatment selection before neoadjuvant therapy with bevacizumab, and indicate DNMT3B as a potential target to improve clinical response.

#816

DNA methylation profiling from circulating tumor DNA for early diagnosis of non-small cell lung cancer.

Naixin Liang,1 Bingsi Li,2 Chenyang Wang,2 Tao Zheng,2 Jiayue Xu,2 Shuai Fang,2 Fujun Qiu,2 Jing Su,2 Lichen Zhang,2 Xin Lu,2 Miaomiao Song,2 Lingjian Yang,2 Han Han-Zhang,2 Xinru Mao,2 Hao Liu,2 Shanqing Li,1 Ke Ma,2 Zhihong Zhang2. 1 _Peking Union Medical College, Beijing, China;_ 2 _Burning Rock Dx, Shanghai, China_.

Introduction: Lung cancer found at an early stage carries much-improved prognosis. Unfortunately, there has been little success in developing blood-based diagnostic method. Profiling somatic mutations from ctDNA has shown great promise for cancer diagnosis, prognosis, and surveillance. Despite the substantial advances made in ctDNA detection techniques, the detection rate remains low for early stage disease. The role of aberrant DNA methylation in the process of tumorigenesis both at individual genes and a genome-wide scale has been well elucidated. It occurs very early in cancer development, thus capable of serving as a diagnostic biomarker. In this prospective study, we evaluated the potentiality of DNA methylation status obtained from ctDNA as an early diagnostic marker for NSCLC.

Methods: Panel Design: Methylation data of tumor samples (12 types, n=4772), adjacent normal (8 types, n=411), and normal white blood cells (WBC, n=656) from TCGA and GSE were compared. Differentially methylated sites were extracted using modified wald-test with an adjusted p-value <0.05 and fold-change>2. Our panel covers 80,672 CpG sites, spanning 1.05Mb of human genome. We performed targeted bisulfite sequencing on plasma samples of 359 early stage Chinese NSCLC patients and 144 healthy individuals to interrogate their methylation statuses with an average sequencing depth of 1,000x. The

Results: The training cohort consisted of 359 early stage Chinese lung cancer patients (266 stage IA, 25 stage IB, 43 stage II and 25 stage III) with a median age of 51 and 144 Chinese healthy individuals with a median age of 59. We constructed a diagnostic classification model using a support vector machine (SVM)-based machine learning classifier based on top 3,000 differentially methylated regions (DMRs) selected by random forest between tumor and normal plasma samples. Subsequently, 5-fold cross-validation with 100-time repeats were performed to gain a robust estimation of model performance, achieving a sensitivity of 79.4%, specificity of 95.4% and area under curve (AUC) of 95.2%. The model was subsequently validated in an independent cohort, consisting of 79 early stage Chinese NSCLC patients and 74 healthy individuals with comparable clinical characteristics as the patients in the training cohort. The model yielded a sensitivity of 77% and specificity of 90% in the validation cohort, suggesting its robustness.

Conclusions: Overall, our findings demonstrated in a large clinical cohort the potential of profiling DNA methylation from ctDNA, which can effectively distinguish cancerous from healthy, for the purpose of diagnosis. This method has potential to serve as a supplementary or alternative approach in lung cancer early detection. The general concept can be further extended to other types of cancer diagnostics.

#817

Assessment and manipulation of DNA methylation in cancer cells using terahertz radiation.

Hee-Jin Yang,1 Hwayeong Cheon,2 Jin Ho Paik,3 Joo-Hiuk Son2. 1 _SMG-SNU Boramae Medical Center, Seoul, Republic of Korea;_ 2 _University of Seoul, Seoul, Republic of Korea;_ 3 _, Seoul National University Bundang Hospital, Seoul, Republic of Korea_.

Carcinogenesis is closely associated with abnormal regulation of gene expression, among which DNA methylation in one of the key epigenetic modifications. Therefore, several studies have been directed towards the evaluation of DNA methylation. Here, we aimed to experimentally observe the specific signal of methylated DNA in various blood cancer cell lines using terahertz (THz) radiation, which is presented as a resonance in the spectrum. THz radiations are electromagnetic waves having frequencies of 0.1−10 THz, which falls between those of infrared radiation and microwaves. Molecular low-energy motions in THz region, such as collective vibrations, torsion of molecular structures, and hydrogen bonds, is correlated with biological characteristics, i.e., vibrational modes of DNA, structural modes in proteins, cell membrane vibrations, or enzyme activity. These features are usually presented as a characteristic resonance of the modes in the THz spectrum. In our study, the degree of global DNA methylation was obtained by the quantification of the resonance around 1.7 THz, and the results were in good agreement with those obtained by enzyme-linked immunosorbent assay (ELISA). Further, DNA demethylation was performed using high-power THz radiation around the resonance frequency of 1.7 THz, which lead to a 10% to 72% decrease in the degree of DNA methylation as shown in Table 1. Thus, our data shows the feasibility of using THz radiation for the assessment and manipulation of DNA methylation. This is the first study wherein the sensing and manipulation of DNA methylation could be achieved using a non-contact and non-invasive optical technique. Use of THz radiation for the assessment of DNA methylation might be helpful for its rapid processing, while THz manipulation of DNA methylation can avoid the toxicity of chemical demethylating agents. We propose that THz radiation may be useful for the diagnosis of cancer and we expect that THz demethylation might find potential application in cancer therapy.

Change of methylation status of blood cancer DNA using high-power THz radiation.

---

Samples | No Exposure | High-power THz exxpsoure | Change (%) THz | Change (%) ELISA

THz | ELISA | Ratio1 | THz | ELISA | Ratio2 | |

1 | 19.62 | 1.85 | 10.61 | 11.66 | 1.06 | 11.00 | 40.57 | 42.70

2 | 11.34 | 1.38 | 8.22 | 10.20 | 1.19 | 8.57 | 10.05 | 13.77

3 | 15.15 | 1.43 | 10.59 | 6.24 | 0.73 | 8.55 | 58.81 | 48.95

4 | 17.73 | 1.47 | 12.06 | 3.51 | 0.44 | 7.98 | 80.20 | 70.07

5 | 11.27 | 1.43 | 7.88 | 10.00 | 1.36 | 7.35 | 11.27 | 4.90

|  | STDEV | 1.58 | |

STDEV | 1.24 | |

THz, the amplitude of terahertz resonance; ELISA, optical density of Enzyme linked immunosorbent assay; STDEV, standard deviation

1 THz/ELISA of no exposure

2 THz/ELISA of high-power THz exposure

#818

Changes in tumor suppressor methylation in SKBR3 cells during treatment with Olaparib or Doxorubicin.

Zuzana Sichmanova, Asta Ottesen, Per Eystein Lonning, Elisabet Ognedal Berge, Stian Knappskog. _University of Bergen, Bergen, Norway_.

DNA methylation is an epigenetic mechanism that can alter gene expression without modifying the DNA sequence. Reactivation of tumor suppressor genes that have been epigenetically silenced in cancer could be a potential therapeutic approach. In this study, we performed long-term treatment of the breast cancer cell line SKBR3, with Olaparib or Doxorubicin in order to assess the methylation status of tumor suppressor genes, after the cell cultures had become resistant to these drugs. Using bisulphite conversion and massive parallel sequencing we examined CpG islands in promoter regions of 283 tumor suppressor genes at several time points under the two treatment regiments. Methylation analysis was carried out for each sample using our in-house bioinformatic pipeline established in collaboration with Roche and publicly available tools. We assessed differentially methylated CpG's for each drug, compared to control samples treated with dimethyl sulfoxide (DMSO) using the R package methylKit. Gene ontology and pathway analysis revealed hypermethylation in genes belonging to homologous recombination pathway and hypomethylation in genes responding to cellular stress and protein kinase signaling in Olaparib-treated samples. In Doxorubicin-treated samples, we identified genes involved in inter-strand cross-link repair to be hypomethylated, while other genes associated with more general cellular response to DNA damage were hypermethylated. While in Olaparib-treated samples, differential methylation seemed to be reversible and dependent on the dosage of Olaparib, methylation patterns in Doxorubicin-treated samples appeared to change in an incremental manner, as a function of exposure time to the drug.

#819

DNA methylation genome-wide analysis in remnant gastric cancer.

Tomoaki Ito,1 Kiichi Sugimoto,1 Hajime Orita,2 Masahiro Maeda,3 Hiroshi Moro,3 Toshikazu Ushijima,3 Hitoshi Katai,4 Ryo Wada,2 Kazuhiro Sakamoto,5 Koichi Sato,2 Malcolm V. Brock1. 1 _The Sidney Kimmel Comprehensive Cancer Center at the Johns Hopkins University, School of Medicine, Baltimore, MD;_ 2 _Juntendo University Shizuoka Hospital, Juntendo University, School of Medicine, Shizuoka, Japan;_ 3 _National Cancer Center Research Institute, Tokyo, Japan;_ 4 _National Cancer Center Hospital, Tokyo, Japan;_ 5 _Juntendo University Faculty of Medicine, Tokyo, Japan_.

Purpose: Although primary gastric cancers (PGC) and remnant gastric cancers (RGC) both originate from the same gastrointestinal organ, they have very distinct clinicopathological behaviors. Due to the importance of chronic inflammation in the carcinogenesis of gastric malignancies, we hypothesized that DNA promoter hypermethylation would play a critical role in the carcinogenesis of RGC as well as PGC.

Methods: We investigated the genome-wide DNA methylation patterns of PGC and RGC tissues from 48 patients from an academic medical center in Japan using the Infinium HumanMethylation450 BeadChip assay. The results were validated by quantitative methylation-specific PCR (qMSP) in separate, independent cohorts.

Results: We found that in our training cohort of 48 patients, genes from the gastric cancer tissues identified by the Infinium HumanMethylation 450 Beadchip clustered into high and low methylation groups on multivariate analysis (p=0.004, OR=12.33). PGCs contributed significantly to the high methylation group suggesting that the DNA promoter methylation status in PGC is higher than that in RGC. Supporting this conclusion was the finding that in a separate qMSP analysis in a test cohort, the gene A had significantly higher DNA promoter methylation in cancer tissues in the validation PGC tissues than in RGC.

Conclusion: This study demonstrated that DNA promoter methylation status in PGC is higher than in RGC. This result may reflect the effects of Helicobacter pylori on the induction of DNA methylation in the remnant stomach.

#820

Enzymatic Methyl-Seq: methylome analysis of challenging DNA samples.

Louise Williams, V K Chaithanya Ponnaluri, Brittany S. Sexton, Lana Saleh, Katherine Marks, Mala Samaranayake, Laurence Ettwiller, Shengxi Guan, Heidi E. Church, Nan Dai, Esta Tamanaha, Erbay Yigit, Bradley Langhorst, Zhiyi Sun, Thomas C. Evans, Romualdas Vaisvila, Eileen Dimalanta, Theodore B. Davis. _New England Biolabs, Ipswich, MA_.

DNA isolated from blood draws (cell-free DNA (cfDNA)) or from archival material like formalin fixed paraffin embedded (FFPE) tissues have advanced the field of cancer genetics. DNA methylation (5-methylcytosines (5mC) and 5-hydroxymethylcytosines (5hmC)) is a key epigenetic factor that plays an important role in cellular processes and it's misregulation results in diseased states like cancer. Advances in the field of sample preparation from biological matrices and genomics have enabled cancer biomarker identification based on methylation profiling.

Bisulfite sequencing is the standard method to detect methylation and has been employed for both targeted and whole genome methylation analysis. However, the chemical based bisulfite conversion of cytosines to uracils also results in DNA damage which subsequently results in shorter DNA insert sizes as well as introducing bias into the data. Robust biomarker detection relies primarily on the ability to profile methylation accurately. Analysis of DNA methylation from cfDNA and FFPE DNA is challenging as the DNA is typically of low quality and quantity. To overcome the drawbacks of bisulfite sequencing, we developed an enzyme based methylation detection technology, called NEBNext Enzymatic Methyl-Seq (EM-Seq). DNA damage is minimized enabling longer insert sizes, lower duplication rates and minimal GC bias resulting in more accurate quantification of methylation in the sample DNA.

Using EM-Seq, we profiled cfDNA and FFPE DNA from multiple tissue types. Results for these challenging DNA types showed that the EM-Seq libraries had longer inserts, lower duplication rates, higher percentages of mapped reads and less GC bias compared to WGBS libraries. These libraries also identified a higher number of CpG's and the estimated global methylation levels were in good agreement with the absolute levels quantified using LC/MS. In conclusion, EM-Seq libraries have superior sequencing metrics resulting in robust methylation profiling for these types of challenging DNA samples.

#821

**DNA hypermethylation of** IRX4 **is a frequent event that may confer growth advantage to pancreatic cancer cells.**

Kanchan Chakma,1 Zhaodi Gu,2 Fuyuhiko Motoi,3 Michiaki Unno,3 Akira Horii,2 Shinichi Fukushige2. 1 _Department of Biochemistry and Molecular Biology, University of Chittagong, Chittagong, Bangladesh;_ 2 _Department of Molecular Pathology, Tohoku University School of Medicine, Sendai, Japan;_ 3 _Department of Gastroenterological Surgery, Tohoku University School of Medicine, Sendai, Japan_.

Epigenetic gene silencing by aberrant DNA methylation is one of the important mechanisms leading to the loss of key cellular pathways in tumorigenesis. Methyl-CpG targeted transcriptional activation (MeTA) reactivates hypermethylation-mediated silenced genes in a different way from DNA demethylating agents. Previous study using microarray coupled with MeTA (MeTA-array) identified seven commonly hypermethylation-mediated silenced genes in pancreatic cancer cell lines. In this study, IRX4 (Iroquois homeobox 4) was focused on because IRX4 was located at chromosome 5p15.33 where one of pancreatic cancer susceptibility loci has been identified through genome-wide association study (GWAS). IRX4 has also been identified as a candidate tumor suppressor gene in prostate cancer. IRX4 was greatly downregulated in all of the analyzed 12 pancreatic cancer cell lines by promoter hypermethylation. In addition, IRX4 promoter region was found to be frequently and specifically hypermethylated in primary resected pancreatic cancers (15/22: 68%); corresponding normal pancreatic tissues were unmethylated. To examine whether IRX4 has the growth inhibitory effect on pancreatic cancer cell lines, colony formation assay using PK-1 and PK-9 cell lines has been performed; compared with empty vector-transfected cells, IRX4 expression vector-transfected cells formed significantly reduced number of colonies. Furthermore, tetracycline-inducible IRX4 expressing vector system was constructed and a pancreatic cancer cell line PK-1 was analyzed for further functional investigation; induction of IRX4 suppressed cell growth in PK-1. Our results suggest that DNA methylation-mediated silencing of IRX4 is a frequent event that may confer growth advantage to pancreatic cancer cells and MeTA may be a powerful tool to search critical hypermethylated genes in tumorigenesis.

#822

PureQuant Methylation Assay for the accurate quantification of immune cell populations.

Suman Pradhan, Carl Dargitz, Jerry Guzman, Uma Lakshmipathy. _Thermo Fisher Scientific, Carlsbad, CA_.

A key challenge during the development and manufacturing of cell based immunotherapies is the reliable estimation of cell purity. Current flow cytometric methods, while effective for the detection of surface antigens such as CD8 that positively identifies Cytotoxic T lymphocytes, are not ideal for intracellular markers used to positively identify Regulatory T (Treg) cells and T Helper 17 (Th17) cells. Additionally, characterization methods that utilize live cells pose a challenge in large scale GMP manufacturing environments due to complicated logistics, limited throughput and difficulty in standardization. Therefore, there is an emerging need for alternative assay methods that address some of these challenges.

Here, we introduce our newly developed PureQuant™ Assays that specifically measure the unique DNA methylation status of specific immune cell types. Assays were developed specifically for Cytotoxic T Cells, Regulatory T cells and T helper 17 cells by detecting methylation status of CD8, FoxP3 and IL17A, respectively, via qPCR of bisulfite converted genomic DNA. These assays are robust with minimal sample requirement, utilizing fresh/frozen cells or isolated gDNA. Results are represented as a percentage of the total cell population similar to flow cytometry based methods. Comparison of the two methods for surface markers such as CD8 show comparable results. Our results demonstrate that this method which combines accuracy, low sample requirement and flexibility, is an ideal measurement system for confirmation of T cell identity and purity that is critical for cell therapy research and development.

#823

**A case-control study of constitutional** BRCA1 **methylation in a mammographically screened cohort.**

Basant Ebaid,1 Hongdo Do,1 Jia Min Pang,2 Lisa Devereux,2 Alexander Dobrovic1. 1 _Olivia Newton-John Cancer Research Institute, Melbourne, Australia;_ 2 _Peter MacCallum Cancer Centre, Melbourne, Australia_.

Introduction: Over 90% of breast cancers occur sporadically with no obvious hereditary factors at play, therefore highlighting the need for biomarkers that can identify women who are predisposed to developing breast cancer. DNA methylation of the BRCA1 promoter region is detectable at low levels in the peripheral blood of some women. This phenomenon is referred to as constitutional BRCA1 methylation. Peripheral blood methylation of the BRCA1 gene has been identified as a predisposition factor to the development of BRCA1 methylated tumors. We performed a case-control study to assess peripheral blood BRCA1 methylation from healthy women, and from women with breast cancer. This study aimed to establish the frequency of peripheral blood methylation of the BRCA1 gene in women with and without breast cancer, to further our understanding of the role of constitutional BRCA1 methylation in breast cancer predisposition.

Methods: Constitutional BRCA1 methylation was assessed in peripheral blood DNA of breast cancer patients and healthy women. Whole blood DNA was obtained from healthy women (n=327 controls) and women with breast cancer (n=300 cases) as part of the LifePool Project. Blood was collected on the day of mammography with BreastScreen Victoria. We used a probe-based droplet digital PCR (ddPCR) assay designed to quantify methylated and unmethylated alleles at the BRCA1 promoter region.

Results: Previous research using less-sensitive methodologies has reported a BRCA1 methylation frequency of 2-4% in peripheral blood of healthy women. In this study, there was no significant difference in the observed BRCA1 methylation frequency between cases and controls (6.6% and 6.4% respectively). The discrepancy between our observed methylation frequencies and previously reported data can be attributed to the higher sensitivity of the ddPCR methodology. Nevertheless, the level of BRCA1 methylation in cases was significantly higher than the level of methylation observed in controls, consistent with previous studies. In addition, the frequency of BRCA1 methylation was higher in women diagnosed with breast cancer under the age of 50 compared to women diagnosed over 50 years old (average methylation frequency = 12.5% and 5.3% respectively).

Conclusions: ddPCR methodology enabled accurate quantification of methylated BRCA1 alleles in peripheral blood DNA down to less than 0.1%. In this selected population of women undergoing mammography, there was no difference in the frequency of detectable BRCA1 methylation between cases and controls. Once age was considered, detectable BRCA1 methylation showed an association with age of onset. Tumours for the women with breast cancer will be retrieved and tested for BRCA1 methylation, to further examine the link between the methylation observed in peripheral blood DNA and development of a BRCA1-methylated tumor.

#824

LKB1 loss in lung adenocarcinoma leads to global hypomethylation and altered FOXA binding.

Michael J. Koenig,1 Joseph Amann,1 Bernice Agana,1 Jacob Kaufman,2 Vicki Wysocki,1 Christopher Oakes,1 David P. Carbone1. 1 _The Ohio State University, Columbus, OH;_ 2 _Duke University, Raleigh, NC_.

LKB1 is functionally lost in 30% of lung adenocarcinoma cases. There are no targeted therapies available for LKB1-loss tumors. Previous studies of LKB1-loss in a mouse model of pancreatic cancer suggested that LKB1 loss drives DNA hypermethylation and serine biogenesis. DNA methyltransferases (DNMTs) maintain CpG methylation, which suppresses gene transcription. The primary substrate for DNMTs is S-adenosyl Methionine (SAMe), and the maintenance of CpG methylation has been shown to be dependent on continued regeneration of SAMe. KRAS status is associated both with LKB1 loss and with a CpG island hypermethylation phenotype (CIMP). Using a previously published 16-gene signature for LKB1 loss, we set out to confirm whether the changes that occur in the mouse model of pancreatic cancer also occur in lung cancer. We applied our signature to samples in The Cancer Genome Atlas (TCGA) lung adenocarcinoma dataset (signature-positive samples are described as "LKB1-loss"), which allowed us to compare both gene expression data as well as methylation data from Illumina 450k microarrays. We then developed a cell-line model for LKB1-loss in order to study the mechanism by which LKB1 regulates DNA methylation.

We observed that LKB1-loss is associated with widespread demethylation of the genome in TCGA samples. Approximately 14,000 CpG sites were significantly hypomethylated in LKB1-loss tumors, while only 200 were hypermethylated (adjusted p-value cutoff = 0.05). Furthermore, Student's t-test showed that LKB1-loss samples have significantly lower average methylation (β-values), regardless of KRAS status. Using motif analysis, we found that demethylated CpG sites are significantly enriched for FOXA1/2/3 consensus binding sites. FOXA transcription factors are well known as pioneering transcription factors that mediate demethylation. We discovered in vitro that FOXA1 localization and turnover is dependent on LKB1 expression, specifically the activity of downstream kinase SIK. Rather than showing enhanced serine biogenesis, LKB1-loss tumors express less DNMT1 and LC-MS/MS analysis shows depletion of SAMe. Finally, in vitro models of LKB1-loss show an increased sensitivity to DNMT inhibitors and FOXA1 silencing.

Together, these observations suggest that LKB1-loss lung adenocarcinoma patients have a distinct hypomethylation profile. This profile may make them uniquely sensitive to DNMT-inhibition in combination with other therapies, a hypothesis which we will test in future studies.

#825

TAPS: A bisulfite-free, base-resolution and quantitative sequencing method for cytosine modifications.

Yibin Liu, Paulina Siejka, Gergana Velikova, Ying Bi, Marketa Tomkova, Benjamin Schuster-Boeckler, Chun-Xiao Song. _Ludwig Institute for Cancer Research, Oxford, United Kingdom_.

Aberrant DNA methylation changes are powerful prognostic and predictive biomarkers in cancer diagnosis and treatment. For decades bisulfite sequencing has been the gold standard for DNA methylation analysis. However, this method relies on harsh deamination reaction of unmodified cytosine to uracil which degrades the majority of DNA and generates sequencing libraries with low complexity. Those drawbacks limit the applications on low-input samples and lead to poor sequencing quality, low mapping rates, uneven genome coverage and increased sequencing cost. Here, we present a novel bisulfite-free and base-resolution sequencing method, TET Assisted Pyridine borane Sequencing (TAPS), for direct detection of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) without affecting unmodified cytosine. We utilised TET enzymes to readily oxidize 5mC and 5hmC to 5-carboxylcytosine (5caC), which was then converted to dihydrouracil by pyridine borane via a previously unknown reductive decarboxylation/deamination reaction. This non-destructive method enables high-throughput sequencing with nanoscale input materials (genomic DNA and cell-free DNA) and preserve DNA up to 10 kbs long which could allow long read sequencing. We demonstrated that TAPS could directly replace bisulfite sequencing with higher mapping rates, more even coverage and lower sequencing costs, and enable more comprehensive, cheaper and faster methylome analyses. This would be a revolutionary tool for epigenetic study and facilitate both academic research and clinical diagnostics.

#826

Global DNA methylation levels regulate PD-L1 expression in melanoma.

Aniruddha Chatterjee,1 Antonio Ahn,1 Euan J. Rodger,1 Peter A. Stockwell,1 Matthew Parry,1 Jyoti Motwani,1 Stuart J. Gallagher,2 Elena Shklovskaya,2 Jessamy Tiffen,2 Peter Hersey,2 Michael R. Eccles1. 1 _University of Otago, Dunedin, New Zealand;_ 2 _The Centenary Institute, University of Sydney, Sydney, Australia_.

The programmed death-ligand 1 (PD-L1) receptor is an important immune checkpoint and is often upregulated in cancer cells to allow immune evasion. In melanoma, the patients with PD-L1 expression and absense of tumour infiltrating lymphocytes (TILs) (i.e. "constitutive PD-L1 or PD-L1CON") show worse response rates and prognosis than patients with PD-L1 expression and the presence of TILs (i.e. "inducible PD-L1 or PD-L1IND"). However, how PD-L1 expression is regulated in melanoma cells remains elusive. Understanding the mechanisms of how PD-L1 is regulated is important for predicting responses for anti-PD-L1 treatment and for developing new combinatorial therapies. We hypothesised that epigenetic state regulates constitutive and inducible PD-L1 expression in melanoma.

To address the hypothesis, we have generated whole-genome scale DNA methylomes (using reduced representation bisulfite sequencing) and transcriptomes (RNA-Seq) for patient derived melanoma cell lines (in PD-L1IND and PD-L1CON groups). We discovered extensive global hypomethylation in the constitutive lines, particularly pronounced in intergenic repeat regions and gene bodies. A high proportion of hypomethylated regions exhibited dichotomous methylation patterns indicating a common regulatory mechanism between the PD-L1IND and PD-L1CON lines. RNA-Sequening data indicated that the hypomethylated state of the PD-L1CON cells was correlated with higher upregulation of the differentially expressed genes at a global-scale and the upregulated genes were associated with cancer hallmark properties. The upregulated genes exhibited signatures of viral mimicry and cytosolic sensing of dsRNA similar to what has been observed after DNA methyltransferase inhibitor (DNMTi) treatment in cancers. DNMTi treatment increased PD-L1 transcription in the PD-L1IND cell lines. PD-L1CON samples showed greater resistance to DNMTi compared to PD-L1 negative samples. This was observed at protein-coding genes, transposable elements and long non-coding RNAs. Moreover, genes involved in the innate immune pathway was strongly increased upon DNMTi mediated hypomethylation in the PD-L1IND cell lines (FDR-adjusted P-value = 0.02), however there was no expression change in the PD-L1 positive cells (Adjusted P-value = 1.0). Furthermore, we found that the IRF1 transcription factor, which plays a critical role in the innate immune pathway and also binds to the PD-L1 promoter, is significantly upregulated upon DNMTi in the PD-L1 negative cell lines (Adjusted P-value = 0.01) but not in the PD-L1 positive cells (Adjusted P-value = 0.9). These results show that global hypomethylation levels regulate PD-L1 expression in melanoma. We believe these results are the first to show that DNA methylation levels play a role in regulating PD-L1 on melanoma and suggest they may have important implications for combined treatments targeting methylation (DNMTi) and PD1/PD-L1 (anti-PD1 antibodies).

#827

Estimating breast tissue-specific epigenetic age using next-generation methylation sequencing data.

James R. Castle,1 Nan Lin,1 Jingpeng Liu,1 Chi Wang,1 Yunlong Liu,2 Chunyan He1. 1 _University of Kentucky, Lexington, KY;_ 2 _Indiana University, Indianapolis, IN_.

Epigenetic age captures both genetic and environmental influences across time on cellular functions and is an indicator of biological aging. Epigenetic age may deviate from chronological age substantially in individuals. Epigenetic age is also tissue-specific. Emerging evidence suggests that female breast tissue ages faster than other parts of the body according to epigenetic age estimation using the "Horvath Clock" model. The Horvath method is based on the DNA methylation of 353 CpG loci on the outdated Illumina microarray platforms. The increasing availability of next-generation sequencing data calls for method development that uses DNA methylation sequencing data to estimate tissue-specific epigenetic age. We developed a new method to estimate breast tissue-specific epigenetic aging using next-generation methylation sequencing data and assessed the difference between epigenetic and chronological ages, known as epigenetic age acceleration (EAA), in different breast tissue types. The Illumina TruSeq Methyl Capture EPIC Sequencing technology was used to obtain DNA methylation profiles of approximately 3.3 million CpG sites in 111 tumor, 48 matched adjacent normal, and 462 normal breast tissue samples. A total of approximately 1.4 million CpG sites remained after quality control. Following the Horvath approach, we used an elastic net penalized regression model to regress chronological age on CpG sites in normal breast tissue and defined a new set of 247 clock CpGs specific to breast tissue with randomly divided training (n = 370) and testing (n = 92) data sets. We estimated breast tissue-specific epigenetic age and EAA in tumor, adjacent, and normal breast tissue. We found that breast tissue-specific epigenetic age was positively correlated with chronological age (r=0.87; P<2.2X10-16). Neither normal nor adjacent normal breast tissue showed a significant EAA. However, tumor breast tissue had a significant and increased EAA (median = 8.0 years; P=6.9X10-9). While triple-negative breast tumors showed no significant EAA, hormone receptive-positive and Her2-positive breast tumors had a significant and increased EAA (median=9.5 and 13.1 years; P=1X10-5 and 0.02, respectively). Results of this new model were compared to similar results using the Horvath Clock model and were found to be qualitatively consistent. Further research is needed to determine whether epigenetic age acceleration in normal breast tissue is predictive of breast cancer risk and how breast cancer risk factors influence the rate of acceleration.

#828

Accelerated epigenetic aging in bladder cancer patients.

Yap Ching Chew, Wei Guo, Xiaojing Yang, Paolo Piatti, Mingda Jin, Keith Booher, Benjamin Jara, Xi-Yu Jia. _Zymo Research Corp., Irvine, CA_.

Aging represents the most important risk factor for many chronic diseases including cardiovascular diseases, diabetes, and cancer, therefore understanding the mechanisms of aging is a fundamental step for designing new treatments for chronic diseases. DNA methylation is the most reliable and accurate molecular marker for aging quantification, however, genome-wide DNA methylation profiling techniques, such as reduced representative bisulfite sequencing and Illumina Bead Array that are widely used in aging research are prohibitively expensive and have poor data quality at low-read coverage sites. Here we report a robust targeted bisulfite sequencing approach, called SWARM® (Simplified Whole-panel Amplification Reaction Method), for the accurate biological age determination. SWARM™ is flexible and low cost, requires relatively low DNA starting material, allows the simultaneous amplification and sequencing of hundreds of loci, and has shown to increase sample throughput. Using the SWARM® approach, we were able to analyze the methylation level of several hundreds of age-associated loci including the published Horvath Clock sites. Gender-specific age-predictive models were built using the elastic net regression of DNA methylation levels of the loci and chronological age of urine DNA samples of over 300 healthy subjects of 18 to 88 years old. Urine samples from bladder cancer patients exhibit significant age acceleration, with an average of >10 years. In brief, our gender-specific urine DNAge® analysis is a tool for the precise biological aging quantification and can be used to address questions in aging and urinary track cancers.

#829

SOX2-mediated 5hmC dysregulation in GBM stem cells.

Sweta Sudhir. _Johns Hopkins University, Baltimore, MD_.

Primary brain tumors are among the most devastating forms of cancer and glioblastoma (GBM) represents the most aggressive and lethal form of the disease. We now know that GBM contains small subsets of cells that display tumor-propagating stem-like phenotypes (i.e. glioma stem cells or GSCs) that act as critical determinants of resistance to current treatments and tumor recurrence for which there is no proven therapy. Altered patterns of DNA methylation are widely reported in human GBM. However, substantial knowledge gaps remain in our understanding of the molecular mechanisms responsible for this epigenetic dysregulation, its downstream consequences and its role in the GBM tumor-propagating phenotype. Understanding and ultimately targeting the epigenetic mechanisms that induce and maintain these tumor-propagating cell subsets is critical to improving GBM therapy and patient outcomes.

DNA methylation generally occurs in cytosine-guanine (CpG) sequences and is established by DNMTs, which catalyze the conversion of cytosine to 5-methylcytosine (5mC). DNA methylation is dynamically regulated and reversible by the ten-eleven translocation (TET) family of deoxygenases that catalyze the conversion of 5mC to 5-hydroxymethylcytosine (5hmC). Levels of 5hmC closely align with the differentiation state of cells; terminally differentiated cells contain high levels of 5hmC and less differentiated cells have very low levels. Multiple studies found negative correlations between 5hmC levels and glioma grade and loss of 5hmC correlates with poor prognosis of GBM patients. This strongly suggests that tumor suppressing epigenetic mechanisms are driven by TET-dependent 5hmC generation.

We now show that SOX2, a reprogramming transcription factor that induces GSCs, represses the TET2 and decreases 5hmC in multiple GSC isolates. TET2 repression using two independent shRNA hairpins efficiently decreased 5hmc levels and significantly enhanced self-renewal capacity and tumor growth capacity of low-passage GSCs. We also show that low concentrations of vitamin C, a TET enzyme cofactor, significantly increases 5hmC in low-passage GSCs. This epigenetic change induced by vitamin C associated with increased GSC death induced by temozolomide (TMZ), the main chemotherapeutic used to treat GBM. These findings support a mechanism by which SOX2 induces loss of 5hmC in GSCs by repressing TET2 and show that vitamin C can reverse this epigenetic tumor promoting pathway and potentially enhance GBM chemotherapeutics.

#830

DNA methylation cytometry reveals cancer survival related to cell composition.

Lucas A. Salas,1 Karl T. Kelsey,2 Devin C. Koestler,3 John K. Wiencke,4 Brock C. Christensen1. 1 _Geisel School of Medicine at Dartmouth, Lebanon, NH;_ 2 _Brown University, Providence, RI;_ 3 _University of Kansas Medical Center, Kansas City, KS;_ 4 _University of California San Francisco, San Francisco, CA_.

Tumor microenvironments are heterogeneous and include epithelial cells, stroma, blood vessels and leukocytes. The interactions between normal and tumor epithelial cells result in responses including angiogenesis, immune regulation and tumor growth. Although flow cytometry and single cell sequencing have been used to investigate cellular composition in tumors, these approaches are dependent upon appropriate substrates that may not be routinely available. Recently, the use of molecular markers of cell type from mRNA (CIBERSORT) and DNA methylation (MethylCIBERSORT) have been used to infer specific cell proportions in tumors. Immune cell type DNA methylation signatures have been well-established for whole blood deconvolution but a challenge in tumors is the potential for carcinoma cells to have signal closely related to nontumor epithelial cells, and heterogeneity of potential tumor distinguishing deconvolution biomarkers. We aim to offer an alternative reference-based approach for tumor deconvolution with DNA methylation using ten normal cell components that we apply to TCGA tumor samples and test the relation of sample composition with patient survival. Ten magnetic sorted cell types with Illumina EPIC DNA methylation data were used as references (B cells, CD4T, CD8T and NK, monocytes, neutrophils, eosinophils, epithelial, endothelial and stromal cells). In total 1256 CpGs were selected for the reference library which was applied to deconvolute 17 TCGA tumor types (n=6417: BLCA, BRCA, COAD, CESC, ESCA, HNSC, KIRC, KIRP, LIHC, LUAD, LUSC, PAAD, PRAD, SKCM, STAD, THCA, UCEC). The most abundant cells were epithelial (median:36.6%), endothelial (median: 17.1%), CD8T (median: 12.1%) stromal cells (median:10.3%), and CD4T (median: 4.4%). We calculated three cell ratios: CD8T to epithelial (CD8T:epith), CD4T to epithelial (CD4T:epith) and endothelial to epithelial cells (vasc:epith) and fit Cox proportional hazards models stratified by cancer stage (I, II, III and IV), and adjusted for sex, age at diagnosis, and the three ratios. For all cancers, increased CD8T:epith was associated with worse survival HR: 1.39 (95%CI: 1.15, 1.68). For KIRC, increased CD8T:epith increased the risk of death HR: 1.54 (95%CI: 1.06, 2.22), whereas survival was improved for BRCA HR: 0.15 (95%CI: 0.03, 0.68), and CESC HR: 0.19 95%CI: 0.04, 0.90. A strong effect of increased vasc:epith ratio on risk of death was observed in BLCA HR: 8.03 (95%CI: 3.01, 21.4), and the CD4T:epith ratio reduced the risk of death HR: 0.11 95%CI: 0.02, 0.56. DNA methylation deconvolution is a promising tool for cancer prognosis and offers several technical and cost advantages. However, additional work to further stratify leukocyte signals by cell state such as CD8 T-cell exhaustion, and CD4T Treg infiltration is required.

#831

Hypermethylation in promoter regions of CpG islands of multiple genes is associated with disease progression and molecular response in chronic myelogenous leukemia.

Mamta P. Sumi, Sameer A. Guru, Imtiyaz A Najar, Mariyam Zuberi, N. Gupta, Alpana Saxena. _Maulana Azad Medical College, New Delhi, India_.

Chronic myelogenous leukemia (CML) has a typical progressive course with transition from a less aggressive chronic phase to a terminal more aggressive blast crisis. The molecular mechanisms leading to CML disease progression remain to be elucidated. However, it has been proposed in many studies that alterations in the methylation status of various genes may contribute to the progression of different malignancies. Hence, in this study we tried to understand the role of aberrant methylation of six tumor suppressor genes in the disease progression and molecular response in CML.

We investigated 100 CML patients and 100 age and sex matched healthy controls for the methylation status of six tumor suppressor genes. The methylation status of these genes was evaluated by MS-PCR.

In this study, 100 CML samples and 100 controls were analyzed for the DAPK1, RASSF1A, RIZ-1, p16, p14ARF and SOCS-1 promoter methylation status. These genes, except p14ARF and SOCS-1 showed significant differences in methylation status among CML patients and healthy controls. Also DAPK1, RASSF1A, RIZ-1, p16 and p14ARF genes were observed to be hypermethylated during progression of CML disease to advanced phases. The methylation frequencies of these five genes were seen to increase progressively during CML disease progression. This increase in hypermethylation of these five genes from was statistically significant with a p-value of <0.0001, 0.001, 0.009,0.002 and 0.01 respectively. Although, there was a progressive increase in the promoter methylation of SOCS1 gene during the CML disease progression (CP= 4%, AP=8% and BC=16%) but this did not reached a statistical significance (p-value 0.1).

We also assessed the molecular response of these 100 CML patients after imatinib therapy and the patients were categorized into two groups; major molecular response group and loss of molecular response group. Out of 100 CML patients on imatinib therapy 52 achieved major molecular response while 48 patients showed loss of molecular response. Out of the above studied genes RASSF1 and RIZ1 promoter hypermethylation was significantly associated with loss of molecular response in CML patients receiving imatininb therapy. Out of 48 CML patients who were in loss of molecular response, 23.0% and 16.77% showed promoter hypermethylation of RASSF1 and RIZ1 genes respectively while out of 52 patients that were in major molecular response only 9.6% and 1.92% of patients showed hypermethylation in RASSF1 and RIZ1 genes respectively.

These results suggest that hypermethylation of tumor suppressor genes plays a significant role in the progression and response in CML.

#832

Comparison of single step to two step PCR for DNA methylation status determination utilizing a system that continuously contains PCR products in plastic consumables.

Wanyuan Ao, Derek Bosh, Dale Emery, Robert Parry, Nils Adey. _KimanTech, LLC, Salt Lake City, UT_.

DNA methylation specific PCR is a commonly used method to determine DNA methylation status. However, a well-known risk for PCR, and particularly methylation specific PCR, is carryover contamination from amplification products that can result in false positive PCR results in subsequent tests. This risk can be mitigated using careful technique, facility design, and biochemical methods such as uracil incorporation and subsequent uracil N-glycosylase (UNG) negative selection. Unfortunately, careful technique is highly user dependent, facility design can be cumbersome and expensive, and uracil incorporation is effective for only one step of two-step (nested) PCR reactions and cannot be used with the DNA bisulfite treatment methods used for methylation specific PCR. Our company has developed a system that mitigates PCR product contamination by continuously containing PCR products in plastic consumables and can interface with existing laboratory robotics, real time thermocycling instruments, and cassette electrophoresis gels. Here we utilize this system to compare the sensitivity and specificity of two-step multiplex nested PCR vs. single-step PCR for DNA methylation status detection applications using both qPCR and gel electrophoresis analysis. Published methylation status specific PCR primers that target CpG nucleotides in promotor regions were used to PCR amplify commercially available fully methylated and fully unmethylated DNA. The results demonstrate that a multiplex preamplification using amplicons for the CHAD, GFI1, MX2, NEU1, VWCE genes followed by monoplex nested methylation specific PCRs resulted in a Limit Of Detection (LOD) of 1 to 10 DNA template copies whereas performing just the monoplex methylation specific PCR (no preamplification) resulted in a LOD of 10 to 300 DNA template copies. If DNA isolated from macro dissected FFPE tissue was utilized, positive signals were identified using multiplex nested PCR whereas no signals were identified using single-step PCR. When mixtures of methylated and unmethylated DNA were utilized along with the GSTP gene amplicons, two-step nested PCR using were able to distinguish as low as 1% unmethylated DNA or 2% methylated DNA in a background of the other form, whereas single-step PCR was able to distinguish just 10% unmethylated or 33% methylated DNA under the same conditions. Furthermore, no PCR product contamination was observed upon amplification of samples collected from the external portion of the device demonstrating the PCR products were well contained.

#833

Integrative analysis of TCGA pancreatic ductal adenocarcinoma data.

Nitish K. Mishra, Siddesh Southekal, Chittibabu Guda. _UNMC, Omaha, NE_.

Pancreatic cancer is a heterogeneous type of cancer that exhibits different histopathological, molecular and genomic phenomena. Pancreatic ductal adenocarcinoma (PDAC) is the deadliest type of pancreatic cancer with a five-year survival of only 8%. Prognostic biomarkers for early detection of PDAC are not available. In this study, we used clinical, DNA methylation, and gene expression profiles of 146 TCGA PDAC patients. We analyzed the global patterns of DNA methylation and correlated the promoter DNA methylation with corresponding gene expression. CpG sites with a difference of 0.2 in the mean β value of the primary tumor and normal samples were considered as differentially methylated. We observed differential methylation of about 12-thousand CpG sites between tumor and normal samples, the majority of which are hypermethylated. Chromosome 1, 20, 18 and 19 are predominately hypermethylated and chromosome 9 is hypomethylated. CpG islands and regions that are in close proximity to CpG islands (shores) have more hypermethylated sites compared to the far away regions (shelf). Pathway enrichment analysis of differentially methylated genes enabled us to understand how changes in methylation affect biological pathways involved in the progression of PDAC. Our analysis reveals enrichment of MAPK signaling, Rap1 signaling, calcium signaling pathway, etc. Promoter DNA methylation is highly associated with its corresponding gene expression. A significant correlation between methylated CpG sites within 1.5kb from TSS and corresponding gene expression was observed between 4,971 CpGs and 1,744 corresponding genes, out of these about 92% of the genes showed a negative correlation. Survival analysis showed a statistically significant correlation between the expression levels of genes, NEK2, and ASPM with overall survival of the patients. In the future, we will further analyze the long non-coding RNA (lncRNA) promoter methylation, and role of lncRNA and miRNA in PDAC patients' survival.

#834

Epigenome-wide profiling of DNA methylation in peripheral blood leukocytes and the prognosis of prostate cancer patients in African Americans.

Junfeng Xu,1 Chia-Wen Tsai,1 Wen-Shin Chang,1 Da-Tian Bau,2 Jian Gu1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _China Medical University Hospital, Taichung, Taiwan_.

Introduction: DNA methylation at CpG sites plays important roles in cancer development and progression. Hypermethylation of the promoter regions of tumor suppressor genes leads to gene silencing whereas global hypomethylation may affect chromosome structure and cause genomic instability. African Americans have higher incidence and mortality rates of prostate cancer than other ethnic groups. The goals of this study are to investigate the role of global DNA methylation in prostate cancer aggressiveness and identify CpG site methylations as predictors of aggressive prostate cancer in African Americans.

Methods: We measured global DNA methylation level of long interspersed nucleotide elements (LINE-1) and subtelomeric repeat (D4Z4) in leukocyte DNA from ~300 African American prostate cancer patients and compared their methylation levels between different clinicopathological variables at diagnosis. We then analyzed the association of the LINE-1 and D4Z4 methylation with the risk of biochemical recurrence (BCR) using a multivariate Cox proportional hazards model. In addition, we used the Kaplan-Meier survival function and log-rank tests to assess BCR-free survival associated with D4Z4 methylation.. We also used Illumina's HumanMethylationEPIC beadchip to profile 850K CpG site methylation in leukocytes and analzyed their associations with prostate cancer aggressiveness.

Results: There was no significant differences in the methylation level of LINE-1 between clinically defined aggressive and non-aggressive PCa at diagnosis and LINE-1 methylation was not associated with BCR either. However, the methylation of subtelomeric region D4Z4 was associated with BCR. Patients with higher methylation of D4Z4 exhibited an increased risk of BCR (HR=3.47, 95% CI, 1.10 - 10.97) compared to patients in the lower methylation after adjustment of age, BMI, smoking status, pack year, D'Amico risk groups, and treatments. In Kaplan-Meier survival analysis, patients with higher D4Z4 methylation level had a significantly shorter BCR-free survival time than those with lower methylation level (log rank P = 0.0071). When analyzing individua CpG site methylation, we identified a number of CpG site that can distinguish aggressive from non-aggressive prostate cancer and found a CpG methylation signature that can identify a subgroup of patients with aggressive prostate cancer in African Americans.

Conclusions: These data suggest that methylation in the subtelomeric region D4Z4 may be able to predict worse prognosis of patients. Individual CpG site methylation may be promising biomarkers for the identification of aggressive prostate cancer in African Americans.

#835

Altered DNA modifications in Barrett's oesophagus and oesophageal adenocarcinoma.

Skirmantas Kriaucionis, Marketa Tomova, Sophie Kirschner, Pijus Brazauskas, Mankgopo Kgatle, Richard Owen, Michael White, Benjamin Schuster-Boeckler, Xin Lu. _University of Oxford, Oxford, United Kingdom_.

Cytosine in human DNA can be modified by DNA methyltransferases (DNMTs) and the Ten-Eleven Translocation (TET) family of oxygenases. Activity of these enzymes results in four modifications, among which 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are the major ones. Different patterns of localisation of both these modifications relate to gene transcription and the developmental origin of different cell types. Cancers have been shown to have globally altered DNA modifications, however it is poorly understood when in carcinogenesis the shift in DNA modifications happens and how it influences tumour growth. Here we mapped 5mC and 5hmC genome-wide at single nucleotide resolution in Barrett's oesophagus, a well-known pre-malignant precursor of oesophageal adenocarcinoma (OAC). To get better insight into alterations in Barrett's oesophagus (BO), we also analysed samples from OAC, adjacent normal oesophagus and gastric and duodenal epithelia. We found substantial global hypomethylation outside genic regions in Barrett's oesophagus. Specific hypermethylation was evident in gene regulatory regions as defined by DNAseI hypersensitive sites. Moreover, we observed elevated methylation at CpG islands in a fraction of genes, which have reduced expression in Barrett's oesophagus. While we find 5hmC depleted both in BO and OAC, distribution of the modification in CpG islands is different. Overall our results demonstrate that cancer-specific epigenetic alterations can already be found in benign Barrett's oesophagus, suggesting early redistribution of DNA modifications in the process of transformation.

#836

From tobacco smoking to mutation signature: The role of epigenetic changes in human cancers.

Zhishan Chen, Wanqing Wen, Qiuyin Cai, Jirong Long, Xiao-ou Shu, Wei Zheng, Xingyi Guo. _Vanderbilt University Medical Center, Nashville, TN_.

Tobacco smoking is associated with a unique mutation signature in the human cancer genome. It is unclear whether tobacco smoking alters DNA methylations and gene expressions affect smoking-related mutation signature. In this study, we evaluated the smoking-related DNA methylation sites reported from five previous studies using peripheral blood cells to identify possible target genes. Using the mediation analysis approach, we evaluated whether the association of tobacco smoking with mutation signature was mediated through altered DNA methylation and expression of these target genes. Based on data obtained from 21,108 blood samples, we identified 374 smoking-related DNA methylation sites, annotated to 248 target genes. Using data from DNA methylations, gene expressions and smoking-related mutation signature generated from ~7,700 tumor tissue samples across 26 cancer types from The Cancer Genome Atlas (TCGA), we found 11 of the 248 target genes whose expressions were associated with smoking-related mutation signature at a Bonferroni-correction P < 0.001. This included four for head and neck cancer, and seven for lung adenocarcinoma. In lung adenocarcinoma, our results further showed that smoking increased the expression of three genes, AHRR, GPR15, and HDGF, and decreased the expression of two genes, CAPN8, and RPS6KA1, which were consequently associated with increased smoking-related mutation signature. Additional evidence showed that the elevated expression of AHRR (cg14817490) and GPR15 (cg19859270), were associated with smoking-altered hypomethylations. Our findings provide novel insight into the contributions of tobacco smoking to carcinogenesis through complex molecular mechanisms of the evaluated mutation signature by altered DNA methylation and gene expression.

#837

Circulating cell-free DNA methylation assay: Towards early detection of multiple cancer types.

Dhruvajyoti Roy,1 David Taggart,2 Lianghong Zheng,3 Dan Liu,3 Gen Li,3 Mingzhen Li,3 Kang Zhang,4 Richard A. Van Etten5. 1 _Laboratory for Advanced Medicine, Inc., Irvine, CA;_ 2 _Laboratory for Advanced Medicine, Inc., West Lafayette, IN;_ 3 _Laboratory for Advanced Medicine, Inc., China;_ 4 _Institute for Genomic Medicine, University of California at San Diego, La Jolla, CA;_ 5 _Chao Family Comprehensive Cancer Center, University of California at Irvine, Irvine, CA_.

Background: Alterations in DNA methylation play a key role in tumor initiation, and methylation markers are well established for detecting various cancer types. Cancer-specific DNA methylation patterns of cell-free DNA (cfDNA) isolated from blood samples is a non-invasive method to obtain representative epigenetic information from solid tumors. In the present study, we identified and validated cancer-specific methylation markers for diagnosis of liver, breast and colorectal cancers with high sensitivity and specificity.

Methods: Three blinded validation studies were performed in order to evaluate individual panels of DNA methylation markers developed for the detection of liver, breast or colorectal cancers. For the liver cancer panel study, samples drawn from 154 subjects were analyzed, including healthy donors (30) and subjects diagnosed with benign liver disease (10), liver cancer stage I-IV (60), or another cancer type (30). For the breast cancer panel study, samples drawn from 151 subjects were analyzed, including healthy donors (39) and subjects diagnosed with benign breast disease (15), breast cancer stage I-IV (65), or another cancer type (32). For the colorectal cancer panel study, samples drawn from 154 subjects were analyzed, including healthy donors (42) and subjects diagnosed with benign colorectal disease (14), colorectal cancer stage I-IV (68), or another cancer type (30). Cell-free DNA was extracted from all samples, bisulfite converted, and DNA methylation was quantified at target sites by using the IvyGene Platform for each of the three cancer-specific marker panel.

Results: By quantifying DNA methylation at the target sites, the cancer-specific markers were able to differentiate subjects diagnosed with cancer from both healthy donors and subjects with benign diseases. The liver cancer cfDNA methylation panel showed an overall sensitivity of 95% and specificity of 97.5%. Whereas, the breast cancer cfDNA methylation panel correctly identified the breast cancer samples for an overall calculated sensitivity of 89% and specificity of 96%. The colorectal cancer cfDNA methylation panel also showed high overall sensitivity of 93% and specificity of 100%.

Conclusions: These results demonstrate the high diagnostic potential of cfDNA methylation markers isolated from blood for the detection of multiple cancers at various stages with high sensitivity and specificity. In addition, a quantitative analysis of cfDNA methylation signature provides an opportunity for monitoring of disease. The benefits of a sensitive and specific assay to identify and monitor cancer is anticipated to aid disease management and ultimately, improve patient outcomes.

#838

Epigenetic reprogramming of different cancer-related pathways at the single cell level after low-dose DNA demethylation therapy.

Hideyuki Takeshima, Yukie Yoda, Naoko Watanabe, Mika Wakabayashi, Toshikazu Ushijima. _National Cancer Center Research Institute, Tokyo, Japan_.

The efficacy of DNA demethylation therapy is highly dependent on the dosing regimen. Administration of a drug at a dose much lower than the maximum tolerated dose (MTD) for a prolonged period shows a high therapeutic efficacy, despite its small demethylating effect. However, the mechanisms underlying the high therapeutic efficacy of low-dose treatment are still unclear. In this study, we aimed to reveal the mechanisms by analyzing decitabine (DAC)-treated individual cancer cells. To reveal the epigenetic reprogramming at the single cell level and its functional consequences, individual DAC-treated HCT116 cells (H3 clones) were cloned from DAC-treated bulk HCT116 cells. Genome-wide DNA methylation was analyzed using InfiniumHuman450 beadChip. In bulk cells, only partial reduction of DNA methylation levels was observed, and no genes were completely demethylated. In contrast, in cloned cells, a much larger reduction of DNA methylation levels was observed, depending upon genes. Promoter CpG islands of 233-470 genes of 1,188 hypermethylated genes in HCT116 were completely demethylated (Δβ ≥ 0.6) in some of the clones analyzed. Demethylated genes were highly variable among individual clones, and different cancer-related genes, such as those involved in the p53 pathway, WNT pathway, and cell cycle regulation, were completely demethylated, depending upon clones. For example, CDKN2A (p16) was completely demethylated only in a H3-32 clone, and this clone showed slower cell growth rate (14.6% of untreated cells), and aneuploid cells were detected possibly due to cellular senescence. These results showed that epigenetic reprogramming at the single cell level involves diverse cancer-related pathways, and this mechanism is likely to underlie the high efficacy of low-dose DNA demethylation therapy.

#839

Whole genome DNA methylation analysis of multiple myeloma identifies pervasive hypomethylation and biomarkers of survival.

Benjamin G. Barwick,1 Doris R. Powell,1 Daniel Penaherrera,2 Sheri Skerget,2 Jonathan J. Keats,2 Daniel Auclair,3 Sagar Lonial,1 Lawrence H. Boise,1 Paula M. Vertino1. 1 _Emory University, Atlanta, GA;_ 2 _Translational Genomics Research Institute, Phoenix, AZ;_ 3 _Multiple Myeloma Research Foundation, Norwalk, CT_.

Multiple myeloma is a malignancy of terminally differentiated, antibody secreting B cells known as plasma cells. Normal plasma cell differentiation and cell fate are coupled to epigenetic and transcriptional reprogramming, including a proliferation-dependent global loss of DNA methylation. However, relatively little is known about the epigenetic changes that underlie myelomagenesis and how these contribute to disease etiology. To this end, we have analyzed the DNA methylome of 119 myeloma specimens from the CoMMpass study (NCT01454297) by whole genome bisulfite sequencing (WGBS) and more than 90% of these same specimens were also characterized for structural variants (long-insert whole genome sequencing) and gene expression (RNA-seq). Unsupervised hierarchical clustering grouped together specimens with t(4;14) translocations that upregulate the H3K36 dimethyltransferase NSD2 (also known as MMSET and WHSC1), which likely impacts the DNA methylation state through epigenetic cross-talk. These data also revealed a dramatic genome-wide hypomethylation where myeloma samples had a median global CpG methylation level of 41% as compared to 71% and 89% in normal plasma cells and B cells, respectively. Demethylation of the myeloma methylome occurred in large megabase domains encompassing genes that were devoid of gene expression. Conversely, DNA methylation remained mostly unchanged in the bodies of genes that were highly expressed. Although the majority of these hypomethylated domains were common across myelomas, many regions of variable methylation exist and these differences corresponded with proximal gene expression differences. These variably methylated regions were compared to PFS and OS and this identified 6,314 CpG loci where the level of DNA methylation was prognostic of outcome (P≤0.00001). These loci were clustered into discrete regions and in the majority of cases (79%), reduced DNA methylation at these loci corresponded with poor outcome. For example, several loci in the gene bodies of PRKCE, MGMT, FHIT, WWOX were prognostic of poor survival. Interestingly, myeloma t(14;16) translocations disrupt the tumor suppressor WWOX and induce the oncogene MAF. These data suggest that WWOX expression may also be lost by epigenetic mechanisms. Finally, we analyzed the DNA methylome of primary and relapsed samples for 22 patients, including multiple relapsed samples for 2 patients. These data identified genome-wide DNA methylation remodeling with a median of 1.9 million differential methylated CpGs between the newly diagnosed and relapsed specimens. These relapse differentially methylated loci coincided at the same regions in several patients and significantly overlapped loci where the DNA methylation level was prognostic of outcome. Integrative genetic, epigenetic, and transcriptional analyses for 120 myeloma samples will be presented.

#840

Identification of novel colorectal tumor suppressor genes through genome-wide promoter hypermethylation analysis.

Sarah Bazzocco,1 Jose Higinio Dopeso,1 Águeda Martínez-Barriocanal,1 Estefanía Anguita,1 Rocio Nieto,1 Alex Sanchez,2 John M. Mariadason,3 Diego Arango1. 1 _Vall d'Hebron Research Institute, Barcelona, Spain;_ 2 _Universitat Autònoma de Barcelona, Barcelona, Spain;_ 3 _Olivia Newton-John Cancer Research Institute, Melbourne, Australia_.

Cancer initiation and progression are driven by both genetic and epigenetic changes. Although recent genome/exome sequencing efforts have significantly contributed to the thorough characterization of the genetic changes associated with the oncogenic process, further investigation is required to systematically identify the driver genes regulated by promoter hypermethylation. Using genome-wide analysis of the levels of promoter methylation (HumanMethylation27, Illumina) and the levels of mRNA expression (microarray analysis) in a panel of 30 colorectal cancer cell lines and 223 primary colorectal tumors (TCGA), we found a subset of 553 (5.6%) genes whose levels of promoter methylation showed a significant negative association with their expression. Higher overall methylation levels were associated with microsatellite instability (MSI), a CpG methylator phenotype (CIMP), faster proliferation and absence of APC mutations. Next, because genes that are epigenetically silenced could represent important drivers of the oncogenic process we investigated the role of the zinc finger transcriptional regulator ZNF238/ZBTB18, a gene silenced by promoter methylation, on the growth of colon cancer cells. Reintroduction of ZNF238 in HCT116 and HT29 colon cancer cells with low endogenous levels and promoter methylation of ZNF238, resulted in a significant reduction of cell proliferation both in vitro and in a subcutaneous xenograft NOD/SCID mouse model. Moreover, using immunohistochemical analysis with a validated antibody we found that ZNF238 is lost or reduced in the majority of the 133 primary colorectal tumors included in a tissue microarray, and that lower ZNF238 expression is associated with lymph node metastasis and shorter survival of patients with locally advanced colorectal cancer. In summary, we identified a set of 553 genes putatively silenced by promoter methylation in colorectal tumors that could significantly contribute to the oncogenic process. Moreover, as a proof of concept, we demonstrate that the epigenetically silenced gene ZNF238 has tumor suppressor activity and is associated with the survival of colorectal cancer patients.

#841

Meningioma subgroups associated with functional genomic elements defined by DNA methylation.

Tathiane M. Malta,1 James Snyder,1 Michael Wells,1 Ana deCarvalho,1 Laila Poisson,1 Camila Souza,1 Gelareh Zadeh,2 Kenneth Aldape,3 Daniela Tirapelli,4 Carlos Carlotti,4 Yan Lee,1 Steven Kalkanis,1 Tobias Walbert,1 Houtan Noushmehr1. 1 _Henry Ford Hospital, Detroit, MI;_ 2 _Princess Margaret Cancer Centre, Toronto, Ontario, Canada;_ 3 _Center for Cancer Research, National Cancer Institute, Bethesda, MD;_ 4 _Ribeirao Preto Medical School, University of São Paulo, Ribeirao Preto, Brazil_.

Although most meningioma are non-malignant, there is a high recurrence rate among atypical and anaplastic (malignant) meningiomas (grades II/III). In addition, malignant meningioma usually progresses after treatment. Recently, based on DNA methylation, two subgroups of meningioma were described with recurrence-free survival differences. Epigenetic deregulation at distinct genomic elements can affect changes in gene expression and alter the transcriptional profile of the cancer cells. We seek to understand the mechanisms of meningioma recurrence and progression after initial treatment. In order to address this, we will use DNA methylation data to identify candidate noncoding elements and their connection with genes that might explain differences in meningioma prognostic subgroups. Using published DNA methylation data we compared favorable and unfavorable meningioma subgroups and identified 3,045 differentially methylated probes (p< 0.0001, difference mean-methylation beta-value> 0.2). Focusing on probes within known functional genomics, we identified 18 highly conserved genomic enhancers known to be activated in cancer that can potentially drive meningioma recurrence. We next investigated links between these enhancers and their targeted genes by incorporating GeneHancer annotation. We found that the unfavorable subgroup of meningiomas presented hypomethylation within enhancer regions that have the potential to target PARK7, ARID4B, and FBH1. ARID4B was previously shown to be highly active in high-grade meningiomas. We also identified 16 enhancer regions that overlap known prognostic cancer enhancer, previously identified in other tumor types. Our findings were validated in independent cohort comprised by public and unpublished DNA methylation datasets. Our preliminary results are the first to suggest that DNA methylation changes can be used to identify noncoding regions associated with meningioma prognosis. Identification of noncoding regions associated with meningioma recurrence will provide knowledge of the role of epigenomics in the development of malignant meningioma and of opportunities for targeted therapy.

#842

Racial disparity in breast cancer: Tissue-specific epigenetic mechanisms.

Nan Lin,1 James Castle,1 Jinpeng Liu,1 Aditi Shendre,2 Chi Wang,1 Yunlong Liu,2 Chunyan He1. 1 _University of Kentucky, Lexington, KY;_ 2 _Indiana University, indianapolis, IN_.

Racial disparity in breast cancer is well recognized. Compared with European American (EA) women, African American (AA) women have higher mortality rate despite low incidence rate, and they are more likely being diagnosed at younger age and with features that are more aggressive. Many risk factors of breast cancer, including social, culture, behavioral, genetics, and environmental factors, are disproportionally distributed across two racial groups. These factors could influence DNA methylation differently, as suggested by recent reports that describe biological differences of breast tumor in different racial group. However, no studies have systematically evaluate tissue-specific epigenetic mechanisms underlying the observed racial disparity in breast cancer development. We conducted a molecular epidemiological study of 276 EA and 186 AA healthy women. Genome-wide DNA methylation profiling was performed in normal breast tissue using the Illumina Methyl Captured EPIC-sequencing. Genome-wide association between race and DNA methylation identified differentially methylated sites near genes implicated in metastatic potential and aggressiveness of tumor, tamoxifen-resistance, immune response, and cancer-related metabolic pathways. Many of these genes have not been reported previously to be associated with breast cancer or racial disparities. Further analyses showed that differentially methylated sites across two racial groups were significantly overlapped with differentially methylated sites in tumor and normal breast tissue, suggesting tissue- and racial-specific epigenetic mechanisms underlying breast cancer development. Furthermore, meQTL analyses between known breast cancer risk loci and DNA methylation also suggested AA-specific risk loci were associated with DNA methylation sites implicated in metastatic potential and aggressiveness of tumor. Our study sheds light on tissue-specific genetic and epigenetic mechanisms underlying racial disparity in breast cancer. Further research is warranted to validate the findings in larger studies and to understand gene regulation network by linking with RNA-seq data.

#843

Epigenomic disruption of adipogenic regulators in dedifferentiated liposarcoma.

Hironori Takamatsu,1 Naoko Hattori,1 Naofumi Asano,2 Naoko Iida,1 Akihiko Yoshida,3 Eisuke Kobayashi,3 Robert Nakayama,2 Morio Matsumoto,2 Masaya Nakamura,2 Akira Kawai,3 Toshikazu Ushijima1. 1 _National Cancer Center Research Institute, Tokyo, Japan;_ 2 _Keio University School of Medicine, Tokyo, Japan;_ 3 _National Cancer Center Hospital, Tokyo, Japan_.

Well-differentiated liposarcomas (WDLPS) and dedifferentiated liposarcomas (DDLPS) are the most common soft-tissue sarcomas in adults. They develop as a result of a disturbance in adipogenic differentiation however, the epigenomic changes underlying liposarcomagenesis are unknown. Here, we aimed to elucidate the role of epigenomic alterations in the initiation of DDLPS. Genome-wide DNA methylation profiles of 15 liposarcoma (6 WDLPS and 9 DDLPS) samples and 6 normal adipose tissue samples were obtained using the Infinium MethylationEPIC BeadChip. After grouping neighboring probes into genomic blocks (GBs, < 500 bp), principal component analysis of all the CpG GBs (n = 535,684) was conducted. Each of normal adipose tissue and WDLPS grouped closely while DDLPS distributed broadly, indicating heterogeneous methylation profiles among the DDLPS samples. Unsupervised hierarchical cluster analysis of enhancers (2,000 GBs with the high SD of 23,478 GBs) classified the samples into three tissue types, whereas that of promoters (1,000 GBs with the high SD of 8,668 GBs) could not classify the DDLPS samples distinctly. The Jonckheere-Terpstra trend test revealed 9,945 hypermethylated (p < 0.01, Δβ > 0.2) GBs in DDLPS compared to those in normal adipose tissues and WDLPS samples. Based upon a report on enhancers in adipocytes (Mikkelsen et al. Cell 143, 156-169. 2010), the hypermethylated GBs in DDLPS were enriched with typical- (2.2%: whole genome 1.2%) and super- (13.5%: whole genome 7.0%) enhancers. Genes involved in adipogenesis, including PPARG2, the master regulator of adipogenesis, and its target genes (FABP4 and PLIN1), were aberrantly methylated at the enhancers and repressed in DDLPS. Conversely, promoters were not enriched among hypermethylated GBs in DDLPS (1.2%: whole genome 4.1%). Taken together, these data indicate that aberrant methylation at the enhancers of adipogenic genes might be crucial for the generation of DDLPS. Evaluation of a demethylating agent in combination with a PPARγ agonist as a potential therapeutic is in progress.

#844

Integrated analysis of genetic and epigenetic alterations in gastric carcinomas.

Menghan Yang,1 Eri Arai,1 Hiromi Sakamoto,2 Hirohiko Totsuka,3 Hirokazu Taniguchi,4 Hitoshi Katai,4 Teruhiko Yoshida,2 Yae Kanai1. 1 _Keio University School of Medicine, Tokyo, Japan;_ 2 _National Cancer Center Research Institute, Tokyo, Japan;_ 3 _Hitachi Goverment and Public Sector System Ltd., Tokyo, Japan;_ 4 _National Cancer Center Hospital, Tokyo, Japan_.

Gastric carcinomas (GCs) have highly heterogeneous features in terms of clinical phenotypes, histological characters and molecular mechanisms. In our previous study based on Infinium HumanMethylation27 BeadChip, unsupervised hierarchical clustering analysis using genome-wide DNA methylation profiles of non-tumorous gastric mucosa (N) samples, being at the precancerous condition of GC, divided 110 GC patients into 3 subclusters. This epigenomic clustering based on DNA methylation profiles of N samples showed significant correlations with clinicopathological features of the corresponding carcinomas: For example, GCs belonging to Cluster B1 showed poorer differentiated histology, tumor aggressiveness and poorer outcome. To gain a further understanding of gastric carcinogenesis, we performed integrative analysis of genomics and epigenomics. Whole exome sequencing and single-nucleotide polymorphism microarray analysis were performed in 66 paired samples of GC and the corresponding N from the same patients of the previous epigenomic study. The datasets were analyzed by MutSigCV and GISTIC2.0 pipelines and significant accumulations of single nucleotide variation, insertion, deletion and copy number variation were identified within each of the epigenomic subclusters. Then we performed a pathway enrichment analysis using the genes genetically and epigenetically altered in each subcluster. Somatic mutation of the TP53, SMAD4, or ARID1A genes, which had been well known to occur in GCs, appeared with the same frequencies in 3 subclusters. On the other hand, a certain gene which has rarely reported before was recurrently mutated in our cohort. Epigenetic Cluster B1 was characterized by a fewer copy number alterations, and particularly by amplifications of 17q12, 10p11.1 and 3q26.2, including the genomic region of oncogene ERBB2. In integrated pathway enrichment analysis, p53-related pathways were enriched in Cluster A, and epithelial-mesenchymal transition (EMT)-related pathways were enriched in this particular Cluster B1. Poorly differentiated histological phenotype, i.e. loss of cell polarity or epithelial cell adhesion, may be attributable to molecular signature relating to EMT in Cluster B1. These data indicate that genetic and epigenetic alterations cooperate on gastric carcinogenesis thus impact on tumor aggressiveness. The consistency of molecular characteristics and pathological phenotypes suggests the possibility that future malignant progression can be predicted based on genetic and epigenetic alterations even at early stage of gastric carcinogenesis.

### Oncogenic Gene Dysregulation and Carcinogenesis

#845

c-MET nuclear transportation via membrane-bounded retrograde trafficking in breast cancer cells.

Mei-Kuang Chen, Yi Du, Linlin Sun, Jennifer L. Hsu, Yu-Han Wang, Yuan Gao, Jiaxing Huang, Mien-Chie Hung. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Reactive oxygen species (ROS) can be induced by anti-cancer treatments, such as ionizing irradiation and chemotherapy drugs. Hydrogen peroxide (H2O2) is one of the ROS species highly produced in cancer cells due to rapid proliferation or anti-cancer treatments. ROS serve as important intracellular messengers in cell growth and proliferation signaling pathways. However, ROS also causes cellular stress in cancer cells through oxidizing DNA, protein and lipids. Therefore, balancing ROS stress between signaling and damages at a tolerable level is important for cancer cell survival. c-MET is one of the receptor tyrosine kinases overexpressed in breast cancer. Full-length c-MET is known to be oncogenic for triggering signal transduction cascade from plasma membrane to upregulate cell proliferation and migration in cancer cells. Moreover, c-MET is reported to phosphorylate PARP1 under H2O2 stimulation, and the phosphorylated PARP1 resulting in PARP inhibitor resistance in breast cancer cells. However, it is not yet clear how c-MET travels from cell membrane to the nucleus. Here, we demonstrate that H2O2 induces retrograde transport of c-MET from plasma membrane into the nucleus and that knocking down either COPI or Sec61β attenuates H2O2-induced full-length nuclear c-MET accumulation. However, c-MET kinase inhibitor did not block the nuclear transport of c-MET, suggesting the transportation mechanisms is independent from kinase activity. Our results indicate that membrane-bound retrograde vesicles involve in membrane-to-nucleus transportation of c-MET in breast cancer cells. Moreover, nuclear c-MET interacts with both PARP1 and Ku proteins in breast cancer cells, suggesting the role of full-length nuclear c-MET in mediating repair of ROS-induced DNA damages.

#846

Progesterone receptor membrane component 1 controls cellular proliferation and plays a key role in the molecular circuitry of both ER positive and triple negative breast cancers.

Diego A. Pedroza, Ramadevi Subramani Reddy, Adriana Galvez, Rajkumar Lakshmanaswamy. _Texas Tech Univ. Health Sciences Ctr. El Paso, El Paso, TX_.

Introduction: The role of progesterone and its receptors in breast cancer progression continue to be studied but remain controversial. Progesterone membrane receptors with the ability to regulate kinase signals, mediating breast cancer proliferation have been demonstrated. Increased expression of the progesterone receptor membrane component 1 (PGRMC1), a heme - binding protein with the ability to interact and stabilize epidermal growth factor receptor (EGFR) is frequently found in breast cancer tissue. The basis of the signaling mechanisms of progesterone membrane receptors remain largely unknown. Both the nuclear and membrane progesterone receptors could play a significant role in the development and progression of breast cancers and both could become viable therapeutic options. We, aim to investigate the molecular circuitry of PGRMC1 in both ER-positive and Triple Negative Breast Cancers (TNBCs).

Materials and Methods: Human breast tissues were utilized to identify the expression of PGRMC1 along with a panel of normal and breast cancer cell lines. PGRMC1 overexpressing ZR-75-1 and MDA-MB-468 breast cancer cell lines were selected and treated with AG-205 (PGRMC1 inhibitor) and PGRMC1 siRNAs, while PGRMC1 was overexpressed in MCF10A non-malignant breast epithelial cells. MTS, qRT-PCR, Western blot, immunofluorescence, immunohistochemistry and flow cytometry were performed to study cell proliferation, apoptosis and key markers involved in these processes. In silico analysis utilizing publicly available gene expression datasets were also performed.

Results: Immunohistochemistry demonstrated strong staining for PGRMC1 in human breast cancer tissue compared to normal breast tissue. Increased PGRMC1 expression was observed specifically in ZR-75-1 and MDA-MB-468 cells by qRT-PCR, Western blot and immunofluorescence, these results were validated and compared to microarray-based gene expression analysis of breast cell lines and breast tumor data sets. Both AG-205 and PGRMC1 targeted siRNAs decreased cell proliferation in ZR-75-1 and MDA-MB-468. Minimal effects of AG-205 were observed in MCF10A non-malignant breast epithelial cells. AG-205 treatment and silencing of PGRMC1 induced apoptosis in both cancer cell lines. Furthermore, PGRMC1 overexpression transformed MCF10A cells into a malignant phenotype. Key markers of cell proliferation (pAKT, CCND1, pEGFR, pmTOR) and apoptosis (PTEN, Bcl2, Bax,) revealed that PGRMC1 inhibition decreases proliferation while overexpression promotes tumorigenesis.

Conclusion: Our data demonstrates that PGRMC1 plays a prominent role in both ER-positive and TNBCs. These initial findings uncover the potential of PGRMC1 as an oncogene and therapeutic target for breast cancer patients who overexpress this gene.

#847

Overexpression of long noncoding RNA FAM promotes cancer invasion and drug resistance in human liver cancer.

Po-Shuan Huang, Kwang-Huei Lin. _Chang-Gung University, Taoyuan County, Taiwan_.

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies worldwide. Most patients with HCC have an established background of chronic liver disease and cirrhosis, with major etiological and risk factors including chronic infection with hepatitis B virus (HBV) and hepatitis C virus (HCV). Long non-coding (lnc)RNAs are a class of non-protein coding transcripts longer than 200 nucleotides that regulate complex cellular functions, such as cell growth, differentiation, metabolism and metastasis. Increasing evidence has shown that lncRNAs regulate gene expression by targeting the production, splicing, decay, or translation of target mRNAs. Although deregulation of lncRNAs expression have been detected in HCC or many tumor types, there are still many novel lncRNAs have not been studied and the mechanisms underlying functional impairment and specific involvement of lncRNAs during hepato-carcinogenesis remain to be established. Here, we aimed to investigate the involvement of specific dys-regulated lncRNA-FAM and its molecular mechanism. FAM is overexpressing in HCC and correlates with tumor size, vascular invasion, and pathology stage. Overexpression FAM accelerates cell proliferation, invasion in HCC cells. Further, FAM is induced in the Doxorubicin (DOX) resistance HCC cells show in the Gene Expression Omnibus (GEO) Datasets analysis. Overexpression FAM did increase DOX-resistant in two HCC cell lines. After overexpression FAM, lysosome-associated membrane protein 2 (LAMP2) expression was enhanced which also correlated with DOX-resistant. From the co-immunoprecipitation analysis, FAM directly interacted with LAMP2 to protect its ubiquitination. Thus, FAM may play an oncogenic role during hepato-carcinogenesis.

#848

Hyper O-GlcNAcylation plays an oncogenic role in hepatocellular cancer.

Yongzhan Nie,1 Yi Chu,1 Daiming Fan,1 Mingzuo Jiang,1 Nan Wu,2 Hongwei Li1. 1 _Fourth Military Medical University, Xi'an, China;_ 2 _Lab of Tissue Engineering, Faculty of Life Science, Northwest University, Xi'an, China_.

Background: It is universally accepted that change of specific proteins is of great significance in the development of tumor and post translational modification (PTM) is a major way to modify protein activity. O-GlcNAcylation,as one of post translational modifications of proteins regulated by OGT and OGA, has linked glucose metabolism disorders with the malignancies. Numerous studies have demonstrated the aberrant activation of O-GlcNAcylation and OGT in tumors. The aim of this study is to investigate the potential role and underlying mechanisms of O-GlcNAcylation in HCC progression.

Methods: The level of O-GlcNAcylation was detected by IHC staining and western blot. Function of O-GlcNAcylationin HCC proliferation was investigated by CCK8, BrdU, colony forming, cell cycle assay and subcutaneous xenograft tumor model. Scratch healing, transwell assay and 3D spheroid invasion assay were employed for testing metastatic ability of cells. Moreover, Mass-spectrum and co-IP assay were performed to explore novel OGT binding proteins.

Results: Total O-GlcNAcylation level of proteins was markedly overexpressed in HCC compared to adjacent normal tissues and the upregulation of O-GlcNAcylation was associated with poor outcome of patients. The results of in vitro and vivoexperiments showed consistently that high level of O-GlcNAcylation could promote HCC proliferation and metastasis. A large number of studies show that the O-GlcNAcylation of key protein is the crucial step for O-GlcNAcylation regulation in tumor. We examined the mass spectrum results of HCC cell line IP-OGT samples and found 214 eligible candidates. Then GO annotation and the KEGG pathway analysis was employed to try to find targets within the tumor related range. Among the potential candidates which could related to tumor transcription misregulation, we find out that SIX1 which could regulate a series of downstream oncogenic factors and glycolysis related genes including c-myc, cyclin D1, GLUT1 and ENO1 to be a possible effect factor of O-GlcNAcylation. The further data demonstrated that SIX1 was modified by O-GlcNAcylation, and O-GlcNAcylation could maintain the high expression status of SIX1 by inhibiting its

ubiquitination degradation.

Conclusions: In conclusion, our data suggest that O-GlcNAcylation significantly increased in HCC compared to normal tissues. The high expression of O-GlcNAcylation could be an independent prognostic factor in HCC and was associated with poor outcome of patients. Further, we find out SIX1 was the underlying executor of O-GlcNAcylation in HCC. By inhibiting SIX1 ubiquitination, O-GlcNAcylation could maintain SIX1 and its downstream factors in a high level persistently and therefore promote HCC progression. Here in our study, we for the first time identified a neo-posttranslation O-GlcNAcytion modification pathway in HCC, which could provide potential therapy for HCC treatment.

#849

Metformin enhances trastuzumab efficacy for HER2 + gastric cancer cells.

Mi Young Kim,1 Jin Hyun Park,1 Sungyoul Hong,2 Jin-Soo Kim1. 1 _Seoul National University Boramae Medical Center, Seoul, Republic of Korea;_ 2 _Seoul National University College of Pharmacy, Seoul, Republic of Korea_.

Background: Trastuzumab (Tmab) is an effective monoclonal antibody against advanced gastric cancers (GCs) with HER2 amplification. In TCGA dataset, high mTOR activity was observed in proliferative cluster by RPPA analysis, and almost half of HER2 amplified GC showed increased mTOR expression. Metformin is a widely used antidiabetic drug and an activator of AMP kinase, which could affect mTOR pathway signaling. Here, we describe a therapeutic synergism between Tmab and metformin in HER2+ GC cells.

Material and Methods: The following GC cells were evaluated: HER2 -positive (HER2+) and Tmab -sensitive NCI-N87, YCC-19, YCC-38 and OE19, HER2+ Tmab -resistant OE33 and HER2 negative AGS. We assessed cytotoxic response of these GC cells to Tmab alone or in combination with metformin by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay and anchorage independent growth in soft agar. MGI (mean growth inhibition) was calculated by dividing the colony number of the treated group by that of the untreated control group. Western blots were performed to assess the cellular activity of Tmab and/or metformin in HER2 signal pathway. GC cells were subjected to the Proteome Profiler Human Phospho-RTK Array Kit (R&D Systems) to evaluate the relative phosphorylation of human receptor tyrosine kinases in GC cells after Tmab and/or metformin exposure. Xenograft studies were performed to evaluate in vivo therapeutic efficacy of trastuzumab and/or metformin with HER2+ human GC cells. The statistical significance of differences were determined by using the Kruskal-Wallis test.

Results: Metformin induced phosphorylation of AMP kinase in all GC cells tested and dephosphorylation of mTOR in Tmab sensitive GC cells. We observed that treatment with Tmab in combination with metformin induced a statistically significant decrease in the number of colonies formed in soft agar by N87, YCC19, YCC-38 and OE19 cells, as compared to the numbers formed by control cells or cells in the single-treatment groups (Kruskal-Wallis test, p < .01). No growth inhibitory effect was detected in OE33 cells with compared to trastuzumab alone. The combination of metformin resulted in decreased phosphorylation of HER2 and downstream targets, such as AKT or ERK, in trastuzumab-sensitive HER2+ cells. Phospho RTK arrays showed that the synergistic decrease of phosphorylation of EGFR, HER2 and HER3 with Tmab and metformin. Furthermore, the combination of Tmab and metformin exhibited enhanced antitumor effects in a xenograft model with N87 cells.

Conclusion: Collectively, these data support that trastuzumab in combination with metformin efficiently suppresses the growth of HER2-positive GC cells. Moreover, metformin synergizes with trastuzumab to induce the therapeutic effect on the HER2+GC cells. Further studies are warranted to test this concept in clinical setting.

#850

Evaluating preclinical efficacy of anti-HER2 drug combinations using ER+/HER2 mutant models.

Shyam M. Kavuri. _Baylor College of Medicine, Houston, TX_.

Vaishnavi Devarakonda1,2, LaTerrica Williams1,2, Sinem seker1,2, Jonathan T. Lei1,2, Purba Singh1,2, Airi Han1,2, Meenakshi anurag1,2, Kimberly R. Holloway1,2, Alana L. Welm3, Matthew. J. Ellis1,2*, Shyam. M. Kavuri1,2* 1Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX, USA 2. Department of Medicine, Baylor College of Medicine, Houston, TX, 77030, USA 3. Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA.

Targeting HER2 is one of the greatest successes in oncology, and has resulted in the generation of a wide array of HER2-targeting agents. Our genomic approaches are revealing other mechanisms of HER2 activation, such as our discovery of activating HER2 mutations in different cancer types. From initial breast cancer and SUMMIT trial data, the pan-HER drug neratinib as monotherapy showed initial clinical response in ER+ breast cancer, but with frequent early relapse. This study investigates the preclinical efficacy of anti HER2 agents alone or in combination with endocrine therapy agents or in combination with CDK4/6 inhibitors using ER+/HER2 mutant cell lines and ex vivo HER2 mutant patient derived xenograft (PDX) model. ER+ breast cancer cell lines (T47D and MCF7) stably expressing HER2V777L, and ER+/HER2 mutant PDX model (HER2G778_P780 dup) were used to examine HER2 signaling. We found that MCF7/T47D cells expressing HER2V777L and HER2G778_P780 dup PDX tumors showed strongly activated autophosphorylation of HER2 and increased expression of CDK4, CDK6, phospho-Rb, and cyclin D1 as compared to MCF7/T47D cells expressing HER2WT or ER+/non-HER2mut PDX model, suggesting that HER2 mutations preferentially depend on CDK4/6 signaling for cell growth. Additionally, we showed that activating MCF7 HER2 V777L cause resistance to endocrine therapy treatment (fulvestrant IC50 >5μM). Further, we show that neratinib alone is effective at higher concentrations (IC50 < 2μM) in MCF7/HER2 V777L cells. Next we showed that abemaciclib alone exhibited moderate activity against MCF7 HER2 V777L cells (IC50 < 0.4μM) and additional activity in combination with neratinib (IC50 < 0.06μM) was seen. Moreover, ex vivo HER2 G778_P780 dup cells are relatively resistant to fulvestrant alone (IC50 < 0.2μM), neratinib alone (IC50 < 0.006μM), abemaciclib alone (IC50 < 0.04μM), and neratinib in combination with abemaciclib (IC50 < 0.005μM), suggesting that patients harboring ER+/HER2-mutant tumors may benefit from neratinib in combination with abemaciclib. Therefore, we propose that simultaneous targeting of both HER2 and the CDK4/6 axis is required for maximal inhibition of ER+ breast cancers harboring HER2 activating mutations.

#851

MicroRNA-124 modulated collagen-prolyl hydroxylase P4HA1 expression regulates colon cancer progression.

Sumit Agarwal, Balabhadrapatruni V. Chakravarthi, Michael Behring, Hyung-Gyoon Kim, Kevin Hale, Abduljalil M. Alsubaie, Sameer Al Diffalha, Shajan P. Sugandha, Sooryanarayana Varambally, Upender Manne. _University of Alabama at Birmingham, Birmingham, AL_.

In the US, colon adenocarcinoma is the third most common cause of cancer-related deaths. Since various genetic and epigenetic alterations contribute to progression of this disease, it is necessary to evaluate these molecular events as markers of progression and as potential druggable targets to develop therapeutics. Analysis of publicly available TCGA transcriptome sequencing data using UALCAN, a bioinformatics web portal (http://ualcan.path.uab.edu/), identified in colon adenocarcinoma, overexpression of collagen prolyl-4-hydroxylase α subunit 1 (P4HA1), an enzyme involved in collagen metabolism. P4HA1 changes the composition of the extra cellular matrix by hydroxylating proline residues in collagen. We validated this in silico finding by measuring P4HA1 RNA expression levels in paired normal (n=105) and tumor (n=105) frozen samples by qRT-PCR. Our investigations showed overexpression of P4HA1 in colon cancers compared to their paired normal colonic epithelial tissues. Expression of P4HA1 was independent of pathologic stage; tumor histologic type; and the race/ethnicity, age, and gender of patients. Furthermore, Western blotting and immunohistochemical analyses showed elevated expression of P4HA1 protein in colon tumor tissues as compared to normal colonic epithelial specimens. To assess the functional role of P4HA1 in colon cancer cells, we performed in vitro and in vivo analyses using RNA interference. The results showed that depletion of P4HA1 led to reduced cell growth and cell invasion, and to less tumor growth in xenograft models. Bioinformatics analysis indicated that microRNA miR-124, a tumor suppressor, could be targeting P4HA1. Forced overexpression of miR-124 in colon cancer cells reduced P4HA1 expression and diminished the malignant phenotypes of colon cancer cells, confirming that, in these cells, miR-124 targeted P4HA1. Further, treatment of the cells with diethyl-pythiDC (100 uM), a small molecule inhibitor of P4HA1, reduced their malignant phenotypes. In sum, the present investigations show that overexpression of P4HA1 is involved in progression of colon adenocarcinomas and that it is a potential therapeutic target that is amenable to small molecule inhibition.

This work was partially supported by a NIH grant (3U54CA118948) and by the ELKUS foundation grant to Dr. Manne.

#852

Determining the roles of ERO1-alpha and ERO1-beta in progression of multiple myeloma and drug resistance.

Weichih Chen, Lori Hazlehurst. _West Virginia University, Morgantown, WV_.

Multiple myeloma (MM) is a hematological malignancy characterized by dysregulated growth and accumulation of monoclonal plasma cells within the bone marrow. It is a treatable but incurable disease. Despite improvement in progression-free and overall survival of MM patients nearly all MM patients will relapse and become resistant to current treatments. Therefore, to find an effective therapeutic target that is applicable to MM, our laboratory analyzed a retrospective study, patients with relapsed MM who had received bortezomib therapy were enrolled in phase 2 and phase 3 clinical trial. Patients who had higher ERO1-alpha expression demonstrated significant poorer outcomes as compared to patients with lower ERO1-alpha expression. Moreover, patients with relapsed myeloma had higher expression of ERO1-alpha protein levels compared to newly diagnosed MM patients, suggesting that ERO1-alpha correlated with disease progress and poor survival of MM patients. Disulfide bond formation is critical for the folding, stability and function of newly synthesized secretory and membrane proteins in endoplasmic reticulum (ER). Endoplasmic reticulum oxidoreductase 1 (ERO1) is an essential component in this process. ERO1-alpha and ERO1-beta are two flavoprotein isomers that have been identified in mammalian cells. ERO1-alpha is widely expressed and up-regulated during oxidative stress and ERO1-beta is only expressed in select tissue and induced by unfold protein response (UPR). We found that knockdown of ERO1-alpha MM cells caused cell growth inhibition and decreased colony formation and lambda secretion. Moreover, overexpression of ERO1-alpha enhanced cell growth. Additionally, reducing the expression of ERO1-alpha resulted in upregulated ERO1-beta by transcriptional and translational levels. Reducing the expression of ERO1-beta in MM cells increased cell growth and induced resistance to current treatments, including the proteasome inhibitor (bortezomib), alkylating agent (melphalan), corticosteroids (dexamethasome) and histone deacetylase inhibitor (panobinostat). We found reducing the expression of ERO1-beta resulted in decreased expression of glucocorticoid receptor (GR). Taken together, our data indicate that ERO1 -alpha and -beta are not functionally redundant as increased alpha expression correlates with MM progression while increased beta expression correlates with increased sensitivity to chemotherapeutic agents. Future studies are aimed at delineation of mechanism underlying the divergent phenotypes that result from reducing ERO1- alpha or -beta in MM cells.

#853

Essential role of autocrine FGF19-FGFR4 signaling in head and neck tumorigenesis.

Liwei Lang,1 Lixia Gao,1 Chloe Shay,2 Xiangdong Zhao,1 Austin Shull,3 Yong Teng1. 1 _Department of Oral Biology, Georgia Cancer Center, Augusta Univ., Augusta, GA;_ 2 _Emory Children's Center, Atlanta, GA;_ 3 _Presbyterian College, Clinton, SC_.

The fibroblast growth factor 19 gene FGF19 has been reported to be amplified in several cancer types and encodes for a key autocrine signaler known to promote tumorigenic growth. Thus, it is imperative to understand which cancers are oncogenically addicted to FGF19 amplification as well as the role it serves in these cancer types. We report for the first time that high FGF19 amplification in head and neck squamous cell carcinomas (HNSCC) is associated with increased autocrine secretion of FGF19 and poor patient outcome in HNSCC. FGF19 amplification corresponded with constitutive activation of FGFR4-dependent ERK/AKT-p70S6K-S6 signaling, and addition of human recombinant FGF19 could promote proliferation and soft agar colony formation in HNSCC cells with low FGF19 expression through activating FGFR4 and downstream signaling cascades. Consistently, FGF19 knockout counteracted the observed effects in HNSCC cells carrying high endogenous FGF19, and significantly suppressed tumor growth in an orthotopic mouse model of HNSCC. This study demonstrates that FGF19 gene amplification corresponds with an increased dependency upon FGF19/FGFR4 autocrine signaling in HNSCC, revealing a therapeutic target for this cancer type.

#854

Combination effect of low dose Cytarabine and pan-RAF inhibitor LY3009120 in AML cells with RAS mutations.

Jihyun PARK, Sung-Soo Yoon, Youngil Koh. _Seoul National University, Seoul, Republic of Korea_.

The alterations of RAS oncogenes were known to be involved in various cancers. In acute myeloid leukemia (AML), there are 6% of alterations in KRAS and 13% in NRAS. The RAS/RAF/MEK/ERK cascade has roles in promoting hematopoietic cell growth, inhibiting apoptosis, and is related to chemotherapy resistance. Fortunately, the recent advance in pharmacology suggests the advent of potentially efficacious RAS inhibitors. However, current RAS inhibitor development is focused on solid tumors. We, therefore, tried to identify if potential RAS targeting molecule would be effective in hematologic malignancies. Here we focused on the combination of LY3009120, a pan-RAF inhibitor that targets RAF with widely used antimetabolite agent cytarabine on AML cell line.

We used AML cell lines, HL-60, NB-4 which have NRAS/KRAS mutation and KG-1 as RAS wild-type control cell line. In the cytotoxicity test, we first performed a single effect of cytarabine and LY3009120 in the AML cell lines using cytotoxicity assay. Based on the single treat experiment, cytarabine and LY3009120 were simultaneously treated on three AML cell lines at the indicated concentration. Above all, there was a strong synergistic effect on combination treat of cytarabine containing LY3009120 in HL-60 (56.8%, p=0.0001) and NB-4 (66%, p<0.0001) compared with KG-1 cell line (8%).

In order to examine the changes in RAF/MAPK pathways, we demonstrated western blotting using antibodies to RAS, RAF, MAPK signaling molecules. In HL-60, the phosphorylation of MEK was slightly increased by the single treat of cytarabine or LY3009120. However, the combination treatment of cytarabine and LY3009120 decreased the phosphorylation of MEK. And more, in NB-4, the phosphorylation of ERK (T202/Y204) was not affected by single treatment while the combination treatment decreased ERK phosphorylation at low concentration of LY3009120. Furthermore, we investigated the underlying mechanisms of the combined effect, PARP protein cleavage indicating cell death was examined. PARP cleavage was observed in HL-60 and NB-4 after treatment, whereas KG-1 did not show any change in PARP after treatment.

In conclusion, we show the synergistic effect between cytarabine and LY3009120 on AML cell lines which have the mutation on RAS gene. Even at the low concentration of cytarabine, combining LY3009120 significantly increases the anti-cancer effect. With our data, we suggest that novel therapeutic strategies for AML patients with RAS mutation.

#855

Analysis of a novel variant of HER2 detected by clinical sequencing in cancer of unknown primary.

Yohei Harada, Tomomi Kashiwada, Akemi Sato, Tomonori Abe, Chiho nakashima, Eisaburo Sueoka, Shinya Kimura, Naoko Sueoka-Aragane. _Saga University, Saga, Japan_.

A patient with cancer of unknown primary underwent an NGS-based multiplex gene assays (OncoPrime). As a result, a novel variant of HER2 (E401G) which is a missense alteration located in the extracellular domain of the HER2 protein was detected. This alteration has not been reported (COSMIC, Nov 2018) and its effect on protein function is unknown. Sanger sequencing using genomic DNA from peripheral blood sample showed that the variant was a somatic variant. HER2 E401G variant was confirmed by Sanger sequencing using genomic DNA extracted from the formalin-fixed paraffin-enbedded tumor tissues. In spite of somatic variant, the variant allele frequency was relatively high (55.8%). This result suggested the amplification of HER2 gene, FISH analysis confirmed the copy number gain of HER2 gene (21 copies). The prediction tools in silico (SIFT, PolyPhen-2 and PROVEAN) suggested pathogenicity of the variant, so we are now performing the functional analysis of the mutant HER2 protein. Many variants of unknown significance have been identified through clinical sequencing, establishing a database of integrated information of variant is essential to implement precision medicine of oncology. This study was approved by the Ethics Committee and the patient provided written informed consent for the use of genomic and clinical data for research purposes.

#856

N terminal glycosylation regulates FGFR4 cleavage in cholangiocarcinoma.

Andrew J. Phillips, Ashley M. Mohr, Mary Anne Phillippi, Justin L. Mott, Keith R. Johnson. _University of Nebraska Medical Center, Omaha, NE_.

Cholangiocarcinoma (CCA) is a terminal diagnosis, with 5-year survival rates averaging less than 10%. Thus, developing therapeutic options for cholangiocarcinoma patients is essential. FGFR4 is a heavily N-glycosylated receptor tyrosine kinase that signals through Akt, ERK and STAT3 pathways, and whose overexpression in cancer has previously been observed. The activation and signaling through FGFR4 involve the intracellular tyrosine kinase domain that is activated upon ligand binding the extracellular receptor region. Examination of expression in the TCGA database and our studies in CCA cells demonstrated a role for FGFR4 in promoting tumor cell survival and proliferation. Further, we have observed the presence of a proteolytic cleavage product of FGFR4, that is comprised of the intracellular kinase domain, which we call R4-ICD. Our data and previous studies on recombinant protein demonstrated that the intracellular kinase domain in the absence of the transmembrane and ligand-binding domains is constitutively active. HuCCT-1 cells (human CCA cell line lacking endogenous FGFR4 expression) stably transfected to express R4-ICD had increased proliferation and pro-survival phenotypes when compared to the parental cell line. Of 14 frozen CCA human tumor samples, all showed expression of R4-ICD. Compared to normal liver tissue, R4-ICD expression was greater in 65% (9 of 14) of tumor samples than in normal liver. In addition, total FGFR4 expression was lower in normal cholangiocytes than human CCA samples. Full length FGFR4 expression was observed in 12 tumor samples (85%). Of note, full-length FGFR4 expression levels were far weaker than R4-ICD expression in the 10 samples that showed both signals. Because R4-ICD is produced from full length receptor, we interpret the predominance and higher prevalence of R4-ICD to reflect efficient processing to a stable intracellular kinase fragment. We demonstrated at least two glycoforms of FGFR4, terminal and core, in cell lines, and these were also observed in human tumors. We hypothesized that glycosylation may regulate R4-ICD proteolytic production. Live CCA cells in culture were treated with PNGase F to remove extracellular N-linked sugars from cell surface proteins, including FGFR4. Deglycosylation of FGFR4 was confirmed by immunoblotting. Upon deglycosylation, R4-ICD levels rapidly increased. In summary, CCA cells in culture and in tumors were demonstrated to have high levels of R4-ICD, a constitutively active kinase that promoted tumor cell survival and proliferation. The stable cleavage product of FGFR4, R4-ICD, was increased on receptor deglycosylation. Whether aberrant FGFR4 glycosylation contributes to R4-ICD predominance in tumor samples or to tumor aggressiveness remains to be tested. Further understanding of R4-ICD formation will allow us to better target CCA, possibly by reducing proteolytic processing in conjunction with kinase inhibition.

#857

Identification and functional analysis of FGFR2 binding proteins in diffuse-type gastric carcinoma.

Takuya Shirakihara, Ryuichi Sakai. _Kitasato University, Kanagawa, Japan_.

Diffuse-type gastric carcinoma (DGC) is highly invasive subtype of gastric adenocarcinomas and frequently exhibit scattered peritoneal metastasis. Previous studies have shown that genes of receptor tyrosine kinases (RTKs) such as FGFR2 or Met are amplified in DGC cell lines with high frequency. Hence, these cells exhibit oncogene addiction to sustained activity of these RTKs for maintenance of malignant phenotype. In order to gain novel insight in the downstream signaling pathway of DGC-specific RTKs, phosphoproteomic analysis was performed. Phosphotyrosine-containing proteins associated with RTKs were purified through two sequential rounds of immunoprecipitation from lysates of RTK-amplified DGC cell lines. Here we will show the initial functional data of transferrin receptor (TFRC), one of the identified proteins by MS analysis. In the DCG cells with FGFR2 amplification, TFRC proteins were found to bind FGFR2 and were phosphorylated at Tyr20. Moreover, shRNA knockdown of TFRC as well as inhibitor of FGFR2 revealed effective impairment in cell proliferation. These results suggest that TFRC has essential roles in malignancy of FGFR2-dependent DGC.

#858

Oncogenic EGFR mutations as genomic biomarkers for cetuximab and panitumumab response in colorectal adenocarcinoma.

Nayoung Kim, Jeonghee Cho. _Dankook university, Cheonan-si, Republic of Korea_.

Somatic mutations of Epidermal Growth Factor Receptor (EGFR) occur in ~3% of colorectal cancer patients. Here, through systematic functional screening of 21 recurrent EGFR mutations selected from public datasets, we show that 11 colon cancer-derived EGFR mutants; G63R, E114K, R165Q, R222C, S492R, P596L, K708R, E709K, G719S, G724S and L858R, are oncogenic, and able to transform cells in a ligand-independent manner. We demonstrate that cellular transformation by these mutants requires receptor dimerization. Importantly, the EGF-induced and constitutive oncogenic potential of these EGFR mutants are inhibited by cetuximab or panitumumab in vivo and in vitro.

Taken together, we propose that a subset of EGFR mutations can serve as genomic predictors for response to anti-EGFR antibodies and that metastatic CRC patients with such mutations may benefit from these drugs as part of the first-line therapy.

#859

Basal Notch4 activation is a druggable marker of aggressiveness in a panel of human cancer cell lines.

Lucile Astorgues-Xerri,1 Matthieu Martinet,1 Eric Raymond,2 Sandrine Faivre,3 Annemilaï Tijeras-Raballand1. 1 _AFR Oncology, Paris, France;_ 2 _Saint-Joseph Paris Hospital, Paris, France;_ 3 _Beaujon University Hospital, Clichy, France_.

Background: Notch pathway has been involved in cell differentiation, proliferation, apoptosis, angiogenesis and drug resistance, as well as in epithelial-to-mesenchymal transition. Notch pathway activation leads to a proteolytic cleavage, releasing the Notch intracellular domain (NICD), which translocates to the nucleus and activates target genes, such as HES1 (interacting with Notch for maintenance of stem cells and progenitor phenotype). Our team has demonstrated the involvement of PlGF/VEGFR1/Notch4 axis in the angiogenesis of hepatocellular carcinoma, but what about the role of Notch in other cancer types? The aim of this work is to characterize the basal activation and the role of Notch4 in a panel of human cancer cell lines.

Materials and Methods: We characterized a panel of 8 pancreatic (PDAC), 8 head and neck (H&N), 5 colorectal (CRC), 5 cholangiocarcinoma (CK), and 10 hepatocellular (HCC) human carcinoma cell lines for intracellular domain of the notch protein 4 (NICD4), HES1, E-cadherin, and Vimentin expression by Western Blot. In each tumor type, cell lines with high and low Notch4 activation were selected to assess basal cell proliferation and migration, using MTT and wound-healing assay, respectively. In high versus low Notch4 cell lines, we assessed NUMB expression (involved in NICD proteasomal degradation) and the effect of a Notch inhibitor, PF- 03084014.

Results: Notch4 activation assessed by NICD4 expression was observed in 6 out of 8 (6/8) PDAC, 7/8 H&N, 2/5 CRC, 4/5 CK, and 5/10 HCC cell lines. In PDAC cells, Notch4 activation was correlated with high Vimentin expression. In PDAC-, H&N-, CRC-, and CK-low NICD4 cells, we observed an overexpression of NUMB, suggesting a correlation between low Notch4 activation and proteasomal degradation. In most tumor types, Notch4 activation was associated with an increased proliferation rate and basal migration. PF-03084014 had pronounced antiproliferative effects in high NICD4 cells in comparison to low NICD4 cells, except for HCC. In H&N cells, PF-03084014 displayed no effect on Notch4 activation. We also observed a decrease in HES1 expression (significant in high NICD4 cells) and an increased in AKT phosphorylation (significant in low NICD4 cells). We will further analyze the effect of PF-03084014 on cell signaling and migration in other tumor types to display the results at the conference.

Conclusions: In this study, we demonstrated a correlation between high Notch4 basal activation and increased proliferation/migration in a panel of human cancer cell lines, as well as higher sensitivity to the Notch inhibitor PF-03084014. In highly sensitive H&N cell lines, PF-03084014 inhibited Notch signaling pathway, whereas we observed an activation of AKT survival pathway in the low-sensitive ones. Since Notch inhibition is an interesting topic for antitumor therapy, this study could help to select tumor types that could be good candidate for Notch inhibition in the clinic.

#860

**Solution structure of a dGMP fill-in G-quadruplex forms in PDGFR-** b **promoter.**

Kaibo Wang, Jonathan Dickerhoff, Guanhui Wu, Clement Lin, Danzhou Yang. _Purdue University, West Lafayette, IN_.

Platelet-derived growth factor receptor beta (PDGFR-β) is an important cell-surface-receptor tyrosine kinase implicated in PDGF signaling pathways. The PDGFR-β signaling pathway has been demonstrated as an essential component in control of the cellular growth, proliferation, survival, motility and differentiation. Overexpression of PDGFR-β is associated with tumor growth, angiogenesis, and migration, making PDGFR-β an attractive target for anticancer therapeutics. In the PDGFR-β gene promoter, a highly GC-rich proximal promoter region -165 to -139 nt upstream of the transcriptional start site is crucial for basal promoter activity, which can form G-quadruplexes (G4s). These structures can be stabilized by the G4-interactive molecules to inhibit the PDGFR-β transcriptional activity. Our previous structure study showed that the G4 structures formed in the PDGFR-β gene promoter always contain a broken G-strand. The broken-strand structure suggests a possibility of the formation of a G-vacancy-bearing G-quadruplex (GVBQ) structure, which can be complemented and stabilized by guanine and guanine derivatives. Elevated levels of guanine and guanine derivatives/metabolites are found in tumor cells, suggesting that the GVBQs may play important regulatory roles in tumor cell pathological processes, such as PDGFR-β transcriptional regulation. Using NMR, CD, EMSA and DMS footprinting experiments, herein, we contributed the first atomic resolution structure of a dGMP fill-in G4 formed in PDGFR-β gene promoter in potassium solution. Surprisingly, our results showed that solution dGMP preferentially inserts from the 5' end tetrad instead of the G-22 position of the previously determined structure, and maybe in dynamic equilibrium with our previously reported G-quadruplex. The dGMP, like other intramolecular guanines, that forms four Hoogsteen hydrogen bonds with adjacent guanines and stabilizes by electronic coordination with potassium cation as well as the π-π stacking interaction with the middle tetrad guanine. Moreover, a series of guanine derivatives were investigated to fill in this GVBQ, and found that only these derivatives that keep the ability to form Hoogsteen hydrogen bonds in G-tetrad can serve as the substrate, providing insights into designing new guanine derivatives anticancer drugs.

#861

Precise excision of an oncogenic allele of tumor by Adenovirus-based CRISPR/Cas9 system induces tumor regression.

Taeyoung Koo,1 A-Rum Yoon,2 Sangsu Bae,2 Jin-Soo Kim,1 Chae-Ok Yun2. 1 _Center for Genome Engineering, Institute for Basic Science, Seoul, Republic of Korea;_ 2 _Hanyang Univ., Seoul, Republic of Korea_.

Approximately 15% of non-small cell lung cancer patients possesses several mutations in the epidermal growth factor receptor (EGFR) gene, which plays a critical role in tumor progression. To this end, we demonstrate that an adenovirus-mediated co-delivery of Cas9 and a guide RNA targeting oncogenic EGFR mutation commonly found in lung cancer can selectively remove oncogenic allele while being inactive against wild-type EGFR allele. An EGFR mutant harboring a single-nucleotide missense mutation (CTG to CGG) was targeted as this mutation leads to generation of protospacer-adjacent motif sequence recognized by the Cas9 protein of S. pyogenes. The CRISPR/Cas9-mediated excision of mutant EGFR allele with high indel rate led to potent lung cancer cell killing. Importantly, intratumoral administration of Ad-based CRISPR/Cas9 system led to potent tumor growth inhibition and complete tumor regressions in number of mice harboring EGFR mutant xenograft tumors. Collectively, our findings show that precise and controlled excision of oncogenic mutation by Ad-mediated expression of CRISPR/Cas9 system offers a powerful genomic surgical strategy to excise oncogenic mutations to treat cancers.

#862

Inhibition of Hippo tumor suppressors MST1/2 slows prostate cancer cell proliferation and invasion.

Amelia Schirmer,1 Alexis B. Stokes,2 Erick J. Maravilla,3 Weiwei Fu,4 Everardo Macias3. 1 _Duke University, Durham, NC;_ 2 _Auburn University, Auburn, AL;_ 3 _Duke University Medical Center, Durham, NC;_ 4 _Affiliated Hospital of Qingdao University, Qingdao, China_.

Serine/threonine kinase 3 (STK3) and its paralog STK4, encode MST2 and MST1 kinases respectively, which are essential members of the highly conserved Hippo Tumor suppressor pathway which regulate Yes 1 Associated protein (YAP1). In normal cells YAP1 activation is beneficial for organ and tissue regeneration, with overgrowth being controlled by MST1/2. In cancer cells, MST1/2 play a role in cancer cell proliferation, differentiation and apoptosis by inhibiting YAP1 , yet frequent deregulation of MST1/2 leads to hyper-activation of YAP1 in various cancers. Th current dogma is that STK3/4 are tumor suppressor genes. Yet, STK3, but not STK4 in prostate cancer (PC) is frequently amplified. The goal of this study was to determine if STK3 and/or STK4 gene products MST1/2 play a role in PC growth and/or metastasis. Approach. To assess if MST1 and/or MST2 play a role in PC proliferation, pharmacological and genetic studies were carried out across varying PC cell lines in vitro. XMU-MP-1 a narrow spectrum kinase inhibitor of MST1/2 was used across five distinct PC cell lines. We utilized 2D proliferation and 3D PC cell spheroids to assess effects through live cell image using Incuctye S3. Invasion assays were performed using a 96 well scratch wound migration with or without matrigel with PC3 and DU145 cells. We queried cbioPortal for PC data sets to look at incidence of STK3 and STK4 copy number alterations. Knockdowns of STK3 and STK4 genes using shRNAs to observe phenotypic differences and growth inhibition. Knockdown of STK3/4 pathways were verified through western blot. Results. Proliferation experiments 5 cell lines found that inhibition of STK3/4 with XMU-MP-1 significantly slows 2D and 3D spheroid PC cell growth. IC50 scores ranged from ~500nM to 5uM. Additionally, STK3/4 inhibition in the extremely invasive PC3 and DU145 cell lines significantly inhibited cell migration and matrigel invasion. PC data sets showed that the STK3 gene is frequently amplified in PC tumors which is contrary to an expected tumor suppressive role. In contrast, STK4 was not found to be amplified suggesting variation in STK3 versus STK4 roles. Depletion of STK3 and STK4 individually by shRNA knockdown also inhibited cell growth PC cells. Phenotypic markers of apoptosis and cell death could be seen 72 hours post viral transduction with shRNA knockdown in 22Rv1 and PC3 PC cells. The shRNA knockdowns showed deceased phosphorylated MOB kinase activator 1(MOB1) a downstream phospho-target of MST1/2. However, mixed results were obtained for subsequent YAP phosphorylation. Conclusions. Our results demonstrate that STK3/4 are essential for PC cell proliferation and migration/invasion. Similar effects were observed by both inhibition of MST1/2 either directly through drug treatment or STK3/4 gene depletion by shRNA knockdown. Whether these paralogs have unique or overlapping effects and if they can be targeted for PC therapy remains to be elucidated.

#863

Functions of FRAS1 as a driver gene of liver metastasis from gastric cancer analyzed by the genome editing technology.

Shinichi Umeda, Mitsuro Kanda, Haruyoshi Tanaka, Fuminori Sonohara, Hideki Takami, Norifumi Hattori, Masamichi Hayashi, Chie Tanaka, Daisuke Kobayashi, Suguru Yamada, Goro Nakayama, Masahiko Koike, Michitaka Fujiwara, Yasuhiro Kodera. _Department of Gastroenterological Surgery, Nagoya University Graduate School of Medicine, Nagoya, Japan_.

Background: The prognosis of patients with liver metastasis from gastric cancer (GC) is dismal and it remains a serious problem in the management of GC. To elucidate complex molecular mechanisms underlying formation of liver metastasis is necessary for development of novel therapeutic modality.

Methods: Transcriptome analysis of 57749 molecules with 4 groups (no recurrence, peritoneal recurrence, liver recurrence and nodal recurrence) was conducted to screen out the candidate molecules specific for liver metastasis. Subsequently, the stable knockout (KO) cells were established using the CRISPR/Cas9 system and cell functions, apoptosis, cell cycle, oxidative stress and cancer stemness were analyzed. PCR and antibody array analyses were conducted to identify the co-regulated molecules. Mouse subcutaneous xenograft models and liver metastasis models were generated to evaluate tumorigenicity of the KO cells in vivo. Tissue mRNA expression levels were determined by qRT-PCR in patients with stage II/III GC and correlated with recurrence patterns.

Results: Fraser extracellular matrix complex subunit 1 (FRAS1) was identified as a candidate molecule overexpressed specifically in the liver metastasis group. Proliferation, adhesion, invasion and migration abilities were suppressed in FRAS1-KO cells compared to the control cells. Cell cycle alternation, increased proportions of apoptotic cells, increased oxidative stress and decreased cancer stemness were also observed in FRAS1-KO cells. PCR array analysis revealed that FRAS1 mRNA expression was positively correlated with TGFB1, MAP1B, AHNKA and negatively correlated with BMP2. Pathway analysis demonstrated that expression of MUC1, Survivin, Ki67 and Met proteins were decreased in FRAS1-KO cells. The tumorigenicity of FRAS1-KO cells was reduced in a mouse subcutaneous model and more clearly in liver metastasis models. Patients with high FRAS1 mRNA expression had a significantly higher incidence of liver recurrences after curative gastrectomy.

Conclusion: Our results indicated that FRAS1 contributes to formation of liver metastasis from GC and is a potential therapeutic target.

#864

Mutant IDH1 is essential for chondrosarcoma growth.

Luyuan Li, Xiaoyu Hu, Josiane E. Eid, Joanna DeSalvo, Jonathan C. Trent. _University of Miami Miller School of Medicine, Miami, FL_.

Chondrosarcomas are malignant bone tumors that produce cartilaginous matrix. Mutations in isocitrate dehydrogenase enzymes (IDH1/2) were recently described in several cancers, including chondrosarcomas. IDH mutations detected in human cancers invariably are heterozygous missense substitutions. These mutations lead to the inability of IDH to convert isocitrate into α-ketoglutarate (α-KG). Instead, α-KG is reduced into 2-hydroxyglutarate (D-2HG), an oncometabolite. Due to the structural similarity between D-2HG and α-KG, it has been reported that high levels of D-2HG competitively inhibit α-KG-dependent dioxygenases such as TET, JHDM and PHD enzymes, thus contributing to tumorigenesis. We sought to determine the role of IDH1 mutations in the tumorigenesis of human chondrosarcomas by inactivating mutant IDH1 using pharmacological and genetic approaches. In our study, we employed two human chondrosarcoma cell lines, JJ012 and HT1080, that carry endogenous IDH1 mutations. IDH mutation analysis was performed by PCR-based DNA sequencing, and D-2HG levels were measured by tandem mass spectrometry. Mutant IDH1 was knocked down via siRNA and knocked out via CRISPR/Cas9. We analyzed the effect of mutant IDH1 on chondrosarcoma growth in murine xenograft models. We found that knockdown of mutant IDH1 via siRNA significantly reduced D-2HG production in chondrosarcoma cells. In addition, mutant IDH1 knockdown dramatically inhibited colony formation in the sarcoma cells. Consistently, genetic knockout of mutant IDH1 almost completely depleted D-2HG production and significantly inhibited colony formation in the chondrosarcoma cells. To assess the significance of these results in vivo, we implanted the mutant IDH1- knockout cells in nude mice and studied their capacity for tumor initiation and growth. In these models, we observed that loss of mutant IDH1 led to a marked attenuation of chondrosarcoma formation. Our findings clearly demonstrate that mutant IDH1 plays a vital role in chondrosarcoma tumor formation. By investigating the role of IDH mutations in the pathogenesis of chondrosarcomas, we aim to uncover the potential therapeutic targets against this aggressive cancer.

#865

Isolation of circulating tumors cells from genetically engineered mouse models of lung adenocarcinoma.

Liza J. Burton, Junghui Koo, Carol Tucker-Burden, Wei Zhou, Melissa Gilbert-Ross, Chunzi Huang, Gabriel Sica, Adam Marcus. _Emory University, Atlanta, GA_.

The major cause of cancer-associated mortality is tumor metastasis, but our understanding of this process is far from complete. During successful dissemination, tumor cells invade the surrounding tissue of the primary tumor, intravasate into blood and lymphatic vessels, translocate to distant tissues, extravasate, adapt to the new microenvironment, and eventually seed, proliferate, and colonize to form metastases. Because dissemination mostly occurs through the blood, circulating tumor cells (CTCs) that have been shed into the vasculature and may be on their way to potential metastatic sites are of obvious interest (1). KRAS mutations are the most frequent oncogenic drivers of non-small cell lung cancer and when associated with co-mutations lead to a decrease in overall survival. We have previously established a lenti-Cre-induced Kras and Lkb1 mutant and Kras and p53 mutant genetically engineered mouse modes (KLLenti) and (KPLenti) that develop 100% lung adenocarcinoma to conduct the first study to isolate and maintain primary, CTC and metastatic cells that are KLLenti or KPLenti. We are able to isolate TTF-1+ and pan-cytokeratin+ CTCs from the KLlenti and KPlenti mice and validated their mutational status by genotyping, western blot, and immunofluorescence. Moreover, lkb1-mutant or p53-mutant primary tumor cells have different 3-D invasive properties and patterns, as well as the respective CTCs and metastatic cells when compared across different genetic sub-types. To further study gene expression patterns between primary, CTC, and metastatic sites RNAseq will be employed to identify pathways that drive metastasis within the genetic sub-types.

#866

IRAK4 or TPL2/COT/MAP3K8 inhibition suppresses MAPK pathway activation and tumor growth in pancreatic ductal adenocarcinoma.

Paarth B. Dodhiawala, Namrata Khurana, Hongmei Jiang, Lin Li, Andrea Wang-Gillam, Kian-Huat Lim. _Washington University School of Medicine, Saint Louis, MO_.

Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival of <8% and effective treatment options remain elusive. Oncogenic mutations of KRAS occur in >95% of PDACs and are well-established as the bona fide driver event. However, direct inhibition of KRAS oncoprotein remains unsuccessful in the clinic. Therefore, the current focus is on targeting effectors of KRAS, particularly the RAF-MEK-ERK MAP-kinase (MAPK) pathway. Yet, MAPK inhibitors have been unsuccessful so far due to multiple resistance mechanisms that sustain MAPK signaling. Furthermore, constitutive activation of the NF-kB pathway poses an additional therapeutic barrier. We recently showed that that NF-kB cascade is driven predominantly by Interleukin-1 Receptor associated kinase 4 (IRAK4). Here, through an unbiased reverse phase protein array screen, we discovered IRAK4 as an upstream kinase of MEK and ERK. Inhibition of IRAK4 potently suppresses MEK and ERK activity even in the presence of oncogenic KRAS in multiple PDAC cell lines. Notably, IRAK4 is critical in oncogenic RAS-induced transformed growth and tumorigenesis. Mechanistically, we uncover MAP3K8 (or TPL2/COT) as the kinase through which IRAK4 drives MEK and ERK. Suppression of TPL2 abrogates KRAS-driven MEK-ERK activity and transformed growth. Furthermore, we find that genotoxic stress-induced ERK activation is TPL2-dependent. Together, our study provides a broader understanding of the complex signaling network weaved by oncogenic KRAS and elucidates novel, practical therapeutic strategies that can effectively curb both MAPK and NF-kB cascade in PDAC.

#867

EGFR kinase domain duplication (EGFR-KDD) is activated by asymmetric intra-molecular dimerization.

Zhenfang Du,1 Jean-Nicolas Gallant,2 Monica Red-Brewer,1 Benjamin Brown,3 Jonathan Sheehan,3 Jens Meiler,3 Christine M. Lovly1. 1 _Vanderbilt University Medical Center, Nashville, TN;_ 2 _Vanderbilt Ingram Cancer Center, Nashville, TN;_ 3 _Vanderbilt University, Nashville, TN_.

Recently, we identified a novel oncogenic EGFR exon 18 - 25 Kinase Domain Duplication (EGFR-KDD) in patients with lung cancer (PMID: 26286086). This novel EGFR alteration contains two tandem, in-frame EGFR TKDs. Computational modeling suggested that the linker between the two TKDs may support the formation of an asymmetric intra-molecular dimer. In this study, we sought to provide experimental support for the model that EGFR-KDD could form ligand-independent asymmetric intra-molecular dimers. We constructed point mutations (Table 1) disrupting the potential intra- (C1, N2) and inter-molecular dimer interfaces (N1 and C2) between each TKD of EGFR-KDD. We also introduced catalytically inactivating mutation into each TKD individually (Dead1 and Dead2). We stably expressed these mutants in YAMC and NR6, two cell lines lacking endogenous wild-type EGFR expression. In the absence of ligand, 'wild-type' EGFR-KDD was phosphorylated on known EGFR auto-phosphorylation sites, Y992, Y1068, and Y1173. However, mutations of the intra-molecular dimer interface (C1, N2) abrogated EGFR-KDD auto-phosphorylation, which is an accepted surrogate for EGFR enzymatic activity. In support of these data, expression of EGFR-KDD, but not the intra-molecular dimer interface mutants (C1, N2), supported growth in soft agar, an assay utilized in support of oncogenic phenotypes. Interestingly, phosphorylation of the dimer interface mutants (C1, N2) was rescued by ligand stimulation, suggesting the formation of EGFR-KDD multimers. Our structural modeling by Rosetta suggests that EGFR-KDD can form high order multimers with itself or with wild-type EGFR, and we observed co-precipitation of EGFR-KDD with itself and wild-type EGFR in vitro. Collectively, our findings define a previously unrecognized model of asymmetric intra-molecular dimers for EGFR, which provides important insights for the understanding of EGFR activation mechanisms and shed light on the exploration of therapeutic benefit.

Table 1: Kinase domain mutations constructed for these studies

---

Lobe | Mutation | Functions

N1 | I706Q | Disruption of the 1st N-lobe interface

Dead1 | D837N | Kinase dead mutation of the 1st kinase domain

C1 | V948R | Disruption of the 1st C-lobe interface

N2 | I1057Q | Disruption of the 2nd N-lobe interface

Dead2 | D1188N | Kinase dead mutation of the 2nd kinase domain

C2 | V1299R | Disruption of the 2nd C-lobe interface

#868

Epigenomic profiling of primary hepatocellular carcinoma reveals super-enhancer-associated chromatin regulator network.

Liangliang XU, Feng WU, Otto K.W. CHEUNG, Lemuel L.M. SZETO, Myth T.S. MOK, Kevin Y.L. Yip, Ka F. To, Alfred S.L. CHENG. _CUHK, Hong Kong_.

Hepatocellular carcinoma (HCC) is the second most common cause of cancer mortality worldwide. Parallel with the growing epidemics of obesity and diabetes, non-alcoholic fatty liver disease (NAFLD) has become the predominant cause leading to HCC in a dysregulated metabolic background. Recent HCC genomics studies present only a paucity of recurrent gene mutations, which lead to a sparkling interest in the epigenetic regulation of hepatic carcinogenesis. Chromatin modifications convert the metabolic insults from NAFLD to transcriptional program that contributes to HCC development. Super-enhancers (SEs) are subclass of regulatory elements with unusually strong enrichment for the binding of transcriptional coactivators to fulfill cell identity. Currently, it has been proposed that dysregulation of SEs underlies the development of diseases including cancers, but the role of SEs in NAFLD-associated HCC is still unknown.

In order to investigate the alterations of histone modifications in NAFLD-associated HCC development, nanoscale chromatin immunoprecipitation sequencing of multiple histone marks were performed. By integrating multiple epigenomic profiles from primary NAFLD-associated HCCs and matched non-tumor tissues, we identified recurrent SEs enriched that contribute to fatty liver, lipid metabolism and metabolic syndrome. Interestingly, we observed significant enrichment of recurrent active SEs in a number of target genes. Notably, through CRISPR/Cas9-induced knockout liver cell lines, we showed that deletion of two distinct SE regions significantly reduced the expression of a candidate target gene and attenuated the tumorigenic potential of HCC cells. In summary, our results show that the dysregulation of SEs contributes to human hepatic carcinogenesis, thus representing novel therapeutic targets for NAFLD-associated HCCs.

#869

Molecular characterization of a pediatric high-grade glioma (pHGG) mouse model harboring the H3.3 G34R mutation.

Matthew J. Whalen, Maria B. Garcia-Fabiani, Felipe J. Núñez, Pedro R. Lowenstein, Maria G. Castro. _University of Michigan-Ann Arbor, Ann Arbor, MI_.

Pediatric high-grade glioma (pHGG) accounts for 8-12% of central nervous system tumors and has a 90% mortality rate. Consequently, novel therapies are needed to advance treatment options and improve survival rates. A means to do this is through designing a mouse model that accurately mirrors pHGG in humans to further study this type of pHGG and design treatment options. We focused on the analysis of pHGG harboring mutations on histone H3.3 at glycine 34 (H3.3 G34R) in combination with ATRX and TP53 mutations. We generated a mouse model for pHGG using the sleeping-beauty transposase system and the delivery of plasmids into the sub-ventricular zone of neonatal mice. This results in the transposon-mediated integration of the genetic lesions into the brain cells, thereby allowing the generation of tumors with the desired molecular alterations. Two groups of mice have been developed; the control group (WT H3.3 group) bears tumors generated with the NRASV12 overexpression and two shRNA that target the Tp53 and Atrx genes. The H3.3 G34R group of mice harbors tumors generated with NRASV12 expression, sh-TP53, sh-ATRX, and H3.3 G34R expression. RNA-sequencing was carried out to analyze the transcriptome signature associated with the H3.3 G34R tumor phenotype. Several genes related to the activation of the immune response were differentially upregulated in the H3.3 G34R tumors, such as Ciita, CD74, and Irfl, amongst others. Some of these deregulated genes were validated by immunohistochemistry. Understanding the tumor transcriptome and characterizing the tumor's immune microenvironment are crucial for the development of gene and immune therapies designed to target H3.3 G34R pHGG.

#870

The role of the intermediate prolactin receptor in breast cancer oncogenesis.

Jacqueline M. Grible, Charles V. Clevenger. _Virginia Commonwealth University, Richmond, VA_.

Epidemiological, cellular, and genetic analyses indicate the hormone prolactin (PRL) and its cognate receptor in humans (hPRLr) are significantly involved in breast cancer pathogenesis. Recent evidence demonstrated that a truncated mouse PRLr (mPRLrT) is oncogenic when expressed alongside its canonical long form counterpart (mPRLrL) [Cell Reports 17, 249-260]. The mPRLrT shares significant sequence homology with a naturally-occurring and widely-expressed hPRLr splice variant, the intermediate hPRLr (hPRLrI) isoform. Given this similarity, we hypothesized hPRLrI may also induce transformation, when expressed alongside wild-type long hPRLr (hPRLrL). Like the mPRLrT, hPRLrI co-expression with hPRLrL in the immortalized but not transformed human breast cell line MCF10A resulted in a significant increase in proliferation, migration, viability, and anchorage-independent growth. These results were not observed following overexpression of either isoform alone, demonstrating that hPRLrL and hPRLrI co-expression is necessary to induce transformation of normal mammary epithelia. To further characterize this transformation, we established MCF10A xenografts using female NOD scid gamma (NSG) mice. Following intraductal injection, we observed rapid tumor growth in the hPRLrL/I co-expression cohort, significantly over that of the cohorts harboring either isoform alone, validating our in vitro findings in vivo. To determine mechanisms of transformation, we examined both differential protein stability and altered signaling events. In analyzing receptor degradation, a cycloheximide assay revealed hPRLrL stability is increased when heterodimerized with hPRLrI. hPRLrL turnover has been reported to be impaired in human breast cancer, indicating this phenomenon may be involved in the observed hPRLrI-mediated transformation. In regards to differential signaling, we examined the Jak2/Stat5a pathway. Jak2 is a promiscuous kinase whose significant oncogenic actions are well-characterized, while Stat5a is a transcription factor whose activities are critical in attenuating the actions of Jak2. Following PRL stimulation, it was observed that hPRLrL/I co-expression induced approximately two-fold greater Jak2-Y1007/1008 phosphorylation (pJak2) compared to that induced by hPRLrL expression alone. Further, it was observed that hPRLrL/I co-expression induced ten-fold less Stat5a-Y694 phosphorylation (pY-Stat5a) than hPRLrL expression alone. These data indicate unchecked pJak2 activity may also be a contributing mechanism in the observed transformation. Overall, these results demonstrate that hPRLrI, alongside hPRLrL, is sufficient for transformation of normal breast tissue.

#871

Regulation of CDC25a expression by the ikaros and casein kinase II (CK2) in T-cell acute lymphoblastic leukemia (T-ALL).

Soumya C. Iyer,1 Shriya Kane,2 Chandrika Gowda,1 Chunhua Song,1 Yali Ding,1 Jon Payne,1 Pavan Kumar Dhanyam Raju,1 Bihua Tan,1 Mary McGrath,1 Yevgeniya Bamme,1 Mario Solimon,3 Nathalia Moreno Cury,1 Dhimant Desai,1 Arati Sharma,1 Kimberly J. Payne,4 Sinisa Dovat1. 1 _Pennsylvania State University College of Medicine, Hershey, PA;_ 2 _Georgetown Medical School, WA;_ 3 _University of New Mexico School of Medicine, NM;_ 4 _Loma Linda University, Loma Linda, CA_.

CDC25A is a member of the CDC25 family of phosphatases that plays a major role in cell cycle progression. Here, we present evidence that expression of CDC25a in T-ALL is regulated at the transcriptional level by oncogenic Casein Kinase II (CK2) via direct phosphorylation of Ikaros, a transcription factor and tumor suppressor protein. Global chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) studies in both primary human acute lymphoblastic leukemia cells and cell lines, demonstrated that Ikaros binds to the promoter of the CDC25a gene. Ikaros functions as a tumor suppressor protein and deletion of which is associated with development of T-ALL. Ikaros binding to CDC25a promoter was confirmed by quantitative chromatin immunoprecipitation (qChIP) in primary T-ALL cells. Ikaros knock-down with shRNA results in increased transcription of CDC25a in T-ALL. In mice, T-ALL cells that were derived from Ikaros-knockout mice express high levels of CDC25a. Transduction of these cells with Ikaros-containing retrovirus results in sharp reduction of CDC25a expression. Overexpression of CK2 via retroviral transduction resulted in increased transcription of the CDC25a gene, as measured by qRT-PCR, as well as increased overall expression of CDC25a, as measured by Western blot. Increased expression of CK2 was associated with a loss of Ikaros binding to the CDC25a gene promoter. Molecular inhibition of CK2 using shRNA, as well as pharmacological inhibition with a specific CK2 inhibitor resulted in reduced expression of CDC25a in primary human T-ALL. CK2 inhibition was also associated with strong reduction in AKT phosphorylation, emphasizing that CK2 inhibition downregulates CDC25a and other cell cycle progression genes. Inhibition of CK2 was associated with increased Ikaros binding at the promoter of CDC25a. Ikaros knock-down restored high expression of CDC25a in T-ALL cells that were treated with CK2 inhibitors. These data showed that CK2 and Ikaros are major transcriptional regulators of CDC25a transcription in T-ALL and that CK2 inhibition represses CDC25a transcription via Ikaros-mediated repression. In conclusion, these results indicate that expression of the CDC25a oncogene in T-ALL is regulated by the CK2 which modulates Ikaros activity. Presented data revealed a novel mechanism of therapeutic action of CK2 inhibitors - repression of CDC25a expression via Ikaros. Results provide a rationale for the use of novel CK2 inhibitors in T-ALL.

#872

Wild-type KRAS exerts tumor suppression via cytoplasmic retention of YAP1.

Han Yan,1 Chih-Chieh Yu,1 Stuart Fine,1 Ayman Lee Youssof,1 Ye-Ran Yang,1 Yun Yan,2 Dario Garcia-Carracedo,1 Dillon Karg,1 Edwin Cheung,1 Richard Friedman,1 Emily Chen,1 Ji Luo,3 Yi Miao,4 Wanglong Qiu,1 Gloria H. Su1. 1 _Columbia University, New York, NY;_ 2 _Tianjin First Center Hospital, Tianjin, China;_ 3 _National Cancer Institute, Bethesda, MD;_ 4 _Nanjing Medical University, Nanjing, China_.

KRAS is the most frequently mutated oncogene in pancreatic ductal adenocarcinoma (PDAC) and it has been shown to be essential for pancreatic tumor initiation and maintenance in both humans and mice. We have previously reported that the wild-type Kras allele was selectively lost in both primary pancreatic tumors and metastases developed in a mouse model of PDAC, and the frequency of this allelic loss increased from primary tumors to metastases in this mouse model and human pancreatic cancer cells. This previously unknown association between wild-type KRAS and metastasis and its underlying mechanism remain to be elucidated. Here we restored the wild-type KRAS allele in a doxycycline inducible manner in human pancreatic cancer cell lines that had undergone spontaneous loss of the wild-type allele, to interrogate its tumor-suppressive functions and mechanisms. We observed that the re-expression of wild-type KRAS significantly attenuated the malignancy of pancreatic cancer cells in vitro and in vivo. We also observed the induction of YAP1 cytoplasmic retention upon the restoration of the wild-type KRAS expression by immunofluoresce microscopy, western blot, reporter assay, and confirmed through unbiased RNA-Seq and proteomic analyses. Expression of constitutively activated YAP1 mitigated the inhibitory effects of the wild-type KRAS allele, confirming that YAP1 is a downstream effector of the pathway. Furthermore, YAP1 nuclear localization was found associated with poor prognosis in PDAC patients. Together these results ascribed a novel mechanism whereas wild-type KRAS, in the context of mutant KRAS, exerts tumor suppression via the inhibition of YAP1 nuclear translocation and transcription of YAP1-TEADs downstream targets. Our results demonstrated the importance to differentiate between mutant and wild-type KRAS in future development of targeted therapies, and inhibition of YAP1 activation could be a plausible therapeutic strategy for treating metastasis in PDAC patients.

#873

Molecular mechanisms of non-small cell lung cancer growth and drug resistance.

SK Kayum Alam,1 Matteo Astone,1 Ping Liu,2 Li Wang,1 Abbygail M. Coyle,1 Erin N. Dankert,1 Dane K. Hoffman,2 Stephanie R. Hall,1 Wei Zhang,3 Rui Kuang,3 Anja C. Roden,2 Aaron S. Mansfield,2 Luke H. Hoeppner1. 1 _University of Minnesota, The Hormel Institute, Austin, MN;_ 2 _Mayo Clinic, Rochester, MN;_ 3 _University of Minnesota, Minneapolis, MN_.

Lung cancer is the deadliest and most frequently diagnosed type of tumor worldwide, with 1.6 million deaths reported annually. Non-small cell lung cancer (NSCLC) represents 85% of all lung cancer cases and carries a poor 5-year survival rate below 15%. Prognoses remain dismal due to the large number of patients diagnosed with advanced stage disease and the development of resistance to current therapies. A better understanding of acquired drug resistance will help to circumvent the progression of lung cancer and make significant strides in improving NSCLC patient treatment. Our recent work demonstrates that dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32) and its amino-terminally truncated splice variant (t-DARPP) promote lung tumor growth in orthotopic mouse models. IHC staining of 62 human lung adenocarcinoma tissues showed that t-DARPP expression is elevated with increasing tumor (T) staging score, which represents the size of the primary tumor and whether it has grown into nearby areas, as a metric of tumor progression and growth. Correspondingly, a computational biology analysis of 513 lung adenocarcinoma patients revealed upregulation of t-DARPP isoform expression correlates with advanced T stage and is associated with poor overall survival. We identified a novel physical interaction between DARPP-32 and inhibitory kappa B kinase-α (IKKα) that promotes NSCLC cell migration through activation of non-canonical NF-κB2 signaling. It has been shown that DARPP-32 overexpression inhibits gefitinib-induced apoptosis in gastric cancer and t-DARPP contributes to the trastuzumab resistance phenotype in breast cancer. Our recent findings suggest DARPP-32 and t-DARPP overexpression in NSCLC promotes resistance to specific molecular targeted inhibitors by enabling tumor cells to evade apoptosis via Akt- and Erk-dependent cell survival mechanisms. Current ongoing studies are focused on manipulating expression of DARPP-32 isoforms in lung tumor cells to prevent resistance to targeted therapies in NSCLC patients.

#874

The CRTC1/MAML2 oncogene induces a PGC-1a splice variant that sensitizes mucoepidermoid carcinoma cells to PPARg inhibition.

Adele M. Musicant,1 Kshitij P. Sharma,1 Erin C. Henry,1 Jason Tasoulas,1 Ricardo Padilla,1 Monideepa Sengupta,2 Colin Flaveny,2 Antonio L. Amelio1. 1 _UNC Chapel Hill, Chapel Hill, NC;_ 2 _Saint Louis University School of Medicine, Saint Louis, MO_.

Mucoepidermoid carcinoma (MEC) is the most common type of malignant salivary gland tumor. MECs frequently exhibit a recurrent t(11;19)(q21;p13) chromosomal translocation that fuses the transcriptional coactivators CRTC1 (CREB-Regulated Transcription Coactivator 1; C1) and MAML2 (Mastermind-Like 2; M2). Though many C1/M2-positive tumors are easily resected, some do progress to advanced, metastatic disease accompanied by poor 5-year survival rates. Thus, it is critical to understand the mechanisms by which C1/M2 drives tumor growth and progression in order to identify therapeutic vulnerabilities. Our lab recently identified a correlation between C1/M2 expression and increased levels of the growth hormone IGF-1. This hormone plays critical roles in the development and homeostasis of normal salivary glands, and its dysregulation has been implicated in several cancers including colon and lung cancers. Interestingly, we demonstrate that IGF-1 is not directly upregulated by C1/M2. Rather, we have discovered a novel C1/M2-driven signaling circuit whereby constitutive CREB activation drives the increased production of a minor PGC-1α (PPARγ Coactivator 1α) splice variant, PGC-1α4, which is responsible for the high IGF-1 levels observed in C1/M2-positive MEC cells. Notably, the PGC-1α4 splice variant is minimally expressed in normal tissues and C1/M2-negative cell lines but is selectively upregulated in C1/M2-positive cell lines. We show that C1/M2 directly binds CREB at the PGC-1α4 promoter to induce expression of this variant, resulting in increased IGF-1 expression. IGF-1 promoter analysis revealed a significant enrichment of PPARγ binding motifs and functional studies confirm that this promoter is regulated by PPARγ. Consequently, we demonstrate that a novel PPARγ inverse agonist is capable of repressing IGF-1 promoter activity and reducing growth and clonogenic potential of C1/M2-positive cell lines that exhibit high PGC-1α4 expression. Collectively, our results reveal a novel therapeutic vulnerability in salivary MEC cells that may extend more broadly to other cancers that exhibit dysregulated CREB activity. 

### Targeting Oxidative Stress and Senescence

#875

**Targeting Nrf2 driven glutathione** de novo **synthesis as a novel strategy to suppresses IDH1-mutated glioma.**

Yang Liu, Di Yu, Sabrina Cai, Chunzhang Yang. _NIH, NCI, Bethesda, MD_.

Background: Isocitrate dehydrogenase (IDH) mutation frequently occurs in low grade glioma and secondary glioblastoma, characterizing by the abnormally catalyzation of alpha-ketoglutarate (α-KG) to D-2-hydroxyglutarate (D-2-HG), which induces CpG island methylator phenotype, as well as metabolic reprogramming and high reactive oxygen species (ROS) generation. Our previous data showed that nuclear factor-erythroid 2-related factor 2 (Nrf2) plays a crucial role in maintaining redox homeostasis of IDH1-mutated glioma cells, while the detail mechanism remains elusive. Our recent data showed that glutathione de novo synthesis is governed by Nrf2, which makes great contribution to ROS scavenging in IDH1-mutated tumors and could serve as a potential therapeutic target.

Methods: In this study, we explored the important role of glutathione de novo synthetic pathway in maintaining ROS homeostasis by small RNA interference of key enzymes GCLC, GCLM and SLC7A11. We also investigated the correlation between Nrf2 and glutathione de novo synthetic pathway by Chromatin Immunoprecipitation. We further investigated the therapeutic value of Nrf2 inhibitor, Brusatol, in IDH1-mutated cancer both in vitro and in vivo.

Results: Our data showed that IDH1 mutation cells exhibit high level steady-state of ROS generation and detoxification. Prolonged Nrf2 activity enhanced transcriptional activity of key enzymes in glutathione de novo synthesis, such as GCLC, GCLM and SLC7A11. Targeting Nrf2 using Brusatol suppressed glutathione synthesis in IDH1-mutated cells, resulted in cytotoxic effect and suppressed xenograft growth in vivo.

Conclusions: IDH1-mutated glioma highly depends on Nrf2 activity to maintain ROS homeostasis. Enhanced glutathione de novo synthesis is an important mechanism that governed by Nrf2 and plays critical role in ROS scavenging. Targeting ROS scavenging might be a novel approach to treat gliomas with IDH1 mutation.

#876

CD40-induced growth inhibition of Burkitt lymphoma: A possible role for NADPH oxidase by upregulation of p67phox.

Mazal Iris Dahan, Smitha Antony, Agnes Juhasz, Guojian Jiang, Mariam Konate, Jiamo Lu, Jennifer Meitzler, YongZhong Wu, Krishnendu Roy, James H. Doroshow. _NIH, Bethesda, MD_.

CD40 stimulation together with IL4 has been shown to promote growth of normal B cells in vitro. In contrast to this, CD40 stimulation in several B cell lymphoma cell lines has been demonstrated to be growth inhibitory. Though the mechanism is not completely known, CD40-stimulated cells exhibit increased levels of reactive oxygen species (ROS). In B lymphocytes, a major source of ROS production is the NADPH oxidase (NOX) that is comprised of a membrane-bound Nox2 catalytic subunit and four cytosolic regulatory components, with the p67phox cytosolic protein being the activator factor. Upon stimulation, by either cross linking of the B-cell receptor or following treatment with PMA, the active NADPH oxidase generates superoxide. We have investigated the role of NADPH oxidase in CD40-mediated growth inhibition in Burkitt lymphoma (BL) cells. For this study, we first evaluated the effect of CD40 stimulation on the expression of Nox2 and p67phox in BL cells that demonstrate different levels of endogenous Nox2 and p67phox. Accordingly, Daudi and Raji cell lines, with endogenously high Nox2 and low p67phox, NAMALWA cells which have high Nox2 and no p67phox expression, and Ramos cells that contain no Nox2 or p67phox were analyzed. Treatment with anti-CD40 or CD40 ligand (CD40L) for 24 h induced up-regulation of p67phox in Daudi, Ramos, and NAMALWA cells, but not in the Raji line. No significant change in the expression of Nox2 was observed. Additionally, Daudi, Ramos and NAMALWA cells showed higher production of ROS after CD40 stimulation and activation with anti-IgM or PMA, in a chemiluminescence assay. To determine whether the change in ROS level was related to the upregulation of p67phox, Daudi cells that express either stable p67phox shRNA or control shRNA were generated. Treatment with CD40L for 24 h demonstrated significant ROS production only in control shRNA and not in the p67shRNA cells, confirming that the ROS production is NOX2-p67phox mediated. This enhanced ROS production was observed following treatment with CD40L alone and was enhanced further in conjunction with anti-IgM or PMA. However, cells with the p67phox shRNA showed detectable level of ROS only after activation with anti IgM, albeit to much lower levels than the control. Co-treatment of Daudi cells with anti-CD40 or CD40L and IL4 further increased the expression of p67phox. Although the basal level of ROS in the cells was unchanged upon co-treatment, the ROS generated following stimulation with anti-IgM or PMA was significantly enhanced. Preliminary MTT studies confirmed the growth inhibition of CD40-stimulated Daudi cells but not of Raji cells, suggesting a role for p67phox-dependent, ROS-related inhibition of lymphoma cell proliferation. Experiments are currently ongoing to delineate further the role of CD40 stimulation and Nox2-p67phox mediated ROS in affecting the growth of Burkitt lymphomas and signaling pathways downstream of CD40.

#877

Novel role of truncated HMGA2 in regulating reactive oxygen species in prostate cancer cells.

Ohuod Hawsawi,1 Veronica Henderson,1 Liza Burton,1 Jodi Dougan,1 Ana Cecillia Millena,1 Vanessa Adams,1 Elshaddai Z. White,1 Guang Di Wang,2 Qiang Zhang,2 Cimona Hinton,1 Shafiq Khan,1 Valerie Odero-Marah1. 1 _Clark Atlanta University, Atlanta, GA;_ 2 _Xavier University, New Orleans, LA_.

High mobility group A (HMGA2), a non-histone protein, is up-regulated in several cancers. Due to chromosomal rearrangement of HMGA2 gene, full-length or wild-type HMGA2 can be truncated leading to loss of the C-terminus and the 3'UTR. Both wild-type and truncated isoforms have been detected in uterine leiomyoma patients. However, the functional role of truncated HMGA2 has not been investigated. We hypothesize that truncated HMGA2 plays a role in prostate cancer progression. We analysed expression of wild-type vs truncated HMGA2 in a panel of prostate cancer cell lines using real-time PCR and western blot analyses. We utilized LNCaP cells stably overexpressing wild-type HMGA2 or truncated HMGA2 in LNCaP cells to detect the basal reactive oxygen species (ROS) levels using DCFDA dye. We analysed Jun-D expression, a putative downstream effector of HMGA2, by western blot analysis, and utilized siRNA to knockdown Jun-D. We performed migration and cell viability assays following Jun-D knockdown. Additionally, we performed proteomic analysis following immunoprecipitation with HMGA2 antibody in nuclear extracts from LNCaP cells overexpressing wild-type HMGA2 WT or cytoplasmic extracts from cells overexpressing truncated HMGA2. Finally, we utilized tissue microarray to analyse expression of HMGA2 by immunohistochemisty. Our results showed that prostate cancer cell lines expressed varying amounts of wild-type and truncated HMGA2. LNCaP cells overexpressing truncated HMGA2 exhibited increased nuclear expression of Jun-D, as well as increased ROS compared to LNCaP cells overexpressing wild-type HMGA2 or empty vector Neo control. Knockdown Jun-D in LNCaP cells overexpressing truncated HMGA2 abrogated ROS induction and cell migration with no effect on cell proliferation. Additionally, truncated HMGA2 interacted with a number of proteins, including Ras GTPase-activating protein-binding protein 1(G3BP1), a cytoplasmic stress granule protein that responds to oxidative stress. Truncated HMGA2 is mainly localized to the cytoplasm, and interestingly we also observed cytoplasmic HMGA expression in prostatic tissue with chronic inflammation. Therefore, truncated HMGA2 may promote prostate cancer progression via Jun-D-mediated ROS. GRANT SUPPORT: NIH 1P20MD002285 and NIH/NCRR/RCMI G12RR003062-22

#878

IGFBP2 is required to activate EGFR-DNA-PKcs pathway to protect esophageal adenocarcinoma cells from acidic bile salts-induced DNA damage.

Zhangjian Zhou,1 Heng Lu,1 Shoumin Zhu,1 Ahmed Gomaa,1 Zheng Chen,1 Jin Yan,2 Wael El-Rifai,1 Chengxue Dang,3 Dunfa Peng1. 1 _University of Miami, Miami, FL;_ 2 _Nanjing Medical University, China;_ 3 _Xi'an Jiaotong University, China_.

The incidence of esophageal adenocarcinoma (EAC) is rising rapidly in the US and Western countries. The development of Barrett's esophagus (BE) and its progression to EAC have been linked to gastroesophageal reflux disease (GERD). Exposure of BE and EAC cells to acidic bile salts (ABS) in GERD conditions induces high levels of DNA damage. In this study, we investigated the role of insulin-like growth factor binding protein 2 (IGFBP2) in regulating ABS-induced DNA double-strand breaks (DSBs). Immunohistochemistry analysis demonstrated frequent overexpression of IGFBP2 in EACs (31/57). Treatment of EAC cells with ABS, to mimic GERD conditions, induced high levels of IGFBP2 expression. Knocking down endogenous IGFBP2 in FLO1 cells (with constitutive high levels of IGFBP2) led to a significant increase in DNA DSBs and apoptosis, following transient exposure (20 min) to ABS. On the other hand, overexpression of exogenous IGFBP2 in OE33 cells (with low endogenous levels of IGFBP2) had a protective effect against DSBs and apoptosis. Using western blotting and immunofluorescence assays, we found that IGFBP2 is required for ABS-induced nuclear accumulation and phosphorylation of EGFR and DNA-PKcs, which are required for DNA damage repair activity. Using co-immunoprecipitation assay, we detected IGFBP2 forms complex with EGFR and DNA-PKcs and co-localizes with EGFR and DNA-PKcs in nucleus, following acidic bile salts treatment. We also demonstrated, using cycloheximide (CHX) chase assay, that IGFBP2 promotes EGFR protein stability in response to ABS exposure. In summary, our data indicate that IGFBP2 protects EAC cells against ABS-induced DNA damage and apoptosis through stabilization and activation of EGFR - DNA-PKcs signaling axis.

#879

Nicotine-induced oxidative stress causes epigenetic alterations during malignant transformation of human kidney epithelial cells.

Yuwei Chang, Kamaleshwar Singh. _Texas Tech Univ., Lubbock, TX_.

Nicotine is a component of cigarette smoke and mounting evidence implicates tobacco smoking in kidney cancer development. Whether nicotine alone can cause kidney cancer is not clear. Moreover, the underlying molecular mechanisms for nicotine-induced carcinogenesis is still not well-understood. Therefore, the objective of this study was to determine if chronic exposure to nicotine results in malignant transformation of HK-2 kidney epithelial cells. The data revealed that chronic exposure to nicotine induced growth and malignant transformation in HK-2 cells. Additionally, the nicotine-exposed cells had inherently increased levels of intracellular ROS, and acquired stem cell-like sphere formation, as well as epithelial-mesenchymal-transition (EMT) changes during malignant transformation. Treatment with antioxidant NAC resulted in abrogation of EMT and stem cell-like sphere in HK-2 cells, suggesting the role of nicotine-induced ROS in these morphological changes. Additionally, epigenetic reagents 5-aza-2'-deoxycytidine and trichostatin A also diminished stem cell-like sphere in nicotine-exposed HK-2 cells. The nicotine also induced changes in the expression of epigenetic genes (DNMT3a, DNMT3b, and HMT1) and these changes were reversed by NAC treatment. This further supports the role of nicotine-induced ROS in epigenetic changes during malignant transformation of HK-2 cells. The altered levels of pAKT in low dose nicotine exposed cells were restored after NAC treatment, suggesting that nicotine-induced ROS, through regulation of AKT pathway, controls the EMT and stemness during early stages of carcinogenesis. In summary, to our knowledge, this is the first report showing that chronic exposure to nicotine induces malignant transformation in human kidney epithelial cells through ROS-mediated epigenetic modifications.

#880

**Genetic ablation of the cystine transporter xCT in pancreatic ductal adenocarcinoma inhibits mTORC1, growth, survival and tumor formation: Implications for potentiating chemosensitivity** via **erastin.**

Milica Vučetić,1 Boutaina Daher,1 Jerome Durivault,1 Scott K. Parks,1 Jacques Pouyssegur2. 1 _Centre Scientifique de Monaco (CSM), Monaco;_ 2 _Centre Scientifique de Monaco (CSM) & University Côte d'Azur, IRCAN, Centre A. Lacassagne, Nice, France_.

The lethality of pancreatic ductal adenocarcinomas (PDAC) calls for improved therapeutic strategies. Chemoresistance remains a primary challenge in PDAC treatment, and exploiting oxidative stress might offer novel therapeutic clues. In this regard, we explored the cystine/glutamate exchanger (SLC7A11/xCT) that contributes to the maintenance of the intracellular glutathione (GSH). We deleted xCT via CRISPR-Cas9 in two PDAC cell lines (MiaPaCa-2 and Capan-2) and cultivated xCT-KO clones in the presence of N-acetylcysteine (NAC). In both cell lines, xCT-deletion abolished >90% of 14C-cystine uptake and induced a rapid depletion of GSH following NAC removal. Although several cystine/cysteine transporters have been identified in human cells, our finding demonstrates that, in vitro, xCT is the major actor for GSH synthesis. Consequently, both xCT-KO cell lines exhibited amino-acid stress with ATF4 and GCN2 kinase activation, mTORC1 inhibition, and proliferation arrest followed by ferroptotic cell death. Importantly, tumor growth was also abolished in both KO cell lines indicating the key role of xCT in cellular cysteine availability in vivo. Moreover, the rapid depletion of intracellular GSH in xCT-KO cells led to accumulation of lipid peroxides and cell swelling, both being prevented by vitamin E or iron chelation, two hallmarks of cell death by ferroptosis. Finally, in vitro pharmacological inhibition of xCT by erastin (1μM) phenocopied xCT-KO and potentiated the cytotoxic effects of both gemcitabine and cisplatin in these PDAC cell lines. In conclusion, our findings strongly support the concept that xCT inhibition, by its dual induction of nutritional and oxidative cellular stresses, has the great potential of a successful anticancer strategy.

#881

A role for microsomal glutathione transferase 1 in melanin biosynthesis in melanoma.

Jie Zhang,1 Zhi-wei Ye,1 Danyelle M. Townsend,1 Ralf Morgenstern,2 Kenneth D. Tew1. 1 _Medical University of South Carolina, Charleston, SC;_ 2 _Karolinska Institutet, Stockholm, Sweden_.

Higher organisms have multiple defenses against toxic chemical insult, ultraviolet light (UVR) and oxidative stress. These include thioredoxin and glutathione dependent enzymes, some of which are selenium (Se) dependent, including thioredoxin reductases, Se-dependent glutathione peroxidases, Se-independent glutathione transferases and peroxiredoxins. Microsomal glutathione transferase 1 (MGST1) shares an important and unique characteristic with Se-dependent glutathione peroxidase GPX4, namely the ability to reduce lipid hydroperoxides directly in membranes. In contrast to most other glutathione transferases and peroxidases, we previously found that deletion of MGST1 in mice was embryonic lethal, signifying some essential functional importance. We found that MGST1 was crucial for embryonic development through regulating early hematopoiesis in both zebrafish and mouse derived hematopoietic stem cells. Interestingly, after partial knock-down MGST1, the number of melanocytes located at the midline of zebrafish embryos was significantly reduced. Melanocytes are specialized cells that produce melanin, contributing to skin color, but also providing an antioxidant defense system that includes UVR - causative in the initiation of melanoma. Both mRNA and protein levels of MGST1 were markedly decreased in several amelanotic melanoma cell lines and melanogenesis was significantly decreased in B16 melanoma cells when MGST1 expression was knocked down by 60%. This was accompanied by increased UVB-induced cell death in these B16 cells. In contrast, in normal human melanocytes, recombinant adenovirus induced over-expression of MGST1 induced melanogenesis, concomitant with an increase of tyrosinase protein levels. UVB light was used to show that stimulus of melanin biosynthesis occurred in different melanoma cells in quantitative agreement with their levels of MGST1. We conclude that within the melanin synthetic pathway, MGST1 is critical in regulating conversion of dopaquinone to glutathionyl-DOPA, a precursor of pheomelanin. In the absence of MGST1, dopaquinone can cause toxicities and result in reduced or absent levels of melanin, leading to a deficiency in protection against UVR-induced DNA damage. Thus, absence of MGST1 produces embryonic lethality and influences the susceptibility of skin melanocytes to melanoma.

#882

Thioredoxin system inhibition using auranofin represents a new therapeutic approach for hepatocellular carcinoma.

Derek Lee, Iris Ming-Jing Xu, David Kung-Chun Chiu, Robin Kit-Ho Lai, Chun-Ming Wong, Irene Oi-Lin Ng, Carmen Chak-Lui Wong. _The University of Hong Kong, Pokfulam, Hong Kong_.

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer and third leading cause of cancer deaths worldwide due to late symptom presentation and ineffective treatments. Currently, tyrosine kinase inhibitors Sorafenib and Lenvatinib are the only FDA-approved first-line treatment for HCC patients. Cancer cells experience distinctly high amount of oxidative stress compared to normal cells. Reactive oxygen species (ROS) are by-products of metabolism. However, cancer cells' metabolic activities are hyper-activated as they have greater demands for energy, thereby also resulting in greater amounts of ROS generated. Besides, oncogenes and properties of the tumor microenvironment like ER stress and hypoxia also contribute to ROS generation in cancer cells. With higher concentrations of ROS, cancer cells also have increased antioxidant production capacity to counteract ROS. NADPH is a major metabolite and antioxidant immensely generated by cancer cells. In human HCC, our group previously found the pentose phosphate pathway and folate cycle to be major metabolic pathways of NADPH production. The thioredoxin system is a ubiquitous mammalian antioxidant system that is activated by the antioxidant system-activating electron donor NADPH. Thioredoxin reductase 1 (TXNRD1) is the sole activating-enzyme of the thioredoxin system through transmission of electron from NADPH to TXN, the ROS-scavenging member of the thioredoxin system. TXNRD1 is imperative for maintenance of intracellular redox homeostasis as confirmed when NRF2 was found to be the transcription activator of TXNRD1. Overexpression of TXNRD1 was found in human HCC with significant correlations with poor clinical prognosis and patient survival. Altogether, these findings are indicative of redox balance being vital for HCC growth. Loss-of-function studies utilizing shRNA-mediated inhibition of TXNRD1 resulted in significant induction of oxidative stress which suppressed HCC growth. The resulting oxidative stress also sensitized HCC cells towards its conventional therapeutic Sorafenib. Translationally, pharmacological TXNRD1 inhibitor auranofin (AUR) also induced oxidative stress which greatly sensitized HCC cells towards Sorafenib. Synergism between AUR and Sorafenib was observed as oxidative stress accumulations dramatically induced apoptosis in vitro and suppressed tumor formation in vivo. Our investigation demonstrated oxidative stress induction through inhibition of the thioredoxin system sensitized HCC cells towards conventional therapeutics. Combination of TXNRD1 inhibitor AUR and Sorafenib represents a novel treatment regimen, with enhanced efficacy, for HCC patients.

#883

PRDX4 as a novel target for pancreatic cancer.

Pallavi Jain,1 Azin Sayad,1 Erik Mollen,2 Michael Xie,1 Kevin Brown,3 Jason Moffat,4 David Hedley,5 Paul Boutros,6 Bradley Wouters,1 Marianne Koritzinsky1. 1 _University Health Network, Toronto, Ontario, Canada;_ 2 _University of Maastricht, Maastricht, Netherlands;_ 3 _Donnelly Centre for Cellular and Biomolecular Research, Toronto, Ontario, Canada;_ 4 _University of Toronto, Toronto, Ontario, Canada;_ 5 _Laboratory of Pathobiology, Toronto, Ontario, Canada;_ 6 _Ontario Institute of Cancer Research, Toronto, Ontario, Canada_.

Pancreatic cancer (PCa) is a fatal malignancy showing a 5% 5-year survival rate, creating an urgent need for new therapeutic targets. Many cancers have high production of reactive oxygen species (ROS), leaving them potentially vulnerable to targeting proteins involved in ROS metabolism. In line with this, we identified Peroxiredoxin 4 (PRDX4) as an essential gene in 26% of pancreatic cell lines by mining functional genomics datasets. PRDX4 is localized in the endoplasmic reticulum (ER) where it metabolizes H2O2. We validated in nine established PCa cell lines and patient derived primary cell lines that depletion of PRDX4 using siRNA or shRNA leads to inhibition of proliferation and cell death in a subset of pancreatic cancer cells. This was accompanied by increased levels of reactive oxygen species (ROS) as measured by flow cytometry. Dual-Depletion of NADPH oxidase 4 (NOX4) and PRDX4 could rescue the cell death phenotype thereby indicating that increase in ROS was a consequence of NOX4 activity. PRDX4 depletion led to focal accumulation of phosphorylated H2AX (γH2AX) in the cellular nuclei, consistent with DNA damage. Phosphorylation of H2AX was exclusively dependent upon activation of the upstream kinase DNA-PKcs. Cells depleted for PRDX4 were also more sensitive to ionizing radiation. Depleting PRDX4 with inducible shRNA in established xenografts inhibited tumor growth. Finally, using transcriptome data accompanying the functional genomics datasets, we identified a 2-gene expression signature that predicts sensitivity to PRDX4 targeting.

This study indicates that depletion of PRDX4 causes increased ROS resulting in DNA damage leading to DNA-PKcs activation, inhibition of proliferation and cell death in a subset of pancreatic cancers. PRDX4 is a potential novel therapeutic target that may act synergistically with radiation therapy.

#884

Interplay between NRF2 and GPX4 is critical for cancer cell survival in 3D spheroids.

Patricia Cho, Nobuaki Takahashi, Laura M. Selfors, Hendrik J. Kuiken, Joan S. Brugge. _Harvard Medical School, Boston, MA_.

Antioxidants have historically been thought of as cancer preventative agents; however, increasing evidence in recent years indicate that antioxidants promote cancer cell survival. Using 3D spheroid models, our lab previously demonstrated that normal cells die in the inner layers of spheroids, whereas cancer cells survive. Multiple lines of evidence indicate that survival of inner cells within tumor spheroids is dependent on escape from apoptosis and reactive oxygen species (ROS)-induced cell death through upregulation of anti-apoptotic and antioxidant programs. While the mechanisms associated with apoptosis are well-established, there is relatively little understood about the cellular processes involved in ROS-induced cell death (origins of ROS, types of oxidative stress experienced, mechanisms of cell death, antioxidants involved etc.). Tumor cells with mutations in the nuclear factor erythroid 2-related factor 2 (NRF2) pathway that lead to hyperactivation of this transcription factor serve as an excellent model to study the role of antioxidant programs in the regulation of survival processes. NRF2 is the master regulator of the antioxidant response directly regulating expression of antioxidant enzymes as well as proteins required for the production of non-enzymatic antioxidants. Furthermore, mutations in the NRF2 pathway are frequent in tumor types that are associated with oxidative stress such as lung cancer. We found that knock-down of NRF2 affected cell survival and growth in 3D by regulating (i) cell proliferation and (ii) inner cell survival. To better understand the specific processes responsible for ROS-induced cell death and the antioxidant program(s) utilized by cancer cells to bypass these death programs, we performed CRISPR screens in 3D using lung cancer cell lines with NRF2 hyperactivation to identify downstream targets of NRF2 and/or antioxidants that collaborate with NRF2 to regulate cell survival and growth in 3D. As predicted, we observed drop-out of sgRNAs targeting NRF2. Notably, a top drop-out hit was glutathione peroxidase 4 (GPX4), an essential antioxidant enzyme that reduces membrane phospholipid hydroperoxides to prevent a form of non-apoptotic cell death known as ferroptosis. We found that inhibition of GPX4 resulted in elevated levels of lipid peroxidation and death in the inner cells of 3D spheroids which were rescuable by the ferroptosis inhibitor Ferrostatin-1, implicating ferroptotic cell death in 3D biology. Surprisingly, knock-down of NRF2 resulted in stabilization of GPX4 at the post-transcriptional level. Finally, knock-down of NRF2 combined with GPX4 inhibition induced cell death in both inner and outer cells of 3D spheroids. These data suggest that cancer cells exploit the functional interplay between NRF2 and GPX4 to survive in high oxidative stress environments. This work was supported by funding from the ROADS Program funded by F. Hoffmann-La Roche Ltd..

#885

Induction of PRDX2 by H. pylori reduces ROS and promotes cancer cell survival and resistance to cisplatin.

Sen Wang,1 Zheng Chen,1 Heng Lu,1 Shoumin Zhu,1 Dunfa Peng,1 Mohammed Soutto,1 Ahmed Gomma,1 Nadeem Bhat,1 Huma Naz,1 Zekuan Xu,2 Wael El-Rifai1. 1 _University of Miami, Miami, FL;_ 2 _The First Affiliated Hospital of Nanjing Medical University, Nanjing, China_.

Background & Aims: The antioxidant enzyme peroxiredoxin 2 (PRDX2) plays a critical role in regulating reactive oxygen species (ROS) levels in several diseases. Helicobacter pylori (H. pylori) infection is a well-known risk factor of gastric cancer. The role of PRDX2 in gastric tumorigenesis remains largely unknown. We investigated the molecular function and regulation of PRDX2 in response to infection with H. pylori and cisplatin treatment in gastric cancer cells.

Methods:Western blots (WB) and quantitative real-time PCR (qPCR) analysis were performed on AGS, SNU-1 and MKN28 gastric cancer cell lines to detect PRDX2 expression levels with or without H. pyloriinfection (7.13 or J166 strain). We evaluated the levels of ROS by H2DCFDA staining. WB analysis was used to determine oxidative DNA damage and double stranded DNA breaks by using antibodies against 8-Oxo-guanine and p-H2AX. WB and NF-ƙb luciferase reporter assay were performed to investigate the link between PRDX2 and NF-ƙb signaling. ATP-GLO cell titer analysis was utilized to determine cell viability.

Results:Our data indicated that PRDX2 mRNA and protein expression levels were induced byH. pyloriin AGS and SNU-1 cells. PRDX2 knockdown significantly increased ROS levels and 8-Oxoguanine staining followingH. pyloriinfection in both cells. These results were further confirmed by Western blot data showing that p-H2AX protein level was strongly induced in PRDX2 knockdown and H. pyloriinfected cells. Interestingly, Western blot data showed that TNF-αtreatment induced PRDX2 protein levels while Bay 11-7082 treatment decreased PRDX2 protein level in AGS and SNU-1 cells. At the same time, PRDX2 transient knockdown in both AGS and SNU-1 cells decreased p-P65 (S536) protein expression levels, nuclear localization of NF-ƙB-p65, and luciferase reporter activity (P<0.05). We also found that inhibition of PRDX2 significantly sensitized AGS and SNU-1 cells to cisplatin treatment.

Conclusion:Our data indicates that knockdown of PRDX2 enhances ROS and DNA damage with H. pylori infection on gastric cancer cells after H. pylori infection and sensitizes gastric cancer cells to cisplatin treatment. Our results suggest a positive feedforward loop between PRDX2 and NF-kB to reduce ROS levels.

KEY WORDS: PRDX2,H. pylori, gastric cancer, reactive oxygen species, DNA damage, NF-κb

#886

Reduced p53 promotes survivorship in response to oxidative stress.

Jada R. Carter, Elshaddai White, Cimona V. Hinton. _Clark Atlanta University, Atlanta, GA_.

Cellular oxidative stress is commonly observed in aggressive cancers and initiates mechanisms that enable adaptation and survival in tumor cells. Traditionally, reactive oxygen species (ROS) have been thought to be cellular damaging agents lacking a physiological function; however, increased ROS generation and oxidative damage are connected to multiple diseases, including neurodegenerative disorders, aging, and cancer. The p53 tumor suppressor gene is often mutated in most cancers, and under normal conditions, functions to initiate apoptosis in response to oxidative stress. Thus, absence of p53, or a mutation rendering it non-functionality, can aid in tumor cell survival. We determined whether prostate cancer cells survived oxidative stress, and whether survivorship was enhanced by the absence of p53. Measurement of ROS generation in serum-starved and serum-treated PC3, DU145 and LNCaP prostate cancer cell lines were performed to demonstrate induction of oxidative stress. We observed that serum-starved cells generated more ROS and oxidative stress compared to cells in serum, and that cells further adapted to stress with the addition of hydrogen peroxide. To further determine adaptation to oxidative stress, we assayed for Annexin-V apoptosis. Previous data in the lab demonstrated that neither cell line demonstrated robust proliferation under oxidative stress; however, DU145 and PC3 cells demonstrated better survival and adaptation to oxidative stress through lower apoptosis profiles compared to LNCaP cells. Survivorship of oxidative stress may be attributed to p53 as LNCaP cells reportedly have functional p53 in literature compared to DU145 and PC3. Therefore, we immunoblotted for phosphorylated p53 expression in response to oxidative stress. To further demonstrate non-functional p53 promotes survivorship in response to oxidative stress, we performed a genomic knockdown of p53 via siRNA and demonstrated survivorship to oxidative stress may be attributed to non-functional p53 via live/dead cell viability assay. Our results correlate with studies demonstrating tumor cell resistance to ROS-mediated cell death contributing to more aggressive phenotypes and activation of anti-apoptotic events. Therefore, our study will extend our understanding of the functionality of p53 in oxidative stress adaptation and cancer cell survival. Moreover, in the future, we will investigate the inverse relationship between p53 and pro-survival factors like nuclear factor kappa B (NF-κB) in oxidative stress adaptation and cancer cell survival.

Acknowledgements: These studies were supported by the NIH/NIGMS/RISE #5R25GM060414-15 and NIH/NIHMD/RCMI #5G12MD007590.

#887

Silver nanoparticles and ionizing radiation induce mitochondrial protein oxidation and effects on cell cycle and proliferation in lung cancer cell lines.

Reetta J. Holmila,1 Stephen A. Vance,2 Ravi Singh,1 S. Bruce King,2 Cristina M. Furdui1. 1 _Wake Forest University Health Sciences, Winston-Salem, NC;_ 2 _Wake Forest University, Winston-Salem, NC_.

Silver nanoparticles (AgNP) are widely applied nanomaterials for both commercial and clinical biomedical applications. AgNP have been shown to have cytotoxicity in various cancer cell lines, as well as radiosensitizing properties. The AgNPs may induce free radicals via Fenton-type reactions, but the cellular mechanisms related to reactive oxygen species (ROS) production remain largely unknown. Both ionizing radiation (IR) and AgNP exposure lead to apoptotic and necrotic cell death through mechanisms that involve DNA damage and induction of oxidative stress, possibly targeting mitochondria. Changes in the thiol redox state of mitochondrial proteins, such as protein sulfenylation, play an important role in a number of cellular processes, and mitochondrial damage and dysfunction along with increased mitochondrial ROS are implicated in a range of human diseases such as cancer. In this work we studied the molecular effects of AgNP exposure with and without IR in a panel of lung cell lines (three cancer cell lines: A549, Calu-1 and NCI-H358 and one immortalized lung epithelial cell line: BEAS-2B). Both AgNP and IR exposures increased mitochondrial protein oxidation detected by a recently developed mitochondria targeted probe for protein sulfenylation (DCP-NEt2C). This increase in DCP-NEt2C labeling was dose and time dependent and the exposures also showed a combined effect. Moreover, the exposures induced changes in cell cycle and decreased cellular proliferation. There were marked differences between different cell lines, likely reflecting differences in the cellular redox state and metabolism. In conclusion, the AgNP and IR exposures increase the mitochondrial ROS as demonstrated by increased protein oxidation and have effects on the cell cycle and cell proliferation. They also appear to have a combined effect which might have implications for clinical applications as well as environmental risk assessment.

#888

Exploring the signaling and activity interactome between oncogenic RAS and the nucleotide pool-detoxifying enzyme MTH1.

Govindi J. Samaranayake, Clara I. Troccoli, Christina Jayaraj, Brittany C. Durden, Nagaraj Nagathihalli, Nipun Merchant, Priyamvada Rai. _University of Miami, Miami, FL_.

MutT Homolog 1 (MTH1) is a NUDIX pyrophosphorylase that hydrolyzes oxidized purine nucleoside triphosphates in the nucleotide pool, thus preventing their incorporation into DNA. Our prior work has shown that MTH1 is critical for the maintenance of multiple pro-tumorigenic phenotypes in oncogenic RAS-driven cancer cells, with its depletion leading to decreased tumor formation in vivo. Our subsequent analyses of TCGA patient datasets showed elevated MTH1 expression to be significantly associated with poorer disease-free survival in RAS-mutated cancers, such as that of the lung and pancreas. We found that MTH1 mRNA levels were positively correlated with KRAS levels even in early-stage non-small cell lung cancer patient tissues, and that the introduction of oncogenic KRAS was sufficient to upregulate MTH1 mRNA and protein levels. The aim of this study is to identify RAS-effector signaling intermediates affecting MTH1 expression and activity.

Chemical inhibitors of the MAPK/ERK, PI3K/AKT and NOX pathways, plus oncogenic RASV12-effector domain mutants (RASV12- S35/ E38/ G37/ C40), were used to identify key signaling molecular mediators of MTH1 expression in the distinct RAS isoforms (H- and K-RAS). The dependencies of the different KRASG12-mutant polymorphisms (KRASG12- C/ D/ V) on MTH1 expression and activity, as well as candidate transcription factors regulating MTH1 expression, were evaluated.

Our work shows MTH1 at the nexus of crosstalk between different effector pathways activated downstream of RAS. Dissecting these signaling intermediates are important in identifying alternate pathways of MTH1 regulation, which may manifest as resistance mechanisms to standard-of-care cancer treatments. Our work will also help understand how to best leverage MTH1 as a therapeutic target in oncogenic RAS-driven cancers driven by the different isoforms, and their respective mutant polymorphisms.

#889

**Biochemical and histopathological evaluation of Al** 2 **O** 3 **nanomaterials in kidney of Wistar rats.**

Anita Patlolla, Paul Tchounwou. _Jackson State University, Ridgeland, MS_.

The present study was investigated to evaluate the response of kidneys in Wistar rats following long term exposure to Al2O3 nanomaterials. To achieve this objective, Al2O3 of three different sizes (30nm, 40nm and bulk) was orally administered for 28 days to 9 groups of 10 Wistar rats each at the dose of 500, 1000 and 2000 mg/kg/rat. A tenth group of 10 rats received distilled water and served as control. After 28 days of exposure the animals were sacrificed and the serum was collected and tested for the activity levels of creatinine and urea following standard methods. Induction of oxidative stress was also investigated by assessing TBARS (MDA), Protein carbonyl, reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) activities. A Histopathological evaluation was also performed to determine the extent of kidney damage. The results showed that both serum creatinine and serum urea levels increased significantly in the treated rats compared to control animals. The increase was found to be more in Al2O3-30nm followed by Al2O3-40nm and Al2O3-bulk treated rats in a dose-dependent manner. Further administration of Al2O3 significantly increased the activities of TBARS, protein carbonyl, catalase and decreased the activities of GSH and SOD in a dose dependent manner in the kidney of rats compared with control group. Histopathological evaluation showed significant morphological alterations in kidney tissue of treated rats in accordance with biochemical parameters. Taken together, the results of this study demonstrate that Al2O3 is nephrotoxic and its toxicity may be mediated through oxidative stress. Further, the results suggest that prolonged oral exposure to Al2O3 NMs has the potential to cause biochemical and histological alterations in kidney of rats at high concentration.

#890

Nuclear factor kappa B (NF-κB) promotes oxidative stress adaptation and survivorship.

Elshaddai White, Jada R. Carter, Cimona V. Hinton. _Clark Atlanta University, Atlanta, GA_.

Cellular oxidative stress is one of the several hallmarks of the aggressive phenotypes observed in prostate cancer. As levels of reactive oxygen species (ROS) increase, oxidative stressors activate mechanisms allowing cancer cells to adapt and survive. It is unclear how cancer cells adapt and survive oxidative stress; however, literature demonstrates that the transcription factor, nuclear factor-kappa B (NF-κB) contributes to survival during oxidative stress. In cancer, several factors resulting in oxidative stress, including chemotherapy, correlate with NF-κB expression and transcriptional activity, enhancing tumor survivorship and resistance to therapies. We examined adaptability and survivorship to oxidative stress in two common prostate cancer models, DU145 and PC3, which display varying degrees of tumorigenicity, respectively. We observed that serum-starvation induced ROS, stimulating an early oxidative stress environment, and cells further conferred adaptability with the addition of hydrogen peroxide (H202). Measurement of cell viability demonstrated a low death profile in stressed cells, while cell proliferation was stagnant. Quantitative measurement of apoptosis showed no significant cell death in stressed cells suggesting an adaptive mechanism to oxidative stress survival. Consistent with stress adaptation, we observed NF-κB in nuclear fractionations of cell lines which were consistent with nuclear localization observed in other diseases in literature that adapt to oxidative stress. Adaptability to oxidative stress may be a precursor to the inflammatory response in prostate tumor tissues, and/or a general survival mechanism to anti-tumorigenic agents.

Acknowledgements: These studies were supported by the NIH/NIGMS/RISE #5R25GM060414-15 and NIH/NIHMD/RCMI #5G12MD007590.

#891

**Plumbagin and atovaquone inhibit Na** + **/K** + **-ATPase through the generation of oxidative stress.**

Yousef Alharbi, Arvinder Kapur, Mildred Felder, Lisa Barroilhet, Bikash Pattnaik, Manish S. Patankar. _Univ. of Wisconsin-Madison, Madison, WI_.

Plumbagin and atovaquone are chemotoxic to ovarian, breast and other tumors. Both molecules inhibit oxidative phosphorylation causing a rapid increase in intracellular oxygen radicals and apoptosis. Oxidative stress is also known to inhibit the activity of Na+/K+-ATPase (NKA), the ion channel that maintains the membrane potential. Here, we investigate if the oxidative stress mediated by plumbagin and atovaquone also leads to inhibition of NKA activity. We confirm that plumbagin and atovaquone inhibit proliferation of three human (OVCAR-3, SKOV-3 and TYKNu) and one mouse (ID8) ovarian cancer cell lines. Using SKOV-3 and OVCAR-3 as models for ovarian cancer, we demonstrate that oxygen radical scavenger, N-acetylcysteine (NAC) attenuated the cytotoxicity of plumbagin and atovaquone. Using whole cell patch clamping we demonstrate that plumbagin and atovaquone inhibit outward and inward current flowing through NKA in SKOV-3 and OVCAR-3. Both drugs decrease cellular ATP. Providing exogenous ATP (5 mM) in the pipet solution used during patch clamping did not recover NKA activity in the plumbagin or atovaquone treated SKOV-3 and OVCAR-3 cells. However, pretreatment of the cells with NAC completely abrogated the NKA inhibitory activity of plumbagin and atovaquone. Exposure of the SKOV-3 cells to either of the drugs for 1-2 h resulted in a significant decrease in the expression of NKA. We conclude that oxidative stress caused by plumbagin and atovaquone degrades NKA and hence the membrane potential cannot be optimally maintained. Evaluation of compounds that induce oxidative stress should therefore consider contribution of NKA inhibition to the cytotoxic activity of such agents.

#892

Alterations of vitamin B1 homeostasis following oxidative stress in breast cancer and impact of supplementation on cellular redox homeostasis.

Hunter C. Jonus, Ashley E. Ray, Jason A. Zastre. _University of Georgia College of Pharmacy, Athens, GA_.

Vitamin B1, or thiamine, has been demonstrated to enhance the development and growth of breast tumors in vivo. In addition, alterations in the expression of critical thiamine homeostasis genes have been extensively described in pre-clinical models of breast cancer as well as in clinical breast tumor tissue. Despite the effort that has been placed on delineating alterations to thiamine homeostasis that occur during breast cancer, there remains little evidence demonstrating a molecular advantage for supplemental thiamine in breast cancer cells. Our previous work in colorectal cancer suggests that the proliferative benefits of thiamine may be related to the maintenance of oxidative stress. This appears to be facilitated through the adaptive regulation of the thiamine activating enzyme thiamine pyrophosphokinase-1 (TPK1) for thiamine pyrophosphate production. Here, using MCF7 cells as a model of breast cancer, we also identified a relationship between thiamine homeostasis and oxidative stress. Similar to previous findings in colorectal cancer, Western blot analysis revealed that TPK1 expression was up-regulated in response to enhanced levels of ROS induced by hypoxia and chemotherapeutic treatment. TPK1 knockdown mediated by shRNA reduced the proliferation of MCF7 cells, while the presence of supplemental thiamine promoted proliferation as determined by cell counting. The effect of supplemental thiamine on MCF7 proliferation corresponded with a direct impact to the antioxidant status of tumor cells measured by reduced nuclear accumulation of the transcription factor nuclear factor- erythroid 2-related factor 2 by Western blot analysis. Molecular probes detecting intracellular ROS revealed that supplemental thiamine did not reduce the basal level of ROS in MCF7 cells. However, enhanced thiamine reduced intracellular superoxide levels following stimulation with the electron transport chain inhibitor Antimycin A. These findings support that during supplemental thiamine conditions thiamine homeostasis may be exploited in breast cancer for a redox advantage contributing to tumor progression.

#893

**The cofilin-TEAD1-p27** Kip1 **pathway ablates cell senescence.**

Yi-Jang Lee, Cheng-Han Tsai, Chun-Yuan Chang. _National Yang-Ming University, Taipei, Taiwan_.

Morphological change is an explicit characteristic of cell senescence, but the underlying mechanisms remain to be addressed. Here we demonstrated, after a survey of various actin-binding proteins, that up-regulation of cofilin-1 was essential for the reduced rate of actin depolymerization in senescent cells, as well as their morphological enlargement . Additionally, the up-regulated cofilin-1 mainly existed in the serine-3 phosphorylated form according to the 2D gel immunoblotting assay. Accumulation of cofilin-1 in senescent cells depended on the post-translational mechanism, as the ubiquitin-proteasomal degradation of cofilin-1 was reduced without any change in mRNA level. Up-regulation of cofilin-1 was not only found in replicative senescence but also in oxidative stress- and K-Ras oncogene-induced senescence. Over-expression of wild-type cofilin-1 and constitutively phosphorylated cofilin-1 promoted cell senescence with increased cell size. Additionally, senescent phenotypes were reduced by knockdown of total cofilin-1. The senescence induced by over-expression of cofilin-1 was dependent on p27Kip1 but not p53 and p16INK4 expression. Knockdown of p27Kip1 alleviated cell senescence induced by oxidative stress or replicative stress. We also found that over-expression of cofilin-1 induced expression of p27Kip1 through transcriptional suppression of the TEAD1 transcription factor. The TEAD1 transcription factor played a transrepressive role on the p27Kip1 gene promoter as determined by the promoter deletion reporter gene assay. Interestingly, TEAD1 down-regulation was accompanied by up-regulation of cofilin-1 in senescence. Knockdown and restoration of TEAD1 in young cells and old cells could induce and inhibit p27Kip1 and cell senescence, respectively. Furthermore, senescence induced by the over-expression of cofilin-1 could be alleviated by ectopic expression of TEAD1. Taken together, current data suggest that cofilin-1/TEAD1/p27Kip1 signaling is involved in senescence-related morphological change and growth arrest.

Keywords: Cofilin-1, TEAD1, p27Kip1, morphology, growth arrest, senescence

#894

**A Self-Immobilizing NIR Probe for** in vivo **Imaging of Chemotherapy Induced Senescence.**

Xiaowei Ma,1 Jun Liu,1 Ying Wang,2 Chao Cui,1 Philip Deenik2. 1 _University of Florida, Gainesville, FL;_ 2 _University of New Mexico, Albuquerque, NM_.

Post-treatment relapse and distant metastasis are significant causes of mortality by cancers. Previous research has demonstrated that cancer treatments, such as chemotherapy, can sometimes stimulate tumor spread. This phenomenon is associated with an essential process that some cells are resistant to apoptosis by entering premature senescence after therapy. Thus, chemotherapy-induced cellular senescence may play a critical role in cancer recurrence and metastasis. To monitor the cellular senescence induced by chemotherapy, we developed a self-immobilizing near infrared (NIR) probe (4-152) that can be activated by senescence-associated beta-galactosidase (β-gal) and get immobilized at site of activation, allowing the monitoring of chemotherapy-induced senescence in vivo. We first verified our approach in vitro, when cervical cancer cells (HeLa) were treated with camptothecin (CPT), followed by fluorescence imaging with 4-152. The results demonstrated that our designed probe exhibited a dramatic NIR fluorescent response in cells receiving CPT treatment. The fluorescent western blot showed that the probe could be activated by β-gal and attached to the enzyme. The confocal microscopy imaging and flow cytometry results further demonstrated the 4-152 could accumulate in the CPT-induced senescent cancer cells. We then investigated the in vivo imaging ability of the probe for senescence detection in tumor xenografts treated with CPT. After treatment with CPT or saline (as control), 4-152 was administrated to the mice by tail vein injection. NIR imaging was then performed on the IVIS Spectrum in vivo imaging system. The imaging showed that the 4-152 could quickly distribute to the tumor and accumulated for over 24 hours. The fluorescence intensity of the CPT treated tumors was significantly higher than the untreatment tumors. After imaging, tumors and major organs were resected for ex vivo imaging. The fluorescent intensity of tumors showed 6.27-fold higher than liver. Ex vivo immunofluorescence staining and X-gal staining further confirmed the correlation of probe 4-150 and senescence cells.In conclusion, we have developed an activatable self-immobilizing NIR probe for the monitoring of chemotherapy-induced cellular senescence in vivo. This probe may be used for the treatment effect monitoring and therapeutic regimen optimization in the future.

Reference1. Ebos JM. Prodding the beast: assessing the impact of treatment-induced metastasis. Cancer Res 2015; 75: 3427-3435.

#895

The identification of molecular mechanism underlying the progression of gastric cancer peritoneal dissemination by senescent fibroblasts.

Tadahito Yasuda, Mayu Koiwa, Tomoyuki Uchihara, Atsuko Yonemura. _Kumamoto University, Kumamoto-shi, Kumamoto, Japan_.

Background: Secreted factors from cancer associated fibroblast (CAFs) is likely to be implicated in cancer progression, but the precise mechanism is not fully understood. It is reported that senescent cells survive and secrete some cytokines, so called senescence-associated secretory phenotype (SASP). The purpose of current study is to identify the molecular mechanism underlying the progression of gastric cancer (GC) peritoneal dissemination by CAFs showing SASP.

Methods: We examined the senescent state of CAFs activated NFkB signaling by inflammatory cytokines (iCAFs) and evaluated the expression of SASP factors. We investigated the growth ability of GC cell lines treated with iCAFs derived condition medium (CM). We transplanted GC cell lines with intraperitoneal administration to generate peritoneal metastasis mouse model and evaluated the influence of SASP factors on cancer progression.

Results: We found that the growth ability of iCAFs decreased and their expression of p16 and p15, which was senescent markers increased. And the comprehensive genomic analysis revealed that iCAFs was highly enriched the senescent related genes and SASP factors related genes. Next, we also found that iCAFs derived CM significantly increased the growth ability of GC cells. Moreover, also in peritoneal metastasis mouse model, iCAFs derived CM remarkably enhanced intraperitoneal tumors.

Conclusion: These findings suggest that SASP factors from senescent CAFs, which activated by inflammatory cytokines can promote peritoneal dissemination of GC cells.

#896

Knocking out the gene (SNCA) that codes for the Parkinson's disease-related protein alpha-synuclein in SK-Mel-28 melanoma cells significantly retards tumor growth in SCID mice.

Sahar Shekoohi, Santhanasabapathy Rajasekaran, Shu Yang, Sureshbabu Nagarajan, Dhaval Patel, Xiuping Yu, Stephan N. Witt. _Louisiana State University Health Sciences Center, Shreveport, LA_.

The Parkinson's disease-associated protein alpha-synuclein (α-syn) is often highly expressed in melanoma. α-Syn appears to exert a pro-survival function in melanoma cells, possibly by maintaining autophagy homeostasis. The long-term goal of our work is to determine the mechanism by which α-syn confers survival in melanoma cells. Herein, we used CRISPR (clustered regularly interspaced short palindromic repeats) to disrupt SNCA, the gene that codes for α-syn, in the melanoma cell line SK-Mel-28. Western blotting and real-time-qPCR verified that we generated four α-syn knockout (KO) clones. Three clones showed no expression of α-syn, whereas one had about 50% less α-syn than the control cells. DNA sequencing is consistent with three of the clones having both alleles of SNCA knocked out, whereas one clone has only one allele knocked out. α-Syn KO clones were tested in a variety of assays to ascertain the function of α-syn. First, the percentage of cells with exofacial phosphatidylserine (PS) was determined by annexin V-FITC/PI staining in conjunction with fluorescence-activated cell sorting. On average, there was a statistically significant 50% increase in exofacial PS (annexin V+/PI-) in three of the four α-syn KO clones compared to control cells. Exofacial PS was increased in the α-syn KO clones without a concomitant increase in apoptotic cells (annexin V+/PI+). Second, the clones were tested for growth in vivo using SCID mice. All four clones showed a ~65% reduction in the rate of tumor growth, followed over 12 weeks. Third, immunohistochemical staining of excised tumors from the SCID mice showed that the proliferation marker Ki-67 was significantly (p< 0.0001) lower in the tumors generated from the KO clones compared to tumors from control cells. In conclusion, given that α-syn avidly binds to PS, one possibility is that α-syn is like molecular Velcro that binds to PS molecules embedded in the inner leaflet, and this binding helps ATP-driven flippases keep PS in the inner leaflet. Loss α-syn then increases exofacial PS, which is a marker of senescence. Our working model is that α-syn is an anti-senescence protein, and that the α-syn KO clones grow slower than control cells in vivo because the clones have entered a senescent state.

#897

Lipid metabolism is involved in mitotic slippage-induced SASP upon treatment with anti-mitotic drugs.

Karen C. Crasta, Alex Wong, Bryan Lim. _Nanyang Technological Univ., Singapore, Singapore_.

Aberrant lipid accumulation is a hallmark of cancer. However, there is little known regarding mechanisms linking lipid metabolism to chemotherapy-induced cell fates. Here, we describe lipid accumulation in cells following antimitotic drug-treatment. Cells arrested in mitosis, as well as cells that escaped mitotic arrest and underwent mitotic slippage, showed elevated cytoplasmic lipid droplets. Interestingly, we found that TOFA, an inhibitor that targets acetyl-CoA carboxylase (ACC) and blocks lipid accumulation enhanced survival of antimitotic-treated cells. Our work previously revealed that cells that survive slippage become senescent and confer pro-tumourigenic effects through non-cell-autonomous effects. Modulating lipid biosynthesis in cells post-slippage by TOFA amplified their inflammatory secretion profiles and accelerated development of tumourigenic behavior. In contrast to TOFA, inhibition of lipid accumulation by C75, a drug targeting fatty acid synthase, significantly reduced the production of pro-tumourigenic factors. This suggests that discrete lipid biosynthesis pathways could contribute differentially to the regulation of pro-tumourigenic inflammation.

#898

Evaluation of cellular senescence through fluorescence characterization.

Scott T. Clarke,1 Taryn Jackson,1 Amanda Kelley,2 Quentin Low,1 Timothy Huang,1 Yi-Zhen Hu,1 Veronica Calderon,1 Rong Wang,2 Michael O'Grady1. 1 _Thermo Fisher Scientific, Eugene, OR;_ 2 _Oregon State University, Corvallis, OR_.

Aging is considered a risk factor for developing many chronic diseases which includes cardiovascular diseases, cancer, and neurodegenerative diseases. Researchers are now showing that many of these diseases are associated with cellular senescence. Cellular senescence is an important process for the removal of damaged cells, but if senescent cells are not removed a chronic pro-inflammatory environment ensues, increasing the risk of many age-related diseases. These cells accumulate as a result of cancer chemotherapy, are associated with age-related diseases, and are important for development. Recent work has shown that specific targeting of senescent cells results in increased life expectancy in a progeroid Ercc1-/Δ mouse model. As such there is great interest in identifying, characterizing and targeting senescent cells. Senescent cells are identified by the release of pro-inflammatory cytokines and chemokines, increased beta galactosidase activity (b-gal), senescence associated heterochromatin foci (SAHF), and changes in morphology which are increased size and a flattened appearance.

There is no single good marker for determining senescence, so these cells are identified through the collective interpretation of multiple markers. Senescence associated b-gal (SA-b-gal) is considered the "gold standard" for identifying senescent cell, and it is detected with the blue-green precipitating colorimetric x-gal substrate. However, this substrate is limited use in that it cannot be used in flow cytometry or other fluorescence based platforms. Even though C12FDG, a fluorescein based b-gal substrate, has been used since the mid 90's, it has limited utility restricted by its lack of fix-ability. Newer b-gal substrates are demonstrated here which do not leak out of cell; enabling researchers to more easily identify and evaluate cellular senescence.

In this study we use a fluorescence approach to assess the senescence phenotype based on a combination of western blots, fluorescence based imaging and ICC, flow cytometry, cell sorting, and RT-qPCR. We examined several cell-based models include replicative senescence characterized by telomere shortening in primary fibroblasts, cellular senescence characterized through DNA damage pathway by doxorubicin, oxidative stress senescence induced by confluency in atmospheric oxygen environment (SIPS), and senescence induced with palbociclib treatment (a CDK 4/6 inhibitor). Overall, we found that phenotypic characteristics of senescence vary depending on the cellular model and that a multiplex fluorescence based approach to identifying senescent cells will contribute to the understanding of cellular senescence and the age-related diseases.

#899

Selective clearance of cells undergoing therapy-induced senescence in a model of triple negative breast cancer.

Liliya Tyutyunyk, Tareq Saleh, David A. Gewirtz. _Virginia Commonwealth University, Richmond, VA_.

Despite the ability of chemotherapy (and/or radiation) to eliminate the majority of tumor cells, some residual surviving cells may be able to escape cell death by entering into a prolonged senescence-based growth arrest. In addition to apoptosis, the majority of anti-cancer therapies induce senescence that has long been considered to be a favorable outcome of treatment due to its perceived irreversibility. However, accumulating studies in the literature demonstrate that therapy induced senescence may reflect one form of tumor dormancy and disease recurrence, when senescent cells are able to regain proliferative capacity. Tumor cells that escape dormancy often demonstrate an aggressive phenotype and are refractory to further treatment. Senescent cells are characterized by a specific (though often heterogeneous) secretory profile (Senescence Associated Secretory Phenotype or SASP), some components of which have been shown to promote tumorigenesis and may assist in neoplastic escape after therapy. In the current work, we evaluated the capacity of the BCL protein family inhibitor, ABT 263, to selectively clear breast tumor cells induced into senescence by doxorubicin. MDA-MB231-231 human breast cancer cells (a model of triple negative breast cancer) were induced into senescence upon exposure to a clinical concentration of the chemotherapeutic agent, doxorubicin (Dox). While a fraction of the MDA-MB-231 cell population underwent cell death, a large subpopulation entered into a state of prolonged growth arrest that lasted for several days, after which time the cells recovered proliferative capacity and formed colonies. At the time points when cells were arrested, prominent Senescence Associated β-Galactosidase staining marker indicative of senescence. In addition, tumor tissue collected from mice that received injections of Dox also were positive for β-Galactosidase staining. Cells induced into senescence and treated with ABT263 for 18 hours showed a significant increase in apoptosis and did not regain colony forming capabilities. We therefore postulate that senescence and escape from senescence may represent a form of tumor dormancy and disease recurrence, respectively. Consequently, if Therapy Induced Senescence is a deleterious outcome of treatment, it is of importance to identify strategies that might prevent senescent cells from neoplastic escape and induce cell death in order to completely eliminate residual tumors and thereby interfere with disease recurrence.

#900

New molecular mechanisms mediating oncogenic Ras-driven malignant transformation and therapeutic potential.

Mengfeng Li, Junchao Cai, Xiaonan Xu. _Sun Yat-sen University, Guangzhou, China_.

Mutations in Ras oncoproteins are the most common oncogenic events found in nearly one-third of human cancer cases. Interestingly, in normal cells oncogenic Ras alone usually induces senescence, and yet how oncogenic Ras-induced senescence is overridden so that a normal cell can be malignantly transformed remains to be investigated. Moreover, it has been well recognized that senescent cells secrete multiple cytokines to maintain and reinforce cellular senescence, and that senescence-associated inflammation also accompanies the bypass of senescence and assist propagation of incipient tumor cells. Hence, it is important to understand how inflammatory cytokines escape their tumor-suppressive functions in cellular senescence and convert into oncogenic inflammatory signaling during malignant transformation. Here, we show that oncogenic Ras elicits β-Trcp-induced degradation of β-catenin, represses miR-199a expression and induces intense secretion of IL1β and IL6 and consequently upregulates two crucial pro-senescent effectors p21 and IGFBP5, a downstream target of the IL6/STAT3 signaling, resulting in premature senescence in lung epithelial cells. During this process, IL1β and β-Trcp respectively represses miR-199a transcription and maturation, and miR-199a targets β-Trcp and IGFBP5 in a feedback manner. Consistently, overexpression of β-Trcp, which suppresses the β-catenin signaling to directly induce p21 transcription and upregulates IGFBP5 through decreasing miR-199a, or co-stimulation with IL1β and IL6 or co-overexpression of p21 and IGFBP5 induces senescent phenotype similar to that caused by oncogenic Ras. Interestingly, unlike IL1β and IL6, which decrease miR-199a expression, IL1β and TNFα triggers formation of a novel inflammatory transcriptional complex p65/c-Jun to directly induce miR-199a expression and resists oncogenic Ras-induced senescence, although IL1β, IL6 and TNFα are upregulated in the sera of both mice and human patients during the development of oncogenic Ras-driven lung tumors. Furthermore, blocking oncogenic Ras-induced IL1β/β-Trcp/β-catenin/p21 or/and IL6/STAT3/IGFBP5 pro-senescent pathways through enforcing miR-199a expression, or through silencing β-Trcp or stabilizing β-catenin, redirect oncogenic Ras-induced senescence to mesenchymal transformation, conferring lung epithelial cells and mouse embryonic fibroblast cells both tumorigenic and metastatic capacity. Importantly, inhibiting the transformation pathway by antagonizing miR-199a or β-catenin potently suppresses lung tumorigenesis driven by KrasG12D and aggressiveness of malignantly transformed cells. Therefore, our study presents a unique model that explains how premature senescence is induced and malignant transformation takes place, providing a foundation for developing promising diagnostic and therapeutic strategies for Ras-mutated cancers.

#901

Elimination of senescent tumor cells by ABT263 interferes with proliferative recovery and provides a two-hit therapeutic approach.

Tareq Saleh,1 Liliya Tyutyunyk-Massey,1 Graeme F. Murray,1 Moureq R. Alotaibi,2 Ajinkya S. Kawale,3 Zeinab Elsayed,1 Scott C. Henderson,4 Vasily Yakovlev,1 Lynne W. Elmore,5 Amir Toor,1 Hisashi Harada,1 Jason Reed,1 Joseph W. Landry,1 David A. Gewirtz1. 1 _Virginia Commonwealth University, Richmond, VA;_ 2 _King Saud University, Riyadh, Saudi Arabia;_ 3 _Yale University, New Haven, CT;_ 4 _The Scripps Research Institute, La Jolla, CA;_ 5 _American Cancer Society, Atlanta, GA_.

Senescence represents a fundamental response to cancer therapy. Accumulating senescent cells contribute to the deleterious outcomes of cancer therapy including cancer relapse, effects that may be largely mediated by the Senescence-Associated Secretory Phenotype (SASP). In this work, we show that tumor cells induced into senescence by etoposide retain proliferative capacity based on their capacity to generate proliferating colonies in culture as well as giving rise to viable tumors in vivo. Using a flow cytometry-based enrichment approach based on enlarged size and expression of Senescence-Associated β-galactosidase (SA-β-gal), we were able to utilize real time imaging to establish the re-emergence of non-small cell lung cancer cells from senescence-based arrest and the generation or proliferating daughter cells (i.e. self-renewal). Moreover, we implemented High-Speed Live-Cell Interferometry (HSLCI) to provide a single-cell lineage tracking of dividing senescent cells. The recovery from senescence was accompanied by resolution of several senescence-associated hallmarks, specifically SA-β-gal activity, p21Waf1/Cip1 and several components of the SASP (IL-1β, IL-6 and CXCL1). Our data suggests that Therapy-Induced Senescence (TIS) may ultimately be a transient process in that at least a subpopulation of tumor cells can recover proliferative capacity. We further demonstrate that the senolytic agent, ABT263, which has been shown to eliminate senescent cells from aging-related animal models can also eliminate senescent tumor cells that persistent after exposure to chemotherapy by shifting the response towards apoptotic cell death. Furthermore, sequential administration of ABT263 interferes with the ability of tumor cells induced into senescence by chemotherapy to recover growth potential. These studies suggest that senescent tumor cells can potentially contribute to cancer relapse by acquiring proliferative properties. The use of senolytic agents after induction of senescence by conventional or targeted therapies allows for the clearance of residual (possibly dormant) senescent tumor cells, which could serve to suppress disease recurrence and cancer mortality.

#902

The antiandrogen bicalutamide induces senescence in LNCaP cells and quiescence in Myc CaP cells.

Valerie J. Carpenter, Tareq Saleh, David A. Gewirtz. _Virginia Commonwealth University, Richmond, VA_.

The survival benefits in prostate cancer (PCa) conferred by androgen deprivation therapies (ADT) and antiandrogens are often blunted by the development of castration resistant disease (CRPC). There is increasing evidence that the primary cellular response to ADT is senescence rather than apoptosis, and this may influence progression to CRPC. However, the majority of in vitro studies employ the charcoal stripped serum model, and very few studies use antiandrogens, despite the fact that antiandrogens are currently one of the primary modes of therapy for prostate cancer. In an effort to enhance our understanding of antiandrogen effects in PCa, we evaluated the cellular response to the clinically relevant antiandrogen, bicalutamide, in the human LNCaP and murine Myc-CaP cell lines. Acute treatment (48 hours) with bicalutamide resulted in a growth arrest followed by proliferative recovery in both cell lines. However, while the growth arrest was prolonged (lasting at least 6 days) in the LnCaP cells, growth arrest was exceedingly brief (lasting approximately 24 hours) in the Myc-CaP cells. Consistent with these observations, we were able to detect increased beta galactosidase staining (a hallmark of senescence) in the LnCaP treated cells but not the Myc-CaP treated cells. Despite the fact that profound morphological changes were evident in the Myc-Cap cells, including an enlarged and elongated cell body, the highly transient nature of the growth arrest suggests that the Myc-Cap cells undergo quiescence rather than senescence. The absence of senescence induction in the Myc-CaP cells may be a consequence of the androgen receptor copy number gain. However, androgen duplication is highly uncommon in human prostate cancers prior to development of CRPC. Thus, it is likely that antiandrogens can induce senescence in treatment-naïve patients, as seen in the LNCaP cell line. The ability of bicalutamide to induce senescence, albeit perhaps conditionally, suggests that some prostate cancers treated with ADT and/or antiandrogens may be susceptible to drugs that target senescent cells (senolytics). We therefore propose that elimination of senescent cells prior to recovery could attenuate or prevent the development of CRPC.

#903

Real-time imaging of cellular senescence in living animals.

Lina Cui. _University of Florida, Gainesville, FL_.

Senescence, a cellular stage that halts proliferation, plays a critical role in cellular processes such as tumor suppression, tumorigenesis and aging. Monitoring of senescence in real-time in living objects not only holds value in basic biological studies, it can also benefit treatments of various diseases such as cancer and age-related conditions. Our lab has developed a series of small molecule based agents for non-invasive imaging of cellular senescence in real-time. We have demonstrated their applications in tumor models receiving chemo- or radiotherapy, as well as aging mice models. These probes will be useful for treatment response monitoring, therapeutic regimen optimization, as well as imaging-guided removal of aged cells.

#904

Hypoxic attenuation of ΔB-Raf-induced senescence.

Soojeong Chang, Hyunsung Park. _University of Seoul, Seoul, Republic of Korea_.

Oncogene-induced senescence (OIS) is natural surveillance mechanisms against tumorigenesis. The in-vivo oxygen concentration around tumor microenvironment has been estimated as 3%~0.5%. We investigate the effects of physiological hypoxia (1.5% O2) and pathological hypoxia (<0.5% O2) on OIS. Expression of active ΔB-Raf in human lung fibroblast IMR90 cells triggered senescence processes such as activation of senescence associated-β-galactosidase (SA-β-gal) and senescence associated heterochromatin foci (SAHF). We found that hypoxic condition delayed onset of activation of SA-β-gal. Furthermore hypoxic condition blocked formation of heterochromatin foci which was identified as a hallmark of OIS with increase of histone methylation. Rearrangement of chromatin organization by activated ΔB-Raf was also inhibited under hypoxic condition. This study shows that tumor hypoxia assists the transformed cells to overcome an obstacle of senescence caused by oncogenes. [This study was supported by the National Research Foundation of Korea (NRF), grant 2016R1A2B4012840 and 2018R1A4A1025985]

## BIOINFORMATICS AND SYSTEMS BIOLOGY

### Tools for Cancer Genome Analysis

#905

Comprehensive immunogenomic profiling of anti-PD-1 treated melanoma patients reveals subject-specific tumor escape mechanisms.

Charles W. Abbott,1 Sean M. Boyle,1 Eric Levy,1 Rena McClory,1 Sekwon Jang,2 Richard Chen1. 1 _Personalis, Inc., Menlo Park, CA;_ 2 _Inova Medical Group, VA_.

Background: Despite the remarkable response of some melanoma patients to checkpoint inhibitor therapy, the majority of patients do not achieve complete response. It is of great interest to identify biomarkers and mechanisms that influence immunotherapy effectiveness. Here we apply our comprehensive tumor immunogenomics platform (ImmunoID NeXT) to identify potential biomarkers of response to checkpoint blockade therapy related to both the tumor and tumor microenvironment.

Methods: We characterized the immunogenomics of 50 stage III/IV melanoma patients who have undergone anti-PD-1 therapy to assess potential factors influencing response. Tumor response to therapy was evaluated using RECIST criteria with a median follow-up of 12 months. Immuno-genomic profiling was performed using Personalis' ImmunoID NeXT platform; an augmented exome/transcriptome platform and analysis pipeline which from a single paired tumor FFPE and normal blood sample yielded comprehensive tumor mutation information, gene expression quantification, neoantigen characterization, TCR repertoire profiling, HLA typing and tumor microenvironment profiling. The molecular information of the tumors was then analyzed together with their corresponding clinical response.

Results: Through comprehensive immunogenomic profiling we demonstrated that higher TCR clonality in pre-treatment biopsy was predictive of response to PD-1 blockade, and significantly associated with improved progression free survival. We also observed increased response to anti-PD-1 treatment in patients with elevated pretreatment neoantigen burden. Further investigation of patients with high neoantigen burden and TCR clonality that failed to achieve complete response revealed potential resistance mechanisms to anti-PD-1 therapy. Specifically, we identified two patients with high expression of IDO1 or CTLA4, which may facilitate immune escape in a PD-1 independent manner. Additionally, we found two patients with mutations in their antigen presentation machinery (APM). The first patient had two independent HLA mutations in HLA-A and HLA-B, leading to the likely loss of surface expression of the proteins. In the second APM mutation patient we observed a high frequency (80% AF) frameshift variant in B2M, which potentially prevents proper HLA class I folding and antigen presentation. These APM mutations suggest reduced neoantigen presentation in these patients, which are probable mechanisms for tumor escape.

Conclusions: In summary, our comprehensive cancer immunogenomic analysis shows that genomic and immune profiling of pretreatment patient samples can identify biomarkers and resistance mechanisms to immune checkpoint blockade, suggesting the potential efficacy of these as a combinatorial biomarker to optimize patient selection for anti-PD-1 therapy.

#906

COSMIC: Describing the world's knowledge of somatic mutations in cancer.

Simon Andrew Forbes, david beare, charalampos boutselakis, sally bamford, kate noble, claire rye, john tate, chai yin kok, charlie hathaway, laura ponting, christopher ramshaw, raymund stefancsik, samantha thompson, bhavana harsha, nidhi bindal, shicai wang, steven jupe, helen speedy, celestino creatore, peter fish, sari ward, charlotte cole, elisabeth dawson, zbyslaw sondka. _Wellcome Trust Sanger Institute, Cambridge, United Kingdom_.

COSMIC, the Catalogue Of Somatic Mutations In Cancer (http://cancer.sanger.ac.uk/cosmic) is a continual effort to integrate all available information on somatic mutations and other molecular alterations causing every form of human cancer. Being the world's largest and most comprehensive database of somatic mutations in human cancer, it also provides web-based tools for exploration and interpretation of collected data. The content of the database is primarily obtained from the scientific literature by the team of experienced post-doctoral curators and combined with information from online sources, including the TCGA and ICGC. During thorough & exhaustive manual curation, all the available information about mutations and tumor samples (e.g. disease type, demographic data, treatments) are collected, standardized and integrated to allow for both creation of wide virtual cohorts and large-scale studies, as well as precise analysis at the level of a single sample, gene or mutation.

The 87th release of COSMIC (Nov 2018) encompasses 5,992,260 coding mutations and 19,574 gene fusions, curated from 1,403,267 cancer samples, including 35,490 whole cancer exomes/genomes, primarily hand-curated data from 26,494 scientific publications. Additionally, COSMIC describes 1,179,545 Copy Number Variants, 9,147,833 gene expression variants, and 7,879,142 differentially methylated CPGs.

In addition to this broad database, COSMIC includes a range of specialized projects highlighting specific aspects of cancer in order to emphasize events with a higher impact in disease etiology. This includes the Cancer Gene Census (http://cancer.sanger.ac.uk/census), which defines and describes genes (currently 719) and their dysfunctions driving oncogenesis, and characterizes their impact on hallmarks of cancer. COSMIC3D (http://cancer.sanger.ac.uk/cosmic3d) provides an interactive view of cancer mutations in the context of 3D protein structures, and predicts potential drug-binding sites.

Significantly updated 4 times a year, COSMIC is available free-of-charge for academic and non-profit users via COSMIC webpage (http://cancer.sanger.ac.uk/cosmic) or to download through COSMIC downloads (http://cancer.sanger.ac.uk/cosmic/download).

#907

Bioinformatic method to define epigenetically regulated enhancer elements associated with cancer.

Thais S. Sabedot,1 Seth H. Cassel,2 Galen F. Gao,3 Caleb A. Lareau,3 Andrew Cherniack,2 Alexander Lazar,4 Cigall Kadoch,2 Houtan Noushmehr1. 1 _Henry Ford Health System, Detroit, MI;_ 2 _Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA;_ 3 _Broad Institute of MIT and Harvard, Cambridge, MA;_ 4 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

BACKGROUND: Several mechanisms involved in gene regulation are altered in cancer. Cataloging these alterations can lead to a better understanding of tumorigenesis. In addition, the alterations can be used to classify patients with similar clinical features and thereby lead to better targeted treatment. Epigenetics (e.g. DNA methylation) is the process by which cells define gene regulation and aberrant DNA methylation patterns have been observed in many cancer types. Alterations in non-promoter (intergenic regions) have been shown to be tightly associated with functional genomic elements such as enhancers or transcription factor binding. In order to identify altered candidate functional elements associated with specific gene or pathways, we developed a method to integrate enhancer, DNA methylation and gene expression data, using tumor and non-tumor data.

METHOD: Using epigenome-wide platform (Illumina 850K), CpG probes were separated into promoters and intergenic regions. The intragenic CpGs were further filtered by overlapping with known functional enhancer database from multiple studies. The nearest genes to each CpG enhancer is further stratified based on differential gene expression. Each CpG/gene pair is classified as methylated or unmethylated by sample, using a 50% methylation cutoff. The mean expression of the methylated samples is calculated and pairs with lower than the bottom 10% (1.28 standard deviation) of the mean expression in the unmethylated group of samples are selected. Finally, CpG/gene pairs with at least 75% of the methylated samples have expression values lower than the mean expression in the unmethylated group of samples are classified as epigenetically silenced. CpG/gene pairs unmethylated and upregulated are called as epigenetically active. By separating samples into different epigenetically deregulated states (silenced or active), we can further characterize each sample by evaluating the association or enrichment for specific clinical features such as outcome, treatment, age at diagnosis, etc.

RESULTS: As a proof of concept, we applied our method across the TCGA PanCan cohort to identify potential enhancers regulating genes encoding subunits of the SWI/SNF protein complex. Our method was able to detect several deregulated enhancers associated with SWI/SNF genes specifically altered in each tumor type, independent of mutation. We validated the results using Hi-C data from primary cancer cell lines.

#908

Comprehensive analysis of alternative polyadenylation across cancer phenotypes.

Eddie L. Imada,1 Diego F. Sanchez,2 Tejasvi Matam,2 Leonardo Collado-Torres,3 Christopher Wilks,2 Wikum Dinalankara,2 Alexey Stupnikov,2 Ben Langmead,2 Shawn E. Lupold,2 Luigi Marchionni2. 1 _Universidade Federal de Minas Gerais, Belo Horizonte, Brazil;_ 2 _Johns Hopkins University, Baltimore, MD;_ 3 _Lieber Institute for Brain Development, Baltimore, MD_.

The three-prime untranslated region (3'-UTR) of a mRNA influences its biological behavior, from stability, post-transcriptional control through miRNAs, and availability for translation. Alternative polyadenylation (APA) can modulate 3' end site selection, and approximately 50% of coding genes are subject to it. Global transcript shortening has been reported in normal and cancer cells. APA can be seen as a regulatory step that controls differential expression of transcript isoforms, hence it can be analyzed similarly to gene expression, comparing relevant phenotypes (e.g., tumor vs. normal, survival) with appropriate statistical methods (e.g., generalized linear models, Cox proportional hazards models).We analyzed APA across 16 cancer types, taking advantage of the following public domain resources: 1) recount2, an annotation-agnostic RNA expression database for over 72,000 human samples (Collado-Torres et al, 2017); 2) Snaptron, a search engine and database that enables one to summarize expression for specific genomic regions and features (Wilks et al, 2017); and 3) APADB, the largest database collection of Human APA sites for coding and non-coding genes (Müller et al, 2014). We leveraged Snaptron to extract expression levels for 100-base-pair windows upstream and downstream APA sites defined in APADB. We annotated these genomic features, corresponding to short and long transcript isoforms, using metadata from recount2. As a proof of concept, we analyzed differential APA isoform expression in TCGA, comparing tumor vs. normal samples, and identifying APA events associated with recurrence and survival, as well as other well-defined clinical, morphologic and molecular classifications.Our preliminary results show hundreds of genes switching PA sites to shorten or extend 3'-UTR length in primary tumors when compared to normal tissues. Some of these genes are associated with cell cycle and proliferation, indicating that PA sites are dynamically used in primary tumors as another mechanism to evade and modulate post-transcriptional control. Even more interestingly, a substantial fraction of these APA isoforms were associated to tumor recurrence and survival independently from standard clinical and pathological variables.In conclusion, by leveraging public domain resources, such as APADB, recount2, and Snaptron, we created a comprehensive resource that enables to detect dynamic usage of PA sites across cancer phenotypes. Furthermore, the association of many APA isoforms with tumor progression suggests that these could serve as clinically useful biomarkers. Most importantly, the comprehensive resource we have built accounts for over 72,000 human samples, hence it is not limited to the cancer phenotypes we explored in this study. Once released in the public domain, our APA expression atlas will empower the scientific community at large to explore APA across many other cancer and human disease phenotypes.

#909

Single-cell transcriptomic profiling of non-small cell lung cancer uncovers inter- and intra-cell population structure across TCGA lung adenocarcinoma and lung squamous cancer subtypes.

Kofi E. Gyan,1 Aditya Deshpande,1 Shaham Beg,1 Huasong Tian,1 Joel Rosiene,2 Marlon Stoeckius,3 Peter Smibert,3 Davide Risso,1 Juan Miguel Mosquera,1 Marcin Imielinski1. 1 _Weill Cornell Medicine, New York, NY;_ 2 _SUNY Downstate College of Medicine, New York, NY;_ 3 _New York Genome Center, New York, NY_.

Lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) are the most prevalent types of non-all cell lung cancer (NSCLC), a leading cause of cancer death worldwide. In this study, we analyzed the transcriptomes of ~45,000 single cells (scRNA) from 13 NSCLC patients, including 5 LUAD cases which were collected and profiled at our institution. To correlate genomes and transcriptomes we performed Whole-Genome Sequencing (WGS) on 3 of these 5 LUAD cases. By comparing tumor tissue with matched adjacent non-malignant lung tissue we are able to confidently distinguish 13 cell-type specific clusters that unambiguously match previously characterized lineages. We developed algorithms for the identification of malignant cells derived from tumor tissue through scRNA analysis of copy number alterations and single nucleotide variants (SNV). Joint analysis of WGS and scRNA confirmed an enrichment of tobacco-associated SNVs among malignant cells of the tumor. Stromal cell types demonstrated consistent expression patterns across cases, while malignant cells demonstrated both inter- and intra-tumoral heterogeneity in their expression of signatures related to GPCR signaling, 3' UTR mediated translational regulation, and cell-cell junction organization. In particular, one case displayed a unique pattern of intra-tumoral heterogeneity, as a subset of malignant cells robustly express a marker of pulmonary neuroendocrine cells, CGRP. Employing immunohistochemistry, the spatial organization of these malignant cells is revealed to be mutually exclusive within the tumor microenvironment and overlapping in expression of clinical markers of small-cell lung cancer. Finally, we deconvolved bulk TCGA LUAD and LUSC gene expression samples and analyzed the relationship between cell type specific gene expression in cell types of the lung and passenger mutation topographies. Our results provide insight into the molecular and clinical correlates of deconvolved NSCLC transcriptomes and provide a novel methodology with which to explore genomic variation at a single cell resolution. Furthermore, our dataset provides a resource for illuminating cancer-cell transcriptional changes and revealing key molecular drivers of tumor-stromal interactions in lung cancer.

#910

The cBioPortal for cancer genomics.

Jianjiong Gao,1 Tali Mazor,2 Adam Abeshouse,1 Ersin Ciftci,2 Ino de Bruijn,1 Benjamin Gross,1 Karthik Kalletla,3 Priti Kumari,2 Ritika Kundra,1 James Lindsay,2 Aaron Lisman,1 Pieter Lukasse,4 Ramyasree Madupuri,1 Angelica Ochoa,1 Oleguer Plantalech,4 Pichai Raman,3 Fedde Schaeffer,4 Robert Sheridan,1 Jing Su,1 S. Onur Sumer,1 Yichao Sun,1 Sander Tan,4 Sjoerd van Hagen,4 Avery Wang,1 Manda Wilson,1 Hongxin Zhang,1 Gaofei Zhao,1 Kelsey Zhu,5 Kees van Bochove,4 Ugur Dogrusoz,6 Trevor J. Pugh,5 Adam Resnick,3 Chris Sander,2 Ethan Cerami,2 Nikolaus Schultz1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Dana-Farber Cancer Institute, Boston, MA;_ 3 _Children's Hospital of Philadelphia, Philadelphia, PA;_ 4 _The Hyve, Utrecht, Netherlands;_ 5 _Princess Margaret Cancer Centre, Toronto, Ontario, Canada;_ 6 _Bilkent University, Ankara, Turkey_.

The cBioPortal for Cancer Genomics is an open-source software platform that enables interactive, exploratory analysis of large-scale cancer genomics data sets with a biologist-friendly interface. It integrates genomic and clinical data, and provides a suite of visualization and analysis options, including OncoPrint, mutation diagram, variant interpretation, survival analysis, expression correlation analysis, alteration enrichment analysis, cohort and patient-level visualization, among others.

The public site (http://www.cbioportal.org) hosts data from more than 200 studies from individual labs and large consortia, including the newly added TCGA Pan-Cancer Atlas data and the Count Me In project. These studies can be explored and queried individually or combined together into "virtual studies". Users are now allowed to login and save virtual studies for query and analysis. The site is currently accessed by approximately 30,000 unique visitors per month. The software is also installed locally at dozens of academic institutions and pharmaceutical/biotechnology companies. A notable instance is the cBioPortal for AACR GENIE (http://www.cbioportal.org/genie/) hosting 60,000 clinically sequenced samples from multiple institutions.

Over the past year, the code base has been fully refactored, resulting in a more responsive and interactive website. A new web API is in beta facilitating easier programmatic access to data. In addition, all public studies are available for download from the new datahub (https://github.com/cBioPortal/datahub/).

The cBioPortal remains under active development. The portal is fully open source (https://github.com/cBioPortal/) under a GNU Affero GPL license. Development is a collaborative effort among groups at Memorial Sloan Kettering Cancer Center, Dana-Farber Cancer Institute, Children's Hospital of Philadelphia, Princess Margaret Cancer Centre, and The Hyve. Ongoing and future development is focused on: (1) building the open source community; (2) continued performance improvements; (3) expanding user support, documentation and training resources; (4) developing novel features to support immunogenomics and immunotherapy; (5) enhancing individual variants and overall patient interpretation; (6) creating a simplified query interface; and (7) enabling comparative analysis of user-defined patient cohorts.

#911

UCSC Xena for cancer genomics visualization and interpretation.

Mary Goldman, Brian Craft, Jingchun Zhu, David Haussler. _UC Santa Cruz, Santa Cruz, CA_.

UCSC Xena (http://xena.ucsc.edu/) is a web-based visual integration and exploration tool for multi-omic data and associated clinical and phenotypic annotations. Our unique Visual Spreadsheet shows multiple data types side-by-side enabling discovery of correlations across and within genes and genomic regions. We offer dynamic Kaplan-Meier survival analysis, powerful filtering and subgrouping, charts, statistical analyses, genomic signatures, comparative transcript views, and bookmarks. We link out to the UCSC Genome Browser, giving users additional genomic context for any gene or coordinate, as well as MuPIT/CRAVAT and TumorMap, to give users complementary views of the same data.

Xena showcases seminal cancer genomics datasets from TCGA, the Pan-Cancer Atlas, PCAWG, GDC, GTEx, ICGC, and more; a total of more than 1500 datasets across 50 cancer types. We support virtually any type of functional genomics data. In addition to the commonly available SNPs, INDELs, copy number variation, and gene expression datasets, we support DNA methylation, exon-, transcript-, miRNA-, lncRNA-expression and structural variants. We also support clinical data such as phenotypes, subtype classifications and biomarkers. A recompute of TCGA, TARGET and GTEx datasets through the same bioinformatics pipeline allows users to compare expression between tumor and normal tissues. All of our data is available for download via our python API or through AWS S3 buckets.

A researcher can host their own data securely via private hubs running on a laptop or behind a firewall, with visual and analytical integration occurring only within the Xena Browser. The lightweight Xena Data Hubs are straightforward to install on Windows, Mac and Linux. Loading data is easy using either our application or command line interface.

Our newest features include:

* a new, more intuitive wizard to load your data into a local Hub

* URL bookmarks to save interactive views for yourself or to share with collaborators

* genomic signatures: dynamically build as a weighted sum over a set of genes

* hierarchically cluster a list of genes, regulons or probes

* upper vs lower quartile in a KM plot

## CLINICAL RESEARCH

### Biomarkers for Early Detection and Biologic Assessment of Cancer

#912

Macrophage inhibitory factor (MIF), Osteopontin (OPN) and anti-Interleukin-8 (IL-8) autoantibodies (AAb) complement CA125 for detection of early stage ovarian cancer.

Jing Guo,1 Wei-lei Yang,1 Joseph Celestino,1 Karen Lu,1 Anna E. Lokshin,2 Zhen Lu,1 Robert C. Bast1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _University of Pittsburgh, PA_.

Background: Early detection of ovarian cancer promises to reduce mortality. At present, only 20 percent of ovarian cancer is found in early stage (I-II). CA125 is found in sera from approximately 60% of patients with early stage disease. Greater sensitivity could be observed with a panel of blood tests.

Methods: A panel of 10 antigen biomarkers (MIF, Leptin, IL-6, IL-8, TNFa, OPN, FGF2, HE4, TGFa and a panel of autoantibodies (AAbs) against 12 antigens (IL-8, MDM2, c-myc, PLAT, EpCAM, HOXA7, ENO1, PDI, HSPA5, HSPA8, ANXA2, Cathepsin D) were analyzed in sera of 77 ovarian cancer patients with early stage (I-II), 50 patients with late stage (III-IV), and 216 healthy women from the Normal Risk Ovarian Cancer Screening Study (NROSS) using Luminex-based immunoassays. Serum levels of serum tumor marker CA-125 were routinely measured in all patients using a Roche assay. The most promising markers (CA125, MIF, OPN, and anti-IL-8 autoantibodies were validated using an independent panel with sera from 69 early stage and 50 late ovarian cancer patients and 200 healthy controls.

Result: Serum level of CA125, MIF, OPN and IL-8 AAb were elevated in early stage (I-II) and late stage (III-IV) ovarian cancer patients compared with healthy controls. Analyzing the positive biomarkers in combination with CA125 increased detection of early stage disease from 62% with CA125 alone to 92% a panel of CA125, MIF, OPN and IL-8 AAb at 98% specificity. For patients with late stage disease sensitivity increased from 90% to 98% with the panel. Complementarity of CA125 with MIF, OPN and IL-8 AAb was confirmed with an independent validation set, where sensitivity for early stage disease was improved from 84% to 91% at 98% specificity.

Conclusion: MIF, OPN and IL8 AAb could be used in combination with CA125 to distinguish ovarian cancer patients from health controls with high sensitivity.

#913

Early detection of metastatic relapse and monitoring of therapeutic efficacy by ultra-deep sequencing of plasma cell-free DNA in patients with urothelial bladder carcinoma.

Emil Christensen,1 Karin Birkenkamp-Demtröder,1 Himanshu Sethi,2 Svetlana Shchegrova,2 Raheleh Salari,2 Iver Nordentoft,1 Hsin-Ta Wu,2 Michael Knudsen,1 Philippe Lamy,1 Sia V. Lindskrog,1 Ann Taber,1 Mustafa Balcioglu,2 Søren Vang,1 Zoe Assaf,2 Shruti Sharma,2 Antony S. Tin,2 Ramya Srinivasan,2 Dina Hafez,2 Thomas Reinert,1 Samantha Navarro,2 Alexander Olson,2 Rosie Ram,2 Scott Dashner,2 Matthew Rabinowitz,2 Paul Billings,2 Styrmir Sigurjonsson,2 Claus L. Andersen,1 Ryan Swenerton,2 Alexey Aleshin,2 Bernhard G. Zimmermann,2 Mads Agerbæk,1 Cheng-Ho J. Lin,2 Jørgen B. Jensen,1 Lars Dyrskjøt1. 1 _Aarhus University Hospital, Aarhus N, Denmark;_ 2 _Natera Inc., San Carlos, CA_.

Background

Circulating tumor DNA (ctDNA) analysis is an emerging field for diagnosis, monitoring of treatment efficacy and detection of disease recurrence in various cancers. In bladder cancer, previous work has demonstrated prognostic value of ctDNA for early stage cancers and early detection of recurrence following radical cystectomy (CX), however more extensive investigation of the potential impact of ctDNA analysis on clinical decision making remains to be carried out.

Methods

A total of 68 patients with localized advanced bladder cancer to be treated with neoadjuvant chemotherapy (NAC) and CX were prospectively included. We performed RNA-Seq to identify RNA subtypes and whole exome sequencing to identify somatic mutations for ctDNA analysis. Based on allele frequency and sequence context, 16 somatic mutations were selected for each patient for multiplex PCR and subsequent ultra-deep sequencing (median coverage of 114,000x). The sample-level analytical sensitivity was determined to be >95% at 0.01% ctDNA concentration level when at least 2/16 mutations were detected. The protocol was applied to detect ctDNA in 618 blood samples procured at diagnosis, during NAC, before CX and during surveillance following CX.

Results

The presence of ctDNA was highly prognostic already at diagnosis before initiation of NAC (HR=31.7, p=0.001). Analysis of ctDNA dynamics during NAC revealed that clearance of ctDNA was associated with pathological downstaging (≤TaN0 at CX) and reduced disease recurrence. Conversely, continually detectable ctDNA during NAC was associated with lack of pathological downstaging and later disease recurrence (p=0.023). Our results suggest that ctDNA dynamics during NAC may offer superior predictive value compared to clinical parameters and tumor centric molecular markers such as DNA damage response mutations and RNA subtypes. Following CX, ctDNA analysis identified 92% of metastatic relapse (100% specificity) during disease monitoring (100% positive- and 98% negative predictive value). The positive lead-time over radiographic imaging was 100 days on average (range: -17-245 days).

Conclusions

ctDNA analysis of blood samples procured throughout the disease course of patients with bladder cancer demonstrates novel evidence of early risk stratification and monitoring of treatment efficacy, which adds to the already strong evidence of ctDNA-based recurrence detection. Collectively, this paves the way for novel ctDNA-based clinical trials.

#914

Development of a whole-urine, next generation sequencing-based assay for early detection of aggressive prostate cancer.

Andi K. Cani, Kevin Hu, Javed Siddiqui, Sumin Han, Daniel H. Hovelson, Chia-Jen Liu, Simpa S. Salami, Ganesh S. Palapattu, Todd M. Morgan, John T. Wei, Arul M. Chinnaiyan, Scott A. Tomlins. _University of Michigan, Ann Arbor, MI_.

Despite advances in biomarker development, early detection of aggressive prostate cancer (PCa) remains challenging. Existing biomarkers show modest improvement over models based on serum prostate specific antigen (PSA). We have previously developed a clinical-grade laboratory-developed test, named MiProstate Score (MiPS), for individualized risk prediction of aggressive prostate cancer. It uses transcription-mediated amplification to quantify the gene-fusion TMPRSS2:ERG (T2:ERG) (T1E4 splice isoform) and the lncRNA PCA3 from whole-urine obtained after a digital rectal exam (DRE), combined with serum PSA. To improve MiPS, we describe here the pre-clinical development and validation of a targeted next generation sequencing assay (NGS-MiPS) using post-DRE urine RNA to asses ~90 PCa transcriptomic biomarkers. These include those in MiPS as well as many isoforms of common PCa gene fusions, mRNA, and lncRNA candidate biomarkers nominated by our large-scale PCa tissue RNAseq and other sources. We have obtained a 98% informative sample rate from 2.5 mL of urine and high technical reproducibility (Pearson r=0.99). Risk scores for having PCa [or high-grade PCa (Gleason Score >6)] on biopsy, as determined by clinical MiPS vs. the clinical MiPS model using NGS data, were highly concordant, Pearson's r=0.74 (and r=0.81). Urine from patients with benign or Gleason 6 vs. Gleason ≥ 4+3=7 cancer on biopsy (extreme design) showed expected differences in the levels of T2:ERG T1E4 (p=0.00003) and PCA3 (p=0.07), with additional T2:ERG splice isoforms and other biomarkers also being significantly different between low vs. high grade cancer. Feature selection and logistic regression trained in an extreme design cohort (n=73) yielded a 29-transcript model that outperformed MiPS and serum PSA in two validation cohorts: 1. A held-out set from the extreme design cohort n=36, AUC 0.81 vs. 0.76 and 0.63 respectively; 2. A separate active surveillance cohort n=45, AUCs 0.66 vs. 0.56 and 0.53 respectively. These results support the potential utility of our urine based targeted NGS assay to supplement serum PSA for the early detection of aggressive prostate cancer.

#915

Pseudogene associated recurrent gene fusion in prostate cancer.

Balabhadrapatruni V. S. K. Chakravarthi,1 Shanker Kalyana-Sundaram,2 Pavithra D. Arachchige,3 Shannon Carskadon,3 Satya S. Pathi,1 Mireya Diaz-Insua,3 Craig Rogers,3 James Peabody,3 Clara Hwang,3 Mani Menon,3 Sean Williamson,3 Gupta Nilesh,3 Sooryanarayana Varambally,4 Nallasivam Palanisamy3. 1 _Crown Bioscience Inc, San Diego, CA;_ 2 _GlaxoSmith Kline, Collegeville, PA;_ 3 _Henry Ford Health System, Detroit, MI;_ 4 _University of Alabama, Birmingham, AL_.

Pseudogenes are a class of non-coding genes that are dysfunctional relatives of known functional genes. Often considered as junk DNA, pseudogenes have recently emerged as functional units in the form of non-coding RNA transcripts and micropeptides with regulatory roles in gene expression. Notably, association of some pseudogenes with oncogenes and tumor suppressor genes have been observed, indicating a possible role in cancer formation. Although many genetic aberrations in prostate cancer (PCa) have been identified, the expression of pseudogenes in PCa has not been well explored. We surveyed the transcriptional landscape of pseudogenes using next generation paired-end sequencing data and observed the formation of a recurrent gene fusion by trans-splicing event between a protein coding gene KLK4 and a noncoding pseudogene KLKP1 and its cognate fusion protein. RNA in situ analysis of 649 cases using tissue microarray identified high Gleason grade specific expression (>6) in 24% of cases compared to benign, high grade prostate intraepithelial neoplasia and atypical lesions, confirming cancer specific expression and with significantly high expression in Caucasian American compared with African American PCa. Furthermore, KLK4-KLKP1 expression was seen to be associated with ERG+ and ETV1+ cases. However, no association was observed with SPINK1+, ETV4+ and ETV5+ tumors, suggesting mutually exclusive expression with distinct PCa markers. Using expression constructs transfected into RWPE, PrEc and HEK293 cells, we confirmed the expression of a ~15 KD KLK4-KLKP1 fusion protein. RT-PCR analysis of 33 PCa derived xenografts identified 15 with endogenous expression of KLK4-KLKP1 fusion transcript. We generated a polyclonal antibody using antigenic peptide from the pseudogene part of the fusion protein and confirmed its specificity by western blot analysis using cell lysates from RWPE, PrEc, HEK293 and PDX models with and without the fusion gene. Immunohistochemistry analysis also confirmed the specificity of the antibody to the fusion protein. To explore the oncogenic properties of KLK4-KLKP1, we analyzed the effects on cell proliferation and invasion using RWPE cells transfected with KLK4-KLKP1. Increased cell proliferation and invasion was observed and Chicken chorioallantoic membrane (CAM) assay also revealed increased cell migration to lower CAM suggesting a role for KLK4-KLKP1 in cancer development. Notably, KLK4-KLKP1 transcripts could be detected in urine samples obtained from PCa patients, indicating the potential for noninvasive detection for early diagnosis. Taken together, we showed the oncogenic role of a new recurrent gene fusion in prostate cancer. In conclusion, our study highlights the underexplored functional roles of pseudogenes and establishes KLK4-KLKP1 as a novel player in PCa development with potential uses as a biomarker and a therapeutic target.

#916

Combined genomic and epigenomic assessment of cell-free circulating tumor DNA (ctDNA) improves assay sensitivity in early stage colorectal cancer (CRC).

Seung-Tae Kim,1 Victoria M. Raymond,2 Joon Oh Park,1 Elena Zotenko,2 Young Suk Park,1 Matthew Schultz,2 Won Ki Kang,1 Oscar Westesson,2 Hee-Cheol Kim,1 Yupeng He,2 Justin I. Odegaard,2 Stefanie A. Mortimer,2 William J. Greenleaf,3 Ariel Jaimovich,2 Jeeyun Lee,1 AmirAli Talasaz2. 1 _Samsung Medical Center, Seoul, Republic of Korea;_ 2 _Guardant Health, San Diego, CA;_ 3 _Stanford University, Stanford, CA_.

Background: ctDNA has the potential to identify patients (pts) with early stage cancer; however, current assays are challenged by limited sensitivity (~50%), reliance on a single analyte (e.g. somatic mutation detection), and/or the need for tumor tissue or genomic DNA sequencing to interpret ctDNA results. Recent studies have demonstrated that ctDNA can be detected using other biomarkers including DNA methylation. We developed a technology in which both somatic mutations and epigenomic alterations can be analyzed in a single assay.

Methods: Using a large database of cell-free DNA (cfDNA) profiles generated from advanced cancer patients, we designed a targeted sequencing assay that detects somatic variants, methylation alterations, and other epigenomic variations at transcription factor binding sites associated with CRC. Total cfDNA was extracted, partitioned based on methylation level, and analyzed. Data were then filtered using a variant classifier to differentiate tumor- from non-tumor-derived alterations without a priori knowledge of tissue or germline sequencing results. A machine learning model was trained on 111 cfDNA samples from 38 late stage and 10 early stage CRC pts and 63 age-matched cancer-free controls. For the independent test set, plasma samples (4-5mL) were collected from 72 pts with stage I-IV CRC prior to and 4 weeks after (N = 50, total of 122 samples) surgical resection. 35 age-matched cancer-free controls were similarly analyzed in the test set.

Results: Of the 72 pts, 62.5% were male, and median age at CRC diagnosis was 61.5 years (range 36-85). Stage distribution was 52.8% stage I/II, 40.3% stage III, and 6.9% stage IV. In the 50 pts with post-surgical samples, clinical follow-up was available for 49 (median post-surgery follow-up: 314 days; range 15-472). Utilizing this assay, pre-surgery ctDNA detection rate was 94% (68/72); 97% in stage I/II, 90% in stage III, and 100% in stage IV. Epigenomic analysis significantly enhanced ctDNA detection relative to somatic mutational analysis alone (94% vs. 56%; p<0.0001). Specificity in age-matched cancer-free controls was 94%.

Discussion: Utilizing a plasma-only sequencing assay incorporating somatic genomic variant detection, epigenomic analysis, and a bioinformatic classifier to filter non-tumor derived variants, ctDNA detection rate in early stage CRC (I-III) is 94% (63/67; 95% confidence interval 86%;98%) with 94% specificity, far outperforming the detection rate of somatic sequence variant detection alone. Clinical follow-up is ongoing to evaluate post-surgery ctDNA detection rate and disease recurrence. These results have significant implications for the clinical utility of ctDNA in early stage cancer management.

#917

Circulating tumor cells accurately detect and characterize lung cancers in high-risk subjects undergoing low-dose CT screening.

YARISWAMY MANJUNATH,1 Sathisha Upparahalli Venkateshaiah,1 Eric T. Kimchi,1 Kevin F. Staveley-O'Carroll,1 Jared Coberly,1 Diego M. Avella,1 Timothy J. Hoffman,2 Chelsea Deroche,1 Klaus Pantel,3 Guangfu Li,1 Jussuf T. Kaifi1. 1 _University of Missouri, Columbia, MO;_ 2 _Haary S Truman VA Memorial Hospital, Columbia, MO;_ 3 _University of Hamburg, Hamburg, Germany_.

Purpose: Lung cancer screening of high-risk subjects with low-dose CT (LDCT) significantly reduces mortality. Integrating liquid biomarkers circulating tumor cells (CTCs) screening could significantly improve the accuracy of findings on LDCT.

Experimental Design: In a prospective, observational clinical trial (NCT02838836; NCT03551951), high-risk screening subjects (≥30 pack-years smoking history, age 55-80) undergoing screening LDCT were enrolled. Peripheral blood (7.5ml) was collected, CTCs were enriched by microfilter isolation and immunofluorescence staining was performed for cytokeratin, EpCAM, CD14/45, PD-L1, vimentin, N-cadherin. Lung cancer tissues were similarly analyzed.

Results: In total, 136 subjects were enrolled prospectively. CTCs/7.5ml of blood were significantly higher in 14 subjects with proven lung cancer identified by screening LDCT (mean 23.57, SEM: ±3.73), in comparison to 37 high-risk subjects with benign-appearing lung nodules on LDCT (3.92 (±0.62)) (p<0.0001). A threshold of ≥12 CTCs in screening subjects with a lung nodule had an accuracy of 96.1% to detect lung cancer. CTCs were absent in 15 healthy controls, and rare in 12 subjects with no lung nodules on LDCT (1.08 (±0.47)). Distinct, large, polymorphonuclear CD14/45+CTCs were at significantly increased ratios in stage III/IV lung cancer patients (p=0.005). PD-L1+, vimentin+, and N-cadherin+CTCs were present in all patients, and expression was found at higher rates in CTCs than in patient-matched lung cancer tissues.

Conclusions: This seminal study suggests that CTC detection can accurately identify lung cancers in high-risk subjects with a nodule on screening LDCT. CTC phenotyping in lung cancer patients allows real-time insights that are potentially highly relevant for personalized treatment strategies.

Table 1. Subjects' characteristics and analysis for total CTCs | |  | |

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

|  | CTCs present  | CTC mean (±SEM); median (range) | p value

Subjects included (total) | 136 | |

|

High-risk LDCT screening subjects  | 63 | 48 (76.2%) | 7.73 (±1.4); 3 (0-56)

|

No lung nodules | 12 | 5 (41.7%) | 1.08 (±0.5); 0 (0-4)

|

Benign lung nodules | 37 | 29 (78.4%) | 3.92 (±0.6); 3 (0-14) | n.s.*

(vs. no lung nodule)

Lung cancers (NSCLC) | 14 | 14 (100%) | 23.57 (±3.7); 19 (13-56) | <0.0001*

(vs. benign lung nodules)

Lung cancer patients (NSCLC) | 72  | 72 (100%) | 25.01 (±1.5); 22 (9-80)

|

Not screened | 58 (81%) | 58 (100%) | 25.36 (±1.6); 23 (9-80) | 0.32† (vs. lung cancers diagnosed by screening)

NSCLC stage (AJCC 8th ed.)

•

I

•

II

•

III

•

IV | 26 (36%)

14 (20%)

19 (26%)

13 (18%) | |

22 (±2.3); 18 (13-48)

21.4 (±2.1); 20 (9-35)

26.4 (±2.2); 27 (10-45)

32.9 (±5.3); 24 (12-80)

|

Healthy controls  | 15 | 0 | 0

|

AJCC: American Joint Committee on Cancer; NSCLC: non-small cell lung cancer

#918

Using machine learning to predict the risk of either having an aggressive form of prostate cancer (PCa) or lower grade PCa/benign prostatic hyperplasia (BPH) based upon the flow cytometry immunophenotyping of myeloid-derived suppressor cells (MDSCs) and lymphocyte cell populations.

George A. Dominguez,1 John Roop,1 Alexander Polo,1 Anthony Campisi,1 Dmitry I. Gabrilovich,2 Amit Kumar1. 1 _Anixa Biosciences, San Jose, CA;_ 2 _The Wistar Institute, Philadelphia, PA_.

The goal of this study was to create a non-invasive confirmatory test for prostate biopsies that objectively analyzes flow cytometry data using machine learning to predict whether a subject is at higher risk for having an aggressive form of prostate cancer (PCa; Gleason ≥ 4+3). The commonly used assay for prostate screening is a prostate specific antigen (PSA) blood test, but due to prostate physiology, PSA testing results in a large frequency of false positives leading to numerous men each year undergoing unnecessary prostate biopsy procedures. Here, we use machine learning to create a neural network (NN) to predict whether a subject has a greater probability in having an aggressive form of prostate cancer (HR-PCa) or is at lower risk (LR-PCa; Gleason < 4+3) or no risk (benign prostatic hyperplasia [BPH]/healthy donor [HD]) based upon the immunophenotyping of myeloid-derived suppressor cells (MDSCs) and various lymphocyte cell populations. MDSCs are known to be key contributors in supporting tumor progression and escape through their ability to suppress anti-tumor responses mediated through T cell and natural killer (NK) cell activity. We used standard multiparametric flow cytometry techniques to immunophenotype the MDSCs and lymphocyte cell populations found in the peripheral blood of 114 biopsy-confirmed PCa and 89 biopsy-confirmed BPH subjects along with 116 healthy donors (HD). Subjects were recommended for biopsy by their physician and excluded if they had a previous history of cancer (not including subjects under active surveillance), had a medical intervention for prostate cancer, or were receiving a dihydrotestosterone (DHT) or alpha-1 blocker for active treatment of benign prostatic hyperplasia (BPH). Machine learning and pattern recognition were used to create a two-network approach to predict whether a subject should be recommended for biopsy (HR-PCa) or be actively monitored (HD or BPH/LR-PCa). Initially, NN1 predicts whether the sample looks more like HD or HR-PCa; if the sample is predicted to be a possible HR-PCa, then it is tested by NN2 and predicted as either BPH/LR-PCa or HR-PCa. The final decision for biopsy would be made by the clinician. By combining immunophenotyping data with machine learning, we achieved a final 90% sensitivity for predicting whether subjects should undergo a prostate biopsy procedure based upon their immunophenotyping. For this study, out of the 203 subjects recommend for biopsy, approximately 105 procedures could have been avoided. In a clinical setting, we believe that this technology, in use with other known clinical risk factors, would allow for clinicians to have a more informed decision when recommending their patients for a prostate biopsy procedure.

## ENDOCRINOLOGY

### Endocrine-Related Cancer Research

#919

Clinical relevance of comprehensive genomic analysis in patients with advanced-stage neuroendocrine neoplasms: Results from the MASTER trial of the German Cancer Consortium.

Simon Kreutzfeldt,1 Leonidas Apostolidis,2 Malgorzata Oles,1 Peter Horak,1 Christoph E. Heilig,1 Christoph Heining,3 Barbara Hutter,4 Laura Gieldon,5 Barbara Klink,5 Mario Lamping,6 Damian T. Rieke,6 Sebastian Uhrig,4 Henning Jann,7 Ulrich F. Pape,7 Albrecht Stenzinger,8 Eva C. Winkler,2 Bertram Wiedenmann,7 Dirk Jäger,2 Benedikt Brors,4 Evelin Schröck,5 Ulrich Keilholz,6 Marianne Pavel,9 Hanno Glimm,3 Stefan Fröhling1. 1 _National Center for Tumor Diseases (NCT) Heidelberg and German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 2 _National Center for Tumor Diseases (NCT) Heidelberg, Heidelberg, Germany;_ 3 _National Center for Tumor Diseases (NCT) Dresden and University Hospital Carl Gustav Carus, Dresden, Germany;_ 4 _German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 5 _Institute for Clinical Genetics, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany;_ 6 _Charité Comprehensive Cancer Center, Charité University Medicine, Berlin, Germany;_ 7 _Department of Hepatology and Gastroenterology, Charité University Medicine, Berlin, Berlin, Germany;_ 8 _Institute of Pathology, Heidelberg University Hospital, Heidelberg, Germany;_ 9 _Department of Medicine, Division of Endocrinology, Friedrich Alexander University Erlangen-Nürnberg, Erlangen, Germany_.

Therapeutic options for neuroendocrine neoplasms (NEN) are limited. Within the MASTER program, a multi-institutional registry trial for prospective stratification of younger adults with advanced-stage cancer across all histologies and patients with rare tumors conducted under the auspices of NCT Heidelberg/Dresden and the German Cancer Consortium, we apply prospective whole-exome/genome sequencing (WES/WGS) and RNA sequencing (RNA-seq) to determine therapeutic choices for individual patients who have exhausted standard therapy options. We here report the clinical and molecular characteristics of the NEN cohort within this study. Between 2013 and 2018, 108 patients (male, n=65; female, n=43) were enrolled. Histologies according to the 2017 WHO Classification were neuroendocrine tumor grade 1 (NET G1), n=8; NET G2, n=31; NET G3, n=9; neuroendocrine carcinoma, n=49; and mixed neuroendocrine/non-neuroendocrine neoplasm, n=11. Primary tumor sites were gastrointestinal tract, n=30; pancreas, n=31; thorax, n=21; genitourinary system, n=11; head and neck, n=5; and other regions, n=10. All patients had advanced-stage disease and had received a median of 2 prior lines of systemic therapy. WES, WGS, and RNA-seq were performed in 69, 40, and 87 patients, respectively. Clinical evaluation of germline and somatic molecular data (single-nucleotide variants, small insertions and deletions, copy number variations, mutational burden, mutational signatures, homologous recombination deficiency scores, gene expression patterns, etc.) from 105 patients by a dedicated molecular tumor board yielded evidence-based recommendations for clinical management in 91 cases (87%). Treatment recommendations were grouped as follows: PARP inhibition, n=35; immunotherapy, n=27; mTOR inhibition, n=22; CDK4/6 inhibition, n=15; tyrosine or serine/threonine kinase inhibition, n=57 (ALK, n=1; ERBB, n=6; FGFR, n=10; MET, n=5; RET, n=12; VEGFR, n=4, MEK, n=9; other, n=10), DNA-crosslinking chemotherapy, n=16; anti-claudin18.2 antibody, n=5; BET inhibition, n=5; and DLL3 antibody, n=5. As of November 2018, at least 18 patients had received molecularly guided treatment (PARP inhibition, immunotherapy, mTOR inhibition, tyrosine or serine/threonine kinase inhibition) of which 11 were evaluable for response (partial response, n=4; stable disease, n=2; progressive diseases, n=5). Twenty-seven patients died before therapy could be started, 16 are currently receiving other regimens, and for the remaining patients no follow-up data are available yet. In conclusion, comprehensive molecular profiling offers valuable insight into to the genomic and transcriptomic landscape of NEN and creates additional therapeutic opportunities in a subset of patients.

#920

p97/VCP: A novel interactor of the sodium iodide symporter, which can be pharmacologically targeted to increase radioiodine uptake in thyroid and breast cancer cells.

Alice Fletcher, Martin L. Read, Vikki L. Poole, Vicki E. Smith, Christopher J. McCabe. _University of Birmingham, Birmingham, United Kingdom_.

By exploiting the canonical function of the sodium iodide symporter (NIS), ablative radioiodine therapy is an effective treatment for papillary thyroid cancer (PTC). Unfortunately, ~25% of PTC patients are unable to accumulate therapeutically sufficient radioiodine due to diminished expression and/or altered plasma membrane localization of NIS. Patients with radioiodine-refractory PTC have reduced mean survival times. Radioiodine therapy has been proposed as a viable treatment for breast cancer but is hampered by low levels of membranous NIS localization. Currently, however, the regulation of NIS membrane localization remains ill-defined.

Mass spectrometry identified the protein p97/VCP as a novel NIS interactor, which was validated through co-immunoprecipitation and proximity ligation assays. p97 siRNA depletion increased NIS-mediated radioiodine uptake by 97% and 141% in the lentivirally-expressing NIS MDA-MB-231 breast and TPC1 thyroid cancer cell lines respectively (p<0.05), and significantly boosted endogenous NIS function in human primary thyrocytes (p<0.05). Conversely, p97 overexpression significantly repressed NIS function in all cell systems, accompanied by lowered membranous NIS localization (p<0.05), as quantified via cell surface biotinylation assays. We next identified five different allosteric p97 inhibitors - Eeyarestatin-1, NMS-873, Astemizole, Clotrimazole and Ebastine - which were all able to overcome p97 inhibition of NIS function in TPC1 and MDA-MB-231 cell lines lentivirally-expressing NIS, significantly increasing NIS function by at least 100%.

TCGA analyses of matched PTCs revealed p97 mRNA expression is highly upregulated in PTC compared to matched normal thyroid (n=58, p<0.05), providing a putative explanation for repressed NIS function. In a wider cohort of 413 PTC patients, high tumoral p97 expression was associated with a worse disease-free survival when compared to low p97 expression (p<0.01, hazard ratio (HR): 2.924 (95% CI, 1.280-6.683)). Strikingly, in patients that did not receive radioiodine, high tumoral p97 expression had no significant effect on disease-free survival (n=136, p=NS, HR: 0.020 (95% CI, 0.000013-31.192)), whereas in patients who received radioiodine, high tumoral p97 expression resulted in a markedly worse disease-free survival (n=194, p<0.01, HR: 6.043 (95% CI, 1.749-20.879)). This is indicative that high p97 expression correlates with a worse response to radioiodine therapy.

Our data therefore highlight a new pathway of NIS regulation. Critically, two of these p97 inhibitors are already FDA-approved, highlighting a novel potential therapeutic strategy for enhancing radioiodine uptake in patients with radioiodine-refractory PTC and increasing the feasibility of radioiodine therapy in breast cancer via the transient inhibition of p97 activity.

#921

**Novel clinical targets revealed by kinome reprogramming in mutant** ESR1 **metastatic breast cancer.**

Derek Dustin, Guowei Gu, Doug Chan, Amanda Beyer, David Edwards, Arnoldo Corona-Rodriguez, Matthew Ellis, Suzanne Fuqua. _Baylor College of Medicine, Houston, TX_.

Background: Approximately half of women with estrogen receptor (ER)-positive breast cancer who undergo adjuvant endocrine therapy (ET) develop disease progression and metastasis. Studies have revealed that up to 40% of metastatic patients develop tumors bearing a mutation in ESR1. ESR1 mutant (ESR1m) tumors are associated with a more aggressive metastatic disease course. Studies have shown that cancers can undergo "kinome reprogramming" in response to therapy and acquired genetic alterations within the tumor. Thus, we hypothesize that expression of ESR1 mutations induces kinome reprogramming, which presents novel potential therapeutic targets to selectively treat ESR1m metastatic breast cancer.

Methods: We performed Kinobeads Precipitation (KiP) using broad specificity kinase inhibitors to capture and quantify by MS the level of active kinases in MCF-7 ESR1 wild-type and ESR1 Y537S mutant cells. We performed targeted siRNA screens and immunoblots to validate the hyperactivity of several of these kinases. Specific kinase inhibitors are being used to test their effects on proliferation and invasion in vitro, and in ex vivo organoid growth assays. Xenograft models are being used to evaluate the effects of the inhibitors on primary tumor growth and distant metastasis.

Results: Hyperactivation of 169 kinases in ESR1m cells was revealed by KiP analysis. Hyperactivation of several growth factor receptors, the PI3K/Akt/mTOR pathway, and the JAK/STAT pathway was demonstrated in Y537S mutant cells. We tested specific inhibitors of these pathways, and have shown that the mutant cells have reduced proliferation when these pathways are targeted. We developed organoid growth assays using primary and metastatic tumors from MCF-7 and T47D Y537S ESR1 xenograft tumors, and the Y537S ESR1 WHIM20 PDX model, and found that ET in combination with these selected targeted kinase inhibitors decreased the growth of organoids. We are currently testing inhibitors in combination with ET in vivo, and preliminary results demonstrate a significant inhibition of distant metastatic frequency. These results suggest that selective inhibition of hyperactivated "reprogrammed" kinase pathways can restore ET sensitivity in ESR1m tumors, and may show promise for use in ER-positive metastatic breast cancer patients.

#922

Preclinical anti-cancer activity of LSZ102, a novel oral selective estrogen receptor degrader targeting wild type and mutant ER.

L. Alex Gaither,1 Choi Lai Tiong Yip,1 Chunrong Wang,1 Weiyi Toy,2 Qing Sheng,1 Jinyun Chen,1 Yuji Mishina,1 Rita Das,1 Stefan Peukert,1 Alice Loo,1 Sarat Chandarlapaty,2 Adam Crystal,1 Tinya J. Abrams1. 1 _Novartis, Cambridge, MA;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Estrogen receptor (ER) blockade is a well-established therapeutic approach in ER+ breast cancer. A novel oral selective estrogen receptor degrader (SERD), LSZ102, is in development. LSZ102 induces proteasome-mediated degradation of both wild type and mutant ERα in MCF-7 cells. LSZ102 also inhibits transcription of ERα target genes and results in a decrease in cell proliferation in a dose dependent manner. Expression of ERα Y537S results in a shift in inhibition of cell proliferation upon incubation with either LSZ102 or fulvestrant; however, the shift with LSZ102 was less severe. Similarly, LSZ102 induced a more pronounced level of ERα degradation than fulvestrant in the Y537S mutant MCF7 cells. Overall, LSZ102 is effective in the wild type and Y537S ERα mutant setting in vitro. In vivo, LSZ102 treatment of ER+ breast cancer xenografts resulted in inhibition of ERα regulated transcripts and a decrease in ERα protein levels. In the MCF7 xenograft model, expression of ERα Y537S resulted in reduced activity by fulvestrant, but not by LSZ102. In vivo, LSZ102 exhibited single agent and combination efficacy upon co-administration with the CDK4/6 inhibitor ribociclib and the alpha-specific PI3K inhibitor alpelisib. LSZ102 is currently in a Phase I clinical trial in patients with ER+ breast cancer in which it is tested as a single agent, and in combination with either ribociclib or alpelisib.

#923

FOXA1 chromatin binding is regulated by LSD1-mediated methylation in prostate cancer cells.

Shuai Gao,1 Sujun Chen,2 Dong Han,1 Wanting Han,1 Jude N. Owiredu,1 Steven P. Balk,3 Housheng H. He,2 Changmeng Cai1. 1 _University of Massachusetts Boston, Boston, MA;_ 2 _Princess Margaret Cancer Center, Toronto, Ontario, Canada;_ 3 _Beth Israel Deaconess Medical Center, Boston, MA_.

Androgen receptor (AR) plays a critical function in driving prostate cancer (PCa) development and androgen deprivation therapy (ADT) is standard of care in treating PCa patients. Although patients generally response well, the cancer eventually relapse and progress to the castration-resistant stage of PCa (called CRPC), which can be further treated with more aggressive ADT. However, the tumors still develop resistance within a year and restored AR activity is found in the majority of relapsed tumors, indicating a pressing need for developing novel AR-targeted therapies.

In PCa cells, AR activity is dependent on the chromatin binding of the pioneer factor, FOXA1. In particular, FOXA1 recognizes the H3K4me1,2 marked enhancers and its binding opens the chromatin for subsequent AR recruitment. Although the role of FOXA1 on AR signaling has been intensively studied, the mechanisms regulating FOXA1 binding to chromatin remain elusive. LSD1/KDM1A was initially identified as a transcription repressor through demethylation of H3K4me1,2. In contrast to its well-established corepressor function, LSD1 has been found to coactivate several transcription factors including AR. This AR coactivator function has been attributed to the phosphorylation of histone 3 on threonine 6 and threonine 11, which may switch LSD1 substrate specificity from H3K4me1,2 to the repressive mark H3K9me1,2. However, we have recently reported that the H3K4 demethylase activity of LSD1 persists at AR-regulated enhancer sites, including sites marked by H3T6ph, arguing against the coactivator function of LSD1 being generally controlled by this mechanism. Importantly, we have shown in a recent study that LSD1 associates with FOXA1 and active enhancer markers, and that LSD1 silencing impaired FOXA1 binding at AR-regulated enhancers, suggesting a role of LSD1 in regulating enhancer availability to AR.

In the current study, we found that LSD1 inhibition globally disrupts FOXA1 chromatin binding prior to androgen stimulation and thus impairs further AR recruitment, resulting in the global inhibition of AR transcriptome. Using immunoprecipitation and mass-spectrometry assays, we discovered that LSD1 maintains FOXA1 binding by demethylating lysine K270, adjacent to its DNA binding domain. We further found that the expression of a methylation-deficient FOXA1 mutant (K270R) stabilizes AR chromatin binding and allows PCa cells resistant to enzalutamide treatment. Using a clinical tested LSD1 inhibitor, we have shown in vivothat LSD1 inhibition dramatically decreases PCa growth alone and in synergy with enzalutamide in AR/AR variants-positive CRPC models. These findings provide fundamental new insights into FOXA1, LSD1, and mechanisms that regulate the activity of AR in PCa cells. As LSD1 inhibitors are moving into the clinic, these new insights into LSD1 function strongly support LSD1 as a therapeutic target in CRPC.

#924

The androgen receptor antagonist darolutamide shows strong anti-tumor efficacy in patient- and cell line-derived xenograft prostate cancer models.

Pascale Lejeune,1 Antje Wengner,1 Simon J. Baumgart,1 Ekaterina Nevedomskaya,1 Eva Corey,2 Dominik Mumberg,1 Bernard Haendler1. 1 _Bayer AG, Berlin, Germany;_ 2 _University of Washington, Seattle, WA_.

Darolutamide is a structurally unique androgen receptor (AR) antagonist which has recently completed a pivotal clinical phase 3 trial. Here we explored the efficacy of darolutamide in different prostate cancer models and in combination with the ATR inhibitor BAY 1895344. Darolutamide was tested in the patient-derived xenografts LuCaP35 and LuCaP96. The studies were performed with SCID male mice implanted subcutaneously. Oral, twice daily treatment with 100 mg/kg darolutamide started at a mean tumor size of about 150 mm3; the study was stopped 3.5 weeks later. In addition the cell line-derived xenograft LAPC-4 was tested after subcutaneous inoculation of SCID male mice. Oral, daily treatment with 100 mg/kg darolutamide started at a mean tumor size of about 50 mm2. The ATR inhibitor BAY 1895344 was applied at 20 mg/kg, twice daily for 3 days followed by 4 days without treatment. X-ray radiation was performed at 5 Gy, once weekly on days 26 and 33 after start of compound treatment. Efficacy was evaluated by calculating the ΔT/ΔC values. Gene expression analysis was performed on RNA extracted from prostate cancer cells treated with darolutamide and/or BAY 1895344, and analyzed using RT2 Profiler PCR arrays (Qiagen). RNA-seq and AR ChIP-seq (06-680, Millipore) datasets of darolutamide-treated cell lines were used for genome-wide studies. γH2AX (ab2893, abcam) levels were evaluated by Western blot analysis. Darolutamide showed strong anti-tumor activity in the LuCaP96 model (6% ΔT/ΔC). Significant anti-tumor efficacy was also observed in the LAPC-4 model following treatment with darolutamide (36% ΔT/ΔC) or with radiation (19% ΔT/ΔC). Stronger efficacy was observed when combining darolutamide with the ATR inhibitor BAY 1895344 (17% ΔT/ΔC) or with BAY 1895344 plus radiation (1% ΔT/ΔC). RNAseq analysis of LAPC-4 and VCaP cells showed that expression of DNA repair genes was regulated by androgen and efficiently blocked by darolutamide. In line with this, we observed androgen-dependent AR binding close to DNA repair genes, which supports a regulatory role of the AR signalling pathway in the regulation of these genes. We also found that γH2AX levels were increased after treatment with BAY 1895344 alone or in combination with darolutamide. Expression of H2AX was also increased following combination treatment. Notably, darolutamide treatment alone did not have comparable effects on H2AX but strongly reduced androgen-driven expression of several DNA damage response genes. In conclusion, darolutamide demonstrated strong in vivo efficacy in xenograft models as single agent, and enhanced activity in combination with radiation and an ATR inhibitor, with the latter involving down-regulation of DNA repair genes. Together these results suggest broad anti-tumor activity of darolutamide in prostate cancer as a single agent or in combination with other anti-tumor modalities.

#925

PDX models of ER+ endocrine resistant metastatic breast cancer identify Polo like kinase 1 (PLK1) as a therapeutic target.

Elodie Montaudon,1 Joanna Nikitorowicz-Buniak,2 Laura Sourd,1 Rania El Botty,1 Ahmed Dahmani,1 Léa Huguet,1 Ludivine Morriset,1 Pierre Painsec,1 Fariba Nemati,1 Sophie Vacher,1 Sophie Chateau-Joubert,3 Sunil Pancholi,2 Camilla Rega,2 Ricardo Ribas,2 André Nicolas,1 Didier Meseure,1 Anne Salomon,1 Zakia Tariq,1 Keltouma Driouch,1 Florence Coussy,1 Guillaume Dutertre,1 Paul Cottu,1 Ivan Bièche,1 Lesley-Ann Martin,2 Elisabetta Marangoni1. 1 _Inst. Curie, Paris, France;_ 2 _Institute of Cancer Research, London, United Kingdom;_ 3 _Alfort Veterinary School, Maisons-Alfort, France_.

Background: Metastatic estrogen receptor positive (ER+) breast cancers (BC) are genetically heterogeneous and a significant proportion of patients display intrinsic or acquired resistance to endocrine treatments, mTOR and CDK4/6 inhibitors. The objective of this study was to identify new therapies in clinically relevant PDX models of metastatic ER+ BC progressing on endocrine treatment and/or palbociclib.

Methods: PDX models were generated by engraftment of spinal bone metastases from 120 BC patients. PDXs were molecularly characterized using SNP-arrays, targeted exome sequencing and global gene expression (GEX) analysis. The anti-tumor efficacy of the PLK1 inhibitor volasertib was evaluated in vivo compared to fulvestrant and palbociclib in two PDX models. PLK1 abundance was analyzed by immunohistochemistry in PDXs and compared with the original primary tumor and bone metastasis from each patient. PLK1 expression measured by RT-PCR was assessed in a cohort of 441 BC patients with 15-year follow-up. GEX analysis of PLK1 was assessed in patients treated with neoadjuvant anastrazole. Mechanistic studies were performed in a panel of endocrine resistant BC cell lines.

Results: Ten PDX have been established, 8 were ER+ and 2 triple-negative (TN). Genomic alterations included mutations in AKT1, PI3KCA, BRCA2, GATA3, NF1, and amplifications of FGFR1, CCND1 and CCNE2. Comparative pathway analysis of bone metastases derived PDX and patients' primary tumors showed enrichment for pathways associated with mitotic nuclear division, chromatid segregation and G2/M transition. PLK1 was the top commonly up-regulated gene within these pathways. Treatment of an endocrine-resistant ER+ PDX, harboring amplification of CCND1, CCNE2, FGFR1 and high expression of CDK1, showed only partial response to palbociclib and resistance to the FGFR inhibitor, AZD4547. However, PLK1 inhibitor volasertib induced rapid tumor shrinkage and complete response within 5 weeks. Importantly, volasertib was also highly effective when tested as second line therapy in palbociclib pre-treated xenografts. In-vitro inhibition of PLK1 by siRNA or volasertib inhibited tumor proliferation without affecting the expression of ER-regulated genes, suggesting an ER-independent function of PLK1 in regulating cell proliferation. Assessment of the clinical validity of PLK1 expression, revealed a strong association with poor metastases free survival (p<0.0001) in ER+ BC, but not in HER2+ or TNBC. Finally, in a cohort of patients treated with neoadjuvant anastrozole, on-treatment gene expression of PLK1 was significantly (p<0.0001) associated with poor response.

Conclusion: We show that inhibition of PLK1 is a new potential treatment strategy for metastatic ER+ BC. Additional experiments are ongoing in PDX and cell lines to investigate G2/M phase dependence of ER+ BC and to identify predictive biomarkers.

## EXPERIMENTAL AND MOLECULAR THERAPEUTICS

### Drug Resistance

#926

Extrinsic and intrinsic activation of RAS/MAPK signaling enables resistance to FLT3 inhibitor, gilteritinib in acute myeloid leukemia.

Sunil K. Joshi,1 Stephen Christy,2 Renata Scopim Ribeiro,2 Shannon McWeeney,2 Jeffrey W. Tyner,2 Cristina E. Tognon,1 Brian J. Druker,1 Elie Traer2. 1 _Oregon Health & Science University (OHSU)/Howard Hughes Medical Institute, Portland, OR; _2 _Oregon Health & Science University (OHSU), Portland, OR_.

Acute Myeloid Leukemia (AML) remains a highly fatal disease due to the development of drug resistance. Mutational activation of kinases is a frequent event in leukemia, driving malignant cell growth. The most commonly mutated kinase in AML is the FMS-like tyrosine kinase 3 (FLT3) receptor. Internal tandem duplication (ITD) events in the juxtamembrane domain of FLT3 have been reported in ~ 20% of patients and lead to constitutive activation of FLT3. Previous work from our laboratory has shown that fibroblast growth factor 2 (FGF2) secreted by mesenchymal stromal cells protects FLT3-ITD AML cells from quizartinib, a FLT3 inhibitor (Cancer Research, 2016). FGF2 binds the FGFR1 receptor and activates MAPK signaling, enabling leukemia cells to become drug resistant. Over months of treatment, FGF2-protected leukemia cells acquired FLT3 resistance mutations. This two-step mechanism of resistance in vitro mirrors what happens in patients treated with quizartinib. Newer FLT3 inhibitors, such as gilteritinib, have been developed that have activity against both ITD and kinase domain mutations that impart resistance to quizartinib. We used a similar approach and treated the FLT3-ITD+ AML cell line, MOLM-14, with 100 nM of gilteritinib in media alone (n=4), or supplemented with 10 ng/ml FGF2 (n=4) or FLT3 ligand (FL, n=4). After 7 weeks, all cultures supplemented with FGF2 or FL eventually resumed growth. In contrast, no MOLM-14 cells cultured in gilteritinib alone resumed growth even after 16 weeks, suggesting the importance of extrinsic factors in facilitating early drug resistance. Selective pressure was then put on the ligand-dependent cultures by removal of FGF2 and FL. This transiently restored sensitivity to gilteritinib, however within a month the cultures resumed exponential growth. To further explore the mechanism of resistance, we performed immunoblots of kinase signaling pathways on MOLM-14 resistant cultures pre- and post-withdrawal of ligands. We saw robust activation of FLT3 receptor in resistant cultures with FL. FGF2 activated the MAPK pathway, circumventing inhibition by gilteritinib. After removal of extrinsic FGF2 and FL, there was re-activation of MAPK signaling but FLT3 itself remained inhibited. Whole Exome Sequencing of resistant cells identified NRAS mutations as the source of MAPK signaling after ligand withdrawal. Two of the four FGF2-supplemented cultures also developed novel FLT3 point mutations, although there was no clear re-activation of FLT3 signaling in these cultures. Our in vitro results suggest that extrinsic signals from the microenvironment can re-activate MAPK pathways enough to keep cells alive but continued selective pressure eventually leads to direct activation of MAPK through RAS mutations. As such, there is a need to consider rational inhibitor combinations early in disease therapy to prevent the emergence of resistance.

#927

Genomic profiling of metastatic breast cancer identifies the Hippo pathway to be a key determinant of CDK4/6 inhibitor sensitivity.

Pedram Razavi,1 Qing Li,1 Zhiqiang Li,1 David N. Brown,1 Christina Ping,2 Ronglai Shen,1 Francisco Sanchez-Vega,1 Nikolaus D. Schultz,1 Jose Baselga,3 Maurizio Scaltriti,1 Jorge S. Reis-Filho,1 Sarat Chandarlapaty1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Stanford University, Stanford, CA;_ 3 _Vall d'Hebron Institute of Oncology, Barcelona, Spain_.

CDK4/6 inhibitors (CDK4/6i) have proven benefits in estrogen receptor-positive (ER+) metastatic breast cancer (MBC), however the activity is variable among patients and biomarkers of sensitivity are lacking. We sought to determine whether any genomic alterations might characterize extreme responders and non-responders from over 500 patients with ER+ MBC who received CDK4/6i plus aromatase inhibitors or fulvestrant.

We first analyzed a cohort of 348 patients who has MSK-IMPACT sequencing performed on a pre-treatment tumor sample and asked whether any somatic mutation or copy number alteration was associated with altered progression-free survival (PFS) on subsequent CDK4/6i-based therapy. As expected, common alterations in CCND1, PIK3CA or ESR1 were not associated with altered PFS on CDK4/6i. Also as anticipated, RB1 loss was associated with resistance to CDK4/6i (median PFS: 3.6 months, 95% confidence interval [CI]: 2.2, not reached, p=0.00041). Unexpectedly, alterations in components of the Hippo pathway (FAT1, YAP, LATS1/2, and NF2) were mutually exclusive with each other and with RB1 and were collectively associated with resistance to CDK4/6i in ER+ MBC (Hazard Radio [HR]: 3.6; 95% CI: 1.7, 7.8, p=0.0044). FAT1 was the most frequently mutated gene in the Hippo pathway with alterations resulting in FAT1 loss associated with a markedly shortened PFS (median: 2.4 months, HR: 11.1; 95% CI: 4.65, 26.62; p=2.2x10-11). No alterations were clearly linked with extremely prolonged PFS on CDK4/6i. Additional analyses from a larger cohort of patients with genomic sequencing prior to starting CDK4/6i are now underway and will be presented.

To further understand the mechanisms of Hippo pathway regulation of cell cycle, cell line and patient derived xenograft models were utilized. These studies revealed that different mechanisms of suppression of Hippo signaling such as FAT1 knockdown (CRISPR or shRNA) and NF2 knockdown (shRNA) lead to YAP activation and YAP-dependent CDK4/6i resistance. Moreover, in all cases, resistance was mediated by upregulation of CDK6 as knockdown of CDK6 prevented drug resistance.

Finally, we have sought to understand the implications of these distinct mechanisms of resistance to CDK4/6i. Clinical follow up on subsequent lines of therapy and duration of treatment after CDK4/6i (e.g. chemotherapy or other hormone therapy) are now being analyzed based on class of resistance alteration and will be presented.

In conclusion, RB1 loss and Hippo pathway alterations mediate resistance to CDK4/6i in patients with ER+ MBC, nominating these as potential biomarkers for this class of drugs.

#928

Preventing melanoma from healing itself after therapy-induced immune attack through IPRES blockade.

Marco Piva, Lu Sun, Gatien Moriceau, Zhentao Yang, Antoni Ribas, Willy Hugo, Roger S. Lo. _UCLA, Los Angeles, CA_.

Over the past few years, therapeutics development for melanoma has led the oncology field, including the development of mutant BRAF-targeted therapies (e.g., BRAF inhibitor (BRAFi)) and immune checkpoint blockade therapies (e.g., anti-PD-1 or aPD-1, aPD-L1, etc). Based on the complementary qualities of aPD-1/L1 immunotherapy (long duration of response) and MAPKi (high rate of initial response), there are currently clinical trials testing the combination of the two. However, despite a more durable tumor suppression, resistance still eventually develops in patients treated by the combination (ref: KEYNOTE-022). My earlier study reported significant intra-patient and intra-tumoral heterogeneity which affect patient-specific responses to therapy. Furthermore, I also observed complex transcriptomic and tumor microenvironment reprogramming associated with resistance to targeted- or immuno-therapies in melanoma. However, the increased immune/stromal cells in the tumor under therapy has prevented us from definitively accounting the source of such reprogramming. To address this need, I utilized the novel single cell transcriptomic analysis to discover therapy-response-associated gene programs across different types of cells within a tumor. In murine mutant BRAF melanoma YUMM1.7, regressing/responding tumors on the 6th day of BRAFi treatment showed significant infiltration of the T cells and other immune cell population and, by the 15th day, the tumor showed activation of IPRES related processes. Combination of BRAFi and aPD-L1 in YUMM1.7 increased the proportion of lymphoid population in the tumor on the 7th day but only incrementally delayed the development of BRAFi resistance. Inhibition of IPRES-related processes (anti-VEGF and/or anti-TGFB) further delayed BRAFi resistance but no complete responses were observed. However, when we employed a UV-mutated subline of the same model, named the YUMMER (Bosenberg lab, Yale), combination of BRAFi, aPD-L1 and IPRES inhibition started to result in durable complete responses in our mouse model. We are now in midst of analyzing the differential response that we observed with this model; our current working hypothesis is that YUMMER induces higher level of antigen-specific antitumor response, which is augmented by the addition of immune checkpoint blockade and IPRES suppression. The results from our study will eventually pave the way to a novel therapeutic combination to overcome melanoma.

#929

Paired Tumor and cfDNA in Patients with HER2-mutant Solid Tumors Treated with Neratinib Reveals Convergence of Multiple On-Target Resistance Mechanisms: Results from the SUMMIT 'Basket' Trial.

Helen H. Won,1 S. Duygu Selcuklu,1 Sarina A. Piha-Paul,2 Cristina Saura,3 Jordi Rodon,2 Ingrid A. Mayer,4 Sherene Loi,5 Geoffrey I. Shapiro,6 Janice Lu,7 Adam Brufsky,8 Catherine Zimel,1 Myra Melcer,1 Maurizio Scaltriti,1 Lisa D. Eli,9 Richard E. Cutler Jr.,9 Alshad S. Lalani,9 Richard P. Bryce,9 Carlos Arteaga,10 Funda Meric-Bernstam,11 Michael F. Berger,1 David B. Solit,1 Alison Schram,1 David M. Hyman1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _MD Anderson Cancer Center, Houston, TX;_ 3 _Vall d'Hebron University Hospital, Barcelona, Spain;_ 4 _Vanderbilt-Ingram Cancer Center, Nashville, TN;_ 5 _Peter MacCallum Cancer Centre, Melbourne, Australia;_ 6 _Dana Farber Cancer Institute, Boston, MA;_ 7 _Keck School of Medicine of USC, Los Angeles, CA;_ 8 _University of Pittsburg, Pittsburgh, PA;_ 9 _Puma Biotechnology, Los Angeles, CA;_ 10 _UTSW Harold C. Simmons Comprehensive Cancer Center, Dallas, TX;_ 11 _MD Anderson, Department of Investigational Cancer Therapeutics, Houston, TX_.

Background: Somatic mutations in ERBB2 occur across various tumor types at relatively low frequencies and can lead to constitutive kinase signaling and oncogenic transformation. SUMMIT is an ongoing basket trial of neratinib, an irreversible pan-HER tyrosine kinase inhibitor, in pts with ERBB2 mutant cancers (NCT01953926).

Methods: Pts with advanced solid tumors and locally documented ERBB2 mutations received neratinib 240 mg daily (N=8) +/- fulvestrant at the labeled dose for pts with ER+ breast cancer (N=6) as part of the global Phase II SUMMIT basket study. Pre- and post-treatment tumor and cfDNA were subjected to NGS using MSK-IMPACT for tissue (410 genes) and MSK-ACCESS for plasma (129 genes) to identify SNVs, indels, and copy number alterations.

Results: In total, 14 pts had successful sequencing of both paired tissue and cfDNA sample (10 breast, 2 gallbaldder, 1 bladder, and 1 unknown primary). All pts achieved clinical benefit on neratinib including 2 CRs, 7 PRs, and 5 SDs. In 1 gallbladder pt, paired tissue sequencing identified loss of the pretreatment clonal ERBB2 mutation but retention of a truncal TP53 mutation, suggesting emergence of a distantly related ERBB2 wildtype clone. In the remaining 13 patients, the pretreatment ERBB2 mutation was retained in tissue at progression. 64% (9/14) pts had at ≥1 acquired alteration in tissue including 3 pts who acquired secondary alterations in ERBB2, both clonal and subclonal ERBB2 mutations as well new focal amplifications. Consistent with this finding, plasma cfDNA sequencing revealed 57% (8/14) of pts acquired ≥1 secondary ERBB2 mutation with the majority occurring at known activating hotspots. Analysis of mutant allele frequencies of these emergent ERBB2 alterations, in comparison to other variants, suggested the majority were subclonal with evidence of multiple independent subclones arising in the same patient. Two pts developed known ERBB2 gatekeeper mutations (T798I and L785F).

Conclusion: In pts with ERBB2-mutant solid tumors with clinical benefit on neratinib, a potential on-target resistance mechanism was identified in 71% (10/14, including 7 with acquired gain-of-function ERBB2 mutations, 2 with ERBB2 both gain-of-function and gatekeeper mutations, and 1 with outgrowth of an ERBB2 wildtype clone). Collectively, these data provide additional evidence that ERBB2 mutations lead to oncogene addiction in solid tumors. We also demonstrate that tumor and cfDNA sequencing provides complementary information that can be integrated to more fully elucidate potential resistance mechanisms.

#930

On-target resistance to mutant selective EGFR inhibitors develops in an allele specific manner dependent on the original EGFR activating mutation.

Yunkai Zhang,1 Benjamin P. Brown,2 David Westover,1 Yingjun Yan,1 Huan Qiao,1 Vincent Huang,1 Zhenfang Du,1 Jarrod A. Smith,2 Jeffrey S. Ross,3 Vincent A. Miller,3 Siraj Ali,3 Lyudmila Bazhenova,4 Alexa B. Schrock,3 Jens Meiler,2 Christine M. Lovy1. 1 _Vanderbilt University Medical Center, Nashville, TN;_ 2 _Vanderbilt University, Nashville, TN;_ 3 _Foundation Medicine Inc., Cambridge, MA;_ 4 _UCSD Moores Cancer Center, La Jolla, CA_.

In-frame deletions within exon 19 (Ex19Del) and the Leu858Arg mutation within exon 21 (L858R) are the two most common oncogenic EGFR mutations found in non-small cell lung cancer (NSCLC). Currently, there is little data differentiating Ex19Del from L858R, and patients whose tumors harbor Ex19Del and L858R are approached similar in clinic when choosing EGFR tyrosine kinase inhibitors (TKIs). Unfortunately, resistance mutations may emerge against all classes of EGFR TKIs, including osimertinib. Recently, G724S has been identified as a novel mutation which is selected in osimertinib resistant tumors. Interestingly, in a large genomic profile database of NSCLC patient samples we found that G724S co-occurs preferentially with Ex19Del (15/19 G724S cases) but not with L858R (0/19 G724S cases). We further combined in silico and in vitro investigations to demonstrate the mechanism of G724S mutation confers resistance to TKIs in an oncogenic variant-dependent manner. Our in silico investigations showed evidence of reduced stability of the osimertinib-bound G724S/Ex19Del complex but not the osimertinib-bound G724S/L858R complex. G724S/Ex19Del-transduced cells are resistant to osimertinib treatment in vitro, while G724S/L858R-transduced cells are sensitive to osimertinib treatment. These results suggest G724S/Ex19Del may emerge under selective pressure from EGFR TKIs, and the underlying oncogenic profile of EGFR can influence the development of drug resistance mutations. Our results have potential direct implications for the treatment of patients with EGFR-mutant NSCLC.

#931

The T790M mutation is acquired through 5-methylcytosine deamination after EGFR TKI treatment in lung cancer.

Najwa El Kadi,1 Luo Wang,1 April Davis,1 Alexander Cooke,1 Varun Vadnala,1 Hasan Korkaya,2 Gregory P. Kalemkerian,1 Khaled A. Hassan1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _Augusta University, Augusta, GA_.

Background: Epidermal growth factor receptor (EGFR) activation mutations occur in 10-50% of lung adenocarcinomas. EGFR tyrosine kinase inhibitors (TKIs) are the mainstay of treatment for stage IV non-small cell lung cancer (NSCLC) patients with EGFR mutations. However, acquired EGFR mutations are the main mechanism of on-target resistance to TKIs. T790M mutation that occurs after first generation TKI treatment, is a cytosine to thymine (C>T) single nucleotide transition leading to a threonine to methionine amino acid change at position 790 (i.e. T790M). Our data suggest that resistant mutations are acquired events secondary to cytosine deamination through Activation Induced Cytosine Deamination enzyme (AICDA). Interestingly, treatment with Osimertinib, that overcomes the T790M mutation, leads to other acquired resistant mutations (C797S, G796S/R and L792F/H) that are mostly cytosine based.

Results: Sub clones of the lung adenocarcinoma cell line PC9 with no evidence of T790M mutation by digital droplet PCR (ddPCR) at baseline, were treated with EGFR TKI. After serially increasing the treatment dose, T790M mutation was detected by ddPCR associated with a significant increase in AICDA expression. Similarly, when the resistant T790M PC9 clones were treated with Osimertinib, the expression of AICDA was also induced. Knocking down AICDA by shRNA or CRISPR-Cas9, decreases the development of T790M in PC9 cell lines after TKI exposure. Using mass spectrometry, we established that cytosine at codon 790 is methylated; thus, deamination of 5-methylcytosine leads to thymine directly, explaining the T790M C>T mutation. In addition, using ChIP assay and pharmacological inhibition we confirm that upon TKI exposure, NFĸB binds AICDA promoter and induces its expression. In a mouse xenograft model, the induction of NFĸB and AICDA after EGFR TKI exposure is abrogated by concurrent use of an NFĸB inhibitor. Finally, patients treated with EFR TKI had an increased expression of AICDA upon progression.

Conclusion: In EGFR driven lung adenocarcinoma, NFĸB pathway is activated upon exposure to EGFR TKIs which induces AICDA expression. AICDA deaminates cytosine into other nucleotides leading to treatment resistance.

#932

Reversing PARP inhibitor resistance by targeting the replication stress response.

Mark J. O'Connor,1 Cristina Cruz,2 Marta Castroviejo-Bermejo,2 Urszula M. Polanska,1 Gemma N. Jones,1 Anderson Wang,1 Zhongwu Lai,3 Josep Forment,1 Krishna Bulusu,1 Alba Llop-Guevara,2 Brian Dougherty,3 Cristina Saura,2 Rachel Brough,4 Chris J. Lord,4 Alejandra Bruna,5 Carlos Caldas,5 Stephen Fawell,3 J Carl Barrett,3 Susan E. Critchlow,1 Judith Balmaña,2 Elaine Cadogan,1 Violeta Serra2. 1 _AstraZeneca, Cambridge, United Kingdom;_ 2 _Vall d'Hebron Institute of Oncology, Barcelona, Spain;_ 3 _AstraZeneca, Waltham, MA;_ 4 _Institute of Cancer Research, London, United Kingdom;_ 5 _Cancer Research UK, Cambridge, United Kingdom_.

PARP1/2 inhibitors (PARPi) are the first approved targeted DNA damage response (DDR) inhibitors and have been shown to have clinical benefit, particularly in tumors harboring mutations in BRCA1/BRCA2 and other genes of homologous recombination repair (HRR). However, resistance to PARPi monotherapy will impact on both the breadth and depth of response. We showed that HRR-mutant TNBC and ovarian cancer patient-derived tumor xenograft (PDX) models frequently exhibit innate or acquired PARP inhibitor resistance and that this resistance is linked to a proficient or reactivated HRR, indicated by the ability to form RAD51 foci. A key component of PARPi mechanism of action results from trapping PARP onto DNA, which has the potential to generate replication stress. Moreover, recent data demonstrate that PARP1, along with components of the HRR, are associated with the re-start and protection of stalled replication forks. Here, we assessed whether combinations of the PARPi olaparib together with inhibitors of the replication stress response (RSR) could reverse PARPi resistance in our PDX cohort and we analyzed the effects on RSR by immunofluorescence and immunoblotting. Our data demonstrate that 7/27 models exhibit WEE1i (AZD1775) single agent activity measured as tumor regressions (≤ -30% change in tumor volume), all of which were PARPi resistant. Of the PARPi and WEE1i resistant models, 5 responded to combination treatment and demonstrated a significant increase in the RSR markers pRPA32 and pan-γH2AX compared to either single agent treatment alone. For monotherapy ATRi treatment, there were three models showing tumor regression with AZD6738, all harbored ATM mutations, two also responding to WEE1i monotherapy and one responded to olaparib monotherapy. There were an additional two models that responded to the combination of PARPi plus ATRi, and one of these also responded to the WEE1i/PARPi combination. Together, our analysis demonstrates that by targeting the replication stress response we could cause tumor regression in 13/20 PARPi resistant PDX models and in 6 of these cases the response required PARP inhibition, with DDR signalling indicating that the PARPi was impacting on the RSR. Further insights will be presented into which genetic backgrounds and acquired PARPi resistant mechanisms are reversed by targeting either WEE1 or ATR, thus highlighting the potential for how PARPi resistance can be reversed by targeting alternative DDR dependencies.

## IMMUNOLOGY

### Adoptive Cell Therapy

#933

Surface-tethered IL-12 improves tumor-specific T cell therapy and enhances inflammatory activity in tumors without inducing systemic toxicities.

De-Kuan Chang, Gulzar Ahmad, Jonathan Nardozzi, Katharine Sackton, Jesse Lyons, Karsten Sauer, Thomas Andresen, Douglas Jones. _Torque Therapeutics, Cambridge, MA_.

Introduction Interleukin-12 (IL-12) is a potent cytokine that augments anti-tumor immune responses by promoting CD4 T cell Th1 differentiation, increasing CD8 T cell and NK cell cytotoxicity, inducing MHC expression on antigen presenting cells, and reprogramming myeloid-derived suppressor cells. However, the clinical utility of IL-12 administered systemically or produced by genetically engineered tumor-specific T cells has been limited by toxicities. DeepTM IL-12 is a fusion protein between IL-12 and a Fab antibody against CD45, an abundant surface receptor on T cells. This tethers IL-12 onto tumor-specific T cells prior to adoptive cell transfer (ACT) with the aim of reducing systemic exposure and focusing IL-12 function on the tumor microenvironment. Here, we describe the superior safety and efficacy profiles of Deep IL-12 tethered T cells in an immune competent adoptive cell therapy model for solid tumors.

Methods The safety and efficacy of Deep IL-12 Primed T cells were evaluated in the PMEL/B16-F10 cancer model. CD8 PMEL T cells contain a TCR that recognizes the gp100 antigen expressed in B16-F10 melanoma cells. Deep IL-12 was loaded onto PMEL CD8 T cells to generate Deep IL-12 Primed PMEL T cells which were then adoptively transferred into mice bearing B16-F10 tumors. We compared tumor size, body weight change, cytokine release, circulating toxicity biomarkers, immune cell activity and histopathology in mice treated with Deep IL-12 Primed T cells and PMEL T cells alone or co-administered with recombinant IL-12.

Results Deep IL-12 Primed PMEL T cells significantly improved tumor growth inhibition and overall survival in mice bearing established B16-F10 tumors as compared with PMEL T cell therapy alone or combined with systemic co-administration of IL-12. Deep IL-12 Primed PMEL

T cells were well-tolerated, as administration of at least 40 million Deep IL-12 Primed PMEL T cells did not cause overt toxicities in non-tumor bearing mice. Deep IL-12 Primed PMEL T cells, but not PMEL T cells combined with systemic IL-12, resulted in elevated and sustained induction of IFNγ in the tumor microenvironment. In contrast, IFNγ induction in serum was transient and returned to baseline levels within four days of ACT. Interestingly, Deep IL-12 Priming increased both accumulation and activity of the adoptively transferred PMEL T cells in the tumor microenvironment but not in off-target healthy tissues such as the spleen. Further toxicological analysis of Deep IL-12 Primed tumor-reactive T cells will be discussed.

Conclusions Deep IL-12 Priming enables tethering of IL-12 to the surface of tumor-specific T cells prior to ACT. This improves anti-tumor efficacy by promoting T cell function selectively in the tumor microenvironment and avoids toxicity by limiting systemic IL-12 exposure, resulting in a highly favorable benefit-risk profile for Deep IL-12 Primed T cells.

#934

γδ CAR T cell therapy significantly mitigates bone metastatic castrate resistant prostate cancer.

Jeremy Steven Frieling, Maria Cecilia Ramello, Ismahene Benzaid, Emiliano Roselli, Chen Hao Lo, Conor C. Lynch, Daniel Abate-Daga. _Moffitt Cancer Center, Tampa, FL_.

Bone metastasis is a frequent complication in advanced prostate cancer, with the resultant lesions significantly contributing to patient morbidity and mortality. While next generation hormone ablation therapies and bone protecting bisphosphonates alleviate these symptoms, the disease remains incurable, and new therapeutic approaches are of urgent need. In this regard, we have focused on tapping the tumor-seeking potential of chimeric antigen receptor (CAR) T cells. CAR T cells have shown remarkable anti-tumor responses in hematologic malignancies, but unique physical and biological challenges have hindered their activity in solid tumors. Interestingly, patients treated with bisphosphonates such as zoledronate exhibit enhanced recruitment and activation of the γδ subset of T cells in bone due intracellular accumulation of isopentenyl pyrophosphate (IPP) phosphoantigen. Whereas conventional CAR T cell therapies utilize αβ T cells, we posit that designing γδ CAR T cells could improve homing to bone metastases when administered with bisphosphonates. First, to test the impact of prostate specific stem cell antigen (PSCA)-specific γδ CAR T cells against bone metastatic prostate cancer in vivo, NSG mice (n=10) were intratibially injected with PSCA/luciferase-expressing C4-2B (2x105) castrate resistant prostate cancer cells. Tumors were allowed to establish for 10 days and then randomized into control or γδ CAR T (1.5x107 via tail vein) groups. Subsequent bioluminescent imaging indicated a rapid and significant (p=0.0006) regression of tumors in the γδ CAR T cell group, leading to increased overall survival (5/5 γδ CAR T vs. 0/5 control after 68 days, p=0.0002). Ex vivo bone morphometry analysis also demonstrated the significant protective effect of γδ CAR T associated bone disease. To determine whether bisphosphonates could further enhance the homing of γδ CAR T to bone, NSG mice (n=30) were intratibially injected with C4-2B (2x105), and randomized into control and zoledronate (30µg/kg) groups. After 10 days, mice received γδ T cells (3x106). Subsets were sacrificed at 1, 3, and 5 days post-T cell administration, and peripheral blood, tibia bone marrow, and spleens isolated. CD3-Vδ2 flow cytometry indicated increased γδ T cells in the tibia bone marrow from zoledronate groups (Day 1=61%, Day 3=32%, and Day 5=57%). Furthermore, decreased tumor growth rates were observed in the zoledronate group, suggesting that increased homing of γδ T cells induced anti-tumor effects. Our data to date demonstrate that γδ CAR T cells significantly mitigate bone metastatic prostate cancer and associated bone disease. Further, bisphosphonates (already used in the clinical setting) enhance the homing of γδ CAR T cells to the tumor-bone microenvironment. We posit that γδ CAR T will be an effective immunotherapy approach for the treatment of men with incurable bone metastatic prostate cancer.

#935

Genetic modification potentiates the anti-tumor activity of human placental CD34+ cells-derived NK cells.

James Li, Xuan Guo, Hemlata Rana, Andrea DiFiglia, Joseph Gleason, Uri Herzberg, Robert Hariri, Xiaokui Zhang. _Celularity, Warren, NJ_.

Celularity is currently developing a proprietary allogeneic NK cells (PNK) derived from human placental CD34+ progenitors for treatment of a broad spectrum of blood disorders and malignant diseases. To augment the anti-tumor function of CD34-derived PNK-cells, a study was conducted to simultaneous knock-out (KO) CBLB (negative regulator of NK cytotoxicity) and TGFβ receptor II TGFBR2 (tumor microenvironment) genes in PNK-cells using CRISPR/Cas9. In DKCT (Double Knockout CBLB TGFBR2) cells, high efficiency of editing (>80%) was achieved for both genes and did not affect expansion and differentiation of CD34 cells into DKCT PNK-cells. The results showed that knocking-out CBLB (CBLB-KO) in PNK-cells led to an increase (2–4 fold) of cytotoxicity and cytokine secretion against a range of hematological and solid cancer cell lines as well as primary tumor cells. However, these enhanced functions from CBLB-KO PNK-cells were still found to be sensitive to TGFβ-mediated suppression. TGFBR2-KO rendered PNK-cells the ability to maintain high-level expression of NK activating receptors and resist inhibition of cytotoxicity after TGFβ exposure. The improved cytolytic function of DKCT cells in vitro was confirmed using xCELLigence platform. Furthermore, DKCT PNK-cells were insensitive to exogenous TGFβ and exhibited improved (2–4 fold) cytotoxicity. Subsequently, we compared the anti-tumor activity of DKCT to controls in an in vivo model of AML, where conditioned NSG mice were engrafted with HL60-GFP-luc cells 3 days prior to PNK-cell infusion. All four PNK-cell groups exhibited increased survival compared to vehicle. In CBLB-KO and DKCT groups, the tumor burden was similar but significantly reduced compared to Cas9 control or TGFBR2-KO. Future studies will address the benefit of DKCT PNK-cells in TGFβ secreting tumor-bearing animal models to pave the way for genetically modified NK cell therapy.

#936

Regulation of CD8+ T cell function and anti-tumor activity by DGKα and DGKζ.

Susan Wee,1 Junchen Gu,1 Cindy Wang,1 Carolyn Cao,1 Sandra Holzhauer,2 Heshani Desilva,1 Erin Wesley,3 Susan Tsai,3 Douglas Evans,3 Matthew Riese3. 1 _Bristol Myers Squibb, Princeton, NJ;_ 2 _Blood Research Institute, Milwaukee, WI;_ 3 _Medical College of Wisconsin, Milwaukee, WI_.

Diacylglycerol kinases (DGKs) are a family of enzymes that metabolize the lipid diacylglycerol (DAG) to form phosphatidic acid. In T cells, two isoforms of DGK, DGKα and DGKζ, serve an important role in dampening signal transduction downstream of the T cell receptor (TCR), where they phosphorylate DAG generated by PLCγ1 after activation of proximal signaling molecules. DAG generated downstream of TCR binds to Protein Kinase C θ (PKCθ) and Ras Guanyl Releasing Protein 1 (RasGRP1), facilitating T cell activation. Elimination of either DGK isoform leads to increased generation of DAG and subsequent strengthened signaling downstream of RasGRP1 and PKCθ. Functionally, this results in formation of CD8+ T cells with enhanced cytokine production and proliferation after TCR stimulation. While mice or CD8+ T cells deficient in DGKα or DGKζ are both known to demonstrate enhanced immune responses to tumor, there has not been, to date, a direct comparison of the anti-tumor activity between mice lacking the two isoforms. We sought to determine which isoform of DGK more intensely attenuated anti-tumor immunity in order to identify which would be a better target for immune-based cancer therapies. We inoculated C57BL/6 mice with a variety of syngeneic tumor cell lines - MC38 colon carcinoma, B16F1 melanoma, and C1498 leukemia - and directly compared survival and tumor growth between mice deficient in DGKα or DGKζ in the presence or absence of anti-PD1 treatment. We found that DGKζ-/- mice more efficiently suppressed growth of subcutaneously implanted tumor cells when compared with DGKα-/- mice across the three model systems and further observed that the combination of DGKζ-deficiency and anti-PD1 were additive in tumor control. Additionally, relative to wildtype CD8+ T cells, we found that DGKζ-/- CD8+ T cells demonstrated qualitatively enhanced mRNA changes as assessed by RNA-seq analysis of cells after TCR stimulation. Pathway analyses from RNA-seq similarly indicated enhanced quantitative changes in DGKζ-/- T cells relative to DGKα-/- T cells, findings confirmed with in vitro biochemical and functional assays. Lastly, using CRISPR-based approaches to eliminate DGKα or DGKζ, we observed similar biochemical changes in human T cells when compared with T cells from gene-deficient mice. Our data, employing genetic approaches to modulate DGKα or DGKζ in both human and mouse models, suggests that DGKζ is a dominant regulator of TCR driven T cell activation and is consistent with the observed superior anti-tumor immune response.

#937

Nr4a transcription factors limit CAR T cell function in solid tumors.

Joyce Chen,1 Isaac F. Lopez-Moyado,1 Hyungseok Seo,1 Chan-Wang J. Lio,1 Laura J. Hempleman,1 Takashi Sekiya,2 Akihiko Yoshimura,3 James P. Scott-Browne,1 Anjana Rao1. 1 _La Jolla Institute for Allergy and Immunology, La Jolla, CA;_ 2 _National Center for Global Health and Medicine, Chiba, Japan;_ 3 _Keio University School of Medicine, Tokyo, Japan_.

In cancer immunotherapy, CD19-targeted CAR T cells have exhibited impressive clinical efficacy against B cell leukemias and lymphomas; however, they have been less effective against solid tumors. This is in part because CAR T cells enter a hyporesponsive ("exhausted" or "dysfunctional") state that is triggered by chronic antigen stimulation and characterized by upregulation of several inhibitory surface receptors and loss of effector function. To identify transcriptional regulators and other candidates contributing to the diminished CAR T cell function in solid tumors, we developed a CAR T cell model in which recipient mice bearing murine melanoma tumors expressing human CD19 were adoptively transferred with CD19-targeted CAR T cells. We demonstrate that in both CD8\+ CAR T tumor-infiltrating lymphocytes (TILs) and endogenous CD8+ TILs expressing high levels of PD-1 and TIM3, the Nr4a proteins Nr4a1 (Nur77), Nr4a2 (Nurr1), and Nr4a3 (Nor1) are prominent effectors of the transcriptional program downstream of NFAT: they promote the expression of inhibitory receptors and genes associated with early hyporesponsive state, and limit effector function. Most importantly, treatment of tumor-bearing mice with CD8\+ CAR T cells lacking all three Nr4a factors (Nr4aTKO) resulted in tumor regression and prolonged survival. Nr4aTKO CD8+ CAR T TILs displayed a transcriptomic profile characteristic of effector function, including upregulation of granzymes and cytokines, and many of these gene expression changes were associated with altered regulatory element accessibility near effector genes. Our data identify Nr4a transcription factors as major players in the cell-intrinsic program of T cell hyporesponsiveness and point to Nr4a inhibition as a promising strategy for cancer immunotherapy.

#938

Disruption of TOX overcomes CAR T cell dysfunction function in solid tumor.

Hyungseok Seo,1 Joyce Chen,1 Arundhoti Das,2 Avinash Bhandoola,2 Anjana Rao1. 1 _La Jolla Institute, San Diego, CA;_ 2 _National Cancer Institute, MD_.

Adoptive cell therapy with chimeric antigen receptor expressing T (CAR T) cells has shown promising therapeutic efficacy against leukemia and lymphoma. However, CAR T cells in solid tumors fail to be as effective as in liquid tumors since they enter into a hyporesponsive (exhausted or dysfunctional) state that is induced by chronic antigen stimulation in cancer. Here, we show that CAR T cells in solid tumors exhibit low effector function and high expression of inhibitory receptors such as PD-1, Tim-3 and Lag 3. We also show that the nuclear factor, Thymocyte selection-associated HMG box protein, TOX - a target of the transcription factor NFAT - is highly expressed in CAR T cells in solid tumors with PD-1 expression. To determine how TOX transcription factors contribute to exhaustion/ dysfunction in CAR T cells, we developed CAR T cells targeting huCD19 and lacking TOX and TOX 2. Surprisingly, treatment of huCD19 expressing melanoma or colorectal tumor-bearing mice with CAR T cells doubly deficient in TOX and TOX2 resulted in the eradication of solid tumors and prolonged survival of the tumor-bearing mice, compared to mice treated with WT CAR T cells or CAR T cells lacking TOX or TOX2 alone. Tumor-infiltrating CAR T cells (CAR TILs) doubly deficient in TOX and TOX2 did not increase their expression of inhibitory receptors such as PD-1, Lag3 and CD160 and showed higher expression of TCF1 compared to WT CAR TILs. Our data identify TOX and TOX2 -high mobility group transcription factors belonging to the TOX family - as critical transcriptional factors for inducing hyporesponsiveness in CAR T cells in solid tumors. Our data suggest that disruption of TOX expression or activity could be a promising strategy for cancer immunotherapy.

#939

**A novel first-in class common-gamma chain cytokine facilitates expansion of CD8** + **T cells and offers a superior cellular source for adoptive T cell therapy.**

Anirban Banerjee,1 Yizhan Guo,1 Sarah Hein,2 Alexander S. Krupnick1. 1 _University of Virginia, Charlottesville, VA;_ 2 _Courier Therapeutics, Houston, TX_.

Isolation, ex vivo expansion followed by reinfusion of activated autologous lymphocytes is a successfully utilized and promising clinical treatment for both solid and liquid tumors. Although it has been routine for most clinical and experimental laboratories to rely on interleukin-2 (IL-2) to support T cell expansion this cytokine can result in substantial expansion and induction of regulatory T cells (Tregs), AICD, exhaustion and terminal differentiation. IL-2 signals when the three subunits of IL-2 receptor, namely α, β and γ form a heterotrimeric protein. We have recently described a novel re-targeted form of IL-2 which utilizes NKG2D rather than IL-2Rα to form the high affinity receptor complex for IL-2 signaling. This cytokine (OIL-2), targets signaling solely to NKG2D-expressing cells but its role in expanding CD8+ T cells for adoptive T cell therapy is unknown

In direct comparison to IL2, OIL-2 resulted in significantly improved expansion of CD8+ T cells (70-fold in vs 345-fold increase in 3 weeks; p<0.002), reduced cell death/apoptosis (% cell viability of 32.73±3.01 vs 73.9±11.28; p<0.017), improved memory formation (%Tcm of 9.867±7.28 in IL-2 vs 25.5±2.78 in OIL-2; p<0.039) and increased effector functions (%IFN-γ+ cells were 43.5±3.15 vs 71.4±1.4; p<0.09). To evaluate the signaling pattern we inhibited PI3K using wortmannin but saw no decrease in the efficiency of OIL-2 (%IFN-γ + cells reduced non-significantly from 70.66±3.154 to 62.1±1.531). We next evaluated phosphorylation of AKT and noted an increase in AktSer473 phosphorylation (MFI of 457±16.58 in IL2 vs 667±75.52 in OIL-2; p<0.003). However, the levels of pSTAT5 after activation was lower in OIL-2 cultured T cells (MFI of 869.2±123.6 vs 574.4±41.42; p<0.001). Taken together our data indicate a pattern of mTOR2 activation by OIL-2 vs mTOR1 by wild-type IL-2. We also found a decrease in %Tregs and FOXO1, and p53 levels in OIL-2 cultured T cells. To fully evaluate our cytokine, we expanded anti-gp100 TCR transgenic CD8+ T cells in either wild-type IL2 or OIL-2 and demonstrated an improvement in the control of B16 melanoma (151.73±40.01vs 62.59±22.49 mm3 on day 13 of growth). We also showed that in-vitro lysis of B16 tumor cells were enhanced by OIL-2 cultured T cells (% B16 lysis of 19.5±7.62 vs 49±7.32; p<0.001).

In conclusion we demonstrate that our first-in class cytokine is more effective and offers a superior method for ex vivo T cell expansion. OIL-2 mediated CD8+ T cell stimulation occur via an altered signaling complex which causes T cell metabolic re-programming. As a result, OIL-2 expanded T cells persist and offers better tumor clearance and provide superior protection against malignancy over T cells expanded through alternative protocols.

### Cancer Vaccines and Intratumoral Immunomodulation

#940

Development of ONCR-177, a miR-attenuated oncolytic HSV-1 designed to potently activate systemic anti-tumor immunity.

Brian B. Haines, Agnieszka Denslow, Michael S. Ball, Jacqueline Gursha, Daniel Wambua, Cecilia Kwong, Lingxin Kong, Prajna Behera, Peter Grzesik, Caitlin Goshert, Allison Colthart, Jennifer S. Lee, Terry Farkaly, Edward M. Kennedy, Lorena Lerner, Christophe Queva. _Oncorus, Cambridge, MA_.

ONCR-177 is a highly modified recombinant oncolytic Herpes Simplex Virus (oHSV) designed to be a safe and efficacious therapy for the treatment of solid tumor indications. Complementary safety mechanisms, such as tissue-specific miR attenuation of essential viral genes and UL37 mutation, were introduced to reduce replication, neuropathic activity, and latency in normal cells, while preserving oncolytic ability in tumor cells. In addition, ONCR-177 is armed with five transgenes: the NK and T cell activating cytokine IL-12, the chemokines CCL4 and FLT3LG (extracellular domain) to allow for expansion and recruitment of classical dendritic cells, and antagonists to the clinically validated immune checkpoint targets PD-1 and CTLA-4 to counter T cell exhaustion. Therefore, ONCR-177 is proposed to have a dual mechanism of action whereby the miR attenuation strategy allows for selective oncolysis of tumors cells and the transgenes mediate potent stimulation of systemic anti-tumor immunity. Since human IL-12 and the anti-CTLA-4 antagonist do not cross-react with the mouse targets, most nonclinical pharmacology studies were conducted with the mouse surrogate mONCR-171, which expresses within the same base vector as ONCR-177 the mouse equivalent to the ONCR-177 transgenes. Intra-tumoral administration of mONCR-171 in the oHSV-sensitive A20 BALB/c lymphoma bilateral tumor model resulted in response rates (partial and complete tumor regressions) of 100% and 80%, respectively, on the injected (ipsilateral) and distant (contralateral) tumor. mONCR-171 was also highly efficacious in the B16F10 melanoma model, an oHSV-resistant C57BL/6 based tumor model engineered to be permissive to oHSV by introduction of Nectin-1, and in the oHSV-resistant MC38 C57BL/6 colon carcinoma model. Abscopal anti-tumor activity could not be explained by propagation of the virus since viral DNA and transgenes were only detectable in the injected tumor. Rather, mONCR-171 treatment resulted in increased numbers of activated NK cells, CD8 and CD4 T cells, and classical dendritic cells. The proportion of Tregs decreased, resulting in large CD8/Treg ratios. These changes in immune contexture occurred in both the ipsilateral and contralateral tumor and were more pronounced with mONCR-171 treatment compared to the base vector without transgenes, indicating that the observed abscopal effects were due to the elicitation of systemic anti-tumor immunity mediated in part by the transgenes. These encouraging preclinical data warrant the clinical investigation of ONCR-177 in patients with metastatic cancer.

#941

B cells are required to generate optimal anti-tumor immunity in response to PD-1 blockade treatment.

Shubhra Singh,1 karishma Bavisi,1 Jason Roszik,1 Patrick Hwu,1 Willem W. Overwijk,2 Manisha Singh1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Nektar, Houston, TX_.

Background: Melanoma is the most deadly form of skin cancer. Although, the immunogenic nature of melanoma makes this disease susceptible to immunotherapy, metastatic melanoma remains highly resistant to established immunotherapies like cancer vaccines. Anti-PD-1 antibodies alone showed great clinical activity but were still ineffective in many melanoma patients. These reports highlight the need to understand the molecular mechanism of immunotherapy induced anti-melanoma immune responses to develop new approaches for effective therapy against melanoma. B cells augment CD8+ T cell-mediated immunity during autoimmunity and allograft rejection, but the role of B cells in anti-tumor CD8+ T cell immunity and in cancer patient survival is controversial. Recently, we reported that intratumoral stimulation of TLR-7/8 in a mouse model of melanoma induced tumor regression and activation of B cells. B cells were required for tumor suppression in an antibody-independent manner. Also, we found that melanoma patients who responded to anti-PD-1 blockade therapy had significantly more B cells and CD8+ T cells in tumors than non-responders. Based on these evidences, we hypothesized that activated B cells induce and/or enhance anti-melanoma CD8+ T cell immunity in response to immunotherapy.

Methods: B16 melanoma mouse model was used. Wild type and B cell Knockout mice were used to determine treatment efficacy and generation of tumor-specific CD8+T cells in response to PD-L1 blockade and/or TLR-7/8 activation. 3M-052 activated B cells were isolated and adoptively transferred into tumor-bearing B cell KO mice, which were treated with anti-PD-L1.Therapeutic efficacy and melanoma-specific CD8+ T cell immunity were determined.

Results: We found that absence of B cells not only decreases the efficacy of TLR-7/8 agonist or anti-PDL-1 abs monotherapy but also TLR-7/8 agonist and anti-PD-L1 abs combination therapy. In addition, mice were unable to develop potent anti- melanoma CD8+T cell immunity in the absence of B cells in response to either mono or combination therapy. Interestingly, we reported prolonged survival of tumor bearing B cell KO mice which received 3M-052 activated B cells compared to inactivated B cells recipients in response to anti-PD-L1 antibody therapy.

Conclusions: These results suggest that activated B cells enhance anti-melanoma CD8+ T cell immunity in response to immunotherapy. We expect our findings to lead the field closer to development of biomarkers based on B cell activation that predict response to immunotherapy. Our findings may also facilitate design of more effective anti-melanoma immunotherapy.

#942

The personalized vaccine, NEO-PV-01 with anti-PD1, induces neoantigen-specific de novo immune responses in patients with advanced metastatic melanoma: Association with clinical outcomes.

Siwen Hu-Lieskoven,1 Patrick A. Ott,2 Aung Naing,3 Rana H. Besada,4 Samantha J. Gates,4 Victoria R. Kohler,4 Riley R. Curran,4 Meghan E. Bushway,4 Julian Scherer,4 Kristen N. Balogh,4 Tracey E. Sciuto,4 Ying S. Ting,4 Michael S. Rooney,4 Dewi Harjanto,4 Zhengping Huang,4 Yuting Huang,4 Yvonne Ware,4 April Lamb,4 Lisa D. Cleary,4 Melissa A. Moles,4 Richard B. Gaynor,4 Matthew J. Goldstein,4 Les H. Brail,4 Joel Greshock,4 Lakshmi Srinivasan4. 1 _University of California, Los Angeles, CA;_ 2 _Dana Farber Cancer Research Institute, Boston, MA;_ 3 _MD Anderson Cancer Center, Houston, TX;_ 4 _Neon therapeutics, Cambridge, MA_.

Background: Neoantigens arise from DNA mutations in cancer cells and are important targets for T cell mediated anti-tumor immunity. NEO-PV-01 is a personal neoantigen vaccine of up to 20 peptides designed based on a patient's neoantigen and HLA profile that is directed at inducing tumor-specific T cell responses to neoantigens. Here, we report relationships between baseline tumor characteristics, immune response and clinical outcome for NT-001, a Phase 1b study of NEO-PV-01 + adjuvant in combination with nivolumab in either first or later line therapy for patients with metastatic melanoma, bladder and non-small cell lung cancer (NCT02897765). This analysis is focused on the melanoma cohort.

Methods: Serial blood and tumor biopsies were scheduled for collection: i) prior to treatment, ii) after 12 weeks of nivolumab monotherapy and iii) after completion of NEO-PV-01 vaccination. Baseline features and immune responses in tumor cells were characterized by immunohistochemistry for multiple immune and tumor markers, gene expression, whole exome and TCR sequencing. Antigen-specific responses by IFNγ ELISpot, intracellular cytokine staining, multi-parameter surface and functional phenotyping by FACS and the presence of cytolytic properties were monitored in serial peripheral blood samples. Durability of immune responses up to 104 weeks post start of treatment will be measured.

Results: A cohort of 10 melanoma patients demonstrated CD4 and CD8 T cell responses against 56% of vaccine peptides that were detected primarily in the post-vaccination samples as measured by IFNγ ELISpot. Analysis of additional patients is ongoing. T cell responses were neoantigen-specific for most peptides tested (86%; 12/14). Vaccine-induced immune responses were durable in patients who reached the week 52 treatment timepoint. Most T cell responses were polyfunctional, as evident by secretion of multiple cytokines, exhibited a memory and effector memory phenotype and were cytolytic. Epitope spreading, defined as post-vaccination T cell responses to neoantigens not included in the vaccine, was observed in multiple melanoma patients analyzed and evaluated for association with post vaccine clinical responses. Further, multi-platform assessments of immune and molecular responses including gene expression and TCR repertoire analysis demonstrate extensive responses in patients continuing study past 52 weeks. Additional correlates of clinical outcomes with molecular and immunologic responses will be presented.

Conclusions: Treatment with NEO-PV-01 + adjuvant in combination with nivolumab induced broad de novo neoantigen-specific immune responses in metastatic melanoma. Immune responses were specific and correlations with clinical outcomes will be discussed.

#943

Filtering out self-like neoantigens improves immune response to cancer vaccines.

Guilhem Richard, Bethany Biron, Christine Boyle, Matthew Ardito, Leonard Moise, William Martin, Gad Berdugo, Anne S. De Groot. _EpiVax Inc, Providence, RI_.

Clinical studies have highlighted the potential of precision cancer immunotherapy to effectively control the tumor of patients across cancer indications. However, recent studies showcase the difficulty of establishing robust CD8 and CD4 T cell responses. We hypothesize that poor cancer vaccine performance may be due in part to the inadvertent inclusion of suppressive T cell neo-epitopes in neoantigen vaccines that may be recognized by regulatory T cells (Tregs).

To test this hypothesis, we used the Ancer™ system to identify and select neo-epitopes from the CT26 syngeneic mouse model. Ancer™ leverages EpiMatrix® and JanusMatrix™, state-of-the-art predictive algorithms that have been extensively validated in prospective vaccine studies for infectious diseases (Moise et al., Hum. Vaccines Immunother 2015; Wada et al., Sci. Rep. 2017). Distinctive features of Ancer™ over other in silico pipelines are its ability to accurately predict CD4 T cell epitopes and to identify tolerated or Treg epitopes.

In a first experiment, optimally selected CT26 neoantigen vaccine candidates were identified with Ancer™ and ranked according to tumor expression level and predicted Class I- and Class II-restricted immunogenicity. Self-like, putative Treg epitopes were removed in this process. Naïve Balb/c mice were immunized subcutaneously with a peptide pool comprised of the 20 highest ranking neoantigens delivered with PolyICLC (Oncovir). Immunization with Ancer™-derived neoantigens induced strong IFNg ELISpot responses compared to controls (p < 0.001). Flow cytometry confirmed the vaccine stimulated multifunctional CD4+ and CD8+ T cells.

In a follow-on experiment, ten self-like neoantigens, from the same CT26 genome, were selected with Ancer™. These neoantigens may be recognized by Tregs due to their high degree of similarity with self, based on JanusMatrix™. Co-administration of the CT26 self-like neoantigens with our optimally designed neoantigen vaccine in naïve Balb/c mice diminished IFNg ELISpot responses by 5-fold compared to vaccination without the self-like neoantigens (p = 0.003).

While it has been well known that Tregs are present in tumors, these results suggest the possibility that tumor-derived neo-epitopes may be recruiting Tregs to the tumor. More importantly, the inadvertent inclusion of Treg driving neoantigens in vaccine formulations may hinder efforts to induce strong T cell-mediated tumor control. In silico screening of neoantigen sequences using specialized tools offers the possibility of enriching and designing new vaccines with higher quality candidates. Efforts are ongoing to determine the effect of Ancer™-derived self-like neoantigens on CD4+ and CD8+ T cells and how the inclusion of self-like neoantigens in vaccines affects their efficacy.

#944

exoSTING: An engineered exosome therapeutic that selectively delivers STING agonist to the tumor resident antigen presenting cells resulting in improved tumor antigen specific adaptive immune response.

Su Chul Jang, Raymond J. Moniz, Chang Ling Sia, Rane A. Harrison, Damian Houde, Nikki Ross, Ke Xu, Nuruddeen Lewis, Raymond Bourdeau, Christine McCoy, Tong Zi, Agata Villiger-Oberbek, Scott Estes, Jorge Sanchez-Salazar, Kevin Dooley, William K. Dahlberg, Sriram Sathyanarayanan, Kyriakos D. Economides. _Codiak Biosciences, Cambridge, MA_.

Background: The Stimulator of Interferon Genes (STING) pathway is an attractive target in immuno-oncology. Selective activation of the STING pathway in antigen presenting cells (APCs) is essential for eliciting a potent and specific anti-tumor immune response. STING is ubiquitously expressed in normal cells including T cells and endothelial cells. Direct intra-tumoral administration of a free STING agonist results in activation of the STING pathway in all cells, resulting in loss of viability of immune cells, tissue damage, and systemic immune activation due to vascular leakage. Exosomes are an efficient natural messenger system that delivers macromolecules between cells. Leveraging exosome biology, we have developed a novel engineered exosome therapeutic, exoSTING, to selectively target the STING pathway in tumor resident APCs.

Results: exoSTING is composed of exosomes engineered to express high levels of Protein X (PrX), a transmembrane glycoprotein naturally occurring on exosomes, and loaded with a STING agonist. Following intra-tumoral injection in checkpoint refractory B16F10 tumors, exoSTING selectively activates STING in APCs, leading to anti-tumor immunity with greater than 100-fold improvement in potency compared to free STING agonist. The activity of exoSTING is substantially diminished in the PrX knock out exosomes, highlighting the importance of this surface glycoprotein for preferential activation of APCs. In contrast to the free STING agonist, exoSTING is retained within the injected tumor, minimizing systemic exposure. Furthermore, exoSTING administration preserves the viability of T cells and APCs, reduces collateral tissue damage, and does not induce systemic cytokine production, resulting in a broader therapeutic window in contrast to free STING agonist. exoSTING produced an increased systemic tumor-specific T cell response as demonstrated by elimination of non-injected abscopal tumors. The specificity of exoSTING activity was demonstrated using a STING knockout mouse (Tmem173gt/J). T cells and myeloid cells play a critical role in exoSTING mediated anti-tumor immunity, in contrast to NK cells which are not required. exoSTING treatment results in significant induction of PD-L1 expression (P<0.005). In combination with PD1 checkpoint blockade, exoSTING shows enhanced efficacy over high-dose free STING agonist on abscopal tumors.

Conclusion: exoSTING is an engineered exosome therapeutic that leverages exosome biology and specifically targets the STING pathway in APCs, resulting in greater potency with preserved viability of T cells and APCs, greater systemic tumor antigen specific immune response, reduced systemic cytokine production, and enhanced efficacy.

#945

PBRM1 loss reduces IFNγ-STAT1 activity and promotes resistance to immunotherapy and antiangiogenic therapy in renal cell carcinoma.

Xiande Liu,1 Wen Kong,1 Christine Peterson,1 Anh Hoang,1 Xuesong Zhang,1 Lijun Zhou,1 Patrick Pilie,1 Truong Lam,1 Haifeng Zhu,1 Sevinj Isgandrova,2 Margie Moczygemba,2 Eric Jonasch1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Texas A &M Health Science Center, Houston, TX_.

PBRM1 is a subunit of SWI/SNF complex that regulates chromatin accessibility for transcription factors, and more than 30% of clear cell renal cell carcinoma (RCC) contain PBRM1 mutations. Nivolumab (anti-PD-1) showed profound and prolonged response only in a subset of patients with advanced RCC. The impact of PBRM1 loss on response to immunotherapy in RCC patients still remains contradictory. It is important to develop immune competent murine RCC models that can precisely isolate the influence of PBRM1 loss on the tumor microenvironment. IFNγ-related gene expression profiles have been reported to predict response to anti-PD-1 blockade. We analyzed the effect of PBRM1 on the IFNγ STAT1 signaling pathway in isogenic murine RCC Renca cells. We observed that Pbrm1 knockout reduced IFNγ-induced STAT1 phosphorylation, and consequently the expression of downstream genes including Irf1, Cxc9 and Stat1 itself. Considering the critical role of CXCL9 in T cell recruitment, we found that Pbrm1 deficiency reduced accessibility to the Cxcl9 promoter by STAT1 and BRG1, the ATP-dependent enzyme subunit of the SWI/SNF complex. Furthermore, a bioinformatics analysis of the Cancer Genome Atlas of human RCC and our murine RCC RNAseq database showed that PBRM1 inactivation was associated with reduced expression of an IFNγ-related mRNA profile, including checkpoint genes CTLA4 and PDCD1. These results revealed that PBRM1 loss reduced IFNγ signaling pathway activation and chromatin accessibility by the transcription factor STAT1. Since Pbrm1 deficiency reduced IFNγ-STAT1 activity and consequently Cxcl9 expression, we then compared the immune cell infiltration in both Pbrm1 proficient and deficient Renca tumors in BalB/C mice. As expected, Pbrm1 deficient tumors harbored fewer infiltrating CD3+ and CD8+ T cells, and expressed less PD-1. It has previously been reported that response to immune checkpoint therapy requires pre-existing T cells, and their intrinsic anti-tumor activity potentiated by blocking immune checkpoints. We found that Pbrm1 deficient tumors demonstrated longer latency but more resistance to immunotherapy with PD-1 blockade. We previously showed that antiangiogenic therapy increased T cell infiltration associated with PD-L1 upregulation, and proposed that antiangiogenic agents could promote the response to checkpoint blockade. Here we confirmed that antiangiogenic agent axitinib reduced Pbrm1 proficient tumor growth, and its combination with anti-PD-1 blockade exhibited synergistic efficacy. In contrast, neither axitinib monotherapy nor combination therapy with PD-L1 antibody exerted obvious control on Pbrm1 deficient tumor, indicating patients with PBRM1 functional mutation were more resistant to immunotherapy and antiangiogenic therapy. This study provides mechanistic insights into therapeutic response of PBRM1 mutated RCC.

#946

Early signs of activity of Tedopi OSE2101, a multiple neoepitope vaccine, in a phase 3 trial in advanced lung cancer patients after failure to previous immune checkpoint inhibitors ATALANTE-1.

Santiago Viteri,1 François-Roger Vanel,2 Werner Hilgers,3 Jordi Remon,4 Guillermo Viteri-Ramirez,5 Elisabeth Quoix2. 1 _Instituto Oncológico Dr Rosell, University Hospital Dexeus, QuironSalud Group, Barcelona, Spain;_ 2 _Service de Pneumologie, Unité de Cancerologie Thoracique, Nouvel Hopital Civil, Strasbourg, France;_ 3 _Department of Medical Oncology, Institut Sainte Catherine, Avignon, France;_ 4 _CIOCC Barcelona - HM Delfos, Barcelona, Spain;_ 5 _Radiology Department, Complejo Hospitalario de Navarra, Pamplona, Spain_.

Introduction: Tedopi is a neoepitope vaccine restricted to HLA-A2 positive patients targeting five tumor-associated antigens frequently expressed in lung cancer cells. Potential enhanced efficacy of cancer vaccines after ICI treatment remains unexploited and its biological basis is poorly understood. The integrity of the antigen presentation pathway is crucial in vaccine activity and genetic alterations such as beta-2-microglobulin inactivating mutation may explain lack of efficacy. Tedopi is currently being tested in the phase III ATALANTE-1 trial compared with chemotherapy in advanced NSCLC patients (pts.) who relapse after ICI. We report early signs of safety and efficacy with Tedopi in the first step of the open-label study.

Methods: 18 pts have been enrolled in the experimental arm and received at least one dose of Tedopi. These pts have been assessed every 6 weeks for radiological response according to RECIST 1.1 criteria as well as toxicity according to CTCAE criteria. Tedopi dose was 5 mg injected subcutaneously every 3 weeks for 6 doses, then every 2 months completing 1 year of treatment and thereafter every 3 months until progression or toxicity. Description of three patients with significant clinical benefit with third-line treatment with Tedopi is presented.

Results: Patients' clinical characteristics are reported in table 1. Treatment duration with Tedopi ranges from 4.9 to more than 12 months, including one patient with brain metastases. One patient achieved a partial response and two patients achieved stable disease by RECIST 1.1 criteria. Safety profile was manageable and no patient withdrawn for toxicity.

Conclusions: Tedopi after failure to previous ICI therapy as third-line treatment has shown long-term clinical benefit and good safety profile. Baseline genomic molecular profile assessment, as well as, immunophenotype searching evidence of vaccination are ongoing.

## MOLECULAR AND CELLULAR BIOLOGY / GENETICS

### Emerging Targets in Cancer Epigenetics

#947

Defining UHRF1 domains that support maintenance of human colon cancer DNA methylation and tumorigenicity.

Xiangqian Kong,1 Jie Chen,2 Wenbing Xie,1 Stephen M. Brown,1 Yi Cai,1 Kaichun Wu,3 Daiming Fan,3 Yongzhan Nie,3 Yong Tao,1 Ray-Whay Chiu Yen,1 Hariharan Easwaran,1 Michael J. Topper,1 Scott B. Rothbart,4 Limin Xia,2 Stephen B. Baylin1. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China;_ 3 _State key Laboratory of Cancer Biology, National Clinical Research Center for Digestive Diseases and Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi'an, China;_ 4 _Van Andel Research Institute, Grand Rapids, MI_.

Reversing the DNA methylation abnormalities by targeting the maintenance DNA methylation machinery represents a sought-after therapy paradigm in both liquid and solid tumors. UHRF1, a multi-domain protein with both chromatin reader and writer functions, is essential for targeting DNA methyltransferase 1 (DNMT1) to replicating DNA to maintain DNA methylation in both normal and cancer cells. Dysregulation of the DNMT1-UHRF1 axis is being increasingly linked to malignant transformation and progression across cancer types. Our recent study shows that UHRF1 depletion, alone or in combination with low doses of a DNMT inhibitor, effectively induces DNA demethylation and tumor suppressor genes (TSGs) reactivation. These findings collectively suggest UHRF1 represents a promising target for developing next-generation DNA demethylation agents for cancer therapy.

Understanding the collective contribution of UHRF1's domains for maintenance methylation is essential for the major translational goal of blocking maintenance of abnormal DNA methylation in established cancers and throughout cancer initiation and progression. While the UHRF1 domains responsible for de novo or re-methylation have been identified, the domain requirements specifically for maintaining normal and cancer-specific DNA methylation are poorly characterized. Herein, by developing a carefully timed genetic complementation assay, we systematically interrogate the roles for each major domain of UHRF1 for maintaining genome-wide DNA methylation in human colorectal cancer (CRC) cells. Distinct from the domain required for establishing methylation, we find that UHRF1 histone-binding and hemimethylated DNA reader domains, but not E3 ligase activity, are essential for maintaining cancer-specific DNA methylation in both HCT116 and RKO cells. Disrupting either one of the essential domains phenocopies UHRF1 depletion for global DNA demethylation and reactivation of epigenetically silenced TSGs. Supporting the essential roles of abnormal DNA methylation in sustaining the key oncogenic properties of CRC cells, we further show genetic perturbation of either histone- or hemimethylated DNA-binding activities of UHRF1 dramatically impairs CRC proliferation, invasion, and metastasis in vitro and in vivo. Moreover, for eight UHRF1 chromatin-reader domain regulated TSGs, we reveal a strong negative correlation between high UHRF1 expression, promoter hypermethylation, and their low expression with disease progression and overall survival in CRC patients in both TCGA and two independent CRC cohorts (n=363 and 390, respectively). Taken together, our data identify important differences from domains dominant for de novo DNA methylation, and provide important implications for the oncogenic functions of UHRF1 in CRC, and for credentialing UHRF1 as a desirable target for cancer drug development and means to personalize this.

#948

Epigenomic correlates of checkpoint blockade immunotherapy resistance.

Alvin H. Shi,1 Li-Lun Ho,1 Stuart Levine,1 Vinod Yadav,1 Jamie Cheah,1 Christian Soule,1 Dennie T. Frederick,2 David Liu,3 Genevieve Boland,4 Manolis Kellis1. 1 _MIT, Cambridge, MA;_ 2 _Massachusetts General Hospital, Boston, MA;_ 3 _Dana Farber Cancer Institute, Boston, MA;_ 4 _Massachussets General Hospital, Boston, MA_.

The development of immune checkpoint inhibitors (ICI) has revolutionized treatment for numerous cancer types. However, a significant fraction (~45%) of patients treated with ICI fail to exhibit a sustainable clinical response to the drug. A number of previous studies have conducted genomic and transcriptomic profiling of patients treated with ICI, yielding a number of promising insights into the mechanisms underlying ICI resistance. However, to our knowledge, there has been no comprehensive epigenomic profiling study of ICI resistance in a clinical cohort. We hypothesize that epigenomic longitudinal profiling - conducted in parallel with transcriptomic and genomic profiling - will provide novel evidence to confirm existing hypotheses regarding ICI resistance mechanisms and potentially uncover novel regulatory pathways involved in ICI resistance. We chose to profile H3K27ac histone mark since it is a marker of active enhancers and promoters. We first optimized a high-throughput "chip-in-a-tip" H3K27ac ChIP-seq experimental pipeline for processing snap frozen melanoma samples. We applied this method along with RNA-seq and Whole Exome Sequencing (WES) to N=30 samples (N=13 pre-treatment, N=17 on/post treatment) from N=11 metastatic melanoma patients (N=5 responders/partial-responders, N=6 progressive disease) treated with anti-PD1. Differential H3K27ac binding & expression analysis between responders and non-responders significanlty implicates MHC class I peptide loading and processing, BAT3 complex, BCL3, JAK/STAT signaling, and NFKB pathways as correlates of ICI resistance. Motif analysis provides significant evidence for STAT1 and several novel binding motifs for mediating changes in H3K27ac binding between responders and non-responders. Joint epigenomic and expression differential analysis between pre-treatment and on/post-treatment samples suggests that a large number of pathways, including TCR signaling, Notch signaling, VEGF signaling, MAPK signaling, NFKB signaling, and IL-3 & IL-6 pathways are significantly changed as a result of ICI treatment. Finally, to address differential tumor purity and immune infiltration in our samples, we develop a Bayesian probabilistic model for ChIP-seq deconvolution model aimed at assigning putative cell-type identity to differential peaks. By leveraging immune ChIP-seq profiles from the Roadmap Epigenomics project and melanoma cell-line ChIP-seq from literature, along with estimates of tumor purity from matched RNA-seq/WES data, we are able to assign a putative cell-type-of-origin for peak clusters. We show in simulations that our model assigns reasonable estimates of cell-type identity. We then apply this method to our data to illustrate potential uses of this model in assisting us with interpreting our ChIP-seq results.

#949

ARID1A is a critical regulator of luminal identity and therapeutic response in estrogen receptor-positive breast cancer.

Eneda Toska,1 Guotai Xu,1 Sagar Chhangawala,1 Emiliano Cocco,1 Pedram Razavi,1 Jordan Otto,2 Yanyan Cai,1 Carmen Chan,1 Drew R. D' Avino,2 Clayton Collings,1 Ross L. Levine,1 Maurizo Scaltriti,1 Jorge S. Reis-Filho,1 Cigall Kadoch,2 Christina Leslie,1 Jose Baselga3. 1 _Mem. Sloan Kettering Cancer Ctr., New York, NY;_ 2 _Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA;_ 3 _Vall d'Hebron Institute of Oncology, Barcelona, Spain_.

Mutations in ARID1A, a subunit of the SWI/SNF chromatin remodeling complex, are the most common somatic alteration of the SWI/SNF complex across all cancers including estrogen receptor positive (ER)+ breast cancer. We have recently reported that ARID1A inactivating mutations are present at a high frequency in advanced endocrine resistant ER+ breast cancer. However, the mechanisms whereby disruption of ARID1A influences breast cancer progression and/or endocrine therapy resistance are unknown. In order to elucidate mechanisms of resistance to endocrine therapy, we performed an epigenome CRISPR/CAS9 knockout screen that identified ARID1A as the top candidate whose loss determines resistance to the ER degrader, fulvestrant. ARID1A knockout cells were found to be less responsive to endocrine therapy compared to intact ARID1A cells in vitro and in vivo. These observations led us to undertake a comprehensive chromatin-based mechanistic characterization of ARID1A loss in ER+ breast cancer and its role in endocrine therapy response. ARID1A disruption in ER+ breast cancer cells led to widespread changes in chromatin accessibility converging on the master transcription factors (TFs) that regulate gene expression programs critical for luminal (ER+) lineage identity. Global transcriptome profiling of ARID1A knockout cell lines and patient samples harboring ARID1A inactivating mutations revealed an enrichment for basal-like (ER-) gene expression signatures. The state of increased cellular plasticity of luminal cells that acquire a basal-like phenotype upon ARID1A inactivation is enabled by loss of ARID1A-dependent SWI/SNF complex targeting to genomic sites of the major luminal-lineage determining transcription factors including ER, FOXA1, and GATA3. Thus, through widespread chromatin reprograming and functional regulation of mater luminal TFs, tumor cells alter lineage fidelity and become less responsive to luminal-specific anti-ER therapy. We also show that ARID1A regulates genome-wide ER-chromatin interactions and ER-dependent transcription. Altogether, we uncover a critical role for ARID1A in the determination of breast luminal cell identity and endocrine therapeutic response in breast cancer.

#950

In vivo **efficacy and pharmacodynamic modulation of JNJ-64619178, a selective PRMT5 inhibitor, in human lung and hematologic preclinical models.**

Hillary J. Millar,1 Dirk Brehmer,2 Tinne Verhulst,2 Nahor Haddish-Berhane,1 Tony Greway,1 Dana Gaffney,1 An Boeckx,2 Erika Van Heerde,2 Thomas Nys,2 Joseph Portale,1 Ulrike Philippar,2 Tongfei Wu,2 Sylvie Laquerre,1 Kathryn Packman3. 1 _Janssen Research and Development, LLC, Spring House, PA;_ 2 _Janssen Pharmaceutica, Beerse, Belgium;_ 3 _Janssen Research and Development, LLC, Cambridge, MA_.

Protein arginine methyltransferase 5 (PRMT5), a type II methyltransferase, is responsible for symmetric arginine di-methylation of multiple cellular proteins involved in the regulation of cellular transcription. PRMT5 is involved in cellular processes such as survival, proliferation, and apoptosis, and an elevated tumor PRMT5 protein level has recently been correlated with poor survival of cancer patients.

JNJ-64619178, a selective PRMT5 inhibitor, showed inhibition of cellular growth in several cell lines representing multiple cancer histologies in vitro. From this, a broad selection of xenograft models was chosen to demonstrate potent anti-tumor efficacy.

Xenograft models representing small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), acute myeloid leukemia (AML), and non-Hodgkin lymphoma were chosen to demonstrate anti-tumor efficacy. Biologically significant tumor growth inhibition up to 99% was observed in both solid and hematological xenograft models, including an aggressive disseminated model of AML, with oral doses of 1 to 10 mg/kg, once daily. Importantly, continued inhibition of tumor growth was observed for several weeks following dosing cessation.

Dosing of JNJ-64619178 results in inhibition of Sym-Arg di-methylation of SMD1/3 proteins, core components of the spliceosome in the tumor, and general Sym-Arg di-methylation of serum proteins. These serve as pharmacodynamic markers of PRMT5 inhibition in xenograft models. Potent and prolonged inhibition of SMD1/3 di-methylation was observed in the SCLC model, during and after the dosing period. This has led to the exploration of alternative dosing regimens preclinically.

PRMT5 inhibitor JNJ-64619178 is currently being investigated in a Phase I clinical trial, based on its high selectivity and potency, favorable pharmacokinetics and safety properties, and strong preclinical efficacy and pharmacodynamic data.

#951

Setdb1 deletion protects against formation of pancreatic ductal adenocarcinoma through inducing apoptosis and upregulating p53 expression.

Satoshi Ogawa,1 Akihisa Fukuda,1 Motoyuki Tsuda,1 Tomonori Masuda,1 Makoto Sono,1 Yuichi Fukunaga,1 Takaaki Yoshikawa,1 Osamu Araki,1 Munemasa Nagao,1 Norihiro Goto,1 Yukiko Hiramatsu,1 Takahisa Maruno,1 Yoichi Shinkai,2 Hiroshi Seno1. 1 _Kyoto University, Kyoto, Japan;_ 2 _RIKEN, Wako, Japan_.

One group of epigenetic regulators known to be altered in cancer development is histone modification. Setdb1, a histone methyltransferase that trimethylates histone H3 on lysine 9 (H3K9), has been implicated in tumorigenesis by altering the expression of oncogenes/ tumor suppressor genes. In this study, we aimed to elucidate the functional role of Setdb1 in pancreatic acinar regeneration and formation of pancreatic ductal adenocarcinoma (PDAC).

Setdb1 was expressed in pancreatic ductal cells and a small subset of pancreatic acinar cells in adult wild type mice. Upon caerulein-induced pancreatitis, Setdb1 was expressed in acinar cells and acinar to ductal metaplasia (ADM). To determine the impact of pancreatic Setdb1 deletion on acinar regeneration, we generated Ptf1a-Cre; Setdb1f/f (CS) mice and induced caerulein-pancreatitis. Pancreatic deletion of Setdb1 resulted in pancreatic atrophy concomitant with increased apoptosis of acinar cells and increased expression of p53 in CS mice after caerulein-induced pancreatitis. In addition, pancreatic acinar regeneration was delayed with persistent inflammation in CS mice.

To investigate the impact of pancreatic Setdb1 deletion on PDAC formation, we next generated Ptf1a-Cre; KrasG12D; Setdb1f/f (KCS) mice and compared to control Ptf1a-Cre; KrasG12D (KC) mice. Pancreatic deletion of Setdb1 dramatically accelerated spontaneous development of ADM and pancreatic intraepithelial neoplasia (PanIN), the most common precursor lesion of PDAC, in KCS mice compared to KC mice at the age of 4 weeks. Acinar cell culture experiments revealed that Setdb1 null acinar cells converted to ADM, at least in part, in a cell autonomous manner. Long term observation revealed massive pancreatic atrophy in KCS mice. Immunostaining revealed a dramatic increase in apoptotic cells and upregulated expression of p53 in KCS mice compared to KC mice at the age of 20 weeks. Consistently, microarray analysis revealed upregulated expression of p53 and apoptotic pathway genes in KCS mice compared to KC mice.

We next examined the impact of pancreatic Setdb1 deletion on PDAC formation in the context of p53 deletion. Surprisingly, PDAC formation was suppressed in KCS mice in the context of heterozygous p53 deletion (KPhereroCS mice). Again, apoptosis was increased and p53 expression was upregulated in KPhereroCS mice. In contrast, in the context of homozygous p53 deletion, PDAC was formed and increased apoptosis was canceled in KPhomoCS mice. These findings suggested that Setdb1 deletion protects against PDAC formation through inducing apoptosis, which is mediated by p53.

In conclusion, Setdb1 is required for normal pancreatic acinar regeneration and protects against PDAC formation though inducing apoptosis, which is mediated by p53. Thus, Setdb1 could be a therapeutic target for PDAC which retains wild p53 expression.

#952

Determining the contribution of cystathionine beta synthase to lung cancer lineage fate.

Mojtaba Bakhtiari, Christine Fillmore Brainson. _University of Kentucky, Lexington, KY_.

The two major pathologies of non-small cell lung cancer, adenocarcinoma (ADC) and squamous cell carcinoma (SCC) are historically treated as separate diseases, even though mounting evidence shows that epigenetic reprogramming from ADC to a more SCC fate allows lung cancers to evade therapies. Our team recently developed an isogenic lung cancer model in which lineage switching from ADC to SCC was epigenetically controlled through loss of Polycomb Repressive Complex 2 (PRC2) gene repression of squamous genes. Levels of S-adenosyl methionine (SAM), the methyl donor for the complex, influence PRC2 stability and activity. Steady state metabolism showed that SAM levels were decreased significantly and cystathionine levels were increased significantly in SCC cells relative to ADC cells. Our hypothesis is that increased activity of the enzyme cystathionine beta synthase (CBS) drives epigenetic reprogramming of lung ADC to an aggressive and therapy-resistant state through reduction of SAM pools and destabilization of PRC2, leading to expression of squamous genes. First, we stained a series of human lung cancer tissues for CBS and H3K27me3 and queried TCGA data to validate that CBS is up-regulated in squamous tumors. In addition, we leveraged 3-dimensional organoid cultures and traditional 2-dimensional human non-small cell lung cancer cell lines to explore the contribution of CBS to lung cancer lineage fate. With both of these systems, we used lentiviral small hairpins targeting Cbs/CBS and a lentivirus encoding a full length Cbs/CBS cDNA to modulate gene and protein levels. Lastly, we tested differential sensitivities to the common chemotherapeutic carboplatin on matched isogenic cultures that differ only in lineage state. In human tumors, SCCs have significantly higher levels of CBS and significantly lower levels of H3K27me3 than ADCs. Knock-down of CBS decreased cystathionine and increased both homocysteine and S-adenosyl methionine levels in 2-dimensional human NSCLC cultures. We also observed a global increase in H3K27me3 mark in shCBS cultures relative to shGFP controls. In contrast, CBS over-expression decreased global levels of H3K27me3. Importantly, we observed that squamous organoids are significantly less sensitive to the common chemotherapeutic carboplatin than are ADC organoids, suggesting that lineage switching driven by increased CBS activity will drive chemotherapy resistance. We are now characterizing changes in lineage fate and carboplatin sensitivity that are caused by these genetic manipulations of CBS. Our data suggest that lung cancer lineage fate may be governed in part through expression and activity of the enzyme CBS. Because CBS is a redox-sensitive enzyme, this could be the missing link between increased oxidative stress and ultimate epigenetic reprogramming to a therapy-resistant squamous state in lung cancer. Funded by ACS IRG-85-001-25, K22 CA201036 and KY LCRP to CFB

#953

JARID1 family inhibitor reduces generation of drug resistant EGFR mutation-positive lung cancer cells.

Shin Ariga, Ichiro Kinoshita, Junko Kikuchi, Yasushi Shimizu, Hirotoshi Dosaka-Akita. _Hokkaido University, Sapporo, Japan_.

Epigenetic alteration contributes to tumor initiation, progression and invasion. Epigenetic machinery has become a major focus for the new development of molecular targeted cancer therapeutics. H3K4 trimethylation (H3K4me3) is epigenetic marks which exist surrounding transcription start site of active genes. JARID1 family is demethylase of H3K4me2/me3. JARID1a and JARID1b have been suggested, especially to have oncogenic properties and associations with anti-cancer drug resistance. We previously showed that JARID1 family inhibitor, 2-4(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one (PBIT), recovers sensitivity of drug-resistant lung cancer cells. Here we examined whether PBIT affect the generation of drug resistant cells. A recent report revealed that "secretomes" induced by targeted therapies paradoxically stimulate the outgrowth of drug-resistant subpopulation, which are driven by downregulation of transcription factor FRA1. We confirmed that EGFR mutation-positive PC9 and H1975 cells treated with EGFR-TKIs produced the secretomes that promoted EGFR-TKIs-resistant cell growth. Interestingly, PC9 and H1975 cells pre-treated with 1 μM PBIT did not produce such tumor-promoting secretomes. IGF-1 was upregulated in the secretomes by the EGFR-TKI treatment, but the upregulation was disrupted by the 1 μM PBIT pre-treatment. Immunoblot assay showed that PC9 and H1975 decreased expression of FRA1 after the treatment with EGFR-TKIs, while PC9 and H1975 pre-treated with PBIT mitigated such decrease in FRA1 expression after treatment with EGFR-TKIs. Consistently, chromatin immunoprecipitation assay showed that H3K4me3 at FRA1 promoter region was decreased after treatment with gefitinib in PC9, while the decrease did not occur in PC9 pre-treated with PBIT. To investigate whether PBIT inhibits generation of drug resistant cells, we treated 100 small groups (100 cells each) of cells with EGFR-TKIs with or without 1 μM PBIT for 4 weeks. EGFR-TKI-resistant cells were significantly reduced in combination with PBIT. Of note, 1 μM PBIT alone did not affect clonogenic cell growth in soft agar colony formation assay. Immunoblot assay showed that expression of IGF-1-AKT-S6 pathway molecules in TKI-resistant cells was mitigated by the PBIT combination therapy. These results indicate that PBIT reduces EGFR-TKI-induced secretomes containing IGF-1 as a relevant mediator by preventing downregulation of FRA1 expression via prevention of decrease of active marker H3K4me3 at FRA1 promoter regions. JARID1 inhibitor may have potential to overcome EGFR-TKI-resistance in lung cancer, especially via off-target mechanisms.

### Receptor Tyrosine Kinase and GTPase Signaling in Cancer

#954

SHP2 inhibition overcomes RTK-mediated pathway re-activation in KRAS mutant tumors treated with MEK inhibitors.

Hengyu Lu,1 Chen Liu,1 Roberto Velazquez,1 Hongyun Wang,1 Lukas M. Dunkl,2 Malika Kazic-Legueux,2 Anne Haberkorn,2 Eric Billy,2 Eusebio Manchado,2 Saskia M. Brachmann,2 Susan Moody,1 Jeffrey A. Engelman,1 Peter S. Hammerman,1 Giordano Caponigro,1 Morvarid Mohseni,1 Huaixiang Hao1. 1 _Novartis Institutes for BioMedical Research, Cambridge, MA;_ 2 _Novartis Institutes for BioMedical Research, Basel, Switzerland_.

Introduction: FGFR1 was recently shown to be activated as part of a compensatory response to prolonged treatment with MEK inhibitor (MEKi) such as trametinib in several KRAS mutant lung and pancreatic cancer cell lines. We hypothesize that other receptor tyrosine kinases (RTKs) are also feedback activated in KRAS mutant cell lines after MEKi treatment.

Experimental procedures: We profiled a large panel (n>32) of KRAS mutant cancer cell lines for the contribution of RTKs to the feedback activation of phospho-MEK following MEK inhibition, using a SHP2 inhibitor (SHP099) that blocks RAS activation mediated by multiple RTKs. We then performed in vitro and in vivo combination efficacy studies and pathway analysis in various KRAS mutant cancer models.

Results: We find that RTK-driven feedback activation widely exists in KRAS mutant cancer cells and involves several RTKs including EGFR, FGFR, and MET. We further demonstrate that this pathway feedback activation is mediated through mutant KRAS in KRAS G12C or G12D models. Finally, SHP099 and MEK inhibitors exhibit combination benefits inhibiting MAPK pathway and KRAS mutant cancer cell proliferation in vitro and in vivo.

Conclusions: Our findings suggest that MAPK inhibition in KRAS mutant cancer provokes feedback re-activation of the pathway that often involves RTK activity and SHP2 inhibition may enhance the efficacy of MEKi in KRAS mutant tumors. These findings provide a rationale for exploration of combining SHP2 and MAPK pathway inhibitors for treating KRAS mutant cancers in the clinic.

#955

Musashi-2 regulates EGFR expression in NSCLC, cell proliferation and survival, response to EGFR inhibitors in EGFR-mutant NSCLC.

Alexander Kudinov,1 Petr Makhov,2 Bulat Faezov,2 Igor Bychkov,2 Alexander Deneka,2 Emmanuelle Nicolas,2 Kathy Q Q. Caid,2 Rohan Brebione,3 Eleanor Avrilf,4 Anna S. Nikonova,2 Ilya G. Serebriiskii,2 Hossein Borghaei,2 Erica A. Golemis,2 Yanis Boumber2. 1 _University of New Mexico, Albuquerque, NM;_ 2 _Fox Chase Cancer Center, Philadelphia, PA;_ 3 _Temple University, Philadelphia, PA;_ 4 _Brown University, Providence, RI_.

Musashi-2 (MSI2) is an RNA-binding protein that regulates mRNA translation. We recently established that MSI2 is elevated in a subset of non-small cell lung cancer (NSCLC) tumors upon progression and drives NSCLC metastasis, in part based on activity supporting a TGF-beta/SMAD3/claudin signaling cascade. Here, reverse phase protein array (RPPA) analysis of MSI2-depleted versus control KrasLA1/+;P53R172HΔG/+ murine NSCLC cell lines identified a significant upregulation of HER3 (ERBB3) upon MSI2 depletion. Negative MSI2-dependent regulation of ERBB3 protein was confirmed in multiple murine and human NSCLC models, based on analysis of MSI2 depletion or overexpression. Further, MSI2 positively regulated expression of the epidermal growth factor receptor (EGFR) protein in the same models. Comparing EGFR and KRAS driven models, we found that MSI2 depletion significantly impairs cell proliferation and clonogenicity only in EGFR-mutant NSCLC cell lines. Using RNA immunoprecipitation analysis coupled with qPCR, we show that MSI2 directly binds to EGFR and, to a lesser extent, to HER3 mRNA. MSI2 mRNA binding was correlating with the presence of predicted MSI2 binding sites in corresponding mRNAs. Taken together, this data suggest direct regulation of EGFR protein expression by MSI2 at post transcriptional level. NSCLC lung tissue microarray immunohistochemistry analysis revealed that MSI2 total positivity by H-score, 2+/3+ and 3+ positivity correlated with EGFR 3+ staining. Finally, EGFR inhibitors erlotinib and afatinib synergized with MSI2 depletion in EGFR mutant models, suggesting that therapeutic targeting of MSI2 could be of clinical value, especially in EGFR-mutant lung cancer. In vivo experiments of erlotinib treatments of wild type or MSI2 depleted tumors are ongoing and these results will be presented.

#956

**An essential role for** Argonaute 2 **in EGFR-KRAS signaling in pancreatic cancer development.**

Ronald F. Siebenaler, Sunita Shankar, Jean C. Tien, Vijaya L. Dommeti, Sylvia Zelenka-Wang, Seema Chugh, Ingrid J. Apel, Malay Mody, Anudeeta Gautam, Chandan Kumar-Sinha, Arul M. Chinnaiyan. _University of Michigan, Ann Arbor, MI_.

The RAS gene family is among the most commonly mutated genes within cancer, but little progress has been made in successfully targeting RAS mutations. Targeting binding partners of mutated RAS presents as a promising alternative therapeutic strategy. With the goal of uncovering novel interactors of RAS, we recently identified Argonaute 2 (AGO2) of the RNA-induced silencing complex (RISC) as a novel partner of the KRAS through its Switch II domain. In order to assess the role of AGO2 in KRASG12D driven disease, we developed a mouse model of pancreatic cancer with conditional loss of AGO2. While AGO2 knockout did not prevent development of early precursor pancreatic intraepithelial (PanIN) lesions, AGO2 null lesions displayed increased activation of the EGFR-RAS signaling axis during PanIN development that failed to progress to late stage PanINs, pancreatic ductal adenocarcinoma (PDAC), and metastatic disease. This resulted in a dramatic increase in the survival of mice with AGO2 ablation. Unlike the PanINs in AGO2 sufficient mice, the early PanIN lesions with AGO2 ablation showed staining for the senescence associated beta galactosidase activity, suggesting that AGO2 loss induces oncogene induced senescence. To extend these observations and explore the role of AGO2 interaction with mutant forms of HRAS and NRAS proteins, we performed co-IP of AGO2 with RAS proteins using isoform specific antibodies. Both HRAS and NRAS bound AGO2 in T24 cells (HRASG12V) and SK-MEL-2 cells (NRASQ61H), respectively. In T24 cells, AGO2 knockdown led to the senescent phenotype and was accompanied with changes in the EGFR-RAS signaling axis, similar to that observed in the PanINs of the mice with AGO2 loss. In this cell line model, AGO2 loss reduced mutant HRAS expression and increased wild type RAS activity. These signaling effects were also consistent with our observation that AGO2 loss increased RAS activation in the mouse embryonic fibroblast (MEF) model. Together with our previous work with mutant KRAS dependent cells, these data suggest that 1) AGO2-wild type RAS binding prevents RAS activation and 2) mutant RAS-AGO2 association regulates oncogenic RAS levels in cell line models. Studies on the mouse model and the close proximity of RAS and AGO2 with EGFR also furthered our understanding of the RAS-AGO2 interaction. Using a variety of cell line models, we observed that EGFR-mediated phosphorylation of AGO2Y393 disrupts the interaction between WT RAS and AGO2. However, the mutant KRAS-AGO2 interaction was recalcitrant to EGFR regulation. This provides the first instance of a nucleotide dependent association of RAS and AGO2 and sheds light on the dynamic nature of the RAS-AGO2 interaction.

#957

**An essential role for** Argonaute 2 **in mouse models of** KRAS **driven cancers.**

Sunita Shankar,1 Jean Ching-Yi Tien,1 Ronald F. Siebenaler,1 Vijaya L. Dommeti ,1 Sylvia Zelenka-Wang ,1 Jessica Waninger ,1 Kristin M. Juckette ,1 Alice Xu ,1 Xiao-Ming Wang ,1 Seema Chugh ,1 Malay Mody,1 Sanjana Eyunni ,1 Andrew Goodrum,1 Grace Tsaloff ,1 Yuping Zhang,1 Ingrid J. Apel,1 Javed Siddiqui ,1 Richard D. Smith ,1 Heather A. Carlson,1 John Tesmer ,2 Xuhong Cao ,1 Jiaqi Shi ,1 Chandan Kumar-Sinha ,1 Arul M. Chinnaiyan1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _Purdue University, IN_.

In 2016, we identified a direct interaction between RAS and Argonaute 2 (AGO2), a key mediator of RNA-mediated gene silencing, that is essential for KRAS-driven oncogenesis using pancreatic and lung cancer cell line models. Recently, we employed a genetically engineered mouse model of pancreatic cancer to define the effects of conditional loss of AGO2 in KRASG12D-driven pancreatic cancer (KC model). Genetic ablation of AGO2 did not interfere with development of the normal pancreas or KRASG12D-driven early precursor pancreatic intraepithelial neoplasia (PanIN) lesions. However, AGO2 loss prevents progression from early to late PanIN lesions, development of pancreatic ductal adenocarcinoma (PDAC), and metastatic progression. This results in a dramatic increase in the survival of KRASG12D mutant mice deficient in AGO2 expression. Mechanistically, lack of PanIN to PDAC progression was due to oncogene-induced senescence (OIS) through activation of EGFR-wild type RAS-phosphoERK signaling in the absence of AGO2.

Using validated pan-RAS and AGO2 antibodies for immunofluorescence (IF) and proximity ligation assay (PLA), we observed increased RAS and AGO2 co-localization at the plasma membrane in mouse and human pancreatic tissues associated with PDAC progression. While AGO2 ablation permits PanIN initiation driven by the EGFR-RAS axis, these lesions undergo OIS rather than progressing to PDAC. Further, we used co-IP and PLA analyses to probe the effects of EGF stimulation in cell lines expressing wild-type and mutant forms of KRAS. In wild-type RAS expressing cells, RAS-AGO2 co-localization and interaction were limited to the intracellular regions of the cells, and dramatically increased and shifted to the plasma membrane under conditions of stress (serum starvation). Interestingly, EGF stimulation disrupted this membrane RAS-AGO2 interaction and restored it to intracellular levels. Using phosphorylation-deficient AGO2 mutants, we further demonstrate that the disruption of wild-type RAS-AGO2 interaction is due to EGFR-mediated AGO2Y393 phosphorylation. Interestingly, mutant KRAS-AGO2 interaction is not subject to EGFR activation, suggesting that although both the wild type and mutant RAS bind AGO2, they are differentially regulated through growth factor receptor activation.

We will discuss our ongoing studies evaluating the effects of AGO2 ablation in the KRASG12Ddriven lung cancer mouse model and PDAC progression with p53 loss (KPC model). Our recent in vivo work supports a biphasic model of pancreatic cancer development: an AGO2-independent early phase of PanIN formation reliant on EGFR and wild-type RAS signaling, and an AGO2-dependent phase wherein the mutant KRAS-AGO2 interaction is critical for PDAC progression.

#958

Tumor-vascular interactions promote STING-driven inflammation in the tumor microenvironment.

Marco Campisi,1 Shriram K. Sundararaman,2 Shunsuke Kitajima,2 Valeria Chiono,1 Roger D. Kamm,3 David A. Barbie2. 1 _Politecnico di Torino, Turin, Italy;_ 2 _Dana-Farber Cancer Institute, Boston, MA;_ 3 _Massachusetts Institute of Technology, Boston, MA_.

The recruitment of T cells following intratumoral administration of Stimulation of Interferon Genes (STING) agonists in the tumor microenvironment (TME) is a critical event in the STING-driven antitumor immune response, a pathway with great relevance in the context of cancer immunotherapy. We have previously demonstrated that LKB1 mutation is associated with suppression of tumor cell STING levels and reduced production of T-cell chemoattractants such as CXCL10 in KRAS-driven non-small cell lung cancer (NSCLC). Consistent with this, immunohistochemical staining of patient samples showed poor infiltration of CD3, CD4, and CD8 T cells into LKB1 negative versus LKB1 intact cancer epithelium, and instead, retention of T-cells in stroma. To examine how LKB1 alters immune cell recruitment in a STING-dependent manner, we used a 3-D microfluidic co-culture system to study interactions between vasculature and tumor spheroids derived from a KRAS/LKB1 mutated (KL) cell line with LKB1 reconstitution +/- STING deletion. To form the vasculature, we co-cultured tumor spheroids with fibroblasts and endothelial cells for 7 days, and identified changes in morphology, cytokine production, and gene expression that occur in co-culture. We first observed that co-culture induced synergistic production of multiple immune cell chemo-attractants such as CXCL10, CCL2, CCL5, and G-CSF. Interestingly, this more physiologic ex vivo tumor model of LKB1 reconstitution revealed particularly strong cooperative production of STING-dependent cytokines such as CXCL10 in the vasculature. Moreover, STING depletion in LKB1 reconstituted tumor cells did not significantly attenuate production of CXCL10 and other cytokines in co-culture, suggesting that tumor/vessel interaction may promote STING activation in the vasculature regardless of cancer cell-intrinsic STING function. Furthermore, although there was no appreciable response after treatment of KL cancer cells with cGAMP based STING agonists, treatment of isolated 3-D vascular networks with cGAMP enhanced vascular permeability and increased production of CXCL10 and CCL5, possibly contributing to defective chemokine gradients that retain T cells near the vasculature. Thus, developing these more complex models that incorporate the vasculature may elucidate important aspects of STING biology and may ultimately aid further development of effective immunotherapies targeting this signaling axis.

#959

Proteomic and genetic interaction mapping of the Ras pathway reveals new effectors and vulnerabilities.

Marcus R. Kelly,1 Kyuho Han,1 Kaja Kostyrko,2 Edwin E. Jeng,1 Nancie Mooney,1 Alejandro Sweet-Cordero,2 Michael Bassik,1 Peter K. Jackson1. 1 _Stanford University, Stanford, CA;_ 2 _University of California San Francisco, San Francisco, CA_.

The RAS oncogenes are mutated in one third of human cancers, but therapies against Ras-driven cancers have been unsuccessful. Ras proteins themselves are not yet druggable, and the Ras pathway contains uncharacterized redundancies, feedback mechanisms, and tributaries that have stymied the development of other targeted therapies. Multiple-agent combination therapies hold some promise, but their development requires a more thorough understanding of Ras cell biology than we currently possess. We therefore pursued creation of a physical and genetic map of the Ras pathway in non-small-cell lung cancer (NSCLC). Using H, K-, and NRas and twelve other Ras pathway proteins as baits, we conducted tandem affinity purification experiments to create a high-confidence protein-protein interaction (PPI) network. We integrated our data with public PPI, genetic susceptibility, and patient data to assemble an interpretable interaction map encompassing 360 proteins and 1000 physical interactions among them. Guided by the topology and annotations of this PPI map, we constructed a library of 1000 sgRNAs covering 120 genes. These were screened for pairwise genetic interactions (GIs) in A549 and H23 NSCLC lines using a dual sgRNA vector system to discover over 250 synthetic lethal genetic interactions. Each suggests a strategy for combination therapy. The combined GI/PPI network also produced myriad mechanistic hypotheses. Pursuing these, we made new discoveries that demonstrate the power of the approach. First, we find that KRas binds the cell adhesion regulator Radil, and that Ras regulates cell morphology by modulating the Rap signaling pathway. Second, we identify the critical guanine nucleotide exchange factor and effector by which KRas up-regulates macropinocytosis. Third, we demonstrate that that the in vivo physical interactions between Ras, Raf, and RalGEF family proteins depend crucially on the specific paralogs involved. The equivalent in vitro interactions are not selective, demonstrating that many undiscovered factors direct the strong specificity observed in vivo. Distinct genetic interaction patterns between paralogs in these same families support the non-equivalence of their members. Fourth, we detect a new synthetic lethal genetic interaction between the GTPase chaperone Rap1GDS1 and the GTPase RhoA. The strength of interaction varies among NSCLC lines, but correlates with the KRas dependence of each. This and other interactions show that several other small GTPases work in concert with Ras signaling to regulate tumor progression. Together, these discoveries show that our multiomic approach furthers two critical goals: it produces testable hypotheses that point to new Ras cell biology and reveals combination susceptibilities for the development of new therapies against Ras-driven cancers.

#960

Structures of oncogenic KRAS mutants in the active state and complexed with RasGAP provide new insights into targeting RAS-driven cancer.

Timothy Tran, Srisathiyanarayanan Dharmaiah, Wupeng Yan, Albert Chan, Timothy Waybright, William Gillette, Dominic Esposito, Andrew Stephen, Dwight Nissley, Frank McCormick, Dhirendra K. Simanshu. _Frederick National Laboratory for Cancer Research, Frederick, MD_.

RAS proteins are responsible for a third of all cancer cases and are involved in both early onset and progression of the deadliest cancers, including lung, colon, and pancreatic cancers. RAS proteins act as binary molecular switches that cycle between active GTP-bound and inactive GDP-bound states. The conversion from inactive to the active form is stimulated by guanine nucleotide-exchange factors (GEFs). Conversion back to the inactive form is mediated by GTPase-activating proteins (GAPs). In the GTP-bound state, RAS proteins interact with a variety of effector proteins such as RAF Kinase, PI 3-Kinase, and RalGDS, leading to activation of several signaling cascades within the cell. Among the three RAS isoforms - HRAS, KRAS, and NRAS, KRAS is the most commonly mutated gene. 98% of oncogenic RAS mutations are found at amino acid positions G12, G13, and Q61, which impair intrinsic and GAP-mediated GTPase function resulting in accumulation of constitutively GTP-bound RAS in cells. To gain insights into the effect of oncogenic mutations on overall structure and GTP hydrolysis, we solved high-resolution crystal structures of wild-type and six oncogenic mutants (G12C, G12D, G12V, G13D, Q61L, and Q61R) of KRAS4b in complex with GMPPNP (a non-hydrolysable GTP analog) and magnesium. Structural comparison of mutants vs. wild-type KRA4b in complex with GMPPNP/Mg2+ and KRAS4b mutants vs. WT-HRAS bound to RASA1-GAP provides a rationale for impaired intrinsic and GAP-mediated GTP hydrolysis in the KRAS mutants. Interestingly, we noticed variations in the conformation of the switch I region which contacts both nucleotide and many downstream effector proteins. In the structure of three KRAS mutants, the switch I region adopts a partially open conformation known as the state I. Importantly, the state I structures reveal new pockets that could be exploited for structure-based drug design. Recently we have also solved the structures of GMPPNP-bound wild-type and Q61 mutants of KRAS in complex with RasGAPs. Structural analysis of KRAS Q61 mutants bound to RasGAPs provides a rationale for impaired GAP-mediated GTP hydrolysis and new insights for designing small molecules that could potentially restore GAP-mediated GTPase activity in these mutants.

## PREVENTION RESEARCH

### Nutrition, Physical Activity, and Cancer Prevention: Current Status and Emerging Strategies

#961

Joint associations of physical activity and body mass index and the risk of excess body fatness-related cancer.

Maret L. Maliniak,1 Susan M. Gapstur,2 Lauren E. McCullough,1 Erika Rees-Punia,2 Mia M. Gaudet,2 Caroline Y. Um,2 Mark A. Guinter,2 Elizabeth A. Fallon,2 Alpa V. Patel2. 1 _Emory University, Atlanta, GA;_ 2 _American Cancer Society, Atlanta, GA_.

Background: Consistent evidence has linked excess body fatness (EBF), typically measured in large epidemiologic studies using body mass index (BMI), with a higher risk of 13 types of cancer. Whether physical activity mitigates any of the excess risk associated with EBF is unclear—as few studies have examined the joint association between BMI and physical activity.

Methods: We examined the joint association of BMI and physical activity on the risk of 12 of the 13 EBF-related cancers (excluding meningioma and including esophagus [adenocarcinoma], gastric cardia, colon and rectum, liver, gallbladder, pancreas, postmenopausal breast, corpus uteri, ovary, kidney [renal cell], thyroid, and multiple myeloma) among 70,658 men and 72,209 postmenopausal women (median age: 63 years) without a history of cancer at study entry in the Cancer Prevention Study II Nutrition Cohort (1992-2013). Multivariable Cox proportional hazards models were used to calculate hazard ratios (HR) and 95% confidence intervals (CI) for the individual and joint associations between BMI (kg/m2) and recreational physical activity (MET-hours/week) at study entry and first cancer incidence.

Results: Between 1992-2013, 3,860 men and 9,001 women were diagnosed with an EBF-related cancer. Compared to normal weight (BMI 18.5-<25 kg/m2), overweight (25-<30 kg/m2) was associated with a modest increased risk of EBF-related cancer (men: HR 1.12; 95% CI: 1.04, 1.21; women: HR 1.13; 95% CI: 1.08, 1.19) and obesity (≥30 kg/m2) with a stronger risk (men: HR 1.48; 95% CI: 1.34, 1.63; women: HR 1.48; 95% CI: 1.40, 1.57). Higher levels of physical activity (>15 vs. >0-<7.5 MET-hrs/wk) were associated with a lower risk for EBF-related cancer among women (HR: 0.90; 95% CI: 0.85-0.95) but less so among men (HR: 0.96; 95% CI: 0.88, 1.04). Among women, there was no evidence of interaction between physical activity and BMI for the risk of EBF-related cancer (P-interaction=0.59); however, there was suggestive evidence that the excess risk associated with overweight was modestly mitigated by the highest level of physical activity (HR: 1.00; 95% CI: 0.91, 1.10). Among men, there was some evidence of an interaction between BMI and physical activity on risk (P-interaction=0.06) with the higher risk associated with overweight limited to those who were inactive (HR: 1.27; 95% CI: 1.08, 1.49). However, risk was higher for obese men irrespective of physical activity level.

Conclusion: Our results suggest that higher levels of recreational physical activity may offset a small amount of the increased risk of EBF-related cancer among overweight men and women. However, physical activity does not appear to mitigate the strong effect of obesity (BMI ≥30 kg/m2) on risk of EBF-related cancers, thus reinforcing the importance of maintaining a healthy body weight even if physically active.

#962

Validation of breast cancer risk model incorporating classical risk factors and polygenic risk scores in 14 prospective cohort studies in 6 countries.

Parichoy Pal Choudhury,1 Amber Wilcox,2 Chi Gao,3 Brian Carter,4 Anika Husing,5 Mark Brook,6 Mikael Eriksson,7 Kara Martin,8 Chris Scott,9 Min Shi,10 Thomas Ahearn,2 Michael Jones,6 Nick Orr,11 Minouk Schoemaker,6 Kamila Czene,7 Jenny Chang-Claude,5 Jacques Simard,12 Doug Easton,13 Marjanka K. Schmidt,14 Dale Sandler,10 Clarice R. Weinberg,10 Celine Vachon,9 Roger Milne,15 Per Hall,7 Anthony Swerdlow,6 Rudolph Kaaks,5 Myrto Barrdahl,5 Mia Gaudet,4 Antonis Antoniou,13 Peter Kraft,3 Montserrat Garcia-Closas,2 Nilanjan Chatterjee1. 1 _The Johns Hopkins University, Baltimore, MD;_ 2 _National Cancer Institute, Rockville, MD;_ 3 _Harvard University, Cambridge, MA;_ 4 _American Cancer Society, Atlanta, GA;_ 5 _German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 6 _Institute of Cancer Research, London, United Kingdom;_ 7 _Karolinska Institutet, Stockholm, Sweden;_ 8 _Cancer Council Victoria, Melbourne, Australia;_ 9 _Mayo Clinic, Rochester, MN;_ 10 _National Institute of Environmental Health Sciences, Durham, NC;_ 11 _Queen's University Belfast, Belfast, United Kingdom;_ 12 _Laval University, Québec City, Quebec, Canada;_ 13 _University of Cambridge, Cambridge, United Kingdom;_ 14 _The Netherlands Cancer Institute, Amsterdam, Netherlands;_ 15 _Cancer Council Victoria, Melbourne, Australia_.

Background: Prospective validation of breast cancer risk models integrating classical risk factors and genetic variants is required for risk-stratified prevention and screening strategies. The objective of this study was to validate a breast cancer risk model integrating classical risk factors and a 313-variant polygenic risk score (PRS) in multiple prospective cohort studies, and to project five-year risk of breast cancer in six different countries.

Methods: The study population included 7,529 cases and 230,103 controls from 14 prospective cohort studies in Australia, Germany, the Netherlands, Sweden, UK, and USA. We used the Individualized Coherent Absolute Risk Estimator (iCARE) tool for risk model building, validation, and risk projection. Expected five-year risk of invasive or in situ breast cancer was compared to observed risk, overall and within deciles of expected risk using goodness of fit statistics. We evaluated calibration of the relative risk through meta-analysis across cohorts, and of the absolute risk within each cohort. Model discrimination was evaluated using the area under the curve (AUC), and percentages of women crossing risk thresholds. Projections of five-year risk distributions were estimated for women of European ancestry aged 50-70 years in the general populations of these six countries.

Results: Analysis showed overall good calibration of the integrated iCARE-based model relative risk for both women younger than 50 years (χ2=14.9, P=0.09) and aged 50 years or older (χ2=14.6, P=0.10), with a small overestimation of risk for women in the highest decile of expected risk (RR = 3.5 expected vs 2.3 (95% CI 1.6 to 3.2) observed for women <50 years; and 2.8 expected vs 2.3 (95% CI 2.0 to 2.7) observed for women 50+ years). The age-adjusted AUCs for the integrated model were 63.1 (95% CI 60.9 to 65.3) and 62.9 (95% CI 61.8 to 64.0), for the two age groups respectively. The calibration of absolute risk showed substantial variation across cohorts, particularly for the older group, but had no systematic bias. Model based projections in the general populations showed that compared to the population average, women in the 1st and 99th percentiles of the integrated risk score had relative risks 0.19 and 3.56 respectively. The proportion of women of European ancestry aged 50-70 years with a five-year risk greater than 3% (threshold for consideration of risk-lowering drugs by U.S. Preventive Services Task Force) ranged from 7.1% in Germany to 18.2% in the US, which corresponds to ~5.5 million women in the US.

Conclusions: Five-year risk predictions from a model with classical risk factors and PRS are well calibrated and provide substantial risk stratification across multiple cohorts in six different countries. Further studies are needed to evaluate the clinical utility of the validated model for risk stratified screening and prevention of breast cancer.

#963

The interplay between lifestyle-related factors and genetics with risk of invasive breast cancer among postmenopausal women from the UK Biobank.

Rhonda S. Arthur, Tao Wang, Thomas Rohan. _Albert Einstein College of Medicine, Bronx, NY_.

Background: Many genetic variants have been associated with increased risk of breast cancer. In contrast, an overall healthy lifestyle has been associated with a reduced risk. However, the degree to which an overall healthy lifestyle may attenuate the impact of genetic variants on risk of breast cancer remains equivocal. In this study, we examined the association of a healthy lifestyle index (HLI) with risk of breast cancer by genetic risk groups.

Methods: The study included 106,814 postmenopausal women of white descent aged 40 to 70 years who were enrolled in the UK Biobank cohort between 2006 and 2010. The HLI was based on a combination of diet, physical activity, smoking, alcohol consumption and anthropometry (ranging from 0 to 20, with the higher scores representing more favorable lifestyle). A polygenic risk score (categorized as low, intermediate and high) was generated as the sum of risk alleles weighted by the logOR of 117 breast cancer associated single nucleotide polymorphisms. A total of 1,949 incident invasive breast cancer cases were ascertained after a median follow-up of 5.7 years. Cox proportional hazards regression models were used to estimate the hazard ratios (HR) and 95% confidence intervals (CI) for the association of a healthy lifestyle risk score with risk of breast cancer by genetic risk groups.

Results: Compared to women with an unfavorable lifestyle (lowest quartile), women with a favorable lifestyle (highest quartile) had a 32% reduced risk of invasive breast cancer (HR: 0.68: 0.59, 0.79). Women with high genetic risk (highest tertile) had an 86% increased risk of invasive breast cancer (1.86: 1.66, 2.08) compared to those with low genetic risk (lowest tertile). Compared with women who had an unfavorable lifestyle, a favorable lifestyle did not significantly reduce risk of invasive breast cancer among women with low genetic risk (0.80: 0.63-1.02), while women with a favorable lifestyle had 32% and 43% reduced risk of invasive breast cancer in the intermediate and high genetic risk categories,

respectively (0.68: 0.53, 0.86 and 0.57; 0.44, 0.74, respectively). However, interaction between the genetic score and the lifestyle score did not achieve statistical significance (pinteraction=0.162).

Conclusion: This study suggests that an overall healthy lifestyle may attenuate the impact of genetics on risk of breast cancer, particularly among women with intermediate and high genetic risk.

#964

The association of meat intake, meat cooking methods, and meat-derived mutagen exposure with the risk of sessile serrated polyps.

Dominique A. Mosley,1 Harvey J. Murff,2 Reid M. Ness,2 Walter E. Smalley,2 Wei Zheng,2 Martha J. Shrubsole2. 1 _Spelman College, Atlanta, GA;_ 2 _Vanderbilt University Medical Center, Nashville, TN_.

Colorectal polyps are the precursors of colorectal cancer, the second leading cause of cancer death in the United States. Red and processed meat intakes have been associated with an increased risk of conventional colorectal adenomas and are classified as carcinogens by the World Health Organization. The mechanism for the relationship between red and processed meat intakes and adenoma risk remains unclear. An explanation may be the exposure to mutagens derived from meat including heterocyclic amines (HCAs) and polycyclic aromatic hydrocarbons (PAHs) which are formed by cooking meats at high temperatures. Although intakes of these meats and mutagens has been associated with risks of conventional adenomas including in our study, no previous study has evaluated risk of sessile serrated polyp (SSPs). SSPs, a more recently recognized precursor lesion for microsatellite instable cancer, were strongly associated with red meat intake in our earlier study. This new study builds on that work by evaluating the associations between meat intake, meat cooking methods, and meat mutagens with risks of SSPs and compares the risks to conventional adenomas and hyperplastic polyps. The analysis is conducted in the Tennessee Colorectal Polyp Study, using polytomous logistic regression models to estimate odds ratios (ORs) and 95% confidence intervals (95% CI). Dietary information was collected by a self-administered food frequency questionnaire and telephone interview to ascertain meat intake and doneness. This information was used to estimate the intakes of meats, well-done meats, PAHs, HCAs, and mutagenic activity. Several measures of meat intake were strongly associated with risk of SSPs. Highest quartile levels of total meat intake (OR=2.04; 95% CI: 1.24-3.36, p for trend=0.002), red meat intake (OR=2.32; 95% CI: 1.37-3.91, p for trend<0.001), processed meat intake (OR=1.99, 95% CI: 1.24-3.20, p for trend=0.004), well-done red meat (OR=2.13, 95% CI: 1.28-3.55, p for trend=0.002), well-done processed meat (OR=1.60, 95% CI: 1.06-2.4, p for trend=0.03), and the HCA MeIQx (OR=2.55, 95% CI: 1.50-4.33, p for trend=0.003) were associated with statistically significantly increased risks of SSP in comparison to the lowest intake levels. Most of these associations were stronger for risk of SSP than for the other types of polyps. In particular, MeIQx intake was not associated with risk of conventional adenomas suggesting that the mechanisms by which red meat intake affects polyp risk may be different between the conventional adenoma and serrated pathways. Future studies are needed to confirm the relationship between meat and meat mutagen intake with SSP risk and to further define the mechanism(s) by which intake of these factors affects SSP risk.

#965

Effects of replacing dietary animal protein with plant-based protein on the formation of intestinal N-nitroso compounds (NOCs) and biomarkers of colon cancer in healthy adults - a 12-week intervention study.

Anne-Maria Pajari, Essi Päivärinta, Vesa Lapinkero, Jessica Manngård, Tiina Pellinen, Suvi Itkonen, Anne Salonen. _University of Helsinki, Helsinki, Finland_.

Epidemiological studies have indicated that high consumption of red and processed meats and low consumption of fiber-rich and plant-based foods are associated with an increased risk for colorectal cancer. Here we aimed to investigate whether partial replacement of dietary animal proteins with plant-based proteins changes gut metabolism and markers for colon cancer risk. We carried out a 12-week partly controlled clinical intervention study with following diets: 1) 70% of proteins from animal-based sources and 30% from plant-based sources, representing habitual Finnish/Western diet at the moment 2) 50% animal and 50% plant-based protein sources and 3) 30% animal and 70% plant-based proteins. Protein intake on all diets was planned to be 17 E% and the actual intake was analyzed using 4-day food records before and at the end of the intervention. Healthy, 20-69-year old participants (n=136) were randomly allocated to intervention groups. Participants collected stool samples before and at the end of the intervention. Fecal water was extracted and its effect on the viability of human colon carcinoma cells after 24h exposure was determined using cck-8 reagent. A 1:5 fecal homogenate was prepared and analyzed for total and heme-derived NOCs using selective de-nitrosation and detection by Ecomedics CLD 88 Exhalyzer. A separate spot stool sample was collected for gut microbiota analyses. At the end of the intervention, the viability of HCA7 colon carcinoma cells exposed to fecal water was significantly lower (P=0.005 ANCOVA) in the most plant-based diet group in comparison to group 1 (29.9% vs. 37.7%, P=0.011) and group 2 (29.9% vs. 37.1%, P=0.021). The concentrations of total NOCs (groups 1: 4.0 ± 2.4, 2: 3.1 ± 1.8, 3: 2.7 ± 1.5 pmol/mg faeces; 1 vs. 2 P=0.056, 1 vs. 3 P=0.001) and heme-derived NOCs (1: 2.5 ± 1.9, 2: 1.8 ± 1.4, 3: 1.4 ± 0.9 pmol/mg faeces; 1 vs. 2 P=0.055, 1 vs. 3 P<0.001) decreased in a dose-dependent manner when dietary animal protein was replaced with plant protein sources. Even though the protein intake was somewhat lower in group 3 (14.5 E%, P<0.001) than in group 1 (17.6 E%), it is unlikely to explain the results. Diets rich in plant-based proteins and scarce in red and processed meat may provide a cancer-preventive metabolic environment in the gut and thus decrease the risk for colorectal cancer.

#966

Coffee consumption and invasive breast cancer incidence among postmenopausal women in the Cancer Prevention Study II Nutrition Cohort.

Susan M. Gapstur,1 Marjorie L. McCullough,1 Rebecca A. Hodge,1 Ying Wang,1 Caroline Y. Um,1 Terryl J. Hartman,2 Mia M. Gaudet1. 1 _American Cancer Society, Atlanta, GA;_ 2 _Emory University, Atlanta, GA_.

Accumulating evidence suggests that coffee consumption may be inversely associated with breast cancer risk among postmenopausal women, with growing evidence of a stronger association for caffeinated than for decaffeinated coffee. However, few studies have examined this association by hormone receptor status or minimized confounding due to cigarette smoking. Recently, we found that caffeinated, but not decaffeinated coffee consumption was inversely associated with breast cancer mortality among nonsmoking women enrolled in the Cancer Prevention Study-II (CPS-II). Subsequently, the 2018 WCRF/AICR Diet, Nutrition, Physical Activity and Cancer Third Expert Report drew no conclusion regarding the association between coffee consumption and breast cancer risk. To provide further evidence, we examined total, caffeinated and decaffeinated coffee consumption in relation to postmenopausal invasive breast cancer incidence, overall, among nonsmokers and by estrogen and/or progesterone receptor (ER/PR) status among women enrolled in the CPS-II Nutrition Cohort, a subcohort of CPS-II. This analysis included 57,120 postmenopausal women who were cancer-free and reported coffee intake in 1999, and who were followed for cancer incidence through June 2013. During follow-up, 2,848 (2,349 ER or PR positive, and 252 ER and PR negative) women with a verified invasive breast cancer were identified. Cox proportional hazards regression was used to estimate multivariable-adjusted hazard ratios (HR) and 95% confidence intervals (CI). Neither total nor decaffeinated coffee consumption was associated with risk of invasive breast cancer among all women or in any subgroup. For caffeinated coffee consumption, the HR (95% CI) for consumption of 2 or more cups/day, compared to less than 1 cup/month, was 0.90 (0.82-1.00) among all women, and 0.88 (0.80-0.98) among nonsmokers. Similarly, caffeinated coffee consumption was associated with a statistically significantly lower risk of ER+ or PR+ breast cancer (HR=0.88, 95% CI 0.79-0.99) and a nonsignificant lower risk of ER- and PR- breast cancer (HR=0.83, 95% CI 0.59-1.16) among all women. These findings underscore the importance of stratifying on type of coffee in studies examining the association between coffee consumption and risk of invasive postmenopausal breast cancer. Additionally, to better understand the role of caffeinated vs. decaffeinated coffee in breast cancer etiology we will examine the potential mediating effects of coffee metabolites on this association.

## TUMOR BIOLOGY

### Novel Approaches to Understand Cancer Stem Cells

#967

Single cell transcriptomics uncovers clonal heterogeneity linked to drug response and cellular phenotype in adult brain tumour stem cells.

Laura M. Richards,1 Owen Whitley,2 Florence M. Cavalli,3 Zhaleh Safikhani,4 Fiona Coutinho,3 H. Artee Luchman,5 Benjamin Haibe-Kains,4 Samuel Weiss,5 Peter Dirks,3 Gary Bader,6 Trevor J. Pugh4. 1 _University of Toronto, Department of Medical Biophysics, Toronto, Ontario, Canada;_ 2 _University of Toronto, Department of Molecular Genetics, Toronto, Ontario, Canada;_ 3 _The Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children, Toronto, Ontario, Canada;_ 4 _Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada;_ 5 _Hotchkiss Brain Institute, Department of Cell Biology and Anatomy, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada;_ 6 _The Donnelly Centre, University of Toronto, Department of Molecular Genetics, Toronto, Ontario, Canada_.

Brain tumours remain a largely incurable disease, with glioblastoma multiforme (GBM) representing the predominant form of malignancy. GBMs contain a rare population of cells, termed brain tumour stem cells (BTSCs) that drive tumour growth and disease relapse. BTSCs can be isolated and expanded in culture from primary GBMs and retain self-renewal and proliferative capacity in vitro. However, the functional diversity within the BTSC fraction, as well as relationship to the tumour bulk remains unknown.

To investigate genotypic and phenotypic states within the stem-like compartment of GBM, we profiled 54,442 patient-derived BTSCs isolated from 25 GBMs using single cell RNA-sequencing (10X Genomics Chromium). Within each BTSC population, we observed diverse transcriptional clonotypes ranging from 1-7 subpopulations per sample. BTSCs grown as adherent monolayers displayed greater heterogeneity (median 4 clusters, range 2-7) compared to sphere-based cultures (median 2 clusters, range 1-3). Across samples, clusters were commonly associated with cell cycling, glial cell development and DNA replication pathways. Within multiple BTSC cultures, we identified transcriptional subclones with variable stemness properties, such as expression of ASCL1, suggesting a hierarchy of differentiation within the tumor-initiating fraction of GBM. Additionally, inference of CNVs from scRNA-seq data revealed subclonal somatic CNVs within BTSCs correspond to distinct transcriptional clonotypes that may partially explain stemness phenotypes and the formation of subpopulations during culture.

To nominate targeted and combination therapies against BTSC populations in GBM, we next mapped transcriptomic features of BTSC subpopulations to high-throughput drug-screening data from central nervous system cell lines aggregated by the PharmacoDB platform.This approach nominated FDA-approved therapies that may be effective against subpopulations within each BTSC culture. In some cases, subpopulations were predicted to be sensitive to different drugs, opening a path for testing combination therapies nominated from single cell RNA-seq in vitro. Overall, these data illustrate the extent to which BTSC cultures functionally recapitulate the stem fraction in bulk primary tumours, as well as define networks of self-renewal, therapeutic resistance and targetable vulnerabilities in glioblastoma. Ongoing efforts include single nuclei RNA-seq of primary GBM tumours to enable comparison of transcriptional programs of BTSC cultures with cellular populations found in primary tissues.

#968

Targeting intratumoral heterogeneity in TNBC with novel combinations of FDA approved drugs.

Nathan A. Truchan, Joseph P. Burnett, Duxin Sun. _University of Michigan, Ann Arbor, MI_.

Triple negative breast cancer, like so many other solid tumors, has intratumoral heterogeneity which makes it difficult to treat. It is paramount to understand the question, "where does the intratumoral heterogeneity come from?" To that end, we developed a dual color reporter cell line using CRISPR/Cas9 to precisely knock in fluorescent proteins, mCherry and EGFP, behind CSC marker ALDH1A3 and differentiation marker CD24 respectively, in the TNBC cell line SUM149. With fluorescent microscopy we are able to track individual cells over time, view symmetrical and asymmetrical self-renewal, differentiation, and lineage restriction. Through direct observation we determined that the intratumoral heterogeneity comes from CSCs. In short, CSCs give rise to all the different cell types found within TNBC. Once we established the reporter cell line we wanted to ensure that any response to therapeutics was the same as in the parental SUM149 cell line. In the past, we have treated parental SUM149 with docetaxel or sulforaphane. We therefore treated our reporter cells with either docetaxel or sulforaphane. The sulforaphane treatment resulted in a significant reduction of CSCs while the docetaxel resulted in significantly more CSCs, both results matched the results from parental SUM149. To our surprise the docetaxel treatment result was due to the significant de-differentiation of mCherrylow to mCherryhigh cells. The de-differentiation and incomplete elimination of the heterogeneous cell populations pose a significant problem for TNBC patients, one that must be addressed. In order to overcome this issue, we will use our reporter cells to test already approved FDA drugs to find and evaluate novel combinations for their ability to completely eliminate the heterogeneous cell populations found within TNBC.

#969

Characterization of murine mammary epithelial populations throughout development and in premalignant tissue via mass cytometry.

Gary K. Gray, Joan S. Brugge. _Harvard Medical School, Boston, MA_.

Single-cell technologies provide a unique opportunity to develop a comprehensive understanding of the dynamic transitions of mammary epithelial cells during normal development, pregnancy, and tumorigenesis. To provide a higher resolution map of epithelial cell dynamics, we developed a mass cytometry panel composed of 39 antibodies targeting cell surface antigens of distinct murine mammary populations. We first utilized this panel to evaluate mammary epithelial composition through puberty and virgin adulthood. This analysis identified subpopulations of the three major mammary epithelial lineages (basal, alveolar progenitor [AP], and hormone-sensing cells [HS]). Interestingly, no clusters expressed markers consistent with their being intermediate between basal and luminal populations or the two luminal populations. We also identified puberty-restricted populations in each of the three lineages. Intriguingly, the luminal pubertal populations expressed basal markers such as CD104 and CD73. We also examined epithelial dynamics during a pregnancy cycle, including mid-pregnancy, lactation, involution, and post-involution. Three hitherto undescribed transit-amplifying clusters were enriched during pregnancy and persisted through involution. These clusters were highly proliferative (as measured by Idu incorporation) and expressed several basal markers, whose expression persisted among AP cells during involution and among a subset of post-involution AP cells. Finally, we analyzed alterations in mammary tissue in the context of loss of tumor suppressors (Apcmut/+, Atm+/-, Cdkn1a-/-, Arf-/-, Trp53+/-, or Trp53-/-) or expression of oncogenes (MMTV-Errb2, -neu, or -PyMT). Myriad alterations were observed across the conditions, the most striking of which was restriction of alterations in MMTV models to cells in the AP lineage. The expression of PyMT increased the frequency and basal-like features of AP cells. Analysis of a PyMT-induced tumor also showed that tumor cells were overwhelming of an AP-like phenotype. In conclusion, our data paint a high-resolution portrait of mammary epithelial dynamics throughout development and in the context of pro-tumorigenic genetic alterations. We report numerous novel populations during puberty and the pregnancy cycle. A particularly intriguing observation is that luminal populations appear to acquire basal-like differentiation features during puberty, pregnancy, and pre-tumorigenesis, suggesting the oft-reported mixed luminal/basal differentiation status of breast cancer could be a coopting of normal developmental programs. This result may provide clues for uncovering and blocking the molecular mechanisms of breast carcinogenesis.

#970

The mutational landscape of normal human endometrial epithelium.

Luiza Moore,1 Daniel Leongamornlert,1 Tim Coorens,1 Mathijs Sanders,1 Peter Ellis,1 Francesco Maura,1 Kevin Dawson,1 Simon F. Brunner,1 Jyoti Nangalia,1 Henry Lee-Six,1 Raheleh Rahbari,1 Patrick Tarpey,1 Yvette Hooks,1 Krishnaa Mahbubani,2 Christine A. Iacobuzio-Donahue,3 Jan J. Brosens,4 Inigo Martincorena,1 Kourosh Saeb-Parsy,2 Peter J. Campbell,1 Michael R. Stratton1. 1 _Wellcome Sanger Institute, Cambridge, United Kingdom;_ 2 _Department of Surgery and Cambridge NIHR Biomedical Research Centre, Cambridge, United Kingdom;_ 3 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 4 _Tommy's National Miscarriage Research Centre/Warwick Medical School, Warwick, United Kingdom_.

Human endometrium is a highly dynamic tissue that undergoes hundreds of cycles of breakdown, rapid repair and remodelling in response to the oscillating levels of oestrogen and progesterone during female reproductive years. The marked regenerative capacity of this tissue's epithelial compartment is maintained by intra-glandular adult stem cells (ASCs) that reside within the stratum basalis which is retained during menstruation. Although the endometrial ASCs were first described over a decade ago, they remain poorly characterised in comparison to their counterparts in other tissues, such as the small and the large intestines. In particular, the size of the stem cell pool within individual glands, the rates of their division, and mutational landscape are largely unknown. In this study, we isolated 215 morphologically normal endometrial glands from women aged 19 to 81 using laser capture microscopy. Analysis of whole-genome sequencing data identified that the overwhelming majority of the glands were clonal cell populations, and thus originating from a single ASC. Somatic mutations were found to accumulate at a linear rate during adult life. Elevated body mass index (BMI), a well-recognised risk factor for endometrial cancer, accelerated the rate of mutation acquisition. Surprisingly, despite the heterogeneity in age, reproductive history and BMI in our cohort, we find relatively homogenous mutational processes within normal endometrium. Comparison with cancer, shows lower somatic mutation burden and fewer operative signatures. Remarkably, we not only identify recurrent acquisitions of certain cancer-associated mutations, particularly those that are advantageous to cell growth, proliferation and migration, but also show that such events occur early in life, potentially even before adolescence. Over time, these mutant ASCs serve as a reservoir for the acquisition of further driver mutations to the extent that in some cases, the entire sampled endometrium becomes 'neoplastic' on the genomic level while still retaining the apparently normal phenotype. In older individuals, we observe a shift in the spectrum of acquired cancer-associated mutations, possibly reflecting post-menopausal changes in the levels of sex-steroid hormones and the resultant tissue microenvironment.

#971

PRMT5 modulates splicing for genome integrity and preserves proteostasis of hematopoietic stem cells.

Darren Qiancheng Tan,1 Ying Li,1 Chong Yang,1 Jia Li,1 Shi Hao Tan,1 Desmond W. Chin,2 Ayako Nakamura-Ishizu,1 Henry Yang,1 Toshio Suda1. 1 _Cancer Science Institute of Singapore, National University of Singapore, Singapore;_ 2 _Center for Hematology and Regenerative Medicine, Huddinge, Karolinska Institutet, Stockholm, Sweden_.

Protein arginine methyltransferase 5 (PRMT5) has recently been identified as a promising therapeutic target in various solid and hematologic cancers, while PRMT5 inhibitors are currently in clinical development. Functionally, PRMT5 has an established role in regulating splicing, and is essential for hematopoiesis. However, the mechanisms underlying PRMT5 dependency in HSCs, particularly PRMT5-mediated splicing, remain unclear. Moreover, how pharmacological inhibition of PRMT5 affects HSCs remains undetermined. To clarify the function of PRMT5 in HSCs, we adopted a loss-of-function approach, coupled with functional and transcriptome profiling.

We report that depletion of PRMT5 activity severely perturbs HSC maintenance. HSCs from Mx1-Cre Prmt5fl/fl mice (PRMT5Δ/Δ), which exhibit severe bone marrow (BM) failure, failed to reconstitute lethally irradiated wild-type recipients. Notably, this was associated with rapid loss of Prmt5Δ/Δ HSCs. Similarly, EPZ015666-mediated PRMT5 inhibition led to a dose-dependent decrease in the growth and viability of EML cells (murine BM-derived hematopoietic precursor cell line). Interestingly, this was associated with increased HSC size and PI3K/AKT/mTOR pathway activity. In addition, protein synthesis rate was also elevated in an mTOR-dependent manner. Thus, we confirm the cell-intrinsic requirement for PRMT5 activity in HSCs, and demonstrate a potential role for PRMT5 in regulating HSC proteostasis.

We also analyzed the splice variant landscape of PRMT5Δ/Δ HSCs, and observed substantial upregulation of intron retention and exon skipping events; which were overrepresented for processes associated with maintenance of genomic integrity. Notably, we show that splicing of key genes involved in base excision repair, telomere maintenance and the Fanconi anemia pathway were dysregulated in PRMT5Δ/Δ HSCs, as well as EPZ015666-treated EML cells. Thus, suggesting that HSCs depleted of PRMT5 activity are potentially deficient in DNA repair.

In agreement, PRMT5Δ/Δ HSCs exhibited elevated γH2A.X levels, replicative stress-associated oxidative DNA lesions and DNA strand breaks. Likewise, EPZ015666-treated EML cells exhibited dose-dependent increments in γH2A.X levels, and increased sensitivity to DNA damaging agents. Corroborating these observations, loss of PRMT5 activity in both PRMT5Δ/Δ HSCs and EPZ015666-treated EML cells led to elevated reactive oxygen species (ROS) levels, p53 activation and induction of apoptosis; which was significantly rescued by antioxidant treatment or co-depletion of p53. Therefore, depletion of PRMT5 activity renders HSCs vulnerable to DNA damage. Collectively, our findings uncover a critical role for PRMT5 in maintaining genomic integrity, and highlight a novel link between PRMT5 and proteostasis in HSCs.

#972

Direct observation in living tumors shows macrophage-dependent induction and dissemination of cancer stem cells in breast cancer.

Ved P. Sharma,1 Yarong Wang,1 Binwu Tang,2 George S. Karagiannis,1 Emily A. Xue,1 David Entenberg,1 Lucia Borriello,1 Anouchka Coste,1 Chinmay R. Surve,1 Dominic Esposito,3 Maja H. Oktay,1 Lalage M. Wakefield,4 John S. Condeelis1. 1 _Albert Einstein College of Medicine, Bronx, NY;_ 2 _National Cancer Institute, Bethesda, MD;_ 3 _Frederick National Laboratory for Cancer Research, Frederick, MD;_ 4 _National Cancer Institute, MD_.

Cancer stem cells (CSCs) play an important role during metastatic progression of breast cancer. However, the in vivo properties and dynamic behavior of CSCs are not well understood. Here, we employed high-resolution intravital multiphoton microscopy using a SOX2/OCT4 responsive fluorescent stem cell biosensor to directly observe CSC dynamics in the living animal using an orthotopic breast cancer xenograft model. We report that CSCs constitute a minority population (1-3%) in the primary tumors, and display the slow-migratory, invasive phenotype that is specifically associated with disseminating tumor cell population. We also report, for the first time, that CSCs are preferentially localized in direct contact with macrophages near and in tumor microenvironment of metastasis (TMEM) sites, the macrophage-containing intravasation doorway for tumor cells and that CSCs metastasize to lung and are strikingly enriched in early lung metastatic colonies. This is explained by our observation that, in vitro and in vivo, direct physical contact with macrophages induces stemness in non-stem cancer cells via juxtacrine Notch-Jagged1 signaling. These data indicate for the first time that macrophages play an actively inductive role in the CSC niche and promote TMEM-mediated CSC intravasation and early metastatic seeding.

#973

The mitochondrial deoxyguanosine kinase regulates lung adenocarcinoma cancer stem like cells through AMPK/YAP1 signaling.

Shengchen Lin,1 Chongbiao Huang,2 Jianwai Sun,3 Xiuchao Wang,2 Jiaxin Kang,1 Matthew Taylor,1 Bin Fang,4 Pankaj K Singh,5 John Koomen,4 Jihui Hao,2 Shengyu Yang1. 1 _Penn State Univ. College of Medicine, Hershey, PA;_ 2 _Tianjin Medical University Cancer Institute and Hospital, Tianjin, China;_ 3 _South China Agricultural University, China;_ 4 _H. Lee Moffitt Cancer Center, Tampa, FL;_ 5 _University of Nebraska Medical Center, Omaha, NE_.

Metastasis involves the dissemination of tumor cells from primary site to distant organs, and the colonization of disseminated cancer cells to establish distant metastases. Some of the disseminated cancer stem-like cells (CSC) are able to remain in dormancy for years before establishing secondary tumor and triggering metastatic recurrence. The accepted paradigm of cancer therapy aims at eradicating rapidly proliferating cancer cells. However, how to eliminate disseminated metastatic cells in dormancy and to prevent metastatic recurrence remains a major challenge in the clinic. Here, we report that the CSC in non-small cell lung cancer (NSCLC) rely on the mitochondrial deoxyguanosine kinase (dGK) to maintain mitochondrial homeostasis and cancer cell stemness. There is a pool of mitochondrial dNTP in the cell maintained by the mitochondrial deoxynucleoside salvage pathway and dedicated for the mitochondrial DNA (mtDNA) homeostasis in slow-cycling and post-mitotic cells. The role of the mitochondrial deoxynucleoside salvage pathway in tumor initiation and progression is poorly understood. Here, we investigated the role of the mitochondrial dGK, a rate-limiting enzyme in the mitochondrial deoxynucleoside salvage pathway, in the self-renewal of lung adenocarcinoma CSC. Our data support that dGK overexpression strongly correlates with cancer progression and patient survival. Depletion of dGK in lung cancer cells remarkably reduced the proportion of active aldehyde dehydrogenase (ALDH+) CSC, inhibited tumor sphere formation in vitro and prevent tumor-initiation ability of CSC in xenograft mouse models. Mechanistically, dGK controls the biogenesis of respiratory Complex I and mitochondrial oxidative phosphorylation (OXPHOS), which in turn regulates CSC self-renewal through AMPK-YAP1 signaling. The restoration of mitochondrial OXPHOS in dGK depleted lung cancer cells using NDI1, a single subunit yeast NADH : ubiquinone oxidoreductase, was able to rescue AMPK-YAP1 signaling and CSC stemness. Genetic targeting of dGK using doxycycline-inducible CRISPR/Cas9 was able to markedly induce tumor regression. Our findings reveal a novel role for mitochondrial dNTP metabolism in lung cancer tumor growth and progression, and indicate that the mitochondrial deoxynucleotide salvage pathway could be potentially targeted to prevent CSC-mediated therapy resistance and metastatic recurrence.

### The Immune Microenvironment in Cancer

#974

The molecular and genomic drivers of immunologically cold tumors: Novel discoveries from pan-cancer analysis.

Guangchun Han,1 Ruiping Wang,1 Jianfeng Shen,1 Pankaj Singh,1 Shaojun Zhang,1 Arvind Rao,2 Jianjun Gao,1 Alexander Lazar,1 Andrew Futreal,1 Guang Peng,1 Kunal Rai,1 Linghua Wang1. 1 _UT MDACC, Houston, TX;_ 2 _University of Michigan, MI_.

Immune-checkpoint blockade (ICB) is able to elicit remarkable immune response but currently the durable response is limited to a minority of cancer patients. Recent evidence suggests that a prerequisite for the efficacy of ICB, for example the PD-1 inhibitor, is T-cells infiltration in the tumor microenvironment (TME). The immunologically 'quite' and T-cell depleted (immune 'desert') tumors are less likely respond to ICB. However, we still lack a detailed understanding of the genomic and molecular determinants that drive the 'cold' immune phenotypes. We here performed a comprehensive pan-cancer analysis to define and characterize the immunologically 'hot' (inflamed) and 'cold' (quite or T-cell depleted) tumors by high-dimensional, multi-platform data integration of 9,887 tumors from The Cancer Genome Atlas (TCGA). This study provided an unprecedented catalog of the molecular and genomic drivers of immune 'cold' tumors by analyzing the immune cell composition, patterns of their spatial organization within the TME, the dysregulated chemokines networks, somatic mutations, DNA copy-number alterations, aneuploidy status, differentially expressed immune regulatory genes, and enriched signaling pathways. We showed that the immunologic status is highly heterogeneous within and across TCGA tumor types and subtypes. We integrated the information of T-cell spatial organization with our immune phenotypes and found that the 'band-like' spatial organization (T cells clustered at the invasive margin of the tumor and didn't get into the tumor body) pattern is strongly associated with the 'cold' phenotype, particularly in colon and stomach cancers. Our analysis revealed a number of other factors that are associated with the 'cold' phenotype by either preventing T-cell infiltration or promoting T-cell dysfunction, and we performed functional validation on several targets of interest. This study provided an in-depth understanding of the genomic and molecular determinants of immunologically 'cold' tumors and suggested potential biomarkers that can be targeted for the development of novel combination therapy strategies to convert the 'cold' tumors into 'hot' ones and improve the odds of cancer immunotherapy success.

#975

Liver metastases (mets) induce systemic immunosuppression and immunotherapy resistance in metastatic melanoma.

Ines Silva,1 Annie Tasker,1 Camelia Quek,1 Robert Rawson,1 Su Yin Lim,2 Kevin Wang,3 Jordan Conway,1 Rebecca Velickovic,1 Tasnia Ahmed,1 Serigne Lo,1 Jean Yang,3 Helen Rizos,2 James S. Wilmott,1 Richard A. Scolyer,1 Alexander M. Menzies,1 Georgina V. Long1. 1 _Melanoma Institute Australia, The University of Sydney, Sydney, Australia;_ 2 _Melanoma Institute Australia, Macquarie University, Sydney, Australia;_ 3 _School of Mathematics and Statistics, The University of Sydney, Sydney, Australia_.

Melanoma patients (pts) with liver mets have a lower response rate (RR), and shorter progression-free (PFS) and overall survival (OS) compared to pts without liver mets when treated with anti-PD-1 (PD1) therapy. The liver microenvironment (ME) induces T-cell tolerance through the interaction of T cells with liver sinusoidal endothelial cells. To explore this further we compared clinicopathological features, circulating cytokines and tumor gene expression (GE) profiles of pts with and without liver mets treated with PD1 combined with ipilimumab (PD1+IPI), and treated with BRAF targeted therapy (TT).

Demographics, disease characteristics and outcome data were collected from 140 pts treated with PD1+IPI and from 76 pts treated with BRAF+/-MEKi. Tumor-infiltrating lymphocytes (TILs) immunoreactivity score (TILs density x % of tumor with TILS), % of tumor content, necrosis and fibrosis were assessed by immunohistochemistry in liver and lung samples. Pre-treatment circulating cytokines and tumor GE data (RNA seq) were compared between pts with and without liver mets.

In pts treated with IPI+PD1, liver mets had the lowest tumor regression (med -7%) compared to all other sites of disease (med -66%), while in TT-treated pts the response was similar across all sites of disease. Pts with liver mets (n=39) had lower RR (44% v 75%), and shorter median PFS and OS (p<0.05) when treated with IPI+PD1 than those without liver mets (n=101). In contrast, in pts treated with TT, RR, PFS and OS were similar between pts with (n=19) vs without (n=57) liver mets. Pts with liver mets had less response in adrenal and LN mets when treated with IPI+PD1 compared to pts without liver mets, but not with TT. In a multivariate analysis performed on the PD1+IPI cohort and validated in the TT group, presence of bone (OR 4.6 p=0.004) and spleen mets (OR 13.5 p=0.01) were associated with the presence of liver mets. Compared to lung mets (n=22), liver mets ME (n=22) had a lower TILs immunoreactivity score (med 30 vs 80, p=0.05), while there was no difference in the % of tumor content, fibrosis or necrosis. The expression of 65 cytokines was measured in plasma of treatment-naive pts; pts with liver mets (n=37) had higher levels of Eotaxin 2 (p=0.01), IP-10 (p=0.02) & IL-8 (p=0.03) compared to pts without liver mets (n=99). GE analysis of melanoma samples showed higher expression of MMP-8 and HIF1a in pts with (n=58) vs without (n=28) liver mets, validated in an independent cohort (n=58).

Pts with liver mets display distinct clinicopathological features, distinct circulating cytokines and melanoma GE profiles, and are less responsive to PD1+IPI compared to pts without liver mets. The levels of Eotaxin-2, IP-10 and IL-8 are higher in melanoma pts with liver mets compared to pts without liver mets, similar to what is seen in pts with colon cancer liver mets. Liver mets' ME may hold unique immunosuppressive mechanisms that are amenable to therapeutic targeting.

#976

Regulation of breast tumor metastasis by the dynamic interaction between the TMEM macrophage, tumor and endothelial cells.

Chinmay R. Surve, Allison Harney, Yarong Wang, Xiaoming Chen, Ved Sharma, Richard Stanley, Maja Oktay, John Condeelis. _Albert Einstein College of Medicine, Bronx, NY_.

Tumor cell intravasation is an essential step in the metastatic cascade, but its exact mechanism is not completely understood. We have previously shown that the direct physical association of a tumor cell over-expressing Mena, a perivascular Tie2hi/Vegfhi macrophage and an endothelial cell, forming a cell triad termed "tumor microenvironment of metastasis" (TMEM), increases vascular permeability, facilitating intravasation of tumor cells. It is only at the TMEM site that intravasation occurs leading to breast tumor metastasis. TMEM density is a clinically validated prognostic marker of distant metastasis in breast cancer patients. The precise molecular mechanisms relating TMEM function had not been elucidated. Here we describe the molecular mechanism. We show here that TMEM function involves the three cells in TMEM: firstly endothelial cell-secreted Ang2 stimulates VEGF build up in the TMEM macrophage, secondly a tumor cell secretes CSF1 which, third, stimulates the TMEM macrophage VEGF secretion, leading to vascular opening and metastasis. In addition, we show that acute blockage of CSF1R and Tie2-Ang2 signaling by inhibitors and blocking antibodies both in vitro and in mammary tumors leads to decreased macrophage VEGF production and secretion, decreased trans-endothelial migration of tumor cells, and decreased TMEM-dependent vascular permeability, circulating tumor cells and lung metastases. We conclude that dynamic interaction between the cells associated with TMEM leads to Ang2 and CSF1-mediated stimulation of macrophage VEGF expression and secretion leading to vascular opening, resulting in tumor cell intravasation. This is the first description of the molecular mechanism behind the predictive power of the clinically used prognostic marker TMEM and represents a major step in defining new biomarkers and targets for the treatment of metastatic tumors.

#977

Nano immunotherapeutics crossing blood-brain barrier to activate local brain tumor immune system.

Anna Galstyan, Antonella Chiechi, Tao Sun, Ekaterina S. Shatalova, Rameshwar Patil, Keith L. Black, Eggehard Holler, Alexander V. Ljubimov, Hui Ding, Julia Y. Ljubimova. _Cedars-Sinai Medical Ctr., Los Angeles, CA_.

Introduction: Glioblastoma multiforme (GBM) has limited treatment options. Checkpoint inhibitors anti-CTLA-4 and/or anti-PD-1 antibodies (Abs) cannot activate anti-tumor immune response in the brain because of their inability to cross blood-brain barrier (BBB) and modulate brain privileged immunity. A new generation of nano immunotherapeutics (NIT) that pass BBB and activate general and local brain tumor immune systems was developed.

Experimental procedures: BBB-crossing NIT were synthesized, based on the poly (β-L-malic acid) polymer (P), P/a-CTLA-4 Ab and P/a-PD-1 Ab, and a-mouse transferring receptor (a-msTfR) Ab or AP-2 peptide for delivery through BBB. Syngeneic GL261 glioma cells were intracranially inoculated into C57/BL mice. Six treatment groups received 5 I.V. injections of PBS, anti-PD1 and anti-CTLA4 Abs (controls), and polymers conjugated with anti-PD-1(P/a-PD-1), anti-CTLA-4 (P/a-CTLA-4) and a combination (P/a-CTLA-4+P/a-PD-1) at 10 mg/kg.

Summary of the data: Single NIT treatment (P/a-CTLA-4 or P/a-PD-1) improved brain tumor bearing mice survival vs. free a-CTLA-4 and a-PD-1 (p=0.0076 and 0.0017, respectively). The combination group (P/a-CTLA-4 + P/a-PD-1) showed the best efficacy for survival (p=0.0001). Flow cytometry (FC) analysis of T cell populations in the brain tumor revealed a reduction of total CD4+ T-cells in animals treated with P/anti-PD-1 and P/a-CTLA-4 + P/a-PD-1 combination vs. free anti-PD-1 Ab. The fraction of Tregs (CD4+FOXP3+) was reduced by all polymer treatments compared to free Abs. Activation of CD8+ T cells (CD8+IFN-γ+ and CD8+CD69+) was significantly increased by P/a-CTLA-4 or P/a-PD-1 and combination therapy, vs. free a-CTLA-4 and a-PD-1 Abs. To independently confirm FC data, the local immune response was investigated for CD8 and CD4+FoxP3 positive T-cells, and IFN-γ, iNOS markers for M1 macrophages by immunofluorescence staining using FIJI software. In P/a-CTLA-4 + P/a-PD-1 group vs. PBS the percentage of CD8+ T cells was significantly increased (p=0.0005), together with reduction of CD4+FoxP3+ T cells (p=0.0006) and increase of iNOS-positive macrophages (p=0.0001). FC and histology results confirmed that the NIT treatment stimulated local brain immune system. FC analysis of T-cell population in blood and spleen also demonstrated significant activation of systemic immune response after combination therapy, based on cytokines level: IL-1β, IL-2, IL-10, IL-4, IL-5, TNF-α, IL-6, IL-12, and IFN-γ after treatment.

Conclusion: GBM treatment with BBB-crossing polymer-conjugated antibodies a-PD-1 and a-CTLA-4 was able to increase tumor-bearing animal survival, which was supported by immunological results indicating the activation of T cell population systemically and locally in the brain. Support: NIH grants R01 CA206220, R01 CA230858

#978

**Tim-4** + **tissue-resident macrophages impair anti-tumor T cell immunity.**

Andrew Chow, Sara Schad, Sai K. Sharma, Sadna Budhu, Aditi Gupta, Corrin Pimentel, Hong Zhong, Jason S. Lewis, John T. Poirier*, Jedd D. Wolchok*, Charles M. Rudin*, Taha Merghoub*. _MSKCC, New York, NY_.

Over 600,000 Americans and 8 million worldwide die of cancer each year. The success of immune checkpoint blockade with anti-PD-1 therapy has been a remarkable clinical advance. However, most patients do not respond to anti-PD-1 monotherapy and the majority of those who initially do respond eventually succumb to the disease. There is currently an unmet need to identify therapeutic strategies to overcome resistance and further enhance the efficacy of anti-PD-1 therapy. Tim-4 is a phosphatidylserine (PS) receptor that has been reported to be expressed on myeloid cells. Prior work has demonstrated that Tim-4 deficiency or blockade in mice is associated with features of autoimmunity and enhanced anti-tumor immunity. Using flow cytometry, immunohistochemistry, and in vivo biodistribution, we determined that Tim-4 is expressed predominantly on tissue-resident macrophages, such as Kupffer cells, peritoneal macrophages, pleural macrophages, and lymph node macrophages, but not tumor-associated macrophages, in mice and humans. We provide evidence that Tim-4+ macrophages produce high levels of the cytokines TNF-α and TGF-β, which have been linked to T cell dysfunction, and also the TGF-β activating integrin αv (CD51). Antibody blockade of Tim-4 enhances the anti-tumor activity of anti-PD-1 therapy after subcutaneous tumor inoculation of B16 melanoma, CT26 colon carcinoma, and MC38 colon carcinoma. Finally, combined blockade of Tim-4 and PD-1 resulted in substantially more curative responses in a MC38 model of peritoneal carcinomatosis, compared to anti-PD-1 monotherapy. This was associated with enhanced CD8+ T cell infiltration into the peritoneal cavity, increased CD8+ T cell/Foxp3+ regulatory T cell ratio, and reduced expression of the exhaustion marker PD-1. Thus, we show that Tim-4+ tissue-resident macrophages impair anti-tumor T cell immunity and antibody blockade of Tim-4 is a strategy that should be considered for clinical studies in cancers resistant to anti-PD-1 monotherapy.

#979

Programmed death-ligand 1 expression and tumor-infiltrating immune cells in cancers associated with Li-Fraumeni syndrome.

Emilie Saucier,1 Claudia Pasqualini,1 Louise Galmiche,2 Frédérique Larousserie,3 Marie Karanian,4 Véronique Minard-Colin,1 Birgit Geoerger,1 Dominique Valteau-Couanet,1 Laurence Brugières,1 Jean-Yves Scoazec1. 1 _Gustave Roussy Cancer Campus, Villejuif Cedex, France;_ 2 _Hopital Necker-Enfants Malades, Paris, France;_ 3 _Hôpital Cochin, Paris, France;_ 4 _Centre Leon Bérard, Lyon, France_.

Purpose: Li-Fraumeni syndrome (LFS), due to TP53 germline mutations, is characterized by high incidence of multiple cancers. Dysregulation of the TP53 pathway is associated with changes in chemokine production and reduced tumor immune infiltration in some cancer types. Recent works also suggested that TP53 negatively regulates PDL1 expression through the non-coding RNA miR-34. To better understand immune evasion of LFS spectrum cancers and to explore the feasibility of PD1/PDL1 targeted therapies as an alternative to genotoxic treatments in LFS patients, we investigated a range of LFS-related tumor types for PDL1 expression and tumor-infiltrating immune cells.

Methods: We retrospectively collected a total of 72 tumor samples (osteosarcoma=32, rhabdomyosarcoma=11, adrenocortical carcinoma=11 and breast carcinoma=18) from 60 pediatric and adult patients identified through the LFS French database and direct contact with oncologic centers. Immunochemistry analyses were performed on whole slide sections. PDL1 staining was performed using the clone E1L3N (Cell Signaling Technology) according to the standard automated protocols. Immune infiltrate was characterized by the following antibody panel: CD8, FOXP3 and CD68. For CD8+ and FoxP3+ T cells, cell density per surface unit was determined by cell counting; for CD68+ macrophages, a semi-quantitative scoring (from 0 to 4) was performed.

Results: Median age of patients was 13 years (range: 6-23) for osteosarcoma, 1.7 years (range: 0.8-5) for rhabdomyosarcoma, 2.3 years (range: 0.4-6) for adrenocortical carcinoma and 27.5 years (range: 22-54) for breast carcinoma. Seven patients presented ≥2 metachronous tumors. Biopsy was performed at diagnosis for 68 patients, at relapse for 3 patients and at early progression for one patient. None of the samples presented any detectable PDL1 expression neither on tumor cells nor on tumor microenvironment. Low/very low amount of CD8+ cytotoxic T-cells was displayed, regardless of the histology. FoxP3+ regulatory T-cell infiltration varied among tumor types. Thirty-five/72 tumors (49%) did not contain regulatory T cells. Breast carcinomas presented significant amounts of regulatory T cells (16/18, 89%), but at low density for 10 out of 16 samples (62%). Sixty-two/72 tumors (86%) presented CD68+ macrophage infiltrates, with high density in 37/62 (60%) of them (score ≥ 2+).

Conclusion: The absence of PDL1 expression on tumor cells and microenvironment as well as the low amount of regulatory T cells do not support the hypothesis of PDL1 induction through p53 inactivation in LFS. The role of innate immunity needs to be explored to identify alternative immunological targets.

#980

Clinical impact of T-cell exhaustion in patients with diffuse large B-cell lymphoma.

Suvi-Katri Leivonen,1 Matias Autio,1 Oscar Bruck,1 Satu Mustjoki,1 Judit M. Joergensen,2 Marja-Liisa Karjalainen-Lindsberg,3 Klaus Beiske,4 Harald Holte,4 Sirpa Leppä5. 1 _University of Helsinki, Helsinki, Finland;_ 2 _Aarhus University Hospital, Aarhus, Denmark;_ 3 _Helsinki University Hospital, Helsinki, Finland;_ 4 _Oslo University Hospital, Oslo, Norway;_ 5 _Helsinki University Hospital Comprehensive Cancer Center, Helsinki, Finland_.

Introduction: Tumor microenvironment (TME) and limited immune surveillance impact lymphoma pathogenesis and survival. Immune checkpoint receptors, such as programmed cell death-1 (PD1), lymphocyte-activation gene-3 (LAG3) and T-cell immunoglobulin and mucin domain-3 (TIM3), mediate signals leading to T-cell exhaustion and immune escape. Here, we have characterized the immunological profiles of diffuse large B-cell lymphoma (DLBCL) and associated the findings with outcome.

Methods: We utilized the NanoString nCounter platform with a 770-gene PanCancer Immune panel to profile gene expression from 81 RNA samples collected from high-risk DLBCL patients. Multiplex immunohistochemistry (mIHC) with digital image analysis was used to characterize the T-cell phenotypes (CD3, CD4, CD8, PD1, LAG3, and TIM3) in two DLCBL patient cohorts (n=52 and n=136). The findings from gene expression profiling and mIHC were correlated with clinical parameters and patient outcome (i.e. progression-free survival (PFS) and overall survival (OS)).

Results: Gene expression profiling revealed a high degree of heterogeneity among DLCBL. Unsupervised hierarchical clustering identified gene clusters differentially expressed between the patients. The distinct clusters contained genes for cytolytic factors and immune checkpoint molecules (GZMB, PRF1, IFNG, TIM3, LAG3), T-cells (CD3, CD2, CD28), macrophages (CD68, CD163), B-cells (MS4A1, CD19, CD79A/B), and extracellular matrix (FN1, ITGA1/5/6, VEGFA). mIHC revealed that the proportion of CD3+ T-cells varied markedly between the patients (median 24.8%, range 0.5-65.1%). On average, 8.8% (0-34.3%) of the CD3\+ T-cells were positive for TIM3, whereas 0.6% (0-5.4%) were positive for LAG3. High content of TIM3+ cells was associated with poor outcome (PFS: HR 1.06, 95%CI 1.02-1.10, P=0.003; OS: HR=1.12, 95%CI 1.04-1.20, P=0.003) in a cohort of 52 DLBCL patients independent of the international prognostic index (IPI). In addition, high proportion of TIM3+CD3+ cells associated with poor survival independently of IPI (PFS: HR 1.07, 95%CI 1.00-1.14, P=0.040; OS: HR 1.087, 95%CI 1.01-1.17, P=0.019). The prognostic impact of TIM3 on survival was validated in a separate mIHC cohort of 136 DLBCL patients (PFS: HR 1.07, 95%CI 1.02-1.14, P=0.014; OS: HR 1.10, 95%CI 1.03-1.16, P=0.002).

Conclusions: Our data demonstrates that the molecular immunological profile of DLBCL is heterogenic and that the expression of T-cell exhaustion marker TIM3 correlates with poor prognosis in patients with DLBCL. A more detailed characterization of immune cell composition in the TME and its impact on survival is ongoing. 

# Monday, April 1, 2019

## CANCER CHEMISTRY

### Innovative Concepts for Drug Discovery

#981

Degradation of immuno-oncology targets via proprietary PROTAC platform integrating DNA-encoded library technology and rational drug design.

Jannik N. Andersen,1 Andrew J. McRiner,2 Lynette A. Fouser,1 Junyi Zhang,2 Shilpi Arora,2 Michael Cordeau,1 Ying Zhang,2 John Cuozzo,2 Michael Briskin,1 Matt Clark,2 Diala Ezzeddine1. 1 _Xios Therapeutics, Inc, Waltham, MA;_ 2 _X-Chem Pharmaceuticals, Waltham, MA_.

Since the first small molecule proteolysis targeting chimera (PROTAC) was reported about a decade ago, great progress has been made in the field of targeted protein degradation. Specially designed, small molecules can recruit the ubiquitin-proteasome system (UPS) to tag a protein of interest (POI) for degradation. Based on the ability to knock down a therapeutic POI (instead of inhibiting the target protein activity), this new modality has emerged as a paradigm-shifting approach and opened new avenues for small molecule drug discovery. At Xios Therapeutics, we have applied targeted protein degradation to a number of immuno-oncology (IO) drug targets and we present here the strategy and lessons learned from building our PROTAC platform in collaboration with X-Chem. Specifically, we have leveraged a vertical integration of DNA-encoded library screening (DEL), structural biology, medicinal chemistry, biochemical binding assays and cellular biomarker readouts for the rapid identification of cell potent degraders. We exemplify a modular, 'fit-for-purpose' PROTAC matrix that allows for rapid exploration of optimal E3 ligase-binders conjugated to a POI-binder using either existing or novel ligands identified via DEL. We delineate the structure-activity/property relationship (SAR and SPR) analysis of linker with VHL- and CRBN-based binders for a promising IO target achieving potent protein degradation (>90% degradation and nM DC50 potency) and pathway inhibition in cancer cells. Notably, our affinity-based screening of chemical libraries of unprecedented size (~200 billion molecules) with a priori knowledge of the vector point of attachment from the DNA barcode directly informs the rational design of bifunctional PROTAC molecules. In conclusion, our integrated approach allows us to find new, unexplored compound binding sites for both E3 ligases- and POI-binders that can be utilized by the PROTAC platform to create potent selective degraders and to access targets that have previously been considered undruggable.

#982

Functionally important hotspot interfaces between immune-oncology targets PD-1 and PD-L1 and between Hippo pathway targets YAP2 and tight junction protein ZO-1 are identified using a protein-protein interaction technique optimized with novel dye chemistries.

Amanda Still, Douglass Dey, Rachel Carter, Angela Dailing, Mikell Paige, Lance Liotta, Alessandra Luchini. _George Mason University, Gainesville, VA_.

Protein-protein interactions are thought to be the next frontier in drug discovery. However, there are several well-known challenges facing development of protein-protein interaction (PPI) inhibitors that lead to slow development in the field, including difficulty identifying PPIs and difficulty designing small molecule inhibitors of relatively flat, featureless PPIs. We address these difficulties with the development of a novel method of discovering PPI hotspots, called protein painting. This technique relies on non-covalent labeling of solvent- accessible protein surfaces using small molecular dyes optimized for protein binding. The dyes block access to trypsin cleavage sites, allowing for digestion only of undyed interface regions following denaturation of the protein complex. Interface regions can be identified using mass spectrometry and used as target sequences for drug development. Here we introduce new dye chemistries and elucidate their mechanism of protein binding for the first time. This allows for rapid identification of functionally-relevant hotspots without the need to screen many dye chemistries to optimize surface coverage of the protein complex. We applied this method to elucidate functional hotspots for immmuno-oncology targets PD-1 and PD-L1 and Hippo pathway targets YAP2 and tight junction protein ZO-1. To further functionally validate the hotspot regions identified, we focused on the case study of PD-1 and PD-L1. We discovered a hotspot of PD-1 Lys 78 in the protein-protein interface, and rationally designed a series of 8 peptide inhibitors to target this hotspot. The most active peptide YRCMISYGGADYKRITV derived from PD-L1 disrupted the PD-1/PD-L1 complex with an IC50 of 5.07 µM. The predicted binding site of this peptide on PD-1 overlaps the binding site of therapeutic anti-PD-1 antibody pembrolizumab; crystal structures of pembrolizumab and PD-1 show hydrogen bonding between the antibody and our identified hotspot Lys 78. Furthermore, we prepared a cyclized analog peptide CYRAMISYGGADYKRITC by disulfide bond stapling to increase peptide stability and found that this did not significantly reduce inhibitor potency, with an IC50 of 8.02 µM. Taken together, this data suggests a specific region of PD-1 found within the larger PD-1/PD-L1 interface that may serve as a target for development of next generation small molecule PD-1/PD-L1 inhibitors. By focusing drug discovery efforts against only the PPI hotspot regions, we may accelerate drug development against these difficult targets.

#983

Antibody-drug conjugates of NAMPT inhibitors: Discovery, optimization, and preclinical characterization.

Chris Neumann, Kathleen C. Olivas, Kung Pern Wang, Andrew B. Waight, David W. Meyer, Luke V. Loftus, Margo C. Zaval, Martha E. Anderson, Steven Jin, Julia H. Cochran, Jessica K. Simmons, Paul G. Pittman, Fu Li, Michelle L. Ulrich, Abbie Wong, Weiping Zeng, Robert P. Lyon, Peter D. Senter. _Seattle Genetics, Bothell, WA_.

Nicotinamide phosphoribosyltransferase (NAMPT) regulates the biosynthesis of NAD from nicotinamide via a salvage biosynthetic pathway. Inhibition of NAMPT depletes cellular NAD levels leading to disruption of energy metabolism and cell death. Non-targeted small molecule NAMPT inhibitors have demonstrated poor tolerability in clinical trials and in preclinical models, including cardiac and retinal toxicities in rats. In an effort to improve the therapeutic window of this drug class, we pursued a targeted-delivery approach using antibody-drug conjugates. Through a medicinal chemistry effort, we identified novel NAMPT inhibitors that incorporate chemical functionality in the solvent-exposed terminus to allow construction of enzyme-cleavable drug linkers. Additionally, we applied a pyridinium-based linker strategy that allows for traceless linker attachment through a conserved nicotinamide-mimetic moiety of NAMPT inhibitors. Candidate molecules were evaluated for NAMPT binding affinity and cellular cytotoxicity as free drugs, and for cellular cytotoxicity as ADCs with the alternate linker strategies. Comparisons across inhibitors and linker strategies provide insight into optimal design of cleavable drug linkers for this class of drugs. In vitro, the ADCs deplete NAD and lead to downstream ATP depletion in a time-dependent manner. In vivo evaluation using human tumor xenografts shows translation of the pharmacodynamic effect resulting in tumor regression in models of Hodgkin lymphoma, non-Hodgkin lymphoma, and acute myeloid leukemia. Toxicology studies in Sprague Dawley rats demonstrate excellent tolerability at active doses, with no observable cardiac or retinal toxicities at the highest tested doses in single- and multi-dose regimens. These findings detail the development of a novel payload class and optimized linker strategy for use with antibody-drug conjugates, and demonstrate a preclinical efficacy and safety profile to support continued efforts toward clinical therapeutics.

#984

**Synthesis of mixed, hypoxia-activated phosphoramidate esters for the inhibition of Enolase in** ENO1- **deleted glioblastoma.**

Florian Muller, Victoria C. Yan, Elliot S. Ballato, Kristine L. Yang, Dimitra K. Georgiou, Kenisha Arthur, Pakriti Shrestha, Sunada Khadka, Jeffrey Ackroyd. _UT MD Anderson Cancer Ctr., Houston, TX_.

Background: Precision oncology is currently restricted to activated oncogenes as drug targets, with tumor suppressor deletions remaining largely in-actionable. We developed a novel method that targets tumor suppressor deletions by exploiting vulnerabilities generated by the co-deletion of neighboring metabolic housekeeping genes with critical but normally redundant functions. Proof-of-principle studies were conducted for the glycolytic enzyme Enolase. Cancers harboring the homozygous deletion of 1p36-localized ENO1 remain metabolically active and viable through redundant action of its paralogue, ENO2. Inhibition of ENO2 in cancer cells harboring ENO1-homozygous deletion discriminately kills ENO1-deleted cells. In accordance, our synthetic efforts have focused on phosphonate chemistry to generate clinically-useful substrate mimics of 2-phosphoglycerate, the natural substrate for Enolase. We have generated a phosphonohydroxamate ENO2-inhibitor termed HEX and a pro-drug of which, POMHEX, kills ENO1-homozygously deleted glioma cells in culture with strong specificity. However, the pharmacokinetics of POMHEX are poor, due to the presence of carboxyesterase in extracellular fluids, including plasma.

Results: We have synthesized mechanistically different pro-drugs of HEX, utilizing nitro-aromatics as protecting groups. Nitroaromatic groups are bioactivated by nitroreductases intracellularly, in a manner that is inversely related to oxygen concentration. As such, removal of nitroaromatic pro-drug moieties and release of active drug is favored under low oxygen (hypoxic) conditions. We synthesized a series of nitroaromatic pro-drugs of HEX (the structures will be disclosed at AACR presentation, pending patent filings) and demonstrate that 1) these exhibit selective toxicity to ENO1-deleted glioma cells in culture and 2) this killing is dramatically enhanced by lowering oxygen concentrations as compared to POMHEX. We further find that redox potential of the nitroaromatic moiety correlates with the degree of hypoxia activation with the lower redox potential being associated with great oxygen dependence. Importantly, unlike POMHEX, nitroaromatic HEX pro-drugs are stable in human plasma ex-vivo. We have thus successfully synthesized a pro-drug inhibitor of Enolase that is hypoxia-activated.

Conclusion and future directions: We are gearing up synthesis for pre-clinical evaluation of nitroaromatic-HEX in intracranial tumor xenograft models. While the primary purpose of Nitroaromatic HEX is for the precision oncology treatment of ENO1-homozygously deleted tumors, it has not escaped our notice that such drugs have wider applicability within the context of targeting treatment-resistant tumors cells hiding in hypoxic niches. Indeed, we have observed that virtually all glioma cell lines can be killed by nitroaromatic HEX under hypoxia.

#985

Development and validation of cell-based TFEB translocation assay in a high-content and high-throughput screening format.

Li Zhang,1 Ruili Huang,1 Wen Xing Ding,2 Menghang Xia1. 1 _NCATS/NIH, Bethesda, MD;_ 2 _University of Kansas Medical Center, KS_.

Transcription factor EB (TFEB) is a master regulator of lysosomal gene expression and mitochondrial biogenesis. Autophagy is known to act as a double edged sword in cancer. Autophagy acts as a tumor suppressor during the tumorigenesis process but it also helps to sustain the survival of already transformed cancer cells. Lysosomes sit at the end of the autophagy pathway, which is critical to meet the needs of autophagy. Therefore, small molecules that inhibit TFEB-mediated lysosomal biogenesis and autophagic degradation may be helpful for inhibiting cancer cell growth and limiting cancer progression. On the other hand, small molecules that enhance TFEB-mediated lysosomal degradation may be used to prevent tumorigenesis. In order to identify compounds that modulate TFEB translocation, an image-based TFEB translocation assay was developed in a 1536-well plate format with a GFP-TFEB stable AML12 cell line, which is an immortalized mouse hepatocyte cell line. Torin 1, a known TFEB translocation inducer, was used as positive control in the study. Torin 1 increased TFEB nuclear translocation in a concentration dependent manner with an EC50 of 150nM. To optimize the assay condition, various cell densities per well and time courses were tested. To validate this assay, a group of 384 known anticancer compounds were screened at 11 concentrations ranging from 0.8 nM to 46 μM. From this screening, we identified 25 compounds that enhanced TFEB nuclear translocation with EC50s ranging from 2 nM to 12 μM. Several known and novel TFEB inducers were among these compounds. These results demonstrate that this high-content and high-throughput assay can be used to screen large numbers of chemicals and provide a robust in vitro system to identify TFEB agonists and antagonists, which may be used to either prevent (agonists) or treat (antagonist) cancer.

#986

High content analysis of 2D and 3D oncology models for target and phenotypic drug discovery.

Servane Lachize,1 Rhea van de Bospoort,1 Marta da Silva da Silva,1 Niki van der Steentraten,1 Ian Gowers,2 Roger Clark,2 Jeroen DeGroot,1 Ian Waddell,2 Hajo Schiewe,3 Shushant Jain1. 1 _Charles River, Leiden, Netherlands;_ 2 _Charles River, Saffron Walden, United Kingdom;_ 3 _Charles River, Wilmington, MA_.

Charles River (CR) has developed many disease-relevant cellular models to screen small molecules and genetic modifiers for target and phenotypic based drug discovery. Our expertise and portfolio in bespoke oncology model development (e.g. patient derived xenografts) covers a wide variety of target classes such as inhibitors of cell cycle, cellular motility and migration and transcription factor translocation. Complex cellular models derived from primary cells, patient derived embryonic stem cells and induced pluripotent stem cells have been adapted for phenotypic screening. Models have been further developed to produce physiologically relevant multicellular structure co-cultures. Combining complex biological systems with multiparametric high content analysis provides detailed single cell quantification of cellular and subcellular biology. Such validated High Content assays can be applied to both target and phenotypic based drug-discovery platforms to support preclinical drug discovery and improve translation of targets and compounds to the clinic.

#987

Glycoengineered MSCs for targeting platinum resistant ovarian tumors.

Buddhadev Layek, Drishti Sehgal, Jayanth Panyam, Swayam Prabha. _University of Minnesota, Minneapolis, MN_.

Tumor-targeted delivery of chemotherapeutics can improve efficacy while mitigating their debilitating side effects. However, current drug delivery approaches rely on inefficient passive accumulation of delivery systems in the tumor and suffer from significant non-specific distribution. Here, we propose a novel two-step tumor targeting strategy that can improve delivery of cytotoxic agents to both primary tumor as well as metastatic lesions, resulting in effective tumor inhibition. This strategy involves introduction of non-natural targets in the tumor tissue via glycoengineered mesenchymal stem cells (MSCs expressing targetable synthetic azide groups) followed by the delivery of drug-loaded polymeric nanoparticles (surface functionalized with dibenzyl cyclooctyne; DBCO) that have high affinity for these synthetic targets. DBCO surface functionalized, paclitaxel (PTX)-loaded nanoparticles (DBCO-PTX NP; 17.6% w/w PTX; diameter 313.7 ± 13 nm; and zeta potential -12.5 ± 1.9 mV) were formulated using poly (DL-lactide-co-glycolide) (PLGA) polymer by emulsion-solvent evaporation method. Glycoengineered MSCs (MSC-Az) were generated by culturing MSCs in N-azidoacetylmannosamine-tetraacylated supplemented media without affecting their viability or tumor homing properties. Binding of MSC-Az to DBCO-PTX NP was confirmed with confocal microscopy and flow cytometry analysis. Anticancer efficacy of two-step targeting strategy was evaluated in murine model of platinum-resistant orthotopic ovarian C200-luc tumors. Treatment with MSC-Az + DBCO-PTX nanoparticles resulted in significant inhibition of the tumor growth (p < 0.05) and improved survival (p < 0.05) compared to that with other controls. In summary, our results demonstrate the potential of two-step tumor targeting strategy to enhance the anticancer efficacy of conventional chemotherapeutic drugs.

#988

Synthesis and preclinical evaluation of dual-stimuli responsive doxorubicin prodrug activated by histone deacetylases and cathepsin L.

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

Doxorubicin (DOX) an anthracycline is a leading anticancer drug with a broad spectrum of activity against numerous solid and hematologic malignancies. However, its clinical application is limited by lower efficacy, severe cardiotoxicity and occurrence of secondary malignancies. There is an urgent need for eliminating the Dox adverse effects while retaining its anticancer efficacy. One of the major goals of cancer therapy is the selective targeting of malignancies over normal tissues. One way to avoid these severe adverse reactions is to develop tumor-targeted prodrugs that are converted to active antitumor drugs at tumor sites in the presence of enzymes that are overexpressed human cancers. Among these, the histone deacetylases (HDACs) and cathepsin L (CTSL) are highly expressed in cancer cells and are considered as potential cancer-specific targets. HDACs are critical enzymes involved in the regulation of histone and non-histone proteins and elevated HDACs in tumor cells are known to be closely associated with tumor initiation, progression, and metastasis. Similarly, the lysosomal cysteine protease CTSL plays key roles at multiple stages of tumor progression and metastasis. In the present study, we developed a new prodrug by coupling an acetylated lysine group to doxorubicin (Lys(Ac)-Dox), a masked cytotoxic agent, which is consecutively activated by HDACs and a CTSL to liberate doxorubicin. We first verified whether Lys(Ac)-Dox could be specifically cleaved by HDACs and CTSL in vitro. The results showed that after incubating with HDACs and CTSL at 37 °C for 20 h, the hydrolysis of Lys(Ac)-Dox reached 99%, suggesting that Lys(Ac)-Dox could be successfully cleaved by the target enzymes. To prove that the Lys(Ac)-Dox would have a much improved growth-inhibitory effect against cancer cells, the cytotoxicity of free DOX and Lys(Ac)-Dox against lung cancer cell lines normal human lung epithelial cell line was determined. The dose-response curves obtained from the cell lines tested indicated that Dox was equally cytotoxic against both cancer and normal cells. In contrast, Lys(Ac)-Dox highly cytotoxic against cancer cells and non-toxic to the normal counterparts. To measure the in vivo anticancer efficacy of Lys(Ac)-Dox vis-vis Dox, we developed subcutaneous xenografts by injecting human lung cancer A549 and H460 cells in nude mice. Lys (Ac)-Dox and Dox were administered daily i.p. at 5 mg/kg. The prodrug showed a significantly higher (>2-fold) tumor regression than Dox. A diminished circulating reticulocyte counts from whole blood after Dox treatment is known to reflect the hematological toxicity caused by the drug. Only Dox caused significant reticulocyte ablation while the prodrug did not, validating our drug design. Biochemical tests involving topo II inhibition and ROS production by the prodrug are in progress (supported by CPRIT grant RP 170207 to KSS).

#989

The relationship between TP53 gene status and carboxylesterase 2 expression in human gastric cancer.

Yoshinori Kohira, Hyeon-Cheol Lee, Momoko Ishimine, Hajime Orita, Toshiyuki Kobayashi, Koichi Sato, Takehiko Yokomizo, Tetsu Fukunaga. _Juntendo University School of Medicine, Tokyo, Japan_.

Carboxylesterases are serine hydrolases that are involved in the metabolisms of various endogenous and exogenous compounds. They are also required for activation of many anti-cancer prodrugs. For example, irinotecan (CPT-11), an anti-cancer prodrug that has been approved for the treatment of many types of solid tumors including gastric cancer, is converted by the carboxylesterase CES2 to its active metabolite 7-ethyl-10-hydroxycamptothesin (SN-38), a very potent topoisomerase I inhibitor. Among carboxylesterase isozymes, CES2 is most highly expressed in the gastrointestinal tract. Thus, the expression of CES2 may play an important role in local (i.e., intratumoral) activation of anti-cancer prodrugs such as irinotecan in the gut. Recent studies with cultured cancer cell lines have shown that CES2 expression is regulated by the tumor suppressor protein p53. However, whether CES2 expression is affected by the presence of p53 mutation in clinical cancer samples still remains unclear. In this study, we focused on the regulatory mechanism of CES2 expression in gastric cancer. First, we examined the relationship between TP53 gene status and CES2 expression using gastric cancer cell lines. Several gastric cancer cell lines expressing wild-type p53 (AGS, NUGC4, MKN74, and HSC58) were treated with nutlin-3a, a drug that inhibits the interaction between p53 and the E3 ubiquitin ligase MDM2 and thereby directly activates p53 signaling without genotoxic side effects. The expression of p21, a downstream target of p53, was increased following nutlin-3a treatment in two p53 wild-type cell lines NUGC4 and HSC58. The expression of CES2 was also upregulated by nutlin-3a in three p53 wild-type cell lines AGS, NUGC4, and HSC58. As expected, the expression levels of p21 and CES2 were not largely affected by nutlin-3a in gastric cancer cell lines with TP53 mutations. These results indicate that CES2 expression is positively regulated by the p53 pathway in most gastric cancer cells. We also investigated the relationship between TP53 gene status and CES2 expression in human gastric cancer samples. Our results may provide useful information for predicting the efficacy of anti-cancer prodrugs that are activated by CES2 in gastric cancer.

#990

Improving TRAIL therapy response in pancreatic cancer by replenishment of miR-145.

Saini Setua, Sheema Khan, Nirnoy Dan, Murali M. Yallapu, Sonam Kumari, Stephen W. Behrman, Meena Jaggi, Subhash C. Chauhan. _Univ. of Tennessee Health Science Center, Memphis, TN_.

Background: Pancreatic cancer (PanCa) is a third leading cause of cancer related deaths in US. Unlike other cancers, PanCa is highly resistant to TNF-related apoptosis-inducing ligand (TRAIL) that emerges as one of the most-promising therapy in clinical trials. Our group has previously identified microRNA-145 (miR-145) is downregulated in PanCa, the restoration of which inhibits tumor growth and enhances gemcitabine sensitivity. In this study, we have observed that miR-145 restoration in PanCa cells renders them sensitive to TRAIL treatment. Therefore, we have engineered unique superparamagnetic nanoparticles (SPs) for co-delivering miR-145 and TRAIL for improving TRAIL response in PanCa cells.

Methods: PanCa cells, HPAF-II and AsPC1 were used in the study. A precipitation approach was employed to develop the SP formulation, which was conjugated with miR-145 and pEGFP-TRAIL. Particles were investigated for size, physico-chemical characterization (Dynamic light scattering) and the complexation of miR-145 and pEGFP-TRAIL by gel retardation assay. The formulation was investigated for functional assays, such as, proliferation (MTT), invasion (Matrigel), migration (Boyden chamber), colony and spheroid formation assays. Western blotting and immunofluorescence assays were used to investigate the effects of miR-145 restoration and death receptor activation in cells. The effect of nanoformulation on the tumor growth was investigated using xenograft mice model.

Results: The results in this study demonstrate that acquired resistance to TRAIL in PanCa cells can be minimized with the replenishment of miR-145 expression. Our SP nanoparticles were engineered to co-deliver miR-145 and TRAIL to PanCa cells, which resulted in simultaneous restoration of miR-145 and inhibition of acquired resistance to TRAIL. Combined actions of miR-145 and TRAIL markedly improve TRAIL-induced apoptotic effects in PanCa cells through the activation of an extrinsic apoptosis pathway as indicated by activation of DR5, FLIP, FADD and enhanced expression of caspase-8/3. The co-delivery of miR-145 and TRAIL using SP nanoparticles inhibited tumorigenic characteristics of PanCa cells, which include proliferation, invasion, migration and clonogenicity. The results were reciprocated and got further confirmed with the inhibition of tumorsphere formation and in vivo tumorigenicity in xenograft mice. Immunohistochemical staining of excised tumor tissues demonstrate an activation of death receptor pathway and subsequent expression of apoptotic markers.

Conclusion: The study provides novel insights on two facades- how resistance of cancer cells to TRAIL-based pro-apoptotic therapies can be tackled, and how efficient intracellular delivery of TRAIL can be achieved using a nanotechnology platform. Our results suggest that TRAIL resistance can be overcome by co-delivery of miR-145 and TRAIL using SP nanoparticles.

#991

Secretory phospholipase A2 as a trigger for drug release in the treatment of triple-negative breast cancer.

Shanese L. Jasper,1 Elena B. Skarupa,1 Brian S. Cummings,2 Robert D. Arnold1. 1 _Auburn University, Auburn, AL;_ 2 _University of Georgia, Athens, GA_.

Triple negative breast cancer describes a subgroup of breast cancers which are negative for the estrogen and progesterone receptors as well as the HER2 protein. As such, triple negative breast cancers have seen little benefit from the targeted therapies developed for the treatment of breast cancer. To address this challenge, we examined an alternative target within the tumor as well as the tumor environment. Secretory phospholipase A2 (sPLA2) cleave phospholipids at sn-2 ester bonds, releasing lysophospholipids and fatty acids, and are over expressed in several pathologies including breast cancer. Herein, we evaluated the therapeutic activity of secretory phospholipase A2 responsive liposomes (SPRL) compared to the clinically used, sterically-stabilized, pegylated liposomes (SSL) for in vitro response in a triple-negative breast cancer (TNBC) model. In these studies, SSL and SPRL formulations were made according to previous studies and resulted in three formulations SSL, SPRL- E and SPRL-G. SPRL were made by the addition of either DSPE (SPRL-E) or DSPG (SPRL-G). Doxorubicin was used as the drug of choice and Dox-loaded liposomes were prepared by remote-loading using an ammonium sulfate gradient. Toxicity studies were performed by the use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) or resazurin while uptake studies were performed by fluorescence microscopy and flow cytometry. Briefly, cells were seeded at a density of 10,000 cells per well and subjected to free doxorubicin or liposomal doxorubicin in concentrations from 0 - 100 µM. At the appropriate time points, 0-72 hours, cells were harvested for studies. Treatment of breast cancer cells with doxorubicin encapsulated in SSL and SPRL resulted in cytotoxicity in the MDA-MB-231 cells line comparable to free drug with an IC50 of approximately 3 µM at the 72 hour time point. In tracking of drug and liposome delivery, we demonstrated that drug uptake was liposome-dependent, as encapsulation of doxorubicin in SPRL resulted in greater intracellular drug levels compared to SSL. These data show the therapeutic activity of SPRL compared to SSL, and suggest that SPRL may be useful for the treatment of TNBC. Two-dimensional models do not fully recapitulate the complexity of barriers to drug delivery, nor the effect of multiple cell types. Therefore, ongoing studies are examining the utility of these liposome formulations in three-dimensional co-cultures that include macrophages and fibroblasts. In this model, we hope to assess the ability of the tumor microenvironment to alter drug release.

#992

Comparative analyses of drug-loaded exosomal preparations from different cell types reveal distinctive loading capability, yield, and anti-tumor efficacies.

RAJASHEKHAR KANCHANAPALLY, Sachin Kumar Deshmukh, Suhash Reddy Chavva, Nikhil Tyagi, Sanjeev Kumar Srivastava, Girijesh Kumar Patel, Ajay P. Singh, Seema Singh. _USA-Mitchell Cancer Institute, Mobile, AL_.

Cancer remains one of the leading causes of death worldwide regardless of technological advancements. Inefficiency of current drug-delivery regimes is one of the important factors that limits the therapeutic efficacy of existing chemotherapeutics thus contributing to cancer mortality. To address this limitation, synthetic nanotechnology-based delivery systems have been developed; however, they raise concern of inducing adverse immunogenic reactions. Exosomes are non-immunogenic nano-sized vesicles that have received significant attention as efficient drug delivery system. Here we evaluated the efficacy of different cell types viz. pancreatic cancer cells (PCCs), pancreatic stellate cells (PSCs) and macrophages (MØs) for drug packaging and release into exosomes. PCCs shed the most exosomes and were the most efficient in drug loading followed by MØs and PSCs as examined by HPLC quantification. However, when compared for anti-tumor efficacy, MØs-derived exosomes-loaded with DOX (MØ-Exo-DOX) showed highest activity followed by PSCs and PCCs in WST-1 and Annexin V staining assays. These varying anti-tumor activities likely resulted from non-drug contents of exosomes since we did not observe any significant differences in their uptake by the cancer cells. Altogether, our data suggest that donor cell-specific differences exist in exosomes, which could influence their utility as drug carrier for therapeutic purposes.

#993

Chemotherapy enhances exosome secretion via upregulation of cholesterol metabolism in acute myeloid leukemia.

Chang-Sook Hong,1 Emily J. Jeong,2 Beatrice Razzo,1 Michael Boyiadzis,1 Theresa L. Whiteside1. 1 _University of Pittsburgh, Pittsburgh, PA;_ 2 _Taylor Allderdice High School, Pittsburgh, PA_.

Therapies for Acute Myelogenous Leukemia (AML) have not been successful largely due to disease recurrence after chemotherapy. Recent studies showed that exosomes play a significant role in leukemia chemo-resistance. Levels of exosomes in patients' plasma are elevated at AML diagnosis relative to those in normal donors' plasma and remain elevated when patients reach complete remission (CR). These exosomes carry immunosuppressive molecules, interfere with anti-leukemia immunity and promote blast chemoresistance. We hypothesized that the development of methods for blocking exosome secretion could benefit AML patients. To understand the mechanisms of enhanced exosome secretion in AML blasts, we focused on cholesterol synthesis in blasts and its relationship to exosome secretion. Cholesterol metabolism in AML cell lines after treatment of chemotherapeutic agents (cytarabine and decitabine) was shown to enhance (p<0.005) intracellular level of cholesterol and the expression of HMGCR (3-hydroxy-3-methyl-glutaryl-coenzyme A reductase), the rate-limiting enzyme in cholesterol synthesizing mevalonate pathway. Consequently, the production of exosomes in the cell culture supernatants increased (p<0.02). Treatment with an HMGCR inhibitor, simvastatin, or siRNAs targeting HMGCR blocked (p<0.02) the chemotherapy- induced enhancement of exosome production. Exosomes carry HMGCR and chemotherapy enhanced HMGCR levels on exosomes. Our results show that chemotherapy enhances exosome secretion by increasing cholesterol synthesis in leukemic blasts. A pharmacologic blockade of HMGCR with simvastatin emerges as a potential future therapeutic for exosomes in AML.

#994

Combining ruthenium metallo-intercalators and targeted radionuclide therapy for EGFR-overexpressing oesophageal cancer.

Martin R. Gill, Jyothi U. Menon, Robert Carlisle, Katherine A. Vallis. _University of Oxford, Oxford, United Kingdom_.

Targeted radionuclide therapy (TRT) combines the specificity of molecular targeting with the potent cytotoxicity of ionising radiation. An emerging question is how to most effectively integrate TRT with radiosensitizing small molecules [1]. Auger electron-emitting radionuclides such as 111In are well-suited as the therapeutic radionuclide as their short-range reduces "cross fire" damage to neighbouring cells. As a novel chemical class of radiosensitizing agent, we have found ruthenium polypyridyl complexes (RPCs) that intercalate between DNA base pairs such as Ru(phen)2(tpphz)2+ (phen = 1,10 phenanthroline, tpphz = tetrapyridophenazine) act to stall replication forks and impede mitotic progression in human oesophageal cancer cells [2,3]. This dual mechanism of action results in preferential growth inhibition of rapidly-proliferating oesophageal cancer cells with elevated mitotic indices. In contrast to cisplatin and other DNA-damaging agents, relatively low DSB levels are generated in response to RPC metallo-intercalation and reduced cytotoxicity towards non-cancer cells is observed. In addition to this, treatment is able to enhance cancer cell sensitivity to IR by increasing IR-induced DNA double-strand break (DSB) damage, decreasing cell survival in a synergistic manner. Based on these discoveries, we explore how nanoparticle-mediated delivery of Ru(phen)2(tpphz)2+ and the Auger electron emitting radionuclide 111In can achieve localised cytotoxicity enhancement specifically in EGFR-overexpressing oesophageal cancer cells, thereby demonstrating both a combinational effect alongside molecular targeting for this novel combination [4].

References:[1] M. R. Gill, N. Falzone, Y. Du and K. A. Vallis, Lancet Oncol, 18, e414 (2017).[2] M. R. Gill, S. N. Harun, S. Halder, R. A. Boghozian, K. Ramadan, H. Ahmad and K. A. Vallis, Sci. Rep., 6, 31973 (2016).[3] M. R. Gill, P. J. Jarman, S. Halder, M. G. Walker, H. K. Saeed, J. A. Thomas, C. Smythe, K. Ramadan and K. A. Vallis, Chem. Sci., 9, 841 (2018).[4] M. R. Gill, J. U. Menon, P. J. Jarman, J. Owen, I. Skaripa-Koukelli, S. Able, J. A. Thomas, R. Carlisle, K. A. Vallis, Nanoscale, 10, 10596 (2018).

#995

A new thiadiazine derivative induces oxidative stress dependent JNK pathway activation and cell death in hepatocellular carcinoma.

Deniz C. Kahraman,1 Ebru B. Guven,2 Birsen Tozkoparan,3 Rengul C. Atalay1. 1 _Middle East Technical University, Ankara, Turkey;_ 2 _Kadir Has University, Ankara, Turkey;_ 3 _Hacettepe University, Ankara, Turkey_.

Hepatocellular carcinoma (HCC) is the fifth most common and the second lethal cancer worldwide. HCC is resistant to conventional chemotherapy and radiotherapy due to its highly heterogenous structure. Therefore, it is crucial to design and develop novel therapeutic strategies against HCC. Non-steroidal anti-inflammatory drugs (NSAIDs) have long been studied for the treatment of acute and chronic conditions with pain and inflammation. In recent years, evidence from various studies suggested that NSAIDs reduce the incidence and mortality of various types of cancers. Here, investigated the possible cytotoxic bioactivities of a series of triazolothiadiazine derivatives on HCC cells, which have been previously reported as potent analgesic/anti-inflammatory compounds. For this purpose, we first investigated the anticancer effect of our compounds bearing a triazolothiadiazine core. Initially, 30 compounds were tested against cancer cells. Among all the compounds, three of them (7a, 7b and 7c) showed noticeable cytotoxic activities within µM concentrations and were selected to be screened against a panel of HCC cell lines. Compound 7b was identified as a promising anti-cancer agent against liver cancer cells with cytotoxic doses (less than 5µM) assessed by both the SRB assay and RT-CES analysis and was analyzed with further in vitro and in vivo experiments to disclose the underlying mechanism of its action in HCC cells. Time- and dose- dependent growth inhibition upon treatment with compound 7b was observed due to cell cycle arrest at the G2/M phase and apoptosis. After detecting the activation of SAPK/JNK pathway, it was shown that ROS accumulation was triggering the downstream events and eventually apoptotic cell death. Furthermore, upon treatment with compound 7b, overall survival of xenografts increased, while the tumor size decreased compared to control groups. Additionally, we investigated the potential of this compound on liver cancer stem cell population (CD133+/EpCAM+) which are known to be responsible for drug resistance in HCC. Compound 7b was bioactive against LCSCs and decreased the ratio of CD133+/EpCAM+ population significantly in both Huh7 and Mahlavu cells. Altogether, the anti-tumor and anti-resistance effects of compound 7b approves that this small molecule can be considered as an anti-cancer agent for liver cancer therapeutics.

#996

In silico **modeling and** in vitro **validation of undefined off-target of drugs in hepatocellular carcinoma.**

Esra Sinoplu, Nurcan Tuncbag, Deniz C. Kahraman, Rengul C. Atalay. _METU, Ankara, Turkey_.

Hepatocellular carcinoma (HCC) is the 5th most common and 2nd deadliest cancer worldwide. The molecular mechanism in HCC involves activated cell survival pathways, but there is a lack of the significant oncogenic drivers for targeted therapies. Thus, novel drugs and targets that can be exploited, are required. The pathways reported to be involved in hepatocarcinogenesis were retrieved from KEGG database (HCC, MAPK, calcium, p53, PI3K-Akt, Wnt, TGF-Β, NAFLD and apoptosis) and a directed network with 801 nodes was created. In parallel, small molecule inhibitor drugs having at least one target protein in this network were collected from DrugBank database and integrated into the merged network. By the means of in silico perturbation attack strategies, target proteins and their interactions were calculated as the drug effectiveness, changes in the efficiency of the signaling network, in the number of feedback cycles, and in the network, functionality were identified and ranked. Finally, Brigatinib, Regorafenib, Sunitinib, Thalidomide, Pranlukast, Lenvatinib, and ChloroquineP were identified to have off-target effects specific to HCC pathways. The selected drugs along with Sorafenib were then tested and compared for their cytotoxicity on HepG2, Huh7, Mahlavu and SNU475 cells. Except Thalidomide and Pranlukast, selected drugs were significantly bioactive on the cells with IC50 values below 10µM. We then examined the expression of PanCancer gene panel (NanoString) in Mahlavu cells treated with the selected 8 drugs. As expected a number of genes and pathways were differentially altered in the presence of the drugs and multikinase inhibitors Sorafenib and Regorafenib had similar DEG patterns. In gene expression correlation analysis matrix, Sorafenib differed from the rest of the compounds particularly from Brigatinib while highest correlation was observed with Regorafenib. Although Brigatinib differs from other drugs in terms of its targets, it resulted in the highest number of differentially expressed genes. Thalidomide gene profile was very similar to Sorafenib. This data supports the possible exploitation of Thalidomide for liver cancer therapeutics. We then merged and filtered cancer-associated pathways (MAPK, STAT, PI3K, RAS, Cell Cycle, Apoptosis, Hedgehog, Wnt, DNA Damage Control, Transcriptional Regulation, Chromatin Modification, and TGF-Β) based on the differentially expressed genes in Mahlavu cells treated with the 8 drugs. Previously reported genes such as APC, AXIN, CTNBB1, CAMK, TGF, NGF, ERBB2 were significantly enriched in the network along with the genes that has not been associated with liver cancer therapeutics. The enriched network analysis can be exploited for both drug repurposing and novel target identification.

#997

Three-dimensional high-throughput drug screening for bladder cancer.

Sang-Cheol Lee,1 Young Saing Kim,2 In Gyu Hwang,3 Su Jin Lee,4 Se Hoon Park4. 1 _Soonchunhyang University Hospital Cheonan, Cheonan-si, Chungchungnam-do, Republic of Korea;_ 2 _Gil Medical Center, Gachon University College of Medicine, Incheon, Republic of Korea;_ 3 _Chung-Ang University, College of Medicine, Seoul, Republic of Korea;_ 4 _Sungkyunkwan University Samsung Medical Center, Seoul, Republic of Korea_.

Recent progress in understanding the molecular mechanisms that underlie bladder cancer carcinogenesis offers the prospect of specifically targeting signaling pathways to achieve more effective and rational treatment. In a novel, 3-dimensional high-throughput drug screening (3D HTS) platform to identify novel agents affecting bladder cancer cells, 24 molecularly-targeted agents were screened in seven human bladder cancer cell lines (UCUM3, T24, SW780, RT4, J82, 5637 and 253J-BV). The efficacy of 24 targeted agents on the bladder cell lines varied dramatically on their genomic alterations. BEZ235 (mTOR and PI3K inhibitor) showed antitumor effect in most of cell lines except UMUC3. Another mTOR inhibitor, AZD2014 (inhibitors of mTORC1 and mTORC2) also had IC50 <2 μM in 3 cell lines (5637, J82 and RT4). However, FGFR targeted agents (AZD4547 and BGJ398) did not show the efficacy in cell lines with FGFR3 alterations (J82, SW780 and RT4). In HER2 mutated/amplified cell line (5637), dacomitinib, lapatinib and neratinib showed anti-tumor effect. Immunoblot assay showed that lapatinib and neratinib suppressed phosphorylated HER2 and downstream molecules such as p-AKT or p-ERK. In conclusion, 3D HTS drug screening platform could be a useful preclinical tool to evaluate various kinds of drugs rapidly, and eventually provide a valuable insight into precision medicine along with comprehensive genomic analysis.

#998

HEx: A computational and synthetic biology platform applied to oncology drug discovery.

Ulrich Schlecht, Pablo Cordero, Maureen Hillenmeyer, Ju Eun Jeon, Dan Kvitek, Brian Naughton, Philipp Wiemann, Colin Harvey. _Hexagon Bio, Menlo Park, CA_.

We present a computational and synthetic biology platform for producing novel compounds targeting proteins relevant to oncology. As eukaryotes, fungi share approximately one third of human genes, including many relevant to core metabolism, cell cycle, DNA repair, and protein degradation. We have developed a computational method for mining fungal genomes to identify genes likely to produce compounds that inhibit specific human target proteins, including undruggable targets such as protein-protein interactions. We engineer these genes using our synthetic biology pipeline to produce the novel compounds in a heterologous host. We present in vitro data for an Aurora Kinase (AURKA) inhibitor developed using this platform as proof of concept for Hexagon Bio's pipeline. These data demonstrate the potential of this system to target the more than 6000 genes shared between fungi and humans.

#999

In vivo breast tumor stiffness and vascular drug delivery recapitulated in a microfluidic tumor-on-a-chip.

Benjamin Anbiah,1 Iman Hassani,1 Nicole Habbit,1 Lani Jasper,1 Deborah Ramsay,2 Balabhaskar Prabhakarpandian,3 Robert Arnold,1 Elizabeth Lipke1. 1 _Auburn University, Auburn, AL;_ 2 _CFD reasearch corporation, Huntsville, AL;_ 3 _CFD research corporation, Huntsville, AL_.

Biomimetic tissue engineered microfluidic cancer models offer a higher degree of spatial, temporal and structural precision in controlling the physical parameters and component characteristics of the native tumor microenvironment (TME). Current models working to establish a biomimetic in vitro breast TME are limited by their ability to recapitulate various degrees of in vivo complexities and poor correlation of the diffusional gradients of oxygen, nutrients and anti-cancer drugs. To establish a model and address these challenges, we have used poly(ethylene glycol)-fibrinogen (PEG-Fb) as our biomimetic material to engineer 3D breast tumor tissues and recapitulate the mechanical stiffness of core, midpoint and peripherial zones of the native tumor in a vascularized microfluidic chip.

To assess the mechanical stiffness, the in vivo breast tumor (MDA-MB-231 flank xenograft in Athymic nude mice) and engineered tumor constructs were subjected to parallel plate compression test using Cell Scale Microsquisher and the resulting force versus displacement data was acquired to calculate Young's modulus. Tumor mimetic ("high perfusion chip" (HPC) and "low perfusion chip" (LPC), differ with respect to the vascular network surrounding their respective primary and secondary tumor compartments were used in this study. Breast cancer-associated endothelial cells (hBTEC) were seeded in the vascular network and allowed to form a lumen. Metastatic breast cancer cells MDA-MB-231/ human foreskin fibroblast BJ5ta (ATCC) cells were mixed with polymer precursor solution containing PEG-Fb and Eosin Y. The precursor was loaded into the primary tumor compartment and cross-linked for 2 minutes under visible light. Stiffness was modulated by adding poly(ethylene glycol) diacrylate (PEGDA) to the polymer precursor for recapitulating the different zones of the in vivo tumor. hBTEC media was perfused through the endothelial cell networks were continuously monitored for cell behavior and metastasis.

In vivo breast tumor stiffness at core, midpoint and periphery was found to be within the range of the 3D engineered breast tumor tissues with time in culture through day 29. In the vascularized microfluidic chip, cell laden biomaterial was incorporated, the cancer cells were observed to undergo key events of the TME such as intravasation, circulating tumor cells in the endothelial vascular channel, adherence and migration to the secondary chamber resulting in metastasis. In the native TME there are regional differences in drug diffusion; TRITC dextran (4.4 kDa) was administered at a constant flow rate through the chips' vascularized networks and found to have vascular network geometry and engineered tumor construct stiffness dependent differences in diffusion into the primary tumor chamber, mimicking the in vivo phenomena.

## ENDOCRINOLOGY

### Endocrine-related Cancers

#1000

Targeting androgen receptors and cyclin-dependent kinases 4 and 6 in breast cancer.

Amy H. Tien, Nasrin R. Mawji, Jun Wang, Marianne D. Sadar. _BC Cancer, Vancouver, British Columbia, Canada_.

Androgen receptor (AR) is a transcription factor that plays dual roles in breast cancer: promoting or inhibiting proliferation depending on expression and activity of estrogen receptor-alpha. Expression of AR is detected in up to 90% of all breast cancers. The duration of treatment with aromatase inhibitor and expression of AR are correlated in circulating-tumor cells (CTCs) from breast cancer patients with bone metastases. Constitutively active splice variants of AR (AR-Vs) are expressed in primary breast cancer specimens, cell lines, and CTCs from breast cancer patients. Detection of AR-Vs in CTCs from breast cancer patients is associated with bone metastases. Hence, targeting AR is a potential therapeutic strategy for AR-positive breast cancers. Anti-androgens such as enzalutamide and bicalutamide are in clinical trials for breast cancer patients. However, these approaches target the AR C-terminal ligand-binding domain, which is not present on AR-Vs. Expression of AR-Vs can drive cancer cell growth and resistance to antiandrogens. Ralaniten is an effective AR antagonist that targets AR N-terminal domain (NTD) to inhibit the transcriptional activities of both AR and AR-Vs. Cyclin-dependent kinases 4 and 6 (CDK4/6) are activated by cyclin D1 to phosphorylate Rb and are critical in the transition from G1 to S phase in cell cycle. Luminal AR subtype of triple negative breast cancer is particularly sensitive to CDK4/6 inhibitors and this sensitivity is associated with AR expression. Our objective is to evaluate efficacy of ralaniten in combination with palbociclib on the growth of AR-expressing breast cancers. AR-expressing breast cancer cell line, SUM159PT, and AR-negative cell line, MDA-MB-468 (negative control) were used. To assess the effects of ralaniten on cell growth, we examined cell proliferation by BrdU incorporation, cell viability by alamarBlue staining, and cell cycle by BrdU and 7-aminoactinomycin D staining. Cells were treated with ralaniten with or without palbociclib, or control vehicle. Each analysis was performed after 1 doubling time. Ralaniten significantly inhibited proliferation of SUM159PT, but not AR-negative MDA-MB-468 cells. Enzalutamide was less effective than ralaniten to block proliferation. Cell viability assays showed an additive effect on SUM159PT cells in combination treatment of ralaniten and palbociclib. Ralaniten disrupted cell cycle in SUM159PT cells and caused cell accumulation in late S phase. Interestingly, combination treatment caused cell accumulation in G1 phase and cell reduction in late S phase. In summary, ralaniten effectively inhibited the growth of AR-expressing breast cancer cells. Additional cell lines are being tested. In vivo efficacy of ralaniten and combinations to inhibit the growth of AR-expressing breast cancer xenografts are under investigation. Targeting AR by ralaniten or an analogue may benefit breast cancer patients whose cancers express AR.

#1001

Functional and mechanistic interrogation of androgen receptor degraders for the treatment of metastatic castration resistant prostate cancer.

Steven Kregel, Chao Wang, Ester Fernandez-Salas, Kari Wilder-Romans, Xin Han, Chong Qin, Weiguo Xiang, Jean Tien, Xuhong Cao, Corey Speers, Shaomeng Wang, Arul M. Chinnaiyan. _University of Michigan, Ann Arbor, MI_.

The clinical development of second-generation androgen receptor (AR) antagonists, including enzalutamide, has confirmed that the AR remains a key oncogene in castration-resistant prostate cancer (CRPC). However, response to enzalutamide is temporary and incremental, and prostate cancer (PCa) cells usually maintain AR expression and signaling upon resistance to AR-targeted therapies. One novel strategy for targeting the AR axis in CRPC is to target proteins through proteasomal degradation, which improves the efficacy and specificity of standard inhibitors. We have developed compounds that bind to AR and engage the proteasomal degradation machinery to specifically degrade the AR protein. Based on this, we hypothesized that the pharmacologic degradation of AR represents an important advance in CRPC treatment, and may provide a novel therapeutic strategy for advanced prostate cancer. These compounds affect AR-positive prostate cancer cells by decreasing AR protein levels as well as disrupting oncogenic AR signaling and enzalutamide resistance. Nanomolar concentrations of AR degraders ultimately lead to decreases in PCa cell growth in vitro and in vivo. In conclusion, we developed highly potent small molecules that induced proteasomal degradation of AR proteins, thus demonstrating degrader compounds to be a potential therapeutic strategy for patients with CRPC.

#1002

An association between Annexin A1 (ANXA1) and loss of progesterone receptor expression in estrogen receptor-positive breast cancer.

Sung Gwe Ahn, Soong June Bae, Chihwan Cha, Chang Ik Yoon, So Eun Park, Joon Jeong. _Gangnam Severance Hospital, Seoul, Republic of Korea_.

Purpose: There is a strong body of evidence that low expression of progesterone receptor has a negative prognostic value in estrogen receptor (ER)-positive breast cancer. To discover key factors associated with PR-loss in ER-positive cancer, we conducted cell line experiments and investigated clinical samples.

Methods: We generated PR knock-down MCF7 and T47D cell lines using lentiviral transfection with PR shRNA. Gene expression microarray analyses were performed using Illumina Chip. Annexin A1 (ANXA1) was evaluated for tissue microarray (TMA) using HER2-negative human breast carcinoma.

Results: When PR-intact and PR-knockdown cells were compared using gene-expression profiles, ANXA1 was commonly identified as a molecule associated PR-knockdown in both MCF7 and T47D cell lines. Further, the mRNA expression of ANXA1 was elevated by PR-knockdown in both cell lines. In HER2-negative patients (n=370), ANXA1-positive tumors were associated with a lack of PR expression (P<0.001). The patients with ANXA1-positivity had a poor survival outcome in terms of overall survival and metastasis-free survival.

Conclusions: Our findings inform that ANXA1 may act a role in tumor progression of PR-lacking breast cancer.

#1003

Modulating estrogen related receptors (ERRs) activity in breast cancer using endogenous ligands.

Faegheh Ghanbari,1 Sylvie Mader,2 Anie Philip1. 1 _Devision of Plastic Surgery, Mcgill University, Montreal, Quebec, Canada;_ 2 _Institute for Research in Immunology and Cancer and Biochemistry, University of Montreal, Montreal, Quebec, Canada_.

Estrogen-receptor related receptors which consists of ERRα, ERRβ and ERRγ belong to the orphan nuclear receptor subfamily of NR3B (nuclear receptor subfamily 3, group B). The ERRs have been shown to actively modulate estrogenic responses, and to play an essential role in pregnancy, and are implicated in breast cancer progression. Despite intensive efforts, no endogenous ligand has been identified for ERRs so far. The discovery of ligands that bind these orphan receptors will allow the manipulation of this pathway and may lead to novel strategies for the treatment of cancer and other diseases. In this study we show two endogenous ligands of ERRs: 1) estradienolone (ED), a novel endogenous steroid during pregnancy which acts as an inverse agonist of ERRs, 2) cholesterol as an agonist of ERRs. Our recent results show that ED acts as an inverse agonist of ERRα and ERRγ by directly interacting with these receptors, and inhibiting their transcriptional activity. We also demonstrate that ED has strong anti-mitogenic properties. ED inhibits the growth of both estrogen receptor-positive (MCF-7) and estrogen receptor-negative (MDA-MB-231) breast cancer cells in a dose dependent manner, while of displaying a little effect on normal epithelial breast cells. In addition, we show that the effect of cholesterol or lovastatin (a drug known to inhibit cholesterol synthesis) in triple negative breast cancer (MDA-MB 231) requires ERRα. These data suggest that both ED-ERR and cholesterol-ERR interactions may represent novel physiologically relevant ERR pathways in the human.

#1004

Leveraging an expanding tool box for ER+ breast cancer: exploring breast cancer dependencies to support optimal therapeutic strategies.

Wei Zhou, Jane Guan, Georgia Hatzivassiliou, Anneleen Daemen, Marc Hafner, Ciara Metcalfe. _Genentech, Inc., South San Francisco, CA_.

Aromatase inhibitors (AIs), the Estrogen Receptor (ER) modulator tamoxifen and the full ER antagonist fulvestrant each target ER signaling and constitute the backbone of standard of care for ER+ breast cancer. The growing appreciation of liabilities associated with each of these modalities, which may limit their clinical potential, has triggered a wave of activity around next-generation ER therapeutics, and there are currently more than 10 new molecular entities targeting ER being evaluated in Phase 1 clinical trials. In addition to a collection of ER-targeted therapeutics either approved or under evaluation, the emergence of CDK4/6 inhibitors as combination partners has meaningfully contributed to the oncologist's tool box for the treatment of breast cancer, while also adding complexity to the treatment landscape.

Although direct ER antagonists are expected to be superior to AIs in patients with tumors expressing constitutively activating ER mutations (e.g. ER.Y537S), it is currently unclear if a subset of ER wildtype patients may likewise derive superior benefit from direct ER targeting. It is also unclear which ER+ tumors require CDK4/6 inhibitor/ER antagonist combinations for maximal anti-tumor activity, versus those where single-agent ER antagonist treatment may achieve full cell cycle control. To address these key questions, we used a panel of 22 ER+ cell lines to evaluate the relative anti-proliferative and cytotoxic effects of estrogen (E2) withdrawal (modeling AIs), ER modulation, and full ER inhibition, as single agents and in combination with the CDK4/6 inhibitor Palbociclib. We find a number of cell lines in which direct ER antagonism is superior to E2 withdrawal and is associated with E2-independent ER signaling not attributable to ER mutations. The impact of E2 withdrawal on cell cycle control and proliferation is significantly enhanced by concurrent CDK4/6 inhibition, in line with clinical observations, however, direct ER antagonism in combination with CDK4/6 inhibition remains most efficacious.

We are integrating our cell response data with detailed molecular analyses and transcriptional profiling to 1) identify cellular contexts, beyond ER mutations, where direct ER antagonism is superior to targeting E2 synthesis, 2) identify tumors where full cell cycle control and anti-tumor efficacy is achievable through single-agent ER targeting, and 3) better understand the basis for the co-operativity of endocrine suppression with CDK4/6 inhibition. Deconvolution of the ER+ breast cancer response to distinct modes of ER signaling inhibition alone and in combination with CDK4/6 inhibitors has the potential to guide clinical decision-making for personalized patient care.

#1005

Tamoxifen treatment induces the epigenetic changes in breast cancer stem cells.

Kanagarj Palaniyandi,1 Dhanavathy Gnasampandapandian,1 Ilangovan Ramachandran,2 Yuvaraj Sambandam,3 Parthasarathy Chandrakesan4. 1 _SRM Institute of Science and Technology, Kanchipuram, India;_ 2 _University of Madras, Chennai, India;_ 3 _University of Madras, University of Madras, India;_ 4 _University of Oklahoma Health Sciences Center, Oklahoma City, OK_.

Purpose: Breast cancer is diagnosed in younger women population and is the leading cause of cancer-related deaths in India. Tamoxifen (TOM) is one of the most effective and widely used first-line endocrine therapy for ER+ breast cancer. However, breast cancer patients often develop TOM resistance and enhanced with caner stem cell (CSC)-like phenotypes. These TOM-resistance (TR) breast cancer phenotypes are highly aggressive and they do not respond to most of the chemotherapies. It is therefore essential to understand the mechanism that resulted in the development of TR in breast cancer. The current study was designed to investigate the epigenetic changes associated mechanism that induces the development of hormone-resistance in breast CSCs.

Experimental Procedure: In this study, we used ALDEFLOUR assay, flow cytometry, mammosphere culture, western blot, Chromatin-immunoprecipitation, and RT-PCR analyses.

Results: Our study showed that breast CSC-like population possesses higher TR cancer cell phenotypes than non-CSCs. We generated MCF7-derived mammospheres (CSCs) utilizing MammoCult media. However, when treated with TOM (1µM)-we observed an increased number of mammospheres (CSCs) than vehicle control. We found a strong interplay between TR and epigenetic changes, in particular, the changes observed in histone demethylase, which is believed to be one of the crucial links that involved in the development of therapy resistance and enhanced CSC population. Furthermore, TR breast CSCs derived from MCF7 cells display an increased expression of the NOTCH-1 protein. Induced overexpression of NOTCH in MCF7 cells increased the number of mammosphere formation ability than control cells. Finally, overexpression of NOTCH-1 also led to transcriptional up-regulation of cyclin genes A, B, and D.

Conclusion: We observed a crucial interplay between TR and breast CSCs. TR associated epigenetic alteration enhanced the expression of NOTCH1, which resulted in increased mitotic signals in the tumor cells including CSCs. Thus, these new phenotypes of breast CSCs with TR characteristics need more understanding for the development of effective combinatorial and adjuvant therapies.

#1006

Non-linear relationship between estradiol-regulated changes in chromatin architecture and gene expression.

Taylor M. Parker, Duojiao Chen, Poornima Bhat-Nakshatri, Xiaona Chu, Yunlong Liu, Yue Wang, Harikrishna Nakshatri. _Indiana Univ. School of Medicine, Indianapolis, IN_.

Gene expression in estrogen receptor (ER)-positive breast cancer cells is predominantly driven by ER-estradiol (E2) signaling. However, the effects of ER-E2 signaling on the overall chromatin architecture and accessibility to transcription regulators are not well understood. Chromatin architecture is a key determinant to gene expression patterns and therefore a comprehensive notation is essential for the interpretation of the ER cistrome. To investigate the effects of E2 signaling on chromatin architecture, we employed Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), a high-throughput method to map chromatin accessibility via a hyperactive transposase that inserts sequencing tags into open regions. ATAC-seq was performed on MCF-7 cells, an ERα+ breast cancer cell line, treated with E2 or vehicle control for 1 or 3 hours. Results from the ATAC-seq were compared to RNA-seq data of 3-hours E2-treated MCF-7 cells to determine which regions of E2-regulated open or closed chromatin coincide with increased or decreased respective gene expression. Interestingly, we observed greater chromatin accessibility changes in genes repressed by E2 compared to genes induced by E2. Integration of ATAC-seq and RNA-seq data revealed distinct categories of E2-induced chromatin remodeling and downstream gene expression including increased chromatin accessibility, but repression of the corresponding gene, as well as decreased chromatin accessibility, but induction of the corresponding gene. Validation of the chromatin configurations predicted by ATAC-seq of select corresponding genes is currently being performed by ChIP-qPCR against specific histone modifications, such as H3K27Ac, a marker of open chromatin. To mechanistically dissect the distinct modes of E2 chromatin remodelling, we performed motif enrichment analysis of chromatin regions affected by E2. This analysis showed expected enrichment of estrogen response element (ERE), and pioneer factor FOXA1 and GATA3 response elements in E2-inducible genes with enhanced chromatin accessibility after E2-treatment. Regions in which the corresponding mRNA showed decreased transcript levels but displayed open chromatin in their promoter or enhancer region were enriched for the PBX3 motif and the ERE motif, respectively. Our results reveal previously unrecognized modes of E2-mediated restructuring of chromatin architecture and its correlation with transcription dynamics. This study provides grounds for future work to study fundamental relationships between chromatin accessibility and gene regulation in breast cancer under E2 and anti-estrogen treated conditions as well as in metastatic breast cancers with ERα mutations.

#1007

Reactive oxygen species are essential for neurotensin receptors to regulate EGFR and HER2 transactivation.

Terry W. Moody,1 Lingaku Lee,2 Robert T. Jensen2. 1 _NCI, Bethesda, MD;_ 2 _NIDDK, Bethesda, MD_.

Neurotensin (NTS) is a 13 amino acid peptide which is present in many lung cancer cell lines (Moody et al., Life Sci 1985; 36: 1727). High expression of the NTS receptor 1 (R1) is associated with poor survival of non-small cell lung cancer (NSCLC) patients (Alfano et al., Clin Cancer Res 2010; 16: 4401). NTS stimulates but the NTSR1 antagonist SR48692 inhibits the proliferation of NSCLC cells (Moody et al., Life Sci 2014; 100: 25). NTSR1 regulates NSCLC proliferation by transactivation of the EGFR. The cellular signaling mechanisms of the NTSR1 were investigated using NSCLC cells. Using NCI-H838 or A549 cells, addition of NTS or NTS(8-13) but not NTS(1-8) increased tyrosine phosphorylation of P-EGFR, P-HER2 and P-ERK 4-, 3- and 2-fold, respectively. The increase in EGFR and HER2 transactivation caused by NTS addition to NSCLC cells was blocked by SR48692, gefitinib (EGFR tyrosine kinase inhibitor (TKI)), lapatinib (EGFR and HER2 TKI), N-acetyl cysteine ((NAC) is an antioxidant), tiron (reactive oxygen species (ROS) scavenger) or diphenyleneiodonium (DPI is an inhibitor of NOX and DUOX enzymes). NTS increased ROS in NSCLC cells which was inhibited by NAC, tiron or DPI. NTS or NTS(8-13) stimulate NSCLC colony growth but SR48692, gefitinib, lapatinib or DPI inhibit growth. The results indicate that the NTSR1 regulates in a ROS-dependent manner the formation of EGFR homodimers or EGFR-HER2 heterodimers in NSCLC cells.

#1008

S-Equol inhibits breast cancer growth by regulating phosphorylation status of estrogen receptor β.

Kumaraguruparan Ramasamy, Cathy Samayoa, Shaorong Chen, Rong Li, Ratna K. Vadlamudi, Rajeshwar R. Tekmal. _UT Health San Antonio, San Antonio, TX_.

Background: Breast cancer is the most common malignancy in females and second most common cause of cancer related mortality in women. Since 70% of all breast cancers are estrogen receptor-positive (ER+ve), endocrine therapy such as anti-estrogens or aromatase inhibitors, targeting the estrogen receptor (ER) pathway is the most common treatment used for ER+ve breast cancers. However, patients will develop de novo or acquired resistance to therapy, leads to tumor progression, and metastasis. It is well documented that ERβ functions as tumor suppressor in different cancers including breast cancer. We recently showed that phosphorylation status of ERβ is important for its antitumor activity. However, little is known about the role of ERβ phosphorylation status in hormone therapy and resistance; therefore, we investigated whether phosphorylation status of ERβ has role in overcoming hormone therapy resistance in ER+ve breast cancers.

Experimental design: To elucidate the importance of phosphorylation status of ERβ, we used CRISPR-Cas9 system to knockout ERβ in MCF7-Aro (therapy-sensitive) and Letrozole resistant (MCF7aro-LTLT) cells. Several mutant clones were identified for both MCF7-Aro cells and LTLT cells and the depletion of ERβ protein in both cell clones was confirmed by immunoblotting. The parental and knockout cells with or without treatment of S-equol were analyzed for cell proliferation, protein (Western) and RNA (RT-qPCR) analysis.

Results: First we analyzed the cell proliferation in parental (MCF7 Aro and LTLT) and ERβ knock out cells. The proliferation rate is increased in the ERβ knockout cells compared to the parental cells. Treatment with ERβ agonist S-Equol to the parental cells inhibited the cell proliferation whereas in the knock out cells, the effect of S-equol is compromised. RNA-seq analysis of S-equol treated parental cells showed the downregulation of ERβ target genes involved in tumor progression and resistance to hormone therapies. In contrast, compared to parental cells, ERβ knock out cells showed diverse effects to S-equol treatment. RT-qPCR analysis revealed that S-Equol could not modulate the ERβ-target genes in ERβ knock out cells compared to parental cells.

Conclusions: Our findings provide evidence that phosphorylation status of ERβ is important for elucidating its antitumor activity in therapy-resistant cells. The differential effects of S-equol on parental and ERβ knockout cells suggest that the antiproliferative action of S-equol is partly mediated by ERβ. We believe that our ongoing studies may further validate the role of phosphorylation status of ERβ by using both ERβ agonists and phosphorylation-regulating compounds in both therapy sensitive and resistant cells.

#1009

On the role of DEAR1 as a novel ubiquitin ligase for ER alpha and predictor of tamoxifen response in ER positive breast cancer.

Seetharaman Balasenthil, Nanyue Chen, Ann Killary. _UTMDACC, Houston, TX_.

Estrogen receptor positive (ER+) breast cancer accounts for over 70% of breast cancers. Targeted adjuvant therapies aimed at estrogen receptor have changed the natural history of the disease. However, over time many tumors develop resistance mechanisms which result in overall poor outcome. Thus, biomarkers that could predict response to antiestrogens are critically needed and would have major clinical impact. Thus, it is imperative that additional novel mechanisms be discovered not only to understand the basic underlying biology of ER alpha (ERα) signaling in order to identify genetic alterations that deregulate ERα and its role in breast cancer. DEAR1 is a tumor suppressor gene which is mutated, undergoes loss of heterozygosity in breast cancer. Previously we observed a subset of DEAR1 mutations in early onset breast cancers from premenopausal women, therefore we hypothesized that DEAR1 may function in the regulation of ERα signaling and conversely that mutations in DEAR1 (observed in human breast cancers) will abrogate that effect and could play a role in resistance to antiestrogen therapies. To test this hypothesis we studied the effect of DEAR1 expression on ERα. DEAR1 overexpression downregulated ERα expression and stability suggestive that DEAR1 may regulate ER signaling. DEAR1 interacted with ERα by immunoprecipitation and GST pulldown assays. Next we studied whether E3 ligase DEAR1 facilitates the degradation of ERα by promoting the ubiquitination of ERα. Our results indicate that DEAR1 promoted the polyubiquitination of ERα. To assess the effect of DEAR1 on estrogen mediated response, we examined the effect of DEAR1 on ERα-mediated transactivation using ERE-luciferase reporters. Results indicate that DEAR1 significantly inhibits estrogen-mediated transcriptional activity of ERα. We also studied the effect of DEAR1 in antiestrogen resistance using tamoxifen resistant cells. Preliminary results suggest that the presence of DEAR1 promoted the action of antiestrogen in tamoxifen resistant cell line. We have identified a new novel E3 ligase with regulatory effect of ERα signaling and potential implications in antiestrogen resistance.

#1010

Extracellular matrix signaling modulates estrogen receptor activity in breast cancer.

Josh W. DiGiacomo,1 Inês Godet,1 Michael Trautmann Rodriguez,1 I Chae Ye,2 Daniele M. Gilkes2. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _Johns Hopkins University School of Medicine, Baltimore, MD_.

The tumor microenvironment plays a crucial role in cancer progression. In breast cancer, the extracellular matrix (ECM) is especially important as it contributes to mammographic breast density. Women with higher mammographic breast densities are at a greater risk for the development of breast cancer. Despite this, much knowledge of the ECM's effects on breast cancer progression remains elusive. To better understand how ECM signaling affects breast cancer, we utilize fibroblast-deposited, decellularized ECM scaffolds. Scaffolds are produced for in vitro use by culturing a confluent layer of fibroblast cells over six days followed by decellularization with a detergent solution. RNA sequencing analysis of MCF7 human breast cancer cells cultured on ECM scaffolds versus uncoated dishes revealed differential expression of hundreds of genes. Most notably, genes within hallmark sets for early and late responses to estrogen were significantly enriched when cells were cultured on ECM. We confirm the expression of estrogen-responsive genes in breast cancer cells cultured on ECM even in the complete absence of estrogen, and also note that the estrogen receptor (ER) itself is downregulated at both the RNA and protein-level, consistent with results of estrogen stimulation. Treatment of breast cancer cells with ER inhibitors hydroxytamoxifen and fulvestrant reduces ECM-induced expression of estrogen-responsive genes. Likewise, culturing breast cancer cells on ECM enhances phosphorylation of Erk. Inhibition of phospho-Erk also reduces the expression of the same estrogen-responsive genes in cells cultured on ECM. Through luciferase, conditioned media, and estrogen-starvation assays, we demonstrate that ECM signaling activates estrogen receptor signaling in an estrogen-independent, nonclassical mechanism. In addition, we show that ER positive cells cultured on ECM are more resistant to ER-targeted therapeutics, but not other chemotherapeutic agents like paclitaxel or doxorubicin. Ultimately, we describe a pathway in which ECM can induce estrogen-independent activation of the ER as well as resistance to estrogen-targeted therapies in breast cancer. Clinical research into the effects of mammographic breast density beyond breast cancer predisposition is sparse, and this work provides incentive for investigators to better understand the relationship between breast fibrosis and clinical outcome.

#1011

Prolactin-induced EDD E3 ubiquitin ligase regulates pro/anti-apoptotic gene expression, and stimulates TORC1 signaling and drug resistance in breast cancer cells.

Tyler MacDonald, Lynn N. Thomas, Emily Daze, Penelope J. Barnes, Catherine K. Too. _Dalhousie University, Halifax, Nova Scotia, Canada_.

EDD E3 ubiquitin ligase or UBR5, first identified as a progestin-inducible gene in human T47D breast cancer cells, mediates DNA damage signal transduction and is a key regulator of various cellular processes in cancer. We identified EDD as a prolactin (PRL)-inducible gene and as a novel partner of the TORC1-associated α4 phosphoprotein-PP2Ac phosphatase complex that regulates the initiation of translation and cell cycle progression. EDD targets PP2Ac for proteasomal degradation. Our aim was to further study the role of EDD in breast cancer cell survival, PRL-stimulated TORC1 signaling and drug resistance. We showed that EDD gene expression varied in breast cancer cell lines of different subtypes, such as luminal-A (MCF7, T47D), HER2-enriched (SkBR3), triple-negative claudin-low (MDA-MB-231) and basal-like (MDA-MB-436, -468). In high EDD-expressing MCF7 and T47D cells, EDD knockdown with siRNA (siEDD) significantly decreased cell viability, increased apoptosis, and arrested cells in the G2-phase of the cell cycle. Specifically, MCF-7 cells in late-stage apoptosis increased significantly (P=0.043, n=5) from a range of 2 - 8% in control cells to 20 - 35 % in siEDD-transfected cells. Although T47D cells were less affected by EDD loss, cells in early-stage apoptosis increased from 4 - 5% in control cells to 13 - 20 % in siEDD-transfected cells. SiEDD-induced apoptosis in MCF7 cells correlated with a significant increase in pro-apoptotic Bim and Bak mRNAs/proteins, leading to increased cleavage of caspase-7 at 6 - 48 h and consequently, cleavage of caspase substrate PARP-1 at 24 - 48 h, as compared to control cells. EDD loss in MCF7 cells decreased PRL-induced phosphorylation of eukaryotic initiation factor 4E-binding protein (4EBP1), a mediator of TORC1 signaling. Specifically, doublet α- and β-bands of 4EBP1 were detected in control cell lysates. A hyper-phosphorylated γ-band appeared in lysates of cells treated with PRL for 1 h but this γ-band disappeared in cells with EDD loss, implicating PRL-induced EDD enhancement of TORC1 signaling. Lastly, in low EDD-expressing MDA-MB-436 cells, EDD gain following pCMV-Tag2.EDD transfection increased cell resistance to chemotherapeutic drugs cisplatin and doxorubicin, and TORC1/TORC2 inhibitors rapamycin and INK128, as compared to controls. In contrast, EDD loss in MCF7 cells increased cell sensitivity to these drugs and to selective estrogen receptor modulator tamoxifen. In summary, PRL-induced EDD promotes breast cancer cell survival, enhances 4EBP1 phosphorylation, and increases drug resistance in vitro. These findings implicate EDD as a potential therapeutic target and support PRL receptor blockade as an additional therapy for breast tumours that express this receptor, including the aggressive HER2+ and triple-negative subtypes.

#1012

Selective estrogen receptor downregulators and immune checkpoint inhibitors in breast cancer immunotherapy.

Diana C. Márquez-Garban, Gang Deng, Begonya Comin-Anduix, Alejandro J. Garcia, Emelyine Diers, Gaoyuan Ma, Nalo Hamilton, Michael E. Jung, Richard J. Pietras. _UCLA, Los Angeles, CA_.

Breast cancers (BC) with expression of estrogen receptor-alpha (ERα) occur in more than 70% of newly-diagnosed patients in the U.S. Endocrine therapy with antiestrogens or aromatase inhibitors is an important intervention for BCs that express ERα, and it remains one of the most effective targeted treatments. However, substantial numbers of patients with localized disease, and almost all patients with metastatic BC, become resistant to current endocrine therapies. ERα is present in most resistant BCs, and in many of these its activity continues to regulate BC growth. Fulvestrant represents a class of ERα antagonists that elicit selective ER downregulation (SERDs), an action that helps overcome several resistance mechanisms. Unfortunately, full antitumor efficacy of fulvestrant is limited by its poor bioavailability in clinic. We have designed and tested a new generation of steroid-like SERDs. Using ERα-positive BC cells in vitro, we find that these compounds suppress ERα protein levels with efficacy similar to fulvestrant. Moreover, these new SERDs markedly inhibit ERα-positive BC cell proliferation in vitro even in the presence of estradiol-17β. In vivo, SERD-128 significantly inhibits tumor growth in MCF-7 xenograft models in a dose-dependent manner (P<0.001). Further, our findings show that new SERDs as well as fulvestrant also interact with immune cells expressing ER such as myeloid-derived suppressor cells (MDSC), dendritic cells and T-lymphocytes. Importantly, MDSCs act to protect tumors from immune recognition and elimination in vivo. Since a fraction of triple-negative breast cancers (TNBC; ERα-/PR-/HER2-) but not other BC subtypes respond to immune checkpoint inihbitors (ICIs), we assessed the antitumor effects of SERDs in murine TNBCs in immune-competent, syngeneic mouse models. Notably, SERD treatment induced a blockade of MDSC populations in tumors and rendered TNBCs in vivo more susceptible to ICIs, thereby leading to enhanced tumor killing. Since monotherapy with checkpoint inhibitors has not been effective for most TNBCs, combination therapies with SERDs that enhance immune recognition may increase immunotherapy responses in TNBC and improve patient survival. In addition, SERDs combined with ICIs may potentially benefit patients with other BC subtypes that are unresponsive to current endocrine treatment strategies. [Funded by Tower Cancer Research Foundation-Jessica M. Berman Fund, NCI U54 CA-14393 and UCLA Innovation Fund].

#1013

Role of mitochondria and glutamine metabolism in therapy resistance: Combined targeting of estrogen receptor alpha and exportin 1 in endocrine resistant breast cancers.

Eylem Kulkoyluoglu Cotul,1 Brandi Patrice Smith,1 Hua Chang,2 Yosef Landesman,2 Kevin Duong,1 Zeynep Madak Erdogan1. 1 _University of Illinois Urbana-Champaign, Urbana, IL;_ 2 _Karyopharm Therapeutics, MA_.

Majority of breast cancer specific deaths in women with ERα (+) tumor occur due to metastases that are resistant to endocrine therapy. There is a critical need for novel therapeutic approaches to prevent or delay recurrence of ERα (+) tumors. The objective of this study was to elucidate the role of mitochondrial pathways that are activated in the presence of single therapies and undermine therapy effectiveness in ER (+) breast cancers. In our previous studies, we identified Exportin 1 (XPO1), a nuclear export protein, as an important player in endocrine resistance progression. Selinexor (SXR), an XPO1 antagonist, has been evaluated in multiple later stage clinical trials in patients with relapsed and /or refractory hematological and solid tumor malignancies. Using a combination of transcriptomics, kinase arrays, metabolomics and metabolic flux experiments, we identified glutamine metabolism pathways to be rewired during endocrine resistance. In limited media conditions mimicking nutrient deprived tumor microenvironment, endocrine resistant cells were more dependent on mitochondria for energy production. Their glucose and fatty acid dependency decreased in the presence of SXR and cells were more dependent on glutamine. The effect of glutamine was dependent on conversion of the glutamine to glutamate and mitochondrial complex 1 activity. In order to examine metabolites that might result in the observed phenotype we performed GC/MS whole metabolite profiling and identified aminoacid metabolism pathways to be upregulated when cells were treated with SXR. We demonstrated that combined targeting of XPO1 and ERα rewires metabolic pathways and shuts down both glycolytic and mitochondrial pathways that would eventually lead to autophagy. Remodeling metabolic pathways to regenerate new vulnerabilities in endocrine-resistant breast tumors is novel, and given the need for better strategies for improving therapy response of relapsed ERα (+) tumors, our findings show great promise for uncovering the role ERα-XPO1 crosstalk plays in reducing cancer recurrences.

#1014

Characterization of breast cancer cells resistant to letrozole and palbociclib combination therapy and the potential of dinaciclib to treat such resistance.

Armina Kazi,1 Emily Shukdinas,1 Saranya Chumsri,2 Gauri J. Sabnis3. 1 _Loyola University Maryland, Baltimore, MD;_ 2 _Mayo Clinic Cancer Center, Jacksonville, FL;_ 3 _West Coast University, Los Angeles, CA_.

Treatment of estrogen receptor positive (ER+) breast cancer have included single-agent endocrine therapy, such as with aromatase inhibitors (AIs) and, more recently, AIs in combination with the cyclin-dependent kinase 4/6 inhibitor palbociclib. While combination therapy is effective, drug resistance is a well-known phenomenon and breast cancer cells can acquire resistance to aromatase inhibitor and palbociclib individually. In anticipation of patient who may become resistant to combination therapy with aromatase inhibitor (letrozole) and palbociclib, this current study has 1) developed MCF7Ca cells resistant to both letrozole and palbociclib (MCF7Calet+palb); 2) characterized them in comparison to drug naïve (MCF7Ca) and letrozole alone-resistant (LTLTCa) cells; and 3) explored potential treatments. Characterization of MCF7Calet+palb cells by phase contrast microscopy, mammosphere assays, and invasion assays, indicate that they have more basal/mesenchymal morphology, produce more mammospheres (an indicator of cancer stem cells; 150±10 vs. 80±10 in MCF7Ca and 123± 8 in LTLTCa cells), and are more invasive (1200±50 cells vs. 791±15 in MCF7Ca cells). The more aggressive phenotype of MC7Calet+palb correlated with changes in protein expression. Compared to the other cell lines, MCF7Calet+palb expressed significantly lower fold expression levels of cell cycle cyclin D1 (0.2±0.1), phosphorylated CDK9 (0.5±0.1), and phosphoRb and total Rb (0.4±0.2), but higher levels of phosphoCDK1 and CDK2 (1.4±0.1 and 3.1±0.6). Regarding growth pathway proteins, MCF7Calet+palb comparably had increased HER2 and phosphorylated Akt (6.2±1.3 and 3.3±1.2, resp.) as LTLTCa vs. MCF7Ca cells, but had even more reduced levels of ERα (0.04 vs. 0.1±0.4 in LTLTCa and 1±0.01 in MCF7Ca). Cancer stem cell marker breast cancer resistance protein (BCRP) was increased further in MCF7Calet+palb cells (2.11-fold). Based on these results, the effect of dinaciclib, a broader range CDK inhibitor, was studied. In MTT assays, dinaciclib was shown to be effective in inhibiting cell viability in all three cell types. Dinaclicib's IC50s were 1.4 nM (MC7Ca) 9.5 nM (LTLTCa) and 12.4 nM in MCF7Calet+palb cells, while palbociclib IC50s were 1.5 μM, 11.2 μM, and 118 μM, respectively. Dinaciclib also significantly reduced mammosphere formation 0.01-fold and cell invasion/migration by 0.3-fold vs. untreated. Lastly, preliminary exome sequencing data suggested highly mutated FGFRL1, ARL11, and FCGBP genes, among others, when MCF7Calet+palb cells were compared to MCF7Ca cells. Overall, these results suggest that acquired resistance to combination therapy with letrozole and palbociclib induces genetic, molecular, and phenotypic changes in breast cancer cells, and that dinaciclib may be an effective treatment to either treat and/or prevent this resistance.

#1015

Molecular dissection of androgen receptor signaling in prostatic endothelial, epithelial, and stromal cells.

Nelson T. Gross, Jianming Wang, Gary Smith, Yue Wu. _Roswell Park Cancer Inst., Buffalo, NY_.

Introduction and objectives: Androgen receptor (AR) is involved in development prostate cancer (CaP), and is a primary target for treatment of CaP. Androgen deprivation therapy (ADT) inhibits AR signaling by reducing AR ligands and/or blocking AR-ligand binding. ADT induces apoptosis of both endothelial cells and epithelial cells of human prostate, with apoptosis of endothelial cells preceding the apoptosis of epithelial cells. Further, the AR response to stimulation by androgen also differs between the 2 cell types. The current understanding of AR signaling was gained predominantly using CaP cell lines and samples prepared from whole tissue specimens. The present study sought to delineate AR signaling specifically in endothelial, epithelial, and stromal cells isolated from fresh clinical prostate tissue specimens and primary xenografts of human prostate tissue. Our results reveal the potentially different roles of AR signaling in different human prostate cell compartments, how AR regulates normal differentiated cell functions, and the biological consequences of ADT.

Methods: Prostate tissue remnants were transplanted to male nude mice that were castrated and implanted with a sustained delivery device to maintain testosterone at human plasma levels. Endothelial and epithelial cells of both fresh prostate tissue and prostate tissue xenografts were isolated sequentially using magnetic beads conjugated with an antibody specific to epithelial cell or endothelial cell surface markers; the remaining cell fraction was defined as stromal cells. Transcriptomes were obtained using RNASeq, and were analyzed for differential expression of AR-regulated genes, AR co-regulators, and androgen metabolism enzymes.

Results: Antibody-mediated cell type-specific enrichment isolated effectively the 3 cell types from the prostate from 16 patients. Among 1263 AR-regulated genes, 399, 313, and 223 were predominantly expressed in epithelial, endothelial or stromal cells, respectively. Among 179 AR co-regulators, 26, 26, and 18 were expressed predominantly in epithelial, endothelial or stromal cells, respectively. Among 128 potential androgen metabolic enzyme genes, 42, 15, and 12 were expressed predominantly in epithelial, endothelial or stromal cells, respectively.

Conclusions: Differences in expression of genes associated with AR-mediated trans-regulation were apparent between the different cell types of prostate, whereas, the difference in expression of androgen metabolism genes was less striking. Therefore, the organ level outcome of ADT can only be understood by determining the response of the different cell types in CaP tissue.

#1016

The androgen receptor splice variant AR-V7 associates with the glucocorticoid receptor following androgen blockade.

Eric Bueter, David Hosfield, Erin McAuley, Phillip Selman, Geoffrey Greene, Suzanne D. Conzen, Russell Z. Szmulewitz. _University of Chicago, Chicago, IL_.

Expression of the constitutively active androgen receptor splice variant AR-V7 is associated with resistance to anti-androgen therapies and worse clinical outcomes in prostate cancer. Dimerization is a requirement for AR-V7 transcriptional activity, but whether that occurs through homodimerization or heterodimerization remains poorly understood. Because NR3C-family nuclear hormone receptors rely on a highly conserved D-box motif within the DNA-binding domain to mediate dimerization, we hypothesized that AR-V7 can heterodimerize with other NR3C-family nuclear hormone receptors, with a particular interest in the glucocorticoid receptor (GR). Like AR-V7, GR is upregulated in prostate cancer following androgen blockade, regulates canonical AR target genes, and is associated with resistance to anti-androgen therapies. Using NanoBRET technology in HEK293 cells, we found that AR-V7 interacts with liganded NR3C-family nuclear hormone receptors, including the full-length androgen receptor, the progesterone receptor, the mineralocorticoid receptor, and, most notably, GR. Using co-immunoprecipitation and proximity ligation assays, we demonstrate that AR-V7 associates with GR following androgen blockade in both the VCaP cell line and the CWR-22RV1 cell line, two models of prostate cancer that express endogenous AR-V7 and GR. Preliminary ChIP-Seq experiments in these cell lines suggest AR-V7 and GR chromatin occupancy at the same genomic sites in a ligand-dependent manner. Taken altogether, this data suggests AR-V7 can utilize GR as an alternative binding partner to initiate heterodimerization and transcriptional activation.

#1017

Novel inhibitors of AR-v7 nuclear import: new therapeutic opportunities for CRPC.

Seaho Kim,1 Mohd Azrin Jamalruddin,1 Eiman Mukhtar,1 Michael Miller,2 Leigh Baxt,2 Stacia Kargman,2 Andrew Stamford,2 Peter Meinke,2 Paraskevi Giannakakou1. 1 _Weill Cornell Medicine, New York, NY;_ 2 _Tri-Institutional Therapeutics Discovery Institute, New York, NY_.

Reactivation of androgen receptor (AR) signaling by active splice variants (AR-Vs) is one of key drivers of castration resistant prostate cancer (CRPC). AR-v7 is the most prevalent AR-V and its expression has been clinically associated with poor overall survival, resistance to the AR inhibitors enzalutamide and abiraterone, as well as taxane resistance. Given that treatment with AR inhibitors and taxanes are the only effective therapeutic modalities in CRPC, development of specific AR-v7 inhibitors is urgently needed. Mechanistically, AR-v7 re-activates AR signaling by being constitutively active in the nucleus. While taxane chemotherapy inhibits the nuclear import of AR which is significantly associated with clinical outcomes in CRPC, it has no effect on AR-v7 nuclear localization and activity. Mechanistically, AR-v7 lacks the microtubule-binding domain and—unlike AR—does not utilize the canonical importin-α/β pathway, or RanGTP for nuclear import. Using wheat germ agglutinin to block active protein nuclear uptake resulted in AR-v7 cytoplasmic sequestration, indicating a requirement for an alternative transport receptor. Further, mutation of AR-v7 dimerization domain (D-box) led to its cytoplasmic sequestration, indicating that the D-box is also required for nuclear import. As inhibition of AR nuclear import is a clinically validated therapeutic strategy, we developed a novel drug discovery platform to identify compounds that specifically inhibit AR-v7 nuclear import. Using cells stably expressing inducible AR-v7 in conjunction with an enzyme complementation assay we tested 166,000 compounds by high throughput screening (HTS). The robust HTS performance (Z>0.8) together with subsequent counter screens including confirmation and compound titration, cell toxicity, a tertiary imaging based screen, led to identification of lead compounds that inhibit AR-v7 nuclear import. The lead compounds share structural features, across two main chemotypes, which are amenable to structure-activity relationship studies to identify the most desirable compound for in vivo studies. Using newly synthesized compounds from each of the two chemotypes, we showed specific dose-dependent inhibition of AR-v7 nuclear import. Currently, we are testing these compounds, on inhibition of AR-v7 transcriptional activity across several cell models including enzalutamide-resistant cells as well as inhibition of tumor growth in AR-v7 xenograft models. In parallel we are testing the lead compound for potential direct binding to the AR-v7 dimerization domain or to candidate alternative nuclear transport receptors. Further development of our lead small molecules will yield novel chemotypes, with desirable pharmacological properties that target the unique AR-v7 nuclear import pathway and can be clinically combined with existing AR therapies.

#1018

GnRH antagonists have direct inhibitory effects on castration-resistant prostate cancer via intracrine androgen and AR-V7 expression.

Vito Cucchiara,1 Joy C. Yang,1 Chengfei Liu,1 Hans Adomat,2 Emma Guns,2 Martin E. Gleave,2 Allen C. Gao,1 Christopher P. Evans1. 1 _UC Davis Medical Ctr., Sacramento, CA;_ 2 _Vancouver Prostate Centre, Vancouver, British Columbia, Canada_.

Hormone therapy is currently the mainstay in the management of locally advanced and metastatic prostate cancer (PCa). Degarelix (Firmagon, Deg), a GnRH receptor antagonist demonstrated some advantages over the LHRH agonist Leuprolide (Leup) by avoiding "testosterone flare" and lower FSH levels. We compared the effect of Deg and Leup on prostate cancer (PC) cell progression in vitro and in vivo. GnRHR2 was readily detectable in PC cells. AR transcriptional activity reported by PSA-Luc assay was modulated by both Leup and Deg. In LNCaP and C4-2B MDVR cells, 20µM Deg significantly reduced the cell viability (p<0.01). GnRH-antagonist (alone or in combination with AA or Enza) counteracted the transactivation activity of AR by reducing AR-FL and AR variants at the protein level. In C4-2BMDVR cells, Deg reduced AR-V7 protein expression by 26 to 40% alone or combined with AA and Enza compared to control. Leup, however, enhanced variant expression. Deg reduced AR-variant levels from 17 to 41% in monotherapy or combinations compared to control. In mice, Leup slightly suppressed tumor growth compared to controls (p>0.05). However, tumors in Deg-treated group were 1.5-fold smaller than Leup-treated group but 1.7-fold bigger than surgical castration-group. Ki67 IHC staining confirmed the difference in tumor proliferation among groups. Measurements of intratumoral steroids by LC-MS from tumors, serum samples or cell pellets confirmed that Deg decreased the levels of testosterone and steroidogenesis pathway intermediates, comparable to surgical castration; while Leup had no inhibitory effect. Our results suggest a selective mechanism of action of Deg against AR-variants. The present study provides additional molecular insights regarding the mechanism of Deg compared to GnRH agonist therapy which may have clinical implications.

#1019

**P300/CBP inhibitor CCS1477 targets 22Rv1 prostate tumor AR and c-myc gene expression** in vivo **.**

Paul Elvin,1 Neil Pegg,2 Simone Daminelli,3 Izabela Eden,4 Barbara Young,5 Amy Prosser,5 Jenny Worthington,6 Nigel Brooks2. 1 _Oncognition Ltd, Market Drayton, United Kingdom;_ 2 _CellCentric Ltd, Cambridge, United Kingdom;_ 3 _Fios Genomics Ltd, Edinburgh, United Kingdom;_ 4 _Hologic Ltd, Manchester, United Kingdom;_ 5 _Sygnature Discovery, Nottingham, United Kingdom;_ 6 _Axis Bioservices, Coleraine, United Kingdom_.

Background: Histone acetyl transferases E1A binding protein (p300) and CREB binding protein (CBP) are known co-activators of several key transcription factors that contribute to tumor progression including HIF1a, BRCA-1, p53, c-myc and androgen receptor (AR). A large proportion of AR regulated gene expression has been shown to be dependent on p300 either through direct regulation of AR interaction with promoters of AR regulated genes or subsequent histone modification events. Both p300 and CBP are highly expressed in advanced prostate cancer and androgen deprivation leads to upregulation of both proteins. CCS1477 is a potent, selective inhibitor of the bromodomain in CBP/p300 that has been shown to inhibit prostate tumor cell proliferation in vitro and tumor growth in vivo.

Methods: 22Rv1 prostate tumor cells that express both AR and AR variants were transplanted in nude mice. Established tumors were treated with CCS1477, 20mg per kg, once daily p.o. for 28 days. CCS1477 treatment resulted in virtually complete inhibition of tumor growth that was maintained up to 24 days after cessation of treatment by which time tumor recurrence was evident. Tumors were excised from vehicle and CCS1477 treated animals at day 7, day 28, and day 52; mRNA was isolated and gene expression analysis was carried using Affymetrix Clarion D microarrays. Differential gene expression based on fold change (FC) >1.5 and FDR-adjusted p-value <0.05 identified a number of genes with significant FC in CCS1477 vs vehicle treated control tumors.

Results: Although ~1.5 fold downregulation of AR was maintained from day 7 to day 52, downregulation of AR target genes ETS2, TMPRSS2 and NKX3.1 recovered after treatment cessation. Similarly, expression of c-myc was significantly reduced at day 7 (-2.7 FC) and recovered by day 28. Of note, among the top downregulated genes were CIART and BHLHE40 circadian clock regulated genes that provide negative feedback loops that in conjunction with downregulation of AR and c-myc would disrupt circadian gene regulation. VEGFA mRNA that was downregulated >1.5 fold at all time points, together with c-myc and p300 are all under circadian regulation. Expression of the histone demethylase KDM3A was reduced >1.5 fold at all time points. KDM3A is known to function as an AR coactivator of key AR target genes including NKX3.1 and c-myc.

Conclusions: Gene expression analysis of CCS1477 treated prostate tumors suggests an underlying mechanism involving the inhibition of key drivers of prostate cancer progression including AR and c-myc and a network of interacting pathways.

#1020

p300 and CBP targeting in castration therapy resistant prostate cancer.

Tobias Furlan, Natalie Sampson, Frédéric R. Santer, Zoran Culig. _Innsbruck Medical Univ., Innsbruck, Austria_.

p300 and CBP interact with transcription factors and other proteins via their histone acetyltransferase domain (HAT) and a bromodomain (Bd). In prostate cancer, p300 and CBP act as oncogenes and are commonly upregulated. p300 and CBP are valid targets for therapy due to their function as androgen receptor (AR) coactivators and involvement in castration resistance. Revealing downstream pathways and their function in cancer cell proliferation and resistance should further identify potential novel therapeutic targets. The effect of the p300/CBP HAT inhibitor C646 and the Bd inhibitor ICBP-112 on viability was tested. To this end several human PCa cell lines with different AR expression levels and resistance status were used. The resistant cells were derived from DUCaP, LAPC4 and LNCaP cells chronically treated with Enzalutamide (DUCaP ENZA, LAPC4 ENZA and LNCaP ABL ENZA). Resistance was verified by viability assay. Regulation of AR downstream targets was studied by qRT-PCR and Western blot. Gene expression in the different DUCaP and LNCaP sublines after inhibitor treatment was analyzed with RNAseq. Both inhibitors exhibited IC50 values on viability at micromolar concentrations with a significantly lesser effect on androgen-insensitive PC3 cells compared to AR sensitive cells. Significantly increased sensitivity to inhibitors was observed in enzalutamide-resistant DUCaP ENZA, LAPC4 ENZA and LNCaP ABL ENZA cell lines relative to parental cells: respectively 1.83- (*), 2- (*) and 2.7-fold (*) for C646 and not significant, 1.8- (p<0.1) and 1.56-fold (*) for ICBP-112. Moreover, ICBP-112 treatment of DUCaP cells significantly reduced expression of the AR target genes FKBP5, PSA and TMPRSS2 0.6- (**), 0.57- (**) and 0.37-fold (***) respectively. Similar results were obtained with DUCaP ENZA cells 0.46- (*), 0.45- (*) and 0.57-fold (ns) for FKBP5, PSA and TMPRSS2, respectively. Whilst less pronounced, this regulation was also observed at the protein level. Similar results were obtained with C646. RNAseq results suggest multiple changes in sublines after chronic enzalutamide treatment in DUCaP and LNCaP cells. Inhibition of p300 and CBP reduced androgen-, myc- and parts of an EMT-signature, which are likely involved in the development of enzalutamide resistance. Taken together, C646 and ICBP-112 are effective inhibitors of AR dependent PCa. Furthermore, differences between cell lines suggest that the AR background and other factors influence the inhibitor effectivity. A significantly increased effect of the HAT and Bd inhibitors in CRPC suggests an important role for p300/CBP in CRPC.

#1021

CD24 dependent inactivation of mutant p53 in metastatic castration resistant prostate cancer.

Baozhu Yi,1 Xin Li,1 Zhifang Xu,1 Sejong Bae,1 Shi Wei,1 Runhua Liu,1 James Lillard,2 Lizhong Wang1. 1 _University of Alabama at Birmingham, Birmingham, AL;_ 2 _Morehouse School of Medicine, Birmingham, AL_.

CD24 is not expressed in normal prostate epithelial cells but is overexpressed in high-stage prostate cancer (PC) and metastases, especially in African American (AA) men, and serves as a marker for poor prognosis. The oncogenic function of CD24 has been demonstrated by ectopic and/or inducible expression, targeted mutations, gene silencing, and antibody blockade. Recently, we found CD24-dependent inactivation of mutant p53 in PC cells. CD24 silencing prevents functional inactivation of mutant p53, a gene frequently mutated in about 30% of PCs. TP53 mutation or loss of function promotes the invasion and metastasis of castration-resistant PC (CRPC) cells. Mutant p53 expression is associated with an increased risk of disease-specific death as well as the development of distant metastasis. In the present study, we assesed the protein expression levels of CD24 and mutant p53 by immunohistochemical staining in 150 formalin-fixed paraffin-embedded CRPC tissue specimens from 78 White and 78 AA patients. The frequency of CD24 expression showed a significant difference (p=0.023) between CRPCs of 66.7% AAs and 48.7% Whites. Likewise, the frequency of mutant p53 expression was significantly higher (p=0.014) in CRPCs of 38.5% AAs compared with 20.5% Whites. These results suggest that overexpression of CD24 and mutant p53 is a frequent event in AA CRPCs. Furthmore, we created CD24-/- p53-null PC3 CRPC cell models by use of CRISPR/Cas9 technology. Next, we transfected the TP53R273H or TP53R175H into CD24+/+ and CD24-/- PC3-luc cells to create TP53R273H or TP53R175H CD24+/+ and CD24-/- PC3-luc cell models. Uisng these cell models, we found that CD24 promotes PC3 cell migration and invasion in TP53R273H cells but not in TP53R175H PC3 cells. The p53 target genes, including p21/CDKN1A, VDR, and TP53INP1, were downregulated after CD24 overexpression in TP53R273H cells but not in TP53R175H PC3 cells, supporting the effect of CD24-dependent inactivation of mutant p53R273H. To address the effect of CD24-dependent inactivation of mutant p53 on CRPC cell colonization in vivo, we intravenously injected NOD-SCID (NSG) male mice with TP53R273H or TP53R175H transfected CD24+/+ and CD24-/- PC3-luc cells. After implantation, the numbers and burden of lung and liver metastases were higher in the mice with TP53R273H CD24+/+ cells compared with TP53R273H CD24-/- cells, but no difference was found between the mice with TP53R175H CD24+/+ cells and TP53R175H CD24-/- cells, further supporting the effect of CD24-dependent inactivation of mutant p53R273H.

#1022

The role of dopamine signaling in prostate cancer disparities.

Isaiah R. Pickett,1 Ava M. Boston,1 Marwah M. Almathkour,1 Camille Ragin,2 Mehmet A. Bilen,3 Adeboye O. Osunkoya,4 Bekir Cinar5. 1 _Clark Atlanta University, Atlanta, GA;_ 2 _Fox Chase Cancer Center, Philadelphia, PA;_ 3 _Emory University Winship Cancer Institute, Atlanta, GA;_ 4 _Emory University Hospital, Atlanta, GA;_ 5 _Winship Cancer Institute, Clark Atlanta University, Atlanta, GA_.

Prostate cancer is the second leading cause of cancer-related deaths among men in western countries. Majority of prostate tumors often evolves as metastatic castration-resistant prostate cancer (CRPC) following hormonal therapy. Metastatic prostate cancer is the major cause of mortalities from this disease. African-American (AA) men have the highest incidence and mortality rate compared to any other race such as Caucasian American (CA). Despite recent advances, the cellular and molecular mechanisms that contribute to this disparity are unknown, obstructing the development of an effective therapy for this disease. Genetic and epigenetic studies have indicated that the biology of prostate cancer in AA men is substantially different from CA. Here, we investigate the dopamine (DA) signaling to better understand the mechanism of prostate cancer disparity. Our analysis of the prostate cancer public data sets showed that the dopamine receptor (DR) genes were amplified in up to 18% prostate cancer clinical cases. We have demonstrated that the levels of DR transcripts were significantly higher in the metastatic prostate cancer cell lines with African genetic ancestry than CA counters. We also demonstrate that the efficacy of pimozide, a potent DRD inhibitor and an FDA-approved antipsychotic, on AA cells was significantly higher than CA cells. In addition, we have found that L-dopa, an immediate precursor of DA differentially regulates the growth of AA and CA prostate cancer cells in a dose dependent manner. We also found that carbidopa, a potent inhibitor of the dopa decarboxylase, suppressed prostate cancer cell growth by L-dopa and showed that carbidopa re-sensitized CRPC cell line to enzalutamide, a potent inhibitor of androgen receptor (AR) signaling. These observations suggest that DA/DR signaling plays a critical role in prostate cancer disparity, making the DA/DR-AR axis a viable therapeutic target to reduce disparities in cancer mortality.

#1023

Combining all-trans retinoic acid therapy with androgen receptor N-terminal domain inhibitors for the treatment of castration-resistant prostate cancer.

Jacky K. Leung, Marianne D. Sadar. _British Columbia Cancer Agency, Vancouver, British Columbia, Canada_.

Androgen receptor (AR) signaling plays an essential role in all stages of prostate cancer. Androgen-deprivation therapy is generally effective for advanced prostate cancer until progression to lethal metastatic castration-resistant prostate cancer (mCRPC). Most CRPC continues to be driven by AR signaling. AR transcriptional activity requires a functional N-terminal domain (NTD). Transactivation of AR is mediated by ligand-independent activation by cross-talk with signal transduction pathways targeting the AR NTD, gain-of-function mutations in AR ligand-binding domain (LBD), or expression of truncated AR splice variants that lack LBD (e.g., AR-V7). The intrinsically disordered AR NTD is essential for its transcriptional activity. It harbors six putative binding sites for Pin1, a proline isomerase that regulates protein conformation at specific phosphorylated-Ser/Thr-Pro motifs. All-trans retinoic acid (ATRA) is a validated and potent Pin1 inhibitor. Since conformational changes within the AR NTD are required for transactivation, perturbation of its structure may be a promising approach to block its activity. The purpose of this study was to assess a therapy that combined ATRA with antagonists of AR NTD, ralaniten (EPI-002) and its analogues. We hypothesized that targeting Pin1 activity with ATRA would disrupt the AR NTD and enhance inhibition by EPI compounds which bind to Tau-5 in the NTD. Using reporter gene assays, proliferation assays, and cell cycle analysis by flow cytometry, we tested ATRA in isolation and in combination with EPI in androgen-sensitive (LNCaP) and androgen-independent (LN95) prostate cancer cell lines. We found that treatment with ATRA decreased the transcriptional activity of AR and androgen-induced expression of PSA. ATRA also attenuated the transcriptional activity of AR-V7 and androgen-independent growth of LN95 cells expressing both full-length AR and AR-V7. Co-immunoprecipitation studies confirmed interactions between Pin1 and specific regions of AR NTD. In combination, ATRA had synergistic interaction with EPI compounds and lowered the effective inhibitory concentrations for blocking AR transcriptional activity. Furthermore, combinations decreased cell cycle progression of LN95 cells through S-phase, leading to accumulation of cells in G1 and induction of senescence. These preclinical findings showed that ATRA enhanced the potency of AR NTD inhibitors and support a novel therapeutic strategy for CRPC. Future studies will determine the in vivo antitumor effect of ATRA in combination with EPI compounds on the growth of CRPC xenografts.

#1024

Retinoid X receptors enhance androgen signaling and promote cell cycle progression in prostate cancer.

Lucas J. Brand, Christopher McNair, Jennifer McCann, Karen E. Knudsen. _Thomas Jefferson University, Philadelphia, PA_.

Androgen receptor (AR) is a master transcriptional regulator of the prostate, and a key driver of disease progression in both early- and late-stage prostate cancer (PCa). While initially effective, tumors eventually circumvent AR-targeted therapies, leading to the onset of lethal castration-resistant PCa (CRPC), for which no curative interventions currently exist. However, the majority of CRPC tumors continue to express AR and rely on AR signaling for survival and proliferation, indicating that the AR signaling axis remains a viable target in late-stage disease. Previous work by our lab identified enrichment of retinoid X receptor (RXR) response motifs near AR chromatin binding sites, suggesting that RXR may play a role in regulation of AR chromatin binding. We found that inhibition of RXR expression or activity resulted in downregulation of androgen-regulated gene sets, including cell cycle progression and DNA damage response pathways. Moreover, stimulation with an RXR agonist resulted in altered AR chromatin binding and enhanced androgen-independent proliferation in multiple PCa cell models. These data support a novel role for RXXR as a crucial AR-collaborating factor in PCa, particularly in response to acute androgen deprivation, and further suggest that co-targeting of the AR and RXR axes could lead to enhanced therapeutic benefit.

#1025

Mechanism of antitumor effect of Sorafenib in anaplastic thyroid cancer cell lines.

SAE ISHIHARA. _Osaka City University Graduate School of Medicine, Osaka, Japan_.

Introduction: Anaplastic thyroid cancer (ATC) is a rare refractory disease, found in 1-3% of thyroid cancer. Because of its highly malignant potential, patients often become lethal within 6 months, nevertheless for intensive multimodal therapies. Recent observations indicated the strong efficacy of multi-kinase inhibitor to control disease progression of ATC. Frequent BRAF mutation (40-60%) in tumors of ATC was reported in Japan, indicating BRAF gene alteration as possible molecular target. Moreover, the secretions of VEGF were often found in ATC cell lines, suggesting the important role of VEGF-mediated tumor neo-vascularization in the aggressive progression of ATC. We, thus, examined the antitumor effect of Sorafenib, a multi-kinase inhibitor demonstrating its effect by inhibiting both BRAF and VEGF signal.

Materials & Methods: Human ATC cell lines having four different gene mutations were used (OCUT-4; BRAF mutation, OCUT-6, ACT-1; NRAS mutation, OCUT-2; BRAS and PI3KCA mutation). The effect of Sorafenib on cellular viability was evaluated by MTT assay. In order to investigate the mechanism of cellular damage by Sorafenib, cell cycle was evaluated by flowcytometry, and the signal transduction of RAF/MEK pathway was examined by Western blotting. The amount of VEGF secretion from cells was measured by ELISA. Expression of VEGF-receptor in cells was evaluated by Western blotting. Furthermore, the effects of Sorafenib on human vascular endothelial cells were investigated by recruiting HUVEC (Kurabo, Japan) stimulated with VEGF and conditioned medium of the cancer cells.

Results: The effect of Sorafenib appears most strongly in OCUT-4 cell with BRAF mutation compared with other cell lines. However, the impairment of cellular viability was shown at relatively high concentration (>100 nM). OCUT-4 showed G1 phase arrest by Sorafenib exposure. The specific effect of BRAF inhibition was confirmed by the suppression of MEK and ERK phosphorylation in OCUT-4. The suppression was not observed in OCUT-6, a cell line with NRAS mutation. The paradoxical increase of MEK phosphorylation was found in this cell line, instead. Every cell line secreted different amount of VEGF, and the proliferation of HUVEC was stimulated by their conditioned mediums similarly as by VEGF. The stimulation of proliferation was suppressed both by anti-VEGF antibody and Sorafenib.

Discussion: In the present study, we clearly demonstrated that Sorafenib inhibits growth of human vascular endothelium mediated by VEGF secretion from cancer cells, as well as direct cell cycle arrest on ATC cells. A high concentration of Sorafenib may be needed to show inhibition on the RAF/MEK pathway. Furthermore, the inhibition of the pathway was not effective when additional growth stimulatory pathway than RAF was activated as in case of OCUT-2, or RAS gene were mutated. However, in any case, cancer cell growth mediated by autocrine system was inhibited clearly by Sorafenib.

#1026

Oncogenic alterations in FGFR3 and ERBB2 lead to ligand-independent activation of PPARG in bladder cancer.

Jonathan T. Goldstein,1 Ashton C. Berger,1 Craig A. Strathdee,1 Matthew Meyerson2. 1 _Broad Institute, Cambridge, MA;_ 2 _Dana Farber Cancer Institute, Boston, MA_.

PPARG is genomically activated in muscle-invasive bladder cancer through focal PPARG gene amplification and hotspot mutations in its heterodimer partner, RXRA. However, more than half of the PPARG-activated bladder tumors and cell lines do not have identifiable somatic alterations in either PPARG or RXRA. Using a PPARG-driven reporter assay in RT112 bladder cancer cell line, we screened probe compounds to identify candidate drivers of ligand-independent PPARG activation. We found that pan-FGFR inhibitors and MEK1/2 inhibitors antagonized the PPARG-driven reporter assay with potency similar to reported values for their cognate targets (1-20 nM) and interestingly, RT112 cells carry an FGFR3-TACC3 oncogenic fusion. In addition to the expected effects of these inhibitors on phospho-MEK1/2 and phospho-ERK1/2, they also inhibited production of canonical PPARG targets, including FABP4. In a second subset of cell lines, a parallel story was also observed for the effects of ERBB2 inhibitors in ERBB2 hotspot mutant bladder cancer. Taken together, these data uncover additional mechanisms for functional activation of PPARG in bladder cancer, suggest potential resistance mechanisms for FGFR and ERBB2 inhibitors, and provide a rationale for therapeutic combinations of PPARG modulators.

#1027

Inhibiting pathways predicted from a steroid hormone gene signature yields synergistic antitumor effects in lung cancer.

Abdulaziz A. Almotlak, Mariya Farooqui, Jill M. Siegfried. _University of Minnesota, Saint Paul, MN_.

Mounting preclinical and clinical evidence suggest an involvement of estrogen signaling in promoting non-small cell lung cancer (NSCLC) progression. In estrogen receptor β-positive (ERβ) cases, we previously found seven genes within the prediction analysis of microarrays 50 gene (PAM50) panel that showed strong association in predicting prognosis of NSCLC patients, with Myc, MIA, FGFR4, CXXC5, Grb-7 and FOXC1 being upregulated while PR was downregulated in highly aggressive lung tumors. These genes described one network containing ERβ and human epidermal growth factor receptor 2/3 (HER2/HER3) signaling, which suggests that these two interacting pathways define lung tumors with more aggressive biology. Here, we sought to evaluate the therapeutic potential of combining the pan-HER tyrosine kinase inhibitor, Dacomitinib, and the ER antagonist, Fulvestrant in NSCLC. Cell viability assay in three different human NSCLC cell lines 201T (EGFR wild-type), A549 (K-ras mutant) and HCC827 (EGFR exon 19 deletion) showed strong synergy between Dacomitinib and Fulvestrant, as determined by a combination index < 1. The combination strongly inhibited p-EGFR, p-HER2, p-HER3, p-HER4, and the downstream signaling p-AKT and p-MAPK compared to single treatments. Mechanistically, the transcription activity of activator protein-1 (AP-1) family members Fos and Jun were significantly reduced, as detected by the ability of AP-1 to bind to DNA. Expression of c-Myc and CyclinD1 were also significantly inhibited by the combination. In NSCLC cell lines, the combination reversed the gene signature associated with poor prognosis with c-Myc, MIA, CXXC5, FGFR4, Grb-7, FOXC1 being downregulated while PR was upregulated. Similarly, the AP-1 inhibitor t-5224 mimicked the effects of the combination in reversing the PAM50 gene signature, suggesting that the combination's effect is largely mediated by AP-1 downregulation. In vivo, the combination also revealed a synergistic effect, measured by the combination ratio method (> 1), and induced tumor regression in 201T and A549 xenografts, with average tumor volume significantly lower than the single treatment groups. The immunohistochemical analysis of the xenografts revealed significant downregulation in Ki67. Western blot analysis of the tumor lysates from the combination treatment groups showed an increase in cleaved caspase-3 and a decrease in both ER and HER signaling. The PAM50 gene signature was also reversed in both xenograft studies detected by real-time-qPCR. Similar effects were seen for c-Myc and PR at the protein levels detected by immunohistochemistry staining. These observations encouragingly support the use of this combination clinically in NSCLC, considering its ability to suppress tumor growth and produce a gene signature that predicts better clinical outcomes, as assessed by the PAM50 genes.

## TUMOR BIOLOGY

### Epithelial/Mesenchymal Transition (EMT and MET) 1 / Actin Cytoskeleton

#1028

Extracellular matrix dimensionality reduces cellular cortical tension to stimulate pro-survival signaling in mammary epithelial cells.

FuiBoon Kai,1 Guanqing Ou,1 Alexandra Long,1 Wei Guo,2 Richard Tourdot,2 Ravi Radhakrishnan,2 Christopher Chen,3 Sophie Dumont,1 Valerie M. Weaver1. 1 _University of California, San Francisco, San Francisco, CA;_ 2 _University of Pennsylvania, PA;_ 3 _Boston University, MA_.

Tumor dormancy is a clinical phenomenon in which disseminated tumor cells remain asymptomatic and undetectable over a long period of time. Dormant cells are able to maintain quiescence in hostile microenvironments, escape frontline cancer therapies and evade the immune system, as well as their propensity to reactivate from latency and cause metastatic relapse. Although tumor dormancy is an important problem in the treatment of cancer, the molecular mechanisms underlying this complex process remain unclear. Because dormant cells are frequently found surrounded by a laminin-rich ECM, we hypothesized that ECM dimensionality intrinsically affects cell behaviors that predispose to dormancy. Accordingly, we assessed the biophysical and biochemical response of mammary epithelial cells (MECs) to compliant polyacrylamide (PA) gels and micropatterned surfaces in which ligand presentation and ECM dimensionality were modulated to recapitulate different ECM landscapes. Using traction force microscopy, atomic force microscopy indentation, and laser ablation studies, we found that a 3D ECM led to a drop in cortical tension of MECs. Computational modeling predicted that reduced cortical tension should lead to an increase in the number and/or residence time of actin protrusions as well as a net increase in negative membrane curvature. To test the prediction, we ectopically expressed F-actin and plasma membrane markers in MECs to examine the plasma membrane topography and actin protrusion dynamics. Indeed, MECs in 3D ECM had longer and more stable actin protrusions and more negative membrane curvature inducing proteins, including Exo70, at the plasma membrane. The enrichment of Exo70 at the plasma membrane accompanied the activation of Arf6, which led to an increase in Rac/p38 pro-survival signaling pathway. Consistently, we found that the non-spread cells were able to survive in 3D ECM but the non-spread MECs plated on either a soft 2D PA gels or rigid micropatterned adhesive islands died. We next examined if reduced cortical tension is sufficient to activate pro-survival pathways in cells in 2D. Importantly, pharmacological inhibition of myosin in non-spread MECs on a 2D ECM, which causes loss of cortical tension, increased negative membrane curvature and cell viability. Conversely, genetic knockdown of the negative curvature-inducing protein Exo70 compromised MEC survival in a 3D ECM. Our results provide the first evidence demonstrating that ECM dimensionality alters the biophysical properties of cells to modulate plasma membrane curvature and activate pro-survival signaling pathways. Our findings also offer a unique perspective for why Arf6 and Rac GTPases have been implicated in cancer aggression and suggest that targeting the tissue ECM or cellular cortical tension may provide a novel therapeutic approach to target dormant cells.

#1029

Metastatic cancer cells activate endothelial RhoA-ROCK pathway for trans-endothelial migration.

Md Sanaullah Sajib,1 Fatema Tuz Zahra,1 Jee Hyun Park,1 Paul Tullar,1 Sanjay K. Srivastava,2 Ulrich Bickel,1 Constantinos M Mikelis1. 1 _Texas Tech University Health Sciences Center, Amarillo, TX;_ 2 _Texas Tech University Health Sciences Center, Abilene, TX_.

Metastasis is the process through which tumor cells disseminate from the primary tumor and colonize in distant parts of the body. Two of the steps in the metastatic process are the trans-migration of cancer cells through the endothelial lining of blood and lymphatic vessels, during entrance (intravasation) and exit (extravasation) from the vascular system. We and others have shown that the endothelial RhoA pathway plays important role in endothelial permeability. Translating these findings in the metastasis context we aim to explore the role of endothelial RhoA signaling pathway on cancer cell trans-endothelial migration and metastasis. In vitro, we have established a quantifiable, highly reproducible, transwell-based, two-cell co-culture model of trans-endothelial migration, where fluorescently-labeled cancer cells transmigrate through an endothelial monolayer. Pharmacological and molecular biology approaches were incorporated to dissect the role of endothelial RhoA signaling pathway. Primary and immortalized endothelial cells were used and cancer cells of both mouse and human origin were tested. In all cases, conditioned media from the cancer cells activated endothelial RhoA. Blockade of the RhoA pathway inhibited cancer cell trans-endothelial migration. In vivo, syngeneic and human cancer cell lines were tested in tail-vein and intra-cardiac models of experimental metastasis. We observed decreased lung metastatic nodules in endothelial-specific RhoA-deficient mice, compared to the littermate controls. Treatment with a clinically-relevant inhibitor of the RhoA pathway, Fasudil, also decreased the metastatic colonization of both human and cancer cells. The above findings demonstrate that the endothelial RhoA pathway has a pivotal role on cancer cell trans-endothelial migration.

#1030

Regulation and role of actin dynamics in promoting premalignant cell migration.

Manash K. Paul,1 Arkaprabha Basu,1 Bharti Bisht,1 Paul Pagano,1 Yari Fontebasso,1 Kostyantyn Krysan,1 Linh Tran,1 Mitchel Alioscha-Perez,2 John Minna,3 Dino DiCarlo,1 Hichem Sahli,2 Shimon Weiss,1 Steven M. Dubinett1. 1 _University of California Los Angeles, Los Angeles, CA;_ 2 _Vrije Universiteit Brussel, Brussels, Belgium;_ 3 _University of Texas Southwestern Med. Ctr., Dallas, TX_.

Lung cancer is a highly metastatic disease. Although commonly considered to be a late event in disease pathogenesis, micrometastasis may also occur as an early phenomenon. Induction of epithelial-mesenchymal transition (EMT) is associated with changes in mechanical properties, predominantly due to increased cell contractility and actin stress fiber formation. The molecular mechanisms regulating actin dynamics during EMT in premalignant cells have not yet been defined. Using a novel constricted migration selection strategy and physomic techniques, including deformability cytometry and atomic force microscopy, we identified a highly motile (HM) subpopulation of HBECs, with enhanced heritable migratory capacity both in vitro and in vivo. The HBECs used for selection were modified with genetic changes that can be found in premalignancy (p53null, activated Kras-G12D). Thus, this sub-population of HM-HBECs offer a unique model to investigate premalignant cell migration. Comparative RNA-seq datasets and confocal live cell imaging technology reveal increased migration and expression of key EMT genes in HM-HBECs. HM-HBECs cells were found to be characterized by the transient accumulation of actin stress fibers as compared to parental-HBECs. We performed high-throughput kinase inhibitor screening to better understand the role of kinases regulating early HBEC migration. Incucyte based secondary screen assays established that ERK-MEK pathway inhibition plays a key role in inhibiting actin stress fiber formation and actin-associated protein network assembly thereby delaying early migration. In the absence of a sensitive quantitative method to detect actin cytoskeleton remodeling in a non-destructive manner, we developed computational methods to extract and quantify the actin stress fibers from Super Resolution Optical Fluctuation imaging (SOFI) and confocal microscopic images. SOFI-based fluorescence imaging based on temporal, stochastic "on" and "off" fluorescence fluctuations in combination with actin-tagged blinking fluorescent proteins such as Dronpa showed advantages over methods that use fixed samples in studying actin filament assembly and disassembly. We extracted the orientations of the fibers and the width of their distribution in each time point to quantitatively distinguish different architectures. Our preliminary data suggest a Rac1-Cortactin-actin dynamic regulatory axis that may lead to enhanced migration. This is accompanied by the orientation of actin fibers favoring EMT. Future studies are anticipated to identify novel targets in premalignancy that could be exploited for lung cancer interception. M.K.P. and A.B. contributed equally to this work

#1031

Single-cell analysis reveals a possibility of epithelial to circulating tumor cell transition (ECT) when tumor cells enter into the bloodstream.

Glenn Deng,1 Stefanie Jeffrey2. 1 _China Three Gorges University, Yichang, China;_ 2 _Stanford University, Stanford, CA_.

Tumor cells face the enviormental changes when leave tumor tissue enter into bloodstream. The environmental changes stimulate tumor cells to adapt the new enviorments with cell functional changes. Epithelial to mesenchymal transition (EMT) is well known and it explains the changes relatively in a portion of the circuating tumor cells (CTC) and in the relative later stage. We use the single-cell molecular analysis on house keeping genes, apoptosis genes, metabolism genes, Epithelial genes, EMT genes, metastasis genes, stem cell genes, and proliferation genes. The results reveal that less than half of individual CTCs express normal level of house keeping genes; apoptosis genes are not expressed at the normal level (reduced or not detectable) in the CTCs without house keeping genes expression; the expression of metabolism genes are relatively low especially in the CTCs without GAPDH expression; the expression level of Epithelial genes are low and the EpCAM and KRT7 are undetectable in most CTCs, but KRT8/18/19 are remaining high expression in the CTCs with GAPDH expressed; EMT genes of VIM, Twist1, MMP-2, TGFB1 are highly expressed in CTCs with GAPDH expressed but no or low expression in the CTCs without GAPDH expression; the expression of metastasis genes are relatively high in the CTCs with GAPDH expressed, but not in the CTCs without the expression of GAPDH; the expression of CD44, CD24, Bmi1, Oct-4, CXCR4, CD9, CXCL2, MAPK14 are highly expressed in the CTCs with GAPDH expressed but not in the CTCs without GAPDH expression; CD44 expressed in some CTCs with higher level than CD24, but much more individual CTCs (with GAPDH expressed) showed higher expression of CD24 than that of CD44 per single-cell level; the expression of proliferation genes are reduced in the CTCs with GAPDH expressed and not detectable in the CTCs without GAPDH expression. Based on the results, we hypthesize "Epithelial to circulating transition (ECT)" at the early stage after the tumor cells enter into bloodstream and followed by "CMT (circulating to mesenchymal transition and then MEC (mesenchymal to epithelial transition)". The ECT may includes the cell changes related to surviving, metabolism level, remaining the characteristics of metastatsis, stem cell signatures, Epithelial related genes and EMT related genes, proliferation genes and others. Studying the early changes of tumor cells enter into bloodstream maybe very improtant for gaining the understanding of the characteristics of survival cells and metastatic tumor cells and also important for basic research and clinical applications.

#1032

**Establishing an** in vitro **model of lipopolysaccharide-induced tumorospheres formation of prostate epithelial cells for drug screening.**

Jiajia Jiang, Xueqi Lian, Sijie Tang, Xiaohua Li. _Affiliated AoYang Hospital of Jiangsu University, Zhangjiagang, China_.

Tumorospheres forming assay not only was utilized to enrich cancer stem cell from bulk cancer cells, also was widely used to analyze the ability of clonogenic growth and self-renewal capability of cancer stem cells. Meanwhile, chronic inflammation-induced epithelial transformation has been documented in a broad range of cells. However, the effect of chronic inflammatory agent on tumorosphere formation has never been tested. In current study, the classic pro-inflammatory agent lipopolysaccharide was utilized to establish an in vitro model through inducing prostate tumoroshpere formation. Then, trichostatin A, a panel inhibitor of histone deacetylases, was tested in this model for its anti-tumor effect. The results showed that treatment with lipopolysaccharide stimulated prostate cancer DU145 and PC3 cells undergoing migration and invasion with decreased the ratio of CK8 and CK18 versus vimentin. It also induced tumorospheres formation in suspension culture with increased a panel of tumor stem cell markers transcription (e.g. CD44, α-intergrin, nestin etc) and promoted phosphorylation of Stat3 (p-Stat3) and pro-oncogene c-Myc expression in vitro. Treatment with trichostatin A showed to restrain the above lipopolysaccharide-induced EMT and tumorosphere formation with decreasing the level of p-Stat3, p-Erk, p-c-jun, c-Myc, CD44 and SOX-2. Finally, blocking Stat3 signaling pathway by treatment with trichostatin A and/or small molecule compound Stattic, an inhibitor of p-Stat3, abrogated LPS-induced cancer stem-like tumorosphere formation. Taken together, our data demonstrated that lipopolysaccharide induced cancer stem-like tumorospheres formation successfully, which could be served as a simple and easy handling in vitro model for anti-tumor drug screening.

#1033

Axl-mediated activation of TBK1 drives epithelial plasticity in pancreatic cancer.

Emily N. Arner, Victoria H. Cruz, Wenting Du, Rolf Brekken. _UT Southwestern Medical Center, Dallas, TX_.

Pancreatic ductal adenocarcinoma (PDA) is characterized by an activating mutation in KRAS. Direct inhibition of KRAS through pharmacological means remains a challenge; however, targeting key KRAS effectors has therapeutic potential. We investigated the contribution of TANK-binding kinase 1 (TBK1), a critical downstream effector of mutant active KRAS, to PDA progression. We report that higher levels of TBK1 mRNA are associated with poorer overall survival in human PDA patients and that TBK1 supports the growth and metastasis of KRAS-mutant PDA by driving an epithelial plasticity program in tumor cells that enhances invasive and metastatic capacity. Further, we identify that the receptor tyrosine kinase Axl induces TBK1 activity in a Ras-RalB-dependent manner. These findings demonstrate that TBK1 is central to an Axl-driven epithelial-mesenchymal transition in KRAS-mutant PDA and suggest that interruption of the Axl-TBK1 signaling cascade above or below KRAS has potential therapeutic efficacy in this recalcitrant disease.

#1034

The role of single chromosome gains in metastasis.

Anand Vasudevan, Jason M. Sheltzer. _CSHL, Cold Spring Harbor, NY_.

Metastatic dissemination is responsible for 90% of cancer patient deaths, however, the genetic differences between metastatic and non-metastatic tumors are poorly understood. My proposal seeks to understand what differentiates primary and metastatic cancers and how aneuploidy affects cancer dissemination. A clue is that metastatic tumors show higher levels of aneuploidy than primary near-diploid tumors. This was observed in myelomas, sarcomas and melanomas where high aneuploidy rates were associated with greater metastases formation. Studies show that DNA ploidy could be a prognostic marker, as diploid tumors show lower risk of metastases and a more favorable prognosis. Though the clinical observations are strong, the functional role of aneuploidy in metastasis is unclear. Thus, studying aneuploidy may shed light on how primary tumors develop into metastatic cancers.

In this work, I employed two models to generate aneuploidy- single whole chromosome gains and complex random karyotypes. For the former, we have studied chromosomally stable near-diploid human colorectal cancer cell line (HCT116) into which single extra chromosomes- 3, 5, 8, 13, 18 and 21 - have been added via microcell-mediated chromosome transfer. However, cancer karyotypes are complex, so to generate random karyotypes, chemical inhibitors of the key spindle checkpoint protein Mps1 were used to generate chromosomal instability. My main aim was to address if aneuploidy (single or complex) induces an EMT expression pattern. I demonstrate that HCT116 cells harboring an extra copy of chromosome 5 showed downregulation of epithelial markers such as E-cadherin, Claudin-7 and Epcam. This phenotype was not observed in the other trisomies and drug treatments. Interestingly, the wildtype and all the HCT116 trisomy cell lines showed a lack of mesenchymal marker expression suggesting that trisomy 5 induces a partial EMT phenotype. Subsequently, I assessed if down regulation of epithelial markers affected metastatic phenotypes by using scratch and migration assays. The trisomy 5 cell lines closed the gap in the scratch assay at a faster rate and had higher migratory capability in the migration assay compared to wildtype HCT116 and trisomy 3 and 8 and drug treated HCT116 cells. I am currently using CRISPRi to study possible gene(s) involved in this phenotype and using in vivo models to detect differences in growth and metastasis between wildtype and trisomy 5 cell line. This work will broaden our knowledge on the relationship between aneuploidy and metastasis to determine specific karyotypes associated with greater metastatic potential.

#1035

PLEK2 promotes epithelial mesenchymal transition through increasing Snail1 in lung cancer cells.

En-Chu Liu,1 Tsai-Tu Lee,1 Shey-Lin Wu,2 Tai-Lin Lee,1 Meng-Feng Tsai1. 1 _Da-Yeh Univ., Changhua, Taiwan;_ 2 _Changhua Christian Hospital, Changhua, Taiwan_.

Pleckstrin 2 (PLEK2), one of the pleckstrin homology domain proteins, have been suggested to modulate actin rearrangement and cell spreading in T lymphocytes. Recently studies also implicated that PLEK2 paly an importance role in human myeloproliferative neoplasms, melanoma, and lung cancers. PLEK2 mRNA genes were related to poor overall survival in lung adenocarcinoma patients. However, the biological functions of PLEK2 in lung cancer is not clear. We detected the expression of PLEK2 in various lung adenocarcinoma cell lines using quantitative RT-PCR methods. Molecular manipulations were performed to investigate the roles of PLEK2 in lung cancer cells. Over-expression of PLEK2 in lung cancer cells exhibited markedly promoting in the proliferation and anchorage independent growth, whereas reduced of PLEK2 was found to suppress lung cancer cell progression. This studies also indicated that PLEK2 may play an important role in the epithelial-mesenchymal transition (EMT) features, including a morphological change, increased invasive and migratory ability, reduction the epithelial marker expression and up-regulated EMT regulators. Our results suggested that PLEK2 expression induce lung cancer cell EMT phenomenon through up-regulation Snail1 expression. These results will provide information and help us to understand the roles of PLEK2 in lung cancer cells.

#1036

WISP-1 promotes epithelial-mesenchymal transition in oral squamous cell carcinoma via the miR-153-3p/Snail axis.

An-Chen Chang, Chih-Hsin Tang. _China Medical Univ., Taichung, Taiwan_.

Oral squamous cell carcinoma (OSCC) is the most common oral malignancy and approximately 50% of patients with OSCC present with regional regional lymph node metastasis. The first step in the 'invasion-metastasis cascade' is the phenomenon of epithelial-to-mesenchymal transition (EMT), whereby epithelial cells of the primary tumor lose their apical-basal polarity and transition to a mesenchymal phenotype, gaining the ability to migrate and invade basement membrane and blood vessels. WNT1-inducible signaling pathway protein-1 (WISP-1/CCN4) is an extracellular matrix-related protein that belongs to the CCN family, capable of stimulating bone remodeling and tumor progression. Our previous research showed that WISP-1 promotes cell migration and VEGF-C-dependent lymphangiogenesis in OSCC cells. This investigation further examined the role of WISP-1 in regulating EMT. First, we analyzed data from the cancer genome atlas (TCGA) database, to determine the clinical importance of WISP-1 in oral cancer. We found that WISP-1 expression was significantly associated with clinical disease stage and regional lymph node metastasis. Moreover, we found higher levels of WISP-1 expression in serum samples obtained from 62 patients with OSCC compared with samples from healthy controls. Treatment of the OSCC cell line SCC4 with WISP-1 increased EMT by downregulating expression of the epithelial marker E-cadherin and upregulating the mesenchymal marker Snail. We also observed that WISP-1 regulated the expression of EMT markers via the FAK, ILK and AKT signal transduction pathways. Computational analysis confirmed that microRNA (miR)-29c directly targets the 3′ untranslated region (3′ UTR) of Snail. Treatment of SCC4 cells with WISP-1 inhibited miR-29c expression, while miR-29c mimic prevented WISP-1-induced enhancement of Snail expression, revealing an inverse correlation between miR-29c and Snail expression during WISP-1 treatment. Our findings provide insight into WISP-1-mediated regulation of EMT in OSCC.

#1037

Axl is critical for pancreatic cancer progression and metastasis.

Wenting Du, Huocong Huang, Jason Toombs, Zhaoning Wang, Abdel Hosein, Natalie Burton, Rolf A. Brekken. _UT Southwestern Medical Center, Dallas, TX_.

Pancreatic ductal adenocarcinoma (PDAC), a leading cause of cancer-related death in the US, has a high metastatic rate that is associated with persistent immune suppression. Axl, a member of the TAM (Tyro3, Axl, MerTK) receptor tyrosine kinase family, has been identified as a critical factor that drives metastasis and immune suppression in many cancer types. We have shown that pharmacological inhibition of Axl significantly reduces tumor progression and metastasis and improves response to chemotherapy in multiple preclinical PDAC models. Here we demonstrate that Axl-deficient PDAC bearing mice have longer median survival, smaller tumors, fewer metastases and higher sensitivity to chemotherapy (gemcitabine) compared to Axl wild-type (WT) PDAC mice. Furthermore, Axl-deficient PDAC displays a more differentiated histology and has a more inflammatory and active immune microenvironment. Single cell RNA sequencing of PDAC genetically engineered mouse models reveals that Axl is expressed highly in tumor cells that have a mesenchymal-like phenotype and Axl expression correlates with classic markers of mesenchymal tumor cells at the RNA and protein level. To investigate the importance of Axl on tumor cells, CRISPR-Cas9 was used to knockout Axl in PDAC cells. Human and murine Axl-deficient PDAC cells displayed a more differentiated (epithelial) phenotype in vitro including reduced migratory capacity compared to Axl WT parental cells. Orthotopic injection of Axl-deficient murine PDAC cells into Axl WT mice resulted in longer median survival and fewer metastases compared to WT mice injected with WT PDAC cells. However, Axl-deficient mice injected with WT PDAC cell injection showed similar median survival and metastases as WT mice injected with WT PDAC cells. These in vivo results indicate that Axl expression on tumor cells is critical for PDAC progression and metastasis. In vitro experiments using WT and Axl-deficient human PDAC cells show that Axl is required for TGFβ-mediated epithelial to mesenchymal transition, probably through regulating the expression of Smad2 and Smad4. Interesting, Axl depletion has no effect on the mRNA level of Smad2 and Smad4, but does decrease the protein level, indicating that Axl might be involved in post-translational regulation (e.g., protein stability) of canonical TGFβ signaling intermediates. In summary, our study demonstrates that Axl expressed by pancreatic cancer cells is critical to tumor cell plasticity, tumor progression and metastasis.

#1038

Metformin inhibits epithelial-mesenchymal transition in human pancreatic cancer cell lines.

Juichiro Yoshida, Takeshi Ishikawa, Yuki Endo, Shinya Matsumura, Takayuki Ohta, Katsura Mizushima, Yasuko Hirai, Tetsuya Okayama, Naoyuki Sakamoto, Ken Inoue, Kazuhiro Katada, Kazuhiro Kamada, Kazuhiko Uchiyama, Tomohisa Takagi, Yuji Naito, Yoshito Itoh. _Kyoto Prefectural Univercity of medicine, Kyoto, Japan_.

Background: Epithelial-mesenchymal transition (EMT) is considered to be a crucial event in the development of cancer metastasis. Metformin is an antidiabetic drug used in treatment of type 2 diabetes. Recently, increasing evidence has indicated that metformin can inhibit tumor growth, however, effect of metformin on EMT remains still well unknown.

Methods: We investigated whether metformin inhibits EMT in the human pancreatic cancer cell lines (PANC-1, BXPC-3, and MIAPaCa-2). Pancreatic cancer cells were stimulated TGF-β1 (10ng/ml) with or without metformin(10mM). After 48h, we evaluated for EMT-related factors using western blot analyses, immunocytochemistry and RT-PCR. Wound healing assay was performed to determine cell migration.

Results: The cells stimulated TGF-β1 acquired elongated and fusiform morphology, and metformin inhibited the TGF-β1-induced morphological changes in PANC-1 and BXPC-3 cells. Wound healing assay of PANC-1 and MIAPaCa-2 cells showed that significant wound healing was seen after 24h in cells treated with TGF-β1 compared with the control, and metformin inhibited wound closure. E-cadherin expression weakened after exposure to TGF-β1, but it was reversed by metformin in these cell lines. Metformin treatment reduced the expression of mesenchymal markers (Vimentin, N-cadherin and αSMA) induced by TGF-β1 in these cell lines. Exposure of cells to TGF-β1 resulted in the phosphorylation of mTOR, and exposure of cells to metformin resulted in the phosphorylation of AMPK and blocked the phosphorylation of mTOR in BXPC-3 cells. Furthermore, cell viability was no significantly change between exposing metformin or not.

Discussion: Our results indicate that metformin inhibits TGF-β1 induced EMT in human pancreatic cancer cells. Moreover, our results suggest that metformin induces the phosphorylation of AMPK and inhibits TGF-β1 induced mTOR activation in BXPC-3 cells.

#1039

c-Met activation promotes epithelial-to-mesenchymal transition (EMT) in renal cancer cells through the upregulation of the chemokine receptor CXCR7.

Samik Chakraborty, Murugabaskar Balan, Soumitro Pal. _Boston Children's Hospital, Boston, MA_.

Purpose: The purpose of this study is to explore how the receptor tyrosine kinase (RTK) c-Met modulates the expression of the pro-tumorigenic molecule CXCR7, a chemokine receptor, in renal cancer cells; and to investigate the role of c-Met and CXCR7 in promoting the epithelial-mesenchymal transition (EMT) of renal cancer cells.

Background: c-Met, a pro-oncogenic RTK, can play a major role in the survival and progression of cancer cells; and it is often overexpressed in both clear cell and papillary renal cell carcinoma (RCC). c-Met, upon activation by its specific ligand hepatocyte growth factor (HGF), can induce a broad spectrum of biological pathways involved in the growth of cancer cells. CXCR family of chemokine receptors and their chemokine ligands were shown to exhibit decisive roles in tumor initiation, promotion, progression, and metastasis. CXCR7 is upregulated in several types of cancers, including renal cancer, and found to be involved in tumor cell development, survival, and metastasis. A global process integral to the cancer metastasis is EMT, which results in the disruption of cell-to-cell junctions and endows epithelial cells with the ability to migrate and invade surrounding tissue. EMT process is associated with an increase of mesenchymal markers like vimentin and a decrease of epithelial markers like E-cadherin in cancer cells. Sustained activation of c-Met induces EMT in renal cancer cells, however, the cross-talk between CXCR7 and c-Met signaling in the promotion of renal cancer EMT remains unclear.

Results: We assessed the expression levels of CXCR7 by Western blot analysis and found that CXCR7 is overexpressed in human renal cancer cells (both 786-O and ACHN) when compared to normal renal epithelial cells (RPTEC). Also, in both 786-O and ACHN cells, HGF-induced activation of c-Met further increased the expression of CXCR7 at both RNA and protein levels. In renal cancer cells (both 786-O and ACHN), siRNA-mediated knockdown of c-Met or treatment with XL-184 (a small molecule inhibitor of c-Met) inhibited c-Met-induced upregulation of CXCR7. c-Met activation increased the expression of pro-EMT markers such as vimentin, N-cadherin, and MMP-9 and suppressed the expression of anti-EMT molecule E-cadherin, as measured by Western blot and flow cytometry. On the other hand, siRNA-mediated silencing of CXCR7 attenuated the induction of EMT, suggesting that CXCR7 plays a major role in c-Met-induced EMT in renal cancer cells.

Conclusion: Together, our findings demonstrate that the c-Met-CXCR7 axis can trigger the EMT, which can play an important role(s) in renal cancer proliferation and migration.

#1040

Characterization of extracellular vesicles in pancreatic cancer: Changing the way we think about tumorigenesis.

Charles P. Hinzman, Michael Girgis, Yaoxiang Li, Meth Jayatilake, Meena Rajagopal, Partha P. Banerjee, Amrita K. Cheema. _Georgetown University Medical Center, Washington, DC_.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second leading cause of cancer-related deaths by 2030. Patients diagnosed with PDAC have a 5-year survival rate ~9%. Detection of pre-neoplastic lesions has the potential to improve treatment and overall survival in pancreatic cancer (PC) by 4-5 fold. However, there is currently no validated screening test for the non-invasive and accurate detection of early stage PC.

Extracellular vesicles (EVs) arise from invagination of, and secretion from, cells of all tissue types. Their role in mediating PC tumorigenesis however, is just beginning to be studied. We have found that EVs derived from PC cells (cancer-derived EVs, CDEVs), can impart significant phenotypic and metabolic transformational effects on normal human pancreas cells (hTERT-HPNE and H6c7), tantamount to an epithelial-to-mesenchymal transition (EMT). The goal of this study was to characterize the biochemical cargo of EVs toward understanding their role in PC tumorigenesis.

Results: We isolated CDEVs from various established and PDX PC cell lines. After validating enrichment of EV fractions with quantitative ELISA, immunoblot and transmission electron microscopy (TEM), we added CDEVs to culture media of normal cells. We found that CDEVs induce a myriad of changes in normal cells, including significant morphological changes, increased proliferation and an uncharacteristic invasive capability. CDEVs also caused a bioenergetic switch in normal cells, from a quiescent, aerobic profile to a highly energetic and glycolytic profile.

Comparative analyses of CDEVs and normal-derived EVs (NDEVs) using 1H- and 13C-NMR showed significantly different biochemical fingerprints. Interestingly, we found that NDEVs contained more amino acids than CDEVs, while CDEVs contained elevated amounts of urea and lactic acid. We next designed a 13C-isotope labeling flux experiment to trace the fate of carbon metabolized from 13C-glucose fed to cells in culture. Using UHPLC/Q-ToF-MS, we identified several differentially labeled isotopes in CDEVs, particularly surrounding lipid biosynthesis and folate.

Conclusions: Our results indicate that CDEVs confer enormous transformational properties to normal human pancreas cells in vitro. We hypothesize that EVs could impart tumorigenic properties to normal cells in vivo and this influence could unveil novel mechanisms underpinning cancer onset and progression. We show that there are robust biochemical differences between CDEVs and NDEVs. Biochemical characterization of EV cargo can uncover abrogated pathways in which signaling is mediated by EVs. These signals may be detectable before progression of PC to PDAC, leading to the development of assays for earlier diagnosis in patients.

#1041

**Casein kinase 2 modulates epithelial mesenchymal transition through** Helicobacter pylori **CagA dependent pathway in gastric cancer cells.**

SoDam Lee, Bo Ram Hwang, Jung Chang Yun, Byeong Min Yu, Yong Chan Lee. _Internal Medicine, Seoul, Republic of Korea_.

Helicobacter pylori is the most important carcinogen in human gastric cancer. However, the pathogenic molecular mechanism which underlies the carcinogenesis in gastric cancer by H. pylori remains largely unknown. In this study, we understand that molecular biological mechanism related to gastric carcinogenesis by investigating the role of CK2 in EMT. Casein kinase 2 is a serine/threonine protein kinase and consists of two catalytic subunits (α or α') and regulatory subunits (β). CK2 regulates many substrates and its involved in cell growth, proliferation, survival, angiogenesis, invasion. Epithelial-to-mesenchymal transition (EMT) is involved in many signaling pathways, but the key regulatory kinases in this process have not been clearly identified. In our previous study, we reported the cytotoxin-associated gene A (CagA) protein of H. pylori induces cell migration and invasion through increased CK2α activity in gastric epithelial cells. Although the role of CK2 catalytic subunits has remained largely uncharacterized, several studies have recently focused on regulator subunits in EMT.

Here, we analyzed the expression level of CK2α did not altered, whereas CK2β decreased in CagA-dependent pathway. Moreover, expression of ectopic CK2β was downregulated by CagA. This suggests that CagA negatively regulates the stability of CK2β protein. We examined the level of ubiquitinated CK2β were higher in HP60190 infected cells than in control cells. Thus, CK2β is degraded by the proteasomal machinery following CagA of H. pylori. In addition, CagA binds both CK2α and CK2β, which results in suppression of CK2β binding by infected HP60190, but does not suppress CK2α binding. Furthermore, downregulation of CK2β increased Snail such as CK2 target genes, EMT-related marker in H. pylori-infected gastric cancer cells. Therefore, the role of Snail regulated by CK2β should be considered when developing cancer therapeutic. Overall, CK2 tetramer subunits might control the function of CagA and EMT related genes, thereby regulating CagA-dependent pathology. Taken together, the results of the present study suggest that the CK2 regulatory subunit has diverse effect on CagA-dependent cellular processes. A pharmacological activator or activator of CK2β may have potential as a therapeutic agent for gastric cancer associated with CagA.

#1042

Cellular plasticity and migratory modalities in high-grade serous ovarian carcinoma.

Sharmila A. Bapat, Sagar Varankar. _National Centre for Cell Science, Pune, India_.

Epithelial-mesenchymal (E-M) switch associated plasticity is crucial for the gradual destabilization of tumor tissue architecture to facilitate metastasis. Several studies towards comprehension of the metastatic cascade employ an overview of the phenotype specific markers and their association with distinct functionalities of migration, invasion, stemness, anoikis resistance, ECM remodeling, etc. Recent work on the dissection of phenotypic transitions has identified the existence of transitionary intermediate states across multiple cancers which exhibit greater plasticity as compared to the two phenotypic extremes. In a previous study from our group, we successfully identified molecular subclasses in high grade serous ovarian carcinoma (HGSC) that were associated with distinct modes of metastatic dissemination viz., collective cell migration (CCM) and EMT. Towards, elucidating contributions of distinct phenotypic states to CCM and EMT along with a comprehension of associated molecular regulators we resolved a phenotypic spectrum along the E-M axis. Identification of five phenotypic states followed by their functional characterization was associated with differential migratory capabilities that were further dissected with the aid of live cell imaging to quantitatively distinguish CCM and EMT. Enhanced visual output and extraction of three quantitative metrics viz., displacement, velocity and nearest neighbours generated an informative tool for assessing migratory modes in vitro. Furthermore, these metrics identified migratory modalities inclusive of wound closure by proliferation (passive CCM), sheet-like cell migration (active CCM) and individual cell migration (EMT). Interestingly, phenotypic status and migratory modes across the spectrum emerged as a consequence of a balance between expression and differential sub-cellular localization of two transcription factors viz. Tcf21 and Slug. Application of the novel migratory analyses in view of chemotherapeutic challenges identified the emergence of an epithelial state across the spectrum facilitated by Tcf21 mediated Slug repression and resulted in a switch from EMT to aCCM-mediated migration. Our study thus provides a comprehensive dissection of the E-M axis in HGSC in view of associated functional attributes.

Acknowledgements: DBT Indo-Finnish Collaborative Grants, DBT Indo-Australia Collaborative Grants and NCCS Intramural Grants to SAB funded this research. SSV received fellowship from CSIR.

#1043

TIP60-dependent acetylation of the SPZ1-TWIST complex promotes epithelial-mesenchymal transition & metastasis in liver cancer.

Shen-Nien Wang,1 Li-Ting Wang,2 Shih-Hsien Hsu2. 1 _Kaohsiung Medical University Hospital, Kaohsiung, Taiwan;_ 2 _Kaohsiung Medical University, Kaohsiung, Taiwan_.

Metastasis is the main cause of cancer mortality. However, the triggering mechanisms and regulation of epithelial–mesenchymal transition (EMT) factors in the commitment of metastasis have not been well characterized. Spermatogenic Zip 1 (SPZ1) acts as a proto-oncogene and an upstream regulator of EMT during tumorigenesis. Here we report that the HIV-1 Tat-interacting protein 60 kDa (Tip60) acetyltransferase mediates acetylation at lysine residues of SPZ1 at positions 369 and 374, and of TWIST1 at positions 73 and 76, which are required for SPZ1–TWIST1 complex formation and cancer cell migration in vitro and in vivo. Ectopic SPZ1 and TWIST1 expression, but not that of TWIST1 alone, enhanced vascular endothelial growth factor (VEGF) expression via the recruitment of bromodomain-containing protein 4 (BRD4), thus enhancing RNA-Pol II-dependent transcription and inducing metastasis. Neutralization of VEGF using humanized monoclonal antibodies such as Avastin, effectively abrogated the EMT and oncogenesis induced by the acetylated SPZ1–TWIST1 complex. Our findings highlight the importance of acetylation signaling in the SPZ1–TWIST1–BRD4 axis in the mediation of EMT and its regulation during tumor initiation and metastasis.

#1044

PKC-ι and PKC-ζ are heavily responsible of up-regulating epithelial-mesenchymal transition (EMT) and activating Vimentin to facilitate cellular motility in prostate cancer cell lines.

Wishrawana Sarathi Ratnayake, Christopher Apostolatos, Sloan Breedy, Robert Hill, Clare Dennison, Mildred Acevedo-Duncan. _Univ. of South Florida, Tampa, FL_.

Prostate carcinoma is the most common cancer among men in terms of number of new cases reported each year. Approximately 26,000 deaths reported in 2018 in the United States and tumor metastasis is responsible for more than 90% of these deaths. Expression of Vimentin is a hallmark of metastatic mesenchymal prostate cancer cells which is initiated by EMT. Our previous studies show that atypical protein kinase C- iota (PKC-ι) and zeta (PKC-ζ) inhibition retards the activation of NF-κB pathway by diminishing NF-κB nuclei translocation {Apostolatos, A.H., et al. Int. J. Oncol. 53(5), (2018), 1836-1846 https://doi.org/10.3892/ijo.2018.4542}. The present study shows that siRNA knockdown of PKC-ι and PKC-ζ downregulate Snail1, PRRX1, Vimentin while upregulating E-cadherin thereby diminishes EMT. In-vitro migration and invasion assays for PC-3 and DU-145 prostate cancer cell lines demonstrated a significant reduction of cellular migration and invasion in PKC-ι and PKC-ζ knocked down samples. Immunoprecipitation experiments suggested direct association of Vimentin with PKC-ι and PKC-ζ separately. Laser stimulated confocal immunofluorescence and immuno-gold transmission electron microscopic techniques were used to further confirm the relationship of Vimentin with aPKCs. Real time qPCR was used to analyze the mRNA levels of targeted markers to further validate transcriptional downregulation of Vimentin which was observed in Westerns upon aPKC siRNA knockdown. Overall results showed stronger relationship between PKC-ι and Vimentin over PKC-ζ with Vimentin. Microscopic results also show PKC-ι concentrated along the cell membrane together with Vimentin in addition to the abundant distribution throughout the cell. In addition, our results suggest that both aPKCs target multiple activation sites (S33, S39 and S56) on Vimentin thereby playing a crucial role in the regulation of Vimentin dynamics which is essential for increased prostate cancer cell motility. We have used a novel PKC-ι specific inhibitor 5-amino-1-((1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S) to conduct in-vivo experiments on murine models. Excised tumors were analyzed for pathways observed in in-vitro experiments. Immunohistochemical and Western blot analysis on tumor samples confirmed the relationship of aPKCs with Vimentin. In addition, these samples will be analyzed for the miRNA expression upon PKC-ι inhibition. Overall results suggest that both aPKCs are essential in upregulation of EMT and for the activation of Vimentin to facilitate metastasis of prostate cancer cells. Finally, results suggest that PKC-ι and PKC-ζ can be effectively targeted using specific inhibitors to develop targeted therapeutics for metastatic prostate carcinoma.

#1045

CRISPR/cas9 mediated generation of an EMT reporter cell line for metastatic breast cancer drug discovery and development.

Metewo S. Enuameh, Weiguo Shu, Robert Newman. _ATCC, Gaithersburg, MD_.

Among women, breast cancer continues to be the most common cancer, with metastasis being the leading cause of mortality in cancer patients around the world (DIZON et al. 2016). Epithelial to mesenchymal transition (EMT) - the process by which epithelial cells shift to the mesenchymal state, has been implicated in many aspects of breast cancer tumorigenesis, metastasis and drug resistance ). The accumulation of a large body of data on the association of EMT with cancer over the years has not resulted in EMT being an active target for therapeutic development. This is due in part to the lack of appropriate in vitro models. Here we have exploited some of the basic biology of EMT, to create an advanced in vitro metastatic breast cancer reporter cell line model for use in both basic research and discovery of new EMT inhibitors.

During EMT, E-cadherin protein expression is down regulated in cancer cells in association with the loss of cell-to-cell adhesion, apico-basal polarity and a change to a spindle-shaped morphology. By installing an emerald green fluorescent protein (EmGFP) tag on the C-terminus of the e-cadherin (ECAD) gene in the epithelial BT-474 breast cancer cell line via CRISPR/Cas9 genome editing, end-point or real-time EMT status of cells can be tracked under defined conditions. The EMT reporter cell line was verified at the nucleic acid (genomic and mRNA) and protein levels as well as in cell-based assays. Bio-functional evaluation of the BT-474 ECAD EmGFP cell line, shows that it responds to EMT induction. The subsequent EMT status of cells can be monitored in real time by observing and measuring ECAD EmGFP expression, in addition, the resulting mesenchymal cells have increased invasive capacities. Furthermore, this EMT reporter cell line shows sensitivity to the MEK1/2 inhibitor U0126 - thereby providing the basis for the use of this cell line in high-throughput screening (HTS) applications including the identification of new anti-EMT drugs for metastatic breast cancer. The BT-474 ECAD EmGFP reporter cell line is also a convenient and sensitive model for basic science research on the mechanisms of metastasis.

References

DIZON, D. S., L. KRILOV, E. COHEN, T. GANGADHAR, P. A. GANZ et al., 2016 Clinical Cancer Advances 2016: Annual Report on Progress Against Cancer From the American Society of Clinical Oncology. J Clin Oncol 34: 987-1011.

HAY, E. D., 1995 An overview of epithelio-mesenchymal transformation. Acta Anat (Basel) 154: 8-20.

#1046

A breast cancer MET reporter cell line model for drug discovery and development.

Metewo S. Enuameh, Weiguo Shu, Robert Newman, Sangeeta Kumari. _ATCC, Gaithersburg, MD_.

World-wide, cancer metastasis continues to be the leading cause of death in cancer (DIZON et al. 2016). Although epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) have been implicated in the incidence of cancer metastasis and drug resistance, their impact in cancer progression and patient survival is not fully understood (NIETO et al. 2016). This is partly due to the lack of suitable in vitro models. Thus, to facilitate the utility of the EMT concept in therapeutic development, we have utilized some of the basic biology of EMT/MET, to create a novel advanced in vitro model for use in both basic research and discovery of new anti-EMT drugs.

In breast cancer, vimentin (VIM) intermediate filament (IF) proteins are generally upregulated during EMT and down-regulated during MET (GILLES et al. 1999; THIERY and SLEEMAN 2006; RICHARDSON et al. 2012; LAMOUILLE et al. 2014). Here we employed CRISPR/Cas9 gene editing to generate a VIM RFP (red fluorescent protein) reporter in the MDA-MB-231 breast adenocarcinoma cell line. The VIM RFP C-terminal fusion gene at the endogenous VIM locus, enables end-point or real-time tracking of the MET status as cells transition from epithelial to mesenchymal phenotype under distinct conditions. We have validated the cell line at the nucleic acid (genomic and mRNA) and protein levels as well as in cell-based assays. Bio-functional evaluation of the MDA-MB-231 VIM RFP cell line shows sensitivity to metastatic breast cancer drugs axitinib (tyrosine kinase inhibitor) and U0126 (MEK1/2 inhibitor) via the inhibition of the inherent signaling pathways which impact EMT. These effects provide the basis for the use of this cell line in high throughput screening (HTS) applications including the discovery of new anti-EMT drugs for metastatic breast cancer. Furthermore, the MDA-MB-231 VIM RFP reporter cell line is also a convenient and sensitive model for studying the mechanisms of metastasis and for basic translational science research.

#1047

Novel roles for manganese superoxide dismutase polymorphisms in prostate cancer.

Janae D. Sweeney,1 Valerie Odero-Marah,1 Channing Paller2. 1 _Clark Atlanta University, Atlanta, GA;_ 2 _Johns Hopkins University School of Medicine, Baltimore, MD_.

Prostate Cancer is the most common non-cutaneous cancer and second leading cause of cancer related death in American men. Epithelial Mesenchymal Transition (EMT), a key event in prostate cancer metastasis, is a process in which polarized epithelial cells undergo biochemical changes, allowing them to assume mesenchymal morphologies, enhancing invasiveness and migration. Reactive Oxygen Species (ROS) are chemically reactive molecules of cellular metabolism that induce oxidative stress, and increase levels of SNAI1 (Snail) transcription factor while also promoting EMT. In Phase II clinical trials, patients were treated with natural product, Muscadine Grape Skin Extract (MSKE), and patients with SOD2 Ala/Ala single nucleotide polymorphism (SNP) responded better than those with Val/Val SNP. Ala/Ala SNP is associated with higher dismutase activity [superoxide (O2-) to hydrogen peroxide (H2O2)] than Val/Val. We hypothesize that SOD2 Ala/Ala genotype is associated with increased H2O2 and EMT, potentially antagonized by MSKE in prostate cancer cells. Prostate cancer cell lines were analyzed for SOD2 SNPs by pyrosequencing. Site-directed mutagenesis was conducted to create the Ala/Ala and Val/Val SNP vectors. LNCaP prostate cancer cells were transiently and subsequently stably transfected with SOD2 Val/Val cDNA, as well as empty vector (Neo) control, and verified through western blot analysis. ROS activity was evaluated for baseline ROS activity. Our results showed that most prostate cancer cell lines were heterozygous for SOD2 SNP (Ala/Val), although several had extra copies, for example, LNCaP had Val/Val/Ala, suggesting multiple copy numbers. Only metastatic MDA PCa 2a and MDA PCa 2b contained Ala/Ala homozygous SNP. LNCaP cells transiently and stably transfected with Val/Val showed partial induction of EMT, concomitant with higher levels of Snail and Vimentin, as compared to LNCaP Neo control. Furthermore, data also suggested that transient transfection marginally affected ROS activity while stable transfection caused significant changes in ROS activity compared to LNCaP Neo cells. We have begun to stably transfect Ala/Ala cDNA into LNCaP cells and are interested to see the differences in ROS activity and EMT induction between the two clone cell types (LNCaP Ala/Ala and Val/Val). As previous data suggests, we believe that Ala/Ala will show a higher expression of SOD2 and mesenchymal markers Snail and Vimentin, demonstrating full induction of EMT. In future we will test ROS activity, EMT marker expression in response to treatment of LNCaP Ala/Ala and Val/Val cells with MSKE. These studies may uncover the differential functions and response to treatment of SOD2 SNPs. Acknowledgements: These studies were supported by the NIH/NIMHD/RCI Grant #5G12MD007590-31, NIH/NIGMS/RISE Grant #5R25GM060414, and https://www.thecommunityfoundation.org/

#1048

CD44 promotes epithelial-mesenchymal transition (EMT) by regulating snail, ZEB1, and Caveolin-1 expression and predicts poor survival of ovarian cancer.

Jiayi Zhou, Yan Du, Baoxin Luan, Yinhua Yu, Hongbo Zhao. _OB/GYN Hospital of Fudan University, Shanghai, China_.

CD44 is a stem cell marker which is involved in the generation of a stem cell niche and maintaining stem cell quiescence. Our data first demonstrated that CD44 knockdown by small silencing RNA abrogated both basal Snail expression and TGF-β1 induced Snail expression in HOPM and HOPM-snail cells. In addition, CD44 knockdown caused an decrease in ZEB1 expression. Further, RPPA data indicated that Caveolin-1 may be another regulative target of CD44, and western blotting analysis confirmed that CD44 knockdown caused an increase in Caveolin-1 expression. However, there was no obvious reciprocal regulation among ZEB1, Caveolin-1, and snail. Moreover, CD44 knockdown caused a decrease in cell migration, cell invasion and clone formation of HOPM and HOPM-snail cells.

We next observed the role of CD44 expression in the prognosis of ovarian cancer. Over-expression of CD44 was associated with advanced FIGO stage (P<0.05). Univariate analysis result showed that histological subtype, FIGO stage, intravascular tumor thrombus, CA125 and CD44 expression were associated with overall survival and disease-free survival of ovarian cancer patients. Multivariate analysis showed that CD44 expression was an independent prognostic factor to predict both overall survival and disease-free survival of ovarian cancer patients. Taken together, our data showed that CD44 may be crucial for EMT process by regulating Snail, ZEB1, and Caveolin-1 expression. And CD44 is a potential prognostic factor as well as a treatment target for ovarian cancer.

#1049

Targeting YAP/TAZ pathway inhibits Ewing sarcoma metastasis.

Lisa Bierbaumer,1 Anna M. Katschnig,1 Branka Radic-Sarikas,1 Jeffrey R. Petro,2 Karin Mühlbacher,1 Dave N. Aryee,1 Anna R. Pötsch,1 Sandra Högler,3 Lukas Kenner,4 Aykut Uren,2 Heinrich Kovar1. 1 _St. Anna Children's Cancer Research Inst., Vienna, Austria;_ 2 _Georgetown University Medical Center, Washington D.C., DC;_ 3 _Veterinary University of Vienna, Vienna, Austria;_ 4 _Medical University of Vienna, Vienna, Austria_.

The term epithelial to mesenchymal transition (EMT) and its reverse (MET) describe the dynamic and reversible metamorphosis of cells from a highly organized to a loose migratory phenotype through cytoskeletal reorganization causing cellular plasticity. EMT/MET are inherent to normal embryonal development and wound healing, but cancer cells hijack the underlying mechanisms to enable oscillations between proliferation, invasion and migration that cause metastasis, the major killer of cancer patients. Recently, fluctuations of the oncoprotein EWS-FLI1 were identified to drive EMT/MET in Ewing sarcoma. We previously demonstrated that, in presence of EWS-FLI1, transcriptional co-activators MRTFB and TAZ are largely blocked from associating with TEAD and its target genes keeping tumor cells in a poorly migratory, highly proliferative state. In contrast, under EWS-FLI-low conditions, MRTFB and TAZ associate with YAP/TEAD complexes on chromatin resulting in cytoskeletal target gene activation and phenotypic transition to a highly migratory and low proliferative state. We therefore hypothesized that pharmacologic inhibition of YAP/TAZ/TEAD protein interaction should prevent Ewing sarcoma cells from EMT and consequently interfere with their metastatic potential. Verteporfin is a small molecule safely used in the treatment of age-related macular degeneration. Independent of its photosensitizing activity exploited in ophthalmology, it is a YAP/TAZ pathway -blocking compound. In vitro treatment of EWS-FLI1-low Ewing sarcoma cells with verteporfin resulted in decreased YAP/TAZ/TEAD complex formation in the nanomolar range, reversal of the de-repression of a EWS-FLI1 controlled EMT transcriptional signature, and inhibition of tumor cell migration in a Boyden chamber assay. Upon orthotopic implantation of TC71 Ewing sarcoma cells into the tibial crest of SCID beige mice, intra-peritoneal treatment of mice with 25mg verteporfin/kg/day before and after amputation of the affected limb led to a drastic decrease of lung metastases without affecting primary tumor growth and without obvious general toxicity. Therefore, YAP/TAZ pathway blockade holds promise as a potential metastasis-preventive strategy in the treatment of Ewing sarcoma patients with primary localized disease.

### Human in Mouse

#1050

Characterization of human cancer cell line xenografts in humanized mice.

Christina Stevens, Christopher Maddage, Kerri Lasky, Jonathan Rios-Doria, Holly Koblish. _Incyte, DE_.

Studying immune-oncology (IO) agents that target the human immune system in mice have posed challenges for oncology researchers. Since the commercial introduction of humanized mice, antitumor efficacy studies can now be performed with human cancer cells within mice bearing the components of a human immune system. However, development and characterization of these models is necessary to understand which model may be best for different agents. To this end, we characterized A375, A549, Caki-1, H1299, H1975, HCC827, HCT116, KU19-19, MDA-MB-231, and RKO human cancer cell lines in CD34+ humanized NSG mice for tumor growth rate and immune cell profiling in tumor and spleen compartments. We found that CD4+ T cell composition in both the spleen and tumor varied among the models, with A375, Caki-1, and RKO expressing higher levels of CD4+ T cells compared to the other models. Many of the models had moderate levels of CD8+ T cells in the tumor. Spleen analysis revealed higher NK cell populations in mice bearing Caki-1 tumors, while A549 tumor samples had a higher percentage of NK cells relative to the other models. Surprisingly, many of the tumor models showed an abundance of myeloid cells in the spleen and tumor compartments, although these were mostly comprised of B cells and dendritic cells and lower levels of macrophages and MDSCs. The expression level and percentage of cells expressing PD-1, and PD-L1 in the models were also determined. Furthermore, the antitumor response of select models to PD-1 and PD-L1 antibodies will be presented. In summary, these data demonstrate that there are tumor-intrinsic factors that influence the immune cell repertoire within tumors and spleen. These data may also aid in selection of tumor models to test antitumor activity of novel IO agents.

#1051

**Dianhydrogalactitol (VAL-083) reduces glioblastoma tumor growth upon bevacizumab-induced hypoxia,** in vivo **.**

Anna Golebiewska,1 Anais Oudin,1 Anne Steino,2 Simone P. Niclou,1 Jeffrey Bacha,2 Dennis M. Brown3. 1 _NorLux Neuro-Oncology Laboratory, Luxembourg, Luxembourg;_ 2 _Delmar Pharmaceuticals Inc., Vancouver, British Columbia, Canada;_ 3 _Delmar Pharmaceuticals Inc., Menlo Park, CA_.

Background: Standard-of-care for glioblastoma (GBM) includes surgery, radiation and temozolomide (TMZ). Nearly all tumors recur and 5-year survival is less than 3%. Unmethylated promoter status for O6-methylguanine-DNA-methyltransferase (MGMT) is a validated biomarker for TMZ-resistance. Second-line treatment with bevacizumab has not only failed to improve survival, but has also been shown to induce intratumor hypoxia, which is implicated in increased chemoresistance. VAL-083 is a bi-functional DNA-targeting agent that readily crosses the blood-brain barrier and accumulates in brain tumor tissue. VAL-083 induces DNA double-strand breaks at N7-guanine and cancer cell-death in GBM cancer stem cells (CSCs) and non-CSCs, independent of MGMT. We have previously shown that bevacizumab treatment upregulates expression of glucose transporters GLUT-1/GLUT-3 on GBM cells. We hypothesized that, based on its unique monosaccharide backbone structure, VAL-083 may benefit from bevacizumab-induced GLUT transporter upregulation leading to enhanced VAL-083 uptake and anti-tumor activity.

Methods: To investigate the in vivo anti-tumor effect of VAL-083+bevacizumab, we used a orthotopic patient-derived xenograft GBM model. All mice carried MGMT-unmethylated, temozolomide-resistant recurrent T16 GBM tumors as detected by MRI 35 days post-implantation. Mice were grouped into control, bevacizumab, VAL-083, and VAL-083+bevacizmab. Tumor progression was measured by MRI on days 49 and 56, and tumor growth rate was calculated for the entire study (day 35 vs. 56) and for the last 7 days (day 49 vs. 56).

Results: Tumors were significantly smaller in VAL-083-treated mice both compared to control (-83%, p<0.001) and compared to bevacizumab-treated (-75%, p<0.001) mice. Additionally, analysis of tumor growth in-time showed significantly reduced tumor growth rate for VAL-083+bevacizumab compared to VAL-083 alone (p<0.01).

Conclusions: These results show strong in vivo anti-tumor efficacy of VAL-083 against MGMT-unmethylated, TMZ-resistant recurrent GBM. This effect was further augmented in combination with bevacizumab, providing rationale of clinical investigation of VAL-083 in combination with bevacizumab in the treatment of GBM.

#1052

The human immune system reconstituted B-NDG mouse models are ideal tools for CAR-T and therapeutic antibody preclinical efficacy evaluation.

Qingcong Lin,1 Madeline Lee,1 Yanan Guo,2 Tian Gan2. 1 _Biocytogen, Wakefield, MA;_ 2 _Biocytogen, Beijing, China_.

The goal of our study is to establish mouse models for fast in vivo efficacy studies of biologics, such as CAR-T and therapeutic antibody candidates. B-NDG mice (NOD-Prkdcscid Il2rgtm1/Bcgen) were independently developed by Biocytogen to knockout the IL2rg gene in the NOD-SCID mouse background. Here we demonstrated that B-NDG, as one of the most severely immunodeficient mice, completely lack mature T, B, and NK cells, and were deficient in cytokine signaling.

We have established more than 24 CDX models using B-NDG mice, including human solid tumor and blood tumor cell lines, such as bladder carcinoma, breast cancer, colon carcinoma, colorectal adenocarcinoma, glioblastoma, liver cancer, lung carcinoma, and various leukemia and lymphoma cell lines. We have previously demonstrated that B-NDG mice inoculated with human tumor cells can be used for CAR-T therapy evaluation.

In addition, B-NDG mice are one of the best models for human immune system reconstitution using either PBMC or CD34+ hematopoietic stem cells. Human PBMCs reconstitute very well in B-NDG mice, with the percentage of hCD45+ cells reaching well over 50% within five weeks; moreover, the majority of these cells were CD3+ T lymphocytes. Human PBMC reconstituted B-NDG mice have also been successfully used for multiple therapeutic antibody efficacy studies. In order to reduce the GvHD, we have generated B-NDG B2M mice, on which the B2M gene was knocked-out. B-NDG B2M mice have a longer therapeutic window for PBMC reconstitution when compared with B-NDG mice, and have been successfully used for antibody efficacy studies.

Other than PBMCs, human CD34+ hematopoietic stem cells have been successfully grafted in B-NDG mice, in which all major human myeloid and lymphoid lineage cell types were fully developed, reconstituting a functional human immune system. At 10 weeks post CD34+ cell implantation, the percentage of hCD45+ cells reached over 25%, containing human T, B, and NK cells. These CD34+ reconstituted B-NDG mice are powerful tools for the fields of hematopoiesis, immunology, and autoimmunity, as well to evaluate clinically-relevant therapeutic antibody drugs in vivo.

Without any significant rejection of human-derived cells, the B-NDG mouse becomes an ideal model for transplantation of PDX, which retain the high heterogeneic properties typical of human tumor tissues. Using B-NDG mice, we have successfully established more than 300 PDX models, covering breast, colon, gastric, leukemia, lung, pancreatic cancers, and particularly blood tumor samples. Here we provide examples that use human immune reconstituted B-NDG PDX models to successfully evaluate the in vivo efficacy of human immune checkpoint antibodies in mono-or in combination therapy. In conclusion, we have developed more powerful and predictive mouse models for preclinical pharmacological evaluation of CAR-T and therapeutic antibodies.

#1053

A patient-derived xenograft model demonstarates the effectiveness of combined oxaliplatin and 5-fluorouracil for advanced small bowel adenocarcinoma.

Tomoki Yamano, Jihyung Song, Kei Kimura, Akihito Babaya, Michiko Hamanaka, Kozo Kataoka, Naohito Beppu, Masafumi Noda, Masataka Ikeda, Naohiro Tomita. _Hyogo College of Medicine, Nishinomiya, Japan_.

Background: Combination oxaliplatin and capecitabine therapy is recommended to treat advanced small bowel adenocarcinoma (SBA). However, SBA is rare, and limited preclinical studies and randomized clinical trials have been conducted.

Methods: A patient-derived xenograft (PDX) was established from an advanced SBA patient with liver metastasis and peritoneal metastasis. Growth inhibition by oxaliplatin, irinotecan, 5-fluorouracil (5-FU), and 5-FU in combination with oxaliplatin, irinotecan, or paclitaxel was assessed using the PDX. Drug sensitivity against oxaliplatin, irinotecan, 5-FU, and paclitaxel was assessed using SBA cells from the PDX. The patient's clinicopathological and genetic features were compared with the PDX to evaluate it as a preclinical model.

Results: 5-FU, but not oxaliplatin and irinotecan showed statistically significant growth inhibition compared to control group (P = 0.0024). Among the combination treatment with 5-FU, oxaliplatin showed the highest tumor growth inhibition rate of 0.84 or 1.1, which was 0.46 for irinotecan and 0.49 for paclitaxel, respectively. IC50 of SBA cells was significantly lower against oxaliplatin and higher against paclitaxel compared with colon cancer cell line HT29. These results corresponded to the clinical response to FOLFOX (folinic acid+5-FU+oxaliplatin) and FOLFIRI (folinic acid+5-FU+irinotecan). PDX pathological and genetic features resembled those of the patient.

Conclusions: We established a preclinical model comprising the PDX from SBA and SBA cells from the PDX. This model showed that the combination of oxaliplatin and 5-FU was a

reasonable standard therapy for advanced SBA. A preclinical model comprising PDX and tumor cells from PDX seems to be useful for rare tumors including SBA.

#1054

Systematic establishment of robustness and standards in xenograft experiments and analysis.

The NCI PDXNet Consortium, Yvonne Evrard,1 Jeffrey H. Chuang2. 1 _Leidos Biomedical Research, Inc, Frederick, MD;_ 2 _The Jackson Laboratory for Genomic Medicine, Farmington, CT_.

Patient-Derived Xenograft (PDX) models are a valuable model system for demonstrating clinical translatability in the preclinical testing of therapeutic agents. Large PDX collections such as the NCI PDXNet Consortium are a critical resource for assays of therapeutic efficacy. However, research groups vary in their procedures for experiments and assessments, limiting conclusions about the robustness of PDX results and their mapping to patient outcomes. To determine the sensitivity of PDX assays to different procedures, the PDXNet conducted a study of temozolomide drug response for PDX models derived from three patients, performed in replicate across four PDX Development and Trial Centers (PDTCs) with all sites blinded to the procedures and results of other groups. The three PDX models were chosen based on prior treatment experiments conducted by the NCI Patient Derived Models Repository that showed that one model was sensitive, one was resistant, and one had intermediate sensitivity. Each PDTC used their internally developed SOPs, which varied in their procedures for establishment of xenografts, treatment dosing and scheduling, time period of study, and response criteria. Additionally all groups followed internal sequencing procedures to generate one exome-seq and one RNA-seq per model. Drug responses were consistent across all five testing groups, with each group identically classifying the sensitive, resistant, and intermediate models. We also trained novel statistical classifiers for assessing sensitivity, resistance, and intermediate response on 105 retrospective cohorts of mice collected among the PDTCs. In addition, we compared and benchmarked exome-seq and RNA-seq analysis pipelines developed by each individual PDTC on synthetic test data. We assessed the robustness of sequencing assessement on PDX tumors by applying the optimized pipeline across the replicate samples generated across the separate PDTC groups. Overall, our results show that drug responses of PDX models are consistent and robust across different academic centers. We are developing standardized SOPs for experimental procedures, response measurement, statistical assessment of response, and sequence analysis based on these results. All SOPs will be released publicly through protocol descriptions and shared computational workflows within the NCI Cancer Genomics Cloud. We expect these PDXNet standards to improve the use of PDX and other in vivo models in advancing cancer precision medicine.

#1055

Evaluation of immune checkpoint inhibitor efficacy in EGFR mutant tumors.

Xuzhen Tang, Li Yang, Hui Qi, Xianzhi Zhai, Fuyang Wang, Xiangnan Qiang, Jie Xu, Xiaoran Qin, Qingyang Gu, Shaoyu Yan, Qunsheng Ji. _WuXi AppTec Co., Ltd., Shanghai, China_.

Tyrosine Kinase Inhibitor (TKI) of Epidermal Growth Factor Receptor (EGFR) is a first-line therapy for non-small cell carcinoma lung cancer patients with EGFR mutations, but patients often develop resistance to such a target therapy. Recently several PD-1 and PD-L1 inhibitors were approved for treating NSCLC, however, it remains unclear whether EGFR mutant NSCLC would benefit from PD-1/PD-L1 treatment. PD-1 response in cancer patients is closely associated with oncogenic mutation status, tumor microenvironment, PD-L1 expression and tumor mutation burden (TMB); however, the lack of pre-clinical models limited the investigation of the checkpoint inhibitors in EGFR mutant tumors. Here we use human PBMC reconstitution system for immune-humanization, and established a series of IO-Xenograft models (IO-CDX and IO-PDX) with cancer cell lines (IO-CDX) or patient-derived tissues (IO-PDX) that contain various EGFR mutations, including EGFR 19 del, L858R, T790M, and EGFR over-expression, amplification and fusion. We treated these models with either EGFR TKI (erlotinib, afatinib or osimertinib) or nivolumab, a PD-1 inhibitor. The predictive biomarkers including PD-L1 expression and TMB were analyzed by whole exom sequencing (WES) and FACS for tumor infiltrating lymphocytes (TILs), respectively. We found that 3 TKI-resistant PDX models bearing EGFR T790M mutation exhibited lower TILs than those bearing EGFR wild type or exon 19del mutation. Of the 8 IO-CDX or IO-PDX humanized models we have examined, those exhibiting higher TILs achieved better responses to nivolumab treatment. The 3 most sensitive models are EGFR amplification (wt), EGFR over-expression (wt) and EGFR exon 19del. In contrast, the EGFR T790M model from H1975 cell line was observed with hyper-progressive disease after nivolumab treatment. We will also discuss the contribution of PD-L1 expression and TMB to nivolumab in other models. In summary, these well-established IO-CDX and IO-PDX models can provide a human-resembling immune system for of immune therapeutics and combination with targeted therapies, and help to facilitate the understanding of relationship among tumor microenvironment, driver oncogenic mutations and drug response.

#1056

Xenograft-associated B cell lymphoproliferative disease (XABLD) as a surrogate model to study Epstein-Barr virus (EBV) driven B cell Diseases.

Tomas Vilimas,1 Gloryvee Rivera,1 Brandie Fullmer,1 Wiem Lassoued,1 Lindsay Dutko,1 Amanda Peach,1 Corinne Camalier,1 Li Chen,1 Rajesh Patidar,1 Suzanne Borgel,1 John Carter,1 Howard Stotler,1 Raymond Divelbiss,1 Jesse Stottlemyer,1 Michelle M. Gottholm-Ahalt,2 Michelle Crespo-Eugeni,2 Sean McDermott,1 William Jacob,1 Liqiang Xi,3 Pallavi Galera,3 Yvonne A. Evrard,1 Melinda G. Hollingshead,2 Elaine S. Jaffe,3 Mark Raffeld,3 Biswajit Das,1 Chris Karlovich,1 Vivekananda Datta,1 James H. Doroshow,4 P. Mickey Williams1. 1 _Frederick National Lab for Cancer Research, Leidos Biomed. Research, Inc., Frederick, MD;_ 2 _Frederick National Lab for Cancer Research, Frederick, MD;_ 3 _National Cancer Institute, Bethesda, MD;_ 4 _National Cancer Institute, Frederick, MD_.

Background: Patient-derived tumor xenografts (PDX) are powerful tools to study cancer biology, cancer genomics and developmental therapeutics. A common problem in the development of PDX models is proliferation of atypical lymphocytes at the implantation site, which often overtake or limit the growth of the original tumor. This atypical lymphocyte proliferation has been described as XABLD in our PDX models. In this study, we characterized XABLD cases by morphology, immunophenotyping and genomic profiling. We hypothesize that XABLD tumors are morphologically and phenotypically similar to EBV-driven post-transplant lymphoproliferative disease (PTLD) and diffuse large B cell lymphoma (DLBCL). XABLD is a surrogate model to study EBV-driven PTLD and DLBCL.

Materials and Methods: Models were generated from patient tissue collected under NCI Tissue Procurement Protocol (clinicaltrials.gov: NCT00900198) and CIRB Tissue Procurement Protocol 9846 for development of models for NCI's Patient-Derived Models Repository (https://pdmr.cancer.gov). Specimens were implanted subcutaneously in NOD/SCID/IL2Rg null (NSG) mice and animal health was monitored throughout the study. Tumors in mice with suspected XABLD were harvested and reviewed by histology and immunohistochemical analysis for CD45, B and T cell markers, EBV status, B-cell clonality assay. All samples were also classified by the Lymph2Cx NanoString cell of origin assay and transcriptome profiling.

Results: XABLD cases were found to originate from both solid tumor and circulating tumor cell implants. XABLD is a rapidly growing tumor positive for CD45, CD20, and LMP1 stains, 36 of 42 cases are strongly positive for PD-L1 stain. 39 of 42 cases exhibited an activated B cell (ABC) phenotype with evidence of elevated NF-kB signaling. Most cases were monoclonal for IGK/IGH and contained high numbers of tumor infiltrating CD8-positive T-cells with associated high mRNA expression of activated T cell markers.

Conclusion: The clinical presentation, morphology and molecular characteristics of XABLD cases were similar to EBV-driven DLBCL. As the XABLD models exhibited frequent PD-L1 expression and marked infiltration of CD8-positive T cells, they may be useful for in vitro evaluation of checkpoint inhibitor response and T cell antitumor activity.

Grant Support: This project has been funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

#1057

PDX models of solid cancer express distinct cytokine profiles in humanized mice relating to their tumor infiltrating lymphocyte landscape.

Eva Oswald, Dorothee Lenhard, Stefanie Schmitt, Anna Edinger, Anne Löhr, Volker Knauff, Anke Behnke, Anne-Lise Peille, Julia Schüler. _Charles River Research Services Germany GmbH, Freiburg, Germany_.

Model selection is one critical step in anti-cancer drug development. In the advent of immune modulatory compounds being part of most of the oncology drug development pipelines, the available preclinical models have to be characterized with respect to those assets. In the current study we analyzed 26 PDX models covering a broad range of solid cancer entities with regard to the phenotypic characteristics in a humanized environment. Well established PDX models from six different entities (breast, colon, renal, pancreatic, NSCL cancer and glioblastoma) were implanted subcutaneously into immune-compromised mice substituted with CD34+ hematopoietic stem cells (hu-mice). Tumor characterization included: tumor growth curves, take rates, human immune cell infiltrates of tumor (=TILs), peripheral blood, spleen and bone marrow (determined by flow cytometry, FC, and IHC) and secretion of human cytokines in murine serum (determined by cytokine array & Luminex based technology). Those data were compared to equivalent data-sets in non-humanized mice. The analysis of TILs revealed a large bandwidth of immune cell infiltration in the respective model ranging from 94% huCD45+ cells (renal cancer RXF 2773) to 3.54% huCD45+ (NSCLC LXFA 1041). Based on the percentage and of huCD4+ cell engraftment and the ratio of their subtypes (memory vs naïve) in untreated tumors the models were grouped into high (=hot), medium and low (=cold) infiltrated tumor models. The infiltration rate was stable across different experiments and specific for one distinct model. IHC analyses confirmed infiltration rates in tumor tissue determined by FC for CD45+, CD4+ and CD8+ cells. Furthermore, the models previously defined as "hot" or "cold" expressed distinct cytokine profiles in serum of tumor bearing mice. Amongst other cytokines, hot tumors secreted high levels of IL-17A, IL-18 BPa, MMP-9, Osteopontin, CD31, TIM-3 and YKL-40. In contrast, levels of GDF-15 and Lipocalin-2 were down regulated in those models compared to their cold counterpart. Of note, neither the amount nor the composition of human immune cell populations in peripheral blood of those mice was suitable to conclude on the amount of TILs in the respective model. Further validation of the cytokine profiles using immune modulating compounds on hot as well as cold PDX models are currently underway. Taken together, the characterization of PDX models with respect to their TIL composition facilitates model selection for innovative drugs in the immune-oncology field. The possibility to discriminate hot vs cold tumors by a minimal-invasive method like serum analysis of cytokine expression profile as potential biomarker will help to characterize the complete PDX collection of more than 500 models but will as well serve as additional read-out in preclinical I-O studies.

#1058

Establishment of patient-derived xenografts from patients with gastrointestinal stromal tumors: Analyses of clinopathological characteristics related with success.

Young-Soon Na,1 Min-Hee Ryu,2 Young Soo Park,2 Chae-Won Lee,1 Ju-Kyung Lee,1 Yangsoon Park,2 Jung Min Park,1 Jungeun Ma,2 Yoon-Koo Kang2. 1 _Asan Inst. for Life Science, Seoul, Republic of Korea;_ 2 _Asan Medical Center, Seoul, Republic of Korea_.

BACKGROUND: The gastrointestinal stromal tumor (GIST) has activating mutations in either KIT or platelet-derived growth factor receptor alpha (PDGFRa) gene, and tyrosine kinase inhibitors (TKIs) such as imatinib, sunitinib and regorafenib remain the mainstay of anti-GIST treatment. Patient-derived xenografts (PDXs) are useful preclinical models in cancer research, owing to their demonstration of more real tumor heterogeneity and complexity, compared with cell lines and cell line-based xenograft models. PDX models have been established using numerous tumor types, however, there are only a few PDX models of GIST because of its very low success rate. As we have been establishing PDXs from GIST patients since 2012, in this study, we report the established PDXs and the clinopathological characteristics related to the successful establishment of GIST PDXs.

MATERIALS and METHODS: PDXs have been established in NOD-scid Il2rg-/- (NSG) mice by implanting GIST tumor fragments from 185 patients who underwent surgical resection prior to and after tyrosine kinase inhibitors from July, 2012 to July, 2017. The established PDXs passaged greater than F2 generation. Chi-square test and logistic regression were used for comparison.

RESULTS: Of a total of 185 patients, 66 (35.7%) patients were TKI-naïve, 21 (11.4%) had residual disease after control with TKIs, and the remaining 98 (53.0%) showed disease progression after TKIs at the time of surgical resection. The success rate of establishment of GIST PDXs was 16.8% (31/185). In univariate analyses, a higher engraftment rate was observed for tumors derived from patients with disease progression after TKIs (TKI-naïve vs residual disease vs progressive disease; p<0.001), larger tumor size (≤50 mm vs 50-100 mm vs >100 mm; p<0.001), more mitotic count (≤10/50 HPFs vs >10/50 HPFs; p<0.001), higher Ki-67 index (<1/3 vs ≥1/3; p<0.001), higher cellularity (low vs high; p<0.001), or tumor necrosis (absence vs presence; p=0.001). In addition, PDX engraftment success rate was higher with tumors harboring primary mutation in KIT exon11 (vs other mutations; p=0.025) or with metastatic tumor lesions (vs primary site; p<0.001). In multivariate analysis including significant factors in the univariate analyses, Ki-67 index (p=0.001) and largest tumor size (p=0.058) were independent factors for success of PDX establishment.

CONCLUSION: Clinicopathologic factors such as disease progression after TKIs, larger tumor size, more mitotic count, higher Ki-67 index, higher cellularity or tumor necrosis were associated with higher success rate of PDX establishment. Especially, largest tumor size and Ki-67 index were independent factors for successful PDX engraftment. These findings will be helpful to establish PDX models more efficiently in GIST.

#1059

A case study: OncoRat is a viable patient avatar for a NSCLC patient with a Y1248H Met activating mutation.

Fallon K. Noto,1 Bisoye Towobola Adedeji,1 Sam Moody,1 Chris Brenzel,1 Jack Crawford,1 Goutham Narla,2 Tseten Yeshi Jamling1. 1 _Hera BioLabs, Lexington, KY;_ 2 _University of Michigan, Ann Arbor, MI_.

Human cancer xenografts in rodents can provide predictive data on the success of candidate drugs in clinical trials and have been a pivotal tool in moving new drugs from the bench to the clinic. However, currently available immunodeficient mouse models have shown some limitation and variability in tumor take rates and growth kinetics in cancer cell lines. In addition, commercially available human cancer cell lines aren't representative of the genomic and molecular diversity of cancers found in patients.

Patient Derived Xenograft (PDX), in which tumor tissue is transplanted directly into rodents after biopsy from the patient, better represents that molecular signature, heterogeneity, and pathology of the original tumor. Therefore, in vivo efficacy studies with PDX models could be highly predictive for treatment sensitivity. Despite the many advantages of PDXs for preclinical research, PDX mouse models are hindered by low engraftment rates and slow tumor growth kinetics. The loss of patient tumor heterogeneity and stromal cells as the PDX is passaged multiple times to generate sufficient tumor tissue to inoculate a cohort of animals for efficacy studies is also a disadvantage in the immunodeficient mouse models.

To address these limitations, we have introduced the OncoRat®; built on the SRGTM Platform, a Sprague-Dawley Rag2/Il2rg double knockout rat that lacks mature B cells, T cells, and circulating NK cells. We have demonstrated that the OncoRat has improved tumor take rate and growth kinetics for non-small cell lung cancer (NSCLC) PDXs. The NSLSC PDXs in the OncoRat have a much larger tumor volume, over 20,000 mm3 in the first passage (P0) in the rat, which provides an ample source of tissue for characterization and/or subsequent passage (P1) into OncoRat for drug efficacy studies. This leads to fewer animals used for study and faster timelines to drug efficacy data, resulting in a reduction in cost. In addition, we have used genomic analysis for guidance in planning in vivo efficacy studies. One of our NSCLC PDX models harbors a novel mutation in the MET pathway, suggesting this tumor would not be responsive to standard of care treatment. An efficacy study we performed in the OncoRat suggests that this particular tumor would respond well to Type II MET inhibitors, such as Cabonzantinib. This proof of concept study demonstrates that genomic and molecular analysis can provide insight into treatment outcomes and that PDX models in the OncoRat could serve as patient avatars for predicting treatment outcomes.

#1060

Establishment of a panel of prostate patient derived xenograft (PDX) models and evaluation of anti-androgen therapy.

Jason Davis,1 Anthony Oakden,1 Jane Wrigley,1 Chira Roberts,1 W Qian,2 Bin Fan,2 A Collins,3 Davy Ouyang,2 Jie Cai,2 Rajendra Kumari,2 Yinfei Yin1. 1 _Crownbio Science UK, Loughborough, United Kingdom;_ 2 _Crownbio Science Inc, Beijing, China;_ 3 _University of York, York, United Kingdom_.

BACKGROUND: Prostate cancer is one of the most common cancer worldwide with limited treatment options and very poor prognosis. Surgery, chemo therapy such as docetaxel (Taxotere®) and targeted therapies such as abiraterone (Zytiga®) and enzalutamide (Xtandi®) are the options available for prostate cancer patients but they all have their limitations. The development of new prostate cancer therapy has been slow due to the lack or preclinical models that adequately represent the spectrum of benign, latent, aggressive, and metastatic forms of the human disease. PDX have been reported to be more clinically relevant that cell lines but the generation of prostate PDX models has always been challenging. Here we report the establishment and validation of a panel of prostate PDX models and their utilization in preclinical studies which will help prostate cancer research.

MATERIAL & METHODS: Primary prostate cancer samples from patients undergoing radical prostatectomy in the UK were collected with ethical consent. These samples were then inoculated subcutaneously in Rag2-/-gC-/- mice (Jackson Laboratory) to generate PDX models. Subcutaneous tumor growth was evaluated and monitored by electronic callipers. Successfully established PDX tissue were collected for RNA sequencing and immunohistochemistry (IHC) for key prostate markers. PDX were also tested for sensitivity to docetaxel (Taxotere®), abiraterone and enzalutamide in vivo in mice bearing subcutaneous tumours. Tumours were samples pre and post dosing as well as blood samples collected.

RESULTS: A panel of prostate PDX models have been established and expanded. Two models were established using tumour tissue from patients diagnosed with castrate resistant prostate cancer (CRPC) and two models were from patients who showed hormone sensitivity. Histologically the structure of the original patient sample was retained in the PDX models. These models also showed high KLK3 (PSA) expression levels by RNA sequencing and IHC staining as well as androgen receptor expression. One of the CRPC models showed a TMPRSS-ETS fusion, response to docetaxel (p<0.0001, Two way ANOVA) in vivo and poor response to abiraterone and enzalutamide, whereas the second CRPC model showed no response to any of the agents tested.

CONCLUSIONS: We have established and characterised a panel of prostate PDX models which provide unique and clinically relevant models for preclinical drug evaluation for prostate cancer.

#1061

Animal models of prostate cancer.

Frida Ponthan, Matthew Brown, Emma Playle, Catherine Booth. _Epistem Ltd, Manchester, United Kingdom_.

Background: Prostate cancer is the most common male malignancy and the second most common cause of cancer-related deaths in the United Kingdom and the United States. It's a heterogeneous disease caused by different genetic mutations where the majority of prostate cancers are indolent and slow growing. However, some tumours are aggressive with the ability to invade local structures and metastasise leading to poor clinical outcome. One of the most important aspects in anti-cancer drug development is the availability of robust preclinical models which allow the efficacy of novel therapies to be examined. We have developed a reproducible, well growing and robust subcutaneous model of LNCaP with an effective response to both conventional standard of care (SOC) chemotherapy and androgen receptor inhibitors.

Materials and Methods: Cells were subcutaneously implanted on the flank of NSG mice and treated with an anti-androgen with or without conventional chemotherapy starting after 15-20 days when the mean tumour volume was 150-200mm3. Animal weight and tumour volume was measured 3x weekly. Androgen receptor (AR) expression was analysed with immunohistochemistry and gene expression analysis before and after treatment.

Results: The LNCaP cell line grew well in vivo with 100% take rate. Vehicle treated tumours doubled in size over the first 6 days of treatment and were 4.5x the mean starting volume by day 17 post-treatment start, when the group was removed from the study. Significant tumour growth inhibition was observed in response to both conventional and anti-androgen receptor treatment consistent with the maintained AR expression confirmed by gene expression analysis.

Conclusion: This high take rate responsive model represents a significant improvement in subcutaneous xenograft models of prostate cancer, and allows the comparison of AR responsive therapies, alone or in combination with conventional SOC or irradiation. Current protocols employing luciferase transfected cells has also enabled evaluation of metastatic disease.

#1062

Development and characterization of patient-derived xenograft models for acute myeloid leukemia.

Michelle Ulrich, Martha Anderson, Robert Thurman, Fu Li. _Seattle Genetics, Inc., Bothell, WA_.

Drug development for acute myeloid leukemia (AML) remains challenging, in part, due to the lack of preclinical models that reflect the biology of AML patients. For example, while the majority of AML patients express both CD33 and CD123, only a small fraction of AML cell lines express CD123. Similarly, most AML cell lines have a differentiated phenotype, which may bias the evaluation towards certain types of AML. To address these challenges, we developed a dozen patient derived xenograft (PDX) models to aid preclinical development of AML drugs. Bone marrow samples from 32 AML patients were implanted intravenously into NSG mice. Thirteen of them led to successful expansion of human CD45+ cells in the bone marrow, as demonstrated by flow cytometry. Twelve of these established disease in a secondary passage. Using multicolor flow cytometry analysis, we found that expression of CD33 and CD123 were preserved from AML patients and engrafted disease in NSG mice. Moreover, RNA-sequencing analysis demonstrated that gene expression largely remained consistent between the first two passages. Detailed genetic analysis demonstrated the preservation AML-relevant mutations, such as FLT3-ITD mutations and IDH2 mutations in the PDX model. In summary, we have successfully established AML PDX models that reflect the antigen expression and molecular genetics of AML patients. These models may facilitate the future development of antibody-based therapeutics.

#1063

In vivo **humanized immuno-oncologymodels for drug discovery.**

Jijun Cheng, Feifei Zhang, Yuan Long, Wenhua Xu, Hongkui Chen, Danyi Wen. _LIDE Biotech, Shanghai, China_.

Reliable translational, especially humanized mouse models are critical to evaluate efficacy of immuno-therapeutics. While transgenic humanized mice and human CD34+ HSC-reconstituted mice are often used, their human cellular or molecular immune components are limited or defective. Human peripheral blood monocytic cell (PBMC)-reconstitution in immunocompromised mice, although, is transient and limited in infiltrating xenografts, one can manipulate to delicately reconstitute tumor microenvironment with one type, human T cells, or a few types of human immune cells, dendritic and macrophages, from human PBMC, through either systemic reconstitution or local reconstitution. We found that optimized stable PBMC models, both CDX and PDX, can often meet needs of targeting by many developing IO antibody therapeutics, including bi-specifics, in comparison to other humanized models. In particular, for efficacy study and for the convenience of mouse trials, we arrayed panels of PDX models expressing PD-L1 at different levels, in lung, liver, endometrial and gastric cancers, and panels of MSI-H and/or dMMR positive PDX models in these cancer types. In aiding studies of efficacy and resistance, we establish clinically drug-resistant PDX models, such as those resistant to checkpoint antibody drugs. These models are valuable to evaluate efficacy not only for antibody agents but also for combinational therapies involving biotherapeutics such as oncolytic virus.

#1064

PDX Like Me: A molecular profile-based search tool.

Dale A. Begley, Debra M. Krupke, Steven B. Neuhauser, Joel E. Richardson, John P. Sundberg, Carol J. Bult. _The Jackson Laboratory, Bar Harbor, ME_.

Patient Derived Xenografts (PDXs) are generated through the engraftment of human tumor tissue into a specialized (usually, immunodeficient) mouse host strain. PDXs are a useful pre-clinical platform for evaluating the efficacy of single agent and combination therapies that are targeted to specific genomic characteristics of a patient's tumor. We have implemented the PDX Like Me search tool to assist researchers in identifying PDX models whose tumors match - or are similar to - molecular profiles comprised of one or more genes and one or more genomic characteristics (e.g., copy number status, mutation, and expression). The PDX Like Me interface uses a search syntax similar to the Onco Query Language used by the popular cBioPortal resource.

PDX Like Me is one of several search interfaces supported by the Mouse Models of Human Cancer database (formerly, the Mouse Tumor Biology database). MMHCdb (http://tumor.informatics.jax.org) is a comprehensive resource of information on both genetically engineered mouse models (GEMMs) and PDX models of human cancer that has been expertly curated from peer-reviewed scientific publications and direct data submissions from individual investigators and large-scale programs. MMHCdb provides an easy-to-use search interface as well as tools for visualizing associated data from these models. Information in the database is standardized using controlled vocabularies and official gene and mouse strain nomenclature. MMHCdb contains data from spontaneous or endogenously induced tumors from genetically defined mice. MMHCdb holds data on over 7,500 different strains including over 93,000 tumor frequencies and over 2,250 pathology reports with over 6,300 images from over 4,600 references. MMHCdb provides access to clinical, pathology, dosing study results, and genomics data from over 450 PDX models distributed by The Jackson Laboratory. MMHCdb in collaboration with EMBL-EBI has also co-developed the PDX Finder resource to provide a comprehensive global catalog of PDX models available for researchers.

MTB is supported by NCI grant CA089713.

#1065

**Comprehensive molecular and experimental characterization of ovarian clear cell carcinoma cell lines for** in vivo **drug development.**

Yasuto Kinose,1 Dorothy Hallberg,2 Kai Doberstein,1 Gordon Mills,3 Tan Ince,4 Victor Velculescu,2 Fiona Simpkins,1 Ronny Drapkin1. 1 _University of Pennsylvania Perelman School of Medicine, Philadelphia, PA;_ 2 _Johns Hopkins University School of Medicine, Baltimore, MD;_ 3 _Oregon Health and Science University, Knight Cancer Institute, Portland, OR;_ 4 _University of Miami, Miami, FL_.

Purpose: Ovarian cancer is heterogeneous with multiple histological subtypes and a wide range of genetic aberrations. While high-grade serous ovarian carcinoma (HGSOC) is the most common, clear cell ovarian carcinoma (CCOC) is the most challenging to treat and exhibits low response rates to standard therapies. However, efforts to understand CCOC and develop new therapies have been limited because it represents a minority of ovarian cancers in the U.S. and Europe. In contrast, CCOC accounts for approximately 30% of all ovarian cancer in Japan. To improve the survival of patients with CCOC, a deeper understanding of the molecular features that define available model systems is needed. Our goal is to comprehensively characterize a panel of CCOC lines using next generation sequencing and functional in vitro and in vivo experiments to define the lines that are most faithful to CCOC and are tractable for subsequent in vivo drug discovery.

Method: We obtained 9 CCOC cells from ATCC, Riken Cell Bank, and University of Miami (ES-2, TOV21G, OVTOKO, OVMANA, OCI-C5x, JHOC-5, JHOC-7, JHOC-9, and OVISE). Genomic DNA, RNA, and protein were isolated and subjected to whole exome DNA-seq, RNA-seq, and reverse phase protein array (RPPA), respectively. We performed in vitro MTT assays to test the sensitivity of these lines to chemotherapies. Tumorigenicity was evaluated by injecting 5 million cells of luciferized CCOC lines into NSG female mice using both the subcutaneous route and the intraperitoneal route. Imaging was performed weekly using the In Vivo Imaging System.

Results: ARID1A mutation, the most prevalent mutation in CCOC, is present in 8 of the 9 cell lines (TOV21G, OVTOKO, OVMANA, OCI-C5x, JHOC-5, JHOC-7, JHOC-9, and OVISE), whereas the second common mutation in CCOC (PIK3CA) is detected in 5 lines (TOV21G, OVMANA, OCI-C5x, JHOC-7, and OVISE). The ES-2 cell line has both TP53 and BRAF mutations and its genomic profile is not typical of CCOC. Principal component analysis of RPPA showed distinct groups between the 9 CCOC lines and the 6 HGSOC lines. Interestingly, we also observed two distinct clusters within the CCOC lines. Consistent with our genomic analysis, the ES-2 cell line correlated more closely with the HGSOC lines based on RPPA data. In our in vitro drug studies, OVTOKO and OCI-C5x exhibited resistance to Carboplatin/Paclitaxel. In xenograft study, 4 cell lines (ES-2, TOV21G, OVTOKO, and OCI-C5x) formed measurable tumor within a month. In contrast, OVMANA, JHOC-7, JHOC-9, and OVISE took over 100 days to form tumors.

Conclusion: Our data suggests that there may exist two functionally distinct groups within CCOC that warrants further study. In vitro and in vivo studies identified 4 cell lines that represent tractable models for rigorous therapeutic studies: ES-2, TOV21G, OVTOKO, and OCI-C5x. However, ES-2 appears to cluster more closely with HGSOC and may not represent the CCOC histotype.

#1066

Drug resistant PDX models for testing targeted drug sensitivity.

Jijun Cheng,1 Feifei Zhang,1 Zhen Zhou,2 Yuan Long,1 Wenhua Xu,1 Shizhu Zhao,1 Hongkui Chen,1 Shun Lu,1 Danyi Wen1. 1 _LIDE Biotech, Shanghai, China;_ 2 _Shanghai Chest Hospital, Shanghai Jiaotong University, Shanghai, China_.

Advanced lung cancer of late stage patients is selectively sensitive to available targeted therapeutics and often develops resistance to early sensitive drug treatment, thus, the patient-derived xenograft (PDX) models are critical to evaluate drug sensitivity and resistance. We established PDX models from routine surgery tissues, as well as from tumor cells in pleural effusion specimens and from biopsies of advanced cancer patients who are refractory to treatments. Genetic profiling of the PDX lung models revealed a group of them with TKI drug-associated EGFR mutations (L858R, T790M, Exon19del, C797S). Among them, are an AZD9291-resistant sub-group with EGFR double mutations (Exon19∆, T790M) in the clinic before AZD9291 treatment, and yet, acquired resistance to AZD9291 either through emergence C797S mutation or T790M loss in EGFR or through mutations in other pathways, providing insights into potential combo therapies. Indeed, combinations of AZD9291 with Cetuximab as well as with inhibitors targeting other signaling pathways such as MEK, are efficacious against an AZD9291-resistant lung PDX model with EGFR triple mutations (Exon19∆, T790M, C797S). We also establish valuable lung models resistant to ALK inhibitors, Avastin, PD-1 antibody drugs, and resistant hematopoietic models. In summary, functional drug sensitivity profiling and genetic profiling with drug resistant PDX models provide insights into resistance mechanisms as well as precise therapeutic options against resistance emergence.

#1067

A patient-derived xenograft (PDX) platform for cancer translational, precision medicine and health disparity research.

Chong-Xian Pan,1 Hongyong Zhang,1 Ai-Hong Ma,1 Shuxiong Zeng,1 Maike Zimmermann,1 Clifford Tepper,1 Paul Henderson,1 Luis Carvajal-Carmona,1 Regina Gandour-Edwards,1 Moon Chen,1 Susan Airhart,2 Ralph de Vere Whtie1. 1 _UC Davis Comp. Cancer Ctr., Sacramento, CA;_ 2 _The Jackson Laboratory, Sacramento, CA_.

Introduction and Objective: This purpose of this program is to develop and characterize PDXs from multiple organ sites and patients of multiple ethnic backgrounds, and to make these resources available to the scientific community for translational, precision medicine, and health disparity research.

Methods: PDXs were directly developed from patient cancer specimens, molecularly characterized with next-generation sequencing, and annotated with clinical information. Various aspects of its applications were tested.

Results: UC Davis, working in collaboration with The Jackson Laboratory (JAX), has developed PDX models, reflecting a wide range of tumor types, from 150 of our patients. Each of these models is annotated with clinical and genomic information. The fidelity of these PDXs was validated by the retention of histopathological features and the conservation of genetic aberrations (i.e., 92-97%) of the original patient tumors in bladder cancer PDXs tested. Extensive preclinical studies showed that these PDXs could potentially be used to screen multiple therapeutic agents simultaneously to identify the most efficacious drugs or drug combinations, re-purpose FDA-approved drugs, decipher the mechanisms of primary resistance, decrypt the mechanisms of secondary resistance, guide drug development, and identify biomarkers. For drug repurposing, the EGFR/ERBB2 dual inhibitor lapatinib effectively prolonged the overall survival (OS) of mice carrying ERBB2+ bladder cancer PDXs from 18.4 days to 25.4 days (p=0.007). For screening of targeted therapy in a PDX carrying PI3K, ERBB2 and Src alterations, only a PI3K inhibitor (BEZ235) prolonged the OS (p<0.0001) while lapatinib and a Src inhibitor ponatinib had no effect. For biomarker development, DNA adduct levels correlated with cancer response to alkylating agents and this project has already been translated into a clinical trial. For determination of the mechanisms of secondary resistance, loss of tumor suppressor gene LSP1 expression was associated with secondary resistance to an inhibitor of the PI3K pathway which is commonly mutated in many cancers. With the support of the Minority PDX Development and Trial Center (M-PDTC) U54 grant, UC Davis will continue to establish over 200 PDX models from bladder, lung squamous cell, gastric and hepatocellular carcinoma, with >60% of the specimens coming from minority patients, for health disparity research.

Conclusions: PDXs have great potential for cancer translational, precision medicine and health disparity research. The NCI-funded U54 center with minority PDXs is open for collaboration through the PDX Development and Trial Centers Research Network (PDXNet).

#1068

How humanized mouse models could be useful for immuno-oncology research.

Jean-Francois Mirjolet, Josselin Caradec, Oliver Duchamp, Francis Bichat, Damien France, Caroline Mignard, Fabrice Viviani. _Oncodesign S.A., Dijon, France_.

Immunotherapy is one of the most exiting recent breakthroughs in the field of cancer treatment. Different approaches are developed such as cancer vaccines, adoptive cellular immunotherapy or immune checkpoint blockade, and a number have been regulatory approved or are currently investigated in clinic. Effective immunity against cancer involves complex interaction between the tumor, the host and the environment. The assessment of cancer immunotherapy approaches in preclinical settings requires the use of appropriate animal models that sufficiently reflect the physiological situation in humans and that must be chosen carefully to address specific mechanisms of action. In light of evaluating the therapeutic potential of different immunomodulatory agents in mice xenografted with cancer cell lines or patient-derived xenografts, we developed multiple humanization strategies on different immuno-deficient mouse strains. Either human PBMCs, Hematopoietic Stem Cells (HSCs), or specific human immune cells such as Dendritic Cells (DCs), T cells, subset of T cells (e.g. gamma9 delta2 T cells) and NK cells were used, but also the combinations of immune subpopulations such as the co-transfer of autologous T cells and DCs. We will highlight some results of our therapeutic efficacy investigation and evaluation of novel immuno-oncology therapies eg. adoptive cell therapy, bispecific T-cell engager antibody, and vaccine immunomodulatory agents in such humanized mouse models, assessing survival, tumor growth and effects on tumors and immune cells by flow cytometry and immunohistochemistry analyses. All these different humanized models can permit to evidence significant antitumor efficacy of immunomodulatory agents in vivo and might help improve the success rate in clinical trials.

#1069

New panel of patient derived lymphoma xenografts (PDX) for preclinical research and immune oncology.

Bernadette Brzezicha,1 Michael Becker,1 Maria Stecklum,1 Teresia Conrad,1 Martin Janz,2 Aitomi Bittner,3 Clemens Schmitt,3 Ulrich Keilholz,4 Jens Hoffmann1. 1 _Experimental Pharmacology & Oncology Berlin-Buch GmbH, Berlin-Buch, Germany; _2 _Max Delbrueck Center for Molecular Medicine and Charité, University Hospital Berlin, Campus Benjamin Franklin, Berlin-Buch, Germany;_ 3 _Charité Universitätsmedizin Berlin Campus Virchow-Klinikum, Berlin, Germany;_ 4 _Charité Comprehensive Cancer Center, Charité, Berlin, Germany_.

Lymphomas represent a very heterogeneous group of hematologic malignancies and pose an important challenge in the clinical routine. They frequently develop resistance to the treatment with standard-of-care (SoC) drugs and have a high incidence of disease recurrence.

Recent progress in molecular high throughput profiling has helped to identify genetic drivers and genetic subtypes of diffuse large B-cell lymphomas (DLBCL). Further target validation and drug development projects are highly dependent on corresponding preclinical models representing the different clinical subtypes. Therefore, we started to establish and characterize new patient derived xenografts (PDX) of non-Hodgkin (NHL) and Hodgkin lymphomas (HL) for drug development, translational and immune oncology research.

All lymphoma PDX were derived from peripheral blood, lymph node extirpations or core needle biopsies, and were usually transplanted subcutaneously into immunodeficient mice.

For further characterization, established lymphoma PDX models were treated with SoC and investigational drugs. In addition, the growth of lymphoma PDX on humanized mice was evaluated to establish new models for the evaluation of novel immune therapy approaches. To gain a deeper insight in the molecular biology of the lymphoma models, RNA sequencing of all was performed.

More than 20 new PDX models from NHL (including 8 DLBCL) and HL have been successfully established and characterized. Heterogeneous individual responses to the treatments were observed. Explorative analysis of RNA sequencing data confirmed the representation of the clinical lymphoma subgroups in our panel of models. Likewise, genetic subtypes of DLBCL COO classes could be confirmed by PCA and hierarchical clustering methods. Patterns of genetic driver mutations as revealed by recently published large genomic studies were identified.

Our newly and extensively characterized lymphoma PDX model panel allows the evaluation on novel targeted and immune therapies in preclinical phase II trial settings. They provide an exceptional platform for the identification and validation of new targets and allow the preclinical screening of new combinations in translational research projects.

#1070

**Evaluation of human NK cell responses to PDX tumors in humanized NOD-** scid IL2rgnull **(NSG) mice expressing human IL15.**

Ken-Edwin Aryee,1 Lisa Burzenski,2 Dale L. Greiner,1 Giles F. Whalen,1 Li-Chin Yao,3 Leonard D. Shultz,2 James G. Keck,3 Michael A. Brehm1. 1 _The University of Massachusetts Medical School, Worcester, MA;_ 2 _The Jackson Laboratory, Bar Harbor, ME;_ 3 _The Jackson Laboratory, Sacramento, CA_.

Human innate immunity plays a critical role in tumor surveillance and in immunoregulation within the tumor microenvironment. Natural killer (NK) cells are innate lymphoid cells that have opposing roles in the tumor microenvironment, including NK cell subsets that mediate tumor cell cytotoxicity and subsets with regulatory function that contribute to the tumor immune suppressive environment. The balance between effector and regulatory NK cell subsets has been studied extensively in murine models of cancer, but there is a paucity of models to study human NK cell function in tumorigenesis. Humanized mice are a powerful alternative to syngeneic mouse tumor models for the study of human immuno-oncology and have proven effective tools to test immunotherapies targeting T cells. However human NK cell development and survival in humanized mice are severely limited. To enhance NK cell development, we have developed NSG mice that constitutively expresses human IL15 using a BAC containing the human IL15 gene: NSG-Tg(Hu-IL15) (NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(IL15)1Sz/SzJ; JAX stock number 030890). NSG-Tg(Hu-IL15) mice express a physiological level of human IL15 (7.1 ± 0.3 pg/ml) and support engraftment of human CD34+ hematopoietic stem cells (HSC). Following HSC-engraftment of NSG-Tg(Hu-IL15) mice, significantly higher levels of human CD56+ NK cells are detectable as compared to NSG mice in peripheral blood and within the spleen and bone marrow. Levels of circulating human CD3+ T cells, CD20+ B cells and CD33+ myeloid cells are similar between the HSC-engrafted NSG-Tg(Hu-IL15) and NSG mice. We have described that the human NK cells developing in HSC-engrafted NSG-Tg(Hu-IL15) mice are functional, mediating direct cytotoxicity and ADCC. We have now extended these observation by evaluating the ability of human NK cells to control growth of a PDX melanoma in humanized NSG-Tg(Hu-IL15) mice. Our observations indicate that the growth kinetics of the PDX melanoma are significantly delayed in HSC-engrafted NSG-Tg(Hu-IL15) mice as compared to HSC-engrafted NSG mice. Importantly PDX melanoma growth is not significantly different in NSG-Tg(Hu-IL15) and NSG that are not engrafted with human immune systems. To determine the immune cell subsets contributing to the delayed PDX growth in HSC-engrafted NSG-Tg(Hu-IL15) mice we performed experiments where either human CD8 T cells or human NK cells were depleted. Depletion of human CD8 T cells did not alter the PDX growth kinetics in HSC-engrafted NSG-Tg(Hu-IL15). In contrast, NK cell depletion abrogated the delayed growth of the PDX melanoma, suggesting that human NK cells are directly suppressing tumor growth in HSC-engrafted NSG-Tg(Hu-IL15) mice. Together these data demonstrate that HSC-engrafted NSG-Tg(Hu-IL15) mice support enhanced development of functional human NK cells and that these NK cells limit the growth of PDX tumors.

#1071

**Targeted therapy evaluation in novel pre-clinical** in vitro **and** in vivo **models of Richter transformation.**

Warren C. Fiskus, Abhishek Maiti, Dyana T. Saenz, Christopher P. Mill, Joseph D. Khoury, Kapil N. Bhalla. _MD Anderson Cancer Center, Houston, TX_.

Richter Transformation (RT) is the development (in up to 15%) of aggressive DLBCL in patients with antecedent or concurrent CLL. By comparing immunoglobin gene rearrangements, 80% of RT-DLBCL (mostly ABC type) are clonally related (CR) to the underlying CLL, exhibiting poor median survival (MS) of 1 year, whereas 20% of RT-DLBCLs are clonally unrelated (CUR), exhibiting MS of 5 years. Lack of availability of in vitro cultured RT-DLBCL cells or PD xenograft models had prevented pre-clinical testing of novel targeted agents against RT-DLBCL. Here, we report the establishment of 3 patient-derived xenograft (PDX) models of RT-DLBCL (CR: RT15 and RT17, CUR: RT5) generated by tail-vein infusion and engraftment of luciferase-transduced CD19+ RT-DLBCL cells in NSG mice. RT-DLBCL xenografts grew in the bone marrow and spleen, causing marked splenomegaly and requiring euthanasia 4 to 6 weeks after engraftment. All three RT-DLBCL cells were EBV-negative by genomic and EBNA2 protein expression analyses. NextGen DNA sequencing of RT5 and RT15 cells showed mutations in TP53, ATM, NOTCH2, TET2 and MLL3 genes with a high VAF. Array-CGH showed DNA copy gains or losses in multiple chromosomes including 3, 8, 9, 11, 12, 17 and 18. Anti-H3K27Ac ChIP-Seq analysis showed increased average, normalized read-densities across all super-enhancers/enhancers (SEs/Es), with several SEs/Es exhibiting high scores. ATAC-Seq showed markedly increased signal intensity in RT-DLBCL cells compared to CD34+ normal progenitors, with high peak numbers in specific loci. Western analyses showed that RT15 and RT17 expressed high levels of BCL2, Bcl-xL and MCL1, whereas RT5 lacked BCL2 expression. RT15 and RT17 cells were more sensitive than RT5 cells to anti-BCL2 venetoclax-induced apoptosis (p < 0.01). RT15 and RT17, but not RT5 cells, expressed NFkB2 (p52), consistent with activation of non-canonical NFkB signaling. This was associated with resistance of RT15 and RT17 cells to ibrutinib-induced apoptosis. RT15 and RT17 cells were also less sensitive than RT5 cells to the BET protein inhibitor (BETi) OTX015-induced apoptosis. However, co-treatment with OTX015 and ibrutinib or venetoclax induced synergistic lethality in all RT-DLBCL cells (combination indices < 1.0). BET-PROTAC ARV-825 and ARV-771 treatment depleted BRD4, leading to marked reduction in c-Myc levels and apoptosis of RT-DLBCL cells. Treatment with the ATP-competitive, CDK9 inhibitor NVP2 dose-dependently induced apoptosis in RT-DLBCL cells which was associated with depletion of c-Myc, Bcl-xL and MCL1 protein levels. These findings highlight the activity and support further evaluation of in vitro and in vivo BETi, BET-PROTAC or CDK9i-based combinations with ibrutinib or venetoclax against genetically profiled RT-DLBCL cells that are clonally-related or clonally-unrelated to the antecedent CLL.

#1072

Development and characterization of patient derived xenograft (PDX) avatars of human cholangiocarcinoma.

Sanhita Rath,1 Sen Peng,1 Lauren Hartman,1 George Reid,1 Harshil Dhruv,1 Shannon Glaser,2 Fanyin Meng,2 Gianfranco Alpini,2 Scott Celinski3. 1 _TGEN, Phoenix, AZ;_ 2 _Baylor Scott & White Health, TX; _3 _Baylor University Medical Center, Dallas, TX_.

Cholangiocarcinoma (CCA) is a fatal primary malignancy with limited treatment options and a dismal 5year survival rate of <10%. Poor prognosis and early recurrence after surgical treatment warrants further molecular characterization of these tumors to evaluate therapeutic sensitivities and identification of biomarkers for CCA. Although several Next Generation Sequencing (NGS) studies have revealed some targetable genomic aberrations, the lack of preclinical and translational models has severely limited the identification of clinically relevant targeted therapies for CCA. So far, very few putative molecular targets/pathways have been identified using patient-derived xenograft (PDX) models, CCA cell derived xenograft models (CDX) and CCA cell lines, only 4 of which have progressed to preclinical testing for therapeutic value. In this study we describe development and comprehensive characterization of patient derived xenograft models of CCA from freshly resected patient tissue. Patient tumor cells from 8 patients were engrafted subcutaneously in highly immunocompromised NSG (NOD.Cg-PrkdcscidIl2rgtm1Wj) mice for PDX development. Two (CCA-1 and CCA-7) of 8 (25 %) engraftments yielded successful PDX model. The PDX avatar and matched patient tumor samples were genomically characterized at the whole exome and transcriptome level to check the fidelity of PDX models. Exome sequencing analysis of PDX tumors demonstrate higher genomic aberrations as compared to their patient counterparts. Preliminary analysis of transcriptomic profiles from CCA TCGA dataset, long term established intra- and extra-hepatic CCA cell lines (Mz-ChA-1, TFK-1, SG231, CCLP-1, HuCC-T1 and HuH-28), CCA CDX model from Mz-ChA-1 cells and CCA PDX models demonstrate that CCA PDX models more faithfully recapitulate patient tumors as compared to long term established cell lines. In summary, this proof of concept study demonstrates that PDX avatars of CCA could serve as a good model system for preclinical target discovery and validation for a deadly and neglected disease such as CCA.

#1073

**Role of CD44 in the transition of ductal carcinoma** in situ **to invasive breast carcinoma.**

Fang Liu,1 Jun Zhou,2 Tanima Kundu-Roy,1 Joseph Wahler,1 Jae Young So,1 Yong Lin,1 You-Rong Lou,1 Nicola J. Barnard,1 Isao Matsuura,3 Nanjoo Suh1. 1 _Rutgers Univ., Piscataway, NJ;_ 2 _Rutgers University and Xiangnan University, Piscataway, NJ;_ 3 _National Health Research Institutes, Zhunan Town, Taiwan_.

Ductal carcinoma in situ (DCIS) transition to invasive tumor is an important issue for breast cancer. The human MCF10DCIS.com cell line (abbreviated as MCF10DCIS) is derived from the human MCF10A breast epithelial cell line. The MCF10DCIS xenografts are similar to human high grade comedo DCIS. In the xenograft assay, MCF10DCIS cells form DCIS-like lesion, remain as DCIS-like lesion in the first few weeks, and then progress into invasive carcinoma. Thus, it is an excellent model to study human breast cancer transition from DCIS to invasive carcinoma. To the best of our knowledge, it is also the only cell model for DCIS transition to invasive carcinoma. We investigated the role of CD44 in breast cancer transition from DCIS to invasive carcinoma by knockdown the expression of CD44 in the MCF10DCIS xenograft model. Our results show that knockdown of CD44 expression in the MCF10DCIS cells enhances DCIS transition to invasive carcinoma. Accordingly, a previous study has shown that CD44 expression is lower in invasive human breast tumors compared with DCIS, especially in luminal A subtype. We have also observed that CD44 expression is lower in the invasive carcinomas compared with DCIS. Furthermore, we provide evidence that CD44 plays a role in maintaining the integrity of the myoepithelial layer. Our study uncovered an unexpected role of CD44 in the prevention of DCIS transition to invasive carcinoma. Our findings have important implications for breast cancer.

#1074

The PDX Data Commons and Coordinating Center (PDCCC) for PDXNet in support of preclinical research.

Jacqueline Rosains,1 Anuj Srivastava,2 Wingyi Woo,3 Vishal Sarsani,3 ZiMing Zhao,2 Javad Noorbakhsh,2 Ogan D. Abaan,1 Christian Frech,1 Jack DiGiovanna,1 Ryan Jeon,1 Steve Neuhauser,3 Peter Robinson,2 Yvonne A. Evrard,4 Carol Bult,3 Jeffrey A. Moscow,5 Brandi Davis-Dusenbery,1 Jeffrey H. Chuang2. 1 _Seven Bridges Genomics, Cambridge, MA;_ 2 _The Jackson Laboratory, Farmington, CT;_ 3 _The Jackson Laboratory, Bar Harbor, ME;_ 4 _Frederick National Laboratory for Cancer Research, Frederick, MD;_ 5 _National Cancer Institute, Bethesda, MD_.

Patient-Derived Xenografts (PDX) are proven models to study novel drugs or drug combinations and test hypothesis in preclinical studies. The overarching goal of the PDXNet is to coordinate the development of appropriate PDX models and methods for preclinical drug testing to advance CTEP clinical development of new cancer agents.

The PDXNet is an NCI-funded consortium of six PDX Development and Trial Centers (PDTCs) and one PDCCC. Four PDTCs are responsible for developing PDXs and executing specific preclinical trials focused on cancer types including breast cancer, melanoma, and lung cancer. The other two recently awarded centers are specifically focused on minority PDX models and preclinical trials. Besides the PDTCs, the NCI Patient-Derived Models Repository (PDMR) at the Frederick National Laboratory for Cancer Research (FNLCR) is also providing models and data to the PDXNet. The PDCCC is responsible for coordination and developing standards for PDX generation as well as data analysis and metadata harmonization. The PDX Data Commons is built on top of existing NCI resources, leveraging the Cancer Genomics Cloud maintained by Seven Bridges Genomics, where PDXNet data is co-located with TCGA and other large-scale datasets. The PDCCC is co-led by experts from the Jackson Laboratory, providing scientific leadership in xenograft methods and cancer biology to ensure the promulgation of standards that are well-suited for the PDX community.

A new portal has been set up at https://www.pdxnetwork.org/ to serve as the point of access to PDXNet resources. In addition, we established ongoing network-wide meetings to facilitate knowledge exchange, held PDXNet portal trainings, and set up working groups to tackle specific challenges. For instance, the Data Ontology working group has been working towards building a common data ontology model specifically for PDX datasets. We are in the process of annotating the very first dataset using this new ontology on the PDXNet portal. Also, the Workflows working group has been working on building and benchmarking various RNA-seq and whole exome sequencing analysis workflows to standardize data processing between PDXNet grantees and create a harmonized PDXNet dataset. These PDX models and the accompanying data will be opened to the community for data mining and/or preclinical research.

The PDXNet is a strong step toward building a consensus around PDX models, so that the power for discovery can be expanded by making multi-institutional PDX cohorts a reality. As the coordination center, we are also working closely with the EuroPDX project to exchange standards and knowledge to support the PDX community with a set of standards going forward. The PDCCC is a central part of this process to systematically capture and analyze the variables most influential to PDX models and share protocols and tools to make PDXs an interchangeable research currency for preclinical discovery.

#1075

Genomic data analysis workflows for tumors from patient-derived xenografts (PDXs): Challenges and guidelines.

Xing Yi Woo,1 Anuj Srivastava,1 Joel H. Graber,2 Vinod Yadav,1 Vishal Kumar Sarsani,3 Al Simons,3 Glen Beane,3 Stephen Grubb,3 Guruprasad Ananda,1 Grace Stafford,3 Jeffrey H. Chuang,1 Susan D. Airhart,3 R. Krishna Karuturi,1 Joshy George,1 Carol J. Bult3. 1 _The Jackson Laboratory for Genomic Medicine, Farmington, CT;_ 2 _MDI Biological Laboratory, Bar Harbor, ME;_ 3 _The Jackson Laboratory for Mammalian Genetics, Bar Harbor, ME_.

Patient-derived xenograft (PDX) models are in vivo models of human cancer that have been used for translational cancer research and therapy selection for individual patients. The Jackson Laboratory (JAX) PDX resource has over 450 models representing more than 20 different types of cancer. The models undergo rigorous quality control and are genomically characterized to identify somatic mutations, copy number alterations, and transcriptional profiles. Bioinformatics workflows for analyzing genomic data obtained from human tumors engrafted in a mouse host (i.e., Patient-Derived Xenografts; PDXs) must address challenges such as discriminating between mouse and human sequence reads and accurately identifying somatic mutations and copy number alterations when paired non-tumor DNA from the patient is not available for comparison.

Here we describe bioinformatics analysis workflows and guidelines (https://github.com/TheJacksonLaboratory/PDX-Analysis-Workflows) that we developed specifically for the analysis of genomic data generated from PDX tumors. Our workflows incorporate commonly used software and public databases but are tailored to address the specific challenges of PDX genomics data analysis through parameter tuning and customized data filters and result in improved accuracy for the detection of somatic alterations in PDX models. We also report a gene expression-based classifier that can identify EBV-transformed tumors. Finally, to demonstrate the effectiveness of our workflows, we show the overall concordance of the genomic and transcriptomic profiles of the PDX models in the JAX PDX resource with relevant tumor types from The Cancer Genome Atlas (TCGA).

Using the reliable results obtained from the PDX genomics data analysis, we are able to compare the patient tumor with different PDX passages, perform classification analysis to verify the annotations of PDX tumors, as well as associate genomic signatures of each PDX tumor with results from dosing studies.

Acknowledgements

The data analysis workflows reported in this publication were partially supported by the National Cancer Institute of the National Institutes of Health under Award Number P30CA034196. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The public portal for JAX PDX data is supported by R01CA089713.

#1076

Neoantigens retention by patient derived xenograft model mediate autologous T cells activation in ovarian cancer.

Muzamil Y. Want,1 Anna Konstorum,2 Ruea-Yea Huang,1 Richard Koya,1 Sebastiano Battaglia1. 1 _Roswell Park Comprehensive Cancer Center, Buffalo, NY;_ 2 _UConn Health, Farmington, CT_.

Ovarian cancer (OC) is the fifth leading cause of cancer death in the US, presenting a low mutational burden and a diverse degree of infiltrating T cells. Neoantigens derived from somatic mutations represent an attractive immunotherapeutic target, however, mouse models for the development of personalized immunotherapies are still poor and do not fully recapitulate the individualized nature of OC in patients. To address this hurdle, our study established a patient-derived xenograft (PDX) of an OC patient as segue to studying neoantigen-driven autologous T cell response. We first evaluated tumor mutations in the primary tumor (Pr) and two successive passages (P0, P1) via WES. As tumors from P0 and P1 have a higher mutational count than Pr, we hypothesize that this difference is caused by the loss of immune-surveillance in immunocompromised mice, which allows for the outgrowth of previously repressed clones. By using the variant allele frequency (VAF) to cluster 123 shared mutations, we identified three clusters with unique VAF dynamics (Fig 1A-D). Tumors from Pr, P0 and P1 had a conserved functional with a strong conservation of antigen presenting pathways (Fig 2A-E). In PDX P0 we identified 184 non-synonymous mutations, leading to 30 potential neoantigens with high affinity for the patient's HLAs (Fig 3A-B). Neoantigens were ranked based on differential predicted HLA affinity of the WT versus mutated peptide (Fig 3C). T cell activation by neoantigens was tested in vitro via ELISA and flow cytometry. Interferon-γ production and upregulation of CD137 identified a core set of six neoantigens selectively recognized by patient's autologous CD8+ T cells (Fig 4A-B). Of those, 3/6 neoantigens were common between PDX and primary tumor, corroborating the role of the patient's own immune system in repressing the expansion of selected tumor clones (Fig 5). In vivo ACT studies showed that mice injected with neoantigen-stimulated autologous PBMCs (ACT_MUT) have reduced tumor growth when compared to mice injected with unstimulated cells (ACT_NP) (Fig 6A). ACT_MUT mice have higher levels of circulating T cells 15 days post-ACT and higher intratumoral T cells at end point than ACT_NP (Fig 6 B-C). We then sought to identify the TCR moieties that determine T cell response. Single cell TCRSeq analyses on the two strongest neoantigens identified multiple TCR activated by a single cancer neoantigen (Fig 7A-B), suggesting oligoclonal T cell activation. We tested this hypothesis by generating a motif with the most expanded clones and comparing it with the motif from clones that did not expend. Results indicate a significant difference in the frequency of amino acid in multiple CDR3 locations, suggesting the presence of an oligoclonal response to neoantigenic T cell stimulation. In conclusion, we have successfully established PDX models of OC that can be used to study and predict autologous T cell response to neoantigens.

#1077

Development of a qPCR cfDNA analysis method for the assessment of tumor burden and metastatic status in live PDX mice.

Sudhir K. Sinha,1 Hiromi Brown,1 Andrew Loftus,1 Margarite D. Matossian,2 Bridgette M. Collins-Burow,2 Matthew E. Burow2. 1 _InnoGenomics Technologies,LLC, New Orleans, LA;_ 2 _Tulane University, School of Medicine, New Orleans, LA_.

We report here a novel, high analytical sensitivity multiplexed-qPCR assay for monitoring human and mouse-specific circulating cell-free DNA (cfDNA) using only 15 microliters of blood harvested from patient-derived xenograft (PDX) mice. PDX tumors maintained in murine models are commonly employed to study the biology and drug sensitivity of patient tumors. Our multiplexed qPCR assay incorporates both human and mouse multi-copy PCR amplifications targets in a single qPCR reaction. The multiplex also includes an internal synthetic DNA positive control to monitor accuracy of PCR amplification. All three targets are quantitated by monitoring three fluorophores in a single qPCR reaction. In PDX mice, the implanted human tumor sheds DNA as circulating tumor DNA (human ctDNA). Select metastatic tumors also shed human circulating tumor cells (CTCs) into the mouse's blood that also can be detected with our assay. Our qPCR assay utilizes an Alu retrotransposon sequence, present at thousands of copies per human genome, to detect and quantitate human ctDNA, and a mouse specific multi-copy target to detect mouse cfDNA in plasma harvested from PDX mice. These high copy, repetitive elements are ideally suited for qPCR targets to quantitate circulating-tumor DNA, which is predominately in low abundance. The low detection limits of the assay (below 1pg/µL of human DNA extracted from mouse plasma) allows for longitudinal studies of mice, as the volume of blood required is minimal (as low as 15 µL) and can be repeatedly collected from live mice without the need for a terminal bleed. With the assay quantitating both human ctDNA and mouse cfDNA, normalization of the amount of tumor DNA can be calculated, allowing for more reliable tracking of the tumor growth in the mouse model. Furthermore, CTC DNA had higher measurable concentrations in the peripheral blood of both invasive TNBC PDX models compared to cfDNA concentrations. We have tested this assay successfully in mice that contain various tumor burdens (CRC, prostrate, SCLC, and TNBC). Data will be presented to show the relative amounts of human DNA present in these mice. The assay also accurately predicted the success or failure of the initial tumor engraftment of a patient tumor in a mouse—this can be used to reduce cost by eliminating unsuccessful grafts, without waiting for months to observe if the tumor grew or not. This innovative assay provides a relatively quick, economic, and easy method to monitor ongoing response to therapy. 

### Immune Cells in the Tumor Microenvironment 1

#1078

An immunosuppressive signature in bone marrow as a potential biomarker for recurrence of metastatic prostate cancer after prostatectomy.

Nan Sethakorn,1 Erika Heninger,1 Jamie M. Sperger,1 Kenneth J. Pienta,2 Joshua M. Lang1. 1 _University of Wisconsin-Madison, Madison, WI;_ 2 _Johns Hopkins University, Baltimore, MD_.

Background: Metastatic prostate cancer is the second leading cause of cancer-related deaths in US men. Nearly one-third of men develop recurrence after curative intent treatment. Bone metastases are common, and cause significant morbidity in addition to driving mortality rates. Early dissemination of tumor cells to the bone marrow has been hypothesized as a contributing factor to recurrence. However, the factors that promote early dissemination and survival in the bone microenvironment are not clearly elucidated. Key components of immune regulation such as cytotoxic T cells and regulatory T cells can regulate growth of cancer cell lines and in mouse models. We hypothesize that an immunosuppressive signature results in a permissive environment allowing the growth of disseminated prostate cancer cells. Therefore, the goal of our project is to evaluate mechanisms of immune evasion that promote the development of metastatic prostate cancer.

Methods: Matched blood and bone marrow aspirates were collected from patients undergoing prostatectomy. Samples from five healthy donors were collected to establish a normal baseline. Using multicolor flow cytometry, samples were subjected to multi-parameter panels designed to assess the composition and function of lymphoid and myeloid immune compartments. These panels include markers of known immune subtypes, co-stimulatory signals, and activation signals. Results were analyzed with Prism software.

Results: Preliminary results show variable T cell to B cell ratios ranging from as low as 1:1 to as high as 9:1. A high CD4 to CD8 T cell ratio was identified in 38% of samples, which corresponded with a higher occurrence of regulatory T cells. These data are consistent with an immunosuppressed bone marrow signature, which importantly was not present in the matched peripheral blood samples. Furthermore, our preliminary data demonstrates altered lymphoid and myeloid subsets in patients with prostate cancer as compared to healthy donors.

Discussion: Overall, these results support the hypothesis that an immunosuppressive signature in bone marrow may promote survival of disseminated prostate cancer cells by protecting them from immune eradication. Furthermore, this immune signature within the microenvironment may exist at the time of prostatectomy and can be correlated with clinical biomarkers to identify factors that promote dissemination and metastatic recurrence. Future work involves using descriptive statistics to correlate the immune signatures identified in this project with patient outcomes, including PSA, Gleason grade, recurrence rates and time to recurrence. Ultimately, the goal is to identify an immune signature that accurately predicts risk of developing recurrent prostate cancer, potentially serving as a biomarker for patients that would benefit from aggressive upfront adjuvant systemic therapy.

#1079

**Prognostic role of tumor microenvironment in correlation with CD8** + **T cells in TCGA pan-cancer.**

Shin Hye Yoo, Changhee Park, Chan-Young Ock, Miso Kim, Bhumsuk Keam, Tae Min Kim, Dong-Wan Kim, Dae Seog Heo. _Seoul National University Hospital, Seoul, Republic of Korea_.

Purpose: Tumor microenvironment (TME) is a complex mixture of cells around tumor, showing different associations with tumor growth and survival outcomes. CD8+ T cell plays a key role in adaptive anti-tumor immunity and can predict favorable prognosis with better outcome from immunotherapy. However, little has been known whether various components of TME in correlation with CD8+ T cells affect prognosis. We aimed to investigate the prognostic role of immune cells in TME in correlation with CD8+ T cells using The Cancer Genome Atlas (TCGA) pan-cancer database.

Experimental Design: The fraction of 21 immune cell subpopulations in TME and CD8+ T cell were extracted from mRNA expression data obtained by RNA sequencing of the TCGA samples to which a deconvolution algorithm (CIBERSORT) was applied. The samples were classified into CD8low, CD8high-Xlow/int, and CD8high-Xhigh groups (X is a component of TME), and survival was compared between them in TCGA pan-cancer and each cancer cohort. To determine the associations between each cell type and treatment outcomes, we also evaluated the immune cell subpopulations with CD8+ T cell in patients treated with programmed death-1 blockades using previously reported data on the nCounter system by the PanCancer 730-Immune Panel (GEO accession number: GSE93157).

Results: Profiles of immune subpopulation in correlation with CD8+ T cell varied between different cancer types. In TCGA pan-cancer cohort with available survival data (N=8903), increased regulatory T cells (Treg) (P=2.48× 10-4), M1 and M2 macrophages (P=9.53× 10-3; P=4.72× 10-6, respectively), resting natural killer cells (P=7.29× 10-4), and activated dendritic cells (P=0.00579) were associated with poor prognosis despite high level of CD8+ T cells, whereas elevated germinal center follicular helper T cells (P=8.44 × 10-5) and naïve B cell (P=2.46× 10-7) were good prognostic subpopulations. By each cancer type, kidney cancers (renal papillary tumors and clear cell carcinoma) showed significantly poor prognostic role of Treg (Hazard ratio (HR) 2.59, 95% confidence interval (CI) 1.13 to 5.93; HR 1.62, 95% CI 1.07 to 2.45, respectively). In patients treated with immunotherapy (N=65), shorter progression-free survival was observed in CD8high-Treghigh group than in CD8high-Treglow/int group (P=0.0476). The fraction of CD8high-Treghigh group was higher in non-responders than in responders (15.6% vs. 5.0%).

Conclusions: Our findings suggest that differences in immune cell subpopulations even with high CD8+ T cell might determine the prognosis. Immunosuppressive components in TME such as Treg can be negative factor for both prognosis and response to immunotherapy. A large prospective study is needed to validate the negative predictive role of immunosuppressive TME for patients treated with immunotherapy.

#1080

Link between M1/M2 human macrophages and epithelial-mesenchymal status in head and neck cancer cell lines.

Lucile Astorgues-Xerri,1 Diane Evrard,2 Matthieu Martinet,1 Eric Raymond,3 Sandrine Faivre,4 Annemilaï Tijeras-Raballand1. 1 _AFR Oncology, Paris, France;_ 2 _Bichat University Hospital, Paris, France;_ 3 _Saint-Joseph Paris Hospital, Paris, France;_ 4 _Beaujon University Hospital, Paris, France_.

Background. Head and neck carcinoma (H&N) is one of the leading causes of cancer deaths worldwide. Despite advances in diagnosis and treatment, the survival rates remain low mainly due to locoregional relapse, possibly triggered by the activation of epithelial-to-mesenchymal transition (EMT). Recently, several studies have demonstrated a positive link between macrophages, EMT and invasion in H&N cancer. The aim of this study is to analyze the interactions between human antitumoral M1/protumoral M2 macrophages and H&N human cancer cells with different EMT status, with the aim of developing new therapeutic approaches for H&N cancer patients.

Materials and Methods. M1 and M2 macrophages were obtained from THP-1 cell line (human monocyte) after 48h exposure to 25nM of PMA followed by 48h of recovery culture medium, and 72h exposure to 1ng/ml LPS + 20ng/ml IFNγ to obtain M1 phenotype or 20ng/ml IL4 + 20ng/ml IL13 to obtain M2 phenotype. Differentiation status was validated by immunofluorescence (IF) using CD14 for monocyte, CD68 for macrophage, CD80 for M1, and CD163 for M2. Eight H&N cell lines were characterized for their EMT status (E-cadherin/vimentin expression) by western blot. SQ20B (epithelial) and Hep2 (mesenchymal) cell lines were selected to study the effect of M1 and M2 conditioned medium (CM) on cell proliferation. In addition, we also analyzed the effects of CM from SQ20B and Hep2 on macrophages differentiation using IF.

Results. We confirmed the differentiation of monocytes into macrophages by a decrease of CD14 expression and an increase of CD68 expression, and the differentiation of macrophages into M1 and M2 by an increase of CD80 and CD163 expression, respectively. Among the 8 H&N cell lines, 3 cell lines showed an epithelial status (high E-cadherin expression), one a mesenchymal status (high vimentin expression), and 4 a mixed status. Based on these results, we exposed SQ20B (epithelial) and Hep2 (mesenchymal) to M1 or M2 CM. M1 CM strongly inhibited the proliferation of SQ20B cells, with moderate effect on Hep2 cells, whereas M2 CM displayed no effect on SQ20B cells and slightly increased the proliferation of Hep2 cells. Moreover, macrophages exposed to SQ20B CM displayed a M1 phenotype with an increased expression of CD80, whereas Hep2 CM induced a M2 phenotype with an increased expression of CD163.

Conclusions. In vitro, we showed that M1 and M2 macrophages displayed opposite effects on H&N cancer cells proliferation via their conditioned medium, M1 being anti-proliferative and M2 pro-proliferative. These effects were dependent on epithelial/mesenchymal status of cancer cells. In addition, we showed that factors secreted by epithelial vs mesenchymal cancer cells induced macrophages differentiation into M1 and M2, respectively. These results open up new perspectives on the role of M1/M2 macrophages in EMT-dependent H&N cancers and other tumor types such as colon, lung, and liver carcinoma.

#1081

Smad3 silences neutrophil anticancer activity in the tumor microenvironment.

Patrick Ming-Kuen Tang, Philip Chiu-Tsun Tang, Jeff Yat-Fai Chung, Xiao-Ru Huang, Ka-Fai To, Hui-Yao Lan. _The Chinese University of Hong Kong, Hong Kong, Hong Kong_.

Tumor microenvironment (TME) is a new therapeutic target for cancer, however it is highly heterogeneous and still largely unknown. Recently, we revealed that Smad3 is essential for TME-driven cancer progression, better understanding of the underlying mechanism may uncover novel therapeutic targets for cancer. Indeed, Smad3-dependent TME (Smad3-TME) is originated from bone marrow, where neutrophils are the most abundant leukocytes but their role in cancer is still controversial. Here, we are the first study to reveal the regulatory role of Smad3 in tumor-associated neutrophils (TAN). Interestingly, we observed that knockout of Smad3 (Smad3-KO) dramatically increased TAN population in mice bearing syngeneic melanoma B16F10 and lung carcinoma LLC, where the anticancer N1 phenotypes were significantly induced in the SMad3-KO TME-isolated TAN. Unexpectedly, adoptive transfer of Smad3-KO bone marrow-derived neutrophils markedly suppressed the progression of LLC-tumor on Smad3-WT mice in vivo, and their enhanced cancer-killing activity was confirmed by co-culture assay in vitro. Furthermore, microarray analysis identified a total of 4416 differentially expressed genes associated with Smad3-KO neutrophils under cancer condition in vitro, but the presence of Smad3 largely decreased the transcriptome diversity; especially on genes associated with virus defense response including an anticancer cytokine IL-28. Silencing of IL-28 significantly reduced the anticancer efficiency of Smad3-KO TAN on LLC-bearing mice in vivo. Mechanistically, we uncovered that IL-28A serves as a direct Smad3 target gene and specifically down-regulated in neutrophils under TGF-β1 or LLC-secretome stimulations; showing by ChIP and dual-luciferase reporter assays in vitro. Thus, targeting Smad3 precisely on TAN may represent as a novel and effective immunotherapy for cancer. Acknowledgments: This study was supported by Lui Che Woo Institute of Innovative Medicine (CARE program), Research Grants Council of Hong Kong (GRF 14117815, 14121816, 14163317, C7018-16G, TRS T12-402/13N), Health and Medical Research Fund (03140486, 14152321), Innovation and Technology Fund of Hong Kong (ITS/068/18), Direct Grant for Research CUHK (2017.002).

#1082

Linking tumor microenvironment properties in murine syngeneic tumors with resistance to immune checkpoint inhibitors: Insights from a quantitative systems approach.

Gabriel Helmlinger,1 Ivan Azarov,2 Yuri Kosinsky,2 Veronika Voronova,2 Lulu Chu,1 Suzanne Mosely,3 Simon Dovedi,3 Kirill Peskov2. 1 _AstraZeneca, Waltham, MA;_ 2 _M &S Decisions LLC, Moscow, Russian Federation; _3 _MedImmune, Cambridge, United Kingdom_.

Objectives: Studies in murine syngeneic tumors are critical in the development of immune-based therapies, yet there still are knowledge gaps in the functional meaning (vs. response and resistance to treatments) of baseline molecular and immunological features in these tumors. We developed a quantitative systems model of immuno-oncology (IO), to (i) understand factors within the TME which may underlie anti PD-(L)1 and CTLA-4 efficacy in 6 syngeneic tumors (4T1, LLC, CT-26, MC-38, B16, RENCA); (ii) identify potential baseline factors which relate to treatment resistance.

Methods: Our IO model [1] was used, firstly to incorporate rich datasets from the 6 syngeneic tumor types [2] and to characterize differences in baseline TME conditions. The model was then used to perform mechanistic population simulations of the initiation and development of anti-tumor T cell immune responses, linked to observed individual animal- and cohort-level tumor size dynamics (TSD) under anti PD-(L)1 and CTLA-4 treatments. Variability in individual tumor size dynamics was taken into account using a mixed-effects technique, implemented in the model at the level of tumor-infiltrating T cell influx.

Results: The model adequately described individual- and cohort-level TSD patterns, for all treatment regimens in all 6 tumor types. The model incorporated in one quantitative framework immune cell count data measured in these tumors, by capturing empirical dependencies between TME properties and model parameters. Anti PD-L1 therapy was incorporated into the model via a direct increase in an immune activation rate (IAR) function in TME, validating our previous results [1]. Interestingly, an optimal model incorporating anti CTLA-4 mechanism of action was one considering an indirect effect on IAR through the decrease of immuno-suppressive cell (ISC) function, which supports the hypothesis that the driving force of anti CTLA-4 effects in syngeneic tumors would go through ISC deactivation, e.g., via regulatory T cell (Tregs) depletion. Also, higher counts of Tregs at baseline (e.g., CT26, RENCA) correlated well with responses to anti CTLA-4 treatment. Higher levels of macrophages and/or MDSC infiltration in lesser "immunologically hot" tumors (e.g., 4T1, MC38, LLC) were shown to be the main immuno-suppressive factors limiting tumor responsiveness to checkpoint inhibitor treatments.

Conclusions: This quantitative model may be used as a platform to analyze immune-based treatment data from various tumor types, while providing mechanistic insights on the contributions of baseline TME conditions to response or resistance to treatment. The model may be further used to perform predictive tumor response simulations (monotherapies and combinations), of untested anti CTLA-4, PD-(L)1 dose schedules and of other novel IO agents beyond these two checkpoint inhibitors.

#1083

Combinatorial strategies for tissue characterization with advanced image analysis and digital spatial profiling.

Raffaele De Filippis. _University of St. Andrews, St Andrews, United Kingdom_.

Characterization of the spatial distribution and molecular profiles of diverse cell populations in the tumor microenvironment is important to understand the conditions required for the development of metastatic disease. The NanoString® GeoMx™ Digitial Spatial Profiling (DSP) platform facilitates these studies by enabling highly multiplexed, spatially resolved characterization of proteins and RNA from FFPE tissue (for Research Use Only). The platform leverages cocktails of fluorophore- and oligonucleotide-conjugated antibodies to visualize and digitally quantify targets from areas of interest (AOI). Here, DSP is combined with advanced tissue profiling using machine learning algorithms to define AOIs based on expression of multiple markers. This strategy enables targeted DSP sampling based on the digital segmentation of heterogeneous cell populations within the tumor microenvironment. We present a series of applications of this combined profiling strategy to enable deep characterization of renal, colon, and bladder cancers to address molecular and cellular interactions between host and tumor that facilitate metastasis and poor patient outcome. Whole slide digital scanning of multiplexed immunofluorescence labelled slides was performed on a Zeiss Axioscan.z1. Digitized scans were imported in Definiens Tissue Studio® software and machine learning-based analysis was performed. The image analysis segmented heterogeneous subpopulations, dependent on biomarker expression and spatial resolution, which acted as a digital spatial map to direct DSP sampling. DSP was performed on a serial section of tissue by selecting AOI within bladder and colorectal cancer sections and a geometric DSP profiling strategy was employed to collect all the protein content from the AOI in a single sample. In another application of the method, a clear cell renal cell carcinoma (ccRCC) tissue microarray comprised of cores from patient matched healthy, primary, venous thrombi and distant metastatic sites was profiled with DSP. We present highly-plexed in situ proteomic data captured from several AOIs across bladder and colorectal cancer tissue sections. This data describes the molecular profile from the densely distributed lymphocytes and macrophages in close proximity to tumor subpopulations such as tumor buds and PDL-1 positive cancer cells. We demonstrate that both tumor and immune cell subpopulation profiles within bladder and CRC alter dependent on their proximity to each other. We further present the altering proteomic landscape of ccRCC as it progresses from primary to metastatic disease. This work demonstrates the feasibility and applicability of combining machine learning and advanced image analysis with multiplexed digital spatial profiling to enable deep characterization of tissues. Analysis approaches such as these will have utility in a variety of pathological research settings.

#1084

The role of tumor-associated neutrophils (TAN) and CD8 positive T lymphocyte in the progression of hepatocellular carcinoma (HCC).

Toshihiko Yusa, Hirohisa Okabe, Yo-ichi Yamashita, Takanobu Yamao, Naoki Umezaki, Tatsunori Miyata, Shigeki Nakagawa, Hiromitsu Hayashi, Katsunori Imai, Akira Chikamoto, Takatoshi Ishiko, Hideo Baba. _Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan_.

[Background]Neutrophils are known as immune cells involved in biological defense against foreign substances. On the other hand, in the tumor microenvironment, it is said that neutrophils affect on tumor progression as a tumor-associated neutrophil (TAN). Although, TAN and other immune cells in the progression of HCC is unclear.

[Method]One hundred twenty eight patients who underwent curative hepatectomy for HCC in our institution were enrolled. Immunohistochemistry was performed with CD66b and CD8 antibody to evaluate TAN and CD8+ T lymphocytes, respectively. Expression high or low was determined by cut-off value of each number of immune cells. We counted the number of each immune cells in both tumor (intra-tumor) and the tumor marginal area (extra-tumor). The relationship between the infiltration of each immune cell and clinicopathological factors was examined.

[Result]Comparing the number of immune cells of intra-tumor and extra-tumor, there were significantly more cells in extra-tumor area (p<0.001). There was no difference in between background clinicopathological factors and the number of TAN or CD8+ T lymphocytes in either intra-tumor or extra-tumor areas. Overall survival was significantly better in the group with high CD8+ T lymphocytes in extra-tumor area compared to the low group (p = 0.0491), intriguingly though CD8+ T lymphocytes in intra-tumor area was not associated with prognosis. In the contrast, overall survival was significantly poor in the group with high TAN in extra-tumor area compared to the low group (p = 0.0296), though TAN in intra-tumor area was not associated with prognosis.

[Conclusion]Infiltration of TAN and CD8+ T lymphocytes not in intra-tumor but in the extra-tumor area may be involved in the progression of HCC.

#1085

Neuropilin-1 deficient macrophages as a treatment strategy for glioblastoma.

Michael D. Caponegro, Stella E. Tsirka. _Stony Brook University, Stony Brook, NY_.

Post-diagnosis survival of grade IV glioblastoma (GB) is a dismal 14 months in patients receiving standard of care therapy. With the development of immunotherapeutic strategies targeting non-cancerous, stromal cells of the tumor microenvironment (TME), new avenues are being pursued in clinical trials and pre-clinical animal models of advanced GB. Glioma-associated microglia/macrophages (GAMs) of the TME promote chemoattraction, immune suppression, neoangiogenesis, and tumor cell survival, which parallels tumor-infiltrating macrophage behavior of other solid malignancies. GAMs are now recognized as a potentially practical immunotherapeutic target in GB. Previously in our lab, we have shown that GAM-specific deletion of the cell surface co-receptor Neuropilin-1 (NRP1) drastically reduces pro-tumorigenic behavior of infiltrating microglia and/or macrophages in murine glioma by reducing immunosuppressive tendencies and neoangiogenesis. As an extension to our genetically-ablated mouse model, we present here a platform by which these NRP1-deficient monocytic cells can be harvested ex vivo and locally delivered into the TME of the bulk glioma lesion, to exert anti-tumorigenic effects. To our knowledge, this is the first attempt to deliver cells of monocytic origin intratumorally as a therapeutic option.

#1086

**Immune checkpoint inhibitors** CD300A **and** LAIR1 **are associated with poor prognosis in clear cell renal cell carcinoma.**

Jonas Sjolund, David Lindgren, Helén Nilsson, Martin E. Johansson, Hakan Axelson, Kristian Pietras. _Lund University, Lund, Sweden_.

Immune checkpoint inhibitors targeting the programmed cell death-1 receptor (PD-1) have been shown to improve the survival of patients with clear cell renal cell carcinoma (CCRCC). However, a benefit for CCCRC patients was observed with the FDA-approved PD-1 inhibitor Nivolumab irrespective of PD-1 ligand (PD-L1) expression status indicating there are potentially additional CCRCC checkpoint molecules worth exploring for targeted therapy. We therefore analyzed RNAseq data (n=534, CCRCCs; n=72, normal kidney specimens) from the TCGA project, and found that PD-L1 was not upregulated in CCRCC when compared to normal tissue whereas other checkpoint members i.e. CD300A and LAIR1 were among the most significantly upregulated genes in CCRCCs. In terms of overall survival, high expression of PD-L1 was associated with better outcome in CCRCCs while high expression of CD300A and LAIR1 were significantly associated with worse outcomes. We confirmed these findings in two independent cohorts of CCRCC patients. Furthermore, we performed cell type enrichment analysis, which showed that CD300A and LAIR1 are most likely expressed by tumor-associated macrophages in the CCRCC tumor microenvironment. Our results suggest that PD1/PDL1 might be suboptimal targets in CCRCC and that there are additional unexplored immune checkpoint molecules that warrant our attention.

#1087

Bone-resident neutrophils are mediators of prostate cancer growth in bone.

Leah M. Cook, Diane Costanzo-Garvey, Tyler Keeley. _University of Nebraska Medical Center, Omaha, NE_.

Bone metastatic prostate cancer (BM-PCa) significantly reduces overall patient survival and is currently incurable. Current standard immune therapies have shown promising results for patients with less advanced disease (fewer than 20 bone lesions) suggesting that PCa growth in bone contributes to immunotherapeutic response. Gaining a better understanding of the immunogenicity of BM-PCa may present a new therapeutic approach for treating mCRPC. In bone, prostate cancer cells hijack the behavior of bone resident cells, inducing excessive bone degradation and release of bone-sequestered growth factors, such as transforming growth factor beta (TGFβ), that promote a "vicious cycle" of tumor progression. Findings from my lab revealed that: 1) BM-PCa stimulates bone stromal expression of Interleukin 8 (IL-8), a potent chemoattractant for polymorphonuclear neutrophils (PMNs), innate immune cells generated in bone marrow, and 2) PMNs heavily infiltrate the prostate tumor-bone microenvironment. Recent studies demonstrated the existence of two distinct PMN populations: anti-tumoral (N1) and immunosuppressive pro-tumoral (N2) PMNs, with emergence of the latter regulated by TGFβ. RealTime qPCR revealed that bone metastatic C42B prostate cancer significantly increases neutrophil expression of TGFβ receptor and MMP9, a trend previously identified as a marker of pro-tumoral neutrophils. Based upon these findings, we hypothesized that bone metastatic prostate cancer skews neutrophils to a pro-tumoral phenotype in a TGFβ-dependent manner. To test this, bone marrow-derived primary neutrophils were treated with conditioned media from metastatic metastatic prostate cancer cells (C42B) and poorly metastatic LNCaP and gene expression and oxidative burst were measured. Similar to previous findings, C42B stimulated reactive oxygen species (ROS) production by PMNs, a mechanism of immunosuppression utilized by N2 PMNs, and induced anti-inflammatory gene expression. In contrast, LNCaP cells failed to stimulate this response. C42B media promoted PMN expansion, a phenomenon that is reversed by TGFβ-blockade. Surprisingly, direct contact with neutrophils significantly increased luminescence of luciferase-expressing C42B cells independently of cell growth, when compared to LNCaP, demonstrating that neutrophils activate alternate protein signaling in metastatic cells compared to non-metastatic. In vivo intratibial models utilizing antibody-mediated neutrophil depletion validated our hypothesis that neutrophils are critical mediators of prostate cancer growth in bone. These results demonstrate the significant role of neutrophils in the progression of bone metastatic prostate cancer.

#1088

Unleashing the potential of HDACi to augment immunotherapy for the treatment of colorectal cancer.

Nisha Holay, Uma Giri, Mihailo Miljanic, Milad Soleimani, Omar Shaikh, Anna Capasso, Carla L. Van Den Berg, Gail Eckhardt, Todd Triplett. _University of Texas at Austin, Austin, TX_.

Immunotherapy (IMT) has produced complete responses in a subset of colorectal cancer (CRC) patients harboring tumors molecularly characterized as microsatellite instable (MSI). However, the majority (95%) of CRC patients with microsatellite stable (MSS) tumors remain virtually unresponsive. There is strong rationale that with the correct approach, IMT can elicit potent anti-tumor responses in CRC-MSS patients. Prior studies have demonstrated that histone deacetylase inhibitors (HDACi) have the potential to enhance tumor immunogenicity as indicated by the decreased effectiveness of monotherapy in the absence of an adaptive immune system and augmentation of responses to IMT. Whether HDACi treatment enhances anti-tumor immune responses by increasing tumor immunogenicity, directly affecting T-cells, or both, remains unknown. Therefore, we explored the differential effects of HDAC inhibitors with distinct specificities have directly on tumor and T-cells which will be critical in designing optimal therapeutic strategies.

Results: First, we compared the direct cytotoxic effects on murine CRC cells and found that pan-HDACi caused cell death, while class I/II and HDAC VI inhibitors did not affect survival even at high concentrations (1μM). Evaluation of changes to immunogenic proteins revealed modest, but similar increases in immunogenic proteins (MHC-I, ICOSL, PD1L, CD80) across HDACi compounds. Strikingly, the co-stimulatory ligand CD86 was increased only with an HDAC VI inhibitor. Next, we sought to determine whether distinct inhibitors also had direct effects on T-cells during stimulation. Interestingly, we found all inhibitors did not affect TNFα production but caused a drastic decrease in IL-2, an important cytokine for T-cell survival and function. Moreover, this effect was found at low concentrations of HDACi and thus appear more sensitive.

Conclusions: These results demonstrate for the first time that different classes of HDAC inhibitors have unique properties in affecting tumor growth and immunogenicity. Conversely, all classes blocked IL-2 production by T-cells but not TNFα, suggesting a common mechanism in affecting select components of T-cell effector responses. Although previous studies showed synergy with IMT, our data suggests that HDACi detrimentally affect T-cells directly. Based on this, an optimal strategy for combination with IMT may entail priming the tumor microenvironment and stopping HDACi during T-cell targeted IMT. Future studies are underway to determine whether HDACi effects on T-cells are reversible, the kinetics of these effects and their impact on T-cell co-receptor expression. Collectively, these studies will help identify the most promising HDACi, immunotherapeutic targets and the sequence of combination therapy.

#1089

Characterization of the immune response to lymph mode metastases.

Dennis Jones,1 Timothy Padera2. 1 _Boston University School of Medicine, Boston, MA;_ 2 _Massachusetts General Hospital, Boston, MA_.

Metastasis remains the principal cause of cancer mortality. Lymph nodes are the most common site of solid tumor metastases and lymph node status is the most significant prognostic indicator for patient outcome in human solid cancer. Although antitumor T cells are generated in lymph nodes, metastatic cancer cells are able to grow in this seemingly hostile microenvironment. Further, we recently found that lymph nodes are a portal for metastatic progression to distant organs. The challenge is how to treat cancer cells that have spread to lymph nodes or distant organs in order to prevent their growth and ideally eradicate them from the body. Most cancer therapies are developed against the primary tumor growing in its native microenvironment. Our previous work has shown that classical sprouting angiogenesis (as observed in the primary tumor) does not occur in metastatic lymph nodes and is therefore not necessary for metastatic outgrowth. Here, our preliminary data show that cancer cells remodel the blood endothelium in metastatic lymph nodes. Although many blood vessels lack smooth muscle cell coverage, intravenous lectin perfusion reveals a functional blood vasculature within metastatic lymph nodes. However, we measured decreased expression of adhesion molecules on the vasculature in metastatic lymph nodes. Adoptive transfer of lymphocytes revealed impaired lymphocyte migration into mice harboring metastatic lymph nodes compared to naïve lymph nodes. Together, these findings suggest that decreased vascular adhesion molecule expression results in lymphocyte exclusion and impaired host immunosurveillance, leading to the progression of lymph node metastases. Our ongoing work seeks to inform therapeutic strategies that will enhance functional antitumor T cell infiltration into metastatic tumors and prevent their spread to distant organs.

#1090

β-Hydroxy-β-Methylbutyrate supplementation preserves muscle mass and reduces tumor growth in obese mice.

Michael F. Coleman,1 Kristyn A. Liu,2 Xiaohu Tang,3 Salvador Fabela,1 Laura M. Lashinger,2 Zhengrong Cui,2 Stephen D. Hursting1. 1 _University of North Carolina, Chapel Hill, NC;_ 2 _University of Texas, Austin, TX;_ 3 _Michigan Tech University, Houghton, MI_.

Background: Pancreatic ductal adenocarcinoma (PDAC) remains very challenging to treat with mean 5-year survival of approx. 6%, leading to PDAC's status as the 4th most deadly cancer in the US. Diet induced obesity (DIO) has been shown to promote both increased incidence and growth of PDAC. Simultaneously DIO promotes muscle loss and the loss of immune surveillance in tumors. Cachexia, a chronic catabolic process in which muscle mass is lost, is a common feature of PDAC. We have previously shown that while leucine driven mTOR activation protects muscle mass in tumor bearing mice it also promotes tumor growth. Hence, we sought to test the potential of a leucine metabolite, β-hydroxy-β-methyl-butyrate, to protect against cachexia in a PDAC model while antagonizing DIO mediated tumor growth. We further sought to determine if HMB treatment would alter gemcitabine response in tumors from obese animals.

Methods: C57BL/6 mice consuming either control or high fat diet, bearing Panc02 tumors, were treated with HMB alone or in combination with gemcitabine. Muscle size was determined using Feret's diameter measured from H&E stained cross sections. Immunohistochemistry staining was performed for Ki-67 and CD3. Tumor transcriptomic analysis was preformed using Affymetrics microarray. In vitro characterization HMB modulated signaling in C2C12 myotubes, Panc02 cells, and bone marrow derived macrophages treated with pro-inflammatory signaling molecules.

Results: a) HMB significantly increases muscle fiber size and mTOR signaling; b) HMB reduces DIO increased tumor volume; c) HMB significantly synergizes with gemcitabine to suppress PDAC growth in obese mice; and d) HMB reverts DIO mediate immune suppression and promotes tumor immune surveillance.

Discussion: Antagonism of DIO promoted PDAC tumor growth, by HMB, and promotion of immune surveillance may offer significant synergy with standard therapy or, immune targeted therapies in PDAC.

#1091

Comparison of TCR repertoires between cancer tissues and lymph nodes in colorectal cancer patients.

Kazuma Kiyotani,1 Tatsuo Matsuda,2 Eisaku Miyauchi,2 Yu-Wen Hsu,2 Satoshi Nagayama,1 Makda Zewde,2 Jae-Hyun Park,2 Taigo Kato,2 Makiko Harada,2 Nobuaki Suzuki,3 Hiroaki Nagano,3 Shoichi Hazama,3 Yusuke Nakamura1. 1 _Japanese Foundation for Cancer Research, Tokyo, Japan;_ 2 _University of Chicago, Chicago, IL;_ 3 _Yamaguchi University Graduate School of Medicine, Tokyo, Japan_.

Tumor draining lymph nodes (TDLNs) are located in the routes of lymphatic drainage from a primary tumor and have the highest metastasis in various types of solid tumors. TDLNs are considered as a tissue to activate the antitumor immunity, where antigen-specific effector T cells are generated and released to the blood stream. These effector T cells then infiltrate into the tumor site and attack cancer cells. However, T cell receptor (TCR) repertoire in TDLNs has not been well characterized. We performed TCR sequencing of cancer tissues, corresponding normal mucosa tissues and a total of 203 regional lymph nodes, including 67 metastasis-positive lymph nodes from 23 colorectal cancer patients with lymph node metastasis. Metastasis-positive lymph nodes showed a significantly lower TCR diversity and shared TCR clones more frequently with primary tumor tissues compared to metastasis-negative TDLNs. Hierarchical clustering and principal component analyses also supported that TCR repertoires in metastasis-positive TDLNs were more similar to primary tumor tissues than metastasis-negative TDLNs. These finding suggest that cancer-reactive T cell clones might expand in the metastasis-positive TDLNs, although we have not tested their reactivity against cancer cells. We are now examining whether the T cells in TDLNs can recognized autologous cancer cells.

#1092

V-ATPase family regulates lysosomal exocytosis and neutralizes with tumor-infiltrating lymphocytes for glioblastoma cells.

Yu-Chan Chang, Michael Hsiao. _Academia Sinica, Taipei, Taiwan_.

Immunotherapy based on checkpoint inhibitors provides substantial clinical benefit, but only to a minority of cancer patients. Targeted immunological checkpoint pathways have demonstrated anti-tumor cytotoxicity in the treatment of refractory cancers. However, cancer cells release specific "drones", such as small vesicles, which increase their ability in the blood and released the proteins such as PD-L1 to block the attack from T cells then they have a change to reach the tumor forming. To characterization of exosomes forming, trafficking and secretion can be a novel therapeutic approach to improve immunotherapy in cancer patients. The ATPase H+ Transporting V0 and V1 subunits (V-ATPase, also called ATP6 family) are critical for acidifying a variety of intracellular compartments and lysosomal exocytosis. Nevertheless, the dysfunctional roles of V-ATPases in glioblastoma has not yet been elucidated. For this purposed, we investigated V-ATPase members in lysosomal vesicles and found several candidates are key factors in regulating lysosomal exocytosis in glioblastoma cells. We established transcriptomics datasets by various glioblastoma cell lines, the results showed V-ATPases are highly expressed in malignant cell lines compared to benign or normal immortalized cells. Immunohistochemistry also performed a trend of consistency that increased of V-ATPase expression in tumor parts than normal adjacent tissues. The expression level of several V-ATPase subunits is also associated with clinical parameters and survival rate. Moreover, we observed the number of tumor infiltrating lymphocyte (TILs) was decreased in clinical cohorts and two-way cell models. Therefore, we established V-ATPases related proteomics datasets and found some interaction partners could stabilized protein structure of V-ATPase. Specific peptide can compete between V-ATPase and the interacting partners and further modulate TILs in glioblastoma. This approach can be a novel therapeutic strategy that combines immunotherapy to against glioblastoma in clinical patients.

#1093

Multichannel immunofluorescence imaging to assess the immune composition of tumor microenvironment in bladder cancer.

Ken Hatogai,1 Danny Kim,2 Yuanyuan Zha,2 Gary D. Steinberg,2 Alexander T. Pearson,2 Thomas F. Gajewski,2 Randy F. Sweis2. 1 _University of Chicago / National Cancer Center Hospital East, Chicago, IL;_ 2 _University of Chicago, Chicago, IL_.

Introduction: A T cell-inflamed tumor microenvironment characterized by the presence of tumor-infiltrating T cells or expression of an immune gene signature by RNA sequencing is linked to improved prognosis and response to immunotherapy in bladder cancer. BATF-3+ dendritic cells have been observed in murine models to be critical for both priming an immune response and recruiting effector CD8+ T cells to the tumor microenvironment. However, the role of BATF-3+ dendritic cells in bladder cancers from patient specimens, especially in relation to effector CD8+ T cells, remains unknown.

Methods: We performed multiplex immunofluorescence imaging on 61 surgically resected muscle invasive bladder cancer specimens to evaluate the population of tumor infiltrating immune cells (TIICs) including CD8+, BATF-3+, PD-1+, and FOXP3+ cells. The relationship of TIICs to a previously described immune gene signature based on RNA sequencing was investigated in a subset of these samples.

Results: In the 61 muscle invasive bladder cancer samples analyzed, the average (range) numbers of each cell type per 1000 cells in tumor area were 15.73 (0-162.33) for CD8+ cells, 0.68 (0-6.32) for BATF-3+ cells, 1.42 (0-27.10) for PD-1+ cells, and 6.10 (0-27.82) for FOXP3+ cells. In the subset (n =3 4) with available RNA sequencing data, the average numbers of each cell type for the tumors with T cell-inflamed phenotype (n = 17) vs the tumors with non-T cell-inflamed phenotype (n = 17) were, 23.39 versus 8.99 (P = 0.024) for CD8+ cells, 1.26 versus 0.22 (P = 0.034) for BATF-3+ cells, 3.48 versus 0.37 (P = 0.005) for PD-1+ cells, and 11.40 versus 2.72 (P = 0.001) for FOXP3+ cells, respectively. The proportion of T cell-inflamed tumors was significantly higher in groups with above median TIICs versus below (73.3% versus 31.6% for CD8 [P = 0.016], 75.0% versus 27.8% for BATF-3 [P = 0.006], and 70.0% versus 21.4% for PD-1 [P = 0.005], and 78.9% in FOXP3high and 13.3% in FOXP3low [P < 0.001], respectively). Analyzing BATF-3 and CD8 jointly identified the group with the highest proportion of T cell-inflamed tumors (90.0% in CD8highBATF-3high versus 33.3% in others [P = 0.003]) and showed a stronger association than with CD8 alone. In a spatial analysis using G function between CD8+ cells and BATF-3+ cells, the median area under the curve was 0.88, indicating that CD8+ cells clustered in proximity to BATF-3+ cells.

Conclusions: Infiltration of each immune cell type correlates with a T cell-inflamed or non-T cell-inflamed tumor microenvironment by gene expression profiling. CD8highBATF-3high tumors are nearly universally T cell-inflamed. Further analyses to assess associations between immune cell types and their spatial relations within the tumor microenvironment, and its correlation with survival outcomes and immunotherapy response are ongoing.

#1094

Lipopolysaccharide-induced chronic inflammation promotes lung tumorigenesis in the context of an immunosuppressive microenvironment.

Chia-Hsin Liu, Kong Chen, Yuanpu Di. _University of Pittsburgh, Pittsburgh, PA_.

Clinical and epidemiological evidence suggest that chronic infection and inflammation increase risk of lung cancer. Pseudomonas aeruginosa infection is frequently found in patients with chronic obstructive pulmonary disease (COPD) and is associated with increased lung inflammation and acute exacerbations. However, the mechanism of chronic bacterial infection-induced lung inflammation in promoting lung tumorigenesis remains unclear. To elucidate this mechanism, we established a murine lung cancer model by treating mice with or without recurrent lipopolysaccharides (LPS) from Pseudomonas aeruginosa in combination with nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Interestingly, combined LPS and NNK exposure significantly increased tumor number, tumor incidence, and tumor area compared to NNK treatment alone. In addition, the inflammatory cell counts in the bronchoalveolar lavage (BAL) including macrophages, neutrophils, and lymphocytes were significantly increased in the LPS/NNK treatment group. The BAL fluid of chemokines/cytokines, as analyzed by luminex assays, revealed higher levels of IL-17, CXCL10, GM-CSF, G-CSF, MIP-1a, and KC in LPS/NNK than in NNK treatment group. Flow cytometry analysis of the mouse lung tissue revealed that combined LPS and NNK exposure significantly increased CD4+ T cells including Th1, Th17, and Tregs and myeloid-derived suppressor cells recruitment in the lung. Real-time polymerase chain reaction of mouse lung tissue showed T cell exhaustion related genes, including Pdcd1, Ctla-4, Tim-3, Lag-3, and Foxp3, were significantly upregulated in the LPS/NNK treatment than NNK treatment. Moreover, immunohistochemical staining of LPS/NNK-exposed lung tumors showed higher PD-L1 expression than NNK-exposed lung tumors. Our data suggest that chronic LPS exposure-promoted and NNK-induced lung tumorigenesis is associated with immunosuppressive tumor microenvironment. The changes include recruitment of Tregs and MDSCs, increased T cell exhaustion, and upregulated PD-1/PD-L1 pathway, which may be used as the therapeutic target for chronic inflammation-associated lung cancer treatment.

#1095

Macrophage is a novel and rich source of cancer-associated fibroblasts in the tumor microenvironment.

Philip Chiu-Tsun Tang, Patrick Ming-Kuen Tang, Jeff Yat-Fai Chung, Xiao-Ru Huang, Ka-Fai TO, Hui-Yao LAN. _The Chinese University of Hong Kong, Hong Kong, Hong Kong_.

Background: Cancer-associated fibroblasts (CAF) are highly heterogeneous and their origins are largely unknown. Recently, we revealed that bone marrow-derived macrophages (BMDM) can further differentiate into myofibroblasts locally at the inflammatory site in a Smad3-dependent manner, but its potential role in the tumor microenvironment (TME) is still unexplored. Thus, we hypothesize that tumor-associated macrophages (TAM) may be able to transit into CAF, termed TAM to CAF transition (TAM-CAF), in TME for cancer promotion. Here, we are the first study to discover TAM-CAF as a common phenomenon in cancer.

Methods: The evidence of TAM-CAF was confirmed on tumor biopsies from several cancer types including non-small cell lung carcinoma (NSCLC) tissue microarray. The occurrence pattern of TAM-CAF was determined in LysM-Cre/Rosa26-tdTomato mice with syngeneic lung carcinoma LLC by the fate-mapping study. The pathogenic role of TAM-CAF was examined by adoptive transferring of TAM-CAF cells onto LLC-bearing NOD-SCID mice. The regulatory mechanism was further elucidated on Smad3-WT/KO mice in vivo and BMDM in vitro.

Results: Interestingly, we observed myofibroblast marker expressing TAM (α-SMA+CD68+) in the tumor biopsies of liver, kidney and lung cancers, which contributed to ~40% of the total CAF and positively associated with the NSCLC mortality. Fate mapping study confirmed the existence of macrophage-derived CAF (α-SMA+ tdTomato+) in LLC-tumor, where the contribution of TAM-CAF on CAF production was progressively increased throughout the tumorigenesis. Furthermore, adoptive transferring of TAM-CAF cells largely promotes tumor progression in LLC-bearing NOD/SCID mice associated with a marked increase of angiogenesis in the TME. Mechanistically, we found that phosphorylation of Smad3 is significantly associated with CAF production in NSCLC-TME (n=120), supported by the dramatic reduction of TAM-CAF in LLC-tumor and BMDM after deletion of Smad3. More importantly, we identified a conserved Smad3 binding site on the 5'UTR of FAP gene by ECR browser and ChIP assay in vitro; revealing its direct regulation on FAP expression at transcriptional level during TAM-CAF.

Conclusion: We are the first study to uncover TAM as a rich source of CAF, which is commonly occurred in TME for cancer promotion via a Smad3-dependent mechanism. Thus, TAM-CAF may represent a novel and precision therapeutic target for cancer.

Acknowledgments: This study was supported by Lui Che Woo Institute of Innovative Medicine (CARE program), Research Grants Council of Hong Kong (GRF 14117815, 14121816, 14163317, C7018-16G, TRS T12-402/13N), Health and Medical Research Fund (03140486, 14152321), Innovation and Technology Fund of Hong Kong (ITS/068/18), Direct Grant for Research CUHK (2017.002).

#1096

Response of pancreatic ductal adenocarcinoma to gemcitabine using human organotypic slice cultures.

Jae Hyuck Chang, Chae Yoon Lim, Weon Sun Lee, Guk Jin Lee. _The Catholic University of Korea, Seoul, Republic of Korea_.

Background/Objective: The oragnotypic tumor slice culture model resembles the in vivo situation of tumor. It may serve as a proper ex vivo platform for response to anti-tumor therapies. We aimed to evaluate the response of pancreatic ductal adenocarcinoma (PDA) cells and helper, cytotoxic, and regulatory T cells to gemcitabine using human PDA slice culture model.

Methods: PDA tissues were obtained from patients who were operated on for pancreatic resection. The tumors were sliced by a vibratome, and the tumor slices were then cultured until 5 days with 0.1 μM, 1 μM, and 10 μM gemcitabine for 1 or 2 days. Tumor responses were evaluated by number of tumor cells and proportion of Ki-67 or caspase-3 positively expressed cells in the tumor. CD4+, CD8+, and FOXP3+ cells were analyzed by immunohistochemistry staining.

Results: Gemcitabine treatment to PDA slices exerted proportional cytotoxic effects in terms of tumor cell number, and Ki-67 and cleaved caspase-3 expression. Mean Ki-67 positive cells in PDA cells tended to decrease according to increase of gemcitabine concentration (case 1, 48 hr treatment; 65.7% in control vs. 27.9% in 0.1 μM, p=0.025). Mean caspase-3 positive cells in PDA cells tended to increase according to increase of gemcitabine concentration (case 1, 48 hr treatment; 8.6% in control vs. 18.6% in 0.1 μM, p=0.042). Mean PDA cells per x200 tended to decrease according to increase of gemcitabine concentration (case 2, 48 hr treatment; 78.3 in 1.0 μM vs. 27.3 in 10 μM, p=0.042). Mean CD4+, CD8+, and FOXP3+ cells/total cells tended to decrease according to increase of gemcitabine concentration; in case 3 (24hr treatment), mean CD4+ cells/total cells decreased as 2.6%, 1.6%, 1.5%, and 0.5%, mean CD8+ cells/total cells decreased as 2.6%, 1.5%, 1.4%, and 1.1%, and mean FOXP3+ cells/total cells were 0.2%, 0.3%, 0.2%, and 0.1% in control, 0.1 μM, 1 μM, and 10 μM gemcitabine, respectively. However, mean CD8+/FOXP3+ cell ratio did not significantly decrease, and even increase from 4.4 in 0.1 μM to 8.6 in 1 μM (p=0.039).

Conclusions: Tumoricidal effect of gemcitabine could be demonstrated using organotypic slice culture model. T cells in the tumor decreased according to increase of gemcitabine concentration; however CD8+/FOXP3+ cell ratio relatively maintained.

#1097

**Establishment of murine immune and stroma reference bench for TME study on** in vivo **models.**

Jia Xue, Xiaoyu An, Xiaobo Chen, Wubin Qian, Henry Q. Li, Sheng Guo. _Crown Bioscience Inc., Taicang, China_.

Cancer is a heterogeneous disease and highly related to immunological system. Experimental animal models such as patient derived xenograft (PDX) are excellent system to understand tumor microenvironment (TME), especially for immunotherapy. Deconvolution analysis on bulk tissue genomics of these in vivo tumor models is one of cost-effective ways to investigate TME. Several groups have released robust deconvolution algorithms with human immune cell signature. Our previous research on ~1600 transcriptomes of PDX collection identified various human immune and stromal contents in 24 different cancer types. However, no appropriate mouse immune signature generated from single standard platform could be used so far to study murine immune and stromal contents. To this end, we established a reference dataset by performing transcriptome sequencing of over 10 types of mouse immune and stromal cells, including different subtypes of B cells, T cells, monocytes, macrophages, dendritic cells, neutrophils, natural killer cells, cancer associated fibroblasts, glial cell and epithelial cell, etc. These cells were isolated from 3 different tissues (bone marrow, lymph node and spleen) on tumor-bearing and non-tumor-bearing mice covering a variety of mouse strains. We identified the transcriptomic landscape of murine immune system as well as difference across source tissues/organs and mouse strains. Based on this reference set, which is generated from single standard protocol, we defined precision gene signatures for studied cells and refined deconvolution algorithms for mouse TME studies. We further evaluated multiple algorithms by flow cytometry. In conclusion, our murine immune and stroma reference dataset will be essential basis for model selection in immunotherapy study in future.

#1098

Enhanced AKT-dependent metabolic fitness and antitumor effect of T lymphocytes in lung cancer model.

Sung Eum Kim,1 Kyoung-Ho Pyo,1 Sang Bin Lim,1 Mi Ran Yun,2 Jae Hwan Kim,1 Ji Min Lee,1 Ha Ni Jo,2 Chun-Feng Xin,1 Jae Seok Cho,2 Byoung Chul Cho1. 1 _Yonsei University, Seoul, Republic of Korea;_ 2 _JEUK Institute for Cancer Research, Gumi, Republic of Korea_.

Introduction: PI3K signaling plays a pivotal role in balancing of immune responses. This study is to elucidate the role of PI3K-AKT signaling in regulating T cell metabolic fitness.

Methods: PI3K/AKT inhibitors including BKM120 were used to evaluate the contribution of PI3K-AKT signaling on the metabolic fitness of T cell. Glucose metabolism and T cell activity were measured by flow cytometry, IHC, RT-PCR. Oxygen consumption rate (OCR) and extra cellular acidification rate (ECAR) were examined by seahorse. In addition, anti-tumor effects of T cells were evaluated by adaptive transfer in LLC2 tumor bearing mouse.

Results: Compare to vehicle, BKM120-treated antigen-specific CD8+ T cells co-cultured with LLC2 cancer cells maintained cytotoxic effects under hypoglycemic and hypoxic conditions. Treatment of BKM120 significantly reduced tumor growth, compared to vehicle in syngeneic models (LLC2, KLN205), whereas it did not affect tumor growth in nude mice, which suggests role of activation of antitumor immunity, augmented by BKM120. Compared to vehicle, BKM120 significantly induced higher infiltration of CD8+ T cells and reduction of Foxp3+ regulatory T cell and CD11b+/Gr-1+ myeloid derived suppressor cells. Intriguingly, increased glucose uptake through overexpression of glucose transporter (GLUT1) was observed upon BKM120 treatment. Glut1 overexpression occurred through the release of feedback inhibition of mTORC2 followed by rebound AKT activation. In addition, glycolysis-related genes (Glut1, Hk, Pgam, Pdk and Pgm) and OCR/ECAR were significantly augmented in BKM120/selective PI3K-delta inhibitor (Idelalisib)-treated group. The enhanced glycolysis via PI3K signaling inhibition was significantly more prominent by Idelalisib, compared to BKM120 or selective PI3K-alpha inhibitor (BYL-719). The antigen-specific T cells pretreated with BKM120 ex vivo exhibited substantially more potent antitumor effects, compared to untreated T cells, in LLC2 tumor-bearing nude mice.

Conclusion: PI3K inhibitor, specifically selective PI3K-delta inhibitor, promotes AKT-dependent metabolic fitness of T cells which sustain anti-cancer effects under nutrient/oxygen-deprived tumor microenvironment. These data support a rationale for combination of selective PI3K-delta inhibitor with checkpoint inhibitors anti-PD1 for patient with Lung cancer.

#1099

Organotypic tumor tissue slices provide versatile platform for immuno-oncology.

Ramya Sivakumar,1 Marina Chan,1 Raymond Yeung,2 Taranjit S. Gujral1. 1 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 2 _University of Washington, Seattle, WA_.

Organotypic tissue slices represent one of the most physiologically-relevant culture systems for studying the tumor microenvironment. The thin slices of tumor cut out using automated vibratome retain the original architecture and cellular composition of the tumor from several days up to weeks depending on the investigated tumor model. The slice culture has been emerging as a versatile platform for studying cancer cell-host cell interactions. However, a thorough characterization of the multicellular organotypic culture system, particularly its immune component, is still unresolved to allow a better understanding of its potential applications. Here, we carried out a comprehensive characterization of immune cell composition in organotypic tumor slices prepared from several different syngeneic mouse tumor models. We generated several syngeneic mouse tumor models and harvested the primary tumors to make tumor slices. We then immunophenotyped the major tumor infiltrating leukocytes (TILs) by flow cytometry to understand the immune makeup of these slices immediately after the harvest, over a period of 7 days and upon treatment of various immuno-modulators. We found that the immune compositions of tumor slices do not deviate from that of the tumor cores, immediately after harvest, across all the syngeneic tumor models. During the 7-day follow-up, despite an initial decline in the overall cell viability which stabilized on day 3, we observed a consistent percentage of TILs out of total viable cells, suggesting that the relative composition of tumor and immune populations can be maintained in the tumor slice over time. Among the TILs, macrophages were the predominant immune cell type detected, similar to that observed in many human solid tumors. Macrophages remained viable till day 7 while other cell types were mostly viable till day 5, providing a window of time sufficient to support many short-term perturbation studies. Further, we found that these immune cell populations were responsive to the treatment with immunomodulators. For example, we observed an upregulation of co-stimulatory markers, CD86 and MHC-II, on macrophages and DCs upon stimulation with the pro-inflammatory cytokine IFN γ, further supporting the application of tumor slice in the study of immuno-oncology. Overall, these data support the approach of utilizing tumor slice culture as a platform for studying tumor microenvironment and evaluating efficacy of immune modulatory drugs.

#1100

Genetic driver-mutations define composition and properties of tumor-associated myeloid cells in glioblastoma.

Zhihong Chen, Cameron J. Hurting, Ben Gabanic, Jyothi Murukuti, Dolores Hambardzumyan. _Emory University, Atlanta, GA_.

The GBM microenvironment is composed of numerous non-neoplastic cells, among which tumor-associated myeloid cells (TAMs) are the most abundant. TAMs are constituted of several different cell types, including brain microglia and bone-marrow-derived infiltrating cells, which can be further divided into inflammatory monocytes, monocyte-derived macrophages, and polymorphnuclear neutrophils. These myeloid cells are believed to promote tumor growth and immune evasion by expressing immune checkpoint proteins. Remarkable progress has been made in treating lung cancer and melanoma by using immune-checkpoint inhibitors; however, mixed results were obtained for its application in GBM. To examine how molecular-subtypes of GBM influence the composition and immunosuppression of tumor-associated myeloid cells, we used the RCAS_tv-a system, a somatic cell-specific gene transfer system, to generate de novo murine GBM (mGBM). By manipulating different known human oncogenic-driver mutations such as overexpressing PDGFB or EGFRVIII, or silencing NF1, we generated Proneural (PN), Classical (CL) and Mesenchymal (MES) GBMs respectively, which phenotypically resemble their human counterparts. We found that the majority (80%) of tumor-infiltrating myeloid cells in PN GBM are bone-marrow derived macrophages, whereas this number is significantly less in CL and MES mGBM. Interestingly, unique to MES, there is a significant presence (16%) of neutrophils in these tumors than in other subtypes (4%). Further characterization for subtype-specific expression of PD-L1, a druggable immune checkpoint molecule, by flow cytometry shows that MES has the highest expression of PD-L1), whereas the expression is lower in PN and CL subtypes. Treating GBM-bearing mice with anti-PDL1 antibodies did not prolong their survival. Concurrent treatment of irradiation and anti-PDL1 antibody did not confer significant survival advantage either compared to controls. These results reflect the unsuccessful trails of recent human clinical studies; however, it also emphasizes the importance of understanding the mechanism of resistance in GBM immunotherapy before effective immune treatment can be established. Our immunocompetent mouse models provide indispensable tools for these studies.

#1101

**Characterization of spatial distribution of tumor-infiltrating CD8** + **T cells refines their prognostic impact in pancreatic cancer.**

Yohei Masugi, Tokiya Abe, Minoru Kitago, Masahiro Shinoda, Michiie Sakamoto. _Keio University School of Medicine, Tokyo, Japan_.

Background: The abundance of cytotoxic T cell infiltrates has important implications for patient outcome and therapeutic design in pancreatic ductal adenocarcinoma (PDAC), although intratumoral heterogeneity remains a major challenge for the better understanding of complicated immune microenvironment of PDAC. We aimed to characterize spatial CD8+ cell distribution within PDAC tissues, which might refine the prognostic role of tumor-infiltrating CD8+ lymphocytes.

Methods: We measured CD8+ cell density in tumor center, tumor margin, or whole tumor area, as well as CD8+ cell proximity to carcinoma cells, using fluorescent multiplex immunohistochemistry-based tissue computation on whole tissue sections of 214 surgically resected PDACs. Multivariable Cox proportional hazards regression analysis was performed to assess the association of CD8+ cell density with pancreatic cancer-specific survival, adjusting for major clinicopathologic and immune-related features, including the programmed cell death 1 ligand 1 (CD274, PD-L1) expression and the extent of tertiary lymphoid structures.

Results: We found a substantial heterogeneity in CD8+ cell density with, on average, a 2-fold-lower density in tumor center than in tumor margin. In multivariable model, the trend of prolonged survival with higher CD8+ cell density was stronger for tumor center, compared to tumor margin or whole tumor area (P for trend = 0.002, 0.07, or 0.009, respectively). Tumor CD274 expression and extensive tertiary lymphoid structures were appeared to be associated with higher CD8+ cell density in tumor margin, but not with CD8+ cell density in tumor center. The mean distance from tumor cells to the adjacent CD8+ cells correlated inversely with CD8+ cell density in tumor center (Spearman correlation efficient, -0.89), although the density of CD8+ cells proximate to cancer cells did not show any prognostic correlation.

Conclusion: Our data have shown a prognostic significance of CD8+ cell density in tumor center of PDAC, where CD8+ cell infiltration has been severely limited, suggesting a major role of pro-tumorigenic effect of immunosuppressive microenvironment in pancreatic cancer.

#1102

Time and spatial distribution of monocytic lineages in the process of carcinogen-induced oral cancer.

Chi Jui Chen. _National Tsing Hua university, Hsinchu City 300, Taiwan_.

Head and neck cancer (HNC) is the sixth among the top ten cancers in the world. Among this group of disease, oral cancer has been one of the top five killers in males for many years in Taiwan. Despite combined treatment, the prognosis for HNC patients is, in general, poor, which has strongly stimulated research on finding new strategies specific for HNC. To find a new treatment protocol for HNC, we examined the tumor microenvironment of a carcinogen (4-Nitroquinolone-1-oxide,4-NQO)-induced oral cancer. The immunohistochemical staining (IHC) results showed that this tumor has relative low density of vessels and high percentage of hypoxia around tumor edge. In addition, we found a layer of α-SMA positive cancer-associated fibroblast (CAF) in the junction of tumor and normal tissues. Further, we also found that a cluster of high density of CD68+ macrophages and Gr-1+ cells in this junction. To our surprise, very few tumor-associated macrophages (TAMs) could be identified in other tumor regions except this junction. In addition, the analysis of blood sample during tumor development found that the population of monocytic and granulocytic myeloid-derived suppressor cells (M-MDSCs and G-MDSCs) in the whole blood increases with the growth of the tumor, which has good correlation with the decrease of CD8+ and CD4+ T cells. The unique spatial distribution of myeloid cells in tumor tissue and consistent dynamic change of MDSC and T cells in the blood provides us an ideal tumor model for designing new strategies specific for HNC. We are currently using this tumor model to study the potential of combining immunotherapy with boron neutron capture therapy (BNCT) for HNC.

#1103

The effects of neo-adjuvant chemotherapy on myeloid cells in high-grade serous ovarian cancer metastases.

Owen M. Heath,1 Eleni Maniati,1 Chiara Belato,1 Ganga Gopinathan,1 Laura Lecker,1 Anissa Lakhani,1 Colin Pegrum,1 Jacqueline McDermott,2 Michelle Lockley,1 Desmond P. Barton,3 Frances Balkwill1. 1 _Barts Cancer Institute, London, United Kingdom;_ 2 _University College Hospital, London, United Kingdom;_ 3 _St. George's Hospital & Royal Marsden Hospital, London, United Kingdom_.

Many tumours have abundant macrophage populations. Tumour-associated macrophages (TAMs) frequently have tumour promoting roles and are associated with poor clinical outcome. We hypothesise that targeting TAMs in high-grade serous ovarian cancer (HGSOC) may improve response to chemotherapy.

We have assessed the effects of chemotherapy on TAM populations in human HGSOC obtained pre- and post-chemotherapy as well as in murine HGSOC models harbouring a relevant mutational profile. We find that chemotherapy treatment decreases TAM density within tumour areas. Furthermore, TAMs expressing markers known to associate with disease progression were decreased following chemotherapy. In vivo and in vitro we have demonstrated an up-regulation of inflammasome activation and TLR signalling in live myeloid cells following chemotherapy and have shown that macrophages are killed by chemotherapy at clinically relevant drug concentrations. These observations suggest a mechanism for TAM depletion and highlight chemotherapy induced activation of innate immunity in HGSOC.

The majority of HGSOC patients respond well to first line chemotherapy but will relapse and succumb to treatment resistant disease. We have developed a murine model of HGSOC relapse after first-line chemotherapy, which has the potential to extend translational studies into this clinically important area. We have found that TAMs are re-established in tumours at relapse, suggesting a clinically defined window of opportunity to target TAMs in HGSOC following first-line chemotherapy.

Overall, our results provide a rationale for targeted re-programming of TAMs in HGSOC after chemotherapy.

#1104

Targeting the stress response kinase GCN2 to restore immunity in the tumor microenvironment.

Buvana Ravishankar, Lavanya Adusumilli, Deepa Pookot Pookot, Emily Huang, Raashi Sreenivasan, Lisa Marshall, Deepika Kaveri, Oezcan Talay, Silpa Suthram, Svetlana Miakicheva, Abood Okal, Mikhail Zibinsky, Jeffrey Jackson, Grant Shibuya, Paul Leger, Parcharee Tivitmahaisoon, Scott Jacobson, Steve Wong, Angela Wadsworth, Jerick Sanchez, Martin Brovarney, David Chian, Sachie Marubayashi, Aparna Jorapur, Delia Bradford, Christophe Colas, Gene Cutler, Jacob Schwartz, David Wustrow, Paul Kassner, Dirk Brockstedt. _FLX Bio, Inc., South San Francisco, CA_.

The tumor microenvironment (TME) is characterized by deficiencies in oxygen and key nutrients, such as glucose and amino acids, resulting in an overall immune-suppressive environment. Key suppressive cell types in the TME include tumor, stromal and myeloid-derived suppressor cells (MDSC) which create a nutrient-poor environment that supports tumor growth and limits immune surveillance. General control nonderepressible 2 (GCN2), a stress response kinase, plays a key role in sensing and modulating the cellular response to amino acid deprivation. GCN2 activation in T cells triggers the integrated stress response pathway and promotes T cell anergy and apoptosis. We have developed small molecule GCN2 inhibitors (GCN2i) that are highly potent and selective in vitro. Culturing primary mouse or human immune cells under low nutrient conditions activates the GCN2 pathway limiting T cell proliferation and function. Treatment of these nutrient-deprived T cells with GCN2i resulted in rescue of CD8+ T cell proliferation and effector functions. In addition, GCN2 inhibition in MDSC alone fully reversed CD33+MDSC-induced T cell suppression and effector functions. Our GCN2 inhibitors are orally bioavailable with drug like in vivo ADME properties. Our GCN2i is currently being evaluated in vivo, in murine syngeneic tumor models. Our results demonstrate that inhibition of GCN2 is an attractive approach for relieving T cell suppression and promoting effector function, demonstrating GCN2 as a promising therapeutic target for the treatment of cancer.

#1105

Cisplatin treatment alters the preference of melanoma metastasis to liver associated with Ly6G positive cells.

Chang Chun-Hsiang. _National Tsing-Hua University, Hsinchu, Taiwan_.

This study examined the influence of cisplatin on host stroma and its consequence on metastasis using a metastatic melanoma tumor model. This preliminary result indicates that Gr-1+ cells could play important role on cisplatin treatment-directed liver metastasis. To further examine the role of Gr-1+ cells on this phenomena, Gr-1Ab was administered at day 16 after tumor inoculation. Results show that Gr-1Ab treatment could effectively decrease the Ly6G+ cells in both lung and liver and without significant reduced in lung-metastasis but, increased the metastasis in lung. To identify the effect of cisplatin treatment on B16-F0 melanoma metastasis, normal mice were treated with cisplatin prior to iv inject of B16-F0 melanoma cells. Cisplatin prevented treatment, similar to Gr-1 Ab therapy, decreased Ly6G+ cell in lung and liver tissue. And, it is minor difference of lung metastasis compare to Gr-1 Ab treatment, but more metastatic site than it. It demonstrates that cisplatin therapy induced liver-metastasis could associate with Ly6G+ cells. This study concludes that the cisplatin treatment could alter the preference of tumor metastasis, which is associated with the reduction of Ly6G+ cells It could be a potential target to enhance therapy efficiency.

#1106

Co-administration of the iRGD tumor-penetrating peptide improves the tumor immunostimulatory effects of low-dose IL-2.

Harri Jarvelainen,1 Gregory P. Botta,2 Tatiana Hurtado De Mendoza,2 Erkki Ruoslahti2. 1 _DrugCendR Inc., La Jolla, CA;_ 2 _Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA_.

Background: Recombinant IL-2 at high doses is an effective immunotherapy treatment for various types of solid tumors. However, its clinical utility has been limited by serious mechanism-based side-effects. The clinical-stage iRGD peptide specifically targets tumors and, via activation of the 'CendR' trans-tissue transport pathway, increases tumor penetration and accumulation of various types of co-administered drugs. By selectively increasing the drug delivery into tumors, but not to normal tissues, iRGD potentiates the pharmacological activity of various anti-cancer agents within tumor tissue, while reducing their toxicities. In this study we investigate whether co-administration of iRGD with low-dose IL-2 can improve the tumor immune microenvironment by increasing in the ratios of tumor-killing effector CD8 cells vs. the immunosuppressive T-regulatory cells.

Methods: Subcutaneous breast tumors (4T1) were generated in immunocompetent mice. Mice were treated with either vehicle control, iRGD, IL-2, or IL-2 + iRGD for 5 days. Tumors were preserved for immunohistochemistry (IHC) or enzymatically digested for fluorescence activated cell sorting (FACS) 16 hours after the last dosing. The FACS and IHC panels were designed to detect the percentage of total T cells, CD4 and CD8 T cells, and Treg cells.

Results: Low doses of IL-2 alone were found to increase Tregs within the tumor but had no effect in CD4 or CD8 effector T cells, compared to vehicle treatment. However, when Low dose IL-2 was co-administered with iRGD, we observed a significantly lower percentage of T regs and an increase in CD4 effector T cells. More importantly the ratio of CD8 / Treg cells was increased by at least 10-fold, compared to low dose IL-2 alone.

Conclusions: The combination treatment with iRGD and low doses of IL-2 is capable of favorably altering the tumor immune microenvironment such that immunosuppressive T reg cells were reduced , while there was an increase in the effector CD4 and CD8 T-cell populations. The tumor-selective IL-2 pharmacology benefit obtained with iRGD co-administration may provide new options for the use of the well-validated IL-2 in solid tumor patients, including a strategy to overcome the primary resistance to PD-1 blockade.

#1107

LAG3 in Solid Tumors as a Potential Novel Immunotherapy Target.

Su Jin Lee, Sun-ju Byeon, Jeeyun Lee, Se Hoon Park, Joon Oh Park, Young Suk Park, Won Ki Kang, Ho Yeong Lim, Kyoung-Mee Kim, Seung Tae Kim. _Samsung Medical Center, Seoul, Republic of Korea_.

We performed prospective immunohistochemical analysis of LAG3 for 430 consecutive patients with advanced gastrointestinal, genitourinary, or rare cancers between June 2012 and March 2016. Most patients (428/430, 99.5%) were evaluable for LAG3 expression by immunohistochemistry. In total, 18.5% (79/428) of the evaluated cancers expressed LAG3, including pancreatic (33.3%, 2 of 6), gastric (24.7%, 21 of 85), colorectal (23.6%, 48 of 203), melanoma (12.5%, 1 of 8), genitourinary (9.5%, 4 of 46), biliary tract (6.3%, 1 of 16), and sarcoma (5.4%, 2 of 37), but not miscellaneous (0.0%, 0 of 14) or hepatocellular (0.0%, 0 of 15) cancer. Among 149 metastatic CRC patients, there was no statistically significant difference in gender, age, primary tumor site, pathologic differentiation, KRAS and NRAS status, BRAF status, and microsatellite instability according to LAG3 status (expressed vs. non-expressed). Among 53 metastatic GC patients, LAG3 was only significantly associated with EBV status (P = .042). Our results add to the emerging literature on LAG3 expression in various cancer types and support the need for extended clinical exploration of this target for immunotherapy.

### Metastasis Promoter and Suppressor Genes

#1108

Intestine-specific homeobox recruits p300/CBP-associated factor and bromodomain-containing protein 4 to promote epithelial-mesenchymal transition.

Li-Ting Wang, Shyh-Shin Chiou, Shih-Hsien Hsu. _Kaohsiung Medical University, Kaohsiung, Taiwan_.

The epithelial-mesenchymal transition is an important process in the cancer progression, but its occurrence and the regulatory mechanism are not fully understood. Intestine specific homeobox acted both as a proto-oncogene and upstream regulator of EMT markers, by which modulates tumorigenic initiation and progression in lung cancer cells. ISX acetylated by PCAF recruits bromodomain-containing protein 4, to initiate chromatin remodeling, and up-regulated EMT downstream regulators in tumors cells. Ectopic expression of ISX was shown to enhance TWIST1, Snail1 expression by recruiting BRD4 to enhance Pol II dependent transcription, lead in to remodeling of the tumor microenvironment. In NSCLC carcinoma, increased ISX expression was noted correlating with distinct clinical metastatic features and poor prognosis. These results suggest that theISX-BRD4 axis mediates EMT signaling and exerts significant regulatory effects on tumor initiation and metastasis.

#1109

CPAP promotes HCC angiogenesis and metastasis via interacting with and enhancing STAT3 signaling.

Ruo Yu Chen, Liang-Yi Hung. _National Cheng Kung University, Tainan City, Taiwan_.

Centrosomal P4.1-associated protein (CPAP) is a centrosomal protein and can as a transcriptional coactivator of STAT5 and NF-κB in cancer. Our previous studies indicated that CPAP is overexpressed in tumor tissue and can increase TNFα-mediated NF-κB activation in HCC. Here, we demonstrated that overexpressed CPAP increases tumor growth, angiogenesis, as well as metastasis ex vivo and in vivo. We found that CPAP increases these malignant abilities of tumor cells are through IL-6/STAT3 signaling. We demonstrated that CPAP directly interacts with C-terminal domain of STAT3 to increase STAT3 activity. Overexpression of CPAP enhances IL-6/STAT3/IL-8-mediated angiogenesis and other metastatic genes expression such as CD44 and MCAM. These results indicated that CPAP leads to HCC malignancy and metastasis by increasing STAT3 activity through directly interaction. Clinically, CPAP positively correlates with IL-8 in HCC with vascular invasion; and also positively correlates with CD44 and MCAM in HCC tissues. In summary, our findings shed light on the importance of CPAP to act as a potential therapeutic target for inhibiting the IL-6/STAT3-mediated angiogenesis pathway and treating metastatic HCC.

#1110

KRAS-responsive lncRNAs control lung cancer metastasis and microRNA processing.

Lei Shi, Peter Magee, Sudhakar Sahoo, Michela Garofalo. _Cancer Research UK Manchester Institute, Manchester, United Kingdom_.

KRAS wild-type amplification has been associated with more invasive lung tumors, especially in combination with a KRAS activating mutation. However, the mechanism by which this occurs has been poorly investigated. Here, we show that focal amplification of the wild-type KRAS gene is a common feature in a subset of lung cancer patients and associates with poor overall survival. KRAS activates two lncRNAs, KIMAT1 and HIF1A-As2, which are essential for cancer cell survival and invasion. Mechanistically, KIMAT1 and HIF1A-As2 are MYC targets and promote EMT and distant metastasis by binding and stabilizing two oncoproteins, DHX9 and NPM1, which in turn regulate pro-metastatic pathways; by controlling microRNA processing through DHX9 stabilization by sequestering microRNAs involved in EMT. Our results demonstrate that silencing of the KRAS-responsive lncRNAs KIMAT1 and HIF1A-As2 may represent an effective cancer-cell specific option in KRAS-driven tumors.

#1111

Fucosyltransferase 4-mediated aberrant glycosylation and cell signaling networks promote lung cancer metastasis.

Hsuan-Hsuan Lu,1 Yi-Hsiu Juan,1 Rueyhung Roc Weng,1 Shu-Yung Lin,2 Yen-Wei Chen,3 Hsin-Han Hou,1 Zheng-Ci Hung,1 Yi-Jhen Huang,3 Tsai-Yu Yang,3 Yi-Chieh Wu,3 Giovanni Audrey Oswita,3 Jin-Yuan Shih,1 Hsing-Chen Tsai,3 Chong-Jen Yu1. 1 _National Taiwan University Hospital, Taipei, Taiwan;_ 2 _National Taiwan University Hospital Jinshan Branch, Taipei, Taiwan;_ 3 _National Taiwan University, Taipei, Taiwan_.

Rationale:Aberrant glycosylation has been known to regulate cancer cell trafficking via promoting intravascular adhesion during metastasis. Clinicopathological studies have also shown a strong correlation between aberrant glycosylation and invasive/metastatic potentials of human cancers. However, the major glycosylation enzymes that drive lung cancer metastases and the signaling networks involved in the process remain incompletely understood.

Methods:We identified fucosyltransferase 4 (FUT4) as a strong predictor of patient prognosis from two independent cohorts of patients with NSCLC (TCGA and NTUH). We analyzed functional roles of FUT4 through over-expression and knock-down studies using lung cancer cell lines in vitro. Metastatic and homing potentials of FUT4-overexpressed and/or knockdown cells were evaluated in vivo in immunodeficient mice. Genome-wide RNA-seq and MS-based immune-precipitation proteomics were performed to study molecular signaling networks.

Results: High expressions of FUT4 were associated with poor survivals in lung adenocarcinomas (p= 0.000681). FUT4-overexpressed lung cancer cells showed significantly increased migration, invasion, and adhesion abilities in vitro, as well as enhanced homing ability to the lungs in vivo. FUT4-overexpressedcells displayed typical characteristics of epithelial-to-mesenchymal transition (EMT) in a protein level-dependent manner. Lewis X and AAL antigens were significantly up-regulated in FUT4-overexpressedcells, which facilitated binding to E-, L- and P-selections. GSEA of RNA-seq data revealed the enrichment of metastasis-related signaling, especially in EGF and TGF-β signalings. Molecularly, FUT4 enhanced EGFR/TGFBR signaling through glycosylation of TGFBR receptors, which lead to increased binding affinity between ligands and its receptors.

Conclusion: FUT4 promoted metastasis through enhancing mesenchymal phenotype mediated by glycosylated EGFR/TGFBR signaling, and through increasing binding affinity of cancer cells to selectins. High levels of FUT4 in tumor tissues significantly correlate with poor prognosis in NSCLC patients. Our study not only provided new insights into the role of FUT4 on molecular signaling as a potential therapeutic target, but also identified FUT4 as a prognostic marker in NSCLC.

#1112

CCDC102B promotes metastatic cascade in breast cancer by activating NF-κB via down-regulating RACK1.

Jing Si,1 Rong Guo2. 1 _Jiaxing No.1 Hospital, China;_ 2 _Fudan University Shanghai Cancer Center, China_.

Background: Breast cancer is one of the leading causes of cancer death among women and the pathological status of axillary lymph nodes is an important predictor of prognosis. However, the mechanism involved in this early stage of metastasis remains largely unknown.

Methods: We used microarray analysis to carry out differential genomics analyses between matched pairs of sentinel lymph node metastatic tissues and breast primary tumors. We found 182 up-regulated genes in metastatic tissues (fold change≥1.5). We used CRISPR/Cas9 gene editing system to knockout these genes, each with 6 unique sgRNAs, and screened in vivo by transplanting loss-of-function cell pool into immunocompromised mice. MAGeCK was used to analyze the screening. Immunohistochemistry was performed to examine protein expressions in 212 cases of breast cancer tissues using tissue microarray (TMA) technology. Gene and protein expressions were monitored by qPCR and western blot. The relationship between coiled-coil domain containing 102B (CCDC102B) and receptor for activated C kinase 1 (RACK1) was examined using Co-IP, mass spectrometry, nuclear protein extraction assay and dual-luciferase reporter assay. Functional studies were detected in vitro by cell proliferation, wound-healing and migration, and in vivo by lung metastasis in immunocompromised mice injected CCDC102B-overexpressed cells into tail vein. The primary biological functions and pathways related to CCDC102B were enriched by RNA sequencing. The regulation of NF-κB signaling by CCDC102B and RACK1 was detected using dual-luciferase reporter assay and western blot.

Results: We identified CCDC102B through screening and found that it was significantly up-regulated in breast cancer patients with poor prognosis. The expression of CCDC102B were independent predictors in both DFS and OS (P=0.009; P=0.014). CCDC102B expression was found to promote the proliferation and migration of breast cancer cells MDA-MB-231 and BT549, and lung metastasis in mice. Moreover, we found that overexpression of CCDC102B decreased RACK1 expression whereas knockdown of CCDC102B increased RACK1 expression at both protein and mRNA levels, and the aggressive behavior of CCDC102B in breast cancer cells could be reversed by the expression of RACK1. According to gene set enrichment analysis (GSEA), CCDC102B was correlated with significant enrichment focused on NF-κB pathway. The mechanistic study revealed that gain of CCDC102B resulted in decreased expression of RACK1, which increased levels of epithelial mesenchymal transition (EMT) and phosphorylated Rel-A (p65), leading to the activation of NF-κB pathway and progression of breast cancer metastasis.

Conclusion: Taken together, our findings suggest that CCDC102B is strongly associated with negative clinical outcomes and may serve as a potential metastasis promotor by regulating the activation of NF-κB pathway via RACK1.

#1113

Targeting PRPK function blocks colon cancer metastasis.

Tatyana Zykova, Ke Yao, Seung Ho Shin, Eunmiri Roh, Weiya Ma, Ann M. Bode, Zigang Dong. _University of Minnesota, Austin, MN_.

The biological functions of the p53-related protein kinase (PRPK) remain unclear. We have previously demonstrated that PRPK is phosphorylated by the T-LAK cell-originated protein kinase (TOPK) and that phosphorylated PRPK (p-PRPK) promotes colon cancer metastasis. Here, we analyzed colon adenocarcinomas from 87 patients and found that higher expression levels of p-PRPK were associated with later stages of metastatic dissemination (stage III and IV) as compared with earlier stages (stages I and II). Indeed, levels of p-PRPK were higher in metastatic versus malignant human colon adenocarcinomas. Knocking down PRPK expression attenuated colorectal liver and lung metastasis of colon cancer cells in vivo. An in vitro kinase assay indicated that active PRPK does not phosphorylate p53 directly. We found that PRPK phosphorylates survivin, a regulator of colon cancer metastasis. PRPK phosphorylates survivin at Thr34, which is important for survivin stability. Taken together, our data strongly suggest that the PRPK signaling pathway promotes colon cancer metastasis by modulating survivin stability, and that PRPK could be a new prognostic marker for the survival of colon cancer patients. In addition, we identified an FDA-approved bacteriostatic antibiotic, fusidic acid sodium salt (fusidic acid or FA) as an inhibitor of PRPK, and show that FA combined with 5-fluorouracil (5-FU) inhibited PRPK activity and colon cancer metastasis to the lung in mice. We contend that the combination of FA with 5-FU could be an alternative therapeutic strategy to traditional chemotherapy for colon cancer patients with poor prognosis.

#1114

Upregulation of ALCAM is a marker for non-small-cell lung cancer brain metastases.

Desiree Loreth, Justine Münsterberg, Cecile Maire, Stefan Werner, Monja Gandrass, Christian Bernreuther, Jolanthe Kropidlowski, Manfred Westphal, Stefan Steurer, Katrin Lamszus, Markus Glatzel, Klaus Pantel, Harriet Wikman. _University Medical Center Hamburg-Eppendorf, Hamburg, Germany_.

Non-small-cell lung cancer (NSCLC) is the most common cause of cancer-related death with still a 5-year survival rate of <10%. Every second patient with advanced NSCLC will develop brain metastases and in 50% of those the brain is the only site of tumor relapse (oligo-metastasis). Cancer cells need to have specific properties to access and colonize the brain microenvironment leading to the question which metastatic factors foster brain metastasis formation. Different cell adhesion molecules (CAMs) have been shown to be involved in the process of metastasis in different tumor entities. The activated leukocyte cell adhesion molecule (ALCAM) is a type I transmembrane protein of the immunoglobulin superfamily and involved in the maintenance of cell-cell contacts and has a possible role in the "homing" of metastatic cells in distant organs. ALCAM is also involved in the leukocyte transmigration across the blood-brain barrier. Here we therefore investigated the role of ALCAM in NSCLC brain metastases formation. Our studies on patient material revealed that the ALCAM protein expression in brain metastases (n=71) is significantly increased (50.7% positive, p=0.023) compared to the primary tumor (21.7% positive, n=47). Analysis of matched pairs of primary tumors and brain metastases confirmed this observation, as in 33% a de novo ALCAM expression in the matching brain metastases could be observed. Additionally, patients with a strong ALCAM expression in either their primary tumors or brain metastases showed a significantly shortened overall survival (p=0.017 and p=0.035 respectively). In vitro analysis of the effect of ALCAM knock out in the H460 lung cancer cell line showed no effect on proliferation, migration or colony-forming behavior. However, cell adhesion was severely hampered in the knock out cells. Currently in vivo mice experiments are running. Together these findings indicate an important role for ALCAM in brain metastasis formation.

#1115

NANOG-AKT signaling increases metastatic capacity via stabilizes HDAC1 protein through Chfr inactivation.

Eunho Cho. _Korea University, Seoul, Republic of Korea_.

Histone deacetylases (HDACs), in particular HDAC1, play an important role in tumor development and progression by modifying histone and non-histone proteins. Overexpression of HDAC1, as a cancer marker associated with poor prognosis. Nevertheless, the regulatory molecular mechanisms of HDAC1 expression remain largely unknown. Previously, we validated the correlation between the key pluripotency transcription factor NANOG and HDAC1 in immune-edited tumor cells by identifying that NANOG upregulates HDAC1 expression through its promotor occupancy. In this study, we further elucidated the mechanism that Nanog could regulate the Hdac1 protein level, as well as transcriptional level. Interestingly, we found that Nanog induces phosphorylation of an E3 ubiquitin ligase Chfr through Akt signaling, which results in the inactivation of Chfr. This decreases not only ubiquitinase activity but proteasomal degradation on Hdac1. Furthermore, accumulated Hdac1 on its own gives rise to metastatic capacity in cancer cells. Moreover, inhibition of Akt signaling is significantly effective at reversing HDAC1 mediated metastatic capacity in refractory cancer types. Taken together, we suggest a mechanistic rationale for the application of Akt inhibitors in HDAC1+ cancer treatment.

#1116

The role of ADAM9 and PI3K-dependent NF-kappa B pathway in promoting metastasis of triple-negative breast cancer.

Rui Zhou,1 Hong Zhang,2 Chi Shing Cho,3 Sze Chuen Wong,1 Mei Tian,2 Wing Chi Chan1. 1 _The Hong Kong Polytechnic University, Hong Kong, China;_ 2 _Zhejiang University, Hangzhou, China;_ 3 _Queen Elizabeth Hospital, Hong Kong, China_.

Objectives:

As a zinc-dependent proteinase family member, A Disintegrin and Metalloproteinase 9 (ADAM9) has the metalloproteinase domain that performs proteolytic function to liberate or shed factors, such as bFGF, VEGF, EGF, TGF-α and IGF, from cell surface and make them more accessible to tumor cells. It was shown in previous studies that the upregulation of ADAM9 in breast cancer is associated with metastasis.

In this study, we examined the effect of ADAM9 on the cell proliferation, migration and invasion and elucidated the molecular mechanism in TNBC. We hypothesize that ADAM9 sheds Tumor Necrosis Factor alpha (TNF-α), which activates TNF receptor and then triggers PI3K-dependent NF-kappa B pathway, affecting metastatic phenotypes of triple negative breast cancer (TNBC).

Materials and Methods:

Correlation of the levels of ADAM9 expression and survival time in triple negative breast cancer patients was analysed by the Kaplan-Meier (KM) plotter integrative data analysis tool. The TNBC subjects were divided into two groups of high and low ADAM9 expression levels with respect to the median. We also compared the protein expression levels of ADAM9 in a highly aggressive, invasive and poorly differentiated TNBC cell line, MDA-MB-231, and a non-tumorigenic epithelial cell line, MCF-10A.

ADAM9 was knocked down in MDA-MB-231 using siRNA. In comparison to negative and blank controls, we examined the cell proliferation, migration and invasion, and the phosphorylation levels of AKT subject to ADAM9 knockdown. We also profiled the mRNA expression levels of ADAM9 and NF-kappa B in breast cancer patients.

Results:

As shown in Figure 1(a), the survival curves of these two groups were plotted and compared using Kaplan-Meier survival analysis, indicating that lower ADAM9 expression is linked to better survival rate (p=0.0038). Figure 1(b) illustrates significantly higher ADAM9 protein level in MDA-MB-231, compared with MCF-10A (p=0.0019).

It is shown in Figure 2 and Figure 3(a) that the cell proliferation, migration and invasion, and the phosphorylation of AKT are significantly suppressed by ADAM9 knockdown in the aggressive TNBC cell line. Figure 3(b) shows significant association between ADAM9 and NF-kappa B in the mRNA expression level (p<0.001).

Conclusions:

The results clearly showed the activation of PI3K-dependent NF-kappa B pathway triggered by ADAM9. This suggests a novel molecular mechanism that the stable homotrimers of TNF-α are released from the membrane through the proteolytic cleavage by ADAM9 and activate the signaling pathway leading to metastatic phenotype of TNBC.

Acknowledgments:

This project was supported by the National Basic Research Program of China (973 program, 2014CB744505).

1

#1117

The molecular basis of LIF-mediated NPC metastasis.

Shu-Chen Liu. _National Central University, Taoyuan City, Taiwan_.

Distant metastasis and local recurrence are frequently observed in nasopharyngeal carcinoma (NPC) but the mechanisms remain largely unclear. Here, we report that higher levels of leukemia inhibitory factor (LIF) and LIF receptor (LIFR) are correlated with poorer metastasis/recurrence-free survival. LIF reprograms the invasive mode from collective to mesenchymal migration via acquisition of EMT and invadopodia-associated characteristics. Higher cytoplasmic LIF enhances cancer vascular dissemination and local invasion mechanistically, both in vitro and in vivo, through modulation of LIFR-YAP1-focal adhesion signaling. Pharmaceutical intervention with AZD0530 markedly reverses LIF-mediated cancer dissemination and local invasion through promotion of cytoplasmic accumulation of YAP1 and suppression of focal adhesion kinases. Given the significant role of LIF/YAP1-focal adhesion signaling in cancer progression, targeting of this pathway presents a promising opportunity to block metastasis.

#1118

FOXQ1 stimulates WNT-signaling through transcriptional activation of WNT5B to promote the epithelial to mesenchymal transition.

Allison V. Mitchell, Guojun Wu. _Karmanos Cancer Institute, Detroit, MI_.

Aberrant induction of the epithelial to mesenchymal transition (EMT) is an acknowledged mechanism of acquired metastatic competence and drug resistance in many epithelial cancer types. Both physiological and pathological forms of EMT are thought to occur through a network of transcription factors (TFs) that drive cell plasticity and the acquisition of stem-like traits. The forkhead box transcription factor, FOXQ1, is a potent driver of EMT. FOXQ1 has been shown to be specifically upregulated in the triple negative subtype of breast cancer (TNBC) where is it associated with worse clinical prognosis. However, the role of FOXQ1 within the EMT transcriptional network has not been extensively characterized. In this study, we employed chromatin immunoprecipitation and RNA sequencing in a human mammary luminal epithelial cell line with ectopic FOXQ1 (HMLE/FOXQ1), as a model of breast EMT. We identified FOXQ1 localization within the promoter regions of prominent EMT-TFs, including TWIST1, ZEB1, FOXC2 and SIX2. Further, FOXQ1 direct gene targets are enriched in signaling pathways previously associated with EMT, including the WNT-signaling pathway. Within the WNT pathway, the WNT5B signaling ligand was a direct target for transcriptional activation by FOXQ1. Gene expression analysis in a panel of breast cancer cell lines revealed both FOXQ1 and WNT5B are enriched in the basal B subtype of TNBC. FOXQ1 and WNT5B expression were also found to be upregulated and correlated downstream of TGFβ induced EMT. Disruption of WNT5B expression attenuated EMT-associated stem-like traits, including decreases in mammosphere formation and in the CD44+/CD24- population, and significantly reduced cell invasion. Analysis of breast cancer gene expression from the TCGA revealed that FOXQ1 and WNT5B expression are correlated across all breast samples (r=0.46) and differentially upregulated in ER- breast cancer. Additionally, both FOXQ1 and WNT5B expression were associated with worse recurrence-free survival. Our data supports that WNT5B is a critical FOXQ1 target for mediating EMT features and suggests that disrupting the FOXQ1-WNT5B axis could be a potential strategy to target the EMT cell population in TNBC patients.

#1119

S100A4 alters mitochondrial metabolism to promote invasion and metastasis of non-small cell lung cancer cells through upregulation of NDUFS2.

Lili Liu, Lei Qi, Teresa Knifley, Dava W. Piecoro, Piotr Rychahou, Jinpeng Liu, Mihail I. Mitov, Jeremiah Martin, Chi Wang, Jianrong Wu, Heidi L. Weiss, D. Allan Butterfield, B. Mark Evers, Kathleen L. O'Connor, Min Chen. _Univ. of Kentucky, Lexington, KY_.

It is generally accepted that alterations in metabolism are critical for the metastatic process; however, the mechanisms by which the metabolic changes are controlled by the major drivers of the metastatic process remain elusive. Here, we find that S100A4, a major metastasis-promoting protein, alters metabolic plasticity to drive tumor invasion and metastasis. Investigating how S100A4 regulates metabolism, we find that depletion of S100A4 decreases oxygen consumption rate, mitochondrial activity, and ATP production, and shifts cell metabolism to be more glycolytically active. We further identify that the 49 KD mitochondrial complex I subunit NADH dehydrogenase (ubiquinone) Fe-S protein 2 (NDUFS2) is regulated in an S100A4-dependent manner and that S100A4 and NDUFS2 exhibit co-occurrence at significant levels in various cancer types (cBioPortal). Importantly, we find that knockdown of NDUFS2 inhibits mitochondrial complex I activity, reduces cellular ATP level, and decreases the invasive capacity in three-dimensional (3D) growth, thus mimicking the biological effect resulting from S100A4 knockdown in A549 cells. Likewise, silencing S100A4 and NDUFS2 dramatically decreases the rate of metastasis as well as tumor growth in vivo. Finally, we provide evidence that cells with S100A4 and NDUFS2 depletion shift metabolism toward glycolysis through upregulating hexokinases expression and that suppressing S100A4 signaling sensitizes lung cancer cells to glycolysis inhibition. Together, our findings uncover a novel function of S100A4 in mitochondrial metabolism and highlight the importance of NDUFS2 in regulating the plasticity of mitochondrial metabolism by S100A4 to promote the invasion and metastasis in lung cancer.

#1120

Proinflammatory milieu promotes leptomeningeal metastasis by activation of LCN2.

Yudan Chi. _Memorial Sloan-Kettering Cancer Center, New York, NY_.

The tumor microenvironment is emerging as a critical regulator of cancer progression, especially in metastatic brain malignancies. The unique properties of leptomeningeal metastasis (LM) require a specific framework for acquisition of metastatic traits within the fluid-filled leptomeningeal space. Here, we discuss a number of the distinct features of the proinflammatory environment, including cancer cells and relevant immune cells. We also highlight the exact role of its contribution to cancer growth in the development of LM. Proinflammatory cytokines induce the secretion of cancer-derived LCN2. Mechanistically, we have uncovered that iron, transported by LCN2, supports cancer cell growth. By pharmacologic interference with iron uptake, we will prove its therapeutic beneficence for targeting LM in pre-clinical models.

#1121

Role of c-Met in colorectal cancer lung metastasis.

Piotr G. Rychahou, Nick Roller, Eun Y. Lee, Nicole Rychagov, Matthew Melton, Carrigan Wasilchenko, B. Mark Evers. _University of Kentucky, Lexington, KY_.

Colorectal cancer (CRC) is the second leading cause of cancer deaths in the US; systemic metastasis to the lungs occurs in approximately 10-20% of patients with CRC. Hepatocyte growth factor (HGF) and its tyrosine kinase receptor Met play a pivotal role in the tumor metastatic phenotype and represent attractive therapeutic targets.

The present study was designed to determine the role of c-Met in growth and metastasis of CRC cells to the lung. We evaluated patient-derived xenografts (PDX) tumors and CRC cells for c-Met activation (phospho-Met [Tyr1234/1235]), established PDX cell lines with high phospho-Met (Tyr1234/1235) expression from lung or liver metastases, and investigated the role of c-Met expression in CRC lung metastasis.

Methods. (1) PDX tumors were implanted into NOD-scid IL2Rgammanull mice; PDX cell lines were established from F3 tumor generation. (2) The protein expression of phospho-Met (Tyr1234/1235) was observed by western blotting and immunohistochemistry. (3). HT29 LungM3 cell line was derived from the human CRC line HT29 following multiple rounds of in vivo selection for lung metastasis in immunodeficient mice. HT29LungM3 cells were transfected with shRNA expression vectors targeting the c-Met gene. HT29LungM3 cells were injected into SCID mice subcutaneously to measure tumor growth or intravenously to examine the role of c-Met in CRC lung metastasis.

Results. (1) High phospho-Met (Tyr1234/1235) expression was identified in 6 out of 20 CRC PDX models. Two PDX cell lines with high phospho-Met (Tyr1234/1235) expression, 2377LM (from liver metastasis) and 2387 (from lung metastasis), were established. Metastatic potential of the 2377LM cell line to the liver was confirmed after intrasplenic injection of cancer cells into SCID mice. The metastatic potential of 2387 cells to the lung was confirmed after intravenous injection of cancer cells into SCID mice. (2) Western blot analysis demonstrated high phospho-Met (Tyr1234/1235) expression in HT29LungM3, 2387LM and 2387 cell lines, compared to HCT116 (human CRC), LS174T (human CRC), Caco-2 (human CRC), SK-OV-3 (human ovarian cancer) and JAR (placenta choriocarcinoma) cell lines. (3) HT29LungM3 cells were transfected with shRNA expression vectors targeting the c-Met gene with c-Met knockdown of at least 90%. Surprisingly, c-Met inhibition in the HT29LungM3 cell line had no effect on subcutaneous tumor growth or lung metastasis.

Conclusions. Aberrant expression of c-Met is associated with the progression of multiple human malignancies and has been identified as a novel target for the treatment of malignant tumors. Here, we examined expression of phospho-Met (Ty1234/1235) in CRC PDX cell samples and determined that c-Met knockdown does not decrease CRC lung metastasis. These results are important for understanding the mechanism of CRC lung metastasis and proper strategy selection for prevention and treatment of patients with advanced disease.

#1122

CD44 as potential therapeutic target for medulloblastoma metastasis.

Dan Qi, Fengfei Wang, Ekokobe Fonkem, Jason H. Huang, Erxi Wu. _Baylor Scott & White Health, Temple, TX_.

Medulloblastoma (MB) is a commonly diagnosed pediatric brain tumor that shows highly variable rates of morbidity and mortality. Metastasis is the leading cause of death in patients with MB, approximately 30% of MB patients have metastatic tumors right at diagnosis. Therefore, there is a critical need to dissect the molecular mechanisms that govern MB progression and metastasis. In our previous work, we found that DHA and VP16 synergistically inhibit MB cell proliferation and induce cell death accompanied with a marked reduction in the expression of CD44 standard form (CD44s). In this study, we show that MB cells express CD44s mainly and the elevated level of CD44s enhances MB cell invasion, suggesting that CD44s may play a crucial role in MB metastasis. In addition, we observed CD44s expression only in MB patients with metastasis using a small cohort of samples. Large database analysis show that MB patients with elevated levels of CD44 have poorer outcomes. Moreover, CD44 knockdown markedly reduces the expressions of transcription factor c-Myc and the platelet derived growth factor receptor (PDGFR) β, both are therapeutic targets for MB. Interestingly, PDGFRβ knockdown abolishes CD44s expression and downregulates the level of c-Myc. However, there is no expression change of CD44s and PDGFR β after the knockdown of c-Myc. These results indicate that the expression and function of CD44s could be critical to MB progression and metastasis, highlighting the potential therapeutic targets for MBs with metastasis.

#1123

Bcl-xL promotes metastasis via a novel nuclear function.

Tiantian Zhang, Sha Li, Joseph Na, Soyoung Chi, George Zhang, Yi-Chieh Nancy Du. _Weill Cornell Medical College, New York, NY_.

Cancer cells often evade apoptosis through upregulation of anti-apoptotic proteins and/or downregulation of pro-apoptotic proteins. Bcl-xL, a negative regulator of mitochondria-mediated apoptosis, is frequently overexpressed in cancer cells. Bcl-xL has long been known for its function in regulating apoptosis during embryonic development and in pathological conditions. Any role that Bcl-xL might play in tumor metastasis has been ascribed to its anti-apoptotic function; i.e. Bcl-xL may increase metastasis by lending survival advantage to the tumor cells during the course of metastasis. However, we have demonstrated that Bcl-xL's metastatic function is independent of its canonical anti-apoptotic activity and instead requires a novel nuclear function in cancer. We found that Bcl-xL promotes migration, even when cells are defective in mitochondria-mediated apoptosis. Consistently, Bcl-xL mutants lacking anti-apoptotic activity can still promote migration, induce epithelial-mesenchymal transition (EMT), upregulate TGFβ, and increase migration and invasion of pancreatic neuroendocrine tumor cells and breast cancer cells. Importantly, our data demonstrated that forcible localization of Bcl-xL outside the nucleus impairs its metastatic function.

As a mitochondrial membrane protein in healthy cells, Bcl-xL's transmembrane domain prevents its nuclear import. This suggests that Bcl-xL enters the nucleus by an active nuclear transport. We hypothesized that unique transporters likely exist so as to transport this normally mitochondria membrane-bound Bcl-xL protein to the nucleus. By using immunoprecipitation in combination with mass spectrometry, we identified a novel interacting partner of Bcl-xL. We are investigating whether this protein transports Bcl-xL into the nucleus to promote metastasis.

#1124

S100A14 suppresses metastasis of nasopharyngeal carcinoma via inhibition of NF-kB signaling.

Dong-Fang Meng, Chao-Nan Qian. _Sun Yat-Sen University Cancer Center, Guang Zhou, China_.

Nasopharyngeal carcinoma (NPC) has a high incidence rate in southern China and Southeast Asia. Distant metastasis is the main reason for treatment failure. By analyzing our previously published whole genomic expression profiling data comparing low- versus high-metastasis NPC cells, we found that S100A14 expression was dramatically suppressed in high-metastasis cells. The reduced expression of S100A14 protein in the primary NPC collated with poorer overall survival of the NPC patients. Our preliminary experiments showed that S100A14 could inhibit the migratory and invasive abilities, as well as the epithelial-mesenchymal translation of NPC cells. RNA sequencing analyses found that NF-kB signaling pathway was the most dramatically influenced pathway by S100A14 with a negative relationship found between S100A14 and NF-kB signaling. We therefore propose a hypothesis that S100A14 inhibits NPC metastasis through inhibition of NF-kB signaling. In the proposed study, we will use a series of in vitro and in vivo approaches to clarify the inhibitory effects of S100A14 in terms of nuclear localization of P50 and P65, expression of IL-1beta, phosphorylation activation of the key molecules in NF-kB signaling pathway, as well as the in vivo metastasis of NPC cells. The accomplishment of the proposed study will reveal a novel mechanism of inhibiting NPC metastasis via inhibitory effects of S100A14 upon NF-kB signaling, which should be composed of drugable targets for metastasis prevention and treatment.

#1125

Secretory pathway calcium ATPase-2 (SPCA2) regulates metastasis by suppressing mesenchymal markers in triple negative breast cancer cell lines.

Monish Ram Makena, Donna K. Dang, Myungjun Ko, Manuj Bandral, Rajini Rao. _Johns Hopkins Medical School, Baltimore, MD_.

Introduction: Over 90% of cancer deaths in breast cancer are associated with metastasis. Epithelial-mesenchymal transition (EMT) is the hallmark of metastasis. Dysregulation of the Ca2+ toolkit has profound consequences for tumor growth and metastasis, raising hopes for novel avenues of therapeutic intervention. The Secretory Pathway Ca2+-ATPase Isoform 2 (SPCA2) transports Ca2+ from cytoplasm to the Golgi, and elicits Ca2+ influx by interacting with plasma membrane Ca2+ channels. Previously, we showed that SPCA2 is implicated in breast cancer progression (Feng et al., Cell 2010). Furthermore, low SPCA2 expression is associated with triple negative breast cancers (TNBC), which are highly metastatic. Therefore, we investigated if ectopic expression of SPCA2 modulates EMT in TNBC cell lines.

Methods: TCGA invasive breast carcinoma project datasets were accessed through cBioPortal. Gene expression was determined by qPCR and protein expression by Immunoblotting and confocal microscopy. Live cell calcium imaging was performed using Fura-2 AM dye. NSG mice were used for in vivo studies.

Results: Low SPCA2 expression was associated with poor survival in TNBC patients (n=255, P < 0.05). TNBC cell lines show low SPCA2 expression compared to receptor positive cell lines, when normalized to non-tumorigenic epithelial cell line MCF-10A. Similarly, significantly low SPCA2 expression (P < 0.001) was observed in TNBC patients compared to receptor positive subtypes. Interestingly, we did not observe these results in SPCA1, the housekeeping SPCA isoform, revealing isoform-specific function of Golgi calcium pumps in breast cancer subtypes. Ectopic expression of SPCA2 in TNBC cell line MDA-MB-231 significantly increased baseline intracellular Ca2+ levels (P < 0.001) and uptake of extracellular Ca2+ (P < 0.001) through store independent calcium entry. Increased SPCA2 expression suppressed mesenchymal gene markers (CDH2, SNAI1, SNAI2, VIM and ZEB1, P < 0.05), and decreased cell migration in vitro (P < 0.05) in TNBC cell lines. Overexpression of SPCA2 in MDA-MB-231-luc-D3H2LN cell line reduced metastasis in vivo (quantified 5-weeks after injection in mammary fat pad, P < 0.01) (n=6) compared to control group. Treatment of metastatic TNBC patients with histone deacetylase (HDAC) inhibitors is currently being evaluated in clinical trials. We found treatment of TNBC cell lines with FDA-approved HDAC inhibitors vorinostat and romidepsin elevated SPCA2 expression in a dose-dependent manner (P < 0.05), simultaneously decreasing mesenchymal gene expression (P < 0.05).

Conclusion: These novel findings point to a causal link between low SPCA2 levels, poor prognosis, and the epithelial-mesenchymal transition required for breast cancer metastasis. Restoration of SPCA2 expression in TNBC by HDAC inhibitors may have therapeutic potential.

#1126

Association of CXCR4 expression with lymph node metastasis in distal cholangiocarcinoma.

Yohsuke Yagawa, Atsushi Aruga, Nobuhiro Takeshita, Ryota Higuchi, Takehisa Yazawa, Keishi Tanigawa, Masakazu Yamamoto. _Tokyo Women's Medical University, Tokyo, Japan_.

BACKGROUND: The standard treatment of resectable distal bile duct cancer is pancreatoduodenectomy. However, prognosis is still poor because of a high rate of lymph node (LN) metastasis, underlining the need to further clarify the mechanism of LN metastasis. Recently accumulated evidence has shown that cancer cells can take advantage of the homeostatic mechanism of lymphocyte homing to metastasize to LNs. The CXCL12-CXCR4 axis modulates lymphocyte homing by inducing continuous migration of dendritic cells and T cells that are expressing chemokine receptor CXCR4 to LNs where chemokine CXCL12 is homeostatically secreted.

OBJECTIVE: The aim of this study is to elucidate whether distal bile duct cancer cells express CXCR4 and investigate a possible association with LN metastasis.

METHODS: This study enrolled 32 patients with resectable distal bile duct cancer including 19 patients with LN metastasis and 13 without. All patients underwent pancreatoduodenectomy at our department between 2002 and 2008 and have been followed up (Median observation period: 50.5 (4-184) months). Histopathologically, 27 cases were tubular adenocarcinoma, and 5 cases were other types of cancer. Immunostaining with resected specimens was undertaken to examine the expression of CXCR4 in cancer cells and determine whether its expression level is associated with lymph node metastasis. When 10% and more of tumor cells were stained, specimens were diagnosed as positive for CXCR4. The result of staining level was visually assessed.

RESULTS: Cancer cells were stained heterogeneously by antibody for CXCR4. Normal cells of the bile duct were not stained, while damaged or desquamated cells were stained. With regard to the staining pattern, the cytoplasm and membrane were stained but the nucleus was not stained. 19 out of 32 cases were positive for CXCR4 expression (59.4%). 15 of 19 cases with LN metastasis were positive whereas 4 of 13 cases without LN metastasis were positive (p=0.0107). CXCR4 expression on cancer cells increased with advancing cancer cell undifferentiation in tubular adenocarcinoma; cancer cells were positive for CXCR4 in 0 of 5 (0%), 6 of 16 (37.5%), and 6 of 6 (100%) cases in tubular 1, tubular 2, and tubular 3 adenocarcinoma, respectively.

CONCLUSIONS: Our results indicated that the expression of CXCR4 in cancer cells may be associated with LN metastasis in distal bile duct cancer.

#1127

**A rare subpopulation of melanoma cells with low expression of the metastasis suppressor gene** NME1 **is highly invasive.**

Devin E. Snyder, Ying Wang, David M. Kaetzel. _Univ. of Maryland, Baltimore, Baltimore, MD_.

Malignant melanoma is an extremely aggressive form of cancer, resulting in the majority of all skin-cancer related deaths. Despite developments in melanoma treatment, tumor recurrence and metastasis, and resistance to therapy remain serious challenges to successful management of the disease. Melanoma tumors contain subpopulations of cells that display genomic and phenotypic variation. NME1 is an established metastasis suppressor protein, which inhibits the metastatic spread of melanoma cells. In the current study, we sought to determine whether melanoma cell lines contain a subpopulation of cells that express reduced expression of NME1 and, thus, potentially exhibit aggressive tumor and metastatic activities. Through immunostaining and flow cytometry, we discovered a rare subpopulation of cells with markedly reduced NME1 expression (NME1Low) in two melanoma cell lines (WM9 and WM278). To enable the isolation and further characterization of NME1-based subpopulations, we employed CRISPR-Cas9 technology to introduce the EGFP coding sequence at the C-terminus of the endogenous NME1 gene in both WM9 and WM278 cell lines. The resulting NME1-EGFP fusion protein is thus expressed under the regulatory control of the endogenous NME1 promoter. Fluorescence-activated Cell Sorting (FACS) was used to isolate cells that expressed the NME1-EGFP fusion protein at different levels of expression, from which multiple clones were isolated and expanded for analysis of their tumor growth and metastatic properties. Clones were sequenced and genotyped to ensure accurate EGFP incorporation at the NME1 locus. Clones derived from cells expressing low levels of NME1-EGFP (NME1Low) retained their low expression phenotype over 10 passages in monolayer culture conditions. NME1Low clones derived from both melanoma cell lines were much more invasive than clones expressing normal levels of NME1-EGFP (NME1Normal) when embedded in Matrigel. In addition, RNA-seq analysis demonstrated that NME1Low and NME1Normal clones differed greatly in their RNA expression profiles (>200 differentially expressed genes), with NME1Low clones expressing elevated levels of genes associated with morphogenesis (GO: 0009653). In particular, genes were identified relating to development of neural, bone and heart tissues, suggesting relevance to neural crest cell differentiation. Studies are ongoing to assess the growth and metastatic properties of these distinct cell subpopulations in vivo using a xenograft approach in immunocompromised mice. Overall, these studies strongly suggest that a subpopulation of cells that expresses low levels of NME1 represents a primitive and malignant component of melanoma lesions. These cells have potential for providing new therapeutic targets and molecular markers in advanced melanoma.

#1128

Metastasis suppressor RKIP modulates gene expression variance in triple negative breast cancer.

Dongbo Yang, Sebastian Pott, Marsha Rosner. _University of Chicago, Chicago, IL_.

Triple-negative breast cancer (TNBC) is the most aggressive type of breast cancer with a highly metastatic phenotype. Conventional chemotherapy and radiotherapy are generally ineffective at treating metastasis. Natural metastasis suppressors such as the Raf kinase inhibitory protein (RKIP) are not functional in many TNBC cases and associated with poorer relapse-free survival. Although tumor cell heterogeneity is strongly implicated in contributing to metastasis, it is unknown whether metastasis suppression by RKIP reduces heterogeneity. Therefore, we performed single-cell RNA sequencing of mouse TNBC tumors as well as cultured cells to study the effect of RKIP overexpression on gene expression variance in tumor cells. Elucidating RKIP's effect on gene expression variance at the single cell level reveals gene sets that are important in tumor cell transitions from non-metastatic to metastatic states. We predict that the gene sets whose variance is decreased by RKIP are effective drug targets for metastatic breast cancer. 

### Novel Imaging Targets

#1129

**Monitoring intratumoral CD8 T cell infiltrates in human stem cell engrafted mice during single agent and combination immunotherapy with T cell bispecific antibodies using the human PET-tracer** 89 **Zr-Df-IAB22M2C.**

Christoph M. Griessinger,1 Alessandro Mascioni,2 Fang Jia,2 Michael Torgov,2 Tapan Nayak,1 Preethi Latha Bhavani Mohan,3 Valeria G. Nicolini,3 Johannes Sam,3 Christina Claus,3 Claudia Ferrara-Koller,3 Marina Bacac,3 Pablo Umana,3 Ian Wilson,2 Christian Klein,3 Jean J. Tessier1. 1 _Roche Pharma Research and Early Development, Basel, Switzerland;_ 2 _ImaginAb Inc., Inglewood, CA;_ 3 _Roche Pharma Research and Early Development, Schlieren, Switzerland_.

A persisting challenge in cancer immunotherapies (CIT) is the determination of patients responding early during therapy. As major effector cells, CD8 cytotoxic T cells are a primary target of many CIT approaches. ImaginAb is developing a human CD8 specific minibody 89Zr-Df-IAB22M2C to monitor CD8 tumor infiltrates non-invasively, longitudinally and throughout the whole body using positron emission tomography (PET). We evaluated pre-clinically the capability of this tracer to quantify CD8 tumor infiltrates upon treatment with T cell activating molecules using both, single agent FOLR1-T cell bispecific (TCB) antibody and the combination of CEA-TCB with a CEA-targeted 4-1BBL (CEA-4-1BBL).

In the first study, HeLa-cervical cancer xenograft bearing CD34+ human stem cell engrafted NSG mice (HSC-NSG) were treated for 2 weeks with FOLR1-TCB (2 mg/kg, once weekly), an untargeted control DP47-TCB and vehicle followed by PET imaging with the CD8 specific 89Zr-Df-IAB22M2C tracer (90-95 µCi/10 µg). In the second study, MKN-45 gastric cancer bearing HSC-NSG mice were treated with CEA-TCB (2.5 mg/kg, 2x/week), CEA 4-1BBL (3.0 mg/kg, 1x/week) or the combination of both compounds for two weeks followed by PET imaging with the CD8 tracer (50-60 µCi/10 µg). Ex vivo γ-counting of tumors and organs of interest as well as CD8 IHC was performed for both studies.

Treatment with FOLR1-TCB induced the highest tumor regression and strongest CD8+ T cell infiltrates. 89Zr-Df-IAB22M2C was able to detect tumor infiltrates by PET and γ-counting (13.6±2.7 %ID/g), whereas treatment with untargeted DP47-TCB induced a low T cell infiltrate (8.7±3.2 %ID/g) as compared to the vehicle group (5.6±1.2 %ID/g). In the second study, combined treatment with CEA-TCB and CEA-4-1BBL induced the highest tumor regression accompanied by the highest intratumoral CD8 T cell infiltrates, which were detectable by PET and γ-counting

(8.95±2.47 %ID/g). Single agent CEA-TCB (6.91±1.61 %ID/g) and CEA-4-1BBL (6.24±1.11 %ID/g) showed higher signals compared to vehicle (4.87±1.18 %ID/g). The differences in CD8 infiltrate upon single and combination treatment was confirmed by IHC.

The 89Zr-Df-IAB22M2 PET-tracer proved to be highly sensitive for the detection of intratumoral CD8 T cell infiltrates upon single and combination treatment with T cell activating compounds. These data correlated with CD8 IHC. These results provide further evidence that the CD8 imaging probe, which is currently in Phase II clinical testing (NCT03107663), may be a promising tool for the monitoring of CD8 T cells in patients treated with immunotherapies.

#1130

PET imaging of tumor-immune status for response prediction and treatment monitoring in syngeneic mouse models.

Lotte K. Kristensen,1 Camilla Christensen,2 Carsten H. Nielsen,1 Andreas Kjaer2. 1 _Minerva Imaging, Copenhagen, Denmark;_ 2 _Department of Clinical Physiology, Nuclear Medicine & PET and Cluster for Molecular Imaging, Department of Biomedical Sciences, Rigshospitalet and University of Copenhagen, Copenhagen, Denmark_.

Introduction: Cancer immuno-therapy has been established as a powerful way of treating several types of cancer and the degree of immune cell infiltration in tumors seems to reflect the clinical outcome. Accordingly, non-invasive PET imaging of key immune cell subsets could become a valuable tool for tailoring cancer immuno-therapy. Therefore, we developed antibody-based PET tracers specific for CD8a+ and PD-L1 enabling visualization of tumor infiltrating T-cells and tumor PD-L1 status in syngeneic mouse models. Furthermore, we evaluated the ability of theses tracers to predict and monitor combinations of immune-stimulating therapies.

Experimental procedures: Radiotracers were generated from intact antibodies (anti-PD-L1, clone 6E11, humanized cross-reactive to mouse) or antibody fragments (R-anti-M-CD8a+) and were radiolabeled with 89Zr or 64Cu. Tracer specificity was evaluated by immuno-reactive fraction assays and blocking studies and compared to radiolabeled isotype controls. Syngeneic mouse models were selected based on their respective tumor microenvironment (TME) as analyzed by flow cytometry and immunohistochemistry (IHC). Treatment efficacy of external radiation therapy (XRT) and anti-CTLA-4 was evaluated by baseline and post-therapy scans with 64Cu-NOTA-CD8a-F(ab)'2 in CT26 tumor-bearing mice. Prediction of response to therapy with XRT and anti-PD-L1 was evaluated with 89Zr-DFO-6E11 in various syngeneic mouse models. In vivo imaging data was compared with IHC and flow cytometry.

Results: Tracers were highly specific for their targets with an immuno-reactivity >85%. Blocking of endogenous CD8a+ and PD-L1 was successfully reflected by PET imaging with 89Zr-DFO-CD8a-F(ab)'2 and 89Zr-DFO-6E11, respectively. Fractionated XRT induced infiltration of CD8a+ cells in spleen and CT26 tumors which was detected by in vivo 89Zr-DFO-CD8a-F(ab)'2 PET imaging and ex vivo by IHC analysis. Studies with 89Zr-DFO-CD8a-F(ab)'2 PET imaging of combination therapies with fractionated XRT and immune checkpoint inhibitors are ongoing. 89Zr-DFO-6E11 successfully detected various degrees of PD-L1 expression in xenograft mouse models as well as the upregulated PD-L1 expression in syngeneic mouse models following fractionated XRT. Studies with 89Zr-DFO-6E11 PET imaging of combination therapies with fractionated XRT and immune checkpoint inhibitors are ongoing.

Conclusions: The developed radiotracers enable whole-body visualization of CD8a\+ T cells and PD-L1 expression in various syngeneic mouse models. These radiotracers can be used as tools to predict efficacy of immuno-therapy in preclinical drug development. In addition, they may be valuable in vivo biomarkers of response to immune-therapy in patients.

#1131

CD8 immuno-PET using a fully human mAb can detect increases in tumor-infiltrating T cells by a T cell activating immunotherapy.

Richard Tavare, Makenzie Danton, Jason T. Giurleo, Marcus P. Kelly, Sosina Makonnen, Carlos Hickey, Tomas C. Arnold, Dangshe Ma, William C. Olson, Gavin Thurston, Jessica R. Kirshner. _Regeneron Pharmaceuticals, Inc, Tarrytwon, NY_.

Detection and quantification of immune cell subsets and changes in the tumor immune microenvironment would be a powerful tool to correlated to response in cancer immunotherapy. Currently, biopsies used to monitor the tumor microenvironment suffer from sampling error, restricted sampling over time and to inaccessible tissues/organs, and do not reflect discordance across tumor lesions of the same patient. Immuno-PET detection of CD8-expressing T cells is a promising translational imaging approach to assess CD8+ tumor-infiltrating lymphocytes (TILs) pre- and post-therapy. We have developed a fully human anti-CD8 antibody that was radiolabeled with the positron emitting radionuclide Zirconium-89 (89Zr) using the bifunctional chelator p-SCN-Bn-Desferoxamine (DFO). CD8-genetically humanized immunocompetent mice were used to validate dose dependent targeting of 89Zr-CD8 to lymphoid tissues. Next, two models were developed to monitor the response of the T cell activating bispecific antibody REGN1979 (CD3xCD20). In the first model designed to lack a CD8 antigen sink, Raji lymphoma cells were co-implanted with human peripheral blood mononuclear cells (hPBMCs) in immune deficient NSG mice and treated with REGN1979. Here, 89Zr-CD8 detected a two-fold increase in CD8+ TILs as quantified by flow cytometry compared to control antibody treated mice. To determine if 89Zr-CD8 could detect changes in CD8+ TILs in the presence of increased circulating and splenic T cells, a different model was developed. Raji lymphoma cells were implanted subcutaneously and hPBMCs were implanted intraperitoneally in immune deficient SRG-15 mice (hSIRP KI, Rag2 KO, IL2Rg KO, hIL-15 KI). Due to the humanization of IL-15, T cells from the implanted hPBMCs rapidly expanded and produced a CD8 antigen sink. In the SRG-15 model, total tumor uptake of 89Zr-CD8 was lower and spleen uptake higher than in the NSG model due to the presence of the antigen sink. These SGR-15 mice treated with REGN1979 also demonstrated an increase in CD8+ TILs by flow cytometry that was specifically detected with 89Zr-CD8. This work demonstrates that 89Zr-CD8 can specifically detect CD8+ T cells in lymphoid tissues of CD8 humanized mice and therapy-induced alterations of CD8+ TILs. Importantly, the detection of CD8+ TILs occurs in both the presence and absence of an antigen sink. This work supports the clinical translation of 89Zr-CD8 immuno-PET to investigate its utility for predicting and monitoring response in patients undergoing immunotherapy.

#1132

Multiplexed immunofluorescence and multispectral imaging-based quantification of tumor and immune cell populations reveals spatial relationships in oral cavity squamous cell carcinoma.

Joao Paulo Oliveira-Costa,1 Anuraag S. Parikh,1 Linda T. Neiman,1 Derin Sevenler,1 Doyeon Koo,1 Chenyue Lu,1 William C. Faquin,1 Itay Tirosh,2 Jeremy D. Richmon,3 Kevin S. Emerick,3 Daniel G. Deschler,3 Mark A. Varvares,3 Derrick T. Lin,3 Sidarth V. Puram,4 Bradley E. Bernstein,1 Shannon L. Stott1. 1 _Harvard Medical School/MGH, Boston, MA;_ 2 _Weizmann Institute of Science, Rehovot, Israel;_ 3 _Harvard Medical School/MEEI, Boston, MA;_ 4 _Washington University School of Medicine in St Louis, St Louis, MO_.

Recent single cell expression profiling of head and neck squamous cell carcinoma (HNSCC) identified a partial epithelial-to-mesenchymal transition (p-EMT) subpopulation of cancer cells. We have previously demonstrated an association of p-EMT subpopulations with adverse clinical and pathologic features, in both TCGA expression data and an independent cohort of single institutional patients. Here, we aim to understand cellular co-expression of EMT markers and spatial relationships with immune infiltrates in oral cavity HNSCC. Paraffin blocks of 10 oral cavity HNSCC patients whose tumors had previously undergone single cell expression profiling were characterized by a 13-marker panel of tumor cell, p-EMT, epithelial differentiation, and immune markers. Multiplexed immunofluorescence was used to perform simultaneous staining within two serial tissue sections. Multispectral imaging was performed using wide field microscopy (Vectra 3), and algorithms were used for spectral unmixing, tissue and cell segmentation, and cell phenotyping. Following user training, tissue and cell segmentation and cell phenotyping were successfully performed in an automated fashion. Whole slides containing entire tissue sections were analyzed, enabling high throughput analysis of hundreds of thousands of individual cells per tumor. p-EMT quantification by tumor, as measured by simultaneous signal for p-EMT markers, showed consistent co-localization, in accordance with prior single cell expression profiling data. We found that individual cells defined as p-EMT positive, based on co-expression of two or more p-EMT markers, co-localized to the leading edge of tumor nests, in close apposition to the stroma. Co-localization of LAMB3 and LAMC2 was the most robust marker combination defining this subpopulation. In addition, helper and cytotoxic T cells were identified across tumors, including activated and exhausted T-cell subsets. The relationships of these cellular subpopulations to other immune cells, which may drive immune cell exhaustion, were explored by radial measurements associated with cell densities and intercellular distances. Tumor and immune cell profiling using 13 markers across serial sections enabled high throughput characterization of individual cells with spatial information in a manner not previously possible. Distinct spatial relationships among p-EMT and epithelial tumor cells were identified, including a potential correlation with immune cells. This technology may have clinical utility in HNSCC, which includes predicting the need for therapy and response to immunotherapy.

#1133

**ImmunoPET and T cell labeling for** in vivo **monitoring of immunotherapy: Development of CD8 Fab** 2 **' and Fab tracers for immunoPET and direct labeling of T cells for** in vivo **tracking.**

Camilla Christensen,1 Lotte K. Kristensen,2 Carsten H. Nielsen,2 Andreas Kjær1. 1 _Dept. of Clinical Physiology, Nuclear Medicine & PET and Cluster for Molecular Imaging, Dept. of Biomedical Sciences, Rigshospitalet and University of Copenhagen, Copenhagen, Denmark; _2 _Minerva Imaging, Copenhagen, Denmark_.

Objectives: Cancer immunotherapy is becoming an increasingly important treatment strategy across a broad spectrum of tumor types. Non-invasive imaging methods for in vivo tracking of T-cells can be used for longitudinal monitoring of tumor infiltrating T-cells to assess the effect of immune checkpoint therapy or cell-based immunotherapies. The objective of the current work was to develop and optimize Fab-based tracers directed at CD8 for PET imaging of T cells. The imaging performance of radiolabeled Fab fragments was compared to Fab2' fragments in terms of kinetics and image contrast. Furthermore, Zirconium-89 (89Zr) labeled T cells were tracked in a murine transplantation model.

Methods: Anti-mouse CD8α antibody was cleaved into Fab2' by enzymatic cleavage followed by reduction of disulfide bridge into Fab fragments. Fab2' was conjugated to NCS-DFO for 89Zr labeling, while both Fab2' and Fab were conjugated to SCN-NOTA for Cupper-64 (Cu-64) labeling.

Radiochemical yield, radiochemical purity (RCP) and plasma/buffer stability was assessed by high-performance liquid chromatography. Furthermore, the immunoreactivity and binding affinity was evaluated for the radiolabeled tracers.

In vivo comparison of radiolabeled Fab2' and Fab fragments was performed by longitudinal PET/CT imaging at various time-points after injection in CT26 (murine colon carcinoma) tumor bearing mice. CD3+ T-cells were directly labeled with 89Zr for tracking experiments. The labeling efficiency and viability of the cells was evaluated ex vivo, and in vivo cell tracking was performed by longitudinal PET/CT imaging after injection of labeled T-cells in naive immune-competent mice.

Results: The cleavage of anti-CD8 into Fab2' and subsequent purification by HPLC was optimized to achieve a product with high yield and purity. The optimal Fab2' reduction conditions were determined followed by conjugation to SCN-NOTA An optimized protocol for 89Zr/64Cu-CD8-Fab2' and 64Cu-CD8-Fab was developed with high radiochemical yield and RCP of >99%. PET/CT imaging with all tracers was performed to compare tumor, spleen and lymph node uptake. The highest uptake was found 24h post injection, but a clear delineation of lymphoid organs and tumors was evident already after 6h for the single Fab allowing same day imaging. T-cells labeled with 89Zr and cell tracking PET/CT imaging showed homing primarily to the spleen.

Conclusions: Different tracers directed at T-cells were successfully developed and evaluated in in vivo PET imaging of T cells. The reduced circulation time of Fab vs. Fab2' and the use of 64Cu instead of 89Zr allows repeated imaging every 3 days due to the shorter physical half-life (12.7 h) of 64Cu. The study furthermore demonstrates the use of direct radiolabeling of T-cells as an attractive tool for investigating the distribution of cell-based therapies.

#1134

**PET-imaging of** 89 **Zr-labeled bispecificT-cell engagers in syngeneic tumor bearing mice.**

Frans V. Suurs,1 Derk J. de Groot,1 Urszula M. Domanska,2 Grit Lorenczewski,2 Sabine Stienen,2 Matthias Friedrich,2 Elisabeth G. de Vries,1 Marjolijn N. Lub-de Hooge1. 1 _University Medical Center Groningen, Groningen, Netherlands;_ 2 _Amgen Research Munich GmbH, Munich, Germany_.

BACKGROUND Bispecific T-cell engagers (BiTE®) harness the immune system against cancer. BiTE® antibody constructs are small proteins of ~53 kDa existing of two connected single-chain variable fragments. MuS110, a BiTE® with affinity for murine CD3 (KD= 2.9 nM) and murine EpCAM (KD= 21 nM), was radiolabeled with positron emission tomography (PET) isotope zirconium-89 (89Zr) to study its pharmacokinetics and involvement of the immune system in an immunocompetent mouse model bearing a syngeneic tumor.

METHODS MuS110 and two control BiTE® antibody constructs (hyS110 and AMG110) were radiolabeled with 89Zr. HyS110 has affinity for murine CD3 and human EpCAM, AMG110 for human CD3 and human EpCAM. The human and murine variants of EpCAM and CD3 are not cross-reactive. Either immunocompetent BALB/c or immunodeficient nude BALB/c mice were all orthotopically engrafted in the lower mammary fat pad with EpCAM-positive murine breast cancer cell line 4T1. PET-imaging was performed at 0.5, 3, 6, 24, 48 and 72 h after intravenous (iv) administration of 10 µg 89Zr-muS110. Distribution of 10 µg 89Zr-muS110, 89Zr-hyS110 and 89Zr-AMG110 was assessed 24 h after administration in BALB/c and nude BALB/c mice with PET-imaging and ex-vivo biodistribution. In addition, a group of BALB/c mice received 10 µg muS110 iv daily for 5 days followed by 10 µg 89Zr-muS110 or 10 µg 89Zr-AMG110 and PET-imaging 24 h after tracer administration. Values are expressed as median (interquartile range).

RESULTS PET-imaging revealed fast renal clearance of 89Zr-muS110 in the BALB/c mice resulting in a blood half-life of 0.93 h (one-phase decay). Tumor- and spleen-to-blood ratios increased to 3.7 (3.0 to 4.5) and 9.3 (7.5 to 11.1) after 72 h. Ex-vivo biodistribution 24 h after tracer administration showed 89Zr-muS110 and 89Zr-hyS110 accumulation in the spleen with 8.2 % injected dose per gram (ID/g) and 8.7 %ID/g, in comparison to 89Zr-AMG110 (2.3 %ID/g; vs 89Zr-muS110 P<0.05, vs 89Zr-hyS110 P<0.05). In mesenteric lymph nodes (mesLNs), 89Zr-muS110 and 89Zr-hyS110 resulted in a higher uptake (3.5 and 6.4 %ID/g) than 89Zr-AMG110 (2.0 %ID/g; vs 89Zr-muS110 P<0.05, vs 89Zr-hyS110 P<0.05). Tumor uptake did not differ for 89Zr-muS110 and 89Zr-AMG110 (2.6 vs 2.2 %ID/g, P>0.05). In nude BALB/c mice spleen and mesLN uptake of 89Zr-muS110 was lower than in BALB/c mice (spleen: 3.4 vs 8.2 %ID/g, P<0.05; mesLNs: 1.7 vs 3.5, P<0.05). In addition, in nude BALB/c mice, tumor uptake was the same for 89Zr-muS110 and 89Zr-AMG110 (1.5 vs 1.7 %ID/g, P>0.05). 89Zr-muS110 uptake was lower in spleen and mesLNs following 5 days of 10 µg muS110 iv compared to control mice

(spleen: 4.2 vs 8.2 %ID/g, P<0.01; mesLNs: 1.9 vs 3.5, P<0.01), likely representing target saturation.

CONCLUSION Distribution of BiTE® 89Zr-muS110 is predominantly mediated by the affinity for CD3, resulting in uptake in lymphoid tissues.

#1135

Dextran based nanoparticles for immunotheranostics of prostate cancer.

Balaji Krishnamachary, Marie-France Penet, Zhihang Chen, Jesus Pacheco-Torres, Sangeeta Ray, Yelena Mironchik, Marty G. Pomper, Zaver M. Bhujwalla. _Johns Hopkins University, Baltimore, MD_.

Introduction:

Targeted nanoparticles (NPs) containing imaging reporters provide exciting options for cancer-specific delivery of siRNA to downregulate specific pathways in theranostic strategies. We recently developed a biodegradable modified dextran NP to deliver effectively siRNA and downregulate the target in vivo (1). Here we have developed a prostate specific membrane antigen (PSMA)-targeted dextran NP to deliver siRNA to downregulate PD-L1 in PSMA expressing prostate cancer (PCa) cells for immunotheranostics. In proof-of-principle studies, the NP was decorated with an optical imaging reporter to allow noninvasive detection in vivo, and evaluated in PC3 human PCa cells genetically engineered to overexpress PSMA (PC3-PIP) and in non-PSMA expressing PC3-Flu cells. Since castration-resistant PCa cells express PSMA, the ability to down regulate PD-L1 specifically may prevent these cancer cells from evading immune surveillance, resulting in their destruction by the immune system.

Methods:

For mRNA and protein analysis, PSMA overexpressing PC3-PIP cells and low PSMA expressing PC3-Flu cells were treated with dextran NPs with PD-L1 siRNA or PBS. 6h later, IFNγ (10 ng/mL) or PBS was added to the medium. Cells were collected, and proteins and mRNA extracted, 24h after NP/PBS treatment. Total RNA was isolated, and cDNA prepared following standard protocols, with q-RT-PCR performed for PD-L1 expression. Western blots were probed with anti-PD-L1, and anti-PSMA antibodies. For in vivo studies, PC3-PIP and PC3-Flu tumors were inoculated bilaterally in the flanks of male SCID mice. Once tumors reached ~200 mm3, dextran NPs siRNA with PSMA binding peptide were injected intravenously. Dosage and frequency of IFNγ injection in vivo was established, and bio distribution studies performed. Tumor samples were harvested and processed for mRNA and protein to detect PD-L1 expression.

Results and Conclusion:

Specificity of the PSMA-targeted dextran NP delivery was confirmed by PSMA-dependent retention of the NPs following fluorescent imaging of PC3-PIP and Flu cells. We next assessed the ability of the dextran NPs carrying PD-L1 siRNA to down regulate PD-L1 expression in PC3-PIP and PC3-Flu cells. Studies were performed with or without pre-activation of the cells with IFNγ. q-RT-PCR and western blots analysis confirmed a clear decrease of PD-L1 after NP treatment in PC3-PIP and PC3-Flu cells. Effective NP delivery was confirmed in tumors with optical imaging, both in vivo and ex vivo in excised organs. Ongoing studies are characterizing PD-L1 downregulation in tumors in vivo following treatment with PSMA-targeted dextran NPs delivering PD-L1 siRNA.

References: 1. Chen et al., Theranostics, 2018.

Acknowledgements: Supported by NIH P41EB024495 and R35CA209960.

#1136

**Tumor uptake and biodistribution of** 89 **Zirconium-labeled ipilimumab in patients with metastatic melanoma during ipilimumab treatment.**

Iris H. Miedema, Gerben J. Zwezerijnen, Guus A. van Dongen, Daniëlle J. Vugts, Marc C. Huisman, Otto S. Hoekstra, Tanja D. de Gruijl, Hendrik M. Verheul, Catharina W. Menke, Alfons J. van den Eertwegh. _Amsterdam UMC, location VUmc, Amsterdam, Netherlands_.

Introduction

Ipilimumab, a monoclonal antibody targeting CTLA-4, is approved for the treatment of metastatic melanoma and significantly improves overall survival. Because of the high costs and the potential serious toxicity of ipilimumab, it is of great importance to identify biomarkers that correlate with clinical activity and that can be used to select patients who will benefit from CTLA-4 blockade therapy.

We hypothesize that patients who do not respond to treatment with ipilimumab have lower drug levels in tumor tissues as compared to patients with a good response to therapy. In addition, we hypothesize that immune related adverse events (irAEs) are associated with high drug levels in the affected tissue. As irAEs usually occur approximately 6-8 weeks after the first injection of ipilimumab, we hypothesize that the drug levels in potentially affected tissues will increase at the second injection.

Experimental procedures

To visualize in vivo localization of ipilimumab in patients diagnosed with metastatic melanoma, 37 MBq, 10 mg 89Zr-labeled ipilimumab was injected within 2 hours after their first ipilimumab dose (3 mg/kg). Whole body PET/CT scans were obtained at 2h, 72h and 144h post injection and this procedure was repeated three weeks later at the second ipilimumab cycle. Biodistribution and tumor uptake were assessed visually by a nuclear physician. Focal uptake in tumor lesions exceeding local background was determined in volumes of interest (VOI) and SUVpeak values were obtained. Biodistribution was quantified by defining vital organs (i.e. lungs, kidneys, spleen, liver) and calculating mean %ID/kg. Blood was drawn for dosimetry and immunophenotyping at several time points during the trial. Presented here are initial results of the first three patients, up to 29 patients are planned to be included.

Results

Biodistribution of 89Zr-labeled ipilimumab showed a pattern distinctive for 89Zr-labeled antibodies with uptake in liver and spleen, as well as prolonged circulating antibody in the bloodstream corresponding to the pharmacokinetics of ipilimumab. Visual evaluation confirmed uptake of 89Zr-labeled ipilimumab in 5/12 evaluable tumor lesions, visible at both first and second injection of ipilimumab. Tumor uptake was comparable for 72h and 144h post injection with a mean of 6.9 %ID/kg (range 3.3-10.1) and a SUVpeak of 4.4 (range 2.3-8.9). There were no significant differences in tumor uptake between first and second dose of ipilimumab (mean 7.31 and 6.54 %ID/kg respectively).

Conclusions

Preliminary data of this ongoing study showed that the tracer is able to visualize and quantify uptake of ipilimumab in tumors. Correlations between tumor uptake and response to treatment will be presented. Furthermore, special interest will be given to uptake in lymphoid organs and locations for irAEs.

#1137

Unraveling spatially-dependent interactions of tumor-associated macrophage in the tumor microenvironment.

Victor Wang, Jan Martinek, Hannah M. Brookes, Kyung In Kim, Karolina Palucka, Jeff Chuang. _The Jackson Laboratory for Genomic Medicine, Farmington, CT_.

Tumor-infiltrating lymphocytes (TIL) are a strong prognostic factor in cancer patient outcomes. This includes immune checkpoint blockade (ICB) of the PD-1/PD-L1 axis, where patients with a higher abundance of pre-treatment TILs respond more frequently. However, tumors often elaborate mechanisms to exclude TILs and/or suppress their function. Identifying negative regulators of TILs is thus of great importance to improve patient outcomes and increase the patient population that may benefit from ICB. Tumor-associated macrophages (TAM) are a known modulator of TIL activity but the interaction between the two cell types is typically characterized with in vitro experiments and spatially-agnostic sequencing, ignoring location-specific factors contributing to the interaction's net effects. Interrogating the TAM-TIL interactions in an intact tumor microenvironment (TME) will uncover novel mechanisms responsible for TAM function and may be key to reversing TIL immunosuppression. Here we leverage bulk RNA-sequencing with histocytometry, a multiplex quantitative tissue imaging method, to spatially-resolve the TAM-TIL interactions in intact human metastatic melanoma microenvironments. Bulk sequencing distinguishes tumor samples by high and low enrichment of lymphocyte gene sets, with additional sub-stratification by macrophage enrichment. Modular repertoire analysis implicates an interferon response in the differential lymphocyte enrichment, but does not address macrophage-dependent effects. Drawing from physical chemistry concepts, our spatial analyses of cell-cell interactions from histocytometry reveals distinct TAM populations potentially regulating TIL activity. Phagocytic TAMs in the tumor and non-phagocytic TAMs in the stroma physically contact T-cells, with the former interaction strongly correlated with lymphocyte enrichment and T-cell infiltration. Local and regional TAM phagocytosis affinities estimated by the Langmuir adsorption model further detail the nature of this interaction within the tumor, as well as the role of macrophage phagocytosis in regulating TILs. We demonstrate linear models integrating bulk sequencing and spatial metrics to quantify the relationships between genetics, cell-cell interactions, and T-cell invasiveness. Our work unravels important TAM interactions that shape the TME which have not been previously appreciated, providing novel insight into the forces driving T-cell exclusion and revealing new TAM biology to explore further.

#1138

Non-invasive detection and quantification of tumor-associated macrophage density with magnetic particle imaging.

Jeffrey M. Gaudet,1 Ashley V. Makela,2 Paula J. Foster2. 1 _Magnetic Insight, Alameda, CA;_ 2 _Western University, London, Ontario, Canada_.

Background: There is increasing interest in the role of tumor-associated macrophages (TAMs) in promoting cancer growth and metastatic potential.(Obeid 2013) Increased TAM density has been correlated with poor prognosis and they can contribute up to 50% of the mass in breast tumors.(Bingle 2002) Previous studies have used cellular MRI to indirectly image the spatial distribution of TAMs, through the in situ uptake of superparamagnetic iron oxide (SPIO) nanoparticles (Makela 2017). However, iron-based MRI is limited by low specificity and challenging quantification. Magnetic Particle Imaging (MPI) is an emerging cellular imaging technique that can directly detect and quantify SPIO in vivo (Zheng 2016). MPI produces a positive contrast signal that is not attenuated by biological tissue. In this study, we investigated the first application of MRI and MPI to detect and differentiate between murine breast cancer models with varying metastatic potentials.

Methods: Female BALB/c mice were implanted with 300,000 4T1 (n=3) or 168FARN (n=3) into the inguinal mammary fat pad. Tumors were grown for 3 weeks. 24 hours prior to imaging, 100μL of ferucarbotran (Magnetic Insight) was administered IV. One mouse from each group was first imaged on a 3T clinical MRI (GE Healthcare) with a custom-gradient insert located at Robarts Research Institute. A balanced steady-state free precession pulse sequence was used with imaging time of 30mins. The mice were then sacrificed and fixed in formalin. MPI was performed at Stanford University with the MPI system (Magnetic Insight) alongside three reference fiducials of known concentration. Iron quantification was performed with VivoQuant (inviCRO). Tumor tissue was extracted for histology and ex vivo imaging.

Results: MRI signal voids were observed throughout the 4T1 tumor, but predominately in the tumor periphery. Fewer voids were observed in the 168FARN model, consistent with previous studies.3 MPI signal was observed in two out of three 4T1 tumors. Signal is seen across the entire tumor since the MPI is a sagittal projection compared to a single MRI slice. MPI quantification of SPIO in the 4T1 tumors indicated a consistent uptake, of 4.7 and 5.9 μg of iron. Signal was also detectable in the liver and tail at the site of injection. Fewer voids were observed in the 168FARN model, consistent with previous studies (Makela 2017). MPI signal was not detected in the tumors of the 168FARN group, consistent with the low iron uptake observed in the MRI. Histological analysis will be performed to verify the SPIO and macrophage distribution in tumor samples.

Conclusions: By combining the high spatial resolution of MRI with the accurate quantification and specificity of MPI, additional information can be obtained on TAM presence and distribution. In addition to aiding therapeutic development, this information could be utilized to more accurately grade cancer.

#1139

CD206 positive M2-macrophage targeting engineered exosomes as a potential diagnostic and therapeutic tool.

Mohammad H. Rashid,1 Thaiz F. Borin,1 Roxan Ara,1 Kartik Angara,2 Achyut Bhagelu,3 Jingwen Cai,1 Yutao Liu,1 Ali S. Arbab1. 1 _Augusta University, Augusta, GA;_ 2 _Michigan State University, Grand Rapids, MI;_ 3 _Winship Cancer Institute of Emory University, Atlanta, GA_.

Tumor initiation and evolution is driven by the reciprocal actions between stromal and immune cells within the tumor microenvironment (TME). Tumor-associated macrophages (TAMs) specifically M2-macrophages, a critical component of TME; participate in immune suppression, epithelial to mesenchymal transition, invasion, angiogenesis, tumor progression and subsequent metastasis foci formation. Given their prevalence in most of the human tumor tissue and correlation of their higher infiltration with lower survival, M2-macrophages represent promising targets for diagnosis, prognosis and anticancer therapy. Hence, early in vivo detection and intervention of M2-macrophages in the TME may boost the clinical outcome. Exosomes are biological nanoparticles sizing 30-150 nm, recently drawing huge attention for their potential application as therapeutic and diagnostic tool because they are more biocompatible and biodegradable with lesser toxicity than other synthetic nanoparticles and can easily percolate through the body's barrier systems or through abnormally formed blood vessels in tumor tissue. We have generated engineered exosomes from HEK293 cells by lentiviral transfection system that carry specific peptide sequence on their external surface for targeting CD206 positive M2-macrophages. To determine the in vivo distribution of M2-macrophages, we adopted 111In-oxine based radio-labeling of the targeted exosomes and single-photon emission computed tomography (SPECT). Up to the time, among the used techniques, nuclear imaging is the foremost method to trace exosomes in vivo for better tissue penetration, sensitivity and quantitative analysis. After the labeling with 111In-oxine, we analyzed the binding efficacy and serum stability of the targeted exosome by thin layer paper chromatography (TLPC) using 100% methanol and 2M Sodium acetate solution (1:1) as eluent. More than 98% of 111In-oxine was bound to exosomes and only 5% of free 111In-oxine was dissociated from the exosomes even 24 hours after the incubation with 20% serum. When we injected these radio-labeled targeted exosomes in to 4T1 breast tumor-bearing mice, they went mostly to the periphery of the primary tumor, metastatic area in the lungs, spleen and to the liver. We performed ex vivo quantification of radioactivity from individual organ by gamma counter after final SPECT scan and observed similar distribution of the radio-labeled targeted exosomes. We also labeled the exosomes with DiI dye and injected into the same mice followed by euthanasia after 3 hours and organ collection. Fluorescent imaging from the tumor and spleen showed the adherence of exosomes to the CD206 positive macrophages confirming the targeting efficacy of the exosomes. Henceforth, we intend to utilize these exosomes as a therapeutic probe for carrying chemotherapeutic or antibody to intervene the actions of M2-macrophages in primary and distal TME.

#1140

A new twist on an old strategy: Can the lymph node environment help cancers escape immune surveillance.

Veronica Estrella,1 Pawel Swietach,2 Kim Luddy,1 Dominique Abrahams,1 Pedro Enriquez-Navas,1 Yana Reshetnyak,3 Mehdi Damaghi,1 Robert J. Gillies1. 1 _H. Lee Moffitt Cancer Center, Tampa, FL;_ 2 _University of Oxford, Oxford, United Kingdom;_ 3 _The University of Rhode Island, Kingston, RI_.

Cancer cells are selected for strategies to proliferate, invade, and metastasize within an organism. One such strategy is an ability to create an acidic microenvironment. This acidity stems from an increase in H+ production associated with elevated glucose metabolism. This, combined with poor perfusion, results in an acidic extracellular pH (pHe) of 6.5 - 6.9 in malignant tumors. The resulting acidic environment is toxic to normal cells, giving tumor cells an adaptive advantage. Further, it inhibits immune surveillance, although the mechanism by which this occurs is not entirely clear. However, it is well known that an acidic pH profoundly inhibits T cell effector function with minimal effect on T cell viability. Lymph nodes are the primary site for naïve T cell activation yet are the most common site of metastasis. Reversible suppression of T cell effector function naturally occurs within the lymph node, creating a site of immune privilege. As a result of these findings we hypothesized that cancer cells exploit this normal physiological function, which may be occurring in the lymph nodes. While lymph nodes are known to be hypoxic, nothing to date is known regarding their pH. Here, we show using four different approaches that the pH of lymph nodes is acidic. All experiments were performed using a murine model under normal, inflammatory and tumor bearing conditions. We interrogated lymph node pH using microelectrodes, chemical exchange saturation transfer (CEST) MRI, imaging with pH-low inserting peptide, pHLIP, and intravital imaging (IVI) of pH sensitive fluorescent dyes; all of which reported an acidic pH between 6.2-6.8. With the aid of a novel lymph node window chamber technique we were able to both image and measure the in vivo pH of a murine lymph node. This novel technique granted us the unique opportunity to acquire enough data to accurately map the pH of the lymph node. These IVI images together with pHLIP immunostaining showed that this acidity was restricted to subcortical regions known to contain antigen presenting dendritic cells and lymphocytes. Because this acidity was so extreme and was not affected by NaHCO3, we hypothesize that its maintenance must be an active process. Collective these data demonstrate that murine lymph nodes sustain an acidic pH under altering physiologic conditions. We hypothesize that cancer cells may be adopting the normal physiological conditions found in the lymph nodes to escape immune surveillance.

#1141

Preclinical analysis of biodistribution and PET imaging of a zirconium-89 labeled PSMA-targeted antibody-chelator conjugate.

Stefanie Hammer,1 Sabine Zitzmann-Kolbe,1 Felix Oden,2 Joerg Jannsen,2 Eva Bickel,2 Andre Mueller,2 Lothar Everz,1 Urs B. Hagemann,1 Jenny Karlsson,3 Olav B. Ryan,3 Hartwig Hennekes,1 Patricia E. Cole,4 Dominik Mumberg1. 1 _Bayer AG, Berlin, Germany;_ 2 _Life Molecular Imaging, Berlin, Germany;_ 3 _Bayer AS, Olso, Norway;_ 4 _Bayer AG, Whippany, NJ_.

The PSMA targeted thorium-227 conjugate PSMA-TTC (BAY 2315497) is a targeted alpha therapy approach for metastatic castration resistant prostate cancer that has shown strong anti-tumor activity in PSMA-positive prostate cancer models (Hammer et al. AACR 2017/2018/2019). PSMA-TTC consists of a fully human IgG1 PSMA-targeted antibody covalently linked to a 3,2-HOPO chelator moiety, stably complexing the alpha emitter thorium-227. As this chelator can also complex zirconium-89, positron emission tomography (PET) imaging using the zirconium-89 labeled variant of the PSMA antibody-chelator conjugate could support clinical development of PSMA-TTC by enabling monitoring of organ distribution.

Herein, we describe radiolabeling of the PSMA antibody-chelator conjugate with zirconium-89 and its quality control using bioanalytical assays. In addition, the zirconium-89 labeled PSMA antibody-chelator conjugate was injected into mice bearing LNCaP xenograft tumors and biodistribution was assessed by measurement of radioactivity in tumor and organs as well as PET imaging at different time points after injection.

The PSMA antibody-chelator conjugate was successfully labeled with zirconium-89 at high yield, purity and specific activity. Target binding was confirmed by determination of the immunoreactive fraction. Biodistribution experiments revealed high, long lasting uptake of zirconium-89 in PSMA-positive LNCaP tumors after single injection of the zirconium-89 labeled conjugate. An uptake of 40-50% of the injected dose per gram of tumor was measured at 24, 48, 72, 168 and 240h after injection. The PET images showed that the zirconium-89 labeled PSMA antibody-chelator conjugate provided an excellent image contrast for the delineation of LNCaP xenografts between 48 and 168 h after administration. Other organs showed only very little background and tumor-to-heart ratio was greater than 10 when PET imaging was performed between 48 and 168 h after injection.

These results indicate that the zirconium-89 labeled PSMA antibody-chelator conjugate shows promise for PET imaging studies. These properties may allow its use to support clinical development of PSMA-TTC.

#1142

An activity-dependent proximity ligation platform for spatially resolved and multiplexed quantifications of active enzymes in single cells.

Gang Li, Raymond E. Moellering. _The University of Chicago, Chicago, IL_.

Integration of chemical probes into proteomic workflows enables the interrogation of protein activity, rather than abundance. Current methods limit the biological contexts that can be addressed due to sample homogenization, signal-averaging, and bias toward abundant proteins. To expand the application scope of chemical proteomics and explore the cellular heterogeneity on enzyme activities, we developed a new platform that integrates family-wide chemical probes with proximity-dependent oligonucleotide amplification and imaging to quantify enzyme activity in native contexts with high spatial and single cell resolution. Application of this method, activity-dependent proximity ligation (ADPL), to serine hydrolase and cysteine protease enzymes enables quantification of differential enzyme activity resulting from endogenous changes in localization and expression. In a competitive format, small molecule target engagement with endogenous proteins in live cells can be quantified. Retention of sample architecture enables interrogation of complex environments such as cellular co-culture and patient samples. We also successfully applied ADPL in xenograft tissue sample, exhibiting the application prospect in primary patient tissue. Additionally, implementation of barcoded antibody-oligo direct conjugation enabled multiplexed readout of active enzymes within and between enzyme families, providing the possibility of simultaneous biomarker detection. Together, this work supports ADPL should be amenable to diverse and multiplexed protein families to detect active enzymes at scale and resolution out of reach with current methods.

#1143

The circulating tumor cell (CTC) microenvironment visualized by color coded imaging.

Atsushi Suetsugu,1 Tomoyuki Satake,1 Miki Nakamura,1 Masahito Shimizu,1 Shigetoyo Saji,1 Hisataka Moriwaki,1 Robert M. Hoffman2. 1 _Gifu University Graduate School of Medicine, Gifu, Japan;_ 2 _AntiCancer, Inc., CA_.

Background/Aim: An important question is whether cancer cells circulate with stromal cells. A color-coded mouse model of metastatic lymphoma was utilized to investigate the microenvironment of CTC clusters using fluorescent-protein imaging.

Materials and Methods: EL-4 mouse lymphoma cells expressing red fluorescent protein (RFP) were injected into the spleen of transgenic C57BL6-green fluorescent protein (GFP) mice. The cellular composition of CTC clusters both in heart blood and portal blood was imaged with confocal microscopy.

Results: CTC clusters comprised 8.8% of CTCs determined by color-coded imaging. Heterotypic CTC clusters containing other types of cells were distinguishable from homotypic CTCs. Heterotypic CTC clusters comprising cancer cells and fibroblasts were more rare than homotypic ones. Heterotypic CTC clusters with fibroblasts were observed only in portal blood, not in heart blood.

Conclusion: CTCs can form clusters, which may contain fibroblast or other stromal cells that may play a role in promoting CTC metastasis.

#1144

Imaging radiotherapy induced pulmonary fibrogenic changes with integrin-PET.

Azeem Saleem,1 Yusuf Helo,1 Graham Searle,1 Fatjon Dekaj,2 Jo Cook,2 Zarni Win,3 Roger Gunn,1 Paula Wells2. 1 _Invicro, A Konica Minolta Company, London, United Kingdom;_ 2 _St Bartholomew's Hospital, London, United Kingdom;_ 3 _Imperial College Healthcare NHS Trust, London, United Kingdom_.

Integrin αvβ6 plays a key role in the fibrotic pathway by activation of Transforming Growth Factor β. Expression of αvβ6 integrin in alveolar epithelium increases after radiotherapy (RT) before onset of fibrosis and anti- αvβ6 therapy prevents fibrogenesis. Post-RT lung fibrosis, a major barrier to improved cure rate presents as progressive pulmonary injury. The utility of PET imaging with a αvβ6 integrin ligand was evaluated in patients with non-small cell lung cancer following pulmonary RT.

Subjects who had received pulmonary RT within 6 months were recruited. PET imaging of the lungs over 60 minutes was performed after administration of a fluorine-18 radiolabelled 20 amino acid αvβ6-specific peptide ligand, [18F]-FBAA20FMDV2 (IMAFIB; Cancer Research Technologies (CRT) patented). PET-CT scans were co-registered with the RT planning (RTP) scans and segmented to regions corresponding to RT doses of > 40 Gy (excluding tumour), 25-40 Gy, 15-25 Gy, 8-15 Gy and < 8 Gy. Time activity curves and the standardised uptake values (SUVs) were calculated and comparisons made between RT dose and PET uptake (SUV). Linear regression between SUV and RT dose was performed for each subject and normalised to an intercept of 1.

6 subjects (3M; 3 F) aged 51-75 years underwent an IMAFIB-PET scan between 6 -22 weeks after RT completion and mean IMAFIB radioactivity of 45.4 (range 8.9 – 122) MBq administered. 5 subjects received conformal external beam RT, while one subject received stereotactic RT (SBRT). A correlation between a higher PET uptake (SUV) and higher RT doses received by the lung was observed in all subjects with a significant relationship (p < 0.05) in 3 of the 6 subjects (table). The averaged normalised SUV across all subjects increased monotonically with RT dose, between 1.05 ± 0.14 at 4 Gy and 1.58 ± 0.31 at 52 Gy. | |  | |  | |

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

Subject No. | 11 | 12 | 16 | 17 | 20 | 21

RT dose and fractionation | 55Gy in 5 fractions. SBRT | 64 Gy in 32 fractions | 45 Gy in 15 fractions | 50 Gy in 20 fractions | 64 Gy in 32 fractions | 64 Gy in 32 fractions

Time between completion of RT and PET scan (weeks) | 8 | 22 | 13 | 6 | 7 | 14

Gradient of relationship between PET uptake and RT dose | 0.029 | 0.005 | 0.010 | 0.006 | 0.003 | 0.008

Intercept | 1.305 | 0.504 | 0.837 | 0.391 | 0.691 | 0.469

r2 | 0.841 | 0.539 | 0.836 | 0.879 | 0.310 | 0.672

p value | 0.028 | 0.158 | 0.030 | 0.018 | 0.163 | 0.089

A relationship between IMAFIB PET uptake and RT dose is consistent with RT dose-related activation of αvβ6 integrin in the irradiated lung and to the induction of lung fibrosis in subjects receiving pulmonary RT.

#1145

Biomarker-driven molecular imaging of prostate cancer using the PEG10 promoter.

Mariya Shapovalova. _University of Minnesota, Minneapolis, MN_.

The retrotransposon-derived paternally expressed gene 10 (PEG10) protein is ordinarily expressed at high levels in the placenta. The over-expression of PEG10 has been documented in a number of malignancies including hepatocellular carcinoma and leukemia. Recently, it was discovered that PEG10 isoforms promoted the progression of prostate adenocarcinoma to a highly lethal non-androgen receptor (AR) driven subtype called aggressive variant prostate cancer (AVPC).

In this study, we found that PEG10 was also expressed in castration-resistant PCa possessing constitutively active AR-splice variants in addition to AR-negative AVPC. We subsequently developed a molecular genetic imaging strategy for the non-invasive imaging of PCa by utilizing the cancer specificity of the PEG10 promoter to drive the expression of reporter genes. The transcriptional output of the PEG10 promoter was enhanced to enable high expression of reporter genes by multiple imaging modalities and immunoassay. By using this PEG10 promoter upstream of a reporter gene in a plasmid, we were able to detect PCa by fluorescence and positron emission tomography (PET) imaging after systemic administration of the plasmid in mice. This method also allowed for the detection of PCa by an immunoassay of mouse urine.

Our study demonstrates a pre-clinical proof-of-concept that the PEG10 promoter is a powerful and specific tool that can be utilized for non-invasive detection of highly lethal PCa subtypes. This biomarker imaging technology has the potential to improve detection and can be further modified for therapeutic applications.

#1146

**[** 18 **F]FPyGal: A novel ß-galactosidase specific PET tracer for** in vivo **imaging of tumor senescence.**

Marcel A. Krueger, Jonathan M. Cotton, Benyuan Zhou, Katharina Wolter, Johannes Schwenck, Anna Kuehn, Kerstin Fuchs, Andreas Maurer, Christian La Fougere, Lars Zender, Bernd J. Pichler. _Eberhard Karls University, Tuebingen, Germany_.

Senescence influences treatment outcome in cancer. Therefore it is of great interest to develop a diagnostic tool to detect senescent cells in cancer patients. Here we describe the evaluation of the PET tracer FPyGal for non-invasive imaging of ß-galactosidase as a surrogate marker for senescence. In vitro, we induced senescence in HCT116 cells by doxorubicin, in a liver progenitor cell line by p53-reactivation and in two liver carcinoma cell lines by a ribosomal checkpoint inhibitor (RCI). The cells were then incubated with FPyGal and tracer uptake was determined in a gamma-counter. For in vivo testing, the described cell lines were injected s.c. in mice. The tumor bearing mice were then treated appropriately to induce senescence. PET/MR scans were performed after i.v. injection of FPyGal for assessment of tracer uptake in the tumors. Ex vivo autoradiographies and X-Gal stainings were prepared to correlate intratumoral distribution of ⇓-galactosidase activity and tracer uptake. After toxicology studies, a pilot first-in-man study was performed in a cancer patient treated with Alisertib. Compared to the respective control cells, in vitro tracer uptake was significantly increased in all four cellular models tested. The strongest increase was observed in one of the liver carcinoma cell lines with a >3-fold increase in senescent cells. FPyGal tracer uptake increased in control HCT116 tumors from 1.1 to 1.7%ID/cc in senescent cells in in vivo PET/MRI studies and in the HRas model from 0.9 to 1.5 %ID/cc. In one of the liver tumor models we could observe a 2-fold increase in treated mice. Ex vivo ⇓-galactosidase staining and IHC of tumor tissue confirmed induction of senescence. Autoradiography and X-Gal staining showed an intratumoral correlation of ⇓-gal activity and FPyGal uptake. The compound passed the toxicology tests. A first patient study revealed high and heterogeneous tracer uptake in a liver metastasis. FPyGal shows increased in vitro and in vivo uptake in senescent cells and tumors. No toxicity of the compound could be detected and a first-in-man study showed promising results. Clinical trials including histological cross-validation are currently being prepared.

#1147

**A phage display identified peptide selectively binds to kidney injury molecule-1(KIM-1) and detects KIM-1-overexpressing tumors** in vivo **.**

Jae-Won Yoon,1 Soo-woong Lee,1 Md. Enamul Haque,1 Rang- Woon Park,1 Moon-Chang Baek,1 Dong-Kyu Kim,2 Sang Kyoon Kim,2 Byungheon Lee1. 1 _Kyungpook National University, Daegu, Republic of Korea;_ 2 _Daegu Gyeongbuk Medical Innovation Foundation, Daegu, Republic of Korea_.

Kidney injury molecule-1 (KIM-1), also known as hepatitis A virus cellular receptor-1 and T cell Ig mucin-1, is a transmembrane protein, which is not overexpressed in normal kidneys, but is significantly upregulated in damaged kidney tubular epithelial cells in toxic and ischemic acute kidney injury. Of interest, KIM-1 is overexpressed in most cases of renal cell carcinoma (RCC) and in other types of tumors including ovarian clear cell carcinoma and lung cancer. The extracellular domain of KIM-1 can be identified in the urine of patients with RCC and can thus be used as a biomarker for the detection of RCC. This study was carried out to identify a peptide that selectively binds to KIM-1 by screening a phage-displayed peptide library and to use the peptide for the detection of KIM-1-overexpressing tumors in vivo. Biopanning of a phage-displayed peptide library was performed on KIM-1-coated plates. Compared to other phage clones selected after biopanning, a phage clone displaying the CNWMINKEC peptide showed higher binding affinity to KIM-1 as examined by fluorometry and KIM-1-overexpressing 769-P renal tumor cells 1 as examined by ELISA. The CNWMINKEC peptide and a NeutrAvidin/biotin-CNWMINKEC multimer selectively bound to KIM-1 over albumin as examined by fluorometry and to KIM-1-overexpressing 769-P cells and A549 lung tumor cells compared to KIM-1-low expressing HEK293 normal cells when examined by flow cytometry. Co-localization and competition assays using an anti-KIM-1 antibody demonstrated that the binding of the CNWMINKEC peptide to 769-P cells was specifically mediated by KIM-1. Whole-body fluorescence imaging demonstrated selective homing of the CNWMINKEC peptide to KIM-1-overexpressing A498 renal tumor compared to KIM-1-low expressing HepG2 liver tumor in mice. The CNWMINKEC peptide was not cytotoxic to cells and was stable for up to 24 h in the presence of serum. These results suggest that the CNWMINKEC peptide is a promising probe for in vivo imaging and detection of KIM-1-overexpressing tumors.

Keywords In vivo imaging, kidney injury molecule-1, peptide, phage display, renal cell carcinoma

#1148

Recombinant cancer-stromal cells Identified in the metastatic tumor microenvironment By color-coded imaging.

Miki Nakamura,1 Atsushi Suetsugu,1 Kana Matsuura,1 Tomoyuki Satake,1 Masahito Shimizu,1 Shigetoyo Saji,1 Hisataka Moriwaki,1 Robert M. Hoffman2. 1 _Gifu Univ. Graduate School of Medicine, Gifu, Japan;_ 2 _AntiCancer, Inc., CA_.

Background/Aim:The relationship of cancer and stromal cells is poorly understood, including their ability to fuse or otherwise their genetic materials. Materials and Methods:Red-fluorescent protein (RFP-expressing) mouse colon 26 cancer cells were initially injected subcutaneously in green-fluorescent protein (GFP) nude mice. The resulting subcutaneous tumors were harvested and cultured. Using the Olympus FV1000 confocal microscope, the cells cultured from tumors were visualized. After 14 days culture, the cells were injected into the spleen. Results:After splenic injection, colon 26 metastases were observed in the liver, ascites, and bone marrow. RFP colon cancer cells, GFP stromal cells derived from host GFP nude mice, and recombinant yellow-fluorescent cells were observed in each organ. In addition, in the liver, areas with only GFP stromal cells were observed and assured to be a pre-metastatic niche. Conclusion:Color-coded imaging demonstrated the dynamics of colon cancer and stromal cells at different metastatic sites including the formation of recombinant cancer stromal cells. The significance of which will be evaluated in future experiments.

#1149

Systems pathology by Optra 30 color multiplex immunofluorescence automated whole slide imaging system integrated with image analysis.

Abhi Gholap,1 Anagha P. Jadhav,2 Suraj Somwanshi,2 Gurunath Kamble,2 Isha Doshi,1 Jiaoti Huang3. 1 _OptraSCAN Inc, San Jose, CA;_ 2 _OptraSCAN, Pune, India;_ 3 _Duke Univ. School of Medicine, Durham, NC_.

Background A paradigm shift from single marker immunohistochemistry (IHC) to multiplexing in molecular histopathology has helped better understand the cellular composition and heterogeneity of sub population of cells in tissues. Presently, one needs to manually exchange filters after each multiplex scan for capturing all fluorophores per staining round. This is very cumbersome and inefficient, as this process requires rescanning the same slide multiple times. Moreover, visual assessment and quantification of biomarkers is limited by inter and intra-observer variability. We hereby present an automated multispectral whole-slide imaging system integrated with image analysis for detection of upto 30-color multiplex assay with spectral unmixing and cell segmentation functionality.

Material and Methods

Multiplex IHC whole slide imaging Formalin Fixed Paraffin Embedded rat brain; indirect IHC stained sections with 10 antibodies using photostable fluorophores were obtained. The staining was based on antigenic integrity and antibody binding affinity. Optra's multispectral fluorescent whole slide scanning process was optimized to image sensitive antigens in early cycles and robust antigens in latter cycles. The fully automated fluorescence scanning system imaged weakly expressing biomarkers in the visible range (480-620 nm), highly expressing targets in the violet/blue (405-440 nm), far-red (650-680 nm) and NIR (700-800 nm) range across the entire usable light spectrum (350-800 nm).

ResultsWe could simultaneously detect up to 30 biomarkers within a single tissue section, using 12 staining-imaging-stripping cycles, with 10 antibodies screened per cycle. Software measures enabled separation of spectrally overlapping markers in the multiplex assay. A computer-controlled X-Y-Z stage used autofocusing at 20X/40X magnification. Integrated image analysis solution performed comprehensive evaluation of antigens of interest with accurate quantification of the target proteins based on parameters including but not limited to hue value/width, intensity threshold scoring, morphological characterization, pixel-count thresholding and color saturation.

Conclusion This proposed solution is efficient compared to the current imaging methods to tackle the aforementioned image acquisition and image analysis challenges associated with multiplexing assays.

#1150

Targeted immunoPET imaging of prostate cancer using a novel CD133 antibody.

Paige Glumac, Aaron LeBeau. _University of Minnesota, Minneapolis, MN_.

Prostate cancer is one of the most frequently diagnosed malignancies and the second leading cause of cancer related death among American men. Aggressive variant prostate cancer (AVPC) accounts for approximately 33% of all prostate cancer-related deaths and is characterized by visceral metastasis, minimal responses to current therapies, and poor overall prognosis. The current disease monitoring strategy for AVPC is positron emission tomography - computed tomography (PET/CT) with [18F]-fluorodeoxyglucose (FDG), which is not specific to prostate cancer cells and thus is inadequate in many cases. As such, novel targeted imaging agents are urgently needed for earlier detection and better monitoring of AVPC. The transmembrane glycoprotein, CD133, is overexpressed in AVPC and we have identified a single chain variable fragment, termed HA10, which preferentially recognizes a glycosylation‐independent epitope on CD133. Further characterization of HA10 revealed that it was able to identify CD133 in cells, patient tissue samples, and in vivo xenograft models using fluorescent imaging. The purpose of this study was to investigate the performance of the radioimmunoconjugate, 89Zr-HA10, for µPET/CT imaging in preclinical models of prostate cancer. HA10 was conjugated to p-SCN-Deferoxamine (DFO) and then radiolabeled with [89Zr]Zr-oxalate at room temperature. Parental (CWR-R1) and CD133-transduced (CWR-R1CD133) prostate cancer cell lines were implanted subcutaneously into male athymic nu/nu mice. Mice were administered 180 µCi of 89Zr-HA10 via tail vein injection and µPET/CT imaging was conducted at 24, 48, 72, and 144 hours post-injection. The 89Zr-HA10 construct was successfully labeled with a radiochemical purity >99% as determined by radio-thin layer chromatography. Specificity of 89Zr-HA10 was demonstrated for CD133-positive tumors with strong tumoral uptake detection from 24-72 hours. Furthermore, the margins of the CD133-positive tumors were clearly defined following 3D reconstruction of the µPET/CT images. These studies suggest that 89Zr-HA10 is a promising immunoPET imaging agent for AVPC and potentially other CD133-positive cancer subtypes. Moreover, the use of 89Zr-HA10 as a radiotracer will facilitate a more selective strategy for diagnosis and disease monitoring of these patients compared to the conventional FDG radiotracer. Further studies seek to evaluate the potential of 89Zr-HA10 to detect small metastatic lesions in vivo.

#1151

**Real-time visualization of brain metastasis** in vivo **.**

Takahiro Tsuji,1 Hiroaki Wake,2 Hiroaki Ozasa,1 Koichiro Haruwaka,2 Hitomi Ajimizu,1 Yuto Yasuda,1 Yuichi Sakamori,1 Takashi Nomizo,1 Young Hak Kim,1 Toyohiro Hirai1. 1 _Kyoto Univ. Graduate School of Medicine, Kyoto, Japan;_ 2 _Kobe University Graduate School of Medicine, Kobe, Japan_.

Research into physiological phenomena has taken a leap forward by recent advanced optical bio imaging techniques. Traditionally, physiological phenomena including cell division, cell function, molecular transport and expression, and signal transduction were detected by biochemical methods. However, this information lacked high resolution spatial or temporal information. Using recent bio imaging techniques, both spatial and temporal information can be integrated across the molecular, cellular and systems levels that allow us to further investigate the hierarchical interaction of organ systems. Here, we have developed an in vivo imaging method to visualize brain metastasis of lung cancer which provides real-time information of their proliferation, metastasis and the immune reaction for cancer cells. CMT167 and LLC1 cell lines which expressed mCherry or Venus were established. The fluorescently labeled cell lines were administered into the brain via the carotid artery or direct injection to the CX3CR1-GFP mice, transgenic mice in which microglia was specifically labeled with GFP. The metastasis cancer and the immune reaction were visualized using 2-photon microscopy for 14 days. Interaction between cancer cells and microglia was observed. We identified heterogeneity of the interaction between microglia and cancer cells. Some microglia cells showed phagocytosis-like response to cancer. Other microglia touched, surrounded, and released cancer cells without phagocytotic response. These interactions between microglia and cancer cells could be observed by 3-6 hours continuous real-time in vivo imaging. Further study to address detailed mechanism for microglia reaction is in progress. This research has potential to provide the information of brain metastasis in vivo and to be a new solution to elucidate the process of immunity and niche.

### Regulation of Cancer Stem Cell Stemness, Epithelial-Mesenchymal Transition, and the Microenvironment

#1152

Disulfiram targets both proliferating cancer cells and cancer stem-like population in ER-positive breast cancer.

Seojin Jang, Eunhye Oh, Yoon-Jae Kim, Tae-Min Cho, Jung Min Park, Soeun Park, Minsu Park, Jae Hong Seo, Ji Young Kim. _Korea University, Seoul, Republic of Korea_.

Drug repositioning can benefit from reduced development risk in terms of safety, toxicity and dosage, as it centers upon drugs that have already been tested in clinical trials. Disulfiram (DSF), a drug for the treatment of alcoholism, has potential anti-cancer activities in a copper (Cu)-dependent manner. In the present study, we investigated the effect of DSF on cell viability, apoptosis, breast cancer stem cells (BCSC)-like properties and p53 activation in ER-positive breast cancer MCF7 and T47D cells. DSF treatment caused a significant suppression of cell viability and marked induction of cell death in a Cu-dependent manner. This response was accompanied by caspase-3/-7 and p53 activation in ER-positive breast cancer cells. The latter phenomenon was associated with G1 phase arrest, coinciding with a significant increase in accumulation of nuclear p21 and downregulation of cyclin D1 and survivin. DSF treatment elicits a significant inhibitory effect on these BCSC-like properties including ALDH1 activity and CD44+/CD24- subpopulations. To examine the effect of DSF on mammosphere-forming ability, MCF7 and T47D cells were cultured in anchorage-independent serum-free culture conditions in the presence or absence of DSF and Cu. DSF considerably suppressed mammosphere formation, as evidenced by significant reduction in the number and volume of mammospheres. Our results demonstrate that DSF induces apoptosis and effectively targets cancer stem-like population, suggesting that disulfiram may be potentially effective for the treatment of ER-positive cancer.

#1153

Hypoxic microenvironments promote stemness and enhance the directed migration of glioma stem cells.

Nitya V. Sharma,1 Monica Chau,2 Subhas Mukherjee,1 James Ross,3 Brandon Miller,2 Changming Zhang,4 Jun Kong,3 Carol Tucker-Burden,3 Emily Kaissi,4 John W. Reitnauer,5 Bilge Dundar,1 Cheryl L. Olson,1 Gregory Bix,2 Daniel J. Brat1. 1 _Northwestern University Feinberg School of Medicine, Chicago, IL;_ 2 _University of Kentucky, KY;_ 3 _Emory University, GA;_ 4 _Emory University, Atlanta, GA;_ 5 _Emory University, IL_.

Glioblastoma (GBM) is a highly malignant primary brain tumor and has a poor prognosis and is resistant to the most advanced therapies. The development of highly hypoxic centrally located necrotic regions is strongly associated with accelerated GBM progression, yet mechanistic explanations are not complete. Hypoxic regions within GBM are highly enriched for glioma stem cells (GSC), which are critical for tumor initiation, recurrence and therapeutic resistance due to their self-renewing capabilites. While most GBM cells migrate outward, away from hypoxia, GSCs accumulate there, forming a hypercellular border called pseudopalisades that promote their survival. Mechanisms that mediate GSC accumulation within pseudopalisades are unknown. We hypothesize that GSCs are enriched in the pseudopalisades due to the homing of a subpopulation towards the supportive hypoxic microenvironment. This in turn activates critical hypoxia-induced drivers to promote stemness, survival and migratory behavior encouraging the recruitment of more GSCs. Using two genetically distinct patient-derived GBM cell lines, we found that exposure to hypoxia in vitro led to enrichment of GSCs among a heterogeneous population of GSCs and non-GSCs. We found that hypoxia enhanced the expression of stem cell, pro-survival and pro-migratory factors in GSCs, such as the pro-migratory chemokine SDF-1α, and its cognate receptor CXCR4. Importantly, we found hypoxia also induced upregulation of OLIG2, one of four core transcription factors necessary and sufficient to induce stemness, in GSCs and non-GSCs exposed to hypoxia. We determined that OLIG2 was necessary for the expression of stem cell markers, and that its knockdown reduced cellular migration in hypoxia vs normoxia in GSCs. This suggests a critical role for OLIG2 in both hypoxia-induced stemness and homing behavior. We also generated a unique model of localized hypoxia in one brain hemisphere through vaso-cclusion and observed the homing of xenografted GBM cells towards hypoxia, regulated by OLIG2 in vivo. These data shed light on how hypoxic tumor microenvironments facilitate mechanisms vital to GBM progression and therapeutic resistance.

#1154

DCLK1 monoclonal antibody-based CAR-T cells as novel treatment strategy against multiple myeloma.

Sripathi M. Sureban,1 Robert Berahovich,2 Hua Zhou,2 Shirley Xu,2 Lijun Wu,2 Vita Golubovskaya,2 Dongfeng Qu,1 Randal May,1 Edwin Bannerman-Menson,1 Courtney W. Houchen1. 1 _COARE Holdings Inc, Oklahoma City, OK;_ 2 _Promab Biotechnologies Inc, Richmond, CA_.

Multiple Myeloma (MM) is characterized by the clonal expansion of malignant plasma cells in the bone marrow, and is the second most common hematological cancer. Cellular therapies including chimeric antigen T receptor cell (CAR-T) have emerged as a promising therapeutic strategy to combat this devastating disease. MM are derived from a rare population of cancer stem cells (CSC) that are responsible for initiation, progression, resistance to conventional therapy and recurrence. To date there have been no CAR-T therapies directed at CSC-specific antigens. In this report, we describe the effects of a novel CAR-T targeting the CSC marker DCLK1 on MM cells. Using mouse models, it has been demonstrated that DCLK1 marks the cell of origin of colon and pancreatic cancers. DCLK1 regulates epithelial to mesenchymal transition (EMT), and its upregulation is associated with poor overall survival in many cancer subtypes. Inhibition of DCLK1 activity following RNA silencing resulted in tumor xenograft growth arrest. These features lead us to explore the therapeutic potential of targeting DCLK1+ cells in MM. In order to create a novel DCLK1 CAR, we generated a new humanized DCLK1 monoclonal antibody, clone CBT-15 #8. This antibody recognizes a small extra cellular C-terminal epitope on human isoform 2 and 4 of DCLK1. Novel DCLK1 CAR-T cells containing the clone were generated and detected with anti-human FAB antibody by flow cytometry. Furthermore, we observed cell surface expression of DCLK1 on >93 percent of MM Cells (RPMI8226) by flow cytometry. The CAR-T cells had significantly higher cytotoxicity in MM (RPMI8226 and MM1S) cells than in leukemia K562 cells. The CAR-T cells secreted significantly higher levels of IFN-γ in vitro than T cells (p=0.0036). Further studies are underway to determine its efficacy in inhibiting tumor xenografts in vivo. This report is the first to link the TSC marker DCLK1 to MM and may represent a novel therapeutic strategy for MM and other hematologic malignancies.

#1155

Stemness control by iron chelator is a novel therapeutic strategy for esophageal cancer.

Toru Narusaka,1 Toshiaki Ohara,1 Kazuhiro Noma,1 Yuki Katsura,1 Noriyuki Nishiwaki,1 Motoyasu Tabuchi,1 Takuro Fushimi,1 Toshihiro Ogawa,1 Sho Takeda,1 Satoshi Komoto,1 Hiroaki Sato,1 Satoru Kikuchi,1 Yasuko Tomono,2 Hiroshi Tazawa,1 Shunsuke Kagawa,1 Yasuhiro Shirakawa,1 Toshiyoshi Fujiwara1. 1 _Okayama University, Okayama, Japan;_ 2 _Shigei Medical Research Institute, Okayama, Japan_.

Background:Esophageal cancer has a poor prognosis and high mortality rate because of recurrence and chemo-resistance. Cancer stem cells (CSCs) have been proposed to be responsible for tumor initiation, drug resistance, and recurrence. Although CSCs are considered to be important therapeutic targets, effective CSC targeted therapy has not yet been established. We previously reported that iron chelator suppressed not only proliferation but also the expression of stemness markers in mouse induced pluripotent stem cells (miPS) and miPS inducible CSC model cells (miPS-LLCcm). Moreover, we revealed that iron chelator also suppressed the expression of stemness markers and function in esophageal cancer cell lines with high stemness potential. Thus we hypothesized that stemness control by iron chelator can be a novel therapeutic strategy for esophageal cancer patients. In this study, we performed clinicopathological analysis using primary tissue samples, verified the possibility of clinical study, and explored the mechanisms in vitro.Method: TE-8 and OE33 were used as esophageal cancer cell lines expressing stemness markers. Deferasirox (DFX) and Deferoxamine (DFO) were used as iron chelator. The stemness markers expression including Nanog, Sox2, Oct3/4, Klf-4 and c-Myc was examined by western blot analysis. Reactive oxygen species (ROS) generation was examined by fluorescence staining. Immunohistochemistry (IHC) of Nanog was performed in 134 clinical esophageal cancer patients who received radical esophagectomy in Okayama university hospital. Result: DFX and DFO suppressed the expression of stemness markers although cisplatin could not suppress. DFX and DFO induced ROS generation. Nanog IHC staining revealed that high Nanog expression was correlated with low overall survival (OS) (HR, 2.53 [95% CI, 1.24 to 5.14]; P = 0.01) and disease-free survival (DFS) (HR, 2.33 [95% CI, 1.19 to 4.58]; P = 0.01). Moreover, high Nanog expression was also correlated with low OS in neoadjuvant therapy group, but not in non-neoadjuvant therapy group.

Conclusion: Iron chelator, DFX and DFO, suppressed proliferation and stemness via ROS generation in esophageal cancer cell lines. High Nanog expression was correlated with poor prognosis, which indicates that stemness control by iron chelator is a novel therapeutic strategy for esophageal cancer patients.

#1156

Skeletal muscle progenitor cell-derived exosomes have therapeutic potential in inhibition of prostate cancer cell proliferation.

Krishna M. Sinha,1 Rozita Yarmand,2 Nermin Kahraman,2 Fahriye Duzagac,2 Gabriel Lopez,2 Bulent Ozpolat,2 Johnny Huard1. 1 _UT Health Science Center at Houston, Houston, TX;_ 2 _UT MD Anderson Cancer Center, Houston, TX_.

Adult stem cells have been used as regenerative medicine for treating human diseases through gene therapy approach. These stem cells secrete cytokines, growth factors and extracellular vesicles including exosomes, which may induce antitumor activities. We have demonstrated that muscle progenitor cells (MPCs) from adult (murine and human) skeletal muscle have a high regenerative potential to repair bone, skeletal muscle, and cartilage. However, to date, the potential therapeutic use of MPCs in cancers has not yet been investigated. The objectives of this study were to identify and examine the roles of secreted factors including exosomes from MPCs on the proliferation/survival, motility/migration and invasive potential of prostate cancer (PCa) cells. Here we showed that co-culture of PCa cell lines, PC3 and C42B, with MPCs in trans-well inserts strongly suppressed PCa cell proliferation and survival by activating CDK inhibitors (p16 and p27) and apoptotic BBC3 and NOXA genes. Mass spectrometry analysis of factors secreted by MPCs identified several proteins with known anti-proliferative functions, including IGFBP6/7, pigment epithelium-derived factor (PEDF), HPCAL1, NO, and SPARC. Conditioned media (CM) from MPCs significantly increased docetaxel-induced cytotoxicity of PCa cells by suppressing the expression of key genes including AR, MLL, FOXA1, TWF1, and TGIF. Next, we isolated exosomes from cultured mouse and human MPCs through step-wise ultracentrifugation methods and tested their effect in the proliferation of cancer cells. We observed that treatment of PCa cells with MPC-derived exosomes inhibited cell proliferation as assayed with MTT and down-regulated the expression of AR and its target NKX3.1 genes. We also observed that the migration ability of ovarian cancer cells (OVAC5) and pancreatic cancer (Panc1), was markedly reduced following MPC-derived exosome treatments compared to untreated cells. Taken together, our data suggest that MPCs can suppress the proliferation and survival of cancer cells through secreted factors including exosomes. Thus, adult stem cell-based therapy along with existing therapies may be a promising strategy in treating various solid cancers as well as regenerating cancer-induced bone and muscle tissue lesions.

#1157

Single-cell transcriptmoics identifies rhamm positive proliferative cells within human colorectal cancer stem cells.

Michitaka Nakano, Kikushige Yoshikane, Kohta Miyawaki, Shinichi Mizuno, Kyoko Yamaguchi, Takuji Yamauchi, Hiroshi Ariyama, Hitoshi Kusaba, Masafumi Nakamura, Takahiro Maeda, Eishi Baba, Koichi Akashi. _Kyushu University, Fukuoka City, Japan_.

Cancer stem cells (CSCs) can be prospectively isolated based on specific surface marker such as CD44, CD133, LGR5, and CD166, in human colorectal cancer. However, the employment of these CSCs' markers are still insufficient to prospectively isolate the colorectal CSCs. To overcome the cellular heterogeneity of previously reported CSCs fraction, we performed single-cell mRNA sequencing (scRNAseq) of 72 cells within organoid. By conducting scRNAseq, we identified CSCs-like sub-population with high expression of MKI67 and Cyclin families, which was generally expressed in proliferative state of the cells within the organoid. Remarkably, high expression of hyaluronan-mediated motility receptor (HMMR) encoding cell membranous and cytoplasmic molecule, receptor for hyaluronan mediated motility (RHAMM), which was reported to associate with CD44 in hyaluronan binding, was observed in the CSCs-like cells. Prospective isolation of RHAMM+ CD44+ cells, that were sub-population of conventional CSCs fraction, revealed higher organoid- and tumor-forming abilities. Furthermore, comprehensive gene expression analysis also demonstrated the proliferative gene expression within RHAMM+ CD44+ cells. Here, we report RHAMM+ proliferative cells that sub-fraction the conventional CSCs population. <!--EndFragment-->

#1158

Seven genes were closely associated with ALDH1 in triple negative breast cancer.

Takashi Ishikawa,1 Kazutaka Narui,2 Akimitsu Yamada,2 Kumiko Kida,2 Hidetaka Shima,2 Chiho Endo,3 Shoko Adachi,4 Sadatoshi Sugae,4 Yohei Miyagi,5 Yasushi Ichikawa,3 Itaru Endo3. 1 _Tokyo Medical University, Tokyo, Japan;_ 2 _Yokohama City University Medical Center, Yokohama, Japan;_ 3 _Yokohama City University, Yokohama, Japan;_ 4 _Yokohama City University, Tokyo, Japan;_ 5 _Kanagawa Cancer Center, Yokohama, Japan_.

Background: Aldehyde dehydrogenase1 (ALDH1) is widely accepted as a marker of normal and malignant breast stem cells. We reported that ALDH1 expression was associated with aggressive phenotypes and poor clinical outcome of breast cancer patients. However, mechanics of how ALDH1 affects cancer stem cell features is not known.

Aim: To examine genes associated with ALDH1 and find novel druggable targets for the future cancer stem cell therapy.

Methods: ALDH1-positive and ALDH1-negative cells were dissected by a laser capture microdissection from 5 ALDH1 positive triple negative breast cancer samples. A differential analysis of mRNA expression was performed between ALDH1 positive and negative breast cancer cells in each sample by Affymetrix GeneChip microarrays. 1) Microarray analysis suite (MAS5) and robust multi-chip average (RMA) were used as preprocessing algorithms to analyze gene expression data and to extract genes differently expressed between ALDH1 positive and negative cells. 2) The network analysis was performed with extracted genes with MAS5 by the comprehensive information platform "KeyMolnetTM".

Results: 1) 54,682 genes were extracted and 32,264 genes were selected after eliminating background noise. One hundred and four genes: 63 up-regulated and 41 down-regulated, were extracted in ALDH1-positive cells by MAS5, while only 2 up-regulated genes by RMA. H19 was a gene that was commonly extracted by both algorithms. 2) The network with 104 genes was constructed and BRD4, SMAD4, Pitx3, RARa, MUC1, HASH1 and C/EBPb were identified as genes with the closest distance to ALDH1.

Conclusions: Most of extracted 7 genes have been already known to exert critical roles for maintaining stem cell features, providing a rationale of ALDH1 for the stem cell marker of breast cancer. These genes including H19 could be targets of the cancer stem cell therapy for treating incurable breast cancer.

#1159

Association of ALDH7A1 overexpression with chemotherapy resistance and cancer stem cell in lung cancer.

Yuto Yasuda,1 Yuichi Sakamori,1 Hiroaki Ozasa,1 Hitomi Ajimizu,1 Tetsuya Oguri,2 Ken Maeno,2 Takahiro Tsuji,1 Takashi Nomizo,1 Tomoko Yamamoto,1 Hironori Yoshida,1 Toyohiro Hirai,1 Young Hak Kim1. 1 _Kyoto University, Kyoto, Japan;_ 2 _Nagoya City University, Nagoya, Japan_.

Aldehyde dehydrogenases (ALDHs) play a major role in the oxidation of aldehydes. Certain isoforms of ALDH is related to cancer stem cells (CSCs) responsible for tumor initiating, drug resistance and metastasis. ALDH1A1 and ALDH3A1 have been known for CSC markers of lung cancer. Since these two isoforms and ALDH7A1 are involved in cellular structural and osmoregulatory function, we focused on function of ALDH7A1 in lung cancer. We established SN-38 resistant DMS53 (DMS53-SR) and SN-38 or gemcitabine resistant NCI-H23 (H23-SR or H23-GR, respectively) by intermittent and dose-escalating exposure of the agent. The expression of ALDH7A1 was upregulated in DMS53-SR and H23-SR/H23-GR compared with DMS53 and NCI-H23, respectively. ALDH7A1 expression was altered using a plasmid to overexpress ALDH7A1 in DMS53. ALDH7A1 overexpression led to a slight increase of IC50 of SN-38, upregulation of SOX2, and tumor sphere formation. Overexpression of ALDH7A1 in DMS53 have enhanced tumorigenic capacity in vivo. These data suggested that ALDH7A1 might be a marker of CSC and a therapeutic target of lung cancer.

#1160

Endoplasmic reticulum stress disrupts stemness-related transcriptional regulatory network: Implication for therapy response in cancer.

Appolinaire Olou, Kamiya Mehla, Pankaj Singh. _University of Nebraska Medical Center, Omaha, NE_.

Pancreatic cancer is the third leading cause of cancer-associated deaths in the United States. We have recently demonstrated that endoplasmic reticulum (ER) stress correlates with improved survival in human pancreatic cancer patients on gemcitabine therapy. Cancer cell stemness is a significant contributor of poor response to therapy and disease recurrence. We also observed that inducing ER stress can diminish stemness in cancer cells. Hence, to investigate the mechanism of ER stress-mediated alterations in diminishing cancer stemness, we investigated the impact of ER stress on transcriptomic alterations by performing RNAseq studies. We subjected cancer cells to treatment with thapsigargin and the harvested mRNA was utilized for RNAseq analysis. We observed significant alterations in mRNA expression levels of the stemness-associated gene set, which also showed significant enrichment in Ingenuity pathway analysis. Hence, our studies demonstrate that ER stress regulates stemness at the transcriptional level. Our ongoing and future studies will target individual genes/pathways to identify their relative contributions to ER stress-mediated regulation of stemness. Considering the role of stemness in disease recurrence, our studies will provide novel mechanistic insights that may lead to novel therapies for targeting disease recurrence in pancreatic cancer.

#1161

Utility of evidence based mesenchymal gene signatures gene expression panel in predicting outcome in stage matched colorectal cancer patients.

Benjamin Johnson, Pankaj Ahluwalia, Chetan Pundkar, Saleh Heneidi, Kimya Jones, Ashis Mondal, Ravindra Kolhe. _Medical College of Georgia, Augusta, GA_.

Colorectal cancer (CRC) is third deadliest cancer in United States, only behind lung and prostate cancer. Although several therapies are available to treat colorectal cancer but there is need for new prognostic biomarkers to tailor appropriate personalized therapy. Mesenchymal Stem cells (MSCs) are multipotent stem cells which are attracted toward inflammatory sites. In colon cancer, they have been shown to assist in tumor progression. To assess the MSC gene signatures in colon cancer patients, we picked 5 genes from nCounter plexset Mesenchymal Stem cell panel. These genes named: SMAD4, PTK2, GTF3A, CASP3 and HDAC1 were quantified on Nanostring platform. The aim of the study is to quantify these Mesenchymal gene signatures in colorectal cancer patients. Under the IRB approved protocol, a total of 750 colon cancer patients at Medical College of Georgia with 5 years follow-up were initially selected. The patients were stratified on the basis of overall survival in two groups, with higher (> 5 years) and lower survival (<1 year) along with AJCC staging (I to IV), grade, gender and age. A total of 88 patients fitted in our inclusion criteria on the basis of survival duration after diagnosis. The FFPE blocks were acquired from Medical College of Georgia, Augusta. Total RNA was isolated and quantified through Nanodrop method. The statistical analysis of data was performed using student t-test and Pearson correlation.

We found PTK2 levels to be high in stage I patients (p = 0.02*). Also, patients with higher survival in stage I had high expression of PTK2 (p=0.04*). Additionally, PTK2 was expressed in higher amounts in lower surviving patient group (0.00*). GTF3 levels were higher in (p = 0.01) stage I. CASP3 showed higher expression in stage III and IV combined. HDAC1 expression was higher in Grade II patients compared to Grade I. HDAC1 expression was also higher in African American population.

#1162

CD24 regulates cancer stem cell like traits in bladder cancer.

Akira Oki,1 Mohammad Obaidul Hoque2. 1 _Cancer Institute Hospital of Japanese Foundation for Cancer Research, Tokyo, Japan;_ 2 _Johns Hopkins University, Baltimore, MD_.

Tumors are hierarchically organized by a rare population of cancer stem cells (CSCs) that contributes to cancer initiation, progression, and treatment failure. A better understanding of the molecular mechanisms underlying urothelial CSC regulation and the identification of key molecules associated with CSC generation and maintenance are pivotal for the molecular-targeting therapy. CD24 is overexpressed and acts as a lynchpin of tumorigenesis and metastatic progression in urothelial carcinoma of the bladder (UCB). However, its contribution to cancer stem-like traits of CD24 have not been determined. The functional relevance of CD24 was evaluated using in vitro and in vivo approaches. The knockdown of CD24 attenuated cancer stemness properties including sphere formation, self-renewal, invasion, chemo-resistance, and tumor initiation. The high-CD24-expressing cells, isolated from patient-derived UCB xenograft tumors, exhibited their enhanced stemness properties. Biologically, CD24 was associated with the expression of several key CSC-related molecules, including CD133, ABCG2, and YAP1. In human UCB samples, CD24 was overexpressed not only in primary tumors, but also in urine from UCB subjects. Conclusion: CD24 plays a crucial role in maintaining the urothelial cancer stem-like traits.

#1163

HR23A expression modulates drug resistance through regulation of autophagy and stem cell properties in cancer cells.

Show-Mei Chuang, Chung-Yun Yu, Bang-Hung Liu, Sheng-Chih Lin, Shao-Yi Tang. _National Chung Hsing University, Taichung, Taiwan_.

DNA-damaging agents are commonly used as anticancer therapeutics. Unfortunately, drug resistance arises through a combination of mechanisms, with the sensitivity of cells to apoptosis depending on the balance between apoptotic and survival signals. Our previous studies show that knockdown of HR23A, an accessory protein involved in nucleotide-excision repair (NER) at an early lesion-recognition step, induces autophagy, increases the resistance to cisplatin, oxaliplatin, 5-FU and docetaxel. However, knockdown of HR23A contributed no effect to the sensitivity of tamoxifen or irinotecan. In present study, we found that knockdown of HR23A displayed more sensitivity to the treatment of doxorubicin in A549 cell line. Knockdown of HR23A enhanced cell death via increased ER stress-mediated apoptosis but not autophagy, suggesting a different role of HR23A in response to doxorubicin. Further investigation revealed that HR23A-knockdown cells appear to undergo epithelial-mesenchymal transition and take on certain attributes of cancer stemness. Our experiments show that depletion of HR23A enhances epithelial-mesenchymal transition, EMT regulator protein levels such as Twist1, Slug and Zeb1, cancer cell migration and various cancer stemness properties, including the expression of major pluripotency factors, the capacity for tumor-sphere formation in culture and the expression of cancer stem cell surface markers. The increases of these stemness properties are reversed by ectopic expression of HR23A or further knockdown of Twist1 in HR23A-depleted cells. Furthermore, HR23A directly targets the Twist1 protein and contributes to regulating Twist1 protein stability without changing its gene transcription. Together, our data suggest that knockdown of HR23A induces autophagy, undergoes epithelial-mesenchymal transition and promotes stemness properties, thus leading to the resistance to DNA-damaging agents.

#1164

**Expression of Notch and Nodal embryonic components in** BRAF **\- and** RAS **-like thyroid cancer cell lines.**

Paola M. Dantonio, Cesar Seigi Fuziwara, Kelly C. Saito, Edna T. Kimura. _University of Sao Paulo, Sao Paulo, Brazil_.

TCGA genomic study in papillary thyroid carcinoma (PTC) samples highlights two groups of tumors: BRAF-like and RAS-like, whose genotype might influence its biological behavior. The mechanisms behind progressive loss of thyroid differentiation, observed mainly in the most aggressive histotype anaplastic thyroid carcinoma (ATC), could be associated with the reactivation of embryonic signaling and acquisition of stem cell-like phenotype. In this study we focused on Notch and Nodal embryonic signaling pathways, which are crucial for stemness maintenance in embryonic cells and has also been linked to cancer cell stemness. By assessing the expression of Notch and Nodal pathway components in BRAF- and RAS-like cell lines, we sought to investigate if the reactivation of these pathways is involved in dedifferentiation mechanisms observed in ATC. For that purpose, RNA and protein were extracted from three PTC (TPC-1, BCPAP and K1), six ATC (KTC2, SW1736, C643, Hth7, Hth74 and Hth83) and non-tumoral (Nthy-ori 3-1) cell lines. For gene expression analysis, cDNA generate from mRNA was amplified by qPCR using specific primers for NOTCH1, NOTCH4, HES1, NUMB, CER1 and LEFTY1/2. For protein expression analysis, SDS-PAGE-fractionated proteins were transferred onto nitrocellulose membrane, incubated with anti-Nodal antibody and visualized by chemoluminescence. NOTCH1 was downregulated in BRAF-like PTC cell lines, while slightly overexpressed in RET/PTC-containing TPC-1. On the other hand, NOTCH1 was overexpressed in Hth83 and C643 ATC lines, both HRAS-mutated, and also in BRAF-mutated SW1736. NOTCH4 was overexpressed in PI3K-mutated K1 and all ATC cell lines. Transcription repressor HES1, which prevents cell cycle arrest by inhibiting p27, was highly expressed in all cell lines compared to Nthy. Levels of NUMB expression were elevated in all ATC cell lines with no difference between BRAF- and RAS-mutated, but were inversely related to those of HES1, as expected for a Notch pathway inhibitor. CER1 and LEFTY1/2, which encode Nodal inhibitors, were highly expressed in ATC cell lines (4/6 and 5/6, respectively) but with no apparent distinction between BRAF- and RAS-mutated cells. At the same time, Nodal protein levels are only upregulated in TP53-mutated BCPAP and C643, suggesting that alterations in MAPK components are not sufficient to promote Nodal overexpression. Our results show re-expression of Notch embryonic signaling components in both PTC and ATC cell lines with either BRAF or RAS oncogenes. Moreover, additional genetic alterations such as in PI3K and TP53 might also interfere with the expression of both Notch and Nodal components. We therefore highlight the importance of tumor genotype, which might determine the modulation of embryonic-related genes and therefore contribute to the undifferentiated phenotype in thyroid cancer. Financial support: FAPESP and CNPq.

#1165

Differential expression of CD44 variants drive the progression, invasion, drug-resistance and stemness characteristics in human oral squamous cell carcinoma.

Tanushree Kashyap,1 Siddavaram Nagini,2 Ajay Rana,3 Rajakishore Mishra1. 1 _Central University of Jharkhand, Brambe, Ranchi, Jharkhand, India;_ 2 _Annamalai University, Annamalainagar, Tamil Nadu, India;_ 3 _The University of Illinois at Chicago, Chicago, IL_.

Purpose: Cluster of differentiation 44 (CD44) is a cell-surface glycoprotein and plays role in the progression and severity of oral squamous cell carcinoma (OSCC). Here we report differential function of CD44 variants in OSCC progression.

Materials and methods: The expression of CD44 standard (CD44s) and variants (CD44v4, CD44v6); the activation of signaling pathways (MAPK, PI3K); the cell viability; and the MMP-9/-2 activity were assessed using RT-PCR, immunohistochemistry, Western blotting, MTT assay and gelatin zymography. These experiments were carried out using fresh human OSCC tissue specimens, including adjacent normal, noninvasive (N0), invasive tumor samples (N1-3) and chemo-radiation resistant tumor samples (RCRT). We also used OSCC cell lines, including parental (SCC9/SCC4) and Cisplatin-resistant (CisR-SCC9/-SCC4) to confirm our observation with human tissues. Knock down of CD44 variants or inactivation of MAPK/PI3K pathways was achieved for in vitro analysis in OSCC cells.

Summary: Differential expressions of CD44 variants (v4and v6) were associated with overall oral cancer aggressiveness. CD44v4 expression was positively correlated with the activation of MAPK pathway causing chemoresistance. Conversely, CD44v6 expression and activation of PI3K-Akt caused invasiveness of OSCC. Finally, an overlapping role of two major signal transduction pathways such as MAPK and PI3K, that impinge on the variants/ isoforms expressions of a single gene i.e. CD44 that leads to the OSCC aggressiveness.

Conclusion: Collectively, these results established that CD44 variants (v4and v6) expression driven by MAPK/PI3K are associated with the overall progression, drug-resistance, invasion, and stemness characteristics leading to the aggressiveness of OSCC. Hence targeting these pathways may be exploited for treating OSCC.

Keywords: OSCC, CD44s, CD44v4/6, MAPK, PI3K, Notch, Chemoresistance, Invasion/migration, stemness, aggressiveness of OSCC.

#1166

TDO2 overexpression is associated with cancer stem cells and poor prognosis in esophageal squamous cell carcinoma.

Thang Quoc Pham, Naohide Oue, Yohei Sekino, Yuji Yamamoto, Naoya Sakamoto, Ririno Honma, Kazuhiro Sentani, Wataru Yasui. _Hiroshima University, Hiroshima, Japan_.

Objective: Esophageal cancer is one of the deadliest cancers in the world, and the main subtype is esophageal squamous cell carcinoma (ESCC), which comprises 90% of cases. We previously analyzed the gene expression profile of spheroid colonies and parental cells derived from gastric cancer (GC) cell lines by microarray analysis. Among the genes upregulated in spheroid colonies, tryptophan 2,3-dioxygenase (TDO2) is dramatically upregulated in MKN-1 cells (derived from adenosquamous cell carcinoma) compared with other GC cell lines. Expression of TDO2, an enzyme involved in tryptophan catabolism, has been linked with tumor survival and poor prognosis of brain and breast cancer. However, no studies have investigated the potential role of TDO2 in esophageal cancer. Here we explored the expression and biological significance of TDO2 in ESCC.

Methods: To explore the expression of TDO2 in cancer and normal samples, we used an online analytical tool, the Broad Institute TCGA Genome Data Analysis Center, http://firebrowse.org/. For quantitative reverse transcription-polymerase chain reaction (qRT-PCR), we used 10 ESCC samples (tumor tissues and the corresponding non-neoplastic tissue). TDO2 protein expression was evaluated in 90 ESCC tissue samples by immunohistochemistry. TDO2 function in ESCC cell lines and spheroid colony formation was evaluated by RNA interference (RNAi). Cell growth was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Modified Boyden chamber assays were performed to examine cell invasiveness.

Results: TDO2 expression was upregulated in most cancers except for liver cancer and pancreas cancer. The fold change expression of TDO2 between normal and cancer tissue was the highest in esophageal cancer. TDO2 overexpression was associated with tumor stage, recurrence status and the CD44 cancer stem cell marker in ESCC. TDO2 overexpression was correlated with poor outcome of ESCC patients. Inhibition of TDO2 expression by RNAi in TE-10 and TE-11 cell lines reduced both the number and the size of spheroid colonies as well as cell proliferation and invasive activity. Knockdown of TDO2 expression also induced inactivation of the EGFR signaling pathway.

Conclusion: Our results imply that TDO2 could play an important role in the progression of ESCC. Furthermore, TDO2 may be a potential therapeutic target in ESCC.

#1167

Identification and targeting of CD34+CD176+IL1RAP+ chronic myeloid leukemia stem cells with bi-specific antibodies.

Raghda Elsawi,1 ChengXiang Wu,2 Soha Younes,1 Eman Abdel-Momen Mohammed,1 James E. Robinson,3 Fouad Mohammed Badr,1 Stephen E. Braun2. 1 _School of Medicine, Suez Canal University, Ismailia, Egypt;_ 2 _TNPRC,Tulane University, Covington, LA;_ 3 _School of Medicine Tulane University, New Orleans, LA_.

Leukemia Stem Cells (LSCs) quiescence in Chronic Myeloid Leukemia (CML) plays a major role in therapeutic resistance and disease progression calling for the need for identifying and targeting such cells (1). LSCs belong to the primitive population; CD34+CD38-Lin-, which can't distinguish normal hematopoietic stem cells (HSCs) from CML LSCs. IL1RAP was successfully identified as a marker for BCR-ABL+ CD34+ LSCs, but was not specific as it was also expressed peripherally on platelets and monocytes (2). Another marker, Thomsen-Friedenreich antigen (TF, or CD176), was targeted by anti-CD176 mAb in CD176+ leukemic cell lines and induced Fas-mediated apoptosis not only in LSCs but also in all CD176+ cells (3). Because CD34 molecule is a major carrier of CD176 antigen (4) and IL1RAP is tightly correlated to BCR-ABL expression (5), we evaluated the co-expression of IL1RAP and CD176 on hematopoietic progenitor CD34+ CML stem cells. Peripheral blood mononuclear cells (PBMCs) from patients with CML (n = 4) or healthy volunteers (n = 1) were analyzed for BCR-ABL expression and stained with monoclonal anti-human CD176 and anti-human IL1RAP antibodies for analysis by flow cytometry analysis. CD34+HSCs displayed highly significant co-expression of these markers (P &It; 0.01). Additionally, flow-sorted CD34+CD176+IL1RAP+ cells displayed colony forming potential compared to CD34+CD176+IL1RAP- cells (CML-2, P &It; 0.01). Therefore, we generated a bi-specifıc antibody (bis-Ab); TF/RAP, that binds both antigens simultaneously. One Fab contained the VH and VL specific for CD176 and the other Fab was specific for IL1RAP. Site-directed mutagenesis was used to induce knob-in-hole mutations. Either a duel-positive cell line or CML samples were treated with bis-Ab for one hour, and increasing binding was observed (p &It; 0.001). Linear regression analysis has shown cooperative binding of the bis-Ab as compared to monoclonal antibodies. Complement-dependent cytotoxicity assay (CDC) was used to demonstrate killing of CML stem cells or duel+ cell lines. Our results have shown that our TF/RAP selectively targeted IL1RAP+ and CD176+ cell population among CML PBMCs, but not corresponding normal cells; providing a novel therapeutic strategy for the depletion of CML stem cells from the bulk population in clinical HSC transplantation.

1. Zhou H & Xu R (2015). Leukemia stem cells: the root of CML. Protein & cell, 6(6), 403-12.

2. Järås M et al. (2010). Isolation and killing of candidate chronic myeloid leukemia stem cells.... PNAS, 107(37), 16280-5.

3. Yi B et al. (2011). Mechanisms of the apoptosis induced by CD176 antibody in human leukemic cells. Int. J of Onc, 38, 1565-1573.

4. Karsten U & Goletz S(2013). What makes cancer stem cell markers different? Springer Plus, 2(1), 301.

5. Zhao K et al. (2014). IL1RAP as a surface marker for leukemia stem cells ... Int. J .of clinical and experimental medicine, 7(12), 4787-98.

#1168

Characterization of tumor initiation cells in mammary tissues of C3-1-TAg transgenic mice.

Qiong Cheng,1 Amanda B. Parris,2 Zhikun Ma,2 Yujie Shi,1 Lingfei Kong,1 Xiaohe Yang2. 1 _Henan Provincial People's Hospital, Zhengzhou, China;_ 2 _North Carolina Central University, Kannapolis, NC_.

Animal models are powerful tools for the understanding of tumor development and cancer stem cell (CSC) origin. C3-1-TAg transgenic mice are a model for human triple negative breast cancer. Although general patterns of tumor development in this model have been well characterized, the tumor initiation cells of this model have not been defined. The objective of this study was to characterize the stemness and growth properties of mammary tumor cells in context with mammary epithelial cell (MEC) subpopulations of C3-1-TAg transgenic mice to identify the origin of tumor initiation cells. We first isolated MECs from mammary tissues of C3-1-TAg mice at various ages (8, 17, and 23 weeks old) and primary tumor cells from tumors of this model. Flow cytometry analysis with CD24 and CD49f as cell markers showed that cells from mammary tissues were separated into three subpopulations, including luminal, basal, and stromal populations. We found that the percentage of luminal subpopulation increased with age. Analysis of tumor derived cells using the same markers indicated that the majority of the cells in the plots overlapped with the position of luminal epithelial cells, suggesting their mutual connections. Next, we performed FACS sorting of mammary epithelial cells from the glands based on CD24 and CD49f and obtained four subpopulations of cells, including P1(stromal), P2(luminal), P3(CD24high/CD49fhigh, top of basal population on the plot), and P4(CD24mid/CD49fhigh, lower part of basal population), followed by the characterization of each subpopulations. Colony formation cell (CFC) assay and mammosphere assay were used for evaluation of luminal progenitor cells and self-renewal potential, respectively. Data from CFC assays showed that CFC colony numbers were the highest from the P2 population, followed by a few colonies from P3 population. Mammosphere assay showed that cells from P1 population formed typical mammospheres, while as cells from P2 and P3 formed cellular clusters different from typical mammospheres. FACS sorting with cells from C3-1-TAg mammary tumors obtained P2 and P3 subpopulations. Cells from P2, not P3, subpopulation were efficient in mammosphere formation. Because cells in P2 population are luminal mammary epithelial cells and are enriched with luminal progenitor cells, integration of data above suggest that C3-1-TAg mammary tumor cells are mainly derived from luminal progenitor cells. Using CD61, which is a luminal progenitor cell marker, we found that the percentage of luminal progenitor cells (CD61+/CD49fhigh) in the mammary glands was increased with age, and most tumor derived cells were CD61+/CD49fhigh. Taken together, our data demonstrate that the tumor initiation cells in the C3-1-TAg mammary tumors were derived from luminal progenitor cells. These findings lay a foundation for further characterization of cellular basis and molecular analysis of tumorigenesis in C3-1-TAg transgenic models.

#1169

Characterization of a small molecule inhibitor for GD3 synthase (ST8SIA1), a novel target in breast cancer stem-like cells.

Appalaraju Jappupilli,1 Khoa Nguyen,2 Stanley Ly,2 Michael Andreeff,2 Prashen Chelikani,1 Venkata Lokesh Battula2. 1 _University of Manitoba, Manitoba, Canada;_ 2 _UT MD Anderson Cancer Ctr., Houston, TX_.

We reported that GD2 selectively identifies breast cancer stem-like cells (BCSCs) in TNBCs and that the enzyme ST8SIA1 regulates GD2 biosynthesis. We have shown that knockout of ST8SIA1 expression in TNBC cell lines inhibits tumor growth and metastasis in vivo, whereas ST8SIA1 overexpression induces epithelial-to-mesenchymal transition, leading to metastasis of TNBC cells. Very recently we have shown that ST8SIA1 regulates focal adhesion kinase (FAK)/AKT/mammalian target of rapamycin (mTOR) signaling in GD2+ BCSCs. Here we hypothesize that inhibition of ST8SIA1 activity using small molecule inhibitors targets BCSC function. Because of a lack of a crystal structure for developing small-molecule inhibitors of ST8SIA1, we developed a homolog of ST8SIA1 using the crystal structure of ST8SIA3 (PDB ID: 5B09) as a template. ST8SIA3 shares 32% identity and 54% similarity with this crystal structure. To this model we docked GM3, the substrate for the ST8SIA1 enzyme and analyzed their interactions. Next, we used this model to dock the compound library to identify potential inhibitors of ST8SIA1. To that end, we performed structure-based virtual screening or virtual ligand screening of 10,000 compounds based on the structure of GM3 (>70% identity), which we retrieved from the ChemSpider database. Similarly, we retrieved 1500 compounds based on the parent structure of flavone (>70% identity) from the ZINC database. After further filtration and selection of these structures, we ranked these compounds on the basis of 11 different scoring functions in DS 4.5. We selected the top candidate (ZINC02886919) for further functional characterization. ZINC02886919 had a predicted binding affinity (-logKd) of 6.17, with which we calculated the predicted Kd as 0.812 μM. Visualization analysis demonstrated that ZINC02886919 is involved in both polar and hydrophobic interactions in the binding pocket. We then synthesized ZINC02886919 and pursued pharmacological and in vitro assays. Treatment of SUM159 cells with ZINC02886919 decreased the percentage of GD2+ BCSCs by 5- to 10-fold within 72h in a concentration-dependent manner (from 31% to 6%). In addition, ZINC02886919 inhibited soft agar colony and mammosphere formation for SUM159 cells by 10- to 20-fold. These data indicated that the ST8SIA1 homolog model that we developed shares structural similarity with native human ST8SIA1 protein and that ZINC02886919 is a potential inhibitor of ST8SIA1 activity and thus BCSC function. In conclusion, inhibition of ST8SIA1 expression or activity inhibits BCSC function, TNBC tumor growth, and metastasis. The ST8SIA1 inhibitor ZINC02886919 inhibits GD2 expression and mammosphere and soft agar colony formation in vitro. The effect of ZINC02886919 on tumor growth and metastasis is currently being tested in vivo.

#1170

Designing a biomarker-based bedside tool-kit for the clinical evaluation of bladder cancer.

Isha R. Dabke,1 Richard Pearce,2 Georgios Kallifatidis,2 Balakrishna Lokeshwar2. 1 _Medical College of Georgia at Augusta University, Augusta, GA;_ 2 _Georgia Cancer Center, Augusta, GA_.

Cancer of the urinary bladder (UBC) ranks second in incidence and mortality among the genitourinary cancers causing over 16,000 deaths annually. Currently, the precise diagnosis of bladder cancer involves cystoscopy and cytology, both expensive and painful procedures. Morphologically, malignancies of the bladder can be divided into two subtypes: luminal and basal, which express distinct cytokeratin and stem cell markers and have differing sensitivities to therapy. Although diagnosis based on molecular signatures has the potential to be effective, a verification of their expression in strictly compartmentalized epithelial subtypes is not presently available. The implications of this project are the formation of a bedside tool-kit based on prognostic stem cell biomarkers that can be used following biopsy to efficiently evaluate cancer subtype and provide a preliminary diagnosis.

Towards that goal, we analyzed mRNA expression profiles in five established bladder cancer cell lines (RT-4, 5637, T24, HT-1376, 253J), ranging from a grade I (RT-4) to a grade IV cancer cell line (253J). Based on existing literature, these cell lines were classified into either the luminal or basal subtype of bladder cancer. Through RT-PCR and Western Blot studies, genomic expression levels of eleven biomarkers (luminal: UPK, GATA-3, RAB-25, E-Cadherin; and basal: CK-6, p63, CD44S, CD44V, CyclinB1, EGFR, CD49) were analyzed in these cell lines.

Cell viability following exposure to two chemotherapy drugs: Gemcitabine and Cisplatin, commonly used to treat bladder cancer, were assayed by colorimetric and clonogenic colony-forming assays. Literature states that though basal cancers are more aggressive, they are paradoxically more sensitive to chemotherapy compared to luminal UBC.

Less aggressive/luminal UBC cell lines showed higher mRNA levels for luminal biomarkers, and expressed GATA-3 100% of the time. The more invasive/basal cell lines showed only a slight upregulation of basal biomarkers, but expressed CD44S 100% of the time.

Furthermore, MTT Assays showed a significant toxic effect of Gemcitabine on basal cell lines earlier (t=24 hr vs 48/72 hr) than luminal cell lines. However, Cisplatin did not show a significant difference in luminal vs. basal UBC suggesting that the two subtypes of UBC have similar sensitivity to Cisplatin.

The study presented here suggests that a PCR-based, biomarker toolkit can aid in an efficient classification of bladder cancers at the bedside, and treatment can be personalized based on the UBC subtype.

#1171

Identifying small molecules that specifically inhibit glioma stem cells.

Anthony R. Sloan, Raffaella Spina, Dillon M. Voss, Sophie Laye, Eli E. Bar. _Case Western Reserve University School of Medicine, Cleveland, OH_.

Glioblastomas (GBM) are the most common primary malignant brain tumor in adults and are recognized as one of the deadliest forms of cancer. Despite aggressive therapy consisting of maximal surgical resection followed by concomitant radiation and temozolomide (TMZ) chemotherapy, GBM remains to have a median survival time of ~ 15 months. Glioblastoma stem cells (GSCs) are suggested to play a critical role in the GBM phenotype; GSC have tumor initiation ability, propagation ability, and have been shown to be a driver in promoting GBM resistance to current therapeutic interventions. Due to these poor clinical outcomes and resistance to current treatments, more potent and preferably more specific pharmacological therapies are needed in order to target cellular-molecular pathways to eradicate GSCs and effectively treat GBM. In an effort to identify agents that specifically inhibit GSC growth, we recently conducted a drug screen of over 3,000 small molecules. HSR020913 patient derived neurospheres were treated for 72 hours with library agents at a concentration of 10 µM and cell growth was observed for a period of 5 days. We identified twelve highly potent compounds in the primary screen that inhibited the growth of HSR020913 neurospheres by over 50%. Subsequently, the potency of these compounds was tested on five additional GBM patient derived neurosphere lines (HSR040622, HSR040822, CCF3691, CCF3832, and CCF08-387) at even lower concentrations. We next sought to determine the specificity of these compounds in killing GSCs but not normal cells. To this aim we tested these compounds against immortalized human neural stem cells (v-Myc hNSCs) and normal human astrocytes (NHA). Of the twelve potent compounds identified in the primary screen, three compounds (AGSC9, AGSC11, AGSC12) showed to have negligible to minimal effect, only at higher doses, on normal neural stem cells. The respective half maximal inhibitory concentration (IC50) was determined for all the neurosphere lines utilized. Subsequent studies determined the inhibitory effect of these compounds on clonogenic capacity and enzymatic activity. Future studies are warranted to identify molecular pathways targeted by these compounds and determine their effect on tumor progression and survival in vivo.

#1172

JAK2V617F-mutant megakaryocytes contribute to stem cell aging in myeloproliferative neoplasms.

Helen Wong,1 Melissa Castiglione,1 Molly Quan,2 Huichun Zhan1. 1 _Stony Brook University, Stony Brook, NY;_ 2 _Wellesley College, Wellesley, MA_.

The myeloproliferative neoplasms (MPNs) are stem cell disorders characterized by hematopoietic stem cell (HSC) expansion and an increased propensity to develop hematologic malignancies. The acquired kinase mutation JAK2V617F plays a central role in MPNs. In this work we tested the hypothesis that the JAK2V617F-bearing megakaryocytes (MKs), an important component of the hematopoietic niche, promotes HSC dysfunction in MPNs.

Methods

JAK2V617F Flip-Flop (FF1) mice and Pf4-Cre mice (Radek Skoda, Switzerland) were crossed to generate MK lineage-specific human JAK2V617F knock-in mouse line (Pf4/FF1). Animal experiments were performed in accordance with the Institutional Animal Care and Use Committee guideline.

Results

We previously reported that, at 28 wks of age, the Pf4/FF1 mice developed a murine myeloproliferative syndrome with moderate thrombocytosis, splenomegaly, increased marrow MKs, and a 3-fold increase of CD45+CD201+CD150+CD48- HSCs with increased donor engraftment in a competitive repopulation assay compared to Pf4-cre control mice. (Zhan Leukemia 2016; Zhang Stem Cells 2018) Rigorous, extremely sensitive RT-PCR assays eliminated the possibility that HSCs in Pf4/FF1 mice express the mutant kinase.

To study the effect of JAK2V617F-bearing MKs on HSC function during aging, we followed the Pf4/FF1 mice up to 2 years of age. At 2 yr, there were significant neutrophilia in Pf4/FF1 mice but no change was observed in hemoglobin, platelet count, or marrow HSC cell numbers compared to age-matched control mice. Indeed, while HSC numbers significantly increased in old (2-yr) control mice compared to young (6-mo) control mice (3.3-fold, p=0.0002, n=4-8), there was no significant increase of HSCs between old and young Pf4/FF1 mice (1.6-fold, p=0.158, n=6-8). A competitive marrow transplantation experiment was performed in which CD45.2 donor marrow cells from 2-yr old Pf4/FF1 or control mice were injected intravenously together with competitor CD45.1 wild-type marrow cells intro lethally irradiated wild-type recipient (CD45.1) (n=8-9 in each group). During a 4-month follow up, old HSCs from the Pf4/FF1 mice displayed hallmarks of murine hematopoietic aging including a lower peripheral blood donor chimerism, increased myeloid blood cell production, and decreased lymphoid cells compared to old HSCs from control mice. To begin to understand how JAK2V617F-bearing MKs promote the aging phenotype of HSCs in old Pf4/FF1 mice, qRT-PCR of purified HSCs identified up-regulation of p21 (2-fold, p = 0.019) in Pf4/FF1 mice compared to controls.

Conclusion

The Pf4/FF1 mice, in which the JAK2V617F expression is restricted to MKs, developed a myeloproliferative phenotype at young age and accelerated stem cell aging at old age. The precise mechanism by which JAK2V617F-bearing MKs affect the MPN marrow niche and stem cell dysfunction during disease progression deserve further investigation.

#1173

A novel high throughput platform for head & neck cancer organoids drug screening.

Karla Queiroz,1 Else Driehuis,2 Silvia Bonilla,1 Henriëtte Lanz,1 Hans Clevers,2 Jos Joore,1 Paul Vulto1. 1 _Mimetas, Leiden, Netherlands;_ 2 _Hubrecht Institute, Utrecht, Netherlands_.

Head and neck (HN) cancer is a broad category of tumor types arising from various anatomic structures including the craniofacial bones, soft tissues, salivary glands, skin, and mucosal membranes. Treatment often involves an intensive combination of surgery, radiotherapy and chemotherapy. Despite this, tumour recurrence rates remain high and survival rates are relatively poor. Here, we describe a novel high throughput drug screening platform combining the OrganoPlate, a microfluidic based 3D culture plate, and HN cancer-derived organoids. MIMETAS develops Organ-on-a-Chip-based models for evaluation of new medicines. Our unique microfluidic technology enables testing of compounds on miniaturized 3D organ models in high-throughput. These models are expected to show better predictivity as compared to laboratory animals and conventional 2D cell culture models, without compromising throughput or ease of use. Hereby we show the establishment of HN cancer-derived organoids in 2-lane OrganoPlate, and its usefulness for phenotypic drug screenings. The aim of the study is to evaluate the 2-lane OrganoPlate as a platform for growing HN cancer organoids and drug screening. Organoid lines (T2, T3 and T4) were embedded in the 2-lane OrganoPlate as single cells in an Extracellular Matrix gel. At day 3, cultures were treated with Cisplatin or Carboplatin for 120 hours. Drug response was evaluated by assessment of morphology (phase contrast), Cell Viability (Alamar blue) and proliferation (EdU incorporation). Organoids cultures grow well under perfusion in the 2-lane OrganoPlate and different sensitivity to cisplatin is captured by the used readouts. The high-throughput, microfluidic 2-lane OrganoPlate platform offers an attractive method for growing HN cancer-derived organoids, supporting development of individualized tumour models for phenotypic drug screenings.

### Tumor-Immune Interactions

#1174

Tumor-specific EphA2 receptor tyrosine kinase inhibits anti-tumor immunity by recruiting suppressive myeloid populations in non-small cell lung cancer.

Eileen Shiuan, Wenqiang Song, Shan Wang, Mark Boothby, Jin Chen. _Vanderbilt University, Nashville, TN_.

Background: Given the success of both targeted and immunotherapies against cancer, there is increasing utility for identifying targeted agents that also promote anti-tumor immunity. EphA2 is a receptor tyrosine kinase that contributes to tumor growth and metastasis in various cancer types and has been identified as a viable target for non-small cell lung cancer (NSCLC). Here, we examine how tumor-specific EphA2 affects the infiltration of different immune populations, the activation or inhibition of those populations, and the cytokine and chemokine milieu in mouse models of NSCLC.

Methods: We generated two murine NSCLC cell lines, one derived from Lewis Lung Carcinoma and the other derived from a primary lung tumor harboring a KRAS mutation, as well as p53 and LKB1 loss of function alterations created using CRISPR-Cas9 gene editing. Effects of EphA2 overexpression in these cells were evaluated in both in vitro assays and in vivo models via subcutaneous tumor implantation and tail vein-injected orthotopic tumor formation in immunocompetent, syngeneic mice. Tumor immune infiltrate was assessed by flow cytometry, and cytokine and chemokine RNA expression levels were evaluated using Nanostring's PanCancer Immune Profiling Panel.

Results: Although EphA2 overexpression in these cell lines did not display significant proliferative advantage in vitro in MTT and colony formation assays, it did confer a growth advantage in vivo. Analysis of lung tumor immune infiltrate revealed decreased NK and T cells in the EphA2-overexpressing tumors, as well as increased myeloid populations, such as macrophages and monocytes. Furthermore, T cell activation markers, such as CD25, CD44, and CD69, in both CD4 and CD8 T cells were decreased in the EphA2-overexpressing tumors, while the percentage of regulatory T cells was increased. These changes in T cell activity were accompanied by increased presence of monocyte-derived CD11b+ Ly6C+ Ly6G- cells and Gr1- F4/80+ MHCII+ tumor-associated macrophages (TAMs). Higher infiltration of these myeloid populations coincided with increased monocyte-attracting chemokine and receptor expression, including CCL2, CCL7, CCL8, CCL12, and CCR2 and increased expression of immunosuppressive proteins, including TGFβ and arginase 1.

Conclusion: Our studies suggest tumor-specific EphA2 inhibits infiltration and activation of key lymphocytic populations, especially T cells, while recruiting monocytes and likely promoting their transformation into myeloid-derived suppressor cells (MDSCs) and TAMs. Further functional studies are needed to validate the role of these myeloid cells. Elucidation of EphA2's role in regulating immune recruitment and function will advance our understanding of tumor immune evasion and the potentially beneficial consequences of targeting EphA2 in cancer on the tumor microenvironment.

#1175

A pro-tumorigenic mechanism of M2 tumor-associated macrophages in triple-negative breast cancer.

Anna Juncker-Jensen, Nicholas Stavrou, Raghav Padmanabhan, Erinn Parnell, Judy Kuo, Eric Leones, Flora Sahafi, Josette William. _NeoGenomics, Aliso Viejo, CA_.

Introduction:

Triple-negative breast cancers (TNBCs) are defined as tumors that are negative for both estrogen, progesterone and HER-2 receptors. It is a heterogeneous subtype of breast cancer with a high propensity for systemic metastases and poor survival with only chemotherapy available for treatment. Compared to other hormone-positive breast cancer subtypes, TNBC features a unique tumor microenvironment (TME) characterized by a large number of tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) (1), and the increase of immune infiltrate with high levels of TILs predicts response to neoadjuvant chemotherapy as well as improved survival.

TAMs can be described as classically activated M1 types with pro-inflammatory antitumor functions, versus alternatively activated M2 types with immunosuppressive pro-tumor functions. It has been reported that the presence of M2 TAMs positively correlates with TNBC, hormone receptor negativity, as well as higher tumor proliferation (2-3). However, the mechanisms by which TAMs interact with TNBC cells and their phenotypes in TNBC versus estrogen receptor positive (ER+) patients is not well understood.

Experimental Procedures:

Using MultiOmyx, a proprietary, immunofluorescence (IF) multiplexing assay that enables visualization and characterization of up to 60 biomarkers on a single FFPE section (4), we have characterized TIL phenotypes, tumor proliferation and TAM activation in 15 FFPE tumors from TNBC and 5 tumors from ER+ patients.

Results:

Using a multiplex panel of 9 markers we found a 10-fold higher number of TILs and a 5-fold higher number of proliferating tumor cells in TNBC versus ER+ tumors. Furthermore, the proportion of T helper cells (CD3+CD4+) that were Tregs (CD3+CD4+FoxP3+) was increased from 26% to 43% in TNBC tumors. Interestingly, when analyzing TAMs we found that while ER+ tumors had a significantly higher proportion of M1 TAMs (CD68+HLA-DR+) than M2 TAMs (CD68+CD163+), this expression pattern was reversed in TNBC. When analyzing a possible correlation between the activation state of TAMs and the proliferation of tumor cells, we found a positive significant correlation between the presence of M2 TAMs and proliferating tumor cells (Pearson's correlation p<0.05), only in TNBC and not in ER+ tumors. Conversely, the presence of M1 tumors showed a weak negative correlation to tumor cell proliferation, in both ER+ and TNBC tumors.

Conclusion:

These data are suggestive of a possible pathway in which an increase in alternatively activated immunosuppressive M2 TAMs in hormone receptor negative breast cancer tumors, are responsible for providing a suitable environment for tumor proliferation.

References:

1. Yu T & Di G, Chin J Cancer Res 2017;29(3):237.

2. Medrek C et al., BMC Cancer 2012;23(12):306.

3. Klingen TA et al., Hum Pathol 2017;(69):72.

4. Gerdes MJ et al. PNAS 2013;(110):11982.

#1176

Immunogenomic profiling identifies a subgroup of squamous cell lung cancers with immunosuppressed tumor microenvironment and correlates TGF-beta and Wnt/beta-catenin signaling as predictive of low PD-L1 expression.

Marcelo V. Negrao, Tatiana Karpinets, Jun Li, Alexandre Reuben, Cara Haymaker, Kyle G. Mitchell, Junya Fujimoto, Chi-Wan Chow, Edwin R. Parra, Lorenzo Federico, Jianhua Zhang, Ara A. Vaporciyan, Chantale Bernatchez, Tina Cascone, Boris Sepesi, Ignacio I. Wistuba, John V. Heymach, Jianjun Zhang, Don L. Gibbons, ICON Team. _MD Anderson Cancer Center, Houston, TX_.

Introduction: immune checkpoint inhibitors have revolutionized the treatment of non-small cell lung cancer (NSCLC), but only ~15% of patients will achieve durable clinical benefit. Our group and others have focused on characterizing the interaction between tumor genomic characteristics and immune microenvironment. Genes such as PTEN, CTNNB1, STK11 and KEAP1 have been associated with intra-tumor immune depletion, but a broader understanding of other key driver genes of NSCLC and their interplay with the immune microenvironment is currently lacking. To prospectively delineate the genomic and immune landscape of NSCLC, we launched the ICON Project (Immunogenomic Profiling of NSCLC).

Methods: we prospectively collected tissue specimens from patients with stage I-IIIA NSCLC undergoing surgical resection. Samples were subjected to extensive immune-genomic profiling, including whole-exome and RNA sequencing, multiplex immunofluorescence (mIF), and flow cytometry.

Results: from 2016-2018, 150 patients were accrued, with 78 samples having undergone genomic profiling. Most patients had stage I-II disease (74%) and non-squamous histology (70%). Most commonly mutated genes were TP53, EGFR, KRAS, CDKN2A, STK11, and mean TMB was 7.6 mut/Mb, which is comparable to TCGA (6.9mut/Mb, p=ns). Unsupervised clustering according to expression of CD8+ T cell related genes segregated tumors into immune-enriched (clusters 1 and 3) and immune-depleted (clusters 2 and 4) subgroups. We validated these findings through flow cytometry (CD8+GzB+ T cells) and mIF (CD8), with both methods showing CD8+ T cell infiltration to be enriched in clusters 1 and 3. Cluster 1 included squamous and non-squamous histology, tumors with epithelial and mesenchymal signatures, and high TMB. Cluster 2 included exclusively squamous histology with predominant mesenchymal tumor signature, high TMB, and was enriched for TP53 mutations. Cluster 3 had predominantly non-squamous histology, epithelial signature, and low TMB. Cluster 4 had exclusively non-squamous histology, predominant epithelial signature, low TMB, and was enriched for KEAP1 mutations. In addition, PD-L1 expression (by mIF) was negatively correlated with TGF-beta and WNT/Beta-catenin signaling pathway, as well as high EGFR and FGFR2 expression on malignant cells.

Conclusion: CD8+ T-cell gene signature identifies immunosuppressed subgroups of lung cancer, especially among squamous cell carcinomas with a mesenchymal signature, that may be associated with resistance to immune-based therapies. TGF-beta and Wnt/beta-catenin pathways are associated with an immunosuppressed tumor microenvironment, and may be potential novel targets to restore anti-tumor immunity in lung cancers with low PD-L1 expression.

#1177

Mechanical strain induces phenotypic changes in breast cancer cells and promotes immunosuppression in the tumor microenvironment.

Yong Wang, Paige E. Severino, Kayla Goliwas, Kenneth Hough, Derek Van Vessem, Hong Wang, Andra R. Frost, Selvarangan Ponnazhagen, Joel L. Berry, Jessy S. Deshane. _Univeristy of Alabama at Birmingham, Birmingham, AL_.

Introduction: Cells within the breast tumor mass are met with a variety of biophysical or mechanical signals that is associated with elevated compression or solid stress within the tumor interior, tension at the tumor periphery, and altered interstitial fluid pressure. Understanding how the mechanical stress within the tumor microenvironment (TME) regulates cancer cell phenotype is of interest, yet the response of breast cancer (BCa) cells to these forces is largely unknown. Our study aims to identify the impact of mechanical stress on BCa cell phenotype by mimicking the tension at the tumor periphery.

Materials and Methods: BCa cells (MCF-7 or 4T1.2) were cultured to confluence on collagen coated FlexCell culture plates. These plates were then subjected to 10% uniaxial cyclic/oscillatory strain at 0.3 Hz, or 10% constant strain, or no strain for 48 hours. Strained or control cells were isolated for analysis of proliferation (MTT assay), and migration (8.0 μm pore transwell). Exosomes from conditioned media were isolated via differential centrifugation and purified exosomes were characterized by ImageStream. 5x105 4T1.2 cells or PKH-labeled strained or control cells were injected into the mammary fat pad of BALB/c mice. Tumor volume was measured over 14 days. Tumor-infiltrating immune cells and the uptake of exosomes were analyzed by flow cytometry on day-14 post implantation.

Results: We determined significant increases in proliferation and migration of 4T1.2 and MCF-7 cells in vitro following exposure to oscillatory forces. The populations of CD63+, CD63+CD24+ and CD63+PD-L1+ exosomes were increased when 4T1.2 cells were exposed to oscillatory strain compared to unstrained control cells. Further, we investigated how oscillatory forces affect tumor growth and tumor-immune cell interactions in vivo by using a syngeneic, orthotopic mouse model of BCa. Mice implanted with 4T1.2 cells that were pre-exposed to oscillatory forces showed a significant increase in primary tumor growth at 8 and 11 days post tumor challenge. The percentages of tumor-infiltrating monocytic myeloid-derived suppressor cells (M-MDSC) and recruited macrophages were increased in the TME of mice implanted with 4T1.2 cells that were pre-exposed to oscillatory forces, while the granulocytic MDSC subset was not significantly different between the two groups. A marginal decrease in the percentage of CD8+ T cells was noted in the TME of mice implanted with strained 4T1.2 when compared to controls, suggesting immune suppression in the TME. Furthermore, exosome uptakes by M-MDSC and recruited macrophages were increased in the TME of mice implanted with PKH-labeled 4T1.2 cells, exposed to oscillatory strain.

Conclusion: Together, these data indicate that exposure to mechanical stress changes BCa cell phenotype to an invasive and protumorigenic phenotype that promotes immunosuppressive effects in the TME.

#1178

Image-based quantification of tumor-immune cell interactions in 3D cultures.

Gera Goverse, Nataliia Beztsinna, Kuan Yan, Leo Price, Lidia Daszkiewicz. _OcellO, Leiden, Netherlands_.

INTRODUCTION Increasing numbers of cancer patients that benefit from immunotherapies and maintain durable response requires understanding of cellular mechanisms that govern anti-tumor immune responses. However, the mechanisms of action of these treatment modalities are not fully understood and the progress in this direction is hampered by a lack of appropriate pre-clinical testing models that are both clinically relevant and suitable for routine screening of drug candidates. Therefore, we developed a robust in vitro assay that allows image based analysis of 3D cultures in a high-throughput set-up. Here, immune cells are co-cultured with cancer cells in a 3D environment which recapitulates the tumor micro-environment and its complex cellular interactions. Functional read-outs, such as active migration of immune cells towards tumoroids, infiltration of immune cells into the tumoroids and their killing lead to a better understanding of the immune-modulatory profile of different immunotherapies.

MATERIAL and METHOD Tumoroids generated from cancer cell lines (e.g. breast, prostate) and colorectal cancer organoids (from HUB Organoid Technology) were cultured in protein hydrogel. Different immune cells, such as PBMCs, T cells, dendritic cells or macrophages, with and without activation were added to the 3D culture and their infiltration into tumoroids and subsequent killing was visualized using high-content microscopy. Quantification of immune cell effects was achieved with morphometric analysis with OMinerTM software.

RESULTS and DISCUSSION Image-based analysis enabled the discrimination of immune-tumor cell interactions in 3D cultures. These results demonstrated the effect of immune cell targeting on the tumor progression. Different levels of immune cells infiltration and killing of tumoroids and organoids were measured depending on the activation status of immune cells. The 3D environment, both for the cell culture and image analysis, allows for measurement of spatially resolved information, not accessible by monolayer cultures or biochemical assays.

CONCLUSION Our image-based platform described here allows for analysis of immunotherapy effects on different cell types that engage in a more physiologically relevant spatial setting than when culturing them in traditional 2D cultures. Visualization and quantification of these tumor-immune cells interactions offer a highly powerful tool for cancer immunotherapy drug developers to understand the mechanism of action of their treatments and ultimately translating to a better clinical performance.

#1179

Deep spatial immuno-profiling through high biomarker colocalization in FFPE tumor tissue sections.

Abdul Mohammed, Douglas Wood, Mael Manesse. _Ultivue, Cambridge, MA_.

Background: The recent emergence of highly multiplexed immunohistochemistry (IHC) has the potential to revolutionize immuno-oncology and pathology research as it enables the identification of complex cell subtypes and their potential interactions in the tumor environment. Comprehensive classification of the different cell types in immuno-oncology presents a unique challenge as many of the relevant biomarkers are common to large subsets of cell types within a particular cell class. Therefore, a key feature for accurate phenotypic classification is the ability to detect a high number of colocalized biomarkers. Current IHC technologies offer a low subtyping level due to steric constraints, spectral overlap between dyes, or depletion of deposition sites. Alternative strategies using mass- or cleavable oligonucleotide- tags can potentially achieve high colocalization but are hindered by their low-throughput, high-cost, and elaborate post-acquisition image reconstruction. In this study, we demonstrate how the Ultivue® InSituPlex® technology enables researchers to reliably label and detect at least 4 markers in the same compartment (cytoplasmic or nuclear) on single cells in tumor tissue samples.

Methods: Different FFPE tumor sections were labeled, imaged, and profiled for 15 targets using InSituPlex technology. Staining was carried out in a single workday on a Leica BOND RX autostainer using a cocktail of the 15 primary antibodies relevant to immuno-oncology. Commercially available multi-color fluorescence slide scanners were used to obtain whole slide images. The resulting images were then segmented and analyzed for different phenotypes and their spatial relationships using Indica Labs HALO® software. Additional dimensionality reduction algorithms were used to visualize the high-dimensional phenotypic data of cells with many overlapping biomarkers.

Results: In this poster, we demonstrate the detection of at least 4 co-localized biomarkers on the same cell in FFPE tissue sections. Analysis of the high dimensional, spatially resolved data obtained from a 15-plex assay provided phenotypic information of different lymphocytes, macrophages, dendritic cells, antigen presenting cells, and tumor cell populations with multiple colocalized biomarkers.

Conclusions: InSituPlex technology empowers pathology research through colocalization analysis of many markers on single cells over entire sections of tissue for accurate differentiation and subtyping of cell populations in the tumor microenvironment. As exploratory research in immuno-oncology expands, advancements in multiplex spatial profiling technologies promise to offer greater insights by providing a systems-level view of the biology.

#1180

Impact of the spatial analysis of tumor-associated lymphocytes and tumor-associated macrophages on recurrence at early stage of non-small cell lung carcinoma.

Alejandro Francisco-Cruz,1 Edwin R. Parra,1 Santhoshi N. Krishnan,2 Souptik Barua,2 Mei Jiang,1 Junya Fujimoto,1 Christine B. Peterson,1 Priyam Das,1 Chi-Wan Chow,1 Jaime Rodriguez-Canales,1 Carmen Behrens,1 Neda Kalhor,1 Annikka Weissferdt,1 John Heymach,1 Stephen Swisher,1 Boris Sepesi,1 Arvind Rao,3 J. Jack Lee,1 Cesar Moran,1 Andrew Futreal,1 Jianjun Zhang,1 Ignacio I. Wistuba1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Rice University, Houston, TX;_ 3 _University of Michigan Ann Arbor, Ann Arbor, MI_.

Introduction. The interaction between malignant cells (MCs), stromal cells, tumor-associated lymphocytes (TILs), and tumor-associated macrophages (TAMs) is relevant for non-small cell lung carcinoma (NSCLC) progression. The spatial distribution of those cells may affect the prognosis and can be related to genetic intra-tumor heterogeneity (ITH). The aim of this study was to characterize the immunologic ITH and the spatial distribution of immune cells to MCs in primary NSCLC tumors at early stages using multiplex immunofluorescence (mIF) and image analysis approaches.

Material and methods. We studied 33 surgically resected NSCLC cases (adenocarcinomas=23; squamous-cell carcinomas=10) with a history of recurrence in a follow-up of at least 60 months (recurrence, N=13; non-recurrence, N=15). Consecutive FFPE tissue sections were stained with two mIF panels (panel 1: cytokeratin (AE1/AE3), PD-L1, PD-1, CD3, CD8, and CD68; panel 2: AE1/AE3, CD3, CD8, granzyme-B, CD45RO, and FOXP3). Three intra-tumor regions (3mm2 each) per case were selected after gridding the whole tumor section. A total of 99 intratumor regions were scanned and analyzed using Vectra Multispectral-Microscope and InForm-software. From each intratumor region, TILs and TAMs densities, as well as the coefficient of variation, were evaluated. The median distance and the G-Cross area under the curve (AUC) for specific radial distances (10µm, 20µm, and 40µm) were obtained between TILs and TAMs phenotypes to MCs.

Results. Recurrence was associated with higher MCs density and TAMs/TILs ratio, and lower TIL densities. A high ITH of cytotoxic T-cells (CTLs) PD-L1+ was associated with worse survival. The distance of TAMs PD-L1+ to MCs PD-L1 negative (60µm vs 25µm) or to MCs PD-L1 positive (25µm vs 13µm) was higher in the non-recurrence group than in recurrence group. Close TAMs PD-L1+ to MCs was associated with worst survival. In a radial distance of 10µm, 20µm, and 40µm, a higher infiltration of CTLs PD-1+, was observed in the group of recurrence than non-recurrence group, surrounding MCs PD-L1 negative (AUC 0.49, 3.80, and 20.03; vs AUC 0.01, 0.16, and 1.29, respectively), and MCs PD-L1 positive (AUC 0.60, 4.35, and 19.90; vs AUC 0.01, 0.20, and 2.20, respectively). A high infiltration of CTLs PD-1+ surrounding MCs, with or without expression of PD-L1, was associated with worse survival. All the differences were statistically significant (P<0.05).

Conclusion. Close spatial proximity of antigen-experienced CTLs and TAMs PD-L1+ to MCs are associated with recurrence and poor survival in early stages of NSCLC. We determined that ITH of immune cell densities is associated with recurrence of surgically resected NSCLC. Tumor-immune cell spatial modeling offers a deep understanding of tumor microenvironment that impacts on clinical outcomes. Supported by CPRITRP160668 and UT Lung SPORE grants

#1181

In-depth validation of UltiMapper™ I/O PD-L1 Assay.

Jamie K. Buell, Courtney Hebert, Aditi Sharma, Sean Downing. _Ultivue, Cambridge, MA_.

Background: Current PD-L1 Immunohistochemistry (IHC) assays utilizing conventional brightfield, chromogenic 3,3′-diaminobenzidine (DAB) staining modalities are the norm in both translational and clinical research. With the rise of immuno-oncology has come a need to fully assess the tumor microenvironment and multiple tissue biomarkers in the same sample applying advancements and acceptance of digital pathology in routine clinical applications. Ultivue has developed the UltiMapper I/O PD-L1 Assay, a fluorescent, multiplexed IHC assay that enables high-throughput and whole-slide data analysis of multiple tissue biomarkers in a single histology workday. This multiplexed IHC assay contains an antibody panel consisting of CD8, CD68, PD-L1, and CK/Sox10 markers. In this study we demonstrate the utility, robustness, and ability to derive meaningful biological insights by validating the accuracy, precision, sensitivity, and specificity of the UltiMapper I/O PD-L1 Assay.

Methods: Validation parameters included measurement criteria that indicate accuracy and precision of the UltiMapper I/O PD-L1 Assay. Accuracy was measured through the staining of CD8, CD68, PD-L1, and CK/Sox10 markers on a pathologist selected sample set. The sample set contains multiple primary tumor types (e.g. lung and melanoma) with various tissue morphologies in addition to standard control tissue samples (tonsil). Sample set images stained with UltiMapper I/O PD-L1 Assay were qualitatively assessed for accuracy through pathology review interpretation examining expected morphology and staining patterns. Precision was measured through multiple reproducibility staining runs of the UltiMapper I/O PD-L1 Assay on the diverse sample set including intra-run, inter-run, and inter-technician experiments to qualitatively and quantitatively determine marker staining concordance over the appropriate staining runs. Sensitivity and specificity of the UltiMapper I/O PD-L1 Assay were evaluated based on staining results utilizing standard DAB assays.

Results: Accuracy was confirmed by qualitative assessment across sample set with a high percentage of concordance. Analysis of intra-run, inter-run, and inter-technician runs revealed that cell counts were within a CV of <15% across all markers, in all tested tissues. Sensitivity and specificity demonstrated high degrees of confidence above 85%.

Conclusion: Results presented here indicate that UltiMapper I/O PD-L1 Assay passes accuracy, precision, sensitivity and specificity metrics. Histological staining standards for coefficients of variation in IHC-based assays are typically 15% and under. Data presented here falls below this threshold indicating the potential for use in a high throughput testing laboratory to aid in collection and interpretation of meaningful biological insights in order to aid further testing or decisions.

#1182

New bioluminescent strategies to measure kinetics of immune cell-mediated cytotoxicity.

Maggie L. Bach, Natasha Karassina, Peter Hofsteen, Donna Leippe, James Cali, Jolanta Vidugiriene. _Promega Corporation, Fitchburg, WI_.

The number of immunotherapeutic strategies for treating cancer have expanded in recent years, increasing the need for assays that can be used to assess their effectiveness. To facilitate studies of these immunotherapies, we developed bioluminescent methods that could rapidly and sensitively measure target cell cytotoxicity. Two approaches were developed. The first method utilizes a bioluminescent enzymatic activity assay for a well-accepted cytotoxicity marker, lactate dehydrogenase (LDH), which is rapidly released from dead cells. With the improved sensitivity of the bioluminescent assay, lower levels of LDH can be measured, resulting in the detection of fewer dead cells (LOD< 10 cells). Cytotoxicity in both 2D and 3D cell cultures can be monitored over time by removing samples of medium at different time points. Here we present examples in which this assay was used to test the effectiveness of immunotherapeutic treatments including antibody-drug conjugates (ADC) and antibody-dependent cell-mediated cytotoxicity (ADCC). In ADCC models, cell killing was detectable at early time points (by 2 hours). Increases in the cytotoxicity of individual samples could be followed over time, from early time points to 24 hours. In the second approach we developed a novel bioluminescent assay based on the specific labeling of target cells. With this method, intracellular proteins in live target cells are covalently labeled. Upon target cell death, the labeled proteins are released from the cell and the label is quantitated using bioluminescence detection reagents. Using this assay, we were able to quantitate the killing of target cells in both ADCC and CAR-T applications. By adding the detection reagents directly to the medium at the start of the ADCC reaction, cell death was monitored in real-time for up to 6 hours. Neither assay requires genetic engineering and both involve no or minimal target cell preparation. They are applicable to multiple cell types and the non-lytic nature of these approaches provides kinetic capabilities and opportunities for multiplexing. The ability to rapidly quantitate cytotoxicity will facilitate the functional analysis of immunotherapies.

#1183

**High-plex spatial profiling of whole FFPE tissue sections using InSituPlex** ® **technology for discovery applications.**

Abdul Majeed Mohammed, Zhixin Xia, Gourab Chatterjee, Kevin Hwang, Mael Manesse. _Ultivue, Cambridge, MA_.

Background: Comprehensive spatial phenotyping is an increasingly important pathology research tool for investigating the highly complex biology of tumors and their micro-environment. High multiplex immunohistochemical (IHC) strategies enable spatial profiling of tumor samples and expand the depth of information obtained from a single tissue section. Conventional IHC technologies overcome the limitations of low multiplexing by testing serial sections; however, this method does not provide evidence of true marker colocalization and results in inaccurate phenotyping. More recent multiplex methods involving iterative staining and stripping steps have been developed but suffer from low-throughput workflow and can potentially damage samples. Alternative strategies using localized release of mass- or oligonucleotide-tags are not only low-throughput but are also high-cost and require specialized instruments. In this study, we developed a highly streamlined 15-plex fluorescence IHC assay to identify and subtype lymphocytes, macrophages, dendritic cells, antigen presenting cells, and tumor cells on single FFPE tissue sections in a single work day.

Methods: InSituPlex® technology was used to perform high-multiplex immune profiling (15 targets) of different FFPE tumor sections. Slides were stained with a Leica® Bond®RX autostainer using a cocktail of 15 primary antibodies. Whole slide images were obtained using commercially available multi-color fluorescence slide scanners. The resulting images were then segmented and analyzed for different phenotypes and their spatial relationships using the Indica Labs HALOTM software. Tissue sections processed with single-antibody staining were used as references to characterize the performance of the 15-plex assay.

Results: We demonstrate a 15-plex immuno-fluorescence assay on whole FFPE tumor tissue sections in a single work day using the InSituPlex technology. Equivalence of assay performance between the 15-plex and individually stained reference samples confirmed assay specificity and sensitivity in the high multiplexed format. High-dimensional data obtained from the 15-plex assay allowed for a detailed sub-classification of various cell types and their spatial relationship on each single section.

Conclusions: InSituPlex technology empowers immuno-oncology research through the staining of 15 different immune and tumor markers on single FFPE tumor sections, with a fast, automated workflow and reproducible assay. The resulting high-dimensional images are then used to spatially profile various sub-populations of lymphocytes, macrophages, and tumor cells. This study demonstrates the promises of the InSituPlex technology for translational research and discovery applications where rapid imaging of a large number of biomarkers over the entire tissue is highly desired.

#1184

Comprehensive analysis with interactive exploration of immune response signatures in 10,000 tumor samples.

Vesteinn Thorsson,1 David L. Gibbs,1 Mary L. Disis,2 Elizabeth G. Demicco,3 Alexander J. Lazar,4 Jonathan S. Serody,5 James A. Eddy,6 Ilya Shmulevich,1 Justin Guinney,6 Benjamin G. Vincent5. 1 _Institute for Systems Biology, Seattle, WA;_ 2 _University of Washington, Seattle, WA;_ 3 _Mount Sinai Hospital, Toronto, Ontario, Canada;_ 4 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 5 _University of North Carolina, Chapel Hill, NC;_ 6 _Sage Bionetworks, Seattle, WA_.

In recent years, analysis of cancer genomics data using methods of immunogenomics has yielded valuable insight into how cancer cells interact with immune cells in the tumor microenvironment. A recent analysis of the multiple molecular platforms by The Cancer Genome Atlas (TCGA) of over 10,000 tumors comprising 33 cancer types identified six immune subtypes, spanning multiple tumor types, that are characterized by differences in: macrophage vs. lymphocyte signatures; Th1:Th2 cell ratio; extent of intratumoral heterogeneity; aneuploidy; extent of neoantigen load; signatures of cell proliferation; expression of immunomodulatory genes; and disease outcome [1]. Particular driver mutations correlate with variation in leukocyte levels across all cancers or with the fraction of individual immune cell types. Intracellular and extracellular networks (involving transcription, microRNAs, copy number and epigenetic processes) are predicted to play a role in establishing the observed tumor-immune cell interactions, both across and within immune subtypes. Additionally, machine learning methods have been applied to H&E images to extract information on which tissue regions contain tumor infiltrating lymphocytes (TILs), yielding TIL maps of whole slide images from digital pathology[2]. Spatial patterns of TILs are associated with a variety of genomic alterations, including cancer subtypes.

The CRI iAtlas (www.cri-iatlas.org) is a cloud-based platform for data exploration and discovery, allowing researchers to study TCGA immune response characterizations, and the relationships among them in individual tumor types, tumor subtypes, and immune subtypes. iAtlas supports the adaptive exploration of correlations within the cellularity of the tumor microenvironment, immune expression signatures, tumor mutation burden, cancer driver mutations, adaptive cell clonality, patient survival, and expression of key immunomodulators. iAtlas was launched in April 2018, and has since been expanded to include new capabilities such as (1) user-defined loading of cohorts, (2) a tool for classifying expression data into immune subtypes, (3) integration of TIL mapping from digital pathology images, and (4) addition of annotated genomics datasets from immunotherapy clinical trials as comparative data sources. As the resource evolves, we expect that the CRI iAtlas will help to accelerate discovery and improve patient outcomes by providing researchers greater access to immunogenomics data to better understand the immunological characteristics of the tumor microenvironment and its potential impact on patient responses to immunotherapy.

[1] Thorsson, V, et al., The Immune Landscape of Cancer; Immunity 48, p812 - 830.e14 (2018)

[2] Saltz, J et al. Spatial Organization and Molecular Correlation of Tumor-Infiltrating Lymphocytes Using Deep Learning on Pathology Images; Cell Reports 23 pp.181-193.e7 (2018)

#1185

A method for enhancing anti-tumor T-cell function.

Shane R. Horman. _GNF-Novartis, San Diego, CA_.

Overcoming tumor-mediated immunosuppression and enhancing cytotoxic T-cell activity within the tumor microenvironment are two central goals of immuno-oncology (IO) drug discovery initiatives. However, exploratory assays involving immune components are often plagued by low-throughput and poor clinical relevance. Here we present a novel ultra-high-content assay platform for interrogating T-cell-mediated destruction of 3D multicellular tumor spheroids. Employing this assay platform in a chemical genomics screen of 1,800 annotated compounds enabled the identification of novel small molecule perturbagens capable of enhancing cytotoxic CD8+ T-cell activity in an antigen-dependent manner. Specifically, cyclin-dependent kinase (CDK) and bromodomain (BRD) protein inhibitors were shown to significantly augment anti-tumor T-cell function. The described biotechnology screening platform yields multi-parametric, clinically-relevant data and can be employed kinetically for the discovery of novel IO therapeutic agents.

#1186

Regulation of inducible nitric oxide synthase at the single cell level modulates the inflammatory microenvironment.

Veena Somasundaram,1 Caroline Gilmore,2 Erika M. Palmieri,1 Debashree Basudhar,1 Will Heinz,3 Robert Y. Cheng,1 Lisa A. Ridnour,1 Stephen J. Lockett,3 Daniel W. McVicar,1 David A. Wink1. 1 _National Cancer Institute, Frederick, MD;_ 2 _Frederick National Laboratory for Cancer Research, Frederick, MD;_ 3 _Frederick National Laboratory for Cancer Research, Leidos Biomedical Research Inc. for the National Cancer Institute, Frederick, MD_.

The anti-cancer versus pro-tumor behavior of cancer tissues is dependent largely on tumor cell-macrophage interactions in the tumor/inflammatory microenvironment and is regulated by Nitric Oxide (NO). High inducible nitric oxide synthase (Nos2) is associated with poor prognosis in breast cancer. Previously, we found that murine macrophages can be activated by different inflammatory cytokines/LPS to produce distinct NO fluxes (entirely Nos2-derived), suggesting flux-specific biological ramifications of NO. However, the effects of these NO fluxes on M1 phenotype have not been delineated. LC/MS analysis of M1 stimulated, wild type (wt) and Nos2-/- macrophages showed that Nos2 was the only source of citrulline. Metabolic analyses and microscopy showed that flattened cell phenotype characteristic of M1 macrophages and mitochondrial respiration are Nos2 dependent and regulated in a NO flux-dependent manner, while proinflammatory cytokine profile and aerobic glycolysis are Nos2 independent. We show for the first time that induction of Nos2 expression occurs only in specific stimulated cells that also harbor depolarized mitochondria. NO production has been linked to decreased oxygen consumption in hypoxic environments. We utilized a novel, in vitro chamber system that forms cell-generated hypoxic and metabolic gradients in two-dimensions by restricting the diffusive exchange of oxygen and metabolites to a monolayer of cells in a small volume- analogous to diffusion between a capillary and nearby tissue. We investigated interactions between Nos2 in M1-stimulated macrophages and hypoxia and demonstrated that treatment with IFNγ+LPS increases Nos2 expression and alters the magnitude and spatial extent of hypoxic gradients. A modified scratch assay revealed that low doses (1-50μM) of NO increased and high doses (1000μM) inhibited the migratory capacity of 4T1 tumor cells. However, in vivo, Nos2-/- mice did not show difference in primary tumor or metastatic burden compared to wt mice but bone marrow derived macrophages (BMDMs) from wt tumor bearing mice produced significantly lower levels of NO compared to BMDMs from tumor bearing- Nos2-/- mice. In summary, we find that the right flux NO is required to tune the inflammatory microenvironment. Nos2 and citrulline are robust intracellular readouts of extracellular NO flux. Nos2 dependent and independent events cooperate to regulate inflammatory macrophages. Autocrine, single cell effects on metabolism build up to cause paracrine effects including alleviation of hypoxia. 4T1 primary tumor and lung metastasis were not Nos2-regulated but host Nos2 hampered the ability of BMDMs to respond to proinflammatory stimuli hinting at possible systemic effects of Nos2. Hence, to make a reliable prognostic prediction, it is important to know the exact NO flux, which cells within the tumor express Nos2 and what other cells associate with Nos2hi cells.

#1187

Toward high-throughput, high-multiplex FFPE tumor tissue assays for translational research: 7-color, whole slide imaging.

Amanda J. Bares, Eloise M. Wheeler, Maël Manesse. _Ultivue, Inc, Cambridge, MA_.

Background: Immuno-oncology research and predictive biomarkers discovery requires the ability to rapidly identify, quantify, and spatially map many cell sub-types of the tumor environment in FFPE tissue sections. Multiplexed immunofluorescence (IF) enables identification of multiple targets within separate spectral, or color, channels, which in turn enables more in-depth analysis of rare tissue material relative to traditional methods. Although many methods for multiplexed IF exist, they are often costly and require time-consuming assay development and long imaging times. The Ultivue® InSituPlex® technology enables rapid, optimized staining of multiple targets utilizing widely-used immunohistochemistry (IHC) staining and fluorescence imaging instrumentation. Here, we demonstrate the application of InSituPlex technology to image 6 targets and a nuclear counterstain in a 7-color single whole-slide scan, without linear unmixing-based image reconstruction. This technique expands the subtyping depth that can be achieved in a single imaging round and on a single section, and also increases sample-to-answer throughput with a streamlined, single-day workflow.

Methods: InSituPlex technology was used to perform 6-plex immune profiling of different FFPE tumor sections. Slides were stained with a cocktail of primary antibodies using a Leica® Bond® RX autostainer and imaged on commercially available fluorescent slide scanners. Subsequent image analysis of the whole slide scans was carried out using Indica Labs HALO® software.

Results: In this poster, we present an assay for pathology research that enables fast immuno-profiling in tumor tissues, using a streamlined staining workflow and 7-color imaging. Newly selected fluorophores were used with corresponding optical filter pairs, which increased the number of spectral channels available on commercially available slide scanners while limiting channel cross-talk. Analysis led to the identification of key immune cell phenotypes through marker colocalization without significant ambiguity between targets tagged with spectrally adjacent fluorescent labels.

Conclusion: The newly-expanded fluorophore configuration of the InSituPlex technology enables high throughput identification of 6 targets and a nuclear counterstain on whole tumor sections, empowering translational and immuno-oncology research.

#1188

Reversing epigenetic gene silencing to overcome immune evasion in CNS malignancies.

Nivedita M. Ratnam, Heather M. Sonnemann, Mark R. Gilbert, Amber J. Giles. _National Cancer Institute, Bethesda, MD_.

Glioblastoma remains a lethal brain cancer, and current treatment provides only modest survival benefit. Immunotherapy is currently being investigated as a potential treatment for this disease, but immune cell trafficking to tumor is hampered. Glioblastoma are immunologically "cold", lacking infiltrating lymphocytes. Tumors epigenetically silence the expression of chemokines that attract lymphocytes to evade immune attack. Previous studies in ovarian cancer demonstrated that the histone methyltransferase inhibitor, GSK126, increased T cell infiltration into the tumor, promoting immune-mediated tumor suppression. Lymphocyte infiltration was driven by elevated expression of interferon gamma (IFNγ)-induced chemokines CXCL9 and CXCL10. In the current study, we evaluate the ability of GSK126 to similarly reverse the silencing of CXCL9 and CXCL10 and thereby improve immunotherapy outcome for glioblastoma. We first determined that treatment of murine and human glioblastoma cell lines with GSK126 increased IFNγ-mediated gene expression of CXCL9 and CXCL10 by qPCR and protein by ELISA. Subsequently, transwell migration assays performed using human and murine T cells demonstrated increased T cell migration when exposed to conditioned medium from tumor cells treated with GSK126 and IFNγ. In addition, T cells exposed to GSK126 and IFNγ up regulated CXCR3, the receptor for CXCL9 and CXCL10 and further increased their expression of IFNγ. The in vivo efficacy of GSK126 was tested in combination with anti-PD-1 antibody in C57Bl/6 mice subcutaneously implanted with the syngeneic glioma cell line CT2A. Mice treated with a combination of GSK126 and anti-PD-1 antibody showed a significant decrease in tumor volume with treatment, providing an overall survival benefit compared the anti-PD-1 or GSK126 alone. Taken together, our data suggests a therapeutic role for GSK126 in glioblastoma that could enhance current immunotherapy regimens by reversing the epigenetic changes that enable immune cell evasion and encourage immune cell trafficking to the tumor.

#1189

Revisiting immune exhaustion in Hodgkin's lymphoma.

Anthony R. Colombo,1 Monirath Hav,1 Erik Gerdtsson,1 Jose Bisnesto-Villasboas,2 Stephen Ansell,2 James Hicks,1 Peter Kuhn,1 Akil Merchant1. 1 _University of Southern California, Los Angeles, CA;_ 2 _Mayo Clinic, Scottsdale, AZ_.

Clinical successes with immune check-point blockers have demonstrated the potency of the immune system in controlling cancers, most strikingly in Hodgkin lymphoma (HL), where overall response rates to PD1/L1 inhibitors approach 90%. Complete or durable responses, however, are uncommon, therefore targeting the PD1/L1 axis alone is not sufficient. Recent work analyzing the spatial arrangement of PD1 and PDL1 expressing cells has given us new insight into the mechanism of action of PD1/L1 inhibitors, however this work limited itself to studying a single check point marker on a subset of cells. We hypothesize that comprehensive profiling of the frequency and spatial arrangement of immune cells in the Hodgkin lymphoma tumor immune microenvironment (TME) will provide new insights into the mechanism of checkpoint blockers and identify novel targets for immune therapy. Until now, multiparameter spatial analysis of the immune microenvironment was limited by technical challenges. Flow and mass cytometry are able to identify immune subsets of interest but spatial information is lost. Multiplex tissue imaging methods are limited to 6-8 simultaneous markers and cannot capture the full complexity of the immune phenotypes. The Fluidigm Hyperion imaging mass cytometry (IMC) system combines a CyTOF mass cytometer with a laser ablation system allowing for 40+ parameter simultaneous immunophenotyping on a single slide of FFPE tissue, with sub-cellular resolution. We have developed a panel of 34 antibodies that allow for comprehensive characterization of CD4, CD8 and myeloid cells components in the TME of Hodgkin lymphoma using IMC. Here we report on our spatial analysis of TIM3 and LAG3 expressing CD4+ lymphocytes. Our data suggests LAG3+CD4+ and TIM3+CD4+ lymphocytes had shorter mean nearest distance to PDL1+Hodgkin Reed-Sternberg (HRS) cells upon comparison to PDL1- HRS cells (t-test, p=1.703e-08,p=1.126e-14). Future studies should explore multiple exhausted marker models that seeks to further understand the presence of simultaneous exhaustion signals in the niche environment. These data suggest that therapies that target TIM3 and/or LAG3 should be tested in Hodgkin Lymphoma and that spatial analysis of immune subsets by IMC should be explored as selective and pharmacodynamic biomarkers.

#1190

Tissue slice culture platform: A slice of reality in drug development.

Shannon Burke, Fabien Garcon, Kelli Ryan, James Harper, Robert W. Wilkinson. _MedImmune, Cambridge, United Kingdom_.

Immuno-oncology (IO) therapies for cancer have recently delivered step-change improvements in patient outcomes, but the major proportion of patients remain unresponsive or become refractory to IO treatment. Because IO-based approaches necessarily rely on the interplay of multiple cell types, including immune cells, and other elements that constitute the tumor microenvironment (TME), innovative assays are critical to generate further translational insight. Indeed, results from in vivo mouse models and in vitro assays with human cell lines often do not correlate with clinical efficacy and as such, do not always represent relevant avatars. We have leveraged recent advances in tumor slice culture (TSC) techniques to enable in vitro mechanistic and novel biomarker testing/identification studies. TSC involves the in vitro culture of thick slices (200-300 μm) of fresh tissue prepared using a vibratome and is currently the only in vitro assay system which retains the 3-dimensional tissue architecture, complete with cell-cell and cell-matrix interactions. We are now regularly using the TSC together with tumor samples derived from patients and human cancer cell line derived xenograft or syngeneic mouse cancer models, to generate exciting pre-clinical data for several Oncology programs, including oncolytic viruses, modified RNA, bispecific antibodies and antibody-drug conjugates. For instance, the TSC platform has allowed us to analyse the strong type I IFN-driven inflammatory response initiated by infection of tumor nests within patient samples and demonstrate the consequent infiltration of immune cells from the surrounding stroma. Thus, using a tissue phenomics approach incorporating a range of endpoint assays, including multiplex whole-mount imaging, transcriptomics and cytokine expression, the data generated using the TSC platform will inform the underlying biology and translational strategies of multiple therapeutic candidates.

#1191

PD-L1 expression and the tumor immune microenvironment in NUT carcinoma.

Lisa M. Rooper,1 Nyall R. London,1 Janis M. Taube,1 William H. Westra,2 Justin A. Bishop,3 Hyunseok Kang4. 1 _Johns Hopkins University School of Medicine, Baltimore, MD;_ 2 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 3 _University of Texas Southwestern Medical Center, Dallas, TX;_ 4 _University of California San Francisco Medical Center, San Francisco, CA_.

Nuclear protein in testis (NUT) carcinoma is a rare, highly aggressive malignancy that is defined by translocations of the NUTM1 gene and has a median survival of less than one year. Although immunotherapy is an attractive novel therapeutic option, the immune microenvironment has not yet been characterized in this tumor type. We performed immunohistochemistry for PD-L1, IDO1, CD8, CD68, FOXP3 and myeloperoxidase on whole-slide sections from 9 cases of NUT carcinoma, quantified immune cell subsets using Halo image analysis software, manually scored percentage of PD-L1 and IDO1 expression in tumor cells, and classified immune cell PD-L1 expression as focal (≤5%) or non-focal (>5%). The 9 NUT carcinomas included 6 sinonasal, 2 mediastinal, and 1 soft tissue primaries. Median survival was 11 months (range 2-19); 7 patients (78%) died of disease while 2 (22%) had no evidence of disease at last follow up (12 and 19 months). All tumors demonstrated a dominant population of myeloperoxidase+ neutrophils, with a mean density of 954/mm2 (range 14-2311). There were also a mean 59/mm2 CD8+ cytotoxic T-cells (range 15-107), 75/mm2 FOXP3+ T-regulatory cells (range 9-349), and 54/mm2 CD68+ macrophages (range 0-417). Immune cell density did not correlate with survival. Four tumors (44%) expressed PD-L1, with mean 17% positivity (range 0-70%). PD-L1 expression was associated with improved survival (p=0.02), with higher mean PD-L1 levels in surviving patients (65% vs. 4%, p<0.001). Two tumors (22%) expressed non-focal PD-L1 on tumor-infiltrating lymphocytes, which was also associated with survival (100% vs. 0%, p=0.03); whereas all tumors showed non-focal PD-L1 on neutrophils and macrophages. PD-L1+ tumors also had more CD8+ T-cells per mm2 (81 vs 41, p=0.07). Three tumors (33%) expressed IDO1, which was associated with improved survival (p=0.01). Overall, NUT carcinoma demonstrates an immunosuppressive microenvironment with a dominant population of tumor-infiltrating neutrophils that also show PD-L1 expression. However, evidence of PD-L1 and IDO1 expression on tumor cells with associated cytotoxic T-cell response and improved prognosis suggests that adaptive immunity plays an important role in NUT carcinoma outcomes, raising the possibility that immunotherapy might be harnessed to augment this response.

#1192

Increased regulatory T cells induced by glycolytic metabolic change in EGFR mutant NSCLC after EGFR TKI therapy.

Sook-hee Hong,1 Nahyeon Kang,2 Seung joon Kim,1 Okrane Kim,2 Jin-hyoung Kang,1 Sook Whan Sung3. 1 _Seoul St Mary's Hospital, Seoul, Republic of Korea;_ 2 _Cancer Research Institute, The Catholic University of Korea, Seoul, Republic of Korea;_ 3 _Seoul St Mary's Hospital, Republic of Korea_.

Background. Studies on the immune microenvironment of EGFR mutant lung cancer have been limited. We analyzed the effect of immune microenvironments on the development of EGFR-TKI resistance in EGFR-mutated lung cancer.

Methods. The EGFR mutant lung cancer cell line was co-cultured with activated PBMC for 72 hours with EGFR-TKI. Changes of cytokines/chemokines in the media, PD-1 expression of CD8+ T cells, regulatory T cells fraction and transcriptome analysis of tumor cells were analyzed. We also performed immune profile analysis of fresh tissues of 21 surgically resected NSCLC (7 EGFR mutant and 14 EGFR wild) by multicolor FACS.

Results. The Cytotoxicity of EGFR-TKI (elrotinib) in EGFR mutant HCC827 and H4006 lung cancer cell lines co-cultured with active PBMC tended to decrease. IL-6, IL-8, VEGF, TGF-B1, CXCL1, and CXCL10 were significantly increased after co-culture with activated PBMC but did not decrease after addtional EGFR-TKI treatment. IFN gamma increased after co-culture with activated PBMC but decreased after EGFR-TKI treatment. PD-L1 expression on tumor cells increased after co-culture with activated PBMC (p = 0.08 in HCC827 and p = 0.09 in H4006) but did not decrease after co-culture with activated PBMC and EGFR-TKI treatment (p = 0.36 in HCC827 and p = 0.45 in H4006). PD-L1 expression of A549 (EGFR wild type) did not change with co-culture or EGFR-TKI treatment. PD-1 expression of CD8 T cell co-cultured with HCC827 or H4006 did not change, however proportion of regulatory T cell increased after co-culture with HCC827 or H4006 (p=0.05 and p=0.08, respectively) and did not decrease during co-culture and EGFR-TKI treatment. Proportion of regulatory T cell in co-cultures with A549 or H1975 (erltinib resistant cell line) did not change during co-culture or EGFR-TKI treatment. Transcriptome analysis by RNA sequencing showed 1747 gene sets were differentially expressed in EGFR-TKI treated EGFR mutant cell line co-cultured with activated PBMC compared to EGFR-TKI treatment alone. Interferon gamma response pathway (NES 2.65, FDR q<0.1) and glycolytic pathway (NES 2.15, FDR<0.1) were most significantly changed. Immune profile analysis of human EGFR mutant lung cancer showed that CD4+/CD3+ T cells in EGFR mutant groups was increased compared to EGFR wild group. Proportion of FOXP3+CD25+CD4+ T reg in EGFR mutant group tended to increase compared to EGFR wild group (1.352±0.4 vs 0.74 ± 0.16%, p=0.256).

Conclusion. The regulator T cell is considered to be important in EGFR mutant NSCLC in induction of immuno suppressive microenvrionement and EGFR-TKI resistance. Study on glycolytic pathway mediated immuno suppressive microenvionment and EGFR-TKI resistance is currently ongoing

#1193

S100A proteins play immune regulatory roles in tumor microenvironment.

Nobuyuki Tanaka, Natsumi Kodake, Naoko Ogama. _Miyagi Cancer Ctr. Research Inst., Natori, Japan_.

S100A proteins are Ca2+ binding multifunctional molecule in different cellular compartments. Dysregulated expression of S100A is often observed in many types of human cancers, and is assoiciated with cancer pathogenesis. Inside a cell, S100A proteins respond to various stimuli, and some typical intracellular functions such as cytoskeleton control and motility have been extensively studied. Extracellular functions of S100A proteins have recently emerged, and they bind innate immune receptors such as TLR4 and RAGE. Nevertheless, the mode of S100A secretion has been obscure. We designed a secretion system to induce spontaneous S100A release from the cell, and examined their roles towards tumor microenvironment. We found S100A proteins are included in exosomes, and the stability of S100A contributes to their extracellular functions. Using a syngeneic tumor model, we investigated some S100A proteins show immune regulatory functions on tumor-associated macrophages. These results suggest that S100A secretion affects tumor immune environment.

#1194

Adoptive cellular therapy overcomes tumor-induced dysregulation of myelopoiesis.

Tyler J. Wildes, Catherine T. Flores, Bayli DiVita Dean, Adam Grippin, Kyle Dyson, Duane A. Mitchell. _University of Florida, Gainesville, FL_.

INTRODUCTION: Hematopoietic stem and progenitor cell (HSPC) transfer during adoptive T cell immunotherapy (ACT) prolongs median survival and generates 30% long-term cures for malignant brain tumors. We recently demonstrated during ACT that intratumoral HSPCs differentiate into immune-activating dendritic cells (DCs) through T cell-released IFN-γ. We also determined that CCR2+ HSPCs are the key immune-activating progenitor population. We previously utilized syngeneic transfer of naive HSPCs that were not endemic to the recipient tumor-bearing host. This is different from the clinical paradigm of autologous transfers. This is important because in peripheral cancers tumor-bearing host (TB) HSPCs possess considerable immunosuppressive potential. It remains unknown if this is true in brain tumor-bearing hosts, and it remains unknown how this would impact HSPC+ACT immunotherapy. We therefore evaluated the immunologic function of naïve or TB HSPCs during immunotherapy.

METHODS: We utilized KR158B and GL261 intracranial gliomas to generate TB HSPCs. HSPC culture experiments were performed in RPMI alone or conditioned with T cell supernatants to study HSPC differentiation. In vivo treatment models utilized intracranial gliomas and intravenous transfer of HSPCs with ACT. Data from the immgen.org database was utilized for hypothesis generation.

RESULTS: Brain tumors promoted a 20% expansion of HSPCs including granulocyte monocyte precursors (GMP) but a loss of DC progenitors. The immgen database revealed that GMPs express high levels of IFN-γR1 while DC progenitors express high levels of IFN-γR2, CCR2, and CXCR4, the chemotactic receptor for HPSC homing. We next determined by flow cytometry that TB HSPCs express 40% more IFN-γR1 and 90% more IFN-γR2 on DC progenitors. To determine the differentiation preference of these cells, we performed in vitro culture. After a 3 day culture, naïve HSPCs differentiated into 30% myeloid-derived suppressor cells (MDSCs; CD11b+Ly-6G/6C+) while TB HSPCs differentiated into 60% MDSCs. When both cells types were cultured in activated tumor-specific T cell supernatants containing IFN-γ, both HSPC types differentiated into 30% MDSCs, 80% MHCII+ antigen-presenting cells, and 20% CD11c+MHCII+ DCs. In vivo, ACT rescued intratumoral TB HSPCs to prolong median survival and generate long-term cures. Additionally, TB HSPC-derived cells in brain tumors maintained higher IFN-γR2 and displayed non-inferior differentiation into DCs when compared naïve HSPCs. We are now investigating the impact of PD-1 blockade on TB HSPC function.

CONCLUSIONS: Gliomas exert an immunosuppressive pressure on HSPCs. However, ACT can overcome dysregulated TB HSPC programming and promote generation of DCs instead of MDSCs. A phase I trial evaluating the impact of HSPC transfer on adoptive immunotherapy in pediatric high-grade gliomas is underway at our center (ACTION; NCT03334305).

#1195

Stochastic co-evolution of the adaptive immune system and an evading cancer population.

Jason Thomas George,1 Jeffrey J. Molldrem,2 Herbert Levine1. 1 _Rice University, Houston, TX;_ 2 _MD Anderson Cancer Center, Houston, TX_.

Recent advances in immunotherapy have revolutionized modern cancer treatment. While encouraging, robust therapies that lead to durable remission outcomes still remain a challenge in curing many malignancies as cancer cells may acquire clones that effectively evade the immune system. Cancer immunotherapeutic treatment strategies are quite complex. In extreme cases, hematopoietic stem cell recipients enlist an entire allogeneic T-cell repertoire to fight against a growing malignancy. Perhaps most importantly, the adaptive nature of the immune system uniquely enables this treatment approach to co-evolve alongside an evasive threat. However, this process is poorly quantified and thus merits further study in order to maximally benefit cancer patients. Here, we develop a theoretical framework to quantify the dynamics between a growing collection of cancer cells capable of acquiring multiple evasive clones and a T-cell repertoire that may eventually recognize the evading populations. We create the first mathematical model of stochastic tumor-immune co-evolution by applying principles of stochastic process theory. We demonstrate that our model agrees with experimental time-course data in solid and liquid cancers and relate differences in branched vs. clonal evolution to the stringency of immunosurveillance present during early cancer progression. We argue that the clonal dynamics observed in leukemia patients treated with T-cell transplant therapy likely arise from the selection of pre-existent immune-evasive clones. In conclusion, our co-evolutionary model recapitulates empirical observations and offers a framework for quantifiable predictions to further improve cancer immunotherapy.

#1196

**Induction of NKG2D ligand expression by CD8** + **T cells through a nuclear factor-kappa B and P300/CBP-associated factor-dependent mechanism.**

Jiemiao Hu, Xueqing Xia, Shulin Li. _UT MD Anderson Cancer Ctr., Houston, TX_.

The ligands for the natural-killer group 2 (NKG2D) protein are frequently expressed on tumor cell surfaces, rendering tumor cells susceptible to NKG2D-dependent immune cell attack. However, cancer cells escape from immune surveillance by downregulating NKG2D ligands. We previously discovered that the engagement of activated CD8+ T cells and tumor cells induces NKG2D ligands on tumor cells, but the underlying mechanism remains to be defined. TCGA database analyses suggested that the expression of CD40 and CD137L (their cognate receptors are CD40L and CD137, respectively, expressed on CD8+T cells) is associated with pan-NKG2D ligand expression in sarcoma and colorectal adenocarcinoma patients (Pearson's correlation coefficient r=0.2225 and 0.1947, respectively; and P=.0003 and .0002, respectively). In agreement with these human data analysis results, both in vivo mouse tumor models and in vitro cell assays validated that upon the engagement with the cognate receptors, the tumor necrosis factor receptor superfamily (TNFRSF), particularly CD137 ligand and CD40, initiates the activation of nuclear factor-kappa B (NF-κB) signaling in tumor cells even in the absence of CD8+ T cells. The same as tumor and CD8+ T contact-dependent NKG2D ligand induction, this CD137L/CD40-mediated signaling activation was associated with elevated levels of acetyltransferase P300/CBP-associated factor (PCAF), whereas inhibition of phosphorylated NF-κB abrogated PCAF induction. Although the stimulation of CD137L/CD40-mediated signaling by cognate receptors is vital for NKG2D ligand upregulation, the inflammatory cytokines, including interferon γ (IFNγ) and tumor necrosis factor α (TNFα) released from CD8+ T cells also facilitate NKG2D ligand-induced immune surveillance. These inflammatory cytokines not only trigger NF-κB signaling and PCAF activation, and they also boost T cell chemotaxis, recruit large numbers of CD8+ T cells to tumor sites, and result in high yield expression of NKG2D ligands. Collectively, our results unveil a novel mechanism of NKG2D ligand upregulation involving reverse signaling of CD40 and CD137L on in tumor cells, which along with inflammatory cytokines IFNγ and TNFα, stimulate downstream NF-κB and PCAF activation. Understanding this mechanism may help to develop induced NKG2D ligand-dependent T-cell therapy against cancers.

#1197

Role of NOS2-COX2 inhibition in radiation-induced tumor growth delay and immuno-modulation in the tumor micro-environment.

Debashree Basudhar, Veena Somasundaram, David A. Scheiblin, Robert Y. Cheng, Stephen J. Lockett, David Wink, Lisa A. Ridnour. _National Cancer Institute, Frederick, MD_.

Triple negative breast cancer (TNBC) is associated with lack of expression of human epidermal growth factor receptor 2 (HER2), estrogen receptor (ER) and progesterone receptor (PR), and do not respond to hormonal therapy. It is one of the most aggressive breast cancer phenotypes and remains a major health hazard among women with drug resistance being a limiting factor in treatment. Inflammation is a key driver of poor survival among TNBC patients through increase in metastasis and chemo-resistance. We recently demonstrated that co-expression of pro-inflammatory enzymes nitric oxide synthase2 (NOS2) and cycloxygenase2 (COX2) is a powerful prognostic marker of poor outcome (HR=21) among ER(-) patients where we showed that inflammatory loops involving these proteins globally drive major oncogenic pathways [1].

Apart from intramural signaling, the crosstalk of tumor cells with immune cells is a key driver of immuno-suppression. Tumor progression is associated with tumor infiltrating M2 macrophages and Th2 cells leading to immuno-suppression, aberrant activation of cytokines, chemokines and growth factors thus creating a conducive environment for tumor growth and metastasis. Our goal is to modulate the tumor micro-environment (TME) to increase efficacy of current radiation- and immunotherapy.

Radiation therapy is a commonly used treatment option in different types of cancer including breast cancer. Focal radiation limits systemic side effects commonly associated with chemotherapy. It also activates the immune system. A key component of the immune system mediated tumor clearance is cytotoxic CD8 T cells. More recently a study found that increased CD8 cells and Th17 cells are specifically associated with TNBC patients [2]. However, they undergo functional reprogramming in the TME evident from decreased cytotoxic (IFN-𝛾;) and proliferation marker (granzyme B).

We used confocal microscopy and flow-cytometry techniques to investigate the role of NOS2 and COX2 in radiation induced tumor growth delay and metastasis. We also examined the ability of NOS2 and COX2 in regulation of the immune profile of the TME, thus emphasizing their importance in tumor growth and immune-surveillance. Lastly, we evaluated the role of COX2 and NOS2 inhibition using commercially available inhibitors on radiation induced tumor growth delay in murine models of ER- breast cancer.

[1] Basudhar, D. et al, Proceedings of the National Academy of Sciences of the United States of America 2017, 114 (49), 13030-13035.

[2] Gil Del Alcazar, et al., Cancer discovery 2017, 7 (10), 1098-1115.

#1198

Neoantigen load, tumor immune infiltration and prediction of survival in HCC patients.

Maria Tagliamonte,1 Angela Mauriello,1 Roberta Zeuli,2 Annacarmen Petrizzo,1 Maria Lina Tornesello,1 Michele Ceccarelli,2 Franco M. Buonaguro,1 Luigi Buonaguro1. 1 _National Cancer Institute, Naples, Italy;_ 2 _Sannio University, Benevento, Italy_.

Predicted neoantigens can be relevant to the clinical outcome only when their affinity to MHC I molecule is significantly higher than the corresponding wild type peptide, and when they do not show any homology to unrelated self antigens (e.g. true predicted neoantigens - TPNAs). In addition, they may show homology to pathogen-derived antigens, representing a "super" neoantigen if the patient is immunologically primed. A novel algorithm for prediction of TPNAs presented by each patient's autologous HLA molecules was applied to datasets of all 377 HCC patients available at The Cancer Genome Atlas (TCGA). The number of neoantigens was highly variable between different HCC patients with an average of 14.5 per sample. However, considering those with the highest predicted antigenic quality, the average of neoantigens per samples with such parameters dropped down to 1.5. Results showed that patients' survival did not correlate with the quantity nor the quality of neoantigens. However, only when the cytotoxic activity of tumor infiltrating lymphocytes was low, a correlation between neoantigens and survival was observed, suggesting that in this setting a higher number of neoantigens is required for a more pronounced efficacy on the patients' survival. Predicted neoantigens with homology to unrelated self antigens or pathogen-derived antigens were identified. Nevertheless, a single neoantigen per patient with such a homology did not influence patients' survival. The same algorithm was applied to a cohort of hepatocellular carcinoma (HCC) patients enrolled at our Institute, confirming the results obtained on the TCGA cohort. Interestingly, TPNAs with highest homology to pathogen's antigens were found in the only HCC long-term survival patient. A pre-existing T cell immunity specific for these TPNAs was revealed in the patient, possibly explaining the favourable clinical outcome. In conclusion, the new algorithm allowed the identification of the very few TPNAs in HCC samples. The relevance of quantity and quality of neoantigens in the evolution of HCC without treatment with checkpoint inhibitors (CI) appears to be less central, unless the tumor cytotoxic lymphocyte infiltration is low. The identification in a single patient, of a primed immunity against a neoantigen homologous to a pathogen-derived antigen, can possibly explain the long term survival. Therefore, the identification of high quality neoantigens remains a priority for developing personalized active immunotherapies also in the HCC model characterized by a low mutational burden.

#1199

**Identification of candidate regulators of genomic instability in human lung adenocarcinoma through a new cross-species** in silico **analysis.**

Chinthalapally V. Rao,1 Mudassir Farooqui,2 Yuting Zhang,1 Gaurav Kumar,1 Janani Panneerselvam,1 Nagendra Yarla,1 Adam S. Asch,3 Hiroshi Y. Yamada1. 1 _Univ. of Oklahoma Health Sciences Ctr., Oklahoma City, OK;_ 2 _Univ. of New Mexico Health Sciences Center, Albuquerque, NM;_ 3 _Stephenson Cancer Center, Oklahoma City, OK_.

(a) Chromosome instability (CIN), a type of genomic instability that is mitotic error-driven, has a deep impact on carcinogenesis and cancer recurrence. High CIN is a marker for poor prognosis in various cancers, including lung adenocarcinoma. In the tumor genome, CIN can be indicated with Copy Number Alterations (CNA) as its surrogate indicator. Although a diverse array of genes can cause CIN at the cellular level, genes responsible for tumor CNA remain elusive. In this study, we set out to identify genes whose expression shows correlation with CNA in human lung adenocarcinoma.

(b) We employed a novel cross-species in silico analysis. Our previous lung RNAseq results from lung cancer-prone CIN mice indicated 348 misregulated genes (92 up-regulated, 256 down-regulated; 2-fold cutoff, P<0.05). We hypothesized that some of the genes misregulated in CIN mice are involved in genomic instability in tumors. With in silico analysis with a human cancer genome database, we identified five human genes as candidate regulators of CNA in human lung adenocarcinoma: MMP13, NUF2, CCL22, ZNF366, and GPR114. High expression of MMP13, CCL22, ZNF366, and GPR114 was correlated with low CNA, suggesting their role in suppressing CIN. High expression of NUF2 was correlated with high CNA, suggesting its role in increasing CIN.

(c) MMP13 (Matrix Metalloprotease 13) is a protease involved in the breakdown of extracellular matrix. NUF2 (yeast NUF2 Kinetochore Protein homolog) is involved in the mitotic process, the misregulation of which directly leads to CIN. CCL22 (C-C Motif Chemokine Ligand 22), ZNF366 (Zinc Finger Protein 366), and GPR114 (G protein-coupled receptor 114, aka ADGRG5) are generally markers for subsets of immune cells (likely dendritic cells). This result led us to further hypothesize that infiltration of CCL22-, ZNF336-, and/or GPR114-positive immune cells inhibits pre-tumor cells and tumor cells with high CNA.

(d) The removal mechanism of cells with genomic instability is poorly understood. This is the first time that candidate markers for the immune cells directly or indirectly involved in removal of CIN cells have been identified. This study may aid in fine-tuning cancer-preventive immunotherapy that targets lung cells with genomic instability.

#1200

AXL targeting enhances lymphocyte-mediated cytotoxicity of lung cancer cells.

Sallem Chouaib,1 Stephane Terry,1 Stephanie Buart,1 Agnete Engelsen,1 Gro Gausdal,2 James Lorens,3 Jean Paul Thiery,1 Fathia Mami-Chouaib1. 1 _Gustave Roussy, Villejuif, France;_ 2 _Biobergen, Bergen, Norway;_ 3 _University of Bergen, Bergen, Norway_.

As immunotherapies are now used to treat a large proportion of NSCLC patients, defining mechanisms of immune resistance is critical. Immune resistance may arise from both genetic instability and tumor heterogeneity driven by microenvironmental stresses such as hypoxia that promotes carcinoma cell plasticity as well as extrinsic or intrinsic mechanisms of immune resistance. AXL, a member of the TAM receptor tyrosine kinase family is widely expressed human cancers and increasingly recognized for its role in cell plasticity and drug resistance. In this study, we used a model of hypoxia-induced tumor plasticity to generate multiple lung cancer clones with mesenchymal and epithelial features to address mechanisms of immune resistance. We demonstrate that AXL expression is dramatically increased in mesenchymal lung cancer clones. Moreover, expression of AXL in the cells was correlated with an increased cancer cell intrinsic resistance to both NK and CTL-mediated killing, Notably, small molecule AXL targeting potently sensitized mesenchymal lung cancer cells to cytotoxic lymphocyte-mediated killing. Mechanistically, we showed that attenuation of AXL-dependent immune resistance to immune cells involved a novel molecular network comprising NF-κB activation, increased ICAM1 expression, and upregulation of ULBP1 expression coupled with MAPK inhibition. Congruently, higher ICAM1 and ULBP1 tumor expression, correlated with improved patient survival in two NSCLC cohorts. These results reveal a novel AXL-mediated immune escape regulatory pathway, suggest AXL as a novel candidate biomarker for tumor resistance to NK and CTL immunity, and support AXL targeting to optimize immune response in NSCLC.

#1201

IL4 receptor mediated metabolic changes contribute to histone acetylation leading to metastatic progression in breast cancer.

Demond Williams, Adam Miranda, Chilesi Uririri, Barbara Fingleton. _Vanderbilt University, Nashville, TN_.

IL-4 has a number of well-established functions in immune cells including proliferation, and survival of lymphocytes, polarization of macrophages to the pro-tumor M2 tumor associated macrophage phenotype. Another role of IL-4 signaling is induction of metabolic changes in immune cells including increased glycolysis in B-lymphocytes, and increased expression of arginase 1 with a switch to fatty acid oxidation in macrophages. However, the effects of IL-4 signaling in tumor cells that express IL-4 receptor is not well understood. Previous work from our lab has shown that loss of IL-4 receptor (IL-4R) significantly abrogates metastasis in experimental metastasis models in mice. Thus, we wondered whether changes in metabolism downstream of IL-4 receptor activation may contribute to metastatic phenotypes. Since altered metabolic pathways have been shown to regulate the epigenetic state of cancer cells, we are pursuing histone modifications as a mechanism for the connection between altered metabolism and increased metastatic capability in IL-4R expressing breast cancer cells. MDA-MB-231 and BT549 triple negative breast cancer cells were treated with IL-4 for 2,4,6, and 8 days. Samples of media were assayed for glutamine and glucose consumption, as well as lactate and glutamate secretion. IL-4 decreased consumption of glucose and glutamine, yet concomitantly increased the release of lactate and glutamate. These data suggest that IL-4 alters the utilization of glucose and glutamine in cancer cells. To study potential links to epigenetic regulation cells were treated for 18, or 48 hours with IL-4 before biochemical fractionation was performed in order to obtain nuclear and cytosolic lysates. The nuclear fractions were assessed for histone acetyltransferase (HAT) activity using commercially available assays, and showed that IL-4 increased the activity of histone acetyltransferases. We also performed pull down of acetylated lysine residue from the nuclear fractions of IL-4 treated cells. The cells were then assessed for abundance of acetylated histone 3. In addition, we used western blotting to measure the abundance of specific histone modifications such as histone 3 lysine 9 acetylation and histone 3 lysine 27 acetylation. Overall, histone acetyltransferase activity and levels of histone acetylation were increased by IL-4. Additionally, we found that IL4 increases the acetylation of histone 3 lysine 9. In order to determine the effects of IL-4 signaling on metabolism in vivo we have initiated PET imaging studies in humanized mice that underwent tail vein injections of BT549 cell lines. These mice were treated with MDNA413, an antagonist of IL-4 signaling, and are being assessed for uptake of glucose and glutamine. Together these suggest that IL-4 alters the metabolism and epigenetic state of cancer cells and may be a viable target for therapy in metastatic breast cancer.

#1202

Tumor cell intrinsic STING signaling demonstrates minimal contribution to the anti-tumor response elicited by the STING agonist ADU-S100 (MIW815).

Leticia Corrales,1 Antony L. Desbien,1 Kelsey E. Sivick Gauthier,1 Weiwen Deng,1 Tamara Schroeder,1 Gabrielle L. Reiner,1 Natalie Surh,1 Brian Francica,1 Ken Metchette,1 Chudi O. Ndubaku,1 Andrea van Elsas,2 Meredith L. Leong,1 Sarah M. McWhirter1. 1 _Aduro Biotech, Inc., Berkeley, CA;_ 2 _Aduro Biotech Europe, Oss, Netherlands_.

Innate immune sensing of tumors is a critical step in generating spontaneous anti-tumor T cell responses. Endogenous activation of the STING pathway in immune cells and the subsequent generation of type I IFN is sufficient to generate spontaneous anti-tumor T cell responses. To trigger STING activation within the tumor, Aduro Biotech, Inc. has developed the first-in-class STING agonist, ADU-S100, a small molecule derivative of the natural cyclic dinucleotide STING ligand. In mouse models, intratumoral administration of ADU-S100 increases systemic tumor-specific T cells and results in substantial antitumor efficacy. ADU-S100 is currently being tested as mono- or combination therapy in Phase 1 clinical studies enrolling patients with cutaneously-accessible treatment-refractory advanced cancers and lymphomas. STING is broadly expressed across different cell types, however several studies have demonstrated that tumor cells regulate expression of STING and other members of the pathway, mainly cGAS, by epigenetic mechanisms. Activation of the STING pathway in innate immune cells is necessary for the generation of anti-tumor T cell responses, but the role of other cell types within the tumor microenvironment in response or resistance to STING agonists is not completely understood. To understand the contribution of tumor-cell STING to the anti-tumor response, we generated STING-deficient 4T1 tumor cells using the CRISPR/Cas9 system. Composition of the tumor microenvironment, endogenous T cell responses and tumor growth were comparable in animals with implanted STING-WT or -KO tumor cells. Similar data was observed in the B16.SIY melanoma model. In order to understand the role of tumor-STING in the context of ADU-S100 treatment, animals implanted with 4T1 STING-WT or -KO tumor cells were treated with a wide range of ADU-S100 doses. Expression of STING within tumor cells did not impact activation of innate cells or generation of tumor-specific T cells among all the tested doses. At the lowest dose, tumor cell expression of STING was required for tumor growth control.

Overall, these results show that ADU-S100 activation of STING in host cells rather than in tumor cells is critical for production of type I interferon and tumor control. The data suggests that defects associated with a lack of tumor-cell STING expression can be overcome with higher doses of ADU-S100. This supports potential treatment of cancers of different histologies regardless of tumor cell-intrinsic STING expression.

#1203

Preclinical development of ADU-1805, a highly selective pan-allele anti-SIRPα antibody that blocks the SIRPα-CD47 innate immune checkpoint.

Erik Voets,1 Joost Kreijtz,1 Paul Vink,1 David Lutje Hulsik,1 Mark Parade,1 Sanne Spijkers,1 Inge Reinieren-Beeren,1 Joost Rens,1 Wout Janssen,1 Peter van Zandvoort,1 Brian Francica,2 Meredith Leong,2 Andrea van Elsas,1 Hans van Eenennaam1. 1 _Aduro Biotech Europe, Oss, Netherlands;_ 2 _Aduro Biotech, Inc., Berkeley, CA_.

Background: SIRPα signaling dependent immunoregulatory activity on myeloid cells is activated by binding of its ligand CD47, and blockade of the pathway may enhance anti-tumor immunity. Hence the pathway is thought to represent a novel immune checkpoint. CD47 has been extensively studied in the context of "don't-eat-me" signaling. Since CD47 is ubiquitously expressed on normal cells, upregulated on many cancer cells, and also has SIRPα-independent functions, the safety of a CD47-targeted antibody has been a concern. Alternative strategies therefore are focusing on directly targeting SIRPα because of its more restricted expression to cells of the myeloid lineage.

Methods: Using Aduro Biotech's B-select platform, we have identified and characterized ADU-1805: a highly selective pan-allele anti-SIRPα antibody (EC50 SIRPαV1/SIRPαV2 ≤ 3nM) that lacks appreciable SIRPβ binding (EC50 > 120nM) but cross-reacts with SIRPγ (EC50 ≤ 5nM). We also demonstrate that ADU-1805 is cross-reactive with SIRP family members in non-human primates (NHPs) including cynomolgus monkeys.

Results: ADU-1805 potently blocks CD47 binding (IC50 ≤ 1.5nM) to SIRPα in all known human SIRPA genotypes (including homozygous and heterozygous genotypes) and antagonizes SIRPα-CD47 interactions on primary SIRPα+ myeloid cells (IC50 ≤ 4nM). In line with its antagonistic properties, ADU-1805 enhances tumor cell clearance by human granulocytes and macrophages. Interestingly, anti-CD47 antibodies but not ADU-1805 inhibit T cell activation in an allogeneic mixed lymphocyte reaction. In addition, anti-SIRPα combines in a synergistic manner with PD-1 blockade to reduce tumor burden in several syngeneic mouse tumor models. Unlike CD47-targeting antibodies, ADU-1805 does not trigger hemagglutination or platelet binding in vitro, suggesting a reduced risk of red blood cell and platelet depletion in vivo. ADU-1805 is tested in a NHP study in cynomolgus monkeys at increasing dose levels to assess safety of the antibody.

Conclusions: We have identified ADU-1805 as a potentially best-in-class antagonistic anti-SIRPα antibody with a unique binding profile as it binds all reported human SIRPα alleles but does not appreciably bind to the activating SIRPβ receptor. Blocking the SIRPα-CD47 innate immune checkpoint with ADU-1805 modulates myeloid cells in the tumor microenvironment and promotes antigen presentation and cross-priming of dendritic cells. We are currently advancing ADU-1805 through preclinical studies to address the safety, pharmacokinetics, and pharmacodynamics profile of this anti-human SIRPα antibody in vivo. 

## EXPERIMENTAL AND MOLECULAR THERAPEUTICS

### Cancer Immunotherapy

#1204

Design and development of anti-linker antibodies for the detection and characterization of CAR T cells.

Stuart A. Sievers, Keith A. Kelley, Stephanie H. Astrow, Adrian Bot, Jed J. Wiltzius. _Kite, a Gilead Company, CA_.

Introduction: Chimeric antigen receptor (CAR) T cell therapy is a transformative treatment modality in B cell malignancies. As next generation CARs are developed to improve durable response rates and expand into other indications of unmet need, the proper tools to identify, characterize, and modulate CAR activity will play a pivotal role in the optimization of this therapeutic option. This study developed and evaluated one approach, that of using monoclonal antibodies (mAbs) against common elements utilized in approved and investigational CARs.

Methods: The mAbs KIP-1 and KIP-4 were raised in rabbits against the linkers of 2 single-chain variable fragments, the Whitlow linker (Whitlow, et al. Protein Eng. 1993) and the G4S linker (Huston JS, et al. PNAS. 1988), respectively, to generate universal detection reagents without adding an exogenous peptide recognition motif. Specificity and sensitivity were assessed by flow cytometric analysis of CAR T cells vs nontransduced T cells, immunohistochemistry (IHC) staining of embedded cell pellets with or without a CAR carrying the relevant linker, and epitope mapping by ELISA. Activation was assessed in a competitive stimulation assay in which nontransduced T cells and CAR T cells were mixed at defined ratios and stimulated broadly with OKT3 or specifically with KIP-1 or KIP-4.

Results: Flow cytometric analysis demonstrated that CAR T cells with the Whitlow linker could be detected by phycoerythrin-conjugated KIP-1 at ≤ 10 ng per million cells with no detectable staining on nontransduced T cells above background levels. To further demonstrate specificity, IHC data revealed that KIP-1 bound to T cell pellets expressing CARs containing the Whitlow linker, but not the G4S linker. Epitope mapping revealed that the minimal KIP-1 epitope was contained within the amino acid sequence SGKPGSGE. Furthermore, KIP-1 was able to effectively identify CAR T cells in patient samples by both flow cytometry and IHC. Having established that KIP-1 was specific and sensitive, activation assays were performed to determine its CAR T cell-specific activating capacity. KIP-1 specifically activated CAR T cells, but not nontransduced T cells, as demonstrated by CAR T cell expansion and upregulation of cell surface activation markers, including CD69 and 4-1BB. KIP-4 was also assessed for specificity, sensitivity, and activation capacity with similar results.

Conclusions: Taking advantage of the linear epitopes within the commonly-used Whitlow and G4S linkers, KIP-1 and KIP-4, respectively, can be used in a wide variety of phenotypic and functional assays with CARs of various specificities that share these linkers. These tools provide the means to realize the full potential of investigational CAR T cell products by supporting research, clinical, and manufacturing efforts.

#1205

Targeting vasoactive intestinal peptide signaling to enhance pancreatic cancer responsiveness to immunotherapy.

Sruthi Ravindranathan, Yiwen Li, Shuhua Wang, Mohammad Y. Zaidi, Gregory B. Lesinski, Bassel El-Rayes, Edmund K. Waller. _Emory University, Atlanta, GA_.

Background: Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second leading cause of cancer related death in the U.S., outranking breast and colorectal cancer by 2030. Being completely refractory to checkpoint therapies, management of PDAC remains an unmet clinical need. Intriguingly, the cancer genome atlas shows elevated mRNA expression of vasoactive intestinal peptide (VIP), a neurotransmitter in exocrine pancreatic cancers. Interestingly, VIP also has immunosuppressive properties leading to decreased T cell proliferation and enhances expansion of myeloid derived suppressor cells (MDSCs) and regulatory T cells (Tregs). Thus, we are investigating the role of VIP in PDAC progression and the effect of inhibiting VIP signaling for improved responsiveness to immunotherapy.

Methods: Level of secreted VIP in 1) serum of healthy volunteers, 2) untreated PDAC patients, 3) cell culture supernatant from murine melanoma, breast and PDAC cells and 4) serum from mice bearing these cell lines were tested via VIP specific enzyme immunoassay. Effect of inhibiting VIP signaling in-vivo was tested using VIPhyb, an antagonist for VIP receptor. Mice were subcutaneously inoculated with MT5 or luciferase transfected KPC (KPC luc), PDAC cell lines and treated with VIPhyb and/or anti-PD1 treatment after the tumors were palpable. The treatment regimen involved administering 10µg VIPhyb subcutaneously, every day and 200µg anti-PD1 or IgG2a, intraperitoneally, every three days, for 10 days. The tumors were measured using calipers or imaged via IVIS imaging system. The mice were sacrificed when tumors ulcerated or when they reached IACUC endpoint. Immunohistochemistry was performed on tumor tissue sections harvested at the time of sacrifice for expression of VIP, CD8 and DAPI.

Results: Consistent with the human genome atlas, cell culture supernatants collected from murine PDAC cell lines had significantly higher levels of VIP as compared to melanoma and breast cancer cells. Similarly, elevated levels of VIP were observed in mice bearing PDAC tumors, and in plasma from untreated human PDAC patients, when compared to naïve mice and healthy volunteer samples, respectively. Treatment of immune competent mice bearing MT5 or KPC luc PDAC cells with the combination of VIPhyb and anti-PD1 produced complete and durable regression of tumors in 20% of the mice and delayed tumor progression in both tumor models. Further, upon analysis of the tumor tissues sections, significant infiltration of CD8+ T cells was observed in mice treated with the combination of VIPhyb and anti-PD1.

Conclusion: VIP is a novel immune checkpoint pathway in PDAC. Blocking VIP signaling during anti-PD1 therapy, shows improved survival and delayed tumor progression in mouse models of PDAC. Experiments are underway to understand the mechanism of action, as well as, improving the potency of VIPhyb for enhanced clinical outcome.

#1206

Inhibition of GPR4 attenuates intestinal inflammation in a mouse colitis model.

Edward J. Sanderlin,1 Mona Marie,1 Juraj Velcicky,2 Pius Loetscher,2 Li V. Yang1. 1 _East Carolina Univ. School of Medicine, Greenville, NC;_ 2 _Novartis Institutes for BioMedical Research, Basel, Switzerland_.

Inflammatory bowel disease (IBD), a chronic inflammatory disorder of the digestive tract, is associated with a higher risk of colorectal cancer development. Current therapeutic approaches for IBD are limited and primarily include biologics and steroids such as anti-TNFα monoclonal antibodies and glucocorticoids. Chronic usage, however, can cause significant adverse drug effects such as increased risk of infection and lymphoma in some patients. GPR4, a pH-sensing G protein-coupled receptor, has recently emerged as a potential therapeutic target for intestinal inflammation. Previous studies demonstrate that GPR4 is a pro-inflammatory receptor expressed in vascular endothelial cells (EC) and stimulates leukocyte-EC adhesion and extravasation. Knockout of GPR4 alleviates intestinal inflammation in IBD mouse models. In this study, we have assessed the effects of a recently developed GPR4 antagonist, 2-(4-((2-Ethyl-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)methyl)phenyl)-5-(piperidin-4-yl)-1,3,4-oxadiazole (GPR4 antagonist 13, also known as NE 52-QQ57), in the dextran sulfate sodium (DSS)-induced acute colitis mouse model. The GPR4 antagonist 13 inhibited intestinal inflammation. All colitis parameters, such as body weight loss, fecal score, colon shortening, splenic expansion, and mesenteric lymph node enlargement, were reduced in the GPR4 antagonist 13 treatment group compared to the vehicle group. Histopathological features of active colitis and inflammatory gene expression of colon tissues were reduced in the GPR4 antagonist 13 treatment group compared to vehicle control. Our results indicate that the GPR4 antagonist 13 provides a protective effect in the DSS-induced colitis mouse model and inhibition of GPR4 can be explored as a novel anti-inflammatory approach.

#1207

Reversing lactate-driven immunosuppression using the novel, potent and selective MCT4 inhibitor AZD0095.

Susan E. Critchlow, Gareth Hughes, Anna Staniszewska, Matt King, Filippos Michopoulos, Lorna Hopcroft, Martin R. Brown, Jenna Bradley, Beverley Hammond, Pablo MorentinGutierrez, Larissa Carnevalli, Elizabeth Hardaker, Frederick W. Goldberg. _AstraZeneca, Cambridge, United Kingdom_.

Metabolic adaptation to a Warburg phenotype is a hallmark of cancer, where tumors rely on glucose to produce ATP and building blocks required to support rapid tumor growth. Glycolytic metabolism requires continuous export of lactate from cells by monocarboxylate transporters (MCTs) resulting in accumulation of lactic acid in the tumor microenvironment. Tumor-derived lactate is immune-suppressive and has been shown to modify immune cell function. Inhibition of the MCT4 lactate transporter has the potential to reduce lactate in the tumor microenvironment and reverse immune suppression. AZD0095 is a highly selective, potent inhibitor of MCT4 with a cellular activity of 1-3 nM with a >1000-fold selectivity over MCT1. We confirm that lactic acid inhibits T cell proliferation and viability. AZD0095 does not inhibit T cell proliferation or survival (IC50 > 10 μM). Oral administration of AZD0095 (10-100 mg/kg BID) modulates lactate transport in mouse syngeneic models which do not express the MCT1 lactate transporter. AZD0095 treatment causes an increase in tumor-infiltrating lymphocytes and an increase in cytotoxic T cells in combination with α-PD1. AZD0095 treatment suppresses myeloid infiltration as a monotherapy and in combination with checkpoint inhibitor. Consistent with the observed changes in immune cell infiltration, the combination of AZD0095 with checkpoint inhibitors α-PD1 or α-CTLA4 (10 mg/kg 3x week) enhances the anti-tumor activity of checkpoint inhibitor monotherapy in MC-38 and EMT6 (MCT1 knock-out) mouse syngeneic models. These data demonstrate that AZD0095 can reverse lactate-driven immunosuppression and enhance response to checkpoint inhibition in pre-clinical models.

#1208

Comparative analysis between DNA vs RNA-based T cell repertoire profiling for cancer immunotherapy.

Shanker Kalyana-Sundaram, Christopher Traini, Wendy Halsey, Heather Jackson, Sabyasachi Bhattacharya, Niranjan Yanamandra, Ganesh Sathe, George Livi. _GlaxoSmithKline, Collegeville, PA_.

The T cell receptor (TCR), a defining structure of T cells is responsible for the recognition of the antigen-major histocompatibility complex, leading to the initiation of an immune response. The extreme diversity of the TCR repertoire represents a major analytical challenge. Massively parallel high throughput sequencing is commonly deployed to characterize various key features of T cell receptors including their clonal expansion and repertoire diversity. Genomic DNA is the most widely employed starting material to characterize the TCR repertoire. DNA is highly stable and contains a fixed copy number per cell, which allows for better quantification of TCR clones. However, the inclusiveness is a major limitation of using genomic DNA along with the PCR amplification challenges and sequencing errors due to recombined gene segments within the context of 'unused' segments and introns. Here we deploy RNA-based TCR profiling as an alternate strategy to overcome the major limitations associated with the DNA-based approach. Employing RNA with a simplified PCR amplification strategy allows a more comprehensive identification of unique TCR variants with greater sensitivity. It also provides information about expression levels of TCR sequences, and more importantly, excludes the non-productive sequences that are functionally irrelevant. In addition, Unique Molecular Indexing (UMIs) enables the determination of the absolute count of RNA transcripts processed in a sample, allowing straightforward error corrections.

#1209

**Screening** ex vivo **conditions that increase memory T cell frequency using high throughput flow cytometry and an optimized multiplexed assay.**

Zhaoping Liu, Andrea Donart-Gomez, John O'Rourke. _Intellicyt, Albuquerque, NM_.

A critical process in bio-manufacturing of adoptive cell therapies such chimeric antigen receptor (CAR) T and tumor infiltrating lymphocyte (TIL) therapies is the ex vivo expansion of T cells. Recent clinical studies show a correlation between in vivo expansion and persistence of infused T cells and patient outcomes. Additional studies show that a subset of functional memory T cells including T memory stem cells (Tscm) and central memory T cells (Tcm) are responsible for the majority of in vivo expansion and persistence leading to increased anti-tumor responses. This suggests that ex vivo protocols generating higher percentages of Tscm and Tcm in the total cell product will lead to significant clinical improvements.

We developed a robust, high-content T cell memory assay to address monitoring requirements for T cell phenotype and function for improved ex vivo expansion protocols and other studies where profiling of memory subsets is crucial. This miniaturized assay uses high throughput flow cytometry to measure cell phenotype, cell viability and effector cytokine release in the same sample well of a 96- or 384-well microtiter plate. The optimized antibody panel includes markers to identify T cells (CD3, CD4, and CD8); markers to discriminate between naive, memory, effector subsets (CD45RA, CD45RO, CD62L, and CD95); and a long-term survival marker (CD27). In addition, secreted cytokine quantitation (INFγ and IL-10) is performed simultaneously with the phenotypic and cell health measurements using a bead-based assay.

Proof of concept studies were performed by stimulating peripheral blood mononuclear cells (PBMC) from multiple donors using anti-CD3/anti-CD28 coated beads. Activated T cells were expanded by culturing in serum-free media supplemented with different combinations and concentrations of IL-4, IL-7, IL-15, and IL-21 in a 96-well plate format. On Days 3 through 7 post stimulation, a small aliquot from each culture well was transferred to an assay plate and assessed for phenotype and function using the T cell memory assay. Data were acquired using the Intellicyt iQue Screener PLUS® VBR high throughput flow cytometer and analyzed using the integrated ForeCyt® software package. These data showed that the various cytokine combinations had a distinct effect on promoting T cell memory frequency, especially the Tscm cell subset, as well as effector cytokine secretion in a temporal and concentration specific manner.

These data show the T cell memory assay, combined with high throughput flow cytometry, is a powerful tool to rapidly screen for cytokine combinations that increase the frequency of T-memory cells during ex vivo expansion of T-cell products. Besides their use in optimizing cell manufacturing protocols, these tools can be easily applied to the profiling of other biologics and drugs where the monitoring of T cell memory subsets is required.

#1210

Targeting the IDO1-Kynurenine-AhR pathway for cancer immunotherapy.

Sheena Pinto, Christoph Steeneck, Michael Albers, Simon Anderhub, Manfred Birkel, Larisa Buselic-Wölfel, Gisela Eisenhardt, Claus Kremoser, Thomas Hoffmann, Ulrich Deuschle. _Phenex Pharmaceuticals AG, Heidelberg, Germany_.

The aryl hydrocarbon receptor (AhR) is a ligand-controlled transcription factor that is primarily known to be a sensor of xenobiotics and tumor-promoting activities of halogenated hydrocarbons and polycyclic aromatic hydrocarbons. Several endogenous ligands such as metabolites produced by commensal microorganisms on skin and in gut and metabolites of L-Tryptophan - produced under control of the Tryptophan dioxygenases IDO1 and TDO2 such as L-Kynurenine and Kynurenic acid are known to modulate the transcriptional activity of AhR. The activity of AhR is predominant in many different immune and epithelial cells, thereby balancing immune responses towards various signals.

Activation of the IDO1-Kyn-AhR pathway and accumulation of nuclear AhR protein is frequently seen in different tumor types and possibly linked to the observed diminished anti-tumor immune response. AhR agonizing ligands produced by cancer cells and/or lymphocytes recruited to the TME reduce the anti-tumor immune response through increasing the numbers and function of regulatory T cells while reducing that of cytotoxic CD8+ T cells.

In order to reduce AhR-mediated immune-suppression in cancer patients, Phenex Pharmaceuticals initiated a program to identify small molecule AhR antagonists to block activated downstream signaling of AhR. To this end, we have identified novel AhR antagonists, which showed strong antagonistic activity against agonist-activated human AhR in a cell based CYP1A1 promoter-driven luciferase reporter assay in HepG2 cells. The compound series display good oral bioavailability and low clearance in mice. Our AhR antagonist molecules show single agent anti-tumor activity and increase the efficacy of Gemcitabine and anti-PD-L1 checkpoint inhibition in different syngeneic mouse tumor models. Overall, AhR antagonist treatment increased CD8\+ T cell and M1 macrophage numbers, and decreased IL22 levels, which might be of functional relevance in the tumor context. Future research is directed to determine PK-PD markers suitable for clinical development and identification of human malignancies likely of responding to AhR antagonist treatment.

#1211

Identification, retrieval, and RNA sequencing of single rare antigen-specific T cells from circulation using the RareCyte® platform.

Nolan Ericson, Eric Kaldjian, Tad George, Lance U'Ren. _RareCyte Inc., Seattle, WA_.

Background. The immune system provides antigen-specific protection against pathogens as well as malignancies, both of which evolve strategies to evade immune surveillance and containment. Effective immune response often depends on activation of rare antigen-specific immune cell sub-types. The RareCyte platform provides integrated multi-parameter imaging and retrieval capabilities that allow phenotypic identification and isolation of rare cells for sequence and transcript level analyses in order to study the complexity of host defense.

Methods. Antigen-specific T cells were identified by immunofluorescent staining of live purified T cells from normal donors with the following panel: CD3; CD8; exclusionary cocktail (CD4, CD14, CD15, CD20, and DAPI); and HLA-A2 restricted tetramer (MBL). Tetramers had specificity against influenza-M1 (GILGFVFTL) and the melanoma antigen Mart-1 (ELAGIGILTV). For differential expression experiments, cells were stimulated overnight with specific target peptide or control irrelevant peptide, stained with the multiparameter panel containing the relevant tetramer, then imaged using the CyteFinder® instrument. Automated image analysis identified candidate cells with positive signal in the tetramer and CD8 channels and negative signal in the exclusion channel. Single cells displaying membrane ring tetramer distributions were retrieved using the CytePicker® module, followed by single cell RNA sequencing using the SMART-Seq® v4 kit (Takara Bio) and the MiSeq® instrument (Illumina). Differential gene expression of stimulated versus unstimulated cells was performed using DESeq2 software (BioConductor). Single-cell RNA seq FASTQ files were analyzed with the TraCeR computational method to identify TCR alpha and beta chains in both stimulated and unstimulated cells.

Results. Single influenza and Mart-1 antigen-specific T cells in donor blood samples were detected and retrieved using the CyteFinder® instrument. Anti-influenza specificity was confirmed by RNA sequencing, which revealed a majority of alpha/beta TCR pairings with identical match to literature reports. Comparative gene expression analysis of activated and control flu tetramer-positive T cells revealed 73 significantly down-regulated and 687 significantly up-regulated genes. Pathway analysis of differential expression revealed involvement of TCR signaling and inflammatory response/cytokine signaling.

Conclusions. The RareCyte platform can be used to visually identify and retrieve rare antigen-specific T cells from a bulk population by using tetramers against influenza-specific T cell receptors. T cell receptor sequencing confirmed the flu-specific identity of the tetramer-positive cells. RNA signatures of activation were identified at the single T cell level after peptide stimulation.

#1212

Anti-SIRPα antibodies as a potential weapon for cancer immunotherapy via accelerated screening in humanized mouse models.

James Jin,1 Benny(Yi) Yang,2 Chaoshe Guo,2 Yuelei Shen,2 Jian Ni3. 1 _Biocytogen, Wakefield, MA;_ 2 _Biocytogen, Beijing, China;_ 3 _Eucure, Beijing, China_.

Signal regulatory protein α (SIRPα) is a transmembrane protein with an extracellular region comprising three Ig-like domains and a cytoplasmic region containing immunoreceptor tyrosine-based inhibition motifs which mediate binding of the protein tyrosine phosphatases SHP1 and SHP2. SIRPα is especially abundant in myeloid cells such as macrophages and DCs, whereas it is expressed at very low levels in T, B, NK, and NK T cells. SIRPα inhibits phagocytosis in macrophages upon interacting with its ligand CD47, which is commonly upregulated on the surface of malignant cells. Thus, antibodies that block the CD47-SIRPα interaction should enhance macrophage phagocytosis in the tumor microenvironment and inhibit tumor growth, making anti-SIRPα antibodies promising as potential new tools for cancer immunotherapy. We are interested in subverting the negative feedback of the CD47-SIRPa axis, in order to treat cancer patients. Using Biocytogen's antibody research platform, we obtained several high-affinity anti-human SIRPα mAb by high-throughput flow cytometry screening. Next, these murine antibodies were screened directly in single or double humanized mice models (B-hSIRPα or B-hSIRPα/hCD47) bearing a syngeneic humanized cancer cell line (MC38-hCD47), where we discovered candidates that have potent anti-tumor activity in vivo. Chimeric antibodies and humanized antibodies based on these candidates were generated and further examined in vivo. In addition, we uncovered synergistic anti-tumor activity of anti-human SIRPα mAbs in combination with anti-mouse PD-1 mAb in B-hSIRPα/hCD47 mice. In brief, our existing studies demonstrate that SIRPα is an ideal target for preclinical cancer models. Clinical evaluation of the translational merits for these novel anti-SIRPα antibodies is pending.

#1213

RNA-based screens in primary human immune cells.

Bronwyn Joubert, Cristina Ghirelli, Isabelle Nett, John Prime, Glynn Martin, Jonathan Moore, Benedict Cross, Nicola J. McCarthy. _Horizon Discovery Ltd, Cambridge, United Kingdom_.

Genetic screens have long been used as an approach to identify and validate new targets for drug discovery. The vast majority of these have been carried out in cell lines: mostly cancer cell lines. However, with improvements in tissue culture techniques, the increasing interest in using the immune system to tackle disease and the discovery of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 mediated genome editing, screening primary cells that have not been subverted by transformation into immortal lines, is both appealing and feasible.

We have successfully carried out a CRISPR-Cas9 screen in primary T cells using a combined lentivirus and electroporation protocol. Freshly isolated primary T cells are stimulated with anti-CD3 and anti-CD28 antibodies and then transduced with a pooled sgRNA library. After antibiotic selection, T cells successfully transduced with sgRNAs are electroporated to introduce Cas9 mRNA.

We chose to validate this approach by carrying out a screen similar to that published by Birsoy et al., 2015, in which they ran a CRISPR-Cas9 screen in Jurkat T cells in the presence and absence of the electron transport chain inhibitor phenformin. In our screen, we exposed the CRISPR-Cas9 edited pool of primary T cells to a dose of phenformin that resulted in growth inhibition to a similar degree to that used by Birsoy and colleagues. Our data are in agreement with the published screen, showing that loss of the cytosolic aspartate aminotransferase GOT1 sensitises primary T cells to phenformin. We took multiple time points in our primary T cell screen and used T cells isolated from three different donors, allowing for the analysis of guide drop-out kinetics and reproducibility between donors.

We anticipate that these data will be useful in building more complex screens that assess T cell biology in the presence of additional cells, such as myeloid derived suppressor cells (MDSCs). With a view to this, we have also carried out an arrayed siRNA screen in MDSCs to look for genes that when knocked down reduce the capacity of MDSCs to inhibit T cell proliferation. The endpoint for this screen is based on co-culture of siRNA transfected MDSCs with proliferating primary T cells.

Using this complex data set, we have identified several potential targets, which when validated could provide new therapeutic targets through which the immunosuppressive nature of MDSCs in the tumour microenvironment can be mitigated.

Birsoy, K., et al. (2015) http://dx.doi.org/10.1016/j.cell.2015.07.016

#1214

Enhancement of anti-PD-1 antitumor efficacy in syngeneic preclinical models by the angiogenesis inhibitor lucitanib.

Rachel L. Dusek, Liliane Robillard, Thomas C. Harding, Andrew D. Simmons, Minh Nguyen. _Clovis Oncology, Inc., Boulder, CO_.

Background: Lucitanib is a small molecule inhibitor of multiple tyrosine kinases, including some that promote angiogenesis. Lucitanib monotherapy has demonstrated potent tumor growth inhibition in xenograft models of various carcinomas, mediated predominately through the inhibition of angiogenesis. In addition to its role in angiogenesis, vascular endothelial growth factor (VEGF) and other angiogenic factors are thought to dampen antitumor immune responses by promoting inhibitory immune subsets, suppressing dendritic cell maturation, and altering lymphocyte development and trafficking. In these studies, we further characterized lucitanib's kinase selectivity and investigated the antitumor efficacy of lucitanib in combination with anti-PD-1 therapy.

Methods & Results: The inhibitory profile of lucitanib was evaluated against 376 wild-type kinases in functional enzymatic assays. At 500 nM, lucitanib demonstrated >35% inhibition of 5.1% (19/376) of the kinases evaluated. The IC50 values of lucitanib against VEGF receptors 1-3 (VEGFR1-3), platelet-derived growth factor receptors alpha/beta (PDGFRα/β), and fibroblast growth factor receptors 1-3 (FGFR1-3) ranged from 8.96 to 95.7 nM. In addition, inhibition was observed against DDR1, CSF1R, RET, and KIT. Cell-based kinase assays confirmed that lucitanib is a potent and selective VEGFR1-3, PDGFRα/β, and FGFR1-3 inhibitor. Lucitanib antitumor activity was evaluated in combination with an anti-PD-1 antibody in a panel of syngeneic murine models. The combination treatment was generally well tolerated. In the H22 hepatocellular carcinoma model, the tumor growth inhibition in animals treated with lucitanib (10 mg/kg once daily), anti-PD-1 (5 mg/kg biweekly), or the combination was 58.6%, 50.4%, and 81.4%, respectively, following 16 days of treatment. The median survival time (MST) for lucitanib and anti-PD-1 treated animals was 35.0 and 46.5 days, respectively, whereas the MST had not been reached for the combination group when the study was terminated on day 63. Importantly, the combination treatment resulted in 2/10 mice that were tumor free at the end of the study; however, there were no tumor-free animals in the groups treated with the single agents. Antitumor efficacy was also greater with the combination of lucitanib and anti-PD-1 therapy than with treatment with the single agents in the MC38 and CT26 syngeneic murine models of colon cancer.

Conclusions: Lucitanib demonstrated potent and selective targeting of VEGFR1-3, PDGFRα/β, and FGFR1-3 and enhanced antitumor activity when combined with PD-1 inhibition in multiple syngeneic models. Results from these studies support the clinical development of combined lucitanib and anti-PD-1 treatments to further evaluate the safety and efficacy of the combination in multiple tumor types.

#1215

Improved T cell activation bioassays for development of bispecific antibodies and engineered T cell immunotherapies.

Pete Stecha, Denise Garvin, Jim Hartnett, Brad Swanson, Frank Fan, Mei Cong, Zhi-jie Jey Cheng. _Promega, Madison, WI_.

T Cell-mediated immunity is driven by activation of T cell which results in long-term, antigen-specific, effector and memory responses. In recent years, a variety of immunotherapy strategies aimed at inducing, strengthening or engineering T cell responses have emerged as promising approaches for the treatment of diseases such as cancer and autoimmunity. Current methods used to measure TCR-mediated T cell proliferation and cytokine production rely on primary PBMCs as a source of T cells, which must be stimulated via co-culture with APCs or anti-TCR/CD3 antibodies. These assays are laborious and highly variable due to their reliance on donor primary cells, complex assay protocols and unqualified assay reagents. As a result, these assays are difficult to establish in quality-controlled drug development settings. To overcome this barrier, we developed two reporter-based bioluminescent T cell activation bioassays that can be used for the development of bispecific antibodies and engineered T cell immunotherapies. The assays consist of Jurkat T cells genetically engineered to express luciferase downstream of either NFAT or IL-2 response elements. The T cell activation bioassays reflect the mechanisms of action of biologics designed to induce TCR and/or CD28-mediated T cell activation, as demonstrated using anti-CD3 and/or anti-CD28 antibodies as well as blinatumomab, a bispecific antibody that simultaneously binds CD3 expressed on T cells and CD19 expressed on malignant B cells. The bioassays are pre-qualified according to ICH guidelines and show assay specificity, precision, accuracy and linearity required for routine use in potency and stability studies. Finally, our data illustrate the use of reporter-based T cell activation bioassays for characterizing and measuring the activity of engineered chimeric antigen receptor T cells.

#1216

Novel strategies in cancer immunotherapy: Harness the hERG1 &#9461 macromolecular complex via a new bispecific antibody and its bifunctional TRAIL.

Claudia Duranti, Jessica Iorio, Stefano Coppola, Giulia Petroni, Tiziano Lottini, Lara Magni, Elena Lastraioli, Annarosa Arcangeli. _University of Firenze, Firenze, Italy_.

Introduction Among hindrances in cancer treatment, the lack of appropriate markers to be exploited for targeted therapy, and the need of new potential drugs are two big challenges.hERG1 potassium channels are a novel class of oncological targets and, in cancer, they are known to interact with integrins. It has been recently demonstrated that macromolecular complexes formed between hERG1 and &#9461 integrins selectively occurs in many types of cancer (Becchetti A et al., 2017). In this scenario, hERG1 could be exploited as a therapeutic target providing non cardiotoxic strategies aimed at blocking hERG1.

Materials and Methods A scDb, a bifunctional single-chain diabody, directed against hERG1/&#9461 complex, was developed via SOE-PCR methodology. Such antibody was tested on HCT116 cells in lateral motility and western blotting experiments. Moreover immunohistochemistry (IHC) was performed on metastatic colorectal cancer (mCRC) paraffin embedded samples using the scDb, an anti-hERG1 and an anti-β1 integrin. Moreover, we improved the pro-apoptotic effects of the nude hERG1/β1-scDb, developing a trifunctional recombinant protein, in which the scDb-hERG1-&#9461 was linked to the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL).

Results and Discussion Performing IHC on sequential sections of mCRC confirmed the specificity of the scDb for both hERG1 and &#9461 integrin. In vitro data provide evidences that the administering of the bispecific antibody has an impact on lateral motility. Moreover, signaling pathways are also affected by the antibody treatment, as AKT phosphorylation and HIF1&#945 levels are decreased when the molecule is administered, suggesting a possible effect of the bispecific antibody on the VEGF-A signaling pathway, which are consistent with our previous hypothesis (Becchetti A et al., 2017) of a possible cross-talk leading to a deep impact on VEGF expression and, thus, on neoangiogenesis. Encouraging results were obtained when testing the scDb-hERG1-&#9461 -TRAIL fusion protein, which was capable to produce a strong induction of apoptosis in ALL cells (32.6 vs 11% at 24h; 62.4% vs 10% at 48h), with no effects on TRAIL- resistant (Panc1) tumour cells.

Conclusions scDb-hERG1/β1 could be used as a potential new therapeutic tool for cancer patients' treatment as well as for early molecular diagnosis. In fact, the selective expression of hERG1/β1 complex in cancer cells and its role in angiogenesis and cancer progression suggests that a molecule selectively targeting the complex will be an invaluable tool for cancer treatment. In this view, we have recently licensed a patent which will be exploited with the final aim to undergo clinical trials. Becchetti A, et al. (2017) Science Signaling 10(473). Patent. Inventors: Arcangeli A, Duranti C. et al. Patent Ref: 102017000083637

#1217

Exploiting host pre-existing immunity for melanoma cancer immunotherapy.

Priscilla S. Briquez, Sylvie Hauert, Grégoire Repond, Melody A. Swartz, Jeffrey A. Hubbell. _University of Chicago, Chicago, IL_.

Vaccines are considered one of greatest successes of modern medicine; indeed, prophylactic vaccinations allow tight control of disease spreading and even eradication of some diseases. Vaccines immunize patients against known antigens derived from infectious agents, so that the immune system can efficiently block pathogen entry and mount a faster and better immune reaction in case of infection. In the context of cancer, finding good antigens for immunization against tumor cells can be challenging since tumor cells are derived from the self, and thus are not always efficiently recognized as malignant by the immune system. Here, we seek to develop a cancer immunotherapy that delivers a pre-encountered antigen to tumor cells to redirect the host pre-existing immunity against cancer. As a vaccine model, we used ovalbumin (OVA) antigen adjuvanted with CpG-B oligodeoxynucleotides to pre-immunize C57BL/6 mice, developing both T cell and B cell immunity against OVA. At least one month later, mice were challenged with B16-OVA melanoma cells. We observed that tumor growth in pre-immunized mice was slightly delayed compared to growth in naïve mice, but this effect was modest, leading to an overall 1-day increased survival. We reasoned that this effect could be improved by modifying the cellular localization of OVA antigen; indeed, OVA is expressed intracellularly in B16-OVA cells, thus excluding potential antibody-mediated anti-tumoral immune response. Therefore, we created a B16 melanoma cell line that overexpresses a membrane-bound OVA (B16memOVA), using similar design as published elsewhere (DiLillo et al., J. Immunol. 2010). In vitro, B16memOVA cells behaved similarly to the parental B16 cell line, and the growth of B16memOVA tumors in OVA-expressing mice (which are tolerant to OVA) was similar to the growth of B16 tumors, thus validating our B16memOVA tumor model. When implanted in naïve mice, B16memOVA tumors were able to grow, despite the onset of a neo-immune reaction against OVA, which slows tumor growth. In contrast, mice pre-immunized against OVA totally rejected B16memOVA tumors. Our efforts are now focusing on the development of an antigen-delivery method to trigger the expression of membrane-bound OVA in B16 tumors after implantation, to apply this approach in a therapeutic setup.

#1218

EMP2 serves as a novel target for immunotoxin mediated ovarian cancer therapy.

Khalid Mohamedali,1 Su-Yin Kok,2 Lawrence H. Cheung,1 Michael G. Rosenblum,1 Madhuri Wadehra2. 1 _MD Anderson, TX;_ 2 _UCLA David Geffen School of Medicine, Los Angeles, CA_.

Ovarian cancer (OC) currently accounts for only ~2% of all new cancer cases per year, but once diagnosed, patients only show a 5 year survival of 30%. Subdivided into four subtypes: serous, endometrioid, clear cell, and mucinous carcinoma, high-grade serous ovarian cancer (HGSOC) accounts for 70-80% of the ovarian cancer deaths. Immunoconjugates represent an innovative therapeutic approach to deliver a deadly payload to a tumor cell with more specificity and potentially less avenues for resistance than either can exert individually. However, it has been a challenge to find candidates as well as implement conjugation strategies to achieve these goals for a number of cancers. Epithelial membrane protein-2 (EMP2) is over-expressed in a number of cancers in women including 70% of ovarian tumors, with up to 88% of advanced serous carcinomas showing high expression of the antigen. With an eye towards therapy, we have recently developed an IgG1 targeting the extracellular domain of EMP2 with single digit nM affinity. As the antibody-antigen complex rapidly internalizes, we sought to improve on the efficacy of the naked antibody. Granzyme B (GrB) is a serine protease that plays a critical role in the body's defense against viral infection and tumor development by initiating the apoptotic cascade via both caspase-dependent and -independent mechanisms. Accordingly, we developed novel fusion proteins composed of the anti-EMP2 backbone as the targeting moiety and granzyme B as the cytotoxic payload. The designs comprised (a) the anti-EMP2 IgG1 with GrB fused to the Fc domain through a cleavable linker, and (b) active GrB fused to the IgG heavy chain (Fc) domain containing 2 single-chain antibodies against EMP2, a format that results in a longer plasma retention time. The constructs were expressed by transient transfection in HEK293E cells and purified to homogeneity. Flow cytometry and ELISA demonstrated binding properties of the fusion protein with high specificity and sensitivity to EMP2 expression. The enzymatic activity of the fusion construct was similar to commercially-available GrB. Internalization of the immunotoxins was confirmed, and they showed high cytotoxicity to EMP2-positive cells compared to EMP2-negative cells. We predict that these designs may improve therapeutic outcomes for EMP2+ tumors.

#1219

Generation of anti-OX40 antibody with different immunization strategies.

Xiaojuan Chai,1 Wenfang Xin,1 Teddy Yang,1 Qing Duan,2 Louis Liu1. 1 _ChemPartner, Shanghai, China;_ 2 _Hyamab Pharmaceutical Limited, Shanghai, China_.

Variety of immunogen such as recombinant protein, DNA or cells has been used for animal immunization to develop hybridoma antibody. However, the effectiveness among different types of immunogen in generating functional antibodies especially antibody with agonistic activity has not been systematically investigated.

OX40 (CD134) is a T cell costimulatory receptor that is found primarily on activated CD4+ and CD8+ T cells, regulatory T (Treg) cells and natural killer (NK) cells. Activation of OX40 by its natural ligand or an agonist antibody augments CD4 and CD8 T cell expansion, differentiation, and survival. Agonistic anti-OX40 antibody holds promise as therapeutics for cancer immunotherapy.

In the present study, different immunization strategies (recombinant protein, cell and DNA) have been used to generate anti-OX40 antibody. Antigen specific immune response, number of binders, epitope bins and the functional activities obtained from different immunogens were analyzed and compared. Based on cross reactivity and cell based functional assay results, 25 clones from protein immunization, 22 from DNA immunization and 29 from cell based immunization were expanded and purified. Among the purified antibodies, antibodies obtained from DNA immunization showed stronger agonist activity in both Jurkat OX40-NFAT luciferase reporter activity and primary T cell activation assay. The antibodies obtained from DNA immunization also demonstrated in vivo efficacy and significantly inhibited tumor growth in MC38 syngeneic mouse model using human OX-40 knock-in mice.

In summary, we have evaluated different immunization strategies using OX-40 as a case study and successfully generated high potency high affinity agonistic anti-OX40 antibody that support further development.

#1220

High-resolution epitope mapping differentiates monoclonal antibodies in the crowded cancer immunotherapy space.

Sharon Willis. _Integral Molecular, Philadelphia, PA_.

Epitope mapping provides essential information for differentiating monoclonal antibodies (MAbs) that bind to proteins targeted by many competing drugs. In cancer immunotherapy, for instance, numerous approved or investigational MAbs target the immune checkpoint protein PD-1. Recent high-profile patent law cases have centered on epitope data, highlighting the importance of distinguishing MAb binding sites at amino-acid resolution. Additionally, epitope data can be used to distinguish the mechanisms by which MAbs exert their therapeutic effects, uncovering important differences between competing drugs. To achieve rapid and comprehensive conformational epitope mapping at the amino-acid level, we developed the Shotgun Mutagenesis Epitope Mapping platform. Every residue in a target protein is individually mutated by alanine scanning mutagenesis, expressed in human cells, and assayed by flow cytometry to determine the critical epitope residues. Data from our platform have been used to differentiate MAbs in terms of their binding residues and therapeutic mechanisms of action. We have used our platform to differentiate the unique binding residues and, in some cases, the mechanisms of action of novel MAbs on the crowded surfaces of cancer targets, such as HER-2, from those of benchmark MAbs.

#1221

Mitomycin C induces programed cell death ligand 1 overexpression but enhances anticancer immunity in none small cell lung cancer.

Min Luo. _Sun Yat-Sen Univ. Cancer Ctr., Guangzhou, China_.

Blockade of the checkpoint inhibitor programmed death 1 ligand (PDL1) has demonstrated remarkable success in the clinic for the treatment of cancer; however, a majority of tumors are progressive with low response rate. To enhance the efficacy of PDL1 blockade immunotherapies, we tried to combine chemotherapy capable of enhancing antitumor immunity. Here, we screened that tumor cells with MMC pre-treatment was significantly killed by co-cultured lymphocytes than without ones. Indeed, tumor cells treated with MMC showed the increased release of granzyme B and TNF-α. And tumor lysis was enhanced by combined with PD1 Ab in vitro. Mice treated with both anti-PDL1 and MMC showed an improved overall survival and inhibition within the tumor, indicating that this combination improves the overall quality of the immune response generated. Mechanically, MMC was observed to upregulate PDL1 expression in a concentration and time manner. MMC activated c-JUN to bind with PDL1 promoter and recruited its co-factor, STAT3 to accelerate PDL1 expression. MMC induced an IFN-γ independent signature within the tumor cell, and ERK signaling pathway was activated. A reason for its enhancement in antitumor immunity was the high expression of MHC-I after MMC treatment which attracted more CD8+ T cells. These findings identified a potential mechanism for the observed benefit of combining MMC and PDL1 blockade, in which MMC induces PDL1 upregulation and MCH-I high expression, thereby converting the tumor cell into an immunogenic one, when block PDL1, the antitumor response was enhanced.

#1222

Caspase mediated prodrugs RGDEVD-DOX with EMC-DEVD-S-DOX in combination with anti PD-1 antibody for triple negative breast cancer.

Ha Rin Kim,1 Seung Woo Chung,2 Youngseok Cho,1 Youngro Byun1. 1 _Seoul National University, Seoul, Republic of Korea;_ 2 _Johns Hopkins University School of Medicine, Baltimore, MD_.

Here we suggest a new effective strategy to overcome tumor heterogeneity for eradicating metastatic triple negative breast cancer (TNBC). As a first strategy, we applied a combination therapy of caspase-3 mediated "targeting" (RGDEVD-DOX) and "maintaining" (EMC-DEVD-S-DOX) prodrugs, given in a sequential manner. RGDEVD-DOX is designed to target integrin αvβ3 and induce apoptosis specifically in tumor cells. EMC-DEVD-S-DOX binds to circulating albumin after administration to show enhanced half-life and accumulates at tumor site. In the apoptotic tumor tissue, caspase-3 releases free doxorubicin from the prodrug and this induces indiscriminate killing of surrounding tumor cells regardless of the integrin αvβ3 expression, resulting in continuous activation of prodrug. As another strategy, RGDEVD-DOX, by specifically targeting tumor cells, shows minimal systemic immunosuppression unlike conventional doxorubicin therapy and we expect that its immunomodulatory effects can synergize the therapeutic benefit of αPD-1 antibody. In order to determine the efficacy of RGDEVD-DOX and EMC-DEVD-S-DOX combination, we performed in vivo efficacy test using two TNBC xenograft models. In case of MDA-MB-231 and 4T1 xenograft models, combination therapy showed 80% (p=0.0006) and 75% (p<0.0001) of tumor growth inhibition respectively compared the control group. In order to evaluate the anti-metastatic effect of combination therapy, we prepared TNBC metastasis model. We found that the combination therapy significantly attenuated metastatic incidence compared to monotherapy groups. This result implies that RGDEVD-DOX targets metastatic tumor and EMC-DEVD-S-DOX maintains the anti-metastatic effects in a consecutive fashion. To identify a synergistic effect of RGDEVD-DOX combining immunotherapy, we carried out combination therapy of αPD-1 and RGDEVD-DOX in TNBC mouse model. We assessed the tumor inhibition and evaluated anti-metastatic effect in lung by measuring bioluminescence intensity of 4T1-Luciferase cells. We found that the combination therapy exerted not only inhibitory effect of primary tumor but also anti-metastatic effect. In conclusion, we verified that combination therapy of RGDEVD-DOX with EMC-DEVD-S-DOX or αPD-1 showed synergistic effect in TNBC mouse model. We demonstrate that the sequential combination therapy successively reduces the incidence of metastasis and prolongs survival in TNBC model. Collectively, we propose that RGDEVD-DOX is a promising caspase-3 mediated prodrug in respect of overcoming tumor heterogeneity and stimulating a tumor-specific immune response, which can synergize the efficacy of immunotherapy.

### Cellular Responses to Anticancer Agents 1: The Microenvironment and Metastasis

#1223

Somatic mutations in collagens impact patient outcomes in cancer.

Alexander S. Brodsky,1 Jay Khurana,1 Dongfang Yang,1 Ece Gamsiz,1 Ian Wong,2 Murray Resnick1. 1 _Rhode Island Hospital/Brown Univ. Medical School, Providence, RI;_ 2 _Brown University, Providence, RI_.

Stomach cancer remains among the most lethal diseases around the world demanding improved treatments. The tumor microenvironment (TME) is a critical driver of tumor

progression and response to therapy and mediates many of the cancer hallmarks. Collagens are major components of the TME that controls disease progression and mediate therapeutic response. Collagens have been associated with stiffening of the tumor stroma, malignancy and therapeutic resistance. Collagenopathies are caused by loss of function collagen mutations that disrupt tissue formation and are dominant negative variants. We have found somatic tumor variants in collagens that are similar loss-of-function mutations as the germline variants observed in collagenopathies, providing guidance to interpret these cancer mutations. Indeed, the role of somatic mutations in collagens has not been addressed. Remarkably, we found multiple independent lines of evidence that collagen variants impact stomach adenocarcinoma tumors, consistent with a model that collagen variants disrupt the extracellular matrix (ECM) leading to improved therapeutic responses. 1) We find that loss-of-function collagen missense and nonsense mutations are associated with improved survival and disease-free progression times in gastric tumors in the TCGA dataset. 2) The same type of mutations observed in cancer are known to cause rare human diseases and therefore have the potential to impact cell function. 3) Independent of the patient outcome data, collagen variants were associated with significant changes in expression of tumor environment factors including ECM glycoproteins and EMT expression signatures. 4) Remapping RNA-seq reads reveals that single nucleotide variants of the collagens, matching the exome sequencing data are expressed. We will present computational and functional data supporting the hypothesis that somatic variants impact gastric tumors as well as analysis of the impact of collagens somatic mutations PanCancer. Somatic mutations in collagens represent a new paradigm for how cancer cells mediate the TME providing new biomarker and therapeutic opportunities to disrupt the ECM and improve outcomes for cancer patients.

#1224

Correlation of tumor angiogenesis with peripheral ring-enhancement on CT: Its predictive value of chemotherapy responses of colorectal metastatic hepatic tumor.

Hirotaka Okamoto,1 Shugo Shiba,2 Hiroshi Iino,2 Makoto Sudoh,1 Hiroyuki Wakana,1 Suguru Maruyama,1 Kenji Kawashima,3 Toshio Fukasawa,3 Daisuke Ichikawa,1 Hideki Fujii2. 1 _Univ. of Yamanashi Faculty of Medicine, Chuo-city, Yamanshi, Japan;_ 2 _Kofu Municipal Hospital, Kofu-city, Yamanshi, Japan;_ 3 _Tsuru-Municipal Hospital, Tsuru-city, Yamanshi, Japan_.

[Background]The treatment of colorectal cancer (CRC) with metastasis is now widely accepted to be systemic chemotherapy of 5-FU base chemotherapy (FOLFOX or FOLFILI) in combination with molecular antibody. The objective of this study was to determine the significance of tumor angiogenesis in metastatic hepatic tumors in relation to the response of systemic chemotherapy.

[Methods]A total of 48 consecutive CRC patients with hepatic metastasis who had been treated with or without hepatic resection were retrospectively reviewed, and factors, such as metastatic tumor angiogenesis and chemotherapy response, were analyzed. Tumor angiogenesis in association with ring-enhancement (RE) on computed tomography (CT) was also microscopically evaluated by microvessel counting (MVD) in sections stained immunochemically with CD34.

[Results]The overall response rate after 6 courses of first-line chemotherapy for the liver metastasis with RE on CT was 64% (23/36), whereas the response rate without RE was 25% (3/12), which was significantly different. The survival rate of patients with or without liver resection of all RE-positive tumors was not different, but the survival rate of patients with liver resection was longer than that of patients without liver resection for all RE-negative tumors. Microscopic examination revealed that peripheral RE on CT of the metastatic tumor was associated with tumor angiogenesis by MVD.

[Conclusions]The peripheral RE on CT of the metastatic hepatic tumor was associated with tumor angiogenesis and may predict chemotherapy response. A combination of liver resection with chemotherapy improves the survival of patients who have multiple hepatic metastasis.

#1225

Aberrant SMAD3 signaling in tumor-associated fibroblasts modulates fibrosis and response to the anti-fibrotic drug nintedanib in non-small cell lung cancer.

Rafael Ikemori,1 Marta Gabasa,1 Miguel Vizoso,2 Paula Duch,1 Sebastian Moran,2 Sabrina Gea-Sorli,3 Paloma Bragado,3 Toni Jauset,4 Manel Esteller,2 Laura Soucek,4 Eduard Monsó,5 Víctor Peinado,3 Cristina Fillat,3 Frank Hilberg,6 Noemí Reguart,7 Jordi Alcaraz1. 1 _Univ. of Barcelona, Barcelona, Spain;_ 2 _Bellvitge Biomedical Research Institute-IDIBELL, Barcelona, Spain;_ 3 _Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain;_ 4 _Vall d'Hebron Institute of Oncology (VHIO), Barcelona, Spain;_ 5 _Hospital Parc Taulí de Sabadell, Sabadell, Spain;_ 6 _Boehringer-Ingelheim Inc, Vienna, Austria;_ 7 _Hospital Clínic de Barcelona, Barcelona, Spain_.

A hallmark of non-small cell lung cancer (NSCLC) is a fibrotic/desmoplastic stroma rich in activated fibroblasts, which are critical regulators of cancer progression, response to therapies and radiotherapy resistance. Paradoxically, we recently reported that the important pro-fibrotic TGF-β transcription factor SMAD3 was epigenetically down-regulated through promoter hypermethylation in tumor associated fibroblasts (TAFs) from NSCLC patients compared to patient-matched control fibroblasts. In addition, we reported that the clinically approved antifibrotic drug nintedanib elicited a stronger inhibition of both the fibrotic phenotype and its associated tumor-promoting effects in TAFs from adenocarcinoma (ADC) patients compared to squamous cell carcinoma (SCC) patients upon TGF-β1 stimulation in vitro, which was consistent with the selective therapeutic response to nintedanib observed in a clinical trial in ADC (but not SCC) patients. Altogether, these previous results suggest that TGF-β1 signaling may be altered in lung TAFs, and that such alteration may depend on their histologic subtype. In this study we addressed these questions by determining the expression and activity of SMAD3 and its closely related homologue SMAD2 in patient-derived TAFs and paired control fibroblasts, and by dissecting their potential contribution to the differential therapeutic responses to nintedanib observed in ADC and SCC. In vitro studies revealed a marked SMAD3 epigenetic repression through promoter hypermethylation, a low pSMAD3/pSMAD2 ratio and a limited fibrotic phenotype selectively in SCC-TAFs. In contrast, ADC-TAFs overexpressed a panel of fibrotic markers upon TGF-β1 stimulation concomitantly with a high pSMAD3/pSMAD2 ratio and a limited SMAD3 promoter methylation. Histologic analysis of a large patient cohort (112 ADC, 96 SCC) confirmed that the extent of fibrosis is larger in ADC than SCC patients. In addition, knocking-down SMAD3 in ADC-TAFs was sufficient to reduce the antifibrotic and antigrowth effects of nintedanib in vitro and in tumor xenografts in vivo. On the other hand, long-term exposure of pulmonary fibroblasts to cigarette smoke condensate was sufficient to hypermethylate the SMAD3 promoter. Since SCC and ADC tumors typically arise in the upper airways and distal pulmonary sites, respectively, it is conceivable that fibroblasts might be more exposed to the smoking epigenetic effects on SMAD3 in SCC. In summary, we report for the first time that tumor fibrosis is higher in ADC than SCC patients, in association with a selective therapeutic response to the antifibrotic drug nintedanib in the former, and identify the subtype-specific extent of SMAD3 epigenetic repression in TAFs and the subsequent aberrant SMAD3/SMAD2 imbalance as major regulatory mechanisms of tumor fibrosis and response to nintedanib in NSCLC.

#1226

A novel polymer-conjugated methionine aminopeptidase 2 (MetAP2) inhibitor SDX-7320 inhibits the growth of EO771 mammary gland tumors and ameliorates the immunosuppressive tumor immune micro-environment (TIME).

Peter Cornelius,1 Benjamin Mayes,1 Sara Little,2 Andrew Slee,3 David Turnquist,1 James Shanahan,1 Bradley Carver1. 1 _Syndevrx Inc., Cambridge, MA;_ 2 _Neosome Life Sciences, Lexington, MA;_ 3 _Cove Bioconsultants, Shrewsbury, MA_.

Obese patients with certain cancers face a greater risk of dying from their cancer compared to non-obese patients. Obesity may contribute to cancer progression via multiple mechanisms: increased leptin, decreased adiponectin, increased adipose tissue estrogen, elevated insulin (secondary to peripheral insulin resistance), increased cytokines, and alteration of the TIME which suppresses host anti-tumor immune responses. Small molecule MetAP2 inhibitors have previously shown clinical anti-tumor activity. However, clinical development in some cases (e.g. TNP-470) has been altered CNS toxicity. SDX-7320 is a polymer-drug conjugate of a novel fumagillin-derived MetAP2 inhibitor (SDX-7539) attached via a cleavable linker to a hydroxypropylmethacrylamide (HPMA) backbone. This is intended to limit CNS penetration and reduce CNS toxicity relative to small molecules. SDX-7320 was tested ± 5-FU in a syngeneic model of post-menopausal, obesity-accelerated breast cancer. Ovariectomized C57Bl/6 mice were fed a high-fat diet (to induce obesity) or low-fat diet for 16 weeks prior to injection of EO771 tumor cells into the mammary gland. Tumors in obese mice grew significantly faster than in low fat-fed (normal) mice. SDX-7320 (8 mg/kg, SC, Q4D) significantly attenuated tumor growth in normal and obese mice relative to vehicles (-33% and -43% respectively). 5-FU (10 mg/kg, IP, 2X/week) had no effect on tumor growth in normal or obese mice, while the combination of 5-FU with SDX-7320 inhibited tumor growth in obese mice greater than SDX-7320 alone (-61% vs -43% respectively), suggestive of a synergistic interaction. In a separate study, the effect of SDX-7320 on the EO771 TIME in obese versus normal-weight mice was assessed. Tumors in obese mice grew significantly faster than in normal mice. SDX-7320 (24 mg/kg, SC, Q4D) decreased tumor growth in both normal and obese mice (-53% and -52% change in tumor volume relative to lean and obese vehicle groups, respectively). SDX-7320 also reduced the immunosuppressive state of the TIME in obese mice. Relative to tumors from untreated mice, MDSC content (flow cytometry), Arginase 1 staining (by IHC) and FoxP3 staining (by IHC) were all decreased. Analysis of cytokines showed that IL-10 was elevated in plasma of obese mice relative to normal mice, and SDX-7320 significantly reduced the plasma levels of IL-10 in obese mice. SDX-7320 exerted favorable effects on the TIME in a model of post-menopausal obesity-accelerated breast cancer suggesting it may potentiate the efficacy of immunotherapy in breast cancer. SDX-7320 is being developed to treat cancers whose growth is accelerated by obesity and metabolic dysfunction. Now nearing completion of Phase I (NCT02743637), further clinical trials with SDX-7320 in combination with other therapies are planned for 2019.

#1227

Immune modulatory effect of pramlintide for cutaneous squamous cell carcinoma treatment.

Leticia Tordesillas, Rebecca Hesterberg, Ivannie Ortiz-Rivera, Brittney R. Sell, Omar Chavez Chiang, Kimberly T. Nguyen, Brian L. Murphy, Pearlie K. Burnette, Elsa R. Flores, Kenneth Y. Tsai. _H. Lee Moffitt Cancer Center, Tampa, FL_.

Cutaneous squamous cell carcinoma (cuSCC) accounts for 15-20% of skin cancers. Treatment of cuSCC with pramlintide, a synthetic analog of the hormone amylin, is currently under investigation in mouse models and human clinical trials. In cancer cells, pramlintide inhibits glycolysis, resulting in cell death and tumor regression. In order to investigate the potential use of pramlintide in combinatory therapies with checkpoint inhibitors, we aimed to study the immune modulatory effect of pramlintide in an immune competent mouse model of spontaneous ultraviolet radiation-induced cuSCC, as well as its effect on human T cells in vitro. To induce cuSCC, SKH-1 hairless mice were subjected to ultraviolet radiation until at least one tumor reached >4mm of diameter. Mice were then treated with 45 µg/kg of pramlintide or vehicle control for 10 days, every other day. Skin-draining lymph nodes, spleen and tumors were harvested for immunophenotyping of the T cell and myeloid compartment by flow cytometry. While the proportion of CD103+ dendritic cells (DCs) and CD3+ T cells remained constant after pramlintide treatment, a significant decrease in the proportion of both monocytic and granulocytic myeloid-derived suppressor cells (m-MDSCs and g-MDSCs) was observed relative to vehicle-control treated mice. Interestingly, pramlintide abolished m-MDSC differentiation from bone marrow progenitors in vitro suggesting that pramlintide may regulate a pathway necessary for m-MDSC development. Purified human CD3+ T cells activated in the presence of pramlintide exhibited a time-dependent increase in intracellular and secreted IFN-γ. Therefore, pramlintide may have immune modulatory effects based on both a reduction in myeloid-dependent suppression and a direct stimulatory effect on T cells that would culminate in superior anti-tumor T cell responses that augment immunotherapeutic approaches for the treatment of cuSCC.

#1228

FF-10850, a novel liposomal topotecan, achieved superior anti-tumor effects and a favorable safety profile via preferential distribution followed by dual payload release mechanisms in tumor.

Susumu Shimoyama, Ken Okada, Toshifumi Kimura, Noriyuki Kasagi, Shinji Nakayama, Keiko Makita-Suzuki, Shun Matsuda, Mikinaga Mori, Kiyohito Takada, Shinji Hagiwara, Takefumi Hara. _FUJIFILM Corporation, Kaisei-machi, Japan_.

[Introduction]

FF-10850, a novel liposomal topotecan, has shown superior anti-tumor effects and a favorable safety profile compared with topotecan or doxorubicin HCl liposome injection (lipo-dox). In the present study, we evaluated the tissue distribution, pharmacodynamics, and payload release of FF-10850 to further characterize mechanisms for enhanced activity and improved safety.

[Methods]

ES-2 ovarian cancer cells were inoculated into nude mice by subcutaneous or intraperitoneal injection. Topotecan concentration and the ratio of p-H2AX/H2AX were measured by LC-MS/MS in tumor and bone marrow, respectively. FF-10850-internalized cell types in tumor were classified by flow cytometry using fluorescent-labeled FF-10850. Topotecan released from FF-10850-internalized cells was quantified in cell culture supernatants by LC-MS/MS. Payload released from the liposomal membranes of either FF-10850 or lipo-dox was quantified by ultracentrifugation/LC-MS/MS following incubation in tumor interstitial fluid and ammonia-containing buffer.

[Results]

Kinetic analysis of topotecan concentration and p-H2AX/H2AX ratio in tumor and bone marrow, after FF-10850 or topotecan injection, revealed that FF-10850 exerted profound drug exposure and DNA damage, preferentially in tumor. These findings were consistent with superior anti-tumor effects and a favorable safety profile of FF-10850 previously observed in both subcutaneous tumor models and in a clinically relevant intraperitoneally disseminated tumor model. Immunophenotyping revealed internalization of FF-10850 by tumor associated phagocytes. Evaluation of payload release confirmed topotecan was released from FF-10850-internalized cells into the extracellular space, and additionally from FF-10850 but not lipo-dox liposomal membranes during incubation in tumor interstitial fluid. A higher concentration of ammonia detected in the tumor interstitial fluid compared to that in plasma. Ammonia-containing buffer was shown to induce payload release from FF-10850 but not from lipo-dox membranes.

[Conclusions]

These data suggest that FF-10850 preferentially accumulates in tumor, presumably via the enhanced permeability and retention effect, and efficiently releases payload through dual mechanisms in tumor. One mechanism is mediated by tumor associated phagocytes, which internalize FF-10850 and release payload into the extracellular space. An additional mechanism is mediated by ammonia, which is reported to be a metabolite in glutaminolysis activated by the metabolic shift in tumor cells. These characteristics of FF-10850 distribution and release may translate to enhanced anti-tumor effects of FF-10850 as observed in subcutaneous as well as intraperitoneally disseminated tumor models closely resembling clinical conditions.

#1229

Neoadjuvant therapy alters collagen architecture of pancreatic cancer.

Kosei Nakajima,1 Yoshinori Ino,1 Sotoshi Nara,2 Toshimitsu Iwasaki,2 Nobuyoshi Hiraoka1. 1 _National Cancer Center Research Institute, Tokyo, Japan;_ 2 _National Cancer Center Hospital, Tokyo, Japan_.

Treatment of pancreatic cancer remains clinically challenging and requires the novel therapeutic interventions to improve patient outcome. Neoadjuvant therapy i.e. preoperative treatments comprising chemotherapeutic agents with/without radiation offers down-staging and improvement of surgical resectability. Here, we report that neoadjuvant therapy alters collagen architecture of pancreatic cancer. Transcriptomic profiles of type 1 to type 28 collagens depicted changes of collagen composition due to neoadjuvant therapy. Immunohistochemically, the expressed area of collagen type 1, 3, 4, and 5 were significantly reduced post treatment. The bioinformatic approaches provided a comprehensive insight into treatment-induced matrix remodeling, which showed that Ephrin-A signaling and Ephrin-A5 appeared as a highly possible pathway and a crucial ligand. Ephrin-A5 co-localized with alpha-SMA(+) cancer-associated fibroblasts, whereby Ephrin-A5 have been implicated to synthesize collagens. The Ephrin-A5 positive cells significantly reduced after neoadjuvant therapy, also inversely correlated with tumor shrinkage rate. Using primary culture cells of cancer-associated fibroblasts, experimental exposure of radiation and chemotherapeutic agents suppressed the proliferation of cancer-associated fibroblasts, collagen synthesis and Ephrin-A5 expression. Thus, our studies demonstrate that neoadjuvant therapy down-regulates Ephrin-A5 expression, leading to a change of extracellular matrix structure. It should be a clinical concern of surgical handling when following resection.

#1230

Therapeutic targeting of hypoxia in experimental esophageal adenocarcinoma.

Md Sazzad Hassan,1 Marisa Lenga,2 Lucia Petrova,1 Urs von Holzen3. 1 _Indiana University School of Medicine-South Bend, South Bend, IN;_ 2 _University of Notre Dame, South Bend, IN;_ 3 _Indiana University School of Medicine-South Bend and Goshen Center for Cancer Care, South Bend and Goshen, IN_.

Introduction: Esophageal adenocarcinoma (EAC) has become the predominant type of esophageal cancer in the United States, and prognosis of EAC remains poor despite modern combination therapies. Therefore, new therapeutic approaches are urgently needed. Hypoxia or insufficient tissue oxygenation contributes to EAC aggressiveness and poor clinical prognosis. Typically, EAC cell lines are used in two-dimensional (2D) cultures for screening of anticancer agents, and they do not represent the hypoxic tumor microenvironment. In this study, we established a novel three dimensional (3D) spheroid co-culture model of EAC. We then observed treatment response of chemo and hypoxia-targeting therapies in 2D, 3D and in vivo models of experimental EAC.

Methods: EAC 3D spheroids were generated from co-culturing human EAC and fibroblast cell lines. NanoCulture® plates and dishes were used for 3D spheroid cultures. Hypoxic status was detected by adding hypoxia probe LOX-1 and fluorescent microscopy. Paclitaxel (PT), carboplatin (CP) and nanoparticle albumin-bound paclitaxel (NPT) were used as chemotherapeutic agents, whereas acriflavin was used as hypoxia-targeting agent. In vitro cell growth was detected by WST-1 assay, in vivo tumor growth was detected by measuring subcutaneous xenograft using OE19 EAC cells, apoptosis was detected by analysis of cleaved caspase 3 and PARP expressions, hypoxia-targeting was detected by analysis of HIF-1α expression and stem cell phenotype was detected by flow cytometric analysis of aldehyde dehydrogenase (ALDH) activity.

Results: The results demonstrated that the 3D culture was more resistant to antiproliferative/proapoptotic effects of chemotherapeutic agents PT, CP, NPT and their combinations over 2D monolayer culture. Contrary to that, hypoxia-targeting agent acriflavin showed stronger antiproliferative/proapoptotic effects in 3D culture than in 2D culture. We observed strong expression of hypoxia inducible factor-1α (HIF-1α) in 3D culture with no expression of HIF-1α in 2D culture, and acriflavin treatment completely abolished the HIF-1α expression in 3D culture. We also observed hypoxia inside the 3D culture spheroids, but not in cells grown in 2D culture. Interestingly, we observed a reduced number of ALDH positive cells in 3D culture after acriflavin treatment compared to that after NPT treatment, indicating the preferential antiproliferative effect of acriflavin over NPT on cancer stem cells. In addition, acriflavin showed significant antitumor efficacy both as monotherapy and in combination with NPT. The xenograft tumor growth inhibition rate after a 2-week treatment with acriflavin, NPT and their combination was 44.97, 58.35 and 67.29 percent respectively (p<0.05).

Conclusion: Thus 3D cultures may be better than 2D cultures in simulating the important in vivo tumor characteristic of hypoxia, and HIF-targeting therapy acriflavin could be a novel treatment strategy for EAC.

#1231

Improving survival in pancreatic cancer using Doxorubicin in combination with L-DOS47.

Sultan Damgaci,1 Heman Chao,2 Marni D. Uger,2 Arig Ibrahim-Hashim,1 Eunjung Kim,1 Pedro Pedro M. Enriquez‐Navas,1 Dominique Abrahams,1 Alexander R. Anderson,1 Albert Guvenis,3 Robert J. Gillies1. 1 _Moffitt Cancer Center, Tampa, FL;_ 2 _Helix BioPharma Corporation, Richmond Hill, Ontario, Canada;_ 3 _Bogazici Universtiy, Istanbul, Turkey_.

BACKGROUND: The tumor microenvironment is acidic, which inhibits the activity of weakly basic drugs such as doxorubicin. In order to test whether neutralization of tumor acidity can improve survival in pancreatic cancer-bearing mice treated with doxorubicin, we tested doxorubicin in combination with L-DOS47. L-DOS47 is a novel therapy comprised of urease conjugated to an antibody against CEACAM6, which raises the pH in the tumor microenvironment by converting endogenous urea to NH3 and CO2. Our findings demonstrate that raising tumor pH with L-DOS47 can improve responses to chemotherapy. As L-DOS47 is currently in clinical testing for NSCLC, current study is clinically relevant. METHODS: Panc02 pancreatic cancer cell line (obtained from Emmanual Zervos, East Carolina University, Greenville, NC) was infected to express CEACAM6. 1 x 106 CEACAM6 Panc02 Clone were injected subcutaneously in the right flank of C57BL/6 mice. Four days later, tumor sizes were measured with calipers and mice were forcibly randomized into 3 groups (10 mice/ group). The 3 groups were treated with Doxorubicin only; L-DOS47 4 hours before Doxorubicin; and L-DOS47 24 hours before Doxorubicin. The rationale for different timings were that MRS measurements of tumor pH indicate that tumor alkalization has been observed both 4 and 24 hours following injection of L-DOS47. Treatments were begun immediately after randomization. Doxorubicin (2.5 mg/kg) was injected every 3-4 days and L-DOS47 (90 μg/kg) was injected either 4 or 24 hour prior to Doxorubicin i.v. RESULTS: All mice were humanely euthanized when tumors either reached 2000 mm3 (endpoint) or had serious ulcerations. For the tumors hadn't reached to the endpoint due to serious ulcerations, times to reach the endpoint were estimated using a mathematical model that describes exponential tumor growth. Kaplan Meier survival curves were generated for each treatment group. According to Log-rank Mantel-Cox, Log-rank for trend, Gehan-Breslow-Wilcoxon test observed mice group survival was significantly different from each other (p<0.008 for all 3 tests). The probability of survival of mice group treated with L-DOS47 either 4 or 24 hours before doxorubicin was significantly better than doxorubicin only group. CONCLUSION: In this study we have observed that neutralizing tumor pH can improve the effect of chemotherapy in vivo. This is consistent with prior observations where neutralization with bicarbonate improved the response to chemotherapy via reversal of an ion-trapping effect [1]. The current work is significant, however, as L-DOS47 can be used clinically, whereas bicarbonate cannot. References: [1] Raghunand, N., Mahoney, B.P., and Gillies, R.J.: 'Tumor acidity, ion trapping and chemotherapeutics. II. pH-dependent partition coefficients predict importance of ion trapping on pharmacokinetics of weakly basic chemotherapeutic agents', Biochem Pharmacol, 2003, 66, (7), pp. 1219-1229.

#1232

Paclitaxel induces immunogenic cell death in ovarian cancer via TLR4-independent and dependent pathways.

Tat-San Lau, Loucia Kit-Ying Chan, Gene Chi-Wai Man, Joseph Kwong. _The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong_.

Immunogenic cell death refers to a phenomenon in which some of the cytotoxic chemotherapeutics kills the tumor cells and induces these dying cells to be immunogenic. The aim of this study is to investigate if the standard-of-care chemotherapy (carboplatin and paclitaxel) induces immunogenic cell death in ovarian cancer. We found that paclitaxel, but not carboplatin, induces multiple molecular determinants of immunogenic cell death (namely calreticulin exposure, ATP secretion, HMGB1 release, ANXA1 expression, and upregulation of CXCL10) in ID8 mouse ovarian cancer cells. By tumor vaccination assay in immunocompetent syngeneic mice, our results showed paclitaxel induces immunogenic cell death in the mice with ID8 ovarian tumor. Since toll-like receptor 4 (TLR4) is a receptor for paclitaxel, we generated isogenic derivatives of ID8 cells with Tlr4 knockout (KO) to investigate if paclitaxel induces immunogenic cell death is dependent on TLR4. Our results showed that the paclitaxel-induced molecular determinants of immunogenic cell death were diminished in the TLR4 KO clones when compared to wild-type controls. Moreover, the tumor vaccination assay showed that paclitaxel was not able to induce immunogenic cell death in mice with ovarian tumor with TLR4 KO. These results indicated that the paclitaxel requires TLR4 to induce immunogenic cell death. To explore the mechanisms of how paclitaxel induces immunogenic cell death through TLR4, we investigated the activation of PERK and phosphorylation of eIF2a in ER stress since they are the first two enumerate elements of the pathways that mediated calreticulin exposure in response to immunogenic anticancer chemotherapeutics. Our results, however, showed that paclitaxel induces activation of PERK and phosphorylation of eIF2a in ovarian cancer cells is independent on TLR4. We then investigate if paclitaxel induces SNARE-dependent exocytosis in mouse ovarian cancer cells because SNARE-dependent exocytosis is the final step of the pathways that mediated calreticulin exposure in immunogenic cell death. Our results showed that paclitaxel induces SNARE-dependent exocytosis of ATP-containing vesicles in mouse ovarian cancer cells via TLR4-mediated IKK2 activation. In summary, our results showed that paclitaxel induces immunogenic cell death in ovarian cancer through TLR4-independent and dependent pathways, in which paclitaxel-induced exocytosis is essential for the release of multiple molecular determinants of immunogenic cell death. Our results provide new evidence that the antitumor effect of paclitaxel occurs in part via activation of an immune response against cancer by inducing immunogenic cell death and provide a rationale for a new combination of paclitaxel and immunotherapies as an anticancer treatment.

#1233

Myeloperoxidase (MPO) activity in myeloid-derived suppressor cells (MDSCs): effects on anticancer drug activity.

Jack C. Yalowich,1 Ragu Kanagasabai,1 Colleen S. Netherby,2 Michelle N. Messmer,2 Scott I. Abrams2. 1 _Ohio State Univ. College of Pharmacy, Columbus, OH;_ 2 _Roswell Park Comprehensive Cancer Center, Buffalo, NY_.

In cancer, suppression of host immunity is related, in part, to immature myeloid-derived suppressor cells (MDSCs) expanded in response to various tumor- or stroma-derived factors. Here, the expression and activity of myeloperoxidase (MPO) was examined in both granulocytic (PMN) and monocytic (M) MDSC subsets recovered from the spleens of 4T1 tumor-bearing mice, as well as interferon regulatory factor-8 (IRF8) knockout mice (IRF8-/-) which develop high levels of MDSCs due to IRF8-deficiency. Phenotypically identical cells from non-tumor-bearing mice served as controls. In addition, the role of MPO in PMN-MDSCs as a determinant of DNA damage induced by the anticancer agents etoposide (VP-16), camptothecin (CPT) and parthenolide (PTL), was evaluated for potential immunomodulatory effects. MPO protein content was ~4-fold greater in PMN-MDSCs from tumor-bearing compared to non-tumor-bearing mice, while MPO specific activity was equivalent in these two populations. MPO protein content in PMN-MDSCs from IRF8-/- mice was at least comparable to levels found in tumor-bearing hosts. In both IRF8-/- and tumor-bearing mice, PMN-MDSCs contained 5-10-fold greater MPO protein and overall enzyme activity compared to isolated M-MDSCs. VP-16 (5 µM)- and PTL (100 µM)-induced DNA strand breaks were 4-fold greater in PMN- compared to M-MDSCs from tumor-bearing mice. In addition, the heme-synthesis inhibitor, succinylacetone (SA) reduced MPO activity 2-3-fold in PMN-MDSCs from tumor-bearing mice with a corresponding decrease in drug-induced DNA damage for both VP-16 and PTL with no effect on CPT-induced DNA damage. The antioxidant dehydroascorbate decreased VP-16-induced DNA damage in PMN-MDSCs containing MPO activity, but not in those cells pre-incubated with SA to deplete MPO. These results indicate that MPO-dependent activation of VP-16 and PTL enhance DNA damage. Antioxidant protection against VP-16-induced DNA damage is consistent with MPO-mediated oxidative activation of VP-16 to a redox-cycling phenoxyl radical (Mol. Pharmacol. 79: 479-487, 2011). Overall, these results suggest that: 1) PTL may be useful to eliminate PMN-MDSCs; 2) MPO activation of VP-16 in PMN-MDSCs may be deleterious via oxidative DNA damage resulting in mixed-lineage leukemia (MLL) gene rearrangements known to be causal for therapy-related myeloid leukemias. Support: CA172105; CA090787; CA140622

#1234

Zoledronic acid-induced suppression of invasive phenotypes of pancreatic cancer cells is mediated through downregulation of CYR61/CCN1.

Arnab Ghosh,1 Sandipto Sarkar,2 Sonia Ghosh,1 Priyanka Ray,3 Mohi Quadir,3 Sushanta K. Banerjee,1 Snigdha Banerjee1. 1 _VA Medical Ctr. Kansas City, Kansas City, MO;_ 2 _Weill Cornell Medical College, New York, NY;_ 3 _North Dakota State University, Fargo, ND_.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cancer in the United States. Till date PDAC has shockingly poor outcome, as it can not be detected at an early stage and has a very aggressive phenotype that is resistant to standard therapies. Previous reports from our lab have depicted that the effect of most commonly used drug to treat PDAC, Gemcitabine, has limited effect and develops resistance due to the presence of a ECM oncoprotein, CYR61/CCN1. Depletion of CYR61/CCN1 protein suppresses invasive phenotypes and remarkably improves the treatment outcome of Gemcitabine in PDAC in-vitro and in-vivo. Hence, a potent synthetic chemical inhibitor of CYR61/CCN1 is essential to make it druggable.

Objective: Zoledronic acid (ZOL) is a FDA approved medication used to treat many bone diseases including osteoporosis, high blood calcium and bone breakdown due to cancer and Paget's disease of bone. Moreover, earlier studies have shown that ZOL significantly blocks pancreatic cancer cell migration and invasion. Thus, in this proposed study, we investigated whether ZOL blocks CYR61/CCN1 expression in PDAC cells to inhibit invasive phenotypes.

Results: Pancreatic cell lines (Panc-1 and AsPC-1) were treated with various concentrations of ZOL for different times points. Cells have shown no signs of stress physically as well as in molecular level. CYR61/CCN1 expression decreased significantly in a time and dose dependent manner at both transcriptional and translational level in PDAC cells and mouse (G12D/+; p53R172H/+; PdxCretg/+ -KPC) primary tumor-derived organoids. We compared the phenotypic alteration resulting due to CYR61/CCN1 downregulation and they reflect reduced stemness, low cell migration and cell proliferation. We treated the cells with exogenous human recombinant CYR61/CCN1 protein along with ZOL treatment and the cells regained/rescued their parental phenotype significantly. This led us to identify the CYR61/CCN1 as a target of ZOL in PDAC cells. Additionally, we found that ZOL treatment or CYR61/CCN1 depletion significantly reduces the expression of PD-L1 (encoded by the CD274 gene), which plays a role in suppressing immune system in many cancers including PDAC. As ZOL can reduce the levels of CYR61/CCN1 and PD-L1, that could act as an added advantage to use this drug in PDAC via suppressing CYR61/CCN1. Additionally, ZOL with targeted-nano-carriers were equally effective in regulation of CYR61/CCN1.

Conclusion: Our results suggest that ZOL could be repurposed as a CYR61/CCN1 inhibitor in PDAC in-vitro and in-vivo. Additionally, ZOL has the ability to block PD-L1 activity that could prove beneficial towards pancreatic cancer treatment. Furthermore, our studies suggest that CYR61/CCN1 is druggable for PDAC and beyond.

#1235

Targeting RNA-binding protein HuR to inhibit human breast cancer invasion and metastasis.

Xiaoqing Wu,1 Gulhumay Gardashova,1 Lan Lan,1 Shuang Han,1 Cuncong Zhong,1 Ragul Gowthaman,1 John Karanicolas,2 Dan A. Dixon,1 Danny R. Welch,3 Ling Li,4 Min Ji,5 Jeffrey Aubé,6 Liang Xu1. 1 _Univ. of Kansas, Lawrence, KS;_ 2 _Fox Chase Cancer Center, Philadelphia, PA;_ 3 _Univ. of Kansas Medical Center, Kansas City, KS;_ 4 _The Air Force Medical University, Xi'an, China;_ 5 _Southeast University, Nanjing, China;_ 6 _University of North Carolina, Chapel Hill, NC_.

The majority of breast cancer-related mortality is due to metastasis. Patients diagnosed with metastatic breast cancer have a dismal 5-year survival rate of only 24%. The RNA-binding protein Hu antigen R (HuR) is overexpressed in breast cancer. Cytoplasmic HuR accumulation correlates with high-grade malignancy, poor distant disease-free survival and serves as a prognostic factor for poor clinical outcome in breast cancer. HuR promotes tumorigenesis by regulating numerous proto-oncogenes, growth factors and cytokines that implicate major tumor hallmarks including invasion and metastasis. Knocking out HuR by CRISPR/CAS9 technology inhibits cell invasion in breast cancer cells. Therefore, HuR is an emerging target for breast cancer therapy, especially the lethal metastatic breast cancer. RNA-binding proteins had previously been considered "undruggable" due to lack of a well-defined binding pocket for target RNAs. Using high throughput screening followed by structure-based rational design and lead optimization, we have identified small molecules that inhibit HuR-mRNA interaction at nM to sub-µM potency. Our lead compound, KH-3, potently inhibits breast cancer cell growth and decreases cell invasion in vitro similar to HuR knockout, as well as increasing the expression of epithelial marker E-cadherin. In the study of mechanism of action, a transcription factor, FOXQ1, which is recently revealed to implicate in breast cancer invasion and metastasis processes, is found for the first time to be a direct mRNA target of HuR and one of the top genes that are reduced by KH-3 treatment. Exogenous introduction of FOXQ1 can rescue cell invasive capability impaired by HuR knockout and abolish the effect of KH-3 on inhibiting cell invasion in breast cancer cells. Moreover, KH-3 disrupts HuR-FOXQ1 interaction in RNP-IP, RNA pull down and FOXQ1 3′-UTR luciferase reporter assays. In vivo efficacy studies show that KH-3 not only exhibits potent antitumor efficacy in an orthotopic xenograft model of breast cancer, but also efficiently inhibits lung metastasis and improves mouse survival in an experimental metastasis model. Our data provide a proof-of-principle that HuR inhibition by KH-3 may be developed as a promising molecular therapy for inhibiting progression and metastasis of breast cancer with high HuR.

#1236

Subclinical doses and choice of replication stress inducer in combination with CHK1 inhibitors for optimal tumor control and immune responses in vitro and in vivo.

Zay Yar Oo,1 Martina Proctor,1 Alex Stevenson,1 Deborah Nazareth,1 Jill Larsen,2 Nikolas Haass,1 Brian G. Gabrielli1. 1 _The University of Queensland, Brisbane, Australia;_ 2 _Queensland Institute of Medical Research Berghofer, Brisbane, Australia_.

Background: Lung cancer and melanoma are responsible for almost 25% of cancer deaths in Australia. Despite recent progress with targeted and immuno-therapies, the 5 year survival rate in late stage patients is still<20%. CHK1 inhibitors are being investigated as chemosensitizing agents with agents that increase replication stress, primarily Gemcitiabine. Clinical trials of this combination have shown some good responses but high levels of normal tissue toxicity.

Materials and methods: Here we have investigated the molecular basis of sensitivity to CHK1 inhibitors as combinations with subclinical doses of hydroxyurea (HU) in melanoma and non-small cell lung cancer (NSCLC) tumor spheres and cell lines in 2% O2 conditions and xenograft models in immune compromised mice and with humanized immune systems.

Results: We report that low dose HU increased the sensitivity of >70% of both melanoma and NSCLC cell lines to CHK1 inhibitor (GDC-0575) triggered apoptosis, with complete loss of viability found with clinically achievable doses of this combination. Similar sensitivity was observed in xenograft models of both melanoma and NSCLC. We also demonstrate that a low dose of Gemcitabine combination with CHK1 inhibitor results in complete loss of proliferative potential in normal tissue, whereas normal tissue retaining proliferative potential after treatment with even high doses of hydroxyurea in combination with CHK1 inhibitor. In vivo, this translates to minimal effect on lymphocyte populations in the blood. The combination also triggers an inflammatory response involving the recruitment of macrophages, associated with increased HMGB1 nuclear staining. Immune responses were also assessed in humanized mouse models.

Discussion: These data indicate that the combination of low dose HU and CHK1 inhibitor have strong anti-cancer activity in the setting of melanoma and NSCLC cancer, and triggers an inflammatory response, and is likely to be better tolerated than current combinations with Gemcitabine. Our data suggest a significant proportion of melanoma and lung cancer patients could benefit from treatment with this drug combination, and this appears to include an immune response to the tumor.

#1237

Down-regulation of chemokine receptor CXCR4 by gambogic acid suppresses metastasis of pancreatic cancer in an orthotopic mouse model.

Sahdeo Prasad, Sanjay Srivastava. _Texas Tech University Health sciences center, Abilene, TX_.

More than 90% of patients die of pancreatic cancer due to metastasis. Among the cytokines, CXCL12 and its CXC chemokine receptor 4 (CXCR4) are known to play a role in invasion and metastasis in many types of cancer. Thus, agents that can interrupt the CXCR4/CXCL12 signaling cascade have the potential to suppress cancer metastasis. In the present study, we demonstrate that gambogic acid (GA), a xanthone from Gamboge hanburyi, down-regulated CXCR4 expression in pancreatic cancer cells in a dose- and time-dependent manner. We found that the decrease in CXCR4 by GA was not specific to cell type since its expression was abrogated in multiple myeloma cell lines and in colorectal, head and neck, prostate, and breast cancer cell lines. Furthermore, down-regulation of CXCR4 was not due to proteolytic degradation since proteasome inhibitors had no effect. Instead, the down-regulation of CXCR4 was due to transcriptional regulation, as indicated by the corresponding down-regulation of mRNA expression. This xanthone also inhibited the invasion of pancreatic tumor cells in vitro. Down-regulation of CXCR4 and prevention of cell invasion by the xanthone were both found to be mediated through the reactive oxygen species (ROS) as N-acetylcysteine, a ROS quencher, abolished these effects. When examined in an orthotopic nude mouse model of human pancreatic cancer, intraperitoneal administration of GA suppressed both tumor growth and metastasis. Immunohistochemical analysis of tumor tissue showed that besides decreasing the level of CXCR4, GA also reduced the expression of VEGF and MMP-9, which are also linked to invasion and metastasis. Thus, our results demonstrate gambogic acid's potential in suppressing tumor invasion and metastasis through down-modulation of CXCR4/CXCL12 signaling pathways.

#1238

The anti-protozoal drug Atovaquone suppresses the metastasis of triple negative breast tumors to brain, lungs and liver by inhibiting integrin-src pathway.

Nehal Gupta,1 Sanjay Srivastava2. 1 _Texas Tech Univ. Health Sciences Ctr., Amarillo, TX;_ 2 _Texas Tech Univ. Health Sciences Ctr., Abilene, TX_.

Triple negative breast cancer (TNBC) is considered to be the most aggressive and malignant neoplasm and is highly metastatic in nature. Metastatic TNBC is difficult to treat and thus leads to poor patient survival. Death of about 90% breast cancer patients is due to metastasis of primary tumor to distant sites. In the current study, we investigated the anti-metastatic potential of atovaquone, a protozoal drug prescribed for Pneumocystis pneumonia. We show that atovaquone induced apoptosis and reduced the survival of several aggressive metastatic TNBC cell lines including few metastatic patient derived cells. The IC50 of atovaquone was around 11-18 µM in the breast cancer cell lines tested. Additionally, atovaquone treatment significantly reduced the expression of integrinα6, integrin β4, FAK, Src, and Vimentin in MDA-MB-231, HCC1806 and 4T1 cells after 72 hours of treatment. In order to study the efficacy of atovaquone in preventing metastasis of breast tumor cells to brain and lungs, we performed three in vivo experiments. We demonstrate that oral administration of 50mg/kg of atovaquone suppressed MDA-MB-231 breast tumor growth by 90% in lungs in an intravenous metastatic tumor model. Further anti metastatic effect of atovaquone was determined by intracardiac injection of 4T1-luc breast tumor cells into the left ventricle of mouse heart. Once breast cancer cells lodged in the brain, mice were treated with 50 mg/kg atovaquone for 15 days. Our results show that atovaquone treatment suppressed the growth of metastatic tumors in brain by 30%. In this model, we also observed 70% and 50% reduced metastasis of breast tumors in lungs and liver, respectively, in atovaquone treated group when compared with control. In intracranial model, the growth of HCC1806-luc brain tumors in atovaquone treated mice was about 55% less than that of control. Angiogenesis is one of the hallmarks of cancer and plays crucial role in metastasis. Interestingly, we observed reduced levels of VEGF in the cells treated with atovaquone. In-vivo angiogenesis was evaluated by performing matrigel plug assay in mice. Our results showed reduced angiogenesis in atovaquone treated mice group. Atovaquone treated tumors from these in-vivo models exhibited reduced metastatic markers and increased apoptosis. Taken together, our results indicate the anti-metastatic effects of atovaquone in-vitro and in-vivo in various breast tumor metastasis models by inhibiting angiogenesis and integrin signaling and provide the rationale for further clinical investigation of atovaquone for breast cancer metastasis. (Supported in part by RO1 grant CA129038, awarded by National Cancer Institute, NIH).

#1239

Structure activity relationship of alexidine analogs for the inhibition of collective invasion.

Jamie Arnst, Rachel Commander, Adam Marcus. _Emory University, Atlanta, GA_.

Lung cancer is the leading cause of cancer-related deaths in the US with an overall 5-year survival of only 15-20%. Metastatic disease is a major contributor to poor survival and novel therapeutics that can selectively target invasive tumor cell populations are lacking. Metabolic reprogramming contributes to tumor progression with effective therapies targeting cancer metabolism available. However, it remains unclear whether targeting cancer metabolism will prove valuable in inhibiting invasive cancer populations. Recent studies have demonstrated anticancer activity by the mitochondria-targeting bioactive small molecule alexidine dihydrochloride. Our group has previously found that alexidine specifically inhibits the a highly invasive subpopulation of H1299 previous identified in our lab by decoupling and decreasing collective invasion. Here we report the structure activity relationship (SAR) between analogs of alexidine on the inhibition of collective invasion in NSCLC cell lines. For these studies, we utilized an in vitro 3D invasion model where NSCLC cell lines were allowed to form spheroids, and then were embedded into Matrigel and allowed to invade over 48h in the presence or absence of the analogs. Using this technique, we identified two novel alexidine analogs with improved efficacy at inhibiting the invasion of H1299 NSCLC cells. Additionally, we examined the phosphorylation of pyruvate dehydrogenase (PDH), the gatekeeper for the entrance of pyruvate into the TCA cycle, following treatment with the alexidine analogs. We observed a significant correlation between PDH phosphorylation and inhibition of invasion following treatment with the analogs. In conclusion, we have identified two novel alexidine analogs with improved inhibition of invasion in NSCLC cell lines in an in vitro model.

### Credentialing of Molecular Targets

#1240

Identification of molecular targets of Tris DBA [Tris(dibenzylideneacetone)dipalladium(0)] for cancer therapy.

Loukik Arora,1 Chris Soon Heng Tan,2 Radoslaw M. Sobota,2 Gautam Sethi1. 1 _National University of Singapore, Singapore, Singapore;_ 2 _Agency for Science, Technology and Research, Singapore, Singapore_.

Cancer is complex disease involving complex genetic and epigenetic heterogeneity making it the second leading cause of death globally after heart disease. While conventional treatments like surgery, radiotherapy, chemotherapy are principal strategies, the focus is shifting to therapies that can target multiple pathways to treat cancers. In the present report, we hypothesized that Tris DBA, an organometallic compound, can inhibit proliferation, tumor growth, and induce apoptosis in multiple myeloma (MM) and hepatocellular carcinoma (HCC) cells, thereby potentially exhibiting significant anticancer effects. Our preliminary results clearly indicated that Tris DBA could substantially inhibit both constitutive as well as IL-6 inducible STAT3 activation and abrogate proliferation/survival in MM and HCC cells. A phospho-proteomic profiling revealed several key phosphorylation changes induced by Tris DBA treatment which further indicate broad anticancer potential of the compound. Additionally, the agent exhibited potent antitumor actvitiy in vivo , with little adverse effects. The ongoing work is aimed at investigating the possible binding target(s) of Tris DBA and further characterize the molecular mechanism(s) underlying STAT3 inhibitory effects of the drug.

#1241

Characterization of a newly developed anti-TIGIT monoclonal antibody by double staining of T cells and NK cells by IHC and FACS.

Weimin Qi, Joseph Chu, Lily Luo, Tatiana Scoggin, Kim Dickinson. _Biocare Medical, LLC, Pacheco, CA_.

T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory domain (TIGIT) is a newly identified surface protein expressed in regulatory, memory, naturalkiller (NK), and activated T cells. Several studies indicate that mouse TIGIT is a vital immunomodulator that can control the activities of both NK and T cells and plays an important role in transplantation tolerance and tumor immune surveillance. In this study, we generated a mouse monoclonal antibody, clone: BC41, by immunizing a recombinant human TIGIT, corresponding to an extra cellular domain. The protein was injected into BALB/c mice for three times and then the splenocytes were electro fused with mouse myeloma cells. The TIGIT-specific hybridoma cells were screened and a TIGIT-specific clone, named as BC41, was selected. The BC41 antibody was characterized by Western blot and immunohistochemistry. In immunoblots, TIGIT was able to detect a 26 kda protein corresponding to human TIGIT on over-expressed cells and native protein in human spleen lysates. Moreover, the BC41 was able to bind mouse TIGIT. FACS result showed that the BC41 antibody bond specifically to CD3/CD28+ T cells. We further analyzed the antibody binding profile by Immunohistochemistry on human T cells and NK cells by double staining. BC41 was able to bond portion of CD8 T cells (EOMES+), Treg cells (FOXP3+), PD1+ T cells and NK (CD57+) cells. Our results demonstrated that the antibody is TIGIT-specific and can be applied for detecting and analyzing tumor-infiltrating cells profile in transplantation and tumor immunotherapy.

#1242

Examining the role of heme and respiratory proteins in the progression of KRAS/LKB1 mutant subtype.

Sanchareeka Dey, Sarada Preeta Kalainayakan, Poorva Ghosh. _The University of Texas at Dallas, Richardson, TX_.

Currently there are few successful targeted therapies for non-small cell lung cancer (NSCLC). Targeting mutations in EGFR, BRAF, ALK etc. provide treatment options only for a defined subset of adenocarcinoma patients leaving majority of adenocarcinoma and squamous carcinoma patients without any options. Activating KRAS mutations are found in 15-30% of all NSCLC patients; however, existing treatment options for KRAS-mutant cancer are not effective in the subset with concomitant loss of LKB1 (kinase that phosphorylates AMPK). Several studies demonstrate the importance of mitochondrial metabolism and oxidative phosphorylation (OXPHOS) in lung tumorigenesis. Components of OXPHOS complexes and markers of mitochondrial biogenesis are highly predictive of reduced overall survival in NSCLC patients. A study using genetically engineered mouse models (GEMMs) for lung cancer (KrasLSLG12D/+Trp53-/- and KrasLSL-G12D/+Lkb1-/-) showed that along with glucose, lactate contributes to the TCA cycle. This highlights the importance of OXPHOS in these models. Also, heme is a prosthetic group for several of these OXPHOS proteins and is involved in oxygen transport and utilization. Hence, we aim to investigate unique therapeutic targets by examining the role of heme and respiratory proteins in the progression of KRAS/LKB1 mutant subtype. We used LSL-KrasG12D LKB1-/- (LSL-KRASG12D; LKBloxP/loxP; LSL-Luciferase (KLLuc)) GEMM model for our study. Briefly, the mice were infected with AdenoCre virus, and sacrificed at different intervals in the progression of the tumors. For histological and immunohistochemical analysis, lung tissues were formalin fixed, paraffin embedded and sectioned (5µm thick). H&E staining showed consistent progression in tumor size over time. We will examine the time-course expression of OXPHOS, heme pathway proteins, and hemoproteins to determine the extent to which this lung tumor subtype depends on heme and respiratory proteins for its progression.

#1243

Effect of antigen retrieval on immuno-based RNA microdissection methods.

Donald J. Johann,1 Meeiyueh Liu,1 Adam Roberge,2 Sarah Laun,2 Michael Emmert-Buck,3 Michael Tangrea2. 1 _UAMS, Little Rock, AR;_ 2 _Sinai Hospital of Baltimore, LifeBridge Health, Baltimore, MD;_ 3 _Avoneaux Medical Institute, Baltimore, MD_.

Introduction: Clinical applications and research studies routinely utilize immunohistochemistry (IHC) approaches on tissue sections. The aim is to selectively target antigens (proteins) in the cells of a tissue section by exploiting the principle of antibody binding, thus enabling target specificity. The antigen retrieval (AR) process employed at the beginning of most IHC protocols increases epitope availability and improves staining characteristics; however, the procedure can damage nucleic acids, in this case RNA, to an unknown extent.

Experimental Procedures: To better understand the effects of AR on RNA quality and quantity, model system samples (lung cancer patient derived xenograft tissue) were subjected to a commonly employed AR method [heat induced epitope retrieval (HIER)] and the effects on RNA were assessed by Qubit, Fragment Analyzer, and digital droplet PCR (ddPCR). The ddPCR experiments were performed following reverse transcriptase (RT) product generation and construction of cDNA, which then served as input to the ddPCR assay. Absolute quantitation of gene expression levels and expressed mutations were evaluated by ddPCR.

Data Summary: The results showed that HIER resulted in optimal staining characteristics, but induced significant damage to RNA, producing extensive fragmentation and decreased yields. In general, fragmentation showed an inflection point at ~200 nt transcript length. Variation in the HIER protocol mitigated but did not eliminate the negative effects. Apart from the AR procedure, the IHC process itself also resulted in a significant decreased yield of RNA. Of note, in these experiments DAB chromogen was used exclusively in the IHC process. However, in spite of the observed deleterious effects on RNA, none of the AR methods combined with IHC negatively affected RT product generation and PCR amplification of small amplicons. Regarding the detection of RT product under different conditions, the size of the amplicon was found to make a difference, with the detection of longer amplicons being more difficult. In addition to a number of successful human and mouse gene expression measurements, KRAS gene mutations were successfully identified in the clinical cases under all conditions.

Conclusions: The data indicate that RNA recovered from histology slides after standard AR and IHC processing can be successfully employed for genomic applications utilizing RT product formation (cDNA) for gene expression and detection of expressed mutations, especially if the detection methods are based on relatively short length nucleic acids. Studies that require larger RNA fragments such as long-read sequencing should avoid the use of HIER.

#1244

Reactivation of mutant p53 by Buxus alkaloids compound.

Jihong Zhang, Yuling Wang, Yongnan Su, Hancuan Mou, Wei Wu. _Kunming University of Science and Technology, Kunming, China_.

The tumor suppressor gene p53 is highly mutated in human cancers, with more than 50% of cancers harboring mutations that abrogate its function. Reactivation of mutant p53 function represents an important anticancer strategy. We present that buxus alkaloids natural compound was more cytotoxic to HT29 cells which harbor R273H mutant. The compound induced mutant p53 protein degradationthrough CHIP and MDM2 pathways and restore the transactivation function of DNA content mutation of p53 R273H and R280K. p53 reactivation was accompanied by the induction of the terget genes, p21, PUMA, NOXA and BAX. The compound alters mutant p53 conformational change detected by conformation-specific antibodies, PAb240 and PAb1620. Electrophoretic mobility shift assay showed a reciprocal loss of mutant protein and generation of wild-type like protein. Similar effects were confirmed in cancer cells expressing exogenous R273H. These results demonstrate that buxus alkaloids natural product can restore transcription function to mutant p53 and leading to cell death.

#1245

Standardized 3D spheroid systems facilitate drug screening and biomarker identification with high relevance for cancer drug discovery.

Julien Duez, Isabelle Fixe, Alexandra Foucher, Eric Mennesson, Nadia Normand. _tebu-bio, Le Perray-en-Yvelines, France_.

Whilst patient-derived cancer organoid culture is not yet compatible with high throughput studies, cell-line-derived multicellular spheroids represent an attractive solution, as they offer the possibility to recapitulate the structural and molecular complexity of cancer cell lesions with higher physiological relevance compared to 2D culture systems, thereby generating more predictable results and reducing the expenses attributed to current preclinical attrition rates. As an illustration of the technological services developed by our team, we focused on colorectal cancer, which is one of the most commonly occurring cancer in humans. When stage I and II tumors are curable, metastatic stages of the disease still represent a clinical challenge. Along the last decades, the use of cytotoxic drugs in association with targeted therapies improved the overall survival of patients with metastatic colorectal cancer. However, a large subset of patients continues to be unresponsive, likely due to the high heterogeneity of colorectal cancer lesions. Sustained efforts are then needed to identify and target relevant biomarkers of crucial interest for the development of treatment strategies. CDK8 is a non-mitotic Cyclin-Dependent-Kinase which functions as an oncoprotein in colorectal cancers. Most published drug discovery projects report attractive binding affinity, selectivity and permeability for CDK8 inhibitors amenable to mouse studies. However these compounds generally display weak antiproliferative or cytostatic activity in conventional cell based assays. The signaletic map involving CDK8 in colorectal cancer has mostly been investigated using RNAi mediated suppression studies and kinase dead mutants. Thus, there is an important need to pursue the exploration of the oncogenic role of CDK8 using complementary pharmacological approaches implemented in the most relevant physio-cellular context. Indeed, CDK8 inhibitors in development are mostly evaluated using colorectal cancer cells in standard culture conditions. In this study we aimed to profile the impact of pro-inflammatory conditions on colorectal cancer response to pharmacological CDK8 inhibition, using HT29 cells cultured as monolayers or multicellular spheroids. A panel of targets were analysed through proteomic multiplex microarrays, comparing 2D and 3D cultures. RTqPCR was used to further validate under/over-expressed genes uncovered through the microarray approach. The resulting signature was compared to previously published RNAi-mediated CDK8 expression inhibition results. Cancer cell line-derived multicellular spheroids are compatible with the identification of biomarkers in pro-inflammatory conditions. Such an approach may be further used to adjust current drug discovery methodologies applied to the selection of CDK8 inhibitors, and more generally to antitumoral drugs.

#1246

Comprehensive RNA interference induced by CRISPR/Cas13a gene editing system in human cancer cells.

Qixue Wang, Yunfei Wang, Yanli Tan, Chunsheng Kang. _Tianjin Medical University General Hospital, Tianjin, China_.

RNA is the intermediate link of central dogma. However, RNA is rarely used as a therapeutic target for its flexible structure and instability. CRISPR/Cas13a is reported as a powerful tool for RNA knockdown. The potential amplification of CRISPR/Cas13a in cancer cells could be further studied. Here we overexpressed the Cas13a protein by lenti-virus vectors in glioma and other cancer cells, and found out that crRNA-EGFP could induce the "collateral effect" after knocking down the target gene. EGFRvIII is a unique EGFR mutant subtype in glioma. CRISPR/Cas13a system could induce cell death of EGFRvIII overexpression glioma cells. Single cell and RNA-seq analysis confirmed the comprehensive RNA interference by CRISPR/Cas13a treatment. Furthermore, CRISPR/Cas13a system could inhibit the formation of glioma intracranial tumor in mice. Our results exhibit the comprehensive RNA interference of CRISPR/Cas13a system in cancer cells, and demonstrated the powerful tumor elimination potential of this system.

#1247

Screening the membrane proteome to determine antibody specificity and discover new immunomodulatory targets.

Duncan Huston-Paterson. _Integral Molecular, Philadelphia, PA_.

The Membrane Proteome Array (MPA) is a cell-based expression array of more than 5,300 membrane proteins that enables the screening of preclinical antibodies for off-target binding and the deorphaning of phenotypic antibodies to identify their associated targets. Each membrane protein is expressed in live cells in its native conformation with appropriate post-translational modifications. Binding interactions are tested and validated using high-throughput flow cytometry, providing a comprehensive assessment of antibody binding across the membrane proteome. Membrane proteins in the MPA are fully functional and can be used for phenotypic screening to identify new therapeutic targets. Using a T-cell co-culture approach, we identified dozens of novel co-stimulatory proteins, as well as many well-validated proteins known to be involved in T-cell activation. The proteins identified in this screen represent novel therapeutic targets across multiple membrane protein classes and likely function on diverse axes of T-cell regulation.

#1248

Not BCL2 but BFL1 is overexpressed and have strong correlation with NF-kB pathway in diffuse large B-cell lymphoma.

Chansub Lee,1 Hyojin Song,1 Seulki Song,1 Daeyoon Kim,1 Jihyun Park,1 Sungyoung Lee,2 Hongseok Yun,2 Sheehyun Kim,2 Sung-Soo Yoon,2 Youngil Koh2. 1 _Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea;_ 2 _Seoul National University Hospital, Seoul, Republic of Korea_.

Venetoclax, anti-apoptotic BCL2-selective inhibitor, has demonstrated efficacy in B-cell multiple lymphoid malignancies. However, several studies have shown that the other anti-apoptotic BCL2 family genes can increase the risk of venetoclax resistance. Accordingly, compounds targeting other BCL2 family are actively being developed. So it is necessary to find which BCL2 family genes is overexpressed in certain tumors. We profiled BCL2 family genes expression with RNA-seq across different types of lymphoid malignancies (follicular lymphoma, burkitt lymphoma, diffuse large b-cell lymphoma, multiple myeloma, and acute leukocyte leukemia). Log2-transformed TMM-normalized RPKM was used for expression levels. As is known, BCL2 and MCL1 was highly expressed in follicular lymphoma and multiple myeloma, respectively. Interestingly, we discovered BFL1, one of anti-apoptotic BCL2 family genes, is overexpressed in both activated B-cell (ABC) and germinal center B-cell (GCB) type diffuse large B cell lymphoma (DLBCL). It has been well known that BFL1 is a pro-survival NF-kB target gene and indeed NF-kB pathway is highly activated in ABC-DLBCL. Also recent studies have shown that activation of this pathway can occur in GCB-DLBCL. Using gene set enrichment analysis, we confirmed that high expression of BFL1 is associated with negative regulation of intrinsic apoptosis and activation of NF-kB in B-cell. Also we discovered that NF-kB pathway activation in correlation with BFL-1 is especially dominant in DLBCL compared to other lymphoid malignancies. Among NF-kB signal related molecules (TNF receptors or Toll-like receptors), CD40 expression significantly correlated with BFL1 expression. In contrast, BCL2, another important apoptosis related gene, showed a negative association with the NF-kB pathway in DLBCL. We could validate our observation using microarray data. In conclusion, BFL1, which may controls intrinsic apoptosis and have strong correlation with NF-kB, is overexpressed in DLCBL. We suggest not venetoclax but BFL1 inhibitor may exert clinical benefit in DLBCL.

#1249

The role of PRMT5 in MYC-driven medulloblastoma.

Nagendra K. Chaturvedi, Varun Kesherwani, Matthew J. Kling, Sutapa Ray, Timothy R. McGuire, Shantaram S. Joshi, J. Graham Sharp, Don W. Coulter. _Univ. of Nebraska Medical Ctr., Omaha, NE_.

Medulloblastoma (MB) is the most common pediatric brain tumor, accounting for about 20% of all brain tumors in children. Children with Group 3 MB (aberrant MYC) show the worst prognosis, with <50% survival. Recently, the role and association of protein arginine methyl transferase (PRMT) 5 have been closely associated with aberrant MYC function in various cancers including other brain tumors such as glioblastoma. However, the role of PRMT5 and its association with MYC in MB have not been explored. Here we provide preliminary data indicating PRMT5 as a novel regulator of MYC and implicating PRMT5 as a potential therapeutic target in MYC-driven MB. Using cell lines, our results showed PRMT5 overexpression in MYC-driven MB compared to non-MYC MB. The results from co-immunoprecipitation experiment in MYC-driven MB cells demonstrated that MYC physically associates with PRMT5 by direct protein-protein interaction. In addition, cycloheximide chase experiment showed that PRMT5 regulates MYC stability. Knockdown of PRMT5 using siRNA in MYC-driven MB cells significantly decreased cell growth and MYC expression. We also tested the therapeutic potential of targeting PRMT5 against MB cell lines using a small molecule inhibitor EPZ015666. We observed a dose-dependent efficacy of EPZ015666 in suppressing cell growth in MYC-driven MB cell lines. We also observed a superior efficacy of this inhibitor against MYC-driven MB cells compared to non-MYC MB cells. Together, our results implicate the regulation of MYC oncoprotein by PRMT5, and suggest that targeting PRMT5 could be a potential therapeutic strategy for MYC-driven MB and other MYC-driven cancers.

#1250

Radotinib induces cell death of multiple myeloma cells.

Jae-Cheol Jo, Sook-Kyoung Heo, Eui-Kyu Noh, Jeong Yi Kim, Jun Young Sung, Ho-Min Yu, Yoo Kyung Jeong, Lan Jeong Ju, Yunsuk Choi. _Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Republic of Korea_.

Background: Radotinib is a medicine for the treatment of some types of cancer. It is approved in South Korea for use as a second-line treatment of chronic myeloid leukemia (CML). Its mechanism of action involves inhibition of the tyrosine kinase Bcr-Abl and of platelet-derived growth factor receptor (PDGFR). It has been little known the effects of radotinib on multiple myeloma (MM) cells.

Methods: First, we examined cytotoxicity of radotinib on MM cell lines, RPMI-8226 and MM.1S. Annexin V positive cell, caspas-3 and -9 activities, cell cycle distribution and mitochondrial membrane potential (MMP, ΔΨm) were observed by analyzed with flow cytometric analysis. And diverse signaling pathways were investigated by Western blotting in MM cells.

Results: Interestingly, radotinib caused cell death of MM cells. Radotinib induced Annexin V positive cells, and caspase pathway activation including caspase-3, -7 and -9. And its treatment remarkably decreased MMP in MM cells. As well as we observed that cytochrome c accumulated dose dependently in the cytosol of radotinib-treated RPMI-8226 and MM.1S cells. Moreover, radotinib decreased the expression of Bcl-xL and Bcl-2, and increased the expression of Bax and Bak in MM cells. Moreover, radotinib significantly suppressed MM cell growth in a xenograft animal model using RPMI-8226 cells.

Conclusion: Radotinib may play an important role as a candidate or chemosensitizer for treatment agent in MM. These data indicate that radotinib has a potential for anti-cancer therapy in MM.

Figure 1. Radotinib significantly suppressed MM cell growth in a xenograft animal model using RPMI-8226 cells.

#1251

Potential tumor suppressor function of polo-like kinase 5 in ovarian cancers.

Mary A. Ndiaye, Shenqin Su, Rebecca M. Baus, Wei Huang, Manish S. Patankar, Nihal Ahmad. _University of Wisconsin-Madison, Madison, WI_.

Ovarian cancer remains one of the leading causes of cancer-related deaths in women worldwide, with an estimated 295,414 new cases and 184,799 deaths to occur in 2018. In the United States, although this malignancy is not in the top 10 for number of estimated new cases, it is the 5th leading cause of cancer-related deaths in women. This disparity is due to the fact that most ovarian cancers are not diagnosed until late stages when only limited options for treatment are available. Therefore, it is important to uncover novel molecular mechanisms that can be targeted to control this deadly cancer. The polo-like kinases (PLKs 1-5) are a family of serine/threonine kinases that that primarily play key roles in cell cycle progression, and have been linked to cancer pathogenesis. Limited information is available regarding the role of PLK5 in cancer. PLK5 is structurally unique, in that it has been shown to be missing the majority of the kinase domain that the other family members have. Interestingly, although PLK5 is not expressed in many tissues, it has been found to be present in appreciable levels in fallopian tube tissue, which suggests that PLK5 may be important in fallopian tube biology. Contrary to previous dogma, recent research has suggested that most ovarian cancers may actually have their origin in the fallopian tubes. Keeping this in mind, we wanted to explore the role of PLK5 in fallopian cancer. To this end, we performed a quantitative immunostaining of PLK5 in a fallopian tube disease tissue microarray (TMA) containing 5 normal tissues, 10 cases of inflammation, 10 fallopian adenocarcinoma, and 4 adjacent normal tissues. Following staining, the TMA was scanned with the Vectra platform and immunostaining was analyzed using InForm software that allows us to quantitatively measure the PLK5 intensity in each tissue and subcellular compartment. Our data demonstrated that PLK5 protein levels were significantly decreased in fallopian tumors compared to normal fallopian tissue (p = 0.01). Interestingly, although there was no significant difference between inflamed tissue and normal tissue, PLK5 levels were significantly reduced in the fallopian tube adenocarcinomas compared to the inflamed fallopian tissue (p<0.001), suggesting a potential role for PLK5 in fallopian tube carcinogenesis. Further, our data demonstrated that PLK5 was enriched in the nucleus (p<0.001) versus the cytoplasm, which is the opposite of what has been previously published in human neurons and glial cells. Taken together, our data suggests that PLK5 may be a tumor suppressor in fallopian tube cancer, and potentially ovarian cancers, as a majority of them are derived from the fallopian tube epithelium. However, further studies are needed to understand the exact roles played by this kinase in these reproductive tissues.

#1252

Intracellular iron chelation by a novel compound, C7, reactivates Epstein-Barr virus (EBV) lytic cycle via the ERK-autophagy axis in EBV-positive epithelial cancers.

Alan K S Chiang,1 Stephanie PT Yiu,1 Kwai F. Hui,1 Chung K. Choi,1 Richard YT Kao,1 Dan Yang2. 1 _Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, Hong Kong;_ 2 _Faculty of Science, The University of Hong Kong, Hong Kong, Hong Kong_.

Pharmaceutical reactivation of lytic cycle of Epstein-Barr virus (EBV) represents a potential therapeutic strategy against EBV-associated epithelial malignancies e.g. gastric carcinoma (GC) and nasopharyngeal carcinoma (NPC). A novel lytic inducing compound, C7, which exhibits structural similarity to Dp44mT, a known chelator of intracellular iron, is found to reactivate EBV lytic cycle in GC and NPC. This study aims to investigate the role of intracellular iron chelation by C7 and other iron chelators in lytic reactivation of EBV in GC and NPC. Testing of 6 structural analogues of C7 revealed only those which had high affinity towards transition metals could induce EBV lytic cycle. Pre-complexing C7 and iron chelators to iron prior to treatment of the cells abolished EBV lytic reactivation. Though hypoxia signaling pathway was activated, it was not the only pathway associated with EBV reactivation. Specifically, C7 and iron chelators initiated autophagy by activating extracellular signal-regulated kinase (ERK1/2) to reactivate EBV lytic cycle since autophagy and EBV lytic reactivation were abolished in cells treated with ERK1/2 blockers whilst autophagy inhibition by 3-MA and ATG5 knockdown significantly abolished EBV lytic reactivation. In summary, we discover a novel mechanism of reactivation of EBV lytic cycle through intracellular iron chelation and induction of ERK-autophagy axis in EBV-positive epithelial malignancies, raising the question whether clinically available iron chelators can be combined with existing therapeutic modalities to treat these cancers.

#1253

MDM2 inhibitor APG-115 synergizes with CDK4/6 inhibitors in a patient-derived xenograft model of dedifferentiated liposarcoma.

Douglas D. Fang,1 Guoqin Zhai,1 Chunhua Xu,1 Qingyang Gu,2 Jingwen Wang,1 Saijie Zhu,1 Dajun Yang,1 Yifan Zhai1. 1 _Ascentage Pharma (Suzhou) Co., Ltd, China;_ 2 _Wuxi Apptec (Suzhou) Co., Ltd, China_.

Liposarcoma is a rare but highly recurrent soft tissue neoplasm. Combination of surgery and radiotherapy is the main treatment for liposarcoma patients while chemotherapy remains experimental and limited depends on disease subtypes. Well-differentiated and dedifferentiated liposarcoma (WDLPS/DDLPS) represents ~50% of all diagnosed liposarcoma and their benefit from chemotherapy has been documented to be minimal. Concomitant amplifications of MDM2 and CDK4 are frequently found in WDLPS/DDLPS patients, suggesting that the combination with two targeted agents may worth further development for the treatment. APG-115 is a potent, selective, orally bioavailable small molecule inhibitor of MDM2-p53 protein-protein interaction and thus activates tumor suppression activity of p53 in p53 wild-type tumors. Currently, APG-115 is in clinical development as a single agent or in combination with PD-1 blockade immunotherapy for solid tumors, including sarcoma. To further explore the sensitive patient population and predictive biomarkers for APG-115, in this study, we evaluated the effect of the combined MDM2/CDK4 inhibition in p53 wild-type, MDM2 and CDK4 amplified liposarcoma using a preclinical xenograft tumor model derived from a dedifferentiated liposarcoma patient (PDX). Tumor-bearing mice were treated with MDM2 inhibitor APG-115, CDK4 inhibitors palbociclib/ribociclib, or their combinations. In the first experiment, APG-115 administered at 40 mg/kg as a single agent demonstrated antitumor activity in the DDLPS PDX model. In comparison with the single agents, the combination treatment with APG-115 and palbociclib or APG-115 and ribociclib exerted synergistic antitumor effects, resulting in T/C values of 16.9% and 27.1%, respectively (T/C value, average tumor volumes in treatment group vs. the vehicle control group). Partial tumor regression (PR) was achieved in 1/5 animals in both combination treatment groups but not in single arms. Another independent experiment with a higher dose of APG-115 (80 mg/kg) combined with palbociclib showed further improved antitumor activity with T/C value of 7.0% (2/5 PR). Collectively, our results suggest that the combination therapy of APG-115 and a CDK4 inhibitor may benefit WDLPS/DDLPS patients with p53 wild-type, and MDM2 and CDK4 amplifications clinically.

#1254

**Predictive models for tumor cell targeting with plinabulin, derived from** in vitro **screening and Affymetrix mRNA expression data.**

James R. Tonra,1 Hagen Klett,2 Chenghao Shen,1 Gerhard Kelter,2 Ramon W. Mohanlal,1 G. Kenneth Lloyd,1 Lan Huang1. 1 _BeyondSpring Pharmaceuticals, New York, NY;_ 2 _Charles River, Germany_.

Tubulin binding drugs are approved for the treatment of many cancer types, without the use of molecular markers to select patients likely to respond. Plinabulin binds β-tubulin in a differentiated pocket and is being tested in a Phase 3 clinical study for the treatment of NSCLC. Additional indications are being considered for plinabulin and an algorithm for selecting especially responsive cancers and patient subgroups would be of significant value. With this in mind, Affymetrix HG-U133 Plus 2.0 array mRNA expression data for 43 human breast, lung, prostate, ovarian or CNS cancer cell lines were utilized to develop mathematical models to predict in vitro plinabulin potency against the same cell lines. Cells were treated for only 24 hours with plinabulin and then cultured for another 48 hours without plinabulin. Viable cell number was then measured with a Cell Titer-Blue Assay, and the plinabulin concentration causing a 70% reduction in viable cells (IC70) versus vehicle treated controls was derived. Cell lines were clearly separable into plinabulin Active (21 cell lines with IC70<1.0 μM) and Inactive (IC70>9.5 μM) groups. Log2 transformed Affymetrix gene probeset signal values, preprocessed with the GeneChip robust multi-array average analysis algorithm, were selected and ranked as predictors of plinabulin activity with a bootstrap forest partitioning technique, utilizing JMP 14.1 statistical software. 56 HIT probesets were identified that also had significantly different expression in responding versus non-responding cell lines (p<0.01, uncorrected t-test). For probesets with gene annotation, only the probeset for each gene with the highest Jetset score was utilized (Li et al., 2011). Top HIT predictor genes include CTNNB1 (β-catenin; oncogene), CALD1 (caldesmon; inhibits myosin ATPase activity), ERI1 (RNA processing), LGR5 (adult stem cell biomarker), SECISBP2L (SLAN; prolongs mitosis), and TRAK1 (mitochondrial/endosome trafficking). Models were constructed from HIT gene probesets in JMP to identify plinabulin responding cell lines, utilizing either one-layer TanH multimode fit neural networks or binary logistic regression. Surprisingly, models incorporating approximately 4-10 probeset values were derived that perfectly predicted plinabulin activity. Importantly, the cell lines tested in the above analyses were not those known to express high levels of multi-drug resistant (MDR) transporters. The importance of MDR status was therefore evaluated separately. Plinabulin activity, unlike that of taxanes, was not significantly affected by the MDR transporter inhibitor verapamil (10 μM), in ovarian cancer cell lines with a known MDR phenotype. To conclude, our work provides novel algorithms that may be of value in selecting cancer patients with tumor cells that are particularly susceptible to the direct cytotoxic effects of plinabulin.

#1255

Identification of mutational landscape predicting efficacy of immune checkpoint blockades and recurrent mutations related to high tumor mutational burden in hepatocellular carcinoma by next generation sequencing.

Li Li,1 Zhaohong Wen,2 Michael J. Overman,3 Xiaosong Rao,1 Yuting Yi,2 Yanfang Guan,2 Jun Liang1. 1 _Peking University International Hospital, Beijing, China;_ 2 _Geneplus-Beijing Institute, Beijing, China;_ 3 _MD Anderson Cancer Center, TX_.

Introduction: HCC is an aggressive and genetic heterogeneous disease. Several disrupted signaling pathways have been identified, though actionable targets are limited. The majority of systemic chemotherapy and targeted therapy (tyrosine kinase inhibitors (TKIs) or monoclonal antibodies) have demonstrated minimal to no clinical activity. Immunotherapy has emerged as a potentially transformative strategy with attractive and long-lasting responses in HCC. However, the clinical efficacy of immune check point blockades (ICB) is limited to a select subset of patients and is hampered by drug resistance.

Methods: In this study, we focused on tumor cell intrinsic factors and conducted a hybridization-capture methodology and targeted 1 Mb of genomic coding sequence ( Gene Plus1021 Panel). Analysis of 81 HCC tumors and matching plasma normal samples was performed to characterize the landscape of somatic mutations especially those related to the sensitivity, resistance and hyper progression of ICB therapy in HCC. For genes identified in our study significantly correlated with tumor mutational burden (TMB), We further did the verification in The Cancer Genome Atlas (TCGA)-LIHC. 363 HCC tumor samples were included in the cohort and the data was downloaded from cBioPortal website (http://www.cbioportal.org/).

Results: We analyzed 81 HCC tumor samples accepted in Peking University International Hospital from January 2015 to Jun 2018 who did not receive ICB therapy at the time of taking specimen. The top quartile (7 muts/Mb) of TMB distribution was used as the cutoff value to define high tumor mutational burden (TMB-H). There were 26 cases (32.10%) with high TMB. Among all the 81 samples analyzed we detected no mismatch repair deficiency (dMMR), but identified five gene mutations having a significant correlation with high TMB including TP53, CTNNB1, ARID1A, MLL and NCOR1, p=0.0439, 0.0196, 0.0023, 0.0247 and 0.0076, retrospectively. We further did the verification in The Cancer Genome Atlas (TCGA)-LIHC. Gene mutations of TP53, CTNNB1, and MLL were proved to be highly associated with TMB-H status, p=0.0009, 0.0016 and 0.0013, retrospectively. Moreover, gene mutations of ARID1A, TP53 and MLL were proved to implicate poor overall survival. We detected 2 mutations (2.47%) in POLE, 2 mutations (2.47%) in PTEN, 1 mutation (1.23%) in DNMT3A, and 1 sample (1.23%) with high microsatellite instability (MSI-H). TMB value for the only one sample in MSI-H status was 9 muts/Mb (TMB-H). Instead of dMMR, one TP53 mutation and one ARID1A mutation were detected in this sample.

Conclusions: TMB-high and crucial driver gene mutations associated with high mutation load should be evaluated as potential predictive biomarkers in datasets of HCC patients treated with ICB therapy.

#1256

Validation of Ubiquitin specific peptidases 37 as a prognostic and diagnostic marker in osteosarcoma.

Mayank Singh,1 Atul Batra,1 Sameer Bakhshi,1 Matthew Summers2. 1 _All India Institute of Medical Sciences, Delhi, New Delhi, India;_ 2 _Ohio State University, Columbus, OH_.

Osteosarcoma has been a challenge for the oncologist because even after surgical removal of tumors 80 percent patients go on to develop metastasis to the Lung. Systematic development of Drug has been literally on a standstill in osteosarcoma. The emergence of Adjuvant therapy after surgery for localized osteosarcoma has increased the long term survival in patients under age 40 by 60 to 70 percent however for patients who go on to develop metastasis the prognosis is poor as there are limited therapeutic options. Protein ubiquitination and deubiquitylation plays an important role in controlling protein stability. Initial studies by us points to a model in which Ubiquitin specific peptidase 37 regulates cell cycle progression. Our data indicates that USP37 expression correlates with transformation by preventing degradation of different oncoproteins. It was found that in osteosarcoma cell lines USP37 is elevated which gives survival advantage to these cells while its depletion leads to enhanced cell death in response to genotoxic stress. USP37 depletion results in reduced resolution of DNA damage foci like gamma H2AX and 53BP1 and increase in no of collapsed replication fork which indicate the reduced ability of cells to carry out constitutive DNA replication. Physically USP37 interacted with proteins involved in DNA damage repair and effected their stability. We tried to correlate our data with archived patient osteosarcoma samples by analyzing if USP37 levels corelate with patient prognosis at our center which will be further discussed. The current data adds a new dimension to this newly explored deubiquitinates and merits development of targeting strategies to explore its therapeutic potential in osteosarcoma.

#1257

The transcription factor CREB is essential in myxoid liposarcoma.

Magdalene Cyra,1 Sebastian Huss,2 Miriam Schulte,1 Ilka Isfort,1 Susanne Hafner,3 Thomas Simmet,3 Thomas Kindler,4 Pierre Åman,5 Eva Wardelmann,2 Wolfgang Hartmann,1 Marcel Trautmann1. 1 _University Hospital Muenster, Gerhard-Domagk-Institute of Pathology, Division of Translational Pathology, Muenster, Germany;_ 2 _University Hospital Muenster, Gerhard-Domagk-Institute of Pathology, Muenster, Germany;_ 3 _Institute of Pharmacology of Natural Products & Clinical Pharmacology, Ulm University, Ulm, Germany; _4 _Department of Hematology, Medical Oncology and Pneumology, University Cancer Center of Mainz, Mainz, Germany;_ 5 _Sahlgrenska Cancer Center, University of Gothenburg, Gothenburg, Sweden_.

Introduction: Myxoid Liposarcoma (MLS) is an aggressive soft tissue tumor, characterized by a high rate of local recurrence and development of distant metastases. Most MLS are driven by a specific reciprocal t(12;16) translocation, and the resulting chimeric FUS-DDIT3 fusion protein acts as an oncogenic transcription factor. Although MLS display a higher chemosensitivity than other liposarcoma subtypes, the substantial rate of recurrence and metastasis in MLS underlines the urgent need for novel, biology- guided therapeutic strategies. As the mechanisms of FUS-DDIT3 mediating MLS pathogenesis are incompletely understood, we here investigate the functional relevance of the transcription factor CREB in MLS cells in vitro and in vivo.

Experimental procedures: In a large cohort of MLS tissue specimens (n=92), CREB (S133) and transcriptional targets such as Rb, Cyclin D1, PCNA and Bcl-xL were analyzed by immunohistochemistry. The functional interplay between CREB and FUS-DDIT3 was analyzed by RNAi-mediated knockdown of the chimeric fusion protein in MLS cells and induced expression of FUS-DDIT3 in human mesenchymal stem cells. CREB-mediated transcriptional activity was modulated by CREB-specific siRNA and treatment with small molecule inhibitors (666-15, KG-501, NASTRp and Ro-318220). The biological effects on MLS cell proliferation were monitored by cell viability, apoptosis and immunoblotting assays. An established myxoid liposarcoma avian chorioallantoic membrane model was used for in vivo confirmation.

Results: Elevated CREB phosphorylation (S133) was demonstrated in 60% of MLS tissue specimens by immunohistochemistry. In MLS cells, RNAi-mediated knockdown of FUS-DDIT3 lead to decreased phosphorylation levels of CREB (S133), accompanied by reduced transcriptional activity. Induced expression of FUS-DDIT3 in human mesenchymal stem cells stimulated phosphorylation of CREB (S133) and CREB target gene expression. Phosphorylation of CREB (S133) was induced by IGF-II stimulation and reduced by RNAi-mediated knockdown of IGF-IR. MLS cell viability was significantly reduced by specific RNAi-mediated knockdown of CREB and treatment with small molecule inhibitors (666-15, KG-501, NASTRp and Ro-318220) in vitro and in vivo.

Conclusions: Our preclinical study demonstrates the essential role of CREB-mediated transcriptional activity in myxoid liposarcoma tumorigenesis and provides a molecularly based rationale for a novel targeted therapeutic approach.

#1258

Pooled-analysis of response markers in cancer cell lines treated with tumor treating fields.

Gitit Lavi Shahaf, Moshe Giladi, Rosa S. Schneiderman, Karnit Gotlieb, Einav Zeevi, Yaara Porat, Mijal Munster, Uri Weinberg, Eilon D. Kirson, Yoram Palti. _Novocure, Haifa, Israel_.

Low intensity, intermediate frequency alternating electric fields (TTFields) exert an inhibitory effect on numerous cancer cell lines with some variability in the response. The goal of the present study is to compare characteristics of cell lines based on their response pattern to TTFields and to identify response markers. Forty-five different human cancerous cell lines were treated for 72 hours with TTFields at optimal cell-specific frequency at the same nominal intensity (1.7 V/cm). Cytotoxicity and clonogenic potential were determined. Functional analysis of differentially expressed genes and mutations associated with response to TTFields was performed based on the Cancer Cell Line Encyclopedia (CCLE) database. Sensitivity to TTFields was compared with pharmacological profiling (CCLE). The cytotoxic response to TTFields was found to be distributed around an average of 50% with (ranging between 14% and 86% reductions in cell counts). The clonogenic effect varied between no effect and 88% reduction in the number of colonies. Increased sensitivity to: Lapatinib, PHA-665752 and PLX-4720 was common in the group of cell lines which were more highly responsive to TTFields. Functional analysis of cell line gene expression and mutation data revealed enriched pathways related to DNA damage repair response, cell migration, hypoxia signaling and oxidative stress. This analysis of cancerous cell line response to TTFields define the optimal frequency to be applied for each cell line and demonstrates the broad effectiveness of TTFields in cells. The role of the specific mutations associated with TTFields response should be further explored in additional studies. Pharmacological profiling may offer a rational for combining specific agents with TTFields.

#1259

HYAL4: The first study on a potential molecular driver and biomarker of invasive bladder cancer.

Daley S. Morera,1 Sarrah S. Lahorewala,1 Soum D. Lokeshwar,2 Andre R. Jordan,2 Vinata B. Lokeshwar1. 1 _Augusta University, Augusta, GA;_ 2 _University of Miami, Miami, FL_.

INTRODUCTION AND OBJECTIVE: The majority of morbidity and mortality in bladder cancer (BCa) patients is due to muscle invasive bladder cancer (MIBC). The prognosis of patients with MIBC could be improved by identification of novel biomarkers and therapeutic agents. Chondroitin sulfate proteoglycans are known to promote tumor growth and metastasis. However, a Chondroitinase (Chase) that degrades chondroitin sulfate has not been identified. HYAL4, a member of the glycosaminoglycan (GAG) degrading enzyme-family, potentially has Chase activity; however, HYAL4 (HY4) has not be studied in any biological system, normal or disease. In this study, we evaluated the expression of all six members of the GAG-degrading enzyme family in BCa specimens and evaluated HY4 functions in preclinical models of BCa.

METHODS: Cohort 1: 79 bladder tissues (normal (NBL) = 31; tumor (TBL) = 52); cohort 2: 40 cystectomy specimens from MIBC patients who received adjuvant Gemcitabine plus cisplatin (G+C) treatment for metastatic disease with chemotherapy. Gene expression was measured by q-PCR in both cohorts. HY4 was either overexpressed (HY4) or knocked-down in immortalized normal urothelial (Urotsa) and 3 BCa cell lines. Transfectants were assayed for Chase activity, anchorage independent growth, motility, invasion, molecular signaling. Tumor growth and metastasis in intravesical examined in HT1376 bladder orthotopic model.

RESULTS: In cohort 1 HYAL1, HYAL4 mRNA levels were significantly (6-13-fold) elevated in TBL tissues when compared to NBL tissues (P<0.001). HY4 levels were 7-fold elevated in MIBC. HY4 levels were an independent predictor of metastasis and death due to BCa (chi-sq: 6.9; P=0.0087; chi-sq: 7.59; P=0.006). In cohort 2, high HY4 levels significantly correlated with G+C treatment failure (chi-sq: 8.7; P=0.003) with > 80% accuracy. HY4-expressing transfectants secreted HY4 and Chase activity in their conditioned media. Overexpression of HY4 in normal urothelial and BCa cells significantly increased, while its knockdown abrogated, anchorage-independent growth, invasion, chemotactic motility (> 3-fold). HY4 expression induced Gemcitabine resistance (IC50: vector: 3.1 nM; HY4: 126 nM). HY4 expression upregulated a stem cell signature. Urothelial cells expressing HY4 formed tumors in NOD/SCID mice. HY4-expressing BCa cells metastasized to lung and spleen in an orthotopic BCa model.

#1260

**Inhibition of DOG1, or TMEM16A, results in antitumoral effects in gastrointestinal stromal tumors (GIST)** in vitro **.**

Robin Fröbom, Erik Berglund, Robert Bränström. _Karolinska Institutet, Stockholm, Sweden_.

DOG1/TMEM16A, over-expressed in >95% of all GISTs, is a calcium-activated chloride channel that has gained attention as a promising drug target due to its involvement in several tumor-physiological processes. Two well-established human GIST cell lines (GIST-T1 and GIST882) were used to study DOG1 inhibitors effect on viability, colony formation, and cell cycle. In this study, we show that specific DOG1/TMEM16A-inhibitors (T16inh-A01 and CaCCinh-A01) decrease chloride currents, and reduces cell viability in GIST cells. However, only CaCCinh-A01 changes the distribution of the cell cycle in GIST cells, putting them into G1 cell cycle arrest, and increases sub-G1 phase population in GIST882 cells. CaCCinh-A01 strongly reduces the colony forming ability of the cells, whereas limited effect by T16inh-A01. Conclusions: DOG1/TMEM16A inhibition has antitumoral effects in GIST cells in vitro, and could potentially serve as a target for GIST therapy. CaCCinh-A01 seems to be the more potent inhibitor, presumably due to its effect not only on chloride currents but also on DOG1/TMEM16A expression.

#1261

Targeting Wee1 in laryngeal squamous cell carcinoma.

Meng-Ling Yuan, Qi-Wei Jiang, Yang Yang, Jia-Rong Huang, Zhi Shi. _Jinan Univ., Guangzhou, China_.

WEE1 is a tyrosine kinase that regulates G2/M cell cycle checkpoint and frequently overexpressed in various tumors. However, the expression and clinical significance of WEE1 in human laryngeal squamous cell carcinoma (LSCC) are still unknown. In this study, we found that WEE1 was highly expressed in LSCC tissues compared with adjacent normal tissues. Importantly, overexpression of WEE1 was correlated with T stages, lymph node metastasis, clinical stages and poor prognosis of LSCC patients. Furthermore, inhibition of WEE1 by MK-1775 induced cell growth inhibition, G2/M cell cycle arrest and apoptosis with the increased intracellular reactive oxygen species (ROS) levels in LSCC cells. Pretreatment with ROS scavenger N-acetyl-L-cysteine could reverse MK-1775-induced ROS accumulation and cell apoptosis in LSCC cells. MK-1775 also inhibited the growth of LSCC xenografts in nude mice. Altogether, these findings suggest that WEE1 is a potential therapeutic target in LSCC, and inhibition of WEE1 is the prospective strategy for LSCC therapy.

#1262

Relationship between RAS, BRAF, PIK3CA and location of primary tumor in urothelial carcinoma.

Young Saing Kim,1 Sang-Cheol Lee,2 In Gyu Hwang,3 Su Jin Lee,4 Se Hoon Park4. 1 _Gil Medical Center, Gachon University College of Medicine, Incheon, Republic of Korea;_ 2 _Soonchunhyang University Hospital Cheonan, Cheonan, Republic of Korea;_ 3 _Chung-Ang University, College of Medicine, Seoul, Republic of Korea;_ 4 _Sungkyunkwan University Samsung Medical Center, Seoul, Republic of Korea_.

Purpose: In urothelial carcinoma (UC), somatic mutations in RAS, BRAF, and PIK3CA genes are suggested for prognostic markers as well as for therapeutic targets. Studies have reported that upper tract (renal pelvis and ureter) and bladder UC differ in factors including prognosis and genetic features.

Methods: We investigated the difference in clinicopathological features and oncogenic mutations (RAS, BRAF, and PIK3CA) according to the location of primary disease. In a prospective biomarker study in patients with metastatic or unresectable UC, tumor DNA was analyzed for mutations in KRAS, NRAS, HRAS, BRAF, and PIK3CA using Ampliseq v2 platform. We further stratified the frequency of the mutations by the location of primary tumor.

Results: Among a total of 210 patients, those with upper tract UC and bladder UC were 90 and 120 patients, respectively. Missense mutations in KRAS, NRAS, HRAS, BRAF, and PIK3CA were present in 9 (4.3%), 1 (0.5%), 18 (8.6%), 4 (1.9%), and 32 (15.2%) patients, respectively. PIK3CA mutations were detected in 16 patients with upper tract UC (17.8%) and 16 patients with bladder UC (13.3%). Likewise, HRAS mutations were detected more frequently in upper tract UC (10/90, 11.1%) than bladder UC (8/120, 6.7%). In metastatic UC setting, PIK3CA or HRAS mutations were not associated with different overall survival.

Conclusions: PIK3CA and HRAS mutations were more frequently observed in patients with upper tract UC compared to those with bladder UC. The different genetic features need to be studied by further studies.

#1263

Targeting of CDCA8 suppresses hepatocellular carcinoma growth by restoring ATF3 tumor suppressor and inactivating oncogenic Akt signaling.

Keon Uk Park,1 Min Ji Ko,1 Ilseon Hwang,1 Hun-Mo Ryoo,2 Yun-Han Lee1. 1 _Keimyung University School of Medicine, Daegu, Republic of Korea;_ 2 _Daegu Catholic University, Daegu, Republic of Korea_.

Background: Hepatocellular carcinoma (HCC) is the third most lethal cancer worldwide which remains a major challenge due to poor prognosis and limited treatment options. Cell division cycle associated 8 (CDCA8) is known as a component of a chromosomal passenger complex required for stability of the bipolar mitotic spindle and it is commonly overexpressed in human HCC. However, the functional role of CDCA8 in HCC progression remains to be clarified. In this study, we hypothesized if targeting of CDCA8 with small interfering (si) RNA could alter the course of HCC progression. We also investigated the molecular mechanism that mediates HCC cell death caused by CDCA8 silencing.

Methods: Human HCC cell lines, Huh1 and Huh7, were transfected with CDCA8 siRNA and tested for growth inhibition and apoptotic induction using MTS, FACS and Western blotting. To obtain insights into the molecular changes in response to CDCA8 knockdown, global changes in gene expression were examined using RNA sequencing.

Results: siRNA silencing of CDCA8 inhibited HCC cell growth and long-term colony formation by blocking cell cycle progression and by inducing apoptotic cell death. RNA sequencing data showed that, representatively, the anti-proliferative effects were driven by a subset of molecular alterations including the upregulation of tumor suppressive ATF3 and GADD34 genes and the downregulation of BGLAP, a key regulator of cell growth and invasiveness. Subsequent Western blot analysis affirmed that CDCA8 silencing induces the increase in the levels of ATF3 and GADD34 and the decrease in phosphorylated Akt in both Huh1 and Huh7 cells. Also, the same condition decreased the levels of PARP-1 and pro-caspase 3, accelerating apoptosis. Of importance, silencing of CDCA8 expression effectively suppressed HCC tumor growth in a murine xenograft model.

Conclusion: These findings suggest that targeting CDCA8 could be an attractive option for molecular therapy of HCC.

#1264

Design, synthesis and biological evaluation of new phenothiazine derivatives as potential Tousled-like kinase 1 inhibitors in prostate cancer treatment.

Siddhant Bhoir,1 Javeena Hussain,1 Vibha Singh,2 Rupesh Chikhale,3 Richard Bryce,3 Sivapriya Kirubakaran,1 Arrigo DeBenedetti2. 1 _IIT Gandhinagar, India;_ 2 _LSU Health Shreveport, LA;_ 3 _University of Manchester, United Kingdom_.

Introduction: The standard therapy for advanced Prostate Cancer (PCa) consists of anti-androgens which provide respite from the disease progression, yet ultimately fail and result in the incurable phase of the disease: mCRPC. Targeting PCa cells before their progression to mCRPC would significantly improve the outcome. Untoward toxicity limits the combination therapies targeting the DNA Damage Response (DDR), and hence the goal of clinical trials is to target the DDR more specifically. Androgen deprivation therapy (ADT) in LNCaP cells results in the increased expression of TLK1B, a critical kinase upstream of NEK1 and ATR, thereby mediating a DDR that typically causes a temporary cell cycle arrest of androgen-responsive PCa cells. Following the DNA damage, the addition of a TLK1 specific inhibitor, thioridazine (THD), impairs ATR and Chk1 activation, establishing the existence of an ADT>TLK1>NEK1>ATR>Chk1 DDR pathway, while its abrogation, leads to apoptosis. However, THD is a known anti-psychotic and has undesirable side-effects. Hence, there is a compelling need to design and develop next-generation TLK1 inhibitors to circumvent the adverse effects and advance them in the clinic.

Methods: We performed immunoblotting of the tumour tissue phosphoproteins (pATR, pChk1 and pNEK1) and immunohistochemistry analysis of the tissue sections from the LNCaP xenograft models. To identify and develop new potent inhibitors against TLK1, we employed an in-silico homology modelling and molecular docking approach. Based on the protein-ligand binding interactions and the docking score, a handful of compounds were shortlisted, synthesised and screened for the TLK1 inhibition potential in-vitro and using cell-based assays.

Results: Our experimental data revealed that the pATR, pChk1, pNEK1, Ki-67 and PCNA were remarkably inhibited when treated with THD in combination with an anti-androgen drug, Bicalutamide (BIC). Moreover, it also induced apoptosis and increased DNA damage as demonstrated by the cleaved PARP, Caspase 3 and γH2AX levels respectively. The new inhibitor screening assay showed J54 compound to be most potent and inhibitory with a logIC50 of 1.1µM. J54 binds to the protein's allosteric site noncompetitively with ATP and interacts with His504 and Gly630 with a corresponding docking score of -6.736. J54 is found to be non-toxic to normal cells and also suppresses the growth of androgen-dependent colonies of LNCaP cells cultured with BIC.

Conclusion: Our preliminary work suggests that targeting the TLK1/NEK1 axis with specific TLK1 inhibitors might be an effective therapy for PCa in combination with standard care, ADT.

#1265

Validation of the interaction between a candidate compound and the intended drug target by a phenotypic rescue approach.

Lieke Geerts,1 Laure Grandmoursel,1 Jamil Aarbiou,1 Jeroen DeGroot,1 Julia Schüler,2 Ian Waddell,3 Anne-Marie Zuurmond1. 1 _Charles River, Leiden, Netherlands;_ 2 _Charles River, Freiburg, Germany;_ 3 _Charles River, Chesterford Research Park, Cambridge, United Kingdom_.

New targets for cancer treatment frequently emerge in literature, but the thorough target validation required to consider these targets for a drug discovery program is often lacking. In pharmacological or genetic perturbation studies using complex biological assays, undesired off-target effects cannot be easily distinguished from the intended mode of action at the desired target. This is especially evident in cancer drug development where it is important to discriminate on-target effects on cell viability from off-target effects resulting in non-specific loss of cellular fitness. Neglecting the possibility of being deceived by off-target effects can have tremendous scientific and financial impact on a drug discovery program. Ideally confidence in a preclinical drug target and a modulating compound is boosted in an early stage by more extensive analysis and validation of the actual drug-target relationship. Rescue of a disease-relevant phenotype by genetic restoration of a target mutation is a gold standard approach in drug discovery by which target validation can be achieved. We aim to follow this approach targeting the BRAF V600E mutation in a number of well described melanoma lines as well as the MAP2K1 Q56P mutation in non-small cell lung cancer cell line H1437. Target validation for both BRAF and MAP2K1 will be addressed by assessing viability, phenotypic changes and sensitivity to compound modulation upon CRISPR/Cas9 repair of the target mutation or by exogenous re-expression of the wildtype variant. Compounds tested will be Vemurafenib for BRAF and Trametinib for MAP2K1. Further investigation into target validity will be done using a physiologically relevant 3D spheroid based co-culture system. Mimicking the tumor microenvironment increases the knowledge about "drug-ability" of a target and sustainability of the target modulation at an early time point in the development process. Such early in-depth validation of the relationship between a compound and the drug target is vital to mitigate the risk of failure at later steps of drug development. 

### Drug Resistance 2

#1266

Co-expression of P-glycoprotein and ABCG2 in a cell line model reveals both independent and cooperative transporter function.

Andrea Robinson, Robert Robey, Michael Gottesman. _National Institutes of Health, Bethesda, MD_.

Although overexpression of multiple ATP-binding cassette transporters has been reported in clinical samples, few studies have examined how expression of multiple transporters might confer resistance to chemotherapy drugs. We therefore examined how P-glycoprotein (P-gp, encoded by the ABCB1 gene) and ABCG2 contribute to drug resistance in a cell line model. HEK-293 cells were transfected with vector encoding full-length ABCB1, ABCG2, or a vector containing both genes under the control of a separate promoter. Cells transfected to express both transporters (B1/G2 cells) expressed high levels of both transporters and were able to transport both the P-gp-specific substrate rhodamine 123 and the ABCG2 specific substrate pheophorbide a when examined by flow cytometry. B1/G2 cells were also cross-resistant to the P-gp substrate doxorubicin, the ABCG2 substrate topotecan as well as mitoxantrone, a substrate of both transporters. The checkpoint inhibitor prexasertib was also found to be a substrate of both P-gp and ABCG2. When B1/G2 cells were incubated with both rhodamine 123 and pheophorbide a, transport of both compounds was observed, suggesting that P-gp and ABCG2 can function independently to transport substrates. P-gp and ABCG2 were also found to function cooperatively to transport the common fluorescent substrates mitoxantrone and BODIPY®-prazosin, as it was necessary to inhibit both transporters to prevent efflux from B1/G2 cells. This was also true in 3-day cytotoxicity assays with mitoxantrone and prexasertib. Thus, P-gp and ABCG2 can independently and cooperatively confer resistance to substrates, underscoring the need to inhibit multiple transporters when they are coexpressed.

#1267

Unusual mechanism of resistance to the novel cytidine analog fluorocyclopentenylcytosine (RX-3117).

Godefridus J. Peters,1 Dzjemma Sarkisjan,1 Btissame El Hassouni,1 Richard J. Honeywell,1 Joris R. Julsing,1 Beatrice Balboni,1 Daniel J. De Klerk,1 Safet Zekanovic,1 Kees Smid,1 Elisa Giovannetti,1 Young B. Lee,2 Deog J. Kim2. 1 _VU Univ. Medical Ctr., Amsterdam, Netherlands;_ 2 _Rexahn Pharmaceuticals, Rockville, MD_.

RX-3117 is a novel cytidine analog showing encouraging results in ongoing Phase 2a studies with gemcitabine-resistant pancreatic ductal adenocarcinoma and advanced urothelial bladder cancer. Cellular uptake of RX-3117 is mediated by the equilibrative nucleoside transporter 1 (hENT1). RX-3117 is activated by uridine-cytidine kinase-2 (UCK2) and is not a substrate for cytidine deaminase (CDA), and shows a good oral bioavailability. In an earlier study accumulation of RX-3117 nucleotides was associated with sensitivity to the drug. RX-3117 is incorporated into RNA and DNA and inhibits DNA methyltransferase 1 (DNMT1). RX-3117 is active against gemcitabine-resistant non-small cell lung cancer (NSCLC) cell lines. In this study we aimed to elucidate potential resistance mechanisms against RX-3117. For that purpose the NSCLC cell lines H460, A549 and SW1573 (including its gemcitabine resistant variant SW-G) were exposed to increasing concentrations of RX-3117. For A549 and SW1573 two variants were obtained: A549/RX1, A549/RX2, SW1573/RX1 and SW1573/RX2. Resistance varied from 10 (H460; SW-G) to >100-fold for the other cell lines. Resistance was stable except for H460 cells. Cross-resistance in the same range was observed for the cytidine analogs ethynyl-cytidine (ETC), cyclopentenylcytosine (CPEC) and aza-cytidine (aza-CR), which are all activated by UCK2; no cross-resistance was observed for gemcitabine and aza-deoxycytidine which are activated by deoxycytidine kinase. No change was observed in the expression of DNMT1, while expression of ribonucleotide reductase 1 was decreased. However, analysis of UCK2 at the enzyme activity level, protein and gene expression did not reveal a decrease in UCK2 while for ETC, aza-CR and CPEC resistance was reported to be mediated by a UCK2 deficiency. No change was found in the other activating enzymes UMP-kinase, CMP-kinase and NME1/NDKA. Despite this lack of change, accumulation of RX-3117 and RX-3117 nucleotides (mono-, di- and triphosphate) was 2-5-fold lower in the resistant cells after incubation with 10 or 100 μM RX-3117 for 4-24 hr. No evidence for accumulation of RX-3117 deoxynucleotides was found in both the parent and the resistant cell lines. Since no change in hENT1 was found, cell lines were further investigated by using RNA-seq, and western blotting focusing on nucleoside/nucleotide degradation enzymes. CDA was decreased in resistant cells, but the cytosolic pyrimidine nucleotidase NT5C3 was increased in the resistant cell lines as well as DCTPP1, which can degrade deoxynucleoside triphosphates. Moreover several DNA repair enzymes SAMHD1, MTH1 and TDP1 were increased. However, inhibition of MTH1 and DCTPP1 did not increase sensitivity to RX-3117. In conclusion, induction of resistance to the novel cytidine analog RX-3117 led to a decreased accumulation of RX-3117 ribonucleotides, which might be associated with an increased degradation.

#1268

The impact of P-glycoprotein, breast cancer resistance protein and CYP3A on pharmacokinetics and metabolism of galunisertib.

Wenlong Li, Matthijs Tibben, Yaogeng Wang, Maria C. Lebre, Hilde Rosing, Jos H. Beijnen, Alfred H. Schinkel. _The Netherlands Cancer Institute, Amsterdam, Netherlands_.

Transforming growth factor-beta (TGF-β) signaling plays a pivotal role in tumorigenesis and contributes to the hallmarks of cancer, including tumor proliferation, invasion and metastasis, inflammation, angiogenesis, and escape of immune surveillance. Galunisertib (LY2157299 monohydrate) is an oral small-molecule inhibitor of the TGF-β receptor I kinase that specifically down-regulates the phosphorylation of SMAD2, abrogating activation of this canonical pathway. Galunisertib showed promising antitumor activity in tumor-bearing animal models for breast, colon, and lung cancers, and for hepatocellular carcinoma.

Potential drug transport mediated by ATP-binding cassette (ABC) transporters and metabolism by CYP3A are of clinical and regulatory concern, as these could modulate the systemic exposure and/or organ distribution of substrate drugs, and thus affect their therapeutic efficacy and toxicity. We here aimed to investigate the roles of two ABC transporters (P-glycoprotein/ABCB1 and Breast Cancer Resistance Protein/ABCG2) and CYP3A enzyme in the pharmacokinetics and tissue distribution of galunisertib.

Transepithelial drug transport was tested using polarized monolayers of Madin-Darby Canine Kidney (MDCK-II) parental cells and its subclones overexpressing human (h) ABCB1, hABCG2, or mouse (m) Abcg2 cDNA. The results suggest that galunisertib is an excellent transport substrate of hABCB1 and endogenous canine ABCB1. mAbcg2 could modestly transport galunisertib, but hABCG2 could not. Based on the in vitro transport experiments, we performed an in vivo pharmacokinetic study in wild-type, ABC transporter knockout (Abcb1a/1b;Abcg2-/-), and Cyp3a knockout (Cyp3a-/-) mice. Following oral administration (20 mg/kg galunisertib), plasma exposure (AUC) in mice was similar to that achieved in humans dosed at 300 mg once daily. Interestingly, the brain-to-plasma ratio was 26-fold higher in Abcb1a/1b;Abcg2-/- mice compared to wild-type mice. However, no significant differences were observed in other tested tissue distributions or in oral availability of galunisertib among these three mouse strains.

Our data suggest that ABC transporters play an important role in restricting the brain penetration of galunisertib, but have little impact on oral availability and other relative tissue distribution. Mouse Cyp3a appears to have little, if any, effect on the pharmacokinetics and metabolism of galunisertib. Our results suggest a potential way to boost brain penetration of galunisertib by pharmacological inhibition of transporters in the BBB with chemical inhibitors, especially for patients with brain metastases. These insights might be used to optimize the clinical application of galunisertib.

#1269

P-glycoprotein expression in refractory gastrointestinal stromal tumors and its implication in the efficacy of paclitaxel as a salvage treatment.

Young-Soon Na,1 Min-Hee Ryu,2 Young Soo Park,2 Chae-Won Lee,1 Ju-Kyung Lee,1 Jung Min Park,1 Yangsoon Park,2 Yoon-Koo Kang2. 1 _Asan Inst. for Life Science, Seoul, Republic of Korea;_ 2 _Asan Medical Center, Seoul, Republic of Korea_.

BACKGROUND: KIT-targeting tyrosine kinase inhibitors (TKIs) such as imatinib, sunitinib and regorafenib are the standard treatment for patients with gastrointestinal stromal tumor (GIST). However, most patients eventually develop treatment resistance to these standard therapies, and new agents must be introduced upon disease progression. Before TKIs were available, most of the conventional cytotoxic agents did not show sufficient clinical activity in GIST patients. However, a recent preclinical study demonstrated that 37 of the 89 FDA-approved anti-tumor drugs including paclitaxel (PTX) possess antitumor effect in at least one GIST cell line. Therefore, in this study, we aimed to evaluate the efficacy of PTX as a salvage treatment for GIST patients who exhibited treatment failure after standard TKI therapy using in-vitro/vivo models.

MATERIALS and METHODS: The effect of PTX in GIST was examined by cell viability assay and rhodamine 123 (Rho123) efflux assay using GIST cells including established patient-derived GIST cell lines, and in animal models with patient-derived xenografts (PDXs) established from patients with GIST tumors refractory to TKIs. Multidrug resistance 1 (MDR1) mRNA expression by reverse transcription-PCR (RT-PCR) and P-glycoprotein (Pgp) expression by Western blotting or immunohistochemistry were evaluated in 20 patients' tumor tissues, 9 GIST cell lines and 21 PDXs. To investigate the role of Pgp on PTX treatment, a stable MDR1-expressing GIST T1 cell line (GIST-T1-ABCB1#17) was prepared and compared with parent GIST T1 cell line. Verapamil was used as a Pgp inhibitor.

RESULTS: Compared to imatinib-sensitive GIST-T1 harboring KIT exon 11 mutation and imatinib-resistant GIST-T1/816 harboring KIT exon 11 and 17 mutations, the patient-derived imatinib- and sunitinib-resistant GIST-R3 cell line harboring KIT exon 11 and 17 mutations was more resistant to PTX. Higher Rho123 efflux was observed in GIST-R3 which had high Pgp expression than in GIST-T1 and GIST-T1/816 which had low Pgp expression. The tumor growth inhibition of PTX was greater in xenografts with low Pgp expression (GIST-T1/816 xenografts and GIST-RX10 PDXs) than in xenografts with high Pgp expression (GIST-R3 xenografts and GIST-RX4 PDX). The GIST-T1-ABCB1#17, a stable MDR1-expressing GIST T1 cell line, exhibited a higher Pgp activity and a less sensitivity to PTX than the parent GIST-T1 cell line. The resistance of GIST-T1-ABCB1#17 to PTX was overcome by verapamil.

CONCLUSION: Pgp expression was an important mechanism of resistance to PTX in preclinical GIST models. PTX is worth being tried clinically as a salvage treatment in patients with refractory GISTs with low Pgp expression.

#1270

Human P-glycoprotein-mediated drug transport: Two aromatic residues in the drug-binding pocket are critical for function.

Megumi Murakami, Eduardo E. Chufan, Sabrina Lusvarghi, Suresh V. Ambudkar. _National Institutes of Health, Bethesda, MD_.

P-glycoprotein (P-gp) is an efflux pump that transports cytotoxic agents, thereby affecting the pharmacokinetics of many drugs and conferring resistance to chemotherapeutic agents in many cancers. P-gp utilizes energy from ATP hydrolysis for the efflux of a number of amphipathic natural products or small molecule anticancer drugs. However, the nature and number of translocation pathways for the transport of substrate-drugs by P-gp is not yet known. Previously we showed that substitution of certain aromatic residues in the drug-binding pocket switched the modulation of ATPase activity from inhibition to stimulation. Molecular modeling studies led to identification of two T-shaped structural motifs formed by F728/Y310 and F978/Y953. In this study, we demonstrate that substitution of all four residues of the T-shaped structural motifs with alanine (Y310A/F728A/Y953A/F978A) results in complete loss of transport of ten fluorescent substrates. Furthermore, alanine substitution of one or two of these residues revealed that when F728 and F978 are both substituted, this has the greatest impact on the binding and/or translocation of drug-substrates. ATPase assays and in silico analyses were performed to gain insight into interactions of these residues with substrates and modulators. These results indicate that the F728 and F978 residues together are critical to maintaining the conformation of the drug-binding pocket for binding and translocation of substrates by P-gp.

#1271

ABCC11 is involved in resistance to alectinib.

Tomoko Yamamoto Funazo,1 Hiroaki Ozasa,1 Takahiro Tsuji,1 Koh Furugaki,2 Yasushi Yoshimura,2 Hitomi Ajimizu,1 Yuto Yasuda,1 Takashi Nomizo,1 Yuichi Sakamori,1 Hironori Yoshida,1 Young Hak Kim,1 Toyohiro Hirai1. 1 _Kyoto University Hospital, Kyoto, Japan;_ 2 _Chugai Pharmaceutical, Co., Ltd., Kamakura, Japan_.

Non-small cell lung cancer is known to have a poor prognosis. One reason for this is resistance to anticancer drugs. Various mechanisms for resistance to anticancer drugs have been reported. Herein we focus on ABCC11, an adenosine triphosphate (ATP)-binding cassette transporter. ABCC11 is ubiquitously expressed in various adult human tissues, including liver, lung, and kidney, and confers drug resistance to some cytotoxic agents such as 5-fluorouracil (5-FU), pemetrexed, and methotrexate. However, the association between ABCC11 and resistance to molecularly-targeted therapeutic drugs is still unknown. We hypothesized that alectinib, a molecularly-targeted therapeutic agent for anaplastic lymphoma kinase (ALK)-rearranged lung cancer, was a substrate for ABCC11. To evaluate the expression of ABCC11 in alectinib-resistant cells, an alectinib-resistant cell line model (AR1S) was established by exposing NCI-H2228, an ALK-rearranged cell line, to alectinib for 3 months. Patient-derived cell lines that were sensitive or resistant to alectinib were also established from a treatment-naïve patient (KTOR-1), and after disease progression (KTOR-1 RE). The protein expression of ABCC11 was increased in both alectinib-resistant cell lines (AR1S and KTOR-1 RE), compared to naïve cell lines (H2228 and KTOR-1). To investigate the role of ABCC11 in alectinib resistance, ABCC11 overexpression cell lines (OE-A and OE-B) were established by introducing an ABCC11 expression construct into H2228. A negative control cell line (mock) was established by introducing the control empty vector into H2228. The gene expression of ABCC11 in OE-A and OE-B was higher than that in mock (133-fold increase, P < 0.0001 and 109-fold increase, P < 0.0001 respectively), and the protein expression of ABCC11 was also higher in OE-A and OE-B. The IC50 for alectinib was higher in OE-A (8.0 times) and OE-B (10.8 times) compared to mock. ABCC11 was knocked down using siRNA in AR1S to evaluate alectinib susceptibility. Knockdown of ABCC11 improved the IC50 for alectinib, compared with a negative control (0.299-fold decrease). Next, the tumor responses to alectinib in OE-A and OE-B were evaluated in vivo. Xenograft models of OE-A, OE-B, and mock on BALB/nu mice were administered daily alectinib (8 mg/kg/day) or vehicle for 10 days. In mice administered alectinib (N = 6-7), the tumor shrinkage rate of OE-A (−23.6%) and OE-B (−34.3%) was significantly lower than that of mock (−76.8%). There results have provided the first of preclinical evidence that ABCC11 is involved in resistance to alectinib.

#1272

Whole genome Crispr screening to identify potential SERD molecule resistance mechanisms.

Christophe Marcireau,1 Karine Berthelot,1 Alice Williart,1 Hamida Fournet,2 Delphine Debono,2 Gilbert Thill,2 Helene Erasimus,1 Dorine Chassin,1 Christophe Lanneau,2 Veeanagouda Yaligara,2 Cecile Orsini,1 Michel Didier,2 Vincent Mikol,1 Monsif Bouaboula,3 Laurent Debussche1. 1 _Sanofi, Vitry sur seine, France;_ 2 _Sanofi, Chilly-Mazarin, France;_ 3 _Sanofi, Cambridge, MA_.

Metastatic breast cancer results in substantial morbidity and mortality for women afflicted with this disease. Approximately 80% of breast cancers express the estrogen receptor. The cancer cell survival and proliferation is driven by the activation of the estrogen receptor. SAR439859 is an effective endocrine therapy for breast cancer selectively and effectively degrading the estrogen receptor. A phase 1/2 study of SAR439859 alone and/or in combination with palbociclib in postmenopausal women with estrogen receptor positive advanced breast cancer was initiated last year by Sanofi.

CRISPR/Cas9 is a recent and revolutionary technology for efficient and directed alterations of the genome. CRISPR allows users to introduce DNA double-strand breaks at precise locations in the genome using complementary guide RNAs (SgRNA). These double strand breaks can be repaired by non-homologous end joining DNA repair mechanism. This DNA repair mechanism is an error prone DNA repair process which can lead to gene invalidation.

We aimed at investigating the potential SAR439859 resistance mechanisms. A better understanding of resistance mechanisms is needed to overcome this potential problem and to propose complementary therapeutic solutions. We conducted a whole genome CRISPR screening in a breast tumor cell line with a thermofisher commercial SgRNA library (ref: M04305) to identified genes involved in potential SAR439859 resistance phenomena. CRISPR screening steps will be presented from the cell line selections to results analysis.

#1273

**Molecular dynamics prediction of the mechanism of acquired resistance to EGFR inhibitors in** EGFR **-mutant lung cancer.**

Youngjoo Lee, Yu-Ra Choi. _National Cancer Ctr. Korea, Goyang-si, Republic of Korea_.

Background Acquired drug resistance is the most challenging problem in treating nonsmall cell lung cancer (NSCLC) patients with EGFR tyrosine kinase inhibitor (TKI). We evaluated whether resistance cell subclones significantly increased shortly after treatment with EGFR-TKIs could determine the final resistance phenotype for EGFR-TKIs in the tumor with EGFR mutations.

Methods We generated 2 EGFR-mutant lung cancer cell lines (PC9GR and HCC827GR) resistant to gefitinib and evaluated the resistance mechanisms in each cell line. In parent cell lines (PC9 and HCC827) the amounts of EGFR T790M mutation and C-MET and VIMENTIN mRNA expression were measured before and after 48 hours treatment with gefitinib or paclitaxel. Using a cell line derived directly from tumors of treatment-naïve NSCLC patient with EGFR mutation, the above tests were repeated. We prospectively collected the bloods from 4 NSCLC patients with EGFR mutations at baseline and eight weeks after EGFR-TKI treatment and measured the level of T790M mutation in circulating tumor DNAs (ctDNA) from their bloods by colourimetric assay.

Results EGFR T790M mutation was detected in PC9GR cells whereas C-MET amplification in HCC827GR cells. The proportion of EGFR T790M mutation significantly increased in the PC9 cells treated with gefitinib whereas not in the same cells treated with paclitaxel or not in the HCC827 treated with gefitinib or paclitaxel. The mRNA expression of C-MET was significantly increased in the HCC827 treated with gefitinib. The VIMENTIN mRNA expression was not changed in both cell lines. When we treated the patient-derived cells with gefitnib, the frequency of T790M significantly increased whereas C-MET mRNA expression decreased and VIMENTIN mRNA expression was not changed. In the corresponding resistance tumor tissue, only T790M mutation was detected without C-MET amplification and VIMENTIN overexpression. Two cases whose T790M mutation level in their ctDNAs increased at 8 weeks by more than 50% revealed T790M mutation in their final resistance tumor tissue. But, in other cases without an early surge of T790M mutation in their ctDNAs, T790M mutation was not detected in posttreatment biopsy sample.

Conclusions Early-on-treatment quantitative change of resistance-related gene alterations may predict the final mechanism of resistance to EGFR-TKI.

#1274

Partial reprogramming of melanoma cells confers drug resistance and increased vulnerability to channel antagonists.

Karol Granados,1 Laura Hüser,1 Aniello Federico,1 Sachindra Sachindra,2 Gretchen Wolff,3 Viktor Umansky,1 Jochen Utikal1. 1 _German cancer research center (DKFZ), Heidelberg, Germany;_ 2 _Charité - University Medicine Berlin, Berlin, Germany;_ 3 _Heidelberg University Hospital, Heidelberg, Germany_.

During melanoma progression, tumor cells undergo phenotypic switching, increasing melanoma plasticity and resistance to mitogen-activated protein kinase inhibitors (MAPKi). We propose a partial reprogramming model to target melanoma therapy resistance. Here we show that partially reprogrammed cells are a less proliferative and more dedifferentiated cell population, expressing a gene signature for stemness and suppressing melanocyte-specific markers. To investigate the development of resistance to MAPKi, cells were exposed to BRAF and MEK inhibitors. Partially reprogrammed cells become less sensitive to MAPKi, showing increased cell viability and decreased apoptosis. Based on genome expression data, we evaluated potential targets to restore sensitivity to MAPKi in partially reprogrammed cells. One of our candidates showed promising results by increasing apoptosis and differentiation after sequential treatment with BRAF and MEK inhibitors, in partially reprogrammed cells and human BRAFi-resistant cells. In summary, we show that partial reprogramming of melanoma cells is a feasible in vitro model to study resistance to MAPKi. Moreover, we suggest our candidate as a target to restore sensitivity of resistant cells to MAPKi which has significant clinical implications in the development of resistance to current treatments against melanoma.

#1275

MK591 (Quiflapon) downregulates c-Myc oncogenic signaling and induces apoptosis in enzalutamide-resistant prostate cancer cells.

Jitender Monga,1 Ajay Bharathan,2 Dhatchayini Subramani,1 Jagadananda Ghosh1. 1 _Henry Ford Health System, Detroit, MI;_ 2 _Salem High School, Canton, MI_.

Background: Enzalutamide is an FDA-approved drug commonly prescribed for advanced prostate cancer. Enzalutamide slows down prostate tumor growth but resistant disease invariably develops which is incurable primarily because currently available therapies cannot effectively kill enzalutamide-resistant prostate cancer cells. Mechanism(s) behind development of enzalutamide-resistance is not properly understood, though overactivation of c-Myc has been found to be a common event which plays an important role in the maintenance and progression of ERPC phenotype. However, direct-targeting of c-Myc poses special problem because of its non-enzymatic nature and certain amount of c-Myc activity is needed by non-cancer cells as well. Thus, c-Myc has emerged as an elusive target which needs to be managed by novel agents and strategies in a cancer-specific way for better control of ERPC.

Methods: We addressed this problem by treating ERPC cells with a variety of cell survival and apoptosis-regulating agents followed by measurement of cell viability, and by analysis of the mRNA and protein levels of c-Myc. Gene expression was analyzed by Illumina Hi-Seq whole genome gene-expression array. Expression of c-Myc was confirmed by RT-PCR and Western blot. Apoptosis was measured by annexin-V binding, PARP-cleavage, and by detecting degradation of chromatin-DNA to nucleosomes. Transcriptional activity of c-Myc was analyzed by nuclear accumulation, DNA-binding, luciferase-reporter assays and expression of c-Myc-target genes.

Results: We found that MK591, a leukotriene biosynthesis inhibitor, dramatically downregulates c-Myc in ERPC cells as revealed by RT-PCR, and Western blot as well as by the reduction in nuclear-accumulation and DNA-binding activities of c-Myc. Treatment with MK591 decreased the Myc-driven E-box-luciferase reporter activity, and substantially reduced the expression of c-Myc target genes (Cyclin D1, CDK4, survivin, Aurora kinase). Moreover, MK591 effectively blocked in vitro invasion and soft-agar colony-formation by ERPC cells. Interestingly, while MK591 strongly inhibits c-Myc function and kills ERPC cells via caspase-mediated apoptosis, it does not inhibit the basal c-Myc function or the viability of non-cancer cells, such as human foreskin fibroblasts (HFF).

Conclusion: Our findings indicate that the expression and oncogenic-function of c-Myc in ERPC cells are severely downregulated by MK591, and suggest that MK591 may turn out to be a suitable new agent to treat advanced, aggressive prostate cancers which are resistant to enzalutamide therapy.

#1276

Inhibition of lipid metabolism decreases proliferation and increases radiosensitvity in EGFR-TKI resistant NSCLC cells with bFGF secretion.

Wei Wang, Shunli Peng, Rong Wang, yueyun Ma, Longhui Zhong. _Nanfang Hospital, Southern Medical University., GuangZhou, China_.

Primary and acquired EGFR-TKIs resistance restricts the benefit of EGFR-TKIs to EGFR mutant NSLCLC patients. Recent studies found that metabolic reprogramming might contribute to develop metastasis and drug resistance of tumor cells. Therefore, illustrating the metabolism change is meaningful and targeting the peculiar metabolic pathways might be considered as potential therapeutic strategies for EGFR-TKIs resistant NSCLC. We separated single clones from gefitnib resistant NSCLC cells, by exposing EGFR-mutant EGFR-TKIs sensitive PC-9 cells into increasing concentrations of gefitnib. Among the numerous clones, clone 5 was resistant to gefitinib or AZD9291 and named as PC-9GR-FGF because of high level of bFGF secretion. We found that bFGF promote the transcription of SREBP-1 and its downstream genes, acetyl-CoA carboxylase alpha (ACC), ATP citrate lyase (ACLY) and fatty acid synthase (FASN) in PC-9 cells. Compared to PC-9 cells, higher ACC expression was detected in PC-9GR-FGF cells. Basing on TCGA database (lung Adenocarcinoma, provisional), patients with high ACC expression showed significant shorter PFS (progression-free survival) and OS (overall survival) compared to those with low ACC expression in the EGFR-mutant NSCLC (p<0.05, N=32). Down-regulation of ACC by siRNA or TOFA, a small molecular ACC inhibitor, mederately decreased the proliferation of PC-9 cells whereas strongly decreased proliferation of PC-9GR-FGF cells in vitro and in vivo. Moreover, Reactive oxygen species (ROS) and oxidative stress scavenger, NAC and ferrostatin-1, failed to reverse the effect of ACC inhibition in PC-9 and PC-9GR-FGF cells. Furthermore, after down-regulation or inhibition of ACC, stronger G2/M arrest and more accumulation of DNA damage marker, γH2AX, were found in PC-9GR-FGF cells compared with PC-9 cells. In addition, ACC expression were increased after irradiation of X-ray in PC-9GR-FGF cells, and inhibition of ACC increased accumulation of γH2AX induced by X-rays in PC-9GR-FGF cells. Our research gives a view sight into lipid metabolism changes in EGFR-mutant NSCLC cells before and after acquired EGFR-TKIs resistance, and explores the possibility of combination strategy with radiotherapy and anti-cancer lipid metabolism therapy.

#1277

Honokiol, a natural product, overcomes acquired resistance of EGFR mutant NSCLC cells to the third generation EGFR inhibitor, osimertinib.

Hongjing ZANG,1 Songqing Fan,2 Jack Arbiser,3 Taofeek K. Owonikoko,3 Suresh S. Ramalingam,3 Shi-Yong Sun3. 1 _Departments of Hematology and Medical Oncology, Atlanta, GA;_ 2 _Central South University, Changsha, China;_ 3 _Emory University School of Medicine and Winship Cancer Institute, Atlanta, GA_.

Osimertinib (AZD9291) is a FDA-approved EGFR inhibitor for treating non-small cell lung cancer (NSCLC) patients with activating EGFR mutations (first-line) or those who have become resistant to 1st generation EGFR inhibitors through the T790M mutation (second-line). The development of acquired resistance to osimertinib limits the long-term benefits of patients, and effective treatment options are urgently needed. We have identified that honokiol, a natural product with potential antitumor activity, may be used to overcome osimertinib resistance. In several osimertinib-resistant cell lines including HCC827/AR, PC-9/AR, PC-9/GR/AR and PC-9/3M (with EGFR 19del, T790M and C797S mutations), the combination of honokiol and osimertinib had synergistic effects on decreasing the survival of these cell lines. The combination inhibited cell colony formation and growth, and augmented induction of apoptosis of resistant cells. This combination enhanced reduction of Mcl-1, a critical mechanism by which osimertinib induces apoptosis in sensitive EGFR mutant NSCLC cells; this effect is likely due to enhanced Mcl-1 degradation since Mcl-1 was degraded much faster in cells exposed to the combination of honokiol and osimertinib than in DMSO-treated control cells. Moreover enforced overexpression of ectopic Mcl-1 abolished the enhanced effect of honokiol and osimertinib combination on induction of apoptosis, supporting a critical role of Mcl-1 reduction in inducing apoptosis of the osimertinib-resistant cells by this combination. Our findings warrant further study of this combination for overcoming osimertinib-resistance in vivo and potentially in the clinic. (This work was supported by the NIH/NCI R01 CA223220 to SYS and Winship lung cancer pilot award to SYS).

#1278

**Structure and activity relationship of disulfiram in inactivating the O** 6 **-methylguanine DNA methyltransferase (MGMT) and potentiation of alkylation DNA damage in brain tumors.**

Viswanath Arutla, Surendra R. Punganuru, Kalkunte S. Srivenugopal. _Texas Tech University Health Sciences Center, Amarillo, TX_.

Disulfiram (DSF), is a well-tolerated, inexpensive, generic drug that has been in use to treat alcoholism since the 1950s. A great volume of evidence has pointed to the strong anticancer activity of this metal-chelating drug against a variety of cancer types. Much of the antitumor effect stems from the affinity of DSF to reactive cysteines present in various regulatory proteins and enzymes, forming conjugates leading to functional inactivation of the targets. Despite its instability and rapid decomposition, copper supplementation has been shown to greatly improve the anticancer efficacy of DSF. Currently, several clinical trials involving DSF and copper gluconate administrations are ongoing, particularly in recurrent glioblastoma. Despite these efforts, the chemistry of DSF interaction with copper and how it leads to cytotoxicity remains unclear in view of the fact that copper-chelated DSF is essentially, an inactivated form of the anti-alcoholic drug. Previously we demonstrated that DSF and the resulting dithiocarbamates conjugate with the active site Cysteine145 of human MGMT in a fashion similar to aldehyde dehydrogenase (ALDH) to inactivate the DNA repair protein and increase the efficacy of alkylating agents (Carcinogenesis 35, 692, 2014). To design more successful therapeutic strategies for DSF, the current study synthesized disulfiram derivatives by keeping the sulfhydryl structure constant but replacing the diethyl groups with different alkyl and cyclic groups. Thus, the ethyl groups were replaced with methyl, n-butyl, cyclohexyl, pyrrolidine, piperidine or benzyl groups to engineer six different DSF analogs. The chemical structures were verified by NMR and mass spectrometry. We investigated the anti-cancer effects and mechanism of DSF analogs in brain cancer cell lines. Compounds with either methyl or pyrrolidine substitutions are highly potent among the DSF analogs specifically in exerting cytotoxicity against the SF188, GBM10, UW228, DAOY, U87MG, and T98G brain tumor cells. The compounds were minimally toxic to normal cells (Astrocytes) when compared to DSF. The DSF analogs also inhibited the MGMT's DNA repair activity with the pyrrolidine substituted analog being the most potent. Through inhibition of MGMT, the analogs also potentiated the cytotoxic activity of temozolomide nearly 10-fold at their sub-toxic concentrations. The compounds also induced high levels of ROS and led to a marked loss of mitochondrial membrane potential and apoptosis. These observations suggest that the growth inhibitory effects of DSF analogs on brain tumor cells involve ROS formation and inhibition of MGMT. Studies to deduce Zn and Cu interactions with the analogs are underway (supported by CPRIT grant 170207 to KSS).

#1279

Calcium-binding properties of tDarpp chemoresistance protein.

Patrycja Magdziarz, Sarwyn Singh, Jamil Momand. _California State University Los Angeles, Los Angeles, CA_.

PPP1R1B (Ch. 17: 39.63-39.64 Mb) produces two proteins (Darpp-32 and t-Darpp) that are frequently overexpressed in bladder, breast, esophageal, gastric, lung and prostate cancer tissue. Overexpression of the shorter protein, t-Darpp, leads to increased AKT phosphorylation and trastuzumab resistance in Her2+ breast cancer through a mechanism that remains unknown. Previously, we showed that the human recombinant protein co-purifies with calcium. To further investigate calcium binding properties, t-Darpp was applied to PVDF membrane and incubated with 45Ca2+. Using this calcium overlay assay we found that binding to Ca2+ was specific as competition with 100x molar excess of Zn2+ or Ni2+ failed prevent calcium binding. To determine if amino acid residue carboxylic acid groups on t-Darpp are critical for calcium binding t-Darpp was pretreated with activating agent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and modifying agent glycine ethyl ester (GEE). GEE modification of protein carboxylic acids was monitored by mass spectrometry. Modified t-Darpp failed to bind to Ca2+, but t-Darpp treated with GEE alone (with no EDC), shows that tDarpp maintains ability to bind Ca2+. Motif analysis shows that the acidic domain of t-Darpp aligns with the sequence of bone sialoprotein II (BSP-II), a known calcium binding protein. In summary, this study indicates that tDarpp is a novel calcium-binding protein.

#1280

Positive PD-L1 expression is associated with unfavorable clinical outcome in EGFR-mutated lung adenocarcinomas treated with EGFR-TKIs.

Seung Hyeun Lee, Myung Jae Park, Boksoon Chang. _Kyung Hee University School of Medicine, Seoul, Republic of Korea_.

Objectives: Clinical implication of programmed death ligand 1 (PD-L1) expression in oncogene-addicted non-small cell lung cancer treated with tyrosine kinase inhibitors (TKIs) is largely unknown. The objective of this study was to determine the frequency of PD-L1 expression and the clinical outcome according to PD-L1 expression in lung adenocarcinomas harboring EGFR mutation and treated with EGFR-TKIs.

Methods: We retrospectively evaluated PD-L1 expression using 22C3 pharmDx assay in lung adenocarcinoma patients with EGFR mutations at two referral hospitals between January 2017 and June 2018. Samples were obtained from surgically resected tumors, small biopsy, or cytologic cell blocks.

Results: Of all 71 patients analyzed, 44 (58.7%) had PD-L1 tumor proportion score (TPS) of < 1%, 23 (30.7%) had PD-L1 TPS of 1%-49%, and 8 (10.7%) had PD-L1 TPS of ≥ 50%. Of the 37 patients treated with first-line EGFR-TKIs, PD-L1 TPS ≥ 1% was associated with a significantly decreased response rate, compared with PD-L1 TPS < 1% (45.7% vs. 67.3%, p=0.005). Furthermore, PD-L1 TPS ≥ 1% was associated with a significantly shorter median progression free survival, compared with PD-L1 TPS < 1% (9.3 months vs. 14.2 months, p = 0.024). Multivariate analysis showed that PD-L1 TPS ≥1% is independently associated with shorter PFS (hazard ratio 1.32, p=0.012).

Conclusion: PD-L1 was positive in approximately 40% of patients with EGFR-mutated lung adenocarcinoma. Postive PD-L1 expression may be associated with unfavorable clinical outcome to EGFR-TKIs among those patients group.

#1281

T790M gatekeeper mutation emerges via de novo at the early stages of erlotinib treament in PC9 non-small cell lung cancer cells.

Sujin Kim, Angela KJ Park, Jeonghee Cho. _Dankook University, Cheonan, Republic of Korea_.

The emergence of the T790M gatekeeper mutation in the Epidermal Growth Factor Receptor (EGFR) gene is an important mechanism that can lead to the acquired resistance to EGFR-targeted tyrosine kinase inhibitors used in a subset of non-small cell lung cancer (NSCLC) patients harboring activating mutations. Here we investigated the paths leading to the acquisition of the T790M mutation by establishing an erlotinib resistant PC9 cell model harboring ectopically introduced EGFR cDNA. We detect the emergence of T790M mutation within the EGFR cDNA in a subset of erlotinib resistant PC9 cell models through Sanger sequencing and droplet digital PCR-based methods, confirming that T790M mutation can emerge via de novo events following treatment with erlotinib. In addition, we show that the de novo T790M bearing erlotinib resistant PC9 cells are sensitive to the 3rd generation EGFR-targeted drug, WZ4002. Furthermore, GFP-based competition cell proliferation assays reveal that PC9 cells ectopically expressing EGFR mutant become more dominantly resistant to erlotinib than parental PC9 cells by acquiring T790M mutation. Taken together, we believe that our findings expand upon the previous notion of evolutionary paths of T790M development, providing an important clue to designing a therapeutic strategy to overcome drug resistance.

#1282

Understanding the kinomic contributions to tyrosine kinase inhibitor resistance in triple negative breast cancer.

Cory Lefebvre, David Litchfield, Alison Allan. _Western University, London, Ontario, Canada_.

There is an increasing need to develop targeted therapies for triple negative breast cancer (TNBC), as conventional chemotherapy, anti-hormone or anti-HER2 therapies are ineffective at combatting systemic disease. TNBC tumors often have increased expression of receptor tyrosine kinases such as EGFR (epidermal growth factor receptor) and the hepatocyte growth factor (HGF) receptor, c-Met; presenting as potential targets for treatment. However, targeted anti-Met and anti-EGFR therapies have faced mixed results in clinical trials due to acquired resistance. We hypothesize that dynamic changes in the kinome contribute to the acquired resistance of TNBC tumors to c-Met and EGFR inhibitors. Using MDA-MB-468 and MDA-MB-231 TNBC cell lines, we investigated the effects of the c-Met inhibitors cabozantinib and tivantinib and the EGFR inhibitor erlotinib on cell proliferation and activation of target receptors and downstream signalling pathways. We observed that cabozantinib and erlotinib significantly inhibited the proliferation (p≤0.05) of one and/or both TNBC cell lines in a cytostatic fashion. Similarly, tivantinib demonstrates growth inhibitory effects but in a mostly cytotoxic manner; consistent with recent reports of its mechanism in inhibiting microtubule polymerization. Exposure to erlotinib (24 hrs) resulted in decreased expression of phosphorylated and total EGFR in both TNBC cell lines and a decrease in activation (phosphorylated:total expression) of ERK1/2 in MDA-MB-468 cells (p≤0.05). Tivantinib and cabozantinib did not appear to have any effects on activation of c-Met or ERK1/2 in MBA-MB-231 or MDA-MB-468 cells following 24-hour exposure. Ongoing studies are aimed at implementing a novel kinomics strategy using SILAC and multiplexed inhibitor beads for kinase enrichment to investigate the dynamic changes in the kinome of susceptible and resistant cell lines in response to erlotinib and cabozantinib. With better understanding of the underlying mechanisms, we can potentially discover targets that could attenuate induced resistance to tyrosine kinase inhibitors.

#1283

Targeting c-MYC and MAPK pathway to overcome pancreatic cancer drug resistance.

Erika Maria Parasido,1 George S. Avetian,1 Jonathan Brody,2 Jordan Winter,2 Eric Londin,2 Michael Pishvaian,1 Eric Glasgow,1 Stephen Byers,1 Goutham Narla,3 Christopher Albanese1. 1 _Georgetown University, Washington, DC;_ 2 _Thomas Jefferson University, Philadelphia, PA;_ 3 _University of Michigan, Ann Arbor, MI_.

Background: Acquired resistance to systemic chemotherapy is the main complication in pancreatic ductal adenocarcinoma (PDAC) treatment. Although there are studies focused on gemcitabine resistance mechanisms, our understanding of the mechanisms of nab-paclitaxel (n-PTX) treatment failure remains extremely limited. To enhance the use of properly powered patient-derived platforms, we adopted the conditionally reprogrammed (CR) cell culture technique in order to develop both parental and nab-PTX-resistant cells. The CR approach allowed us to identify the critical role of c-MYC and ERK in the PDAC drug response. Small molecule activators of PP2A (SMAPS) have showed activity in inhibiting lung KRAS-mutant tumor growth. We used SMAPS as new therapeutic agents in PDAC, for its ability to alter c-MYC activity through PP2A dysregulation and enhance PDAC sensitivity to n-PTX.

Methods: Long-term cultures of PDAC CRs were established from treatment-naive PDAC patients' biopsies, and used to generate drug-resistant cells. Zebrafish and mouse model were used to test the cells' ability to form tumors and to verify the drug resistance in vivo. Molecular analyses were used to characterize the drug-resistant cells and to identify key pathways involved in the drug resistance evolution. Genomic and chemical alterations of the key proteins were used to confirm the involvement in the drug resistance mechanism. We regulated the expression of c-MYC and ERK using SMAPS as a new targeting agent and trametenib to verify the direct correlation between c-MYC and ERK and the drug resistance mechanism.

Results: Using the credentialed KRAS-mutant CR cultures, we generated n-PTX-resistant cell lines. The parental and nab-PTX resistant cells were subjected to subcutaneous injections in nude mice, and formed tumors in 2-3 weeks. Histological evaluation showed that the CRs self-assembled into ductal structures, surrounded by a desmoplastic stromal microenvironment that faithfully recapitulates human PDAC. Resistant profiles were verified both in mouse and Zebrafish model. RNA microarrays identified a sustained induction of a pro-inflammatory pathway leading to c-MYC overexpression. c-MYC silencing and overexpression confirmed the role of c-MYCin the evolution of nab-PTX resistance. Treatment of the resistant CRs with either trametenib or with SMAPS resulted in enhanced sensitivity to nab-PTX. We furtherverified that the enhanced sensitivity was commensurate with a reduction in p-Erk and c-Myc.

Conclusion: The CR methodology addresses the need for a reliable method for generating primary cell lines on a single patient basis. The ability to rapidly model in vitro, and verify in vivo, that the overexpression of c-MYC contributes to the development of n-PTX resistance is a significant advancement in the field. Our data showed that SMAPs or trametanib overcome a significant component of the n-PTX resistance providing new hope for refractory PDAC.

#1284

**Resistance to BRAF and MEK inhibitors in BRAF** V600E **mutant melanoma.**

Hima Patel, Rosalin Mishra, Garrett Joan. _University of Cincinnati, Cincinnati, OH_.

Melanoma five year survival rates are only 15% for patients with advanced disease. About 50% of melanomas contain the BRAFT1799A transversion which encodes for the constitutively active BRAFV600E oncoprotein. This opened up avenues for development of BRAF inhibitors which resulted in a response rate of about 50% and median progression-free survival of about 5 months. Co-targeting the downstream MEK protein with a MEK inhibitor along with BRAF inhibitor resulted in response rates of about 70% and are now approved as standard of care for patients harboring the BRAF-mutant melanoma. In spite of these successes, their long-term efficacy has been limited due to acquired resistance to these inhibitors. In order to study mechanisms underlying resistance, we generated dabrafenib (BRAF inhibitor) and trametinib (MEK inhibitor) resistant WM115 (WM115 TDR) and WM983 (WM983 TDR) cell lines by adding increasing concentration of the drugs to WM115 and WM983 cells with BRAFV600E mutation (parental cells). We observed that the resistant cells undergo a change in their morphology and continue to remain viable over increasing doses of dabrafenib and trametinib as compared to parental cells demonstrated by MTT cell proliferation assay and Matrigel 3D assay. TDR cells demonstrated maintenance of p-ERK levels at 4 and 24 hours which under the same conditions p-ERK was inhibited in the parental cells. Current studies underway include RNA sequencing in order to decipher differentially expressed genes in the parental and TDR which could mediate resistance to BRAF and MEK inhibitors. Additionally we will examine phosphorylated receptor tyorsine kinases (RTKs) using an array to identify divergences among activation of RTKs in parental versus TDR cells. Deciphering causes of resistance to BRAF and MEK inhibitors will help in the development of novel mechanism-based interventions aiding long-term patient prospects in BRAF-mutant melanoma.

#1285

Regulation of docetaxel resistant cancer stem-like cells by RENCA macrobeads.

Prithy Caroline Martis,1 Atira T. Dudley,1 Pradeep R. Dumpala,1 Hunter L. Gazda,1 Melissa A. Bemrose,1 Barry H. Smith,2 Lawrence S. Gazda1. 1 _The Rogosin Institute, Xenia, OH;_ 2 _The Rogosin Institute, New York, NY_.

Despite the initial efficacy of chemotherapy, resistant cells often persist and are thought to contribute to disease relapse. Resistant cells, characterized by stem-like properties, exhibit numerous survival mechanisms, making eradication of these rare cells a significant clinical challenge. We have previously shown that murine renal adenocarcinoma (RENCA) cells encapsulated in agarose macrobeads (MB) form tumor colonies over several months that secrete diffusible factors, which regulate their own growth as well as the growth of freely growing tumor cells outside the macrobead. Young RENCA MBs produce factors necessary for growth and differentiation while mature macrobeads secrete tumor inhibitory proteins. In the current study, we evaluated the effect of young and mature RENCA MBs on the in vitro regulation of docetaxel (DTX) resistant cell populations and in vivo on tumor burden in BALB/cJ mice induced with DTX resistant cells.

RENCA MBs were exposed to DTX (5 μg/mL) and maintained in culture for 6 wks to establish resistant cells, followed by culture alone (control) or co-cultured with young (3 wks) or mature (>18 wks) normal RENCA MBs. Incidence and time of macrobead tumor recurrence were evaluated. Also, six weeks following DTX treatment, 10 resistant cells recovered from RENCA MBs were embedded in a fibrin clot and placed under the kidney capsule along with abdominal implantation of young or mature macrobeads. Animals that did not receive macrobeads served as the control.

In vitro, 21.5% of macrobeads treated with DTX developed recurrent tumor colonies by 24 weeks (control group) with first evidence at 12 weeks. Following co-culture with young RENCA MBs, 86.8% of macrobeads showed evidence of recurrence with earliest indication at 4 weeks. Culture with mature RENCA MBs significantly reduced the incidence of macrobead tumor recurrence to 1.1% with first evidence noted at 22 weeks.

Consistent with in vitro data, 4 of 10 mice in the control group developed palpable tumors 27-29 days following tumor induction while 8 of 10 mice showed evidence of tumors with young macrobead implantation. Remarkably, to date (36 days), none of the mice implanted with mature RENCA MBs developed tumors. Preliminary data suggests that mean tumor volumes (cm3) were smaller (2.6 ± 0.8) in the control group vs. the young macrobead implanted group (6.3 ± 2.3). Furthermore, metastases were localized to the abdomen in the control group (1 of 4 mice) while they were diffuse and prevalent in the abdominal cavity, liver, diaphragm and lungs following implantation of young macrobeads (4 of 4 mice).

These data support the notion that RENCA macrobeads function as a biological system that could be utilized as a novel tool to eradicate or maintain dormancy of resistant cancer cell populations. Phase 2 clinical trials of RENCA macrobeads for the treatment of resistant colorectal cancer have been completed (Trial Nos. NCT01053013 and NCT02046174) with a Phase 3 trial planned.

### Novel Targets and Pathways

#1286

ONC201 inhibits RET and IGFBP2 signaling through ATF4 mediated- Integrated stress response in medullary thyroid cancer.

Rozita Bagheri-Yarmand,1 Ling Li,1 Rohinton Tarapore,2 Joshua E. Allen,3 Steven I. Sherman,1 Robert F. Gagel1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Oncoceutic Inc, Hummelson, PA;_ 3 _Oncoceutic Inc, PA_.

Medullary thyroid cancer (MTC) is an aggressive tumor with frequent lymph node and distant metastasis at diagnosis. Activating mutations of the RET receptor tyrosine kinase occur frequently and prevent apoptosis through inhibition of ATF4, a key transcriptional regulator of endoplasmic reticulum stress. Prior studies have demonstrated shRNA depletion of ATF4 increases RET protein levels and cause resistance to tyrosine kinase inhibitor-mediated cell death. Conversely, forced expression of ATF4 in MTC cells promotes RET ubiquitination and degradation. Here, we report the anti-cancer efficacy of ONC201 in MTC models. ONC201 is a selective DRD2 antagonist that is in clinical trials for a range of advanced cancers. Downstream signaling studies have found that ONC201 induces ATF expression and activates the integrated stress response (ISR) in a variety of cancers. ONC201 decreased cell survival of MTC cell lines (TT and MZCRC1) harboring activating RET mutations and increased the percentage of sub-G1 DNA content. Moreover, ONC201 decreased, in a dose-dependent manner, the abundance of RET protein and induced the mRNA and protein levels of ATF4 and its target genes. In our attempt to uncover RET targets, we performed reverse phase protein array analysis using TT- shRNA-RET knockdown cells. Among the top 5 proteins regulated by RET in this screen, we found insulin-like growth factor-binding protein IGFBP2, to be downregulated in shRNA-RET cells. ELISA based cytokine array of MTC-conditioned media showed high levels of secreted IGFBP2. IGFBP2 have been reported to be an oncogene in many human cancers and we show that elevated IGFBP2 protein levels are associated with poor prognosis of primary MTC. The treatment of MTC cells with ONC201 or overexpression of ATF4 inhibited the secretion of IGFBP2 into the media and decreased IGFBP2 protein levels, suggesting that ONC201 targets IGF signaling through activation of ISR. To investigate the efficacy of ONC201 in vivo, established MTC xenografts (TT or MZCRC1) were treated with a weekly dose of 120 mg/kg orally for 8 weeks and tumor burden was assessed every week. We observed that ONC201 decreased tumor growth of TT and MZCRC1 xenografts by 80-90%. There was no evidence of toxicity, as indicated by weight loss in mice. Immunohistochemical and western blot analysis of tumors at the termination of the study demonstrated decreased IGFBP2 protein levels in mice treated with ONC201. These studies identify IGFBP2 as a novel target of the RET proto-oncogene and demonstrate that ONC201, through its effect on ATF4, reverses RET-mediated signaling. These results support the further investigation of ONC201 as a therapeutic agent for the treatment of MTC alone or in combination with tyrosine kinase inhibitors.

#1287

Targeting pleckstrin-2 for the JAK2-STAT and PI3K-Akt pathways in cancer therapy.

Xu Han,1 Yang Mei,1 Gary E. Schiltz,2 Rama K. Mishra,2 Atul D. Jain,2 Peng Ji1. 1 _Northwestern University, Chicago, IL;_ 2 _Northwestern University, Evanston, IL_.

Pleckstrin-2 (Plek2) is a widely expressed PH domain containing protein that binds to phosphoinositide with unclear functions. Our published reports reveal that Plek2 is overexpressed in JAK2 V617F mutation positive myeloproliferative neoplasms (MPNs). We identified that Plek2 is a downstream effector of the JAK2-STAT5 pathway. Furthermore, through a mouse genetic approach, we discovered that knockout of Plek2 significantly ameliorated the MPN phenotypes and reverted lethality and thrombosis in JAK2 V617F knockin mice. These studies demonstrate that Plek2 is critical for the pathogenesis of MPNs with the activated JAK2-STAT5 pathway, and form a strong foundation for the development of Plek2 inhibitors for the treatment of MPNs. Importantly, our published study shows that Plek2 knockout mice do not develop anemia or cytopenia, indicating Plek2's oncogenic potential is only in the disease background, which makes Plek2 inhibitors less likely to cause severe side effects compared to JAK inhibitor ruxolitinib.

Based on these studies, we used an in silico approach to screen for putative Plek2 binding small molecules and identified hit compounds that bind to the DEP domain of Plek2. Further medicinal chemistry studies identified lead compound NUP-17d that inhibited proliferation of the hyperproliferative hematopoietic cells with potency comparable to ruxolitinib. In addition, NUP-17d also blocked proliferation of several Plek2-overexpressing solid tumor cell lines. Our biochemical assays showed that NUP-17d inhibited Akt phosphorylation. Further mechanistic studies revealed that Plek2 functions as a central hub to mediate the JAK2-STAT and PI3K-Akt pathways to promote tumor cell proliferation. Specifically, Plek2 binds to and recruits PI3K-produced PtdIns(3,4)P2 and PI3K effector proteins including Akt, PDK1, PDK2, and mTOR, which forms a complex and significantly enhances the PI3K signaling. In addition to the biochemical assays, we also demonstrated Plek2's function through a mouse genetic approach using a Pten hematopoietic specific knockout mouse model. Knockout of Plek2 significantly reverted the myeloproliferative phenotype in these mice and markedly extended their survival. Therefore, NUP-17d blocks tumor cell proliferation through the disruption of the Plek2 complex and inhibition of the PI3K-Akt pathway. Indeed, treatment of an erythropoietin-induced myeloproliferative mouse model with NUP-17d also demonstrated its potent inhibitory effect in myeloproliferation in vivo.

These study establishes Plek2 as a oncoprotein mediating JAK2-STAT and PI3K-Akt signaling pathways. In addition to hematologic malignancies, Plek2 is also found to be highly upregulated with an associated worse prognosis in many solid tumors (oncomine, kmplot). Therefore, our Plek2 inhibitors will have a broad impact in cancer therapy, especially cancers with upregulated JAK2-STAT or PI3K-Akt pathway.

#1288

Blocking tumor-associated immune suppression with BAY-218, a novel, selective aryl hydrocarbon receptor (AhR) inhibitor.

Ilona Gutcher,1 Christina Kober,2 Lars Roese,1 Julian Roewe,3 Norbert Schmees,1 Florian Prinz,1 Matyas Gorjanacz,1 Ulrike Roehn,1 Benjamin Bader,1 Horst Irlbacher,1 Detlef Stoeckigt,1 Rafael Carretero,2 Katharina Sahm,3 Iris Oezen,3 Hilmar Weinmann,1 Ingo V. Hartung,1 Bertolt Kreft,1 Michael Platten3. 1 _Bayer AG, Pharmaceuticals Division, Berlin, Germany;_ 2 _DKFZ-Bayer Joint Immunotherapy Laboratory, Heidelberg, Germany;_ 3 _German Cancer Research Center (DKFZ), Heidelberg, Germany_.

Tumor cells co-opt multiple pathways in order to evade attack by infiltrating immune cells. One such mechanism is the upregulation of indole-2,3-dioxygenase (IDO1) and/or tryptophan-2,3-dioxygenase (TDO2), both of which are first-step, rate-limiting enzymes degrading tryptophan to the immunosuppressive metabolites kynurenine (KYN) and kynurenic acid (KA). KYN and KA bind and activate the aryl hydrocarbon receptor (AhR), which is expressed in many cell types and is well known for its immunosuppressive effects. Targeting of the AhR with an inhibitor may therefore provide a novel immunotherapeutic approach for enhancing anti-tumoral immune responses and treating cancer. Here we describe the identification and functional immune characterization of BAY-218, a novel, selective and potent AhR small molecule inhibitor. Mechanistically, BAY-218 inhibited AhR nuclear translocation, dioxin response element (DRE)-luciferase reporter expression and AhR-regulated target gene expression induced by both exogenous and endogenous AhR ligands. In vitro, BAY-218 rescued TNFα production from KA-suppressed LPS-treated primary human monocytes. Furthermore, BAY-218 enhanced T cell cytokine production in a human mixed lymphocyte reaction (MLR) and a mouse antigen-specific bone-marrow-derived dendritic cell (BMDC)-OT-I T cell co-culture. In the MLR, BAY-218 increased anti-PD1 antibody-mediated IL-2 and IFNγ secretion, while an IDO inhibitor did not, indicating that BAY-218 is able to block AhR activation mediated by ligands outside of the IDO-KYN pathway. In vivo, BAY-218 enhanced anti-tumoral immune responses and reduced tumor growth in the syngeneic mouse tumor models CT26 and B16-OVA. FACS analysis of leukocytes infiltrating B16-OVA tumors demonstrated that administration of BAY-218 increased the frequency of tumor-infiltrating CD8+ T cells and NK cells while decreasing GR1+ myeloid cells and CD206+ M2 macrophages. Furthermore, BAY-218 enhanced therapeutic efficacy of an anti-PD-L1 antibody in the CT26 model. In summary, AhR inhibition with BAY-218 stimulates pro-inflammatory monocyte and T cell responses in vitro and drives anti-tumor immune responses, resulting in decreased tumor growth, in vivo. Thus, inhibiting AhR represents a novel immunotherapeutic approach for blocking AhR-mediated tumor-associated immunosuppression.

#1289

AICAR inhibits protein kinase D1 activity leading to epigenetic downregulation of immediate early genes via the NF-kB pathway in acute lymphoblastic leukemia.

Anna Shvab, Guangyan Sun, Bin Li, Felipe Beckedorff, Guy J. Leclerc, Ramin Shiekhattar, Julio C. Barredo. _University of Miami Miller School of Medicine, Miami, FL_.

Acute lymphoblastic leukemia (ALL) is the leading cause of cancer related death in children, and cure rates for relapsed/refractory ALL remain dismal, highlighting the need for novel targeted therapies. We previously uncovered that ALL are vulnerable to metabolic/energy stress and ER-stress via AMP-activated protein kinase (AMPK) activation. In order to identify genome wide metabolic stress and AMPK transcriptionally regulated genes, we used RNA-Seq and compared mRNA profiles in ALL cells treated with the adenosine analog AICAR, an activator of AMPK. RNA-Seq data indicated that high dose AICAR (15 mM/45 min) induced a robust downregulation of a cluster of genes known as the immediate early genes (IEGs), which are critical for cell survival, proliferation and adaptation. AICAR-induced downregulation on IEG expression was dose- and time-dependent, and observed in other cell types (HEK293T, Hela, MEF), indicating this mechanism is conserved in mammalian cells. Using MEF AMPKα2 and AMPKα1/α2 knockout cell lines, we found that these alterations were AMPK-independent. Characterization of AICAR's mechanism of action identified the protein kinase D1 (PKD1) as responsible for these effects. PKD1 is a Ser/Thr protein kinase involved in many cellular processes important for cancer development and progression, including proliferation, survival, apoptosis, invasion, cell adhesion and angiogenesis. We uncovered that high dose AICAR significantly inhibited PKD1 activation (Ser-910) at the plasma membrane which prevented its nuclear translocation. When PKD1 activity was pharmacologically inhibited by CRT0066101 or downregulated by shRNA, we observed similar IEGs' downregulation in ALL cells. Conversely, the effect of AICAR on IEGs' expression was abrogated by PMA, a direct activator of PKD1. In addition, when PKD1 was overexpressed in HEK293T cells, AICAR-induced IEG's downregulation was partially restored. Using a kinase assay, we found that AICAR, but not ZMP, directly inhibited PKD1 kinase activity. Further, we determined that AICAR suppressed phosphorylation and nuclear export of PKD1-targeted histone deacetylases HDAC4/5, which led to decreased histone H3 acetylation at the IEGs' promoter region. Finally, ChIP-qPCR indicated that AICAR-induced PKD1 inhibition prevented NF-κB recruitment to IEGs' promoters. Inhibition of PKD1 activity led to decreased cell proliferation and promoted apoptosis in ALL cells. To confirm the in vivo relevance of our data, single agent and combination experiments using our NSG ALL mouse model are underway. Taken together, we have identified a novel AMPK-independent mechanism leading to AICAR's inhibition of PKD1-mediated ALL survival. Consequently, co-targeting PDK1 and other pro-survival stress response pathways in ALL cells offers novel strategies to overcome therapeutic resistance.

#1290

Development of anti inflammatory agents targeting TonEBP for treatment of chronic inflammatory diseases.

Byeongjin YE, Hyug Moo Kwon, Soo Youn Choi, Jun Ho Lee, Hyun Je Kang, Cheol-Min Park. _Ulsan National Institute of Science and Technology (UNIST), Ulsan, Republic of Korea_.

Tonicity-responsive enhancer binding protein (TonEBP), also known as nuclear factor of activated T cells 5 (NFAT5), belongs to the Rel family of transcriptional factors which include NF-κB and NFAT. TonEBP is a key regulator in chronic inflammatory diseases such as rheumatoid arthritis, atherosclerosis, diabetic nephropathy and hepatocellular carcinoma. TonEBP is a rate-limiting component of "pro-inflammatory enhanceosome" in which TonEBP provides a physical tether between activated transcription factors NF-κB and AP-1 to p300 and RNA polymerase II. Assembled in response to inflammatory signals, the pro-inflammatory enhanceosome drives the transcription of TNF-α, IL-1β, IL-6, MCP-1, iNOS, and COX-2. We discovered a class of compounds (CXs) targeting TonEBP. CXs blocked the assembly of the pro-inflammatory enhancesome and potently suppressed the inflammation-induced expression of TNF-α, IL-1β, IL-6, MCP-1, iNOS, and COX-2. Orally administered CXs exhibited remarkable therapeutic activities in mouse models of inflammatory arthritis. Using an alkynyl derivative of a CX and click reaction, we found that CX was covalently attached to TonEBP by alkylation but not to other components of the enhanceosome. Cells harboring a site-directed mutant TonEBP molecule whose amino acid residues for the alkylation were removed exhibited a markedly reduced sensitivity to CXs. These data demonstrate that CXs are a new class of anti-inflammatory agents with a novel mode of action.

#1291

Notch2 inhibition as a therapeutic intervention in osteosarcoma.

Sankaranarayanan Kannan, John A. Livingston, Michael Roth, Jonathan Benjamin, Yifei Wang, Zhongting Zhang, Wendong Zhang, Chia-Chin Wu, Hannah Beird, Andrew Futreal, Richard Gorlick. _The Univeristy of Texas M.D.Anderson Cancer Center, Houston, TX_.

Notch signaling pathway is a mediator of cell differentiation and is critical for normal bone development. Four functional Notch ligands DLL1 and DLL4, and JAG1 and JAG2 showed various levels of affinity for Notch1-4 receptors. Dose dependent Notch signaling activation or blocking is attributed to anchored or soluble form of Notch ligands binding. Interestingly, Notch pathway is shown to play a dual role, either oncogenic or tumor suppressive, depending on signal dose and tissue-context. To understand Notch signaling activities in osteosarcoma (OS), we first studied the cell surface levels of Notch receptor expression in OS cell lines and patient derived xenograft (pdx) models. Cytometry based screening of NOTCH1-4 receptors showed NOTCH2 as a predominant surface expressed protein in most of the samples. Interestingly, the canonical Notch target genes HES1 and DTX1 were higher (~5-fold) in OS cells compared to mesenchymal cells (MSC). Subsequently, we evaluated selected Notch pathway gene expression in 48 patients with recurrent/metastatic OS by RNAseq analysis. High expression of Notch pathway-related genes was seen in a subset of patients. NOTCH2 expression was comparable between primary and metastatic OS specimens, highest as compared to other Notch receptors and was significantly higher than normal tissue. Thus we aimed to elucidate soluble Notch ligands mediated blocking of Notch signaling using immunoglobulin-G bound DLL-1, -3, and -4 and JAG-1, and -2 in in vitro studies using OS cells. Soluble DLL1-Fc treatment increased OS cell death as compared to the other Notch ligands as well as control-Fc cells. Addition of soluble DLL1 resulted in the decreased expression of Notch downstream genes such as HES1, DTX1 and HEY1. Taken together, these findings identify soluble DLL1 as a potent Notch pathway inhibitor in OS in vitro and support further exploration of NOTCH2 as a potential therapeutic target in OS.

#1292

A new generation of N-terminal domain androgen receptor inhibitors, with improved pharmaceutical properties, in castration-resistant prostate cancer models.

Ronan Le Moigne,1 Nasrin R. Mawji,2 C. Adriana Banuelos,2 Jun Wang,2 Kunzhong Jian,3 Raymond J. Andersen,3 Marianne D. Sadar,2 Han-Jie Zhou,1 Peter Virsik1. 1 _ESSA Pharmaceuticals Inc, Houston, TX;_ 2 _BC Cancer Agency, Vancouver, British Columbia, Canada;_ 3 _University of British Columbia, Vancouver, British Columbia, Canada_.

Introduction: The androgen receptor (AR) pathway continues to drive castration-resistant prostate cancer (CRPC) even in late stages of the disease, and ligand binding domain (LBD)-linked resistance inevitably emerges. Selective inhibition of the N-terminal domain (NTD) of the AR can inhibit its' transcription even in the presence of anti-androgen resistance. A Phase I clinical trial of the first-generation AR NTD inhibitor, EPI-506 (EPI-002 pro-drug), demonstrated PSA declines in anti-androgen resistant metastatic CRPC patients. However, these declines were minor and of short duration, revealing the need for more potent and metabolically stable NTD inhibitors. A new generation of NTD transcriptional inhibitors (Anitens) has been generated. Examples of this new class, EPI-7170 and EPI-7245, demonstrate improved potency, metabolic stability and pharmaceutical properties, and are potent against anti-androgen resistant prostate cancers in in vitro and in preclinical models.

Methods: Chemical structure activity relationships were developed to identify more potent molecules as measured by both cellular and in vivo assays, while metabolic stability improvements were assessed in in vitro ADME assays and in animal pharmacokinetic studies. In addition, the on-target activity and selectivity were also optimized using a variety of cellular experiments.

Results: These next generation Anitens demonstrated a 10-20-fold improvement on AR-driven cellular potency, with IC50's of 0.5-1 uM compared to 10 uM for EPI-002. Aniten AR inhibition was specific to the NTD, as demonstrated by the absence of LBD binding and the inhibition of the AR-V7-driven transcription inLNCaP95 model. In vitro proliferation assays demonstrated AR-dependent activity, with an IC50 ~ 1 uM in LNCaP and >10 uM in the AR-independent cell model PC-3. The antiproliferative effect aligned with the inhibitory effect on a subset of AR-driven genes. In vivo, next generation Anitens demonstrated PSA serum decreases along with significant tumor growth inhibition in mice bearing LNCaP tumors. While EPI-7170 represents a major advance, subsequent chemistry efforts led to the generation of EPI-7245 and other next generation Anitens which exhibit IC50's <500 nM. These new compounds exhibited favorable ADME and PK profiles, with half-life ~ 8 hrs in mice, and predicted low human clearance.

Conclusions: Promising next-generation Aniten compounds have been identified which retain the NTD specificity of first-generation agents. These new molecules demonstrate major improvements in potency and metabolic stability in comparison to the first-generation clinical compound EPI-002. These next generation Anitens, by selectively inhibiting AR transcription in both hormone sensitive and castrated resistant prostate cancer, represent an important next step in AR targeted therapeutics.

#1293

HM43239, a novel FLT3 inhibitor in overcoming resistance for acute myeloid leukemia.

JiSook Kim, InHwan Bae, JaeYul Choi, MinJeong Kim, JooYun Byun, MiJin Moon, EunYoung Lee, Yu-Yon Kim, Hyun Jeong Kang, Eunyoung Kim, SunYoung Jung, YoungGil Ahn, YoungHoon Kim, Kwee Hyun Suh. _Hanmi Research Center, Hwaseong, Republic of Korea_.

Introduction: Acute myeloid leukemia (AML) with FMS-like tyrosine kinase 3 (FLT3) mutation is associated with poor prognosis with a high risk of relapse after therapy and reduced overall survival. Currently, FLT3 inhibitors have shown clinical benefits in the corresponding AML patients. Activating mutations within internal tandem duplication (ITD) and tyrosine kinase domain (TKD) point mutations of FLT3 have been reported as oncogenic driver mutations in about 30% of AML. The acquired D835Y and F691L point mutations of FLT3-TKD are associated with resistance to FLT3-targeted AML therapy. In this study, we have characterized HM43239, a novel FLT3 inhibitor, and assessed its potential as a novel therapy in overcoming resistance for AML patients.

Materials and Methods: In vitro site-directed competition binding assay was performed to measure interactions between HM43239 and FLT3 mutations. Standard proliferation assay, immunoblotting, and apoptosis analysis were carried out to validate the potency of HM43239 in AML resistance cell lines. In vivo study, HM43239 was evaluated in Ba/F3 cells expressing FLT3 ITD/F691L or FLT3 ITD/D835Y xenograft mice models. Combination studies were evaluated in Acute Myeloid Leukemia xenograft mice models.

Results: HM43239 potently inhibited both FLT3 ITD/D835Y and FLT3 ITD/F691L mutations in preclinical evaluation. It showed high in vitro binding affinity to both mutations, and exhibited potent inhibitory activity in in vitro and in vivo models using Ba/F3 cells expressing FLT3 ITD/D835Y or FLT3 ITD/F691L. Moreover, HM43239 could overcome the FL-induced drug resistance with a higher cytotoxic potency in MOLM-14 cells harboring FLT3 ITD. In KG-1a cells, HM43239 potently inhibited phosphorylation of SYK, STAT3 and STAT5. In addition, it inhibited the proliferation and induced the apoptosis of leukemic stem cell (LSC) marker-expressing KG1a cells (CD34+/CD38- cells), suggesting the possibility of targeting LSC. Also, HM43239 significantly inhibited p-FLT3 and p-STAT5 under normal human plasma milieu in a dose-dependent manner in Ba/F3 and MOLM-14 cell line harboring FLT3 ITD. Furthermore, the combination treatment of HM43239 with various reagents (e.g., IAP inhibitor, chemotherapy, etc) demonstrated synergistic efficacy in mouse models, xenografted with both MV-4-11 and MOLM-13 cell lines without any significant toxicity.

Conclusion: Taken together, HM43239 demonstrated the potential therapeutic efficacy for the treatment of AML patients, and implicated the mechanism of overcoming resistance and preventing relapse.

#1294

Differential sigma-2 receptor-mediated metabolic stimulative and apoptotic effects of monovalent CM571 and its homo-bivalent counterpart, MAM03055A.

Cheri Z. Liu,1 Bridget M. McVeigh,1 Marco Mottinelli,2 Hilary E. Nicholson,1 Christopher R. McCurdy,2 Wayne D. Bowen1. 1 _Brown University, Providence, RI;_ 2 _University of Florida, Gainesville, FL_.

Sigma-2 receptors, recently identified as TMEM97, are highly expressed in various types of cancer cells. Sigma-2 receptor agonists have traditionally been characterized as ligands that induce programmed cell death in various cancer cell types. Sigma-2 receptor-mediated cell death involves a number of mechanisms including caspase activation, mitochondrial depolarization, and autophagy. Recently, we reported a novel metabolically stimulative function of the sigma-2 receptor with stimulation of glycolytic hallmarks, including increase in cellular ATP level, reduced ROS, and stabilization of HIF-1α. We have investigated a family of 6-substituted analogs of the canonical sigma-2 receptor antagonist, SN79, members of which can produce both the metabolic stimulative and the cytotoxic effects. Here we compare the activities of two closely related compounds. CM571, the 6-amino derivative, bound with high affinity to both sigma-1 and sigma-2 receptors (sigma-1 Ki = 15.5 nM; sigma-2 Ki = 21.7 nM). MAM03055A (MAM) is essentially a homo-bivalent dimer of CM571, linked by a thiourea moiety. MAM exhibited high affinity and selectivity for sigma-2 receptors (sigma-1 Ki = 3,371 nM; sigma-2 Ki = 55.9 nM). The functional effects of the two compounds were compared in human SK-N-SH neuroblastoma cells. CM571 caused a dose-dependent stimulation of MTT reduction, indicating the metabolic stimulative-effect, producing 40% stimulation at 30 µM. By contrast, MAM induced dose-dependent cell death with an EC50 = 4.24 µM. MAM induced time-dependent cleavage of proapoptotic Bid, a key component of the programmed cell death mechanism of sigma-2 ligands in these cells. Previous studies had shown that only irreversibly binding, isothiocyanate analogs of SN79 induced programmed cell death, while other analogs produced metabolic stimulation. Rat liver membranes were treated with compound, washed to remove unbound ligand, and then subjected to radioligand binding to determine recovery of sigma-1 receptors (3H-pentazocine) and sigma-2 receptors ([3H]DTG + (+)-pentazocine). Treatment with CM571 resulted in full recovery of both sigma-1 and sigma-2 binding. However, MAM treatment resulted in loss of sigma-2 binding and full recovery of sigma-1 activity, indicating irreversible binding to the sigma-2 receptor. Since there is no chemically reactive moiety in MAM to react covalently, MAM binds "pseudo-irreversibly". Treatment of cells with MAM for 60 min, followed by washout produced nearly comparable levels of cell death 24 h later as continuous exposure of cells for 24 hours, consistent with pseudo-irreversible action at the sigma-2 receptor. The results show that mono-valent and bivalent sigma-2 ligands in this series show different modes of interaction with the receptor, resulting in divergent effects on cell viability.

#1295

First-in-field small molecule inhibitors targeting BRN2 as a therapeutic strategy for small cell prostate cancer.

Daksh Thaper,1 Ravi Munuganti,1 Shaghayegh Nouruzi,1 Sahil Kumar,1 Soojin Kim,1 Olena Sivak,1 Adeleke Aguda,1 Dwaipayan Ganguli,1 Sepideh Vahid,1 Loredana Puca,2 Himisha Beltran,3 Amina Zoubeidi1. 1 _Vancouver Prostate Ctr., Vancouver, British Columbia, Canada;_ 2 _Weill Cornell Medical College, New York, NY;_ 3 _Dana Farber Cancer Institute, Boston, MA_.

Introduction: Resistance to newly developed androgen receptor pathway inhibitors (ARPIs), such as Enzalutamide (ENZ), rapidly emerges and patients generally die within two years. In particular, a subset of patients who relapse following ARPI therapy exhibit lineage switching whereby tumours shed their dependence on AR signaling and emerge with neuroendocrine features. These tumours, termed treatment induced neuroendocrine prostate cancer (t-NEPC), carry an extremely poor prognosis and, to date, treatment remains decades old cytotoxic chemotherapy which carries a short-lived response at the cost of significant toxicity. Therefore, targeted therapies for this deadly disease are desperately needed. Thus, the need to develop targeted treatments for this devastating disease is of paramount importance. Recently our group identified the neural transcription factor BRN2 as a major clinically relevant driver of NEPC and aggressive tumor growth, both in vitro and in vivo, suggesting targeting BRN2 is a promising strategy to prevent neuroendocrine differentiation or treat NEPC.

Methods: Study the effects of BRN2 inhibition using siRNA, small molecule inhibitors and CRISPR K/O models.

Results: Inhibition of BRN2 by siRNA and by CRISPR/Cas9 knockout drastically reduced cell proliferation in 42DENZR (NEPC) cell lines. This data was re-capitulated in human NEPC NCI-H660 cells. Loss of BRN2 initiated drastic epigenetic changes in NEPC cell lines as well as in G1 arrest through up-regulation of CDKN1A/1B. This was confirmed using our first in field BRN2 inhibitors. Targeting BRN2 also lead to downregulation several known targets in NEPC like EZH2, AURKA, SOX2 and Peg10. Treatment with BRN2i reduced recruitment of BRN2 to the chromatin by approximately 93% within 16 hours. Moreover, these BRN2 inhibitors displayed adequate pharmacokinetic properties and reduced NEPC proliferation in vivo.

Conclusion: No therapies exist for highly lethal NEPC. Hence, the described work aims to verify BRN2, a central driver of NEPC, and lay the pre-clinical foundation for the integration of targeted therapies into the treatment landscape to improve survival and quality of life for patients suffering from deadly form of prostate cancer.

#1296

Arylsulfonamide KCN1 suppresses primary and metastatic growth of uveal melanoma through anti-angiogenic and anti-invasion mechanisms.

Stefan Kaluz, Qing Zhang, Hua Yang, Satoru Osuka, Jiyoung Mun, Narra S. Devi, Mark Goodman, Hans E. Grossniklaus, Erwin G. Van Meir. _Emory University, Atlanta, GA_.

Uveal melanoma (UM) is the most prevalent primary intraocular malignancy in adults with a survival time less than 1 year for patients that develop metastases (about 50%). HIF-1 plays a critical role in UM adaptation to the hypoxic microenvironment, treatment failure, and metastasis. In this study, we evaluated biodistribution and anti-cancer efficacy of arylsulfonamide KCN1, a lead compound in a novel class of small-molecule inhibitors of the hypoxia-inducible factor (HIF-1) pathway in UM models. PET with 11C-labeled KCN1 showed that KCN1 preferentially localizes to the eye and liver, the organ where UM preferentially metastasizes. In survival studies, UM cells were injected into the suprachoroidal space of the right murine eye using a transscleral technique and the eye was enucleated 7-9 days post-inoculation. Starting on Day 1, KCN1 or vehicle only (cremophor EL/ethanol 1:1, diluted with sterile PBS 1:5) were administered daily i.p., 5 x week, until animals reached the endpoint. Main organs were collected and evaluated for metastatic load. We found that KCN1 potently inhibited the growth of primary eye tumors, VEGF expression, angiogenesis in the primary tumor, the number and size of hepatic metastases, and extended mice survival. KCN1 inhibited invasion of UM cells in in vitro models, such as scratch-wound and Boyden chamber migration assays. To identify possible mediators of hypoxia-HIF-induced metastatic processes inhibited by KCN1 in UM, we tested a series of 20 candidates by qRT-PCR. Among the most consistently HIF-induced/KCN1-inhibited genes were prolyl-4-hydroxylases P4HA1 and 2 that modulate physical properties of the tumor microenvironment by post-translationally modifying collagen. Expression of P4HA1 and 2 is significantly higher in UM patients with metastatic disease and high level of expression correlates with poor prognosis. In summary, our studies suggest KCN1 exerts anti UM activity by inhibiting the primary tumor growth, reducing vascular density in the primary tumor, and the number and size of the hepatic metastases; KCN1 has therefore therapeutic potential for treatment of UM and further clinical development.

#1297

Casein kinase II (CK2) as a potential therapeutic target in acute myeloid leukemia (AML).

Badhauria Preeti,1 Soumya Iyer,1 Elanora Dovat,2 Pavan Kumar Dhanyam Raju,1 Jonathan Payne,1 Chunhua Song,1 Yali Ding,1 Claxton David,1 Sharma Arati,1 Chandrika Gowda1. 1 _Penn State Univ. Hershey Medical Ctr., Hershey, PA;_ 2 _Penn State Harrisburg, Harrisburg, PA_.

Casein Kinase II (CK2) is a pro-oncogenic kinase that is overexpressed in a majority of human malignancies, including leukemia. One third of AML cases without detectable high risk molecular alteration have high expression of CK2 which is strongly associated with poor outcome. Previously published data showed that CK2 directly phosphorylates several transcription factors including Ikaros (encoded by IKZF1) resulting in reduced DNA-binding affinity and loss of Ikaros function as a tumor suppressor. Here we present the evidence demonstrating role of Ikaros and CK2 in regulation of apoptosis in AML. We show that CK2 inhibitor, CX4945 has strong anti-leukemic effect in AML and one of the novel mechanisms of action involves restoration of Ikaros function as a transcriptional regulator of genes involved in apoptosis. Results: Treatment of AML cell lines and primary cells with CK2 inhibitor, CX4945 show cytotoxicity (IC50 3-5uM), increased apoptosis, cell cycle arrest and poor colony formation. Treatment of murine xenografts of AML with CX4945 showed significant anti-tumor effect. Further studies dissecting the mechanism of action of CX4945 are as follows: Genome-wide binding studies using chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) demonstrated that treatment of AML cell line, U937 with CX4945 at IC50 concentration enhances binding affinity of Ikaros transcription factor at the promoter regions of several target genes. Among the significantly affected genes (more than two fold change) are anti-apoptotic genes such as BCL2A1 (B Cell Lymphoma 2 related protein A1) and Bcl-xL (B Cell Lymphoma extra-Large). Dysregulation of apoptotic pathway is hallmark of cancer cells. These findings were confirmed qChIP where CX4945 treated AML cells showed increased binding of Ikaros to the BCL2A1 and Bcl-xL promoters. We used gain-of-function and loss-of-function experiments to determine how Ikaros regulates BCL2A1 and Bcl-xL genes in AML. Ikaros overexpression using lentiviral transduction results in reduced expression of BCL2A1 and Bcl-xL both at mRNA and protein level. Similar results were seen with silencing of CK2 using CK2 SiRNA as well as CX4945. Increased expression of BCL2A1 and Bcl-xL was noted after Ikaros silencing using SiRNA which did not revert with CX4945 treatment.In conclusion, (1) Ikaros represses transcription of anti-apoptotic genes BCL2A1and Bcl-xL, (2) CK2 directly phosphorylates tumor suppressor Ikaros and inhibits its function as transcriptional regulator, (3) Inhibition of CK2 enhances Ikaros-mediated repression of BCL2A1 and Bcl-xL genes resulting in increased apoptosis, (4) CX4945 has strong anti-leukemia effect in AML xenograft models. CK2 is a promising therapeutic target in AML. Our results unveal a novel mechanism of action of CK2 inhibitor, CX4945 in AML which involves restoration of Ikaros mediated regulation of apoptosis.

#1298

The anticancer effects of RX-5902 result from inhibition of phosphorylated p68-mediated β-catenin nuclear translocation.

Young B. Lee, Christina George, Deog J. Kim. _Rexahn Pharmaceutical, Inc., Rockville, MD_.

Phosphorylated p68 (p-p68 or phospho-DDX5) has been shown to be associated with cell transformation, epithelial mesenchymal transition (EMT) and cell migration, and furthermore play a vital role in cell proliferation and tumor/cancer progression by promoting the nuclear translocation of β-catenin. RX-5902 is an oral, small molecule inhibitor of β-catenin nuclear translocation mediated by p-p68. Previous studies have shown that RX-5902 inhibits the growth of cancer cells at low nanomolar IC50 (10 to 24nM) and disrupts the p-p68-β-catenin signaling pathway through an interaction with p-p68 on Tyr 593. By interfering with the p-p68-β-catenin signaling pathway cancer cells undergo apoptosis. In this study, we expanded our investigation to study the effect of RX-5902 on nuclear β-catenin in several types of cancer cell lines that include colon, gastric, hepatic, head and neck, melanoma, breast, pancreatic, ovarian, and prostate. Western blot data, in the cell lines tested, showed a decrease of nuclear β-catenin protein as well as p-p68 and c-myc in a concentration-dependent manner. Furthermore, an in vivo study of the MDA-MD-231 xenograft model demonstrated the ability of RX-5902 to inhibit the tumor growth by decreasing p-p68, β-catenin, cyclin D1 and c-myc proteins. Collectively our findings in both in vitro and in vivo studies support the potential therapeutic effect of RX-5902 in multiple cancer indications through the disruption of the phospho-p68/nuclear β-catenin interaction and blocking the nuclear translocation of β-catenin.

#1299

**Potential antitumor effects of a Golgi disrupting agent, M-COPA,** via **targeting cell-extracellular matrix interaction under the spheroid culture conditions.**

Yoshimi Ohashi,1 Kazuma Takeuchi,1 Mutsumi Okamura,1 Akinobu Akatsuka,1 Isamu Shiina,2 Kentaro Yoshimatsu,3 Shingo Dan1. 1 _Japanese Foundation for Cancer Research, Tokyo, Japan;_ 2 _Tokyo University of Science, Tokyo, Japan;_ 3 _Eisai Co., Ltd., Tokyo, Japan_.

The Golgi apparatus plays an essential role in the transport, processing, and sorting of cell surface proteins. We previously demonstrated that a Golgi disrupting agent, 2-methylcoprophilinamide (M-COPA), exhibited antitumor effect in human cancer cells in vitro and in vivo; however, in vivo antitumor efficacy of M-COPA did not simply reflect its in vitro efficacy, probably due to the lack of microenvironmental features of tumor tissues in the monolayer culture conditions. To bridge the gap between in vitro and in vivo efficacies, we exploited 3-dimensional (3D) spheroid culture models, which can recapitulate physiological characteristics of tumor tissues; i.e., nutrient/oxygen gradient, cell-cell interaction and cell-extracellular matrix (ECM) interaction, if cancer cells synthesize their own ECM molecules. Interestingly, human gastric cancer MKN1 cells displayed hypersensitivity to M-COPA under the 3D culture conditions compared to those in the monolayer culture conditions. Especially, we found that M-COPA caused loss of spheroid integrity. We previously demonstrated that M-COPA exerted antitumor effects via inhibiting intracellular transport of receptor tyrosine kinases onto the cell surface. Therefore, we examined the involvement of cell adhesion molecules in the antitumor effect of M-COPA. As a result, we found that specific integrins, which serve as cell surface receptors responsible for cell-ECM adhesion, were overexpressed and their downstream signaling molecules such as Src, Akt and MEK/ERK were highly phosphorylated in the cells under the 3D culture conditions compared to those under the monolayer culture conditions. Upon exposure to M-COPA, cell surface expression of these integrins was efficiently decreased and the downstream signaling molecules were dephosphorylated along with loss of spheroid integrity. Moreover, expression knockdown or neutralizing antibody of the integrins clearly suppressed sphere formation of MKN1 cells. These results suggest that M-COPA exerted in vivo antitumor effects via targeting specific integrins that mediate cell-matrix adhesion and its downstream intracellular signaling pathway observed in 3D spheroid culture models.

#1300

Novel thyrointegrin αvβ3 antagonist in the treatment of glioblastoma multiforme.

Shaker A. Mousa,1 Thangirala Sudha,1 Kavitha Godugu,1 Mehdi Rajabi,1 Stewart Sell,2 Paul J. Davis1. 1 _Albany College of Pharmacy and Health Sciences, Rensselaer, NY;_ 2 _Wadsworth Center Empire State Plaza, Albany, NY_.

Background: The proliferative and pro-angiogenic actions mediated by L-thyroxine, pro-inflammatory and all known growth factors in glioblastoma multiforme (GBM) are initiated at cell surface thyrointegrin αvβ3 receptors for thyroid hormone on the extracellular domain of integrin αvβ3. Thyrointegrin αvβ3 receptors are over-expressed on cancer and rapidly dividing blood vessel cells, but quiescent on normal cells. A macromolecule Polyethylene glycol-conjugated bi-TriAzole Tetraiodothyroacetic acid (P-bi-TAT) acts with high affinity (Ki 3.1 nM) and specificity for the thyrointegrin αvβ3 receptors without any significant nuclear translocation.

Methods: In the present studies, three primary human GBM cells and U87 glioma cell line were implanted orthotopically or subcutaneously (s.c.) into nude mice that were treated daily for up to 21 days with P-bi-TAT at different doses. Additionally, preclinical Pharmacokinetic and Safety assessments were carried out in multiple species.

Results: Pharmacokinetic profiles indicated once-a-day dosing, and safety assessment studies demonstrated high safety and tolerability at 100-fold over the anticipated human equivalent dose. Fluorescence labeled P-bi-TAT administered s.c. demonstrated high biodistribution to GBM tumor in the brain that was comparable to peripherally implanted tumor. P-bi-TAT administered daily for 10-21 days s.c. at 0.3-10 mg/kg resulted in a dose-dependent suppression of GBM tumor growth and viability monitored with IVIS imaging (p <0.001). Histopathological analysis of tumors revealed 95% loss of the vascularity of treated tumors at 10 days (p <0.001) along with extensive cellular necrosis and apoptosis, without intratumoral hemorrhage. GBM tumors had a 97% volume loss and maximal loss of GBM cell viability during a subsequent 22 days off-treatment period (p < 0.001). Genomic micro-array studies with human primary GBM revealed that over 30 pathways relevant to the progression of GBM are modulated by P-bi-TAT.

Conclusions: P-bi-TAT is a promising lead clinical candidate effective in the treatment of human GBM.

#1301

Targeting protein-DNA interactions in the DNA damage response: Lead identification and optimization for novel inhibitors of RPA and Ku.

John J. Turchi,1 Pamela S. VanderVere-Carozza,1 Navnath S. Gavande,1 Katherine S. Pawelczak2. 1 _Indiana Univ School of Medicine, Indianapolis, IN;_ 2 _NERx Biosciences, Indianapolis, IN_.

Recent advances in our understanding of the mechanisms of the cellular response to DNA damage (DDR) have opened up an exciting array of opportunities to treat human cancer. The majority of drug discovery efforts have focused on the DDR kinases, ATM, ATR and DNA-PK, each of which is activated in response to different types of DNA damage or genomic perturbations. We have taken the novel approach of drugging this pathway by targeting the DNA binding proteins Replication protein A (RPA) and Ku, which are required to activate the ATR and DNA-PK kinases, respectively. Chemical inhibition of the DNA binding activity functionally abrogates kinase activity and provides a novel mechanism by which DDR kinases can be blocked.

The identification of a potent inhibitor of the Ku-DNA interactions was gleaned from a specificity screen and was found to effectively block the Ku-DNA interaction and this inhibits DNA-PK phosphorylation activity. We identified a series of pharmacophores and moieties for potent and specific inhibition of Ku and demonstrate cellular inhibition of Ku DNA binding and DNA-PK activity. Third generation Ku inhibitors were developed to optimize for cellular bioavailability and are able to sensitize cells to DNA double strand beaks inducing agents, consistent with the role of DNA-PK in the non-homologous end joining repair pathway.

We initially identified a series of RPA-DNA interaction inhibitors from an in vitro high throughput screen. Extensive SAR was conducted to optimize for potency, specificity and physicochemical properties and led to the promotion of a lead candidate. We have determined the mechanism of action for single agent anti-cancer activity and combination activity involves chemical exhaustion of RPA. In vivo analysis of the lead candidate in pre-clinical models of lung and ovarian cancer has led to the advancement of a clinical candidate and represent a first in class RPA inhibitor poised for clinical advancement.

Together, these data demonstrate the ability to chemically target structure specific protein-DNA interactions. The molecules synthesized to inhibit these interactions share some common features, but can be optimized to obtain high potency and specificity. In addition, chemical modifications can be employed to allow cellular and in vivo bioavailability while retaining potent inhibitory activity. These data reveal the potential to target a new range of macromolecular interactions to expand the anticancer armamentarium to protein-DNA interactions.

#1302

Targeting hormone receptor-dependent cancers with potent, selective and orally-available small molecule inhibitors of eIF4E.

Gary G. Chiang, Gregory S. Parker, Ivy N. Hung, Vikas K. Goel, Jocelyn Staunton, Maria Barrera, Eric Sung, Ana Parra, Craig R. Stumpf, Joan Chen, Peggy A. Thompson, Andreas Nevarez, Christopher J. Wegerski, Cody Parker, Jeff Clarine, Samuel Sperry, Alan Xiang, Christian Nilewski, Garrick K. Packard, Kaveri Urkalan, Takasuke Mukaiyama, Theo Michels, Justin T. Ernst, Paul A. Sprengeler, Siegfried H. Reich, Kevin R. Webster. _eFFECTOR Therapeutics, San Diego, CA_.

The PI3K/mTOR pathway is commonly dysregulated in many hormone receptor-dependent tumors and plays a key role in promoting tumor growth and mediating drug resistance. In particular, PI3K and mTORC1/2 inhibitors have been intensively studied in the treatment of hormone receptor-dependent cancers and have shown benefit in some clinical settings. However, issues such as dose-limiting toxicities and emergent resistance limit the broader utility of these inhibitors. The translation initiation factor eIF4E is essential for the translation of m7G-capped mRNA and is a key point of convergence for both the PI3K/mTOR and MAPK signaling pathways. We have designed a series of potent, selective and orally-available m7G cap-competitive inhibitors of eIF4E (eFT-4Ei) with favorable drug-like properties. These inhibitors bind to eIF4E either as its free form or with eIF4E-4EBP and eIF4F complexes within tumor cells and downregulate hormone receptor-dependent signaling. Ribosomal profiling of eIF4E inhibitor-treated tumor cells identified a subset of translationally regulated target genes that overlap with mTORC1/2 regulated genes, but also a unique set of translationally regulated target mRNAs. Consistent with this observation, our eIF4E inhibitors show some similarities yet several important differences from existing mTORC1 or mTORC1/2 dual inhibitors in both cellular and physiological assays. Finally, significant anti-tumor efficacy was observed with eIF4E inhibition in vitro and in vivo. Taken together, these results highlight the potential for targeting eIF4E as a novel therapeutic strategy to treat hormone-receptor dependent cancers.

#1303

Targeted inhibition of galectin-1 suppresses glioblastoma growth.

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

Background: Galectin-1 (Gal1), a carbohydrate-binding protein is implicated in cancer cell proliferation, invasion and tumor angiogenesis. An earlier report from our laboratory demonstrated that glioblastoma (GB) cells express high levels of Gal-1, which promotes tumor progression through upregulation of CXCR4 via NF-κB in in vitro GB models. Recently, several Gal1-targeting compounds have emerged. OTX008 is a calixarene derivative designed to bind the Gal1 amphipathic β-sheet conformation. Our study aimed to validate the effect of the non-peptidic galectin-1 inhibitor OTX008 in human GB in relevant pre-clinical models.

Methods: In vitro cell culture and in vivo GL261 murine models of GB were utilized to assess efficacy of treatment with OTX008 via Alzet osmotic pumps. To investigate the mechanistic effect of OTX008 on GB cells, we used RT-PCR and western blots techniques. Magnetic resonance imaging (MRI), and immunohistochemistry (IHC)-staining methods were used to detect tumor growth in in vivo GL261 murine model following OTX008 treatments.

Results: In cultured human and mouse GB cells, OTX008 inhibited proliferation and invasion at micromolar concentrations. We also found that the anti-proliferative effects correlated with Gal1 expression in human and mouse GB cell lines. Furthermore, OTX008 inhibited Gal1 expression and AKT-dependent survival pathways, and decreased CXCR4 expression. In vivo, continued 100ng/kg OTX008 treatment via Alzet osmotic pumps over 14 days reduced tumor growth of GL261 murine model after 3 weeks of implantation. OTX008 treatment was associated with downregulation of Gal1 and Ki67 in treated tumors, as well as decreased micro-vessel density and VEGFR2 expression.

Conclusion: These findings suggest that targeting galectin-1 may be an effective approach in the treatment of GB patients.

#1304

Aryl hydrocarbon receptor ligands 5F 203 and 3,3'-Diindolylmethane disrupt mammospheres derived from MCF-7 cells and induce tumor suppressor miR125b-2 expression.

Eileen Brantley, Nicole Mavingire, Jonathan Wooten, Petreena Campbell. _Loma Linda Univ. School of Medicine, Loma Linda, CA_.

While anti-estrogen tamoxifen (Tam) effectively treats many patients with estrogen receptor positive (ER+) tumors, up to 40% experience relapse following resistance. Breast cancer stem cells (CSCs) within tumors greatly contribute to tamoxifen resistance (TamR) and exhibit unique molecular signatures that drive metastasis and promote relapse. Tumor suppressor miRNAs aid in suppressing breast cancer progression. We have previously shown that aryl hydrocarbon receptor (AhR)-ligand Aminoflavone disrupts the formation of spheres and inhibits the expression of putative stemness marker α6-integrin and α6-integrin-src-Akt signaling. We hypothesize that two AhR ligands, 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203) and 3,3'-Diindolylmethane (DIM), exhibit anticancer properties in MCF-7 breast cancer cells by targeting the CSC population in an AhR-dependent fashion. We found that DIM and 5F 203 disrupted mammospheres derived from MCF-7 cells but demonstrated a reduced capacity to do so in mammospheres derived from AHR100 cells (MCF-7 variants that are AhR-unresponsive). Wound healing and colony forming assays respectively revealed that 5F 203 and DIM also decreased cell migration and cell proliferation in MCF-7 cells and to a much lesser extent in AhR100 cells. 5F 203 and DIM induced miR125b-2 expression and suppressed the expression of stemness-regulating genes such as α6-integrin, a predicted miR125b-2 target. The reduction in stemness-gene expression in MCF-7 cells was attenuated following pretreatment with AhR antagonist CH223191. These data suggest that AhR ligands such as DIM and 5F 203 confer their anticancer actions including those against breast CSCs in an AhR-dependent manner. Our data is expected to provide a rationale for the development of anticancer AhR ligands designed to combat ER+ breast cancer and decrease the risk of relapse.

#1305

Identification of signal transduction kinases inhibited by pan-RAF inhibitor belvarafenib using FRET imaging technique.

Dong-Jun Bae,1 Sang-Yeob Kim,1 JooYun Byun,2 InHwan Bae,2 YoungGil Ahn,2 YoungHoon Kim,2 Kwee Hyun Suh2. 1 _Asan Medical Center, Seoul, Republic of Korea;_ 2 _Hanmi Research Center, Hwaseong, Republic of Korea_.

The mitogen-activated protein kinase (MAPK) pathway plays an important role for the survival and proliferation of tumor cells. For example, the activation of the MAPK pathway due to mutations in BRAF, NRAS and KRAS kinases is known to be the causes of various solid tumors and hematologic malignancies. Accordingly, there is a strong need for effective therapeutic drugs for treating cancers caused by genetic mutation of BRAF, NRAS or KRAS. Belvarafenib (HM95573 or GDC5573), currently in Phase 1 clinical trials, is a selective and orally bioavailable pan-RAF kinase inhibitor. When biochemically assayed for hundreds of kinases, Belvarafenib showed selectivity toward RAF family kinases, including strong inhibition activities for BRAF WT, BRAF mutants and CRAF. Previously, we reported that Belvarafenib strongly inhibits the growth of cancer cell lines with BRAF, NRAS or KRAS mutations and also shows excellent anticancer efficacy in animal models using the corresponding cancer cells. In this study, employing fluorescence resonance energy transfer (FRET) imaging, we examined which protein kinases involved in signal transduction are regulated by Belvarafenib and investigated the mechanism of action for the regulation in typical cancer cells harboring BRAF, NRAS or KRAS mutations such as A375 (melamona), SK-MEL-30 (Kwmelamona), HCT116 (CRC), and Calu-6 (NSCLC). Among the various protein kinases that can be involved in signal transduction, we identified with time-lapse FRET imaging that the phosphorylation of ERK, AKT and S6K are inhibited after the treatment of Belvarafenib in the above cancer cells of BRAF, NRAS or KRAS mutations. Of note, BRAF mutant selective inhibitors, Vemurafenib and Dabrafenib were not active in cancer cells of NRAS or KRAS mutations. These results correlate with the in vitro and in vivo anticancer activity in cancer cells of BRAF, NRAS or KRAS mutations. In conclusion, unlike Vemurafenib and Dabrafenib, the pan-RAF inhibitor Belvarafenib could be effective for treating the cancer caused by NRAS or KRAS mutations, as well as BRAF mutations.

#1306

SEL120, a potent and specific inhibitor of CDK8 induces complete remission in human patient derived xenograft models of acute myeloid leukemia.

Milena Mazan,1 Eliza Majewska,1 Michal Mikula,2 Katarzyna Wiklik,1 Michal Combik,1 Aniela Golas,1 Magdalena Masiejczyk,1 Elzbieta Fiedor,1 Anna Polak,3 Magdalena Cybulska,2 Aleksandra Grochowska,2 Michal Kopczynski,2 Urszula Kuklinska,2 Zuzanna Sandowska-Markiewicz,2 Malgorzata Statkiewicz,2 Agnieszka Paziewska,4 Michalina Dabrowska,2 Arkadiusz Bialas,1 Maciej Mikulski,1 Renata Windak,1 Jerzy Ostrowski,2 Przemyslaw Juszczynski,3 Krzysztof Brzozka,1 Tomasz Rzymski1. 1 _Selvita S.A., Krakow, Poland;_ 2 _Department of Genetics, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Poland;_ 3 _Institute of Hematology and Transfusion Medicine, Poland;_ 4 _Department of Gastroenterology, Hepatology and Clinical Oncology, Medical Center for Postgraduate Education, Poland_.

Cyclin-dependent kinase 8 inhibitors (CDK8i) have anti-cancer activity in human acute myeloid leukaemia (AML) cell lines both in vitro and in vivo. Activity of CDK8i often involves deregulation of super-enhancer-associated genes in AML cell lines. Previous studies established SEL120 as a specific CDK8 inhibitor active in AML cells with increased STAT1/5 signalling pathways. Differential gene expression analysis demonstrated high enrichment of leukaemia stem cell (LSC) signatures in responding cells, linked to resistance to standard therapies and relapsed disease. Cells sensitive to SEL120 treatment were positive for CD34 and negative for lineage commitment surface markers. SEL120 markedly reduced STAT5 phosphorylation on serine 726 (STAT5 pS726) in sensitive cell lines. Prolonged SEL120 treatment led to significant downregulation of CD34 and induction of lineage commitment markers. Transcriptomic analysis revealed that SEL120 regulated many genes involved in differentiation and apoptosis. We observed synergistic effects of SEL120 with standard of care cytotoxic drugs such as cytarabine. Treatment of AML cells with cytarabine spared many CD34+ cells, which could be effectively eradicated by subsequent treatment with SEL120. Many cell lines which were resistant to SEL120 treatment could be sensitized by concomitant treatment with BH3 mimetic agent ABT-199. Combination of both compounds resulted in potent induction of apoptosis in AML cells in vitro and in vivo. Treatment of mice bearing subcutaneously implanted human leukaemia cell lines resulted in significant tumour growth inhibition, whereas cotreatment with ABT-199 led to complete regressions at doses which were vey well tolerated by animals. Next we have selected patient derived primary AML cells using gene expression signatures identifying SEL120 -responder cell lines. In these cells SEL120 significantly reduced viability, induced apoptosis and lineage commitment. Further, the same cells were implanted into NOD scid gamma mice. Animals succumbed to AML, diagnosed by a significant presence of human CD45/CD34 positive leukaemia cells in a peripheral blood and splenomegaly. Stand-alone treatment with SEL120 resulted in the complete remission of AML cells in a peripheral blood and bone marrow, and reduced spleen weight, without symptoms of compound-related toxicity. These results validate SEL120 as a promising agent in the treatment of AML.

#1307

Inhibition of ERK by LY3214996 augments nab-paclitaxel and gemcitabine combination chemotherapy efficacy in preclinical models of pancreatic cancer.

Shripad V. Bhagwat, Wenjuan Wu, Baohui Zhao, Weihua Shen, Lisa Kindler, Jennifer Stephens, Jason Manro, William McMillen, Sajan Joseph, Sheng-Bin Peng, Ramon V. Tiu. _Lilly Research Laboratories, Eli Lilly & Company, Indianapolis, IN_.

Pancreatic ductal adenocarcinoma (PDAC) is currently the third most frequent cause of cancer deaths in the US and is expected to be the second deadliest cancer by 2030. The 5-year survival rate in PDAC patients is only 6% and the poor prognosis is attributed to several factors including late-stage diagnosis, aggressive disease progression and high resistance to conventional therapies. Therefore, there is an urgent need for more effective systemic treatment strategies in PDAC patients. Nab-paclitaxel (Nab-pac) combined with gemcitabine (Gem) or FOLFIRINOX combination chemotherapy regimen are approved as standard of care treatment for frontline metastatic PDAC. Due to limited clinical efficacy of current cytotoxic chemotherapy regimens for PDAC patients, novel approaches are needed to further improve patient survival outcomes. In PDAC, activating mutations in KRAS occur at a frequency of ~90% rendering this a potential therapeutic target of interest. Developing drugs that directly target mutant KRAS protein remains challenging and alternative strategies focus on inhibition of downstream targets of the RAS-MAPK cascade. The addition of ERK inhibitor LY3214996 to the chemotherapy regimen (Nab-pac+Gem) was tested in MIA PaCa-2 (KRAS G12C), Capan-2 (KRAS G12V) and SW1990 (KRAS G12D) pancreatic cancer xenograft models. In the Capan-2 model, combination of LY3214996 with Gem alone was statistically additive (p<0.001) and resulted in 14% tumor regressions (%dT/C =-14). In the MIA PaCa-2 model, LY3214996 has shown dose-dependent single agent activity resulting in 7 out of 7 partial responses at 100 mpk QD. In the combination study, LY3214996 enhanced the efficacy of Nab-pac+Gem and demonstrated 42% tumor regression (%dT/C =-42), and combination effect was additive (p=0.008). Combination of LY3214996+Gem+Nab-pac resulted in 1 complete response (CR) out of 6 whereas Nab-pac+Gem resulted in only 1 partial response (PR) out of 6. In SW1990 model, combination efficacy of LY3214996 and Nab-pac+Gem was additive (p=0.011) and resulted in ~88% tumor growth inhibition (%dT/C=12). Our data suggest that the efficacy of Nab-pac+Gem based chemotherapy can be enhanced through specific inhibition of ERK1/2 kinase activity in KRAS mutant PDAC, and supports the clinical testing of this combination in the current Phase I JUAB (LY3214996) study in metastatic PDAC (NCT02857270).

#1308

APC1 drug: The key for transforming human triple negative breast cancer (TNBC) to HER2+/estrogen receptor (ER)+ cells.

Yoram Devary. _ISK Ltd, Jerusalem, Israel_.

Background: Approximately 15-18% of breast cancer cases are TNBC. This subtype does not express HER2 or ER and therefore cannot be treated with anti-HER2 or endocrine therapy. We have isolated a peptide, named anti pancreatic cancer factor 1 (APC1) from human pancreas. APC1 has a novel sequence and a strong signal peptide. The aim of this study was to investigate APC1 as a potential treatment for TNBC.

Methods: APC1 was originally developed as a treatment for pancreatic cancer. Using anti-APC1- antibodies, we detected it human blood (level in healthy patients: 500 pg/ml). We optimized and stabilized the peptide and used it in our in-vivo experiments. We applied APC1 to human cancer cells and measured differentiation and apoptosis using FACS analysis, Western blot analysis and Elisa. In vivo studies we done with nude mice inoculated SC with human pancreatic cancer cells and human TNBC.

Results: Mice inoculated with human ductal adenocarcinoma cells: Panc-1 cells or human BxPC3 were subjected to treatment with APC1. As opposed to control mice, treated with PBS, mice treated with APC1 were strongly benefited from the treatment. Tumors volumes of mice treated with APC1 were statistically smaller then control mice. Interestingly, pathology assessment of tumors at end of experiments showed that ducts we formed in the tumors of mice treated with APC1 as opposed to mice treated with PBS. This imply that cells in tumors exposed to APC1 differentiated into ducts. Therefore, we tested the impact of APC1 on differentiation of TNBC cells. When applying APC1 to DA231/MDA468 human TNBC cells in-vitro and in-vivo, strong expression of HER2 (100-fold over control) was induced. Treatment of these cells with trastuzumab emtansine after treatment with APC1 induced apoptosis in the cells. APC1-treated tumors were found to be strongly positive for ER. In-vitro experiments demonstrated tamoxifen- induced death in these cells. The mechanism-of-action of APC1 is novel and involves activation of Notch receptor family members. Using γ-secretase inhibitor, we were able to inhibit HER2 induction. Animal toxicology in rats were successfully finalized and the drug has a safe profile. Maximum tolerated dose (MTD)/ Dose Range Finding (DRF) studies in monkeys were also successfully finalized and demonstrated a safe profile for this drug. We are currently working on planning additional experiments in monkeys and on Phase 1 clinical trial with TNBC and pancreatic cancer patients.

Conclusion: Our results suggest that APC1 can turn TNBC into a curable disease.

#1309

Targeting GluN2C ion channels as a promising approach in the treatment of melanoma.

Hessam Tabatabaee, Yuchen Feng, Hamed Yari, Ting La, Simonne Sherwin, Lei Jin, Rebecca Lim, Alan Brichta, Xu Dong Zhang. _University of Newcastle, Callaghan, Australia_.

Ion channels are pore-forming integral membrane proteins that are known to have an impact on all hallmarks of cancer. Due to their expression on the surface of the cells, ion channels provide a distinct therapeutic target with more facilitated access from the extracellular milieu. NMDA receptors are a subtype of ligand-gated ion channels that express unique features including high calcium permeability as well as the requirement for binding of glutamate and the co-agonist glycine in order to be activated. Here we show that a glutamate ionotropic NMDA (N-methyl-D-aspartate) type ion channel, GluN2C is critically required for survival of human melanoma cells. Publicly available microarray data from Gene Expression Omnibus (GEO) were acquired in order to identify the differentially expressed ion channel genes in 127 melanoma patient samples relative to benign nevi or human epidermal melanocytes. Data exists into two major cohorts: First cohort consisted of 18 benign nevi and 45 melanoma (GSE3189), while second cohort contained 8 melanocytes and 82 melanoma (GSE29377). A bioinformatics approach was undertaken to determine commonly upregulated ion channels between the two cohorts. Subsequently, functional enrichment analysis along with pathway evaluation of these genes revealed GluN2C as a novel candidate for further investigation in this study. Upregulation of GluN2C was confirmed in a panel of melanoma cell lines and fresh melanoma isolates. Strikingly, silencing of GluN2C markedly reduced viability and clonogenicity in melanoma cells. Consistently, a pharmacological inhibitor of GluN2C potently killed melanoma cells with minimal effects on melanocytes and fibroblasts. Killing of melanoma cells by inhibition of GluN2C was not due to induction of apoptosis, since a general caspase inhibitor Z-VAD-FMK failed to rescue cells from dying. In contrast, the late stage autophagy inhibitor Bafilomycin A1 blocked the GluN2C induced cell death suggesting that killing of melanoma cells following inhibition of GluN2C is in part associated with induction of autophagy. Collectively, these results uncover a potential role for GluN2C ion channels in the pathogenesis of melanoma and suggest that these ion channels may constitute a promising target for melanoma treatment.

#1310

Abrogation of KRas-addicted tumors by GSK3 suppression-mediated upregulation of β-catenin and c-myc.

Aslamuzzaman Kazi,1 Shengyan Xiang,1 Hua Yang,1 Daniel Delitto,2 Jose Trevino,2 Rays H. Jiang,3 Muhammad Ayaz,1 Harshani Lawrence,1 Perry Kennedy,1 Said M. Sebti1. 1 _Moffitt Cancer Center, Tampa, FL;_ 2 _University of Florida, Gainesville, FL;_ 3 _University of South Florida, Tampa, FL_.

The significant involvement of mutant KRas in human cancer development underscores the need to develop approaches that disable mutant KRas-driven tumors. Targeting KRas directly is challenging, and such identifying vulnerabilities specific for mutant KRas tumors is an important alternative approach. In this study, we discovered that glycogen synthase kinase 3 (GSK3) is required for the in vitro and in vivo growth and survival of human mutant KRas-dependent tumors but not for mutant KRas-independent tumors. Pharmacological inhibition with the GSK3 inhibitor SB as well as siRNA depletion of GSK3 lead to tumor suppression that is mediated at least in part by inhibition of the phosphorylation of the GSK3 substrates c-Myc on T58 and β-catenin on S33/S37/T41. CRISPR/Cas9 targeted knock out studies demonstrated that c-Myc and β-catenin upregulation mediate the antitumor activity of SB. Importantly, GSK3 blockade inhibits the in vivo growth of G12D, G12V, and G12C mutant KRas primary and metastatic patient-derived xenografts from pancreatic cancer patients who progressed on chemo- and radiation therapies. This discovery warrants advanced pre-clinical and clinical investigations of GSK3 inhibitors in mutant KRas-dependent cancers.

#1311

Inhibition of gankyrin-tumor suppressor protein interactions due to small molecule induced conformational change.

Abbas Kabir, Aaron Muth. _St. John's University, Queens, NY_.

Introduction: Gankyrin is a highly conserved, 25 kDa protein containing 7 ankyrin repeats that binds to the S6 ATPase subunit of the 26S proteasome. It plays a crucial role in cellular growth, proliferation and metastasis. It is found to be largely overexpressed in a variety of malignancies and its abnormal overexpression makes it a biomarker in certain cancers. Gankyrin promotes cellular growth and proliferation through a variety of protein-protein interactions. Specifically, it binds to and promotes degradation of numerous tumor suppressor proteins (TSPs) such as p53 and retinoblastoma protein by promoting their proteasomal degradation. In 2016, a small molecule binder of gankyrin (cjoc42), demonstrated an ability to inhibit gankyrin activity in a dose dependent manner by inhibiting gankyrin's interaction with certain TSPs as well as the proteasome.

Methods: In this research, we hypothesize that small molecule binding of gankyrin brings about a conformational change that inhibits its interaction with certain TSPs as well as the proteasome. In order to test this hypothesis, we have performed a series of fluorescence based experiments. We investigated the effect cjoc42 binding to gankyrin has on its intrinsic tryptophan fluorescence as well as its effect on fluorescently conjugated gankyrin. Follow up experiments consisted of both simple western blotting and co-immunoprecipitation assays to further investigate the ability of cjoc42 to induce a conformational change in gankyrin.

Discussion: We observed a diminished fluorescence intensity for both the intrinsic and labeled gankyrin experiments when in the presence of cjoc42. Western blot and Co-IP experiments are currently underway to further support our findings.

Conclusion: Cjoc42, a small molecule binder of gankyrin, appears to induce a conformational change. This conformational change appears to play a prominent role in regulating certain protein-protein interactions. Furthermore, this suggests that gankyrin's binding to specific TSPs and the proteasome are modulated by this mechanism.

### Tyrosine Kinase and Phosphatase Inhibitors 1

#1312

**A novel, highly selective, and extremely potent FLT3 tyrosine kinase inhibitor DFL-0059 exhibits** in vitro **and** in vivo **antitumor activity against AML with FLT3 mutation.**

Chu-Bin Liao, Shao-Zheng Peng, Ying-Shuan Lee, Hung-Jyun Huang, Yuan-Ting Cho, Yu-Chih Pan, Yi-Mei Chang, Yen-Chun Kuo, Pao-Chiung Hong. _Development Center for Biotechnology, Taipei City, Taiwan_.

FLT3 oncogenic mutations including internal tandem duplication (ITD) and point mutation are the most frequently detected in approximately 30% of patients with acute myeloid leukemia (AML), resulting in abnormal ligand-independent activation to promote aberrant proliferation as well as leukemogenesis. Currently, FLT3 has been emerged as a validated target for therapy of AML patients harboring FLT3 mutation. Despite the numerous FLT3 inhibitors under clinical investigation, issues such as selectivity, toxicity, bioavailability, or low potency, warrant the development of novel agents conferring higher potency and selectivity toward the FLT3 with less toxicity. Herein, we presented a novel compound DFL-0059 exhibited extremely potent for FLT3 kinase as well as FLT3 D835A mutant with IC50 value of sub-nM. DFL-0059 also has highly selectivity for FLT3 over 403 other non-mutant kinases with S(35) score of 0.03 at 1 μM in KINOMEscan. Moreover, DFL-0059 only toxic to the cells growth relied on FLT3 signal and significantly inhibited FLT3 singling pathway. The overall selectivity of DFL-0059 in biochemical and cell-based assay was superior to well-known FLT3 inhibitors Quizartinib (AC220) and Gileritinib (ASP2215). Notably, DFL-0059 showed dose-dependent oral activity in FLT3-ITD positive AML xenograft murine model without observed toxicity, especially complete remission was noted. Those results presented here support that DFL-0059 is an effective agent for treatment of FLT3-associated AML patients. The IND enabling study of DFL-0059 has been conducted.

#1313

CPI-818: A selective inhibitor of interleukin-2-inducible T-cell kinase (ITK) that inhibits T-cell receptor signaling, promotes Th1 skewing, and achieves objective tumor responses when administered to dogs with T cell lymphomas.

James W. Janc,1 Craig M. Hill,1 Patrick P. Ng,1 Andrew N. Hoston,1 Antonett Madriaga,1 Trang P. Dao-Pick,1 Kitman S. Yeung,1 Ryan Hudson,2 Anne-Marie Beausoleil,2 Erin Bradley,1 Erik Verner,1 Douglas H. Thamm,3 Richard A. Miller,1 Joseph J. Buggy1. 1 _Corvus Pharmaceuticals, Burlingame, CA;_ 2 _Unity Biotechnology, Brisbane, CA;_ 3 _Colorado State University, Fort Collins, CO_.

Background: ITK is a non-receptor tyrosine kinase that modifies T cell receptor (TCR) signaling. Mice deficient in ITK, but not resting lymphocyte kinase (RLK), exhibit defects in Th2 differentiation while retaining the ability to differentiate into Th1 cells and secrete IFNγ. Combined disruption of ITK and RLK in mice leads to more severe T cell functional defects compared to disrupting ITK alone, and paradoxically allows for normal Th2 responses. Thus, selective pharmacologic inhibition of ITK versus RLK is necessary to inhibit Th2 responses without affecting Th1-dependent immunity. ITK is widely expressed in T cell malignancies, and activation of ITK upregulates GATA-3, a transcription factor that drives Th2 differentiation and is associated with poor survival. Here we report the discovery and characterization of CPI-818, an irreversible inhibitor of ITK. CPI-818 is highly selective for ITK over RLK allowing for an assessment of pure ITK inhibition on normal and malignant T cells.

Results: CPI-818 irreversibly inhibited ITK (IC502.3 nM) with >100-fold selectivity over RLK (430 nM) and BTK (850 nM). The mechanism of ITK inhibition involves covalent binding to CYS-442 confirmed by mass spectrometry. Irreversible inhibition of ITK in vitroand in vivowas demonstrated using an active site competitive probe. CPI-818 inhibited anti-CD3/28 induced phosphorylation of ERK (T202/Y204) and PLCγ (Y783) in PMBCs, and inhibited IL2 secretion in Jurkat T cells (IC5075 nM). CPI-818 demonstrated dose dependently inhibition of TCR-induced proliferation of malignant T cells from Sezary Syndrome patients. In mice orally treatedwith CPI-818 an increase in the ratio of IFNγ/IL-4 (p<0.05) upon antigen-specific re-stimulation in an OVA transgenic adoptive transfer model was observed, consistent with Th1-skewing. Additionally, toassess the impact of CPI-818 on Th-biasing, activated human PBMCs were cultured for 6 days with CPI-818 and inhibition of IL-4 production (average 64% inhibition, n=12) was significantly greater than IFNγ production (average 19% inhibition), supportive of Th1 skewing. To assess the potential of CPI-818 to treat human T cell lymphoproliferative disorders, the safety and efficacy of CPI-818 in companion dogs with spontaneously-occurring T cell lymphomas were evaluated. CPI-818 was dosed orally at 20 mg/kg BID for 2 weeks to 5 months. Three animals were treated: 1 with peripheral T cell lymphoma and 2 with cutaneous T cell lymphoma. Full ITK occupancy in peripheral blood was confirmed using a probe assay. Evidence of anti-tumor activity was seen in all dogs including complete and partial responses. CPI-818 was well tolerated with no change in normal lymphocyte counts. These data support evaluation of CPI-818 in clinical trials in patients with T cell malignancies.

#1314

MERTK and BCL-2 as potential therapeutic targets in early T-precursor acute lymphoblastic leukemia.

Ryan J. Summers,1 Katherine A. Minson,1 Xiaodong Wang,2 Steven V. Frye,2 H. Shelton Earp,2 Deborah DeRyckere,1 Douglas K. Graham1. 1 _Emory University, Atlanta, GA;_ 2 _University of North Carolina Chapel Hill, Chapel Hill, NC_.

Background: Early T-precursor acute lymphoblastic leukemias (ETP-ALL) account for 15% of pediatric T-cell ALL (T-ALL) cases and are characterized by an immature phenotype, resistance to therapy, and high relapse rates. MERTK receptor tyrosine kinase is ectopically expressed in ~50% of T-ALLs, particularly those with an immature T cell phenotype, suggesting a role in ETP-ALL. The anti-apoptotic protein B-cell lymphoma-2 (BCL-2) is specifically expressed in immature T cell precursors, is preferentially expressed in ETP-ALL compared to other T-ALLs, is essential for ETP-ALL cell survival, and is regulated downstream of MERTK in acute leukemia cells. Thus, combination therapies targeting these two proteins may be particularly effective to treat ETP-ALL.

Methods: MERTK and BCL-2 mRNA expression was assessed in T-ALL patient samples using publicly available data. Loucy and PEER ETP-ALL cell lines were cultured with vehicle or MRX-2843, a dual MERTK/FLT3 inhibitor, alone or in combination with the BCL-2 inhibitor venetoclax. Phosphorylated and total MERTK were assessed by immunoblot. Cells were stained with PoPro-1-iodide and propidium iodide dyes and analyzed by flow cytometry to assess cell death. Relative cell numbers were assessed using Presto Blue reagent. Orthotopic xenografts were established in NSG or NSGS mice using luciferase-expressing Loucy cells or an ETP-ALL patient sample and leukemia burden was monitored by bioluminescence imaging or flow cytometry. MRX-2843 or saline vehicle were administered orally once daily. Median survival was determined by Kaplan-Meier analysis.

Results: MERTK and BCL-2 mRNAs were expressed at significantly higher levels in ETP-ALL patient samples relative to other T-ALLs. MRX-2843 mediated a dose-dependent decrease in phosphorylated MERTK and induced dose-dependent cell death (43.2% vs 16% in vehicle-treated cultures, p<0.01) in PEER cells. Moreover, in a patient-derived ETP-ALL xenograft model, treatment with MRX-2843 reduced peripheral blood disease burden by 83% (p<0.001) and decreased spleen weight by 64% (p<0.001) compared to vehicle-treated mice. Furthermore, treatment with MRX-2843 prolonged median survival by 11 days compared to control mice (n=8/group, p=0.0016). In a cell line-derived xenograft model, MRX-2843 reduced disease burden by 60% (n=10/group, p<0.0001) compared to vehicle. Treatment with a combination of MRX-2843 and venetoclax decreased cell density more effectively than either single agent in cultures of the Loucy and PEER cell lines (p<0.05).

Conclusions: MERTK and BCL-2 are preferentially expressed in ETP-ALL relative to T-ALL and MRX-2843 has robust therapeutic activity in cell culture and xenograft models of ETP-ALL. These data validate MRX-2843 as a novel agent with potential for clinical application in patients with ETP-ALL. Combined targeting of MERTK and BCL-2 may be particularly effective.

#1315

Combination of Wee1 inhibition with targeted and standard chemotherapy in preclinical models of pancreatic ductal adenocarcinoma.

Sarah J. Hartman, Stacey M. Bagby, Betelehem W. Yacob, Dennis M. Simmons, Tonia E. Tse, Christopher H. Lieu, S. Lindsey Davis, Alexis D. Leal, Jennifer R. Diamond, Wells A. Messersmith, Todd M. Pitts. _University of Colorado Denver AMC, Aurora, CO_.

Introduction: Pancreatic ductal adenocarcinoma (PDA) is the fourth leading cause of cancer death and has a 5-year survival rate of less than 7%. The poor prognosis associated with PDA is related in part to a lack of screening tests to promote early detection and ineffective systemic targeted therapies. Adavosertib (AZD1775, MK1775) is a selective Wee1 inhibitor with promising preclinical activity in PDA and synergy with cytotoxic chemotherapy in other cancer types. Wee1 is a tyrosine kinase that activates in the G2M cell cycle checkpoint in response to DNA damage. Inhibition of Wee1 with adavosertib prevents the phosphorylation of CDC2, thus allowing unrepaired DNA to enter mitosis and ultimately succumb to mitotic catastrophe. The purpose of this study was to investigate adavosertib in combination with standard chemotherapy and other targeted agents in preclinical models of PDA.

Methods: Athymic nude mice were implanted with PDA PDX models on the right and left flanks. When the average tumor volume reached 100-300 mm3, mice were randomized into one of the following treatments: vehicle, adavosertib, irinotecan, navitoclax, capecitabine, adavosertib + irinotecan, or adavosertib + navitoclax, adavosertib + capecitabine. Tumor volume was calculated using the following equation: volume = (length × width) × 0.52. Four pancreatic cancer cell lines were plated in 96-well plates and Cell Titer-Glo proliferation assays were performed to determine the most effective combination doses of irinotecan, 5FU, or navitoclax with adavosertib in vitro. Combination effects were analyzed using CalcuSyn software. The most effective doses within each cell line were selected and used for Caspase 3/7 apoptosis assays and cell cycle analyses by flow cytometry. Western blots were performed to evaluate changes in downstream effectors.

Results: In vivo, the combination of adavosertib with either irinotecan or navitoclax resulted in decreased tumor growth compared to the respective single agents. The combination of adavosertib with irinotecan, 5FU, or navitoclax in vitro resulted in greater antiproliferative effects in all cell lines, and the several combinations were synergistic in all cell lines as determined by CI values less than 1. Navitoclax increased apoptosis in several cell lines both as a single agent and was enhanced in combination with adavosertib. Irinotecan proved to be more cell cycle dependent and significantly altered the cell cycle in all cell lines. Irinotecan increased phospho-CDC2 and decreased PHH3, while adavosertib increased gamma-H2AX as a single agent and in combination.

Conclusions: The combination of adavosertib with either irinotecan, 5FU, or navitoclax in vivo decreased tumor growth and had enhanced antiproliferative effects in vitro. These data support future studies with adavosertib in combination with standard therapies or navitoclax to treat PDA.

#1316

Preclinical evaluation of TUL-001, an EGFR inhibitor for the treatment of NSCLC with brain metastasis (BM).

Xile Liu,1 Liwei Mu,2 Bin Liu,2 Shouting Wu,2 Degang Wang,2 Lu Zhang,1 Charles Z Ding,3 Lihong Hu,1 Weifeng Mao,3 Zhenzhen Zhu,3 Chen Chen,3 Yuanfeng Xia,3 Chichung Chan,3 Shuhui Chen3. 1 _WuXi AppTec, Wu Han, China;_ 2 _Zhuhai United Laboratories Co., Ltd., Zhu Hai, China;_ 3 _WuXi AppTec, Shang Hai, China_.

Objective: Non-small-cell lung cancer (NSCLC) patients with epidermal growth factor receptor-activating mutations (EGFRm+) have a much higher risk of developing CNS metastases. The cumulative incidence of BM and LM in these patients is more than 50%. These patients have poor prognosis as there are no approved drugs for these indications. Currently approved drugs can neither effectively treat brain metastases nor prevent development of the brain metastases due to their limited blood-brain-barrier (BBB) penetration. TUL-001 is a potent oral EGFR inhibitor, which has excellent potency and BBB (blood brain barrier) penetration ability, it can potently induces tumor regression both in the brain metastases and subcutaneous animal models.

Methods: The anti-proliferative activity of TUL-001 was evaluated in PC-9 (EGFRExon19 Del), and A431 (EGFRWT) NSCLC cell lines. The anti-tumor activity was evaluated in PC-9-luc Lung cancer brain metastases model, which was monitored by an IVIS Xenogen imaging system. The in vivo anti-tumor activity also evaluated in PC-9 subcutaneous transplantation tumor model. Effects on EGFR signaling pathway were assessed by western blot analysis of the downstream effector proteins p-EGFR (Y1068), p-AKT (Ser473) and p-ERK (Thr202/Tyr204).

Results: TUL-001 displayed potent anti-proliferative activity in the EGFR mutant cell line PC-9 (IC50 =3.74 nM), and less potent activity in the A431 (EGFRWT) cell line (IC50 = 54.1 nM), it has about 14 fold selectivity for EGFRExon19 Del over EGFRWT. TUL-001 significantly inhibited tumor growth at all doses tested in the PC-9-luc brain metastases model, and significantly prolong the overall survival for the test animals. In the PC-9 subcutaneous transplantation tumor model, TUL-001 can also shrinkage the tumors, and the TGI for the lower dose group (2.5mpk) was more than 120%. Western blot analysis of the tumor samples in the brain metastases model study showed that TUL-001 inhibited p-EGFR (Y1068), p-AKT (Ser473), and p-ERK (Thr202/Tyr204), indicating that the tumor growth inhibition was through inhibition of the resistant mutant EGFR.

Conclusion: TUL-001 is a potent brain penetrant EGFR inhibitor, and showed excellent antitumor activity in preclinical tumor models. These results warrant TUL-001 going to the clinic study for exploration of its potential for treating EGFRm+ NSCLC cancer with and without brain metastasis.

#1317

Exceptional responses to crizotinib in breast cancer patients with somatic MET and ROS1 alterations.

Benjamin M. Parsons, David R. Meier, Sreeja Sreekumar, Craig S. Richmond, Kyle J. Ernzen, Kristopher A. Lofgren, Jake A. Deviley, Grzegorz T. Gurda, Paraic A. Kenny. _Gundersen Medical Foundation, La Crosse, WI_.

Crizotinib is FDA approved in lung cancers with alterations in MET, ROS1 or ALK. Alterations in these genes are also reported in breast cancer, but are very rare and not routinely evaluated. Here we describe two heavily pre-treated patients with metastatic breast cancer treated with off-label single-agent crizotinib following NGS panel detection of alterations in MET and ROS1 in their tumors. Both patients had strong responses to crizotinib. Patient I was a stage IV triple-negative breast cancer patient in her early forties, previously treated with doxorubicin/cyclophosphamide, carboplatin/paclitaxel and with capecitabine. PET/CT imaging after progression on capecitabine revealed extensive metastatic disease. A NGS assay detected a 30-fold amplification of MET. MET amplification, overexpression and hyperactivity was confirmed by FISH and by IHC for total MET and phospho-MET, respectively. Single agent crizotinib (250 mg, twice daily) was well-tolerated. Immunostaining of a biopsy taken after 9 days of treatment showed elimination of active phosphorylated MET, demonstrating in-tumor crizotinib efficacy at the clinical dose. CT imaging at 10 weeks showed a resolution of her metastatic disease, meeting RECIST 1.1 criteria for a complete response. This response was sustained at 22 weeks. At 37 weeks, the patient progressed in the pleura. An ultrasound guided biopsy revealed a robust resurgence of MET phosphorylation while still taking crizotinib, suggesting an alteration rendering MET recalcitrant to crizotinib. NGS revealed a MET D1228N mutation, previously reported in crizotinib resistant lung cancer. This mutation was not detected in the pre-crizotinib biopsy (locus sequenced at 6300x) suggesting de novo acquisition in response to crizotinib. Mechanistic studies in HEK393 cells indicated that this mutation is sufficient to confer crizotinib (Type I MET inhibitor) resistance but remains sensitive to cabozantinib (Type II MET inhibitor). Transition to cabozantinib (initially 60 mg/day, later 100 mg/day) resulted in clinically stable disease for a period of 7 weeks, at which point the patient again progressed. Patient II had ER+ PR+ HER2- breast cancer in 2014 and developed metastatic disease in 2017. She was subsequently treated with fulvestrant/palbociclib before an NGS panel revealed a GOPC-ROS1 fusion. She had a brisk response to crizotinib, documented by both imaging and reduction in circulating CA27-29. After experiencing crizotinib-related pneumonitis, she switched to ceritinib (an alternative ROS1 inhibitor) and continues to have an excellent response following six months of anti-ROS1 treatment. Together, these cases underline the value of NGS panel sequencing in patients with metastatic breast cancer. Although alterations in crizotinib-sensitive pathways are quite rare in breast cancer, very substantial clinical responses may be observed if these patients can be identified.

#1318

Preclinical characterization of quizartinib and AC886, a metabolite of quizartinib, in AML models, and anti-leukemic activity of quizartinib on midostaurin-resistant AML cells.

Tomoya Aikawa,1 Noriko Togashi,1 Koichi Iwanaga,1 Hiroyuki Okada,1 Yumi Nishiya,1 Shinichi Inoue,1 Mark J. Levis,2 Takeshi Isoyama1. 1 _Daiichi SANKYO CO., LTD., Tokyo, Japan;_ 2 _The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University, Baltimore, MD_.

FLT3 mutations have been found in about 30% of acute myeloid leukemia (AML) cases, and the most common form of FLT3 mutation is internal tandem duplication (ITD) in the juxtamembrane domain, which occurs in approximately 25% of adult AML patients and 10-15% of pediatric patients. FLT3-ITD is a driver mutation in AML and the FLT3-ITD presence is associated with poor prognosis in AML patients. Quizartinib is a second-generation small-molecule inhibitor of FLT3, and a phase 3, QuANTUM-R trial showed significantly prolonged overall survival in patients with FLT3-ITD-mutated relapsed/refractory AML. In this preclinical study, we characterized quizartinib and AC886, a metabolite of quizartinib, compared with other FLT3 inhibitors including midostaurin, gilteritinib, crenolanib and sorafenib, and then evaluated the anti-tumor effect of quizartinib on midostaurin-resistant AML cells. Selectivity profiling of the FLT3 inhibitors against over 400 kinases and over 80 non-kinases showed that quizartinib and AC886 were highly selective against FLT3. Quizartinib and AC886 clearly inhibited FLT3 signaling pathways such as STAT5, RAS/MAPK and PI3K/AKT cascades in FLT3-ITD-mutated AML cells, leading to potent growth inhibition of the AML cells with the IC50 values of <1 nM. When quizartinib was orally administered once a day to mice bearing MV4-11 cells harboring FLT3-ITD mutation, AC886 was rapidly detected and the tumor regression was observed at doses of 1 mg/kg and over without severe body weight loss, while midostaurin and gilteritinib also showed the tumor regression at 30 mg/kg and 10 mg/kg, respectively. In addition, we evaluated the effect of quizartinib on midostaurin-resistant FLT3-ITD-mutated AML MOLM-14 cells, which were established after long term treatment of midostaurin, and acquired KRAS (G12A) or NRAS (G12C) mutation. Quizartinib inhibited the viability of the midostaurin-resistant MOLM-14 cells with the IC50 values of <10 nM, and exerted potent anti-tumor activity in the xenograft models without severe body weight loss. On the other hand, midostaurin and gilteritinib did not show significant anti-tumor effect in the models. This is the first report on the detailed characterization of quizartinib and its metabolite AC886 in comparison with other FLT3 inhibitors. Preclinical anti-leukemic activity in midostaurin-resistant FLT3-ITD-mutated AML cells suggests the potential value of quizartinib for midostaurin failure in AML patients with FLT3-ITD mutations. Further clinical investigation is warranted to find the optimum treatment sequence with FLT3 inhibitors for the AML patients.

#1319

Repotrectinib, a new generation ROS1 inhibitor, is highly potent against fusion ROS1s and emerging resistance mutations.

Wei Deng, Dayong Zhai, Xin Zhang, Dong Lee, Evan Rogers, Jeffrey Whitten, J. Jean Cui. _TP Therapeutics, Inc., San Diego, CA_.

Chromosome rearrangements of ROS1 have been identified as oncogenic drivers in many malignancies, especially non-small cell lung cancer (NSCLC). Crizotinib is the only approved treatment for ROS1+ NSCLC. The efficacy of crizotinib varies among different types of ROS1 fusion partners in patients with ROS1-rearranged NSCLC, and the most dominant fusion CD74-ROS1 was associated with a higher rate of brain metastases and shorter overall survival. Unfortunately, the emergence of drug resistance to crizotinib and other ROS1-targeted therapies represents a major obstacle. The most common resistance mutations to crizotinib in ROS1+ NSCLC are the solvent front mutation (SFM) ROS1 G2032R and gatekeeper mutation ROS1 L2026M. Repotrectinib was rationally designed to overcome resistance mutations. The activity of repotrectinib against multiple fusion ROS1s and corresponding resistance mutations is outlined in the Table. Repotrectinib potently inhibited both wildtype and mutated fusion ROS1s including SFMs and gatekeeper mutations. In cell growth assays using engineered Ba/F3 cells expressing ROS1 fusions with several different partners, such as SDC4, CD74, TPM3 and EZR, repotrectinib demonstrated superior potency in comparison to other ROS1 inhibitors against multiple ROS1 mutations, especially the solvent front and gatekeeper mutations. In xenograft tumor model studies, repotrectinib resulted in tumor regression in the tumors carrying WT or SFM ROS1 fusion genes. Overall, repotrectinib demonstrated a strong inhibition profile against WT and various mutated ROS1s across different fusion partners when compared to many other ROS1 TKIs. A Phase 1/2 clinical trial of repotrectinib is currently enrolling ROS1+ NSCLC patients (NCT03093116). | Ba/F3 Cell Proliferation IC50 (nM)

---|---

|

No Kinase Domain Mutation | ROS1 G2032R | ROS1 L2026M

Inhibitor | CD74-ROS1 | SDC4-ROS1 | EZR-ROS1 | TPM3-ROS1 | CD74-ROS1 | SDC4-ROS1 | EZR-ROS1 | TPM3-ROS1 | EZR-ROS1 | TPM3-ROS1

Repotrectinib | <0.2 | 0.2 | <0.1 | <0.1 | 3.3 | 3 | 5 | 16.3 | <0.2 | <0.1

Crizotinib | 14.6 | 19.6 | 19.4 | 31.1 | 266.2 | 4661 | 660 | 500.6 | 95.6 | 236.2

Lorlatinib | 0.2 | 0.3 | 0.2 | 0.3 | 160.7 | 352.9 | 190.5 | 434.9 | 1.6 | 1.9

Entrectinib | 10.5 | ND | 1.5 | 9.4 | 1813 | ND | 2947 | 1093 | 13.3 | 40.7

Ceritinib | 42.8 | 59.8 | 33.1 | 105 | 1391 | 1883 | 885.8 | 543.7 | 12.6 | 66.5

Brigatinib | 21 | 38.7 | 25.8 | 61 | 1172 | 1473 | 360.6 | 3000 | 24.4 | 41.3

Cabozantinib | 0.5 | 3 | 0.4 | 4.5 | 11.3 | 169.4 | 39.5 | 60.7 | 3.4 | 12.6

Ensartinib | 39.5 | ND | 118.6 | 433.1 | 371.8 | ND | 1757 | 4814 | 543.3 | 1463

#1320

Preclinical evaluation of TQB3804, a potent EGFR C797S inhibitor.

Xile Liu,1 Xiquan Zhang,2 Ling Yang,2 Xin Tian,2 Tiantian Dong,2 Charles Z Ding,3 Lihong Hu,1 Lingyu Wu,3 Lele Zhao,3 Jun Mao,3 Qusheng Ji,3 Shaoyu Yan,3 Zhenzhen Zhu,3 Yuanfeng Xia,3 Chichung Chan,3 Shuhui Chen3. 1 _WuXi AppTec, Wu Han, China;_ 2 _Chia Tai Tianqing Pharmaceutical Group Co., Ltd., Nanjing, China;_ 3 _WuXi AppTec, Shang Hai, China_.

Objective: Osimertinib is the 3rd generation EGFR inhibitor, which has been approved for the treatment of NSCLC patients with EGFRT790M. More recently, a tertiary EGFRC797S mutation was reported as the dominant resistance (40~20%) mechanism to Osimertinib. The emergence of C797S mutation prevent covalent bond formation with Osimertinib, and caused the drug resistance. So, it's an urgent demand for new EGFR inhibitors that can effectively inhibit EGFR triple mutant, d746-750/T790M/C797S & L858R/T790M/C797S. Here, we disclose our clinical candidate, TQB3804, as a novel 4th generation inhibitor that potently inhibits triple mutants.

Methods: The enzyme activities of TQB3804 for EGFRd746-750/T790M/C797S, EGFRL858R/T790M/C797S, EGFRd746-750/T790M, EGFRL858R/T790M, and EGFRWT were measured with corresponding kinase assays. The anti-proliferative activity was evaluated in Ba/F3 (EGFRd746-750/T790M/C797S), NCI-H1975 (EGFRd746-750/T790M/C797S), PC9 (EGFRd746-750), and A431 (EGFRWT) cell lines, and the phosphorylation of EGFR was also evaluated in Ba/F3 (EGFRd746-750/T790M/C797S) cell line. Antitumor activity of TQB3804 was evaluated in three triple mutant cell-derived tumor xenograft (CDX) models Ba/F3 (EGFRd746-750/T790M/C797S), NCI-H1975 (EGFRd746-750/T790M/C797S), and PC9 (EGFRd746-750/T790M/C797S) and one Osimertinib resistant patient-derived xenograft (PDX) model of NSCLC (LUPF104, EGFRd746-750/T790M/C797S).

Results: TQB3804 displayed potent enzymatic activities for EGFRd746-750/T790M/C797S, EGFRL858R/T790M/C797S, EGFRd746-750/T790M, and EGFRL858R/T790M with IC50 of 0.46, 0.13, 0.26, and 0.19 nM respectively, and has similar enzymatic activity for EGFRWT (IC50 = 1.07) to Osimertinib. It also showed expected anti-proliferative activity in 4 cell lines, Ba/F3 (EGFRd746-750/T790M/C797S), NCI-H1975 (EGFRd746-750/T790M/C797S), PC9 (EGFRd746-750), and A431 (EGFRWT), with IC50 of 26.8, 163, 45, and 147 nM, respectively. The phosphorylation for EGFR in Ba/F3 (EGFRd746-750/T790M/C797S) cell line was potently inhibited with IC50 =18.5 nM. TQB3804 significantly inhibited tumor growth in the triple mutant Ba/F3 (EGFRd746-750/T790M/C797S), NCI-H1975 (EGFRd746-750/T790M/C797S), and PC9 (EGFRd746-750/T790M/C797S) CDX models, as well as in the LUPF104 PDX model. Western blot analysis of the tumor samples in the Ba/F3 (EGFRd746-750/T790M/C797S) CDX model showed that TQB3804 inhibited p-EGFR, p-AKT and p-ERK indicating that the tumor growth inhibition was through inhibition of the resistant triple mutant EGFR.

Conclusions: We have identified a potent orally active 4th generation EGFR inhibitor, TQB3804. It can inhibit the activity of Osimertinib resistant triple mutant EGFR, and showed strong antitumor activity in corresponding in vitro and in vivo preclinical assays. These results are considered highly promising and warrant moving the compound forward to clinical investigation.

#1321

TPX-0022, a polypharmacology inhibitor of MET/CSF1R/SRC for treatment of cancers with abnormal HGF/MET signaling.

Dayong Zhai, Evan Rogers, Wei Deng, Xin Zhang, Dong Lee, Jane Ung, Han Zhang, Jing Liu, Yuelie Lu, John Huang, Armin Graber, Zach Zimmerman, John Lim, Jeffrey Whitten, J. Jean Cui. _TP Therapeutics, Inc., San Diego, CA_.

Aberrant activation of the HGF/MET pathway has frequently been found in human cancers via MET mutation, gene amplification and translocation, as well as HGF paracrine or autocrine upregulation. The abnormal HGF/MET signaling not only acts as an oncogenic driver but also confers resistance to many cancer therapies, such as EGFR targeted therapy in NSCLC. One key downstream effector for activated MET is SRC, which is also involved in malignancy formation, tumor metastasis and drug resistance. In the tumor microenvironment, CSF1R plays an important role in regulation of tumor associated macrophages, which promote tumor progression and angiogenesis. Therefore, the polypharmacological inhibition of MET/SRC/CSF1R has great potential for more effectively targeting cancers with abnormal HGF/MET signaling via targeting both tumor intrinsic signaling and the tumor microenvironment. TPX-0022, a novel macrocyclic compound, has been designed and optimized to inhibit MET/CSF1R/SRC with enzymatic kinase inhibition IC50s of 0.14, 0.76 and 0.12 nM, respectively. TPX-0022 potently inhibited cell proliferation of the MET-amplified MKN-45 and SNU-5 gastric cancer cells, with IC50s <0.2 nM, which ranked TPX-0022 as one of the most potent MET inhibitors. TPX-0022 caused suppression of MET auto-phosphorylation at an IC50 of approximately 0.3 nM in MKN-45 cell line. TPX-0022 also potently inhibited the phosphorylation of MET downstream signaling effectors, including AKT, ERK, STAT3 and PLCγ2 in a dose-dependent manner. In the cancer cell line- and patient-derived xenograft tumor models from gastric, lung and liver cancers harboring MET amplification or MET exon14 skipping mutations, TPX-0022 caused dramatic tumor regression and tumor growth inhibition, without overt abnormality and body weight loss in treated mice. Furthermore, the tumor inhibitory effect was associated with drastic inhibition of MET activity. Overall, TPX-0022 is a novel and potent MET inhibitor and has demonstrated desirable drug-like properties, a good preclinical safety profiles, that warrants further clinical development and an IND submission is currently planned.

#1322

Maximizing the therapeutic potential of SHP2 inhibition with rational combination strategies in tumors driven by aberrant RAS-MAPK signaling.

Grace J. Lee, Carlos Stahlhut, James Evans, Denise F. Reyes, Edward G. Lorenzana, Shaoling Li, Elena S. Koltun, Dong Lee, Zhengping Wang, Robert J. Nichols, Jacqueline A. Smith, Mallika Singh. _Revolution Medicines, Redwood City, CA_.

SHP2 (PTPN11) is a protein tyrosine phosphatase and scaffold protein that functions as a convergent node downstream of multiple receptor tyrosine kinases (RTKs), integrating growth factor signals to promote RAS activation. Recently we reported that a SHP2 allosteric inhibitor suppresses RAS activation and proliferation in cancers driven by nucleotide cycling oncogenic KRAS, e.g., KRASG12C and other RAS-GTP dependent downstream mutations, e.g., NF1LOF, BRAFclass3. Daily oral administration of a SHP2 allosteric inhibitor at the maximally tolerated dose achieved tumor cytostasis in cell line-derived and patient-derived xenograft models harboring these mutations, and in some instances, tumor regressions. In most models, tumor cytostasis was followed by regrowth of tumors, reminiscent of patterns observed with MEK inhibitors. In the NCI-H358 KRASG12C model of non-small cell lung cancer, the maximum daily dose of the SHP2 inhibitor resulted in ~75% inhibition (relative to control) of pERK1/2, a downstream indicator of RAS-MAPK signaling in tumor lysates. These results support the hypothesis that targeting a single node results in incomplete RAS-MAPK pathway inhibition, which may permit tumor persistence in these models. To achieve deeper suppression of oncogenic flux through the RAS-MAPK pathway in tumors driven by nucleotide cycling KRAS, NF1LOF, or BRAFclass3, we evaluated combinations of MEK and ERK inhibitors with a SHP2 inhibitor as rational "in-pathway", anti-proliferative combinations. Additionally, we examined the combination with CDK4/6 inhibition as a strategy to impact RAS-MAPK signaling both directly and via cell cycle effects. We found that concurrent targeting of SHP2 and MEK or ERK drives greater anti-tumor efficacy than that observed with monotherapy with either agent. Comparable synergy was observed when SHP2 inhibition was combined with CDK4/6 inhibition. Given the complementary anti-tumor mechanisms triggered by MEK/ERK and CDK4/6 inhibition, we further interrogated a triple combination of SHP2, MEK, and CDK4/6 inhibition to drive complete elimination of RAS-MAPK oncogenic signals. We found that this combination regimen was tolerated, resulted in more significant responses in all tumor models we tested, and delayed the evolution of on-treatment resistance. Investigation of the downstream effects of the triple therapy on tumor cells revealed profound combinatorial effects on cell cycle progression, proliferation and induction of apoptosis. These observed synergies are likely due to a combination of RAS pathway extinction and the inhibition of parallel escape mechanisms. In summary, our findings reveal rational combination strategies to maximize the therapeutic potential of a potent SHP2 inhibitor in patients with tumors harboring nucleotide cycling oncogenic KRAS or other RAS-GTP-dependent downstream mutations.

#1323

CG-806, a pan-FLT3 / pan-BTK inhibitor, demonstrates superior potency against cells from IDH-1 mutant and other non-favorable risk groups of AML patients.

Hongying Zhang,1 Andrea Local,1 Jeffrey W. Tyner,2 Stephen E. Kurtz,2 Beth Wilmot,2 Shannon Mcweeney,2 Brian J. Druker,2 Stephen B. Howell,3 William G. Rice1. 1 _Aptose Biosciences, Inc, San Diego, CA;_ 2 _Knight Cancer Institute, Oregon Health & Science University, Portland, OR; _3 _UC San Diego Moores Cancer Center, San Diego, CA_.

CG-806 is a pan-FLT3 / pan-BTK inhibitor that is more potent (IC50 = 0.08 µM, n=265, p < 0.001) than other FLT3 inhibitors including midostaurin, sorafenib, sunitinib, dovitinib, quizartinib, crenolanib and gilteritinib against acute myeloid leukemia (AML) primary patient samples containing wild-type or mutated FLT3. CG-806 has significant activity against AML cell lines with or without FLT3 internal tandem duplication (ITD) / tyrosine kinase domain (TKD) mutations and in mouse AML xenograft models. Oral CG-806 has a desirable safety profile in the pre-IND studies of rodent and dog 28-day GLP toxicology, rodent respiratory and central nervous system safety, and the bacterial reverse mutation assay. The current study explored the relationship between genetic abnormalities in bone marrow and peripheral mononuclear cells isolated from AML patients and sensitivity to CG-806 using an ex vivo cytotoxicity assay. To correlate CG-806 sensitivity with clinical status, gene abnormalities and expression levels, whole exome sequencing (n=118) and RNA sequencing (n=111) were performed. CG-806 was equally potent against cells from patients in the adverse, intermediate and favorable risk groups (2017 ELN risk stratification), and cells from patients with relapsed or transformed AML (WHO classification) were as sensitive as those from patients with de novo AML. CG-806 had equivalent potency in cases of TP53 WT and TP53 mutations, whereas cases with TP53 mutations were resistant (FDR-corrected p<0.1) to most other FLT3 inhibitors including midostaurin, sorafenib, sunitinib, dovitinib, quizartinib and crenolanib. CG-806 had similar potency in cases with ASXL1 or SRSF2 mutations compared to WT, whereas sunitinib and crenolanib appeared resistant to ASXL1 and SRSF2 mutations, respectively. As expected, patient samples with FLT3-ITD mutation were more sensitive to CG-806 as compared to FLT3 WT (FDR-corrected p<0.01); in addition, cases with high ITD allelic ratio, including concurrent mutations with NPM1 and DNMT3A, had greater sensitivity than cases with low allelic ratio. Most unexpectedly, all 6 specimens containing IDH1 R132 mutations demonstrated significantly greater sensitivity to CG-806 relative to WT (FDR-corrected p<0.01), yet there was no increased sensitivity of IDH-2 mutant cells to CG-806. In conclusion, CG-806 demonstrated potency in primary AML patient samples across all AML subgroups including relapsed/refractory/transformed AML and those with genetic abnormalities related to poor prognosis. While patient samples with FLT3-ITD mutations were expected to have greater sensitivity to CG-806, the most surprising correlation was the sensitivity of patient samples with IDH1 R132 mutations. These features of CG-806 warrant investigation in the clinical setting.

#1324

NMS-P088, a novel FLT3, KIT and CSF1R inhibitor, is a promising clinical candidate for AML and CMML treatment.

Marina Ciomei,1 Elena Ardini,1 Laura Gianellini,1 GianMaria Borleri,2 Gemma Texido Romero,1 Roberta Ceruti,1 Wilma Pastori,1 Nilla Avanzi,1 Daniele Casero,1 Paola Gnocchi,1 Andrea Lombardi Borgia,1 Federico Lussana,2 Alessandro Rambaldi,2 Arturo Galvani,1 Antonella Isacchi1. 1 _Nerviano Medical Sciences, Nerviano, Italy;_ 2 _ASST Papa Giovanni XXIII, Bergamo, Italy_.

NMS-P088 is a potent and selective inhibitor of FLT3 and KIT kinases, including variants with both primary as well as secondary resistance mutations, in particular retaining potent activity against residue 691 gatekeeper mutation which still represents an unmet medical need. Different form most clinically advanced selective FLT3 inhibitors, NMS-P088 was also shown to strongly inhibit CSF1R in biochemical assays (Ki= 4.5 nM; Kd 0.88nM). FLT3, KIT and CSF1R are members of the class III receptor tyrosine kinase family. Activating rearrangements of the JM domain of FLT3 (FLT3 ITD) occur in 20-25% of Acute Myeloid Leukemia (AML) and represent a driver of disease and a negative prognostic factor. Another 5-7% of AML cases harbor an activating D835 mutation in the activation loop of the kinase domain. KIT is also found mutated in circa 8.0% of AML, primarily in core binding factor (CBF) AML. CSF1R has been shown to polarize macrophages towards an immunosuppressive and tumor-promoting phenotype. CSF1 and/or CSF1R genes are expressed in AML blasts and CSF1R mediates supportive interactions between AML and stromal cells in the AML microenvironment. CSF1R has also been found to be highly expressed in blast samples from chronic myelomonocytic leukemia (CMML), a clonal hematopoietic stem cell disorder with poor survival rates post- blast transformation. Hypomethylating agents or standard induction with chemotherapy are the most commonly used therapeutic intervention for CMML and no targeted therapy is currently approved. We found that in vitro NMS-P088 has potent activity on CSF1-dependent macrophages, inhibiting CSF1-stimulated proliferation and cell signalling. In mice NMS-P088 efficiently and dose-dependently decreased tissue infiltration of CSF1R expressing macrophages in liver, consistent with potent in vivo inhibition of this kinase. Furthermore, it showed single agent efficacy in a syngeneic mouse tumor model, with robust reduction of CSF-1R positive intratumoral macrophages. Importantly, we observed high expression of CSF1R in blast samples derived from CMML patients, and the compound was able to inhibit their proliferation and CSF1R signalling. It has been reported that treatment with CSF1R inhibitors induces upregulation of circulating CSF1 ligand as compensatory feedback modulation. Accordingly, during preclinical studies of NMS-088 conducted in non-human primates, a dose-related increase of circulating CSF1 levels was consistently observed. These data confirm in vivo CSF1R inhibition by NMS-P088 and support the opportunity to monitor CSF1 levels as a pharmacodynamic biomarker of CSF1R modulation in the clinical setting. Based on its original kinase targets profile, including FLT3 and KIT gatekeeper resistance mutations as well as CSF1R, preclinical efficacy and safety NMS-P088 was selected to initiate in a clinical trial to potentially address unmet medical needs in AML and CMML.

#1325

TPX-0022, a polypharmacology inhibitor of MET/CSF1R/SRC inhibits tumor growth by promoting anti-tumor immune responses.

Wei Deng, Dayong Zhai, Evan Rogers, Xin Zhang, Dong Lee, Jane Ung, Han Zhang, Jing Liu, Yuelie Lu, John Huang, Armin Graber, Zach Zimmerman, John Lim, Jeffrey Whitten, J. Jean Cui. _TP Therapeutics, Inc., San Diego, CA_.

In the tumor microenvironment, tumor associated macrophages (TAMs) support tumor growth by suppressing antitumor immune responses and promoting angiogenesis, which is associated with disease progression and poor clinical outcomes. In contrast to the classic phagocytic and cytotoxic pro-inflammatory M1 phenotype of macrophages engulfing and digesting pathogens, TAMs often adopt the anti-inflammatory and immune regulatory M2 phenotype in response to colony stimulating factor 1 (CSF1), which is produced by either tumor cells or stroma cells. Signaling through colony stimulating factor 1 receptor (CSF1R), a receptor tyrosine kinase expressed on the surface of mononuclear phagocytes, is involved in the recruitment of TAMs and has been associated with tumor progression and suppression of the immune response. Thus, CSF1R represents a key therapeutic target. TPX-0022, a type I kinase inhibitor with a novel macrocyclic structure, has been designed and optimized to inhibit MET/CSF1R/SRC with enzymatic kinase inhibition IC50s of 0.14, 0.76 and 0.12 nM, respectively. In a Ba/F3 ETV6-CSF1R cell model, TPX-0022 inhibited both autophosphorylation of CSF1R with an IC50 <3 nM and cell growth with an IC50 of 14 nM. In addition, TPX-0022 effectively inhibited the growth of Ba/F3 ETV6-CSF1R xenograft tumors in vivo. In the CSF1/CSF1R signaling-dependent M-NFS-60 model, TPX-0022 not only exhibited potency with an IC50 of 0.3 nM under baseline condition, but also potently inhibited the growth of M-NFS-60 cells with an IC50 of 11.6 nM in the presence of exogenous CSF1 at 1 ng/mL concentration, a condition mimicking typical in vivo conditions in the presence of advanced tumors. In contrast, in our study, the potency of the type II CSF1R inhibitor PLX-3397 demonstrated a strong dependency on the concentration of mouse CSF1, as the anti-proliferation IC50 shifted from <0.1 nM to 146.4 nM when CSF1 concentration changed from baseline to 1 ng/mL. Finally, in the MC38 syngeneic mouse model, TPX-0022 effectively reduced TAMs, altered the polarity of TAMs toward a more M1 phenotype, increased cytotoxic T cells and inhibited the growth of MC38 tumors. These preclinical results demonstrated a potent CSF1R inhibitory activity of TPX-0022 and the ability of TPX-0022 to inhibit tumor growth and promote a pro-inflammatory anti-tumor microenvironment.

#1326

WSD0922: A BBB penetrable EGFR/EGFRVIII inhibitor for the treatment of GBM and metastatic CNS tumor.

Wei Zhong, Jinqiang Zhang, Ahihua Mu, Claire Sun. _Wayshine Biopharm (Shanghai), Shanghai, China_.

Aberrant activity caused by EGFRm+ (EGFRvIII/Del19/L858R) is implicated in the brain tumors such as GBM and DIPG, and metastatic CNS tumors of NSCLC, and cancer of HNSCC. To date, approved EGFRi has limited efficacy against brain tumors due to insufficient BBB penetration and/or poor activity against EGFRvIII. Moreover, GBM is a whole-brain disease and needs a BBB penetrable therapy for killing tumor cells beyond intact BBB. We report that WSD0922, a novel selective EGFRi, displays potent activity, excellent CNS penetration, good safety profile and preclinical anti-tumor efficacy. The IC50 of WSD0922 against EGFRm+ <10nM and the ex-vivo GI50 in GBM PDX with EGFRvIII is ~20nM. WSD0922 is highly selective over other kinases. WSD0922 treatment resulted in dose dependent inhibition on EGFRvIII phosphorylation and AKT phosphorylation in GBM PDX model and the in vivo PD modulation correlated well with PK and efficacy. WSD0922 treatment significantly prolonged the survival of mice with intracranial tumor of EGFRm+ NSCLC. WSD0922 treatment resulted in tumor regression on cetuximab resistant PDX model of HNSCC. In screening platform of squamous lung cancer PDX models, WSD0922 demonstrated remarkable tumor regression in 3 PDX models (platinum sensitive/insensitive/EGFRm+). WSD0922 is well tolerated in GLP tox at dose ~10x clinical equivalent dose. In addition, WSD0922 is predicted to be CNS penetrable with decent PK in human. The phase 1 clinical trial in US will be initiated in early 2019 with a design of 3+3 basket trial. Taken together, our data provide a good rationale for WSD0922 to be developed toward clinic for the treatment of patients with GBM, HNSCC (EGFR and/or EGFRvIII) and CNS metastasis of NSCLC (EGFRm+).

#1327

UM-9107: A selective wild-type and T315I Bcr-Abl inhibitor with in vivo activity against chronic myelogenous leukemia.

Sameer Phadke, Lluis Lopez-Barcons, Taylor K. Johnson, Eric J. Lachacz, Sofia D. Merajver, Matthew B. Soellner. _University of Michigan, Ann Arbor, MI_.

Purpose: Long-term treatment of chronic myelogenous leukemia (CML) with the Bcr-Abl inhibitor imatinib, is initially successful but gives rise to several drug-resistant mutations. The most prevalent of these is the T315I 'gatekeeper' mutation that is sensitive to the second-line therapy, ponatinib. However, ponatinib causes serious vascular adverse events, including fatalities, in over 25% of patients, in part due to its target promiscuity. We have designed a novel Bcr-Abl inhibitor (UM-9107) that shows significant in vitro and in vivo anti-CML activity against wild-type and drug-resistant mutants, including the gatekeeper mutant. Furthermore, UM-9107 has excellent selectivity across the kinome and in vitro surrogate toxicity assessment models demonstrate a low likelihood of vascular adverse events as compared to ponatinib.

Experimental Design: We designed a series of compounds to specifically interact with a specific kinked conformation of the phosphate-binding loop of Abl. Since only a small proportion of kinases display this kinked conformation, we hypothesized that our compounds would be selective for Abl. We have analyzed the compound series biochemically and in vitro. The most potent compound, UM-9107, has been comprehensively characterized including: biochemical inhibition, cellular target engagement, kinome-wide profiling, in vitro cell proliferation, pharmacokinetic studies, and in vivo activity in xenograft models of CML.

Results: We demonstrate that UM-9107 potently inhibits all clinical mutants of Bcr-Abl, including the T315I gatekeeper mutant. UM-9107 shows robust activity against CML cell lines, comparable to ponatinib, with significantly fewer off-targets, as determined by kinome-wide profiling. Phenotypic profiling of UM-9107 in human primary cells shows low risk of vascular adverse events as indicated by specific biomarker readouts. UM-9107 demonstrates good oral bioavailability and in vivo activity in xenograft models of CML, with low toxicity.

Conclusions: The success of imatinib has been revolutionary and has led to an increase in the number of patients on long-term therapy. This inevitably gives rise to drug resistance through point mutations in the Bcr-Abl protein. Our results suggest that UM-9107 is a safe and efficacious preclinical candidate for the treatment of imatinib-resistant CML.

#1328

Exploring the pharmacokinetic-pharmacodynamic relationships of AZD3229, a novel and selective inhibitor of cKIT, in a range of mouse xenograft models of gastrointestinal stromal tumors.

Venkatesh Pilla Reddy,1 Aaron Smith,1 Michael Grondine,2 Rana Anjum,2 Rhys D. Jones1. 1 _DMPK, Oncology, IMED Biotech Unit, Astrazeneca, Cambridge, United Kingdom;_ 2 _Bioscience, Oncology, IMED Biotech Unit, Astrazeneca, Boston, MA_.

cKIT belongs a family of transmembrane tyrosine kinase growth factor receptors and gain of function mutations result in constitutive c-KIT activation that provide an important pathogenic role in gastrointestinal stromal tumors (GIST). AZD3229 is a highly potent and selective oral best-in-class KIT inhibitor that is active against a wide spectrum of primary and secondary KIT mutations that are known to confer resistance to standard of care agents. AZD3229 demonstrates rapid, extensive and sustained inhibition of KIT signaling in preclinical xenograft models with KIT primary exon 11 del mutations and primary/secondary ATP-binding pocket mutations (GIST 430/654A and patient-derived xenograft (PDX) model of KIT V654A, HGiXF-106) and A-loop mutations (PDX model of KIT Y823D, HGiXF-105 and Ba/F3 KIT D816H). In these models, AZD3229 results in anti-tumour activity (tumour regressions ranging from -60% to -99 % TGI) when dosed orally up to 20 mg/kg BID.

The objective of this PK-PD modeling work was to (1) establish the PK-PD-Efficacy relationship for AZD3229 across the four cell-lines and PDX mouse models tested; (2) to confirm the extent and duration of pKIT inhibition required for optimal efficacy, as defined by tumor regressions.

Inhibition of KIT phosphorylation observed with AZD3229 shows a direct relationship to free mouse plasma concentration and an estimated in vivo EC90 was estimated for each model (Ba/F3 D816H = 20 nM; GIST 430/V654A= 43 nM; HGiX-106 = 76 nM; HGiX-105 = 4 nM). No change in the extent of inhibition of KIT phosphorylation was observed on repeat dosing. Optimal efficacy is observed when sustained inhibition of pKIT, >90% is achieved over the dosing interval.

Conclusion: Exploring the in vivo activity and PK-PD relationship in multiple cell-line derived xenograft and PDX models harboring different mutations provides a benchmark from which to anchor human dose predictions that partly reflect the spectrum of responses likely to be seen in GIST patients.

#1329

Preclinical evaluation of TAS6417 as a highly effective, pan-mutation-selective EGFR tyrosine kinase inhibitor.

Hibiki Udagawa,1 Shinichi Hasako,2 Akihiro Ohashi,3 Naomi Abe,2 Tomonori Haruma,2 Toshiharu Komori,2 Miki Terasaka,2 Ryoto Fujita,2 Kaoru Funabashi,2 Hiroyuki Yasuda,4 Kazutaka Miyadera,2 Koichi Goto,1 Daniel B. Costa,5 Susumu S. Kobayashi5. 1 _National Cancer Center Hospital East, Kashiwa, Japan;_ 2 _Taiho Pharmaceutical, Japan;_ 3 _National Cancer Center, Kashiwa, Japan;_ 4 _Keio University, Japan;_ 5 _Beth Israel Deaconess Medical Ctr., Brookline, MA_.

Objectives: Three generations of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have developed to treat advanced non-small cell lung cancer (NSCLC) with EGFR mutations. However, each generation of TKI has different spectrum of activity against the different mutations. Currently, no EGFR TKIs are approved for NSCLC with EGFR insertion 20 mutation. Previously, we have shown that TAS6417, a novel EGFR TKI, is effective for cells harboring EGFR insertion mutations in vitro and in vivo. In this study, we asked whether TAS6417 is effective for tumors harboring other EGFR mutations observed in clinic.

Methods: Genetically engineered Ba/F3 cells expressing EGFR mutants, lung cancer cell lines including newly established cell lines established from tumors harboring EGFR insertion 20 were used to evaluate efficacy of erlotinib, afatinib, osimertinib, poziotinib, and TAS6417 by cell viability analyses. The selectivity indexes (the wild type EGFR/mutant EGFR ratio) were calculated to predict a therapeutic window for each TKI. Effects of each EGFR TKI on EGFR signaling and apoptosis were evaluated by western blotting. Finally, murine xenograft models were used to evaluate efficacy of TAS6417 and all generation of EGFR TKIs for the treatment of NSCLC with EGFR mutations.

Results: Cell viability assays demonstrate that TAS6417 was as potent as poziotinib against common EGFR mutations (L868R and exon 19 deletions) and the most potent against cells harboring the T790M resistant mutations. In addition, TAS6417 was effective in cells harboring EGFR rare mutations (G719X L861Q, and S768I). Even though IC50s were higher than those of poziotinib in cells harboring EGFR insertion 20, the selectivity indexes (the wild type EGFR/mutant EGFR ratio) were higher in TAS6417, indicating a wider therapeutic window. TAS6417 was effective in mice injected with cells harboring EGFR mutations with no body weight loss.

Conclusions: Our preclinical results confirm that TAS6417 is a potent EGFR TKI with a broader spectrum of activity and a wider therapeutic window and support clinical trials in the near future.

#1330

Osimertinib, an irreversible next generation EGFR tyrosine kinase inhibitor, exerts anti-tumor activity in various preclinical NSCLC models harboring G719X mutant-EGFR.

Nicolas Floch, Sue Bickerton, Matthew J. Martin, Darren A. Cross, Paul D. Smith. _AstraZeneca, Cambridge, United Kingdom_.

Two first-generation (erlotinib & gefitinib), two second generation (afatinib & dacomitinib) and a third generation (osimertinib) EGFR-TKIs are currently available for the management of EGFR mutation-positive NSCLC. All these TKIs are effective in patients with NSCLC whose tumors harbor the in-frame deletions in exon 19 and the L858R point mutation in exon 21. These two mutations represent 90% of all EGFR mutations. Osimertinib, when used in the front-line setting, has shown more favorable treatment outcomes than first-generation EGFR-TKIs. In approximately 50% of patients, resistance to first and second generation TKI is mediated by the acquisition of the 'gatekeeper' mutation T790M. Currently, osimertinib is the only registered EGFR TKI that is active against exon 19 deletions and L858R mutation, regardless of the presence of T790M mutation.

While the efficacy of EGFR TKIs for the common EGFR mutations is well established, much less is known about rare EGFR mutations such as exon 20 insertions, G719X, L861Q, S768I, as most of the data consist of single case reports or small case series. This work describes the therapeutic potential of osimertinib in tumors harboring the G719X mutation alone or in combination with L861Q and S768I.

Using available patient-derived xenografts (PDX) and cell lines derived from two of these PDX which habor the G719X mutation, we have evaluated in vitro and in vivo the pre-clinical activity of osimertinib.

We show that osimertinib inhibits signalling pathways and cellular growth of G719X cell lines in vitro. This translates into sustained tumor growth inhibition in vivo in 3 out of 4 PDX explored (87%, p<0.001, 158% p<0.001 & 181%, p<0.001 respectively at day 14) when compared to the control group. Importantly, in these in vivo models, osimertinib achieved exposure consistent with those observed in patients treated with an 80 mg clinical dose. Moreover, osimertinib demonstrates superior activity to afatinib (145% , p<0.001, & 58% p<0.01 respectively at day 14) in a model harboring the G719A. Out of the 4 PDX models explored in vivo, one model appeared to be inherently resistant to osimertinib. Further genomic characterisation revealed that the model displayed amplification of c-MET, a well know mechanism of resistance to EGFR-TKI. Subsequent in vivo pre-clinical study shows that the model is sensitive to c-MET inhibition. In addition, we performed pharmacodynamic studies to explore the relationship between efficacy and target/pathway modulation. These studies establish a clear relationship between depth and duration of inhibition of the phopshorylation of EGFR and anti-tumor efficacy.

The work presented herein demonstrates that osimertinib has the potential to improve upon the current treatment options for NSCLC patients whose tumors harbor a G719X mutation, and warrants further clinical investigation.

#1331

Receptor tyrosine kinases are differentially phosphorylated in metastatic castration resistant prostate cancer.

Raven A. Williams, Joakin Mori, Hui-Xian Lin, Alahni Becks, Clayton Yates, Honghe Wang. _Tuskegee University, Tuskegee, AL_.

Metastatic castration resistant prostate cancer (mCRPC) is the leading cause of death among prostate cancer (PCa) patients. Patients of African American (AA) origin are particularly disproportionately affected as they are less responsive to current therapies, mainly androgen deprivation therapy (ADT) that target androgen receptor signalling, which is less expressed in AA patients. Receptor tyrosine kinases (RTKs) have been implicated in cancer development and progression and inhibition of their activities has been effective in inhibiting cancer progression in other cancers. However, targeting RTK signalling has not yielded satisfactory results in PCas. The objective of this study was to identify precise RTKs that may be targetable in mCRPCs. An in-vitro model for primary vs mCRPC was developed from AA PCa cell lines: RC77T and RC43T. Briefly, invasive RC77T and RC43T cells were isolated in a cell invasion chamber. The invasive sublines were gradually exposed to increasing concentration (up to 10µM) of MDV3100 over a nine-month period to develop castration resistance. The resulting invasive and castration resistant sublines: RCMI77-CR3 and RCMI43-CR3 were subsequently cultured and maintained in K-SFM containing 10µM of MDV3100. Human Receptor Tyrosine Kinase Phosphorylation Antibody Array and Co-Immunoprecipitation protocols were used to identify differences in RTK activation. TCGA patient data was analysed using R to identify correlation between RTK mRNA expression and PCa progressiveness. MTT assay was used to evaluate effect of RTK inhibitors and MDV3100 treatment on cell survival. The RC77T and RC43T cells were epithelial in appearance, while RCMI77-CR3 and RCMI43-CR3 cells tended to be mesenchymal. The RCMI77-CR3 and RCMI43-CR3 sublines were more resistance to MDV3100 compared to their primary PCa counterparts. Human RTK Array identified four RTKs: KIT, LYN, ITK and RYK to be hyperphosphorylated in RCMI77-CR3 and RCMI43-CR3 relative to RC77T and RC43T, while three RTKs including EphA3, EphB1 and ErbB3 were hypo-phosphorylated. Immunoprecipitation and western blot analysis revealed differential expression of phosphotyrosine kinases between RC77T and RCMI77-CR3 cells. Analysis of TCGA patient data showed, mRNA expression level of only LYN and RYK differed significantly (p<0.05) significantly with disease progression. Analysis of effect of tyrosine kinase inhibitors on cell proliferation showed RCMI77-CR3 to be more responsive to dasatinib and axitinib compared to RC77T. No similar differences in response was observed for the inhibitors; SU6656, IWR-1, ICG-001 and BMS. The current study suggests, activation of specific RTKs are involved survival and drug resistance in mCRPC cells. Inhibiting these specific RTK signalling pathways in segments of patients with specific RTK hyperactivation could significantly improve survival. 

## CLINICAL RESEARCH

### Circulating and Cell-free Biomarkers for Diagnosis and Monitoring of Cancer 2

#1332

High Throughput isolation and expansion of circulating tumor cells (CTCs) from Non-small cell lung cancer (NSCLC) patients for personalized treatments.

Mina Zeinali,1 Maggie Lee,1 Arthi Nadhan,1 Anvya Mathur,1 Wei Huang,1 Eric Lin,1 Ramdane Harouaka,1 Max S. Wicha,1 Nallasivam Palanisamy,2 Mathias Hafner,3 Rishindra Reddy,1 Gregory P. Kalemkerian,1 Bryan J. Schneider,1 Khaled A. Hassan,1 Nithya Ramnath,1 Sunitha Nagrath1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _Henry Ford Health System, Detroit, MI;_ 3 _Institute for Medical Technology of Heidelberg University & University of Applied Sciences Mannheim, Mannheim, Germany_.

Background: Circulating tumor cells (CTCs) have emerged as important blood-based surrogate markers of primary tumors. Current methods for isolation of lung CTCs mostly rely on biomarker dependent antibody-based capture, missing populations that may be stem-like in nature. Results: We have applied the microfluidic Labyrinth device for high throughput, label-free, size-based isolation of CTCs from non-small cell lung cancer patients (NSCLC). The Labyrinth device was optimized and tested for inertial separation of cancer cells using the human lung cancer cell line H1650. The recovery and purity were >82% and >78%, respectively, operating at a flow rate of 2.5 mL/min. Using the biomarker-independent Labyrinth separation device, heterogeneous CTC populations were isolated from metastatic NSCLC patients (n=21). Heterogeneous CTC populations were detected, including CTCs (PanCK+ and CD45-), CTCs expressing EpCAM or Vimentin, and CTCs expressing both markers representing an EMT-like population of CTCs. Using Labyrinth, we were able to isolate CTCs from 100% of patients with an average yield of 180±168 CTCs/mL. Among the captured CTCs, EpCAM- CTCs were significantly more common than EpCAM+ CTCs (115.7 vs. 39.1 CTCs/mL respectively). Cell clusters of 2 or more CTCs were also observed in 95% of patients; 79% of these clusters were negative for EpCAM expression, whereas 35% expressed Vimentin, suggestive of an EMT phenotype. Recovered CTCs from patients with RET, ROS1 and ALK rearranged tumors showed aberrations matching with the primary tumor for each gene using FISH analysis. We have successfully expanded the recovered CTCs from 2 patients and screened for therapeutic targeting. We have found that TPX-0005 might be effective in these patients and would direct them to a clinical trial using this compound. Conclusion: The label-free Labyrinth device demonstrated the capability of collecting recovered CTCs from the device using a continuous processing technique while in a suspension state. This advantage opens the opportunities not only for CTC expansion off-chip, but also for ex-vivo drug testing to direct patient-specific therapies.

#1333

Isolation, characterization and expansion of circulating tumor cells in solid cancers.

Arutha Kulasinghe,1 Joanna Kapeleris,1 Liz Kenny,2 Majid Warkiani,3 Ian Vela,1 Jean-Paul Thiery,4 Ken O'Byrne,1 Chamindie Punyadeera5. 1 _Queensland University of Technology, Kelvin Grove, Australia;_ 2 _Royal Brisbane and Women's Hospital, Herston, Australia;_ 3 _University of Technology Sydney, Sydney, Australia;_ 4 _Comprehensive Cancer Center Institut Gustave Roussy, Villejuif, France;_ 5 _Queensland University of Technology, Kelvin Grove, Austria_.

Metastasis in cancer patients is reflected by measurable levels of circulating tumor cells (CTCs) in the blood of cancer patients. CTCs represent cancer cells from the primary and metastatic sites, thereby providing a comprehensive representation of the tumor burden of an individual patient. Recent advancements have shown that PD-1/PD-L1 immune checkpoint therapies have durable responses in a number of solid tumor types.

Our study was designed to use multiple CTC enrichment platforms for the capture of CTCs and novel culture formulations for the ex vivo expansion of CTCs. Head and Neck cancer (n=300) and lung cancer (n=80) patients were recruited to investigate the prognostic role of CTCs.

We evaluated multiple CTC isolation technologies (CellSearch, filtration, CD45 depletion, inertial microfluidics) using matched patient samples which showed that epitope-independent CTC isolation captured a greater proportion of CTCs. Molecular alterations present in the primary tissue were confirmed in the CTCs by 3D-DNA FISH (EGFR-amplification, ALK-translocations). In HNC, the presence of CTC clusters associated with the development of distant metastatic disease (P=0.0313). HNC CTC-positive patients had shorter progression free survival (PFS) (Hazard ratio [HR]: 4.946; 95% confidence internal [CI]:1.571-15.57; P=0.0063) and PD-L1-positive CTCs were found to be significantly associated with worse outcome ([HR]:5.159; 95% [CI]:1.011-26.33; P=0.0485).

In a proof of principle study, we were able to demonstrate for the first time, short-term patient derived CTC cultures outside the patient's body from 7/18 HNC samples (4/7 HPV-positive). Recently, we have preliminary data that suggests that PD-L1 is frequently expressed on CTCs in HNC and lung cancer and an immunoscore may be able to identify patients likely to benefit from immunotherapy-a current unmet clinical need. Expanding CTCs outside the patient's body allows for the recapitulation of the molecular diversity present within the tumor, understanding of the disease progression and testing of therapies.

#1334

Dual-isolation of CTCs and cancer exosomes from blood samples with melanoma using immunoaffinity based OncoBean microfluidic devices.

Yoon-Tae Kang, Ting-Wen Lo, Thomas Hadlock, Emma Purcell, Aaron Kramer, Monica De Reguera, Scott Alan McLean, Sunitha Nagrath. _University of Michigan, Ann Arbor, MI_.

Introduction: Melanoma is among the most aggressive cancers, and its incidence continues to grow. Due to the lack of promising markers to predict the disease and onset of metastasis, early detection and evaluation of treatment efficacy have been hampered. Recent advances in liquid biopsy have proposed alternatives for diagnosing disease with the merits of enabling continuous monitoring and non-invasiveness. CTCs and cancer exosomes are evolving as promising biomarkers due to their innate capability of having cancer-associated molecules and signatures. However, simultaneous isolation of CTCs and exosomes using the same methods from the identical samples has not been studied yet. Here, we propose the use of the OncoBean microfluidic device conjugated with melanoma specific antibodies, MCAM and MCSP for the simultaneous isolation. Using whole blood samples from patients, CTCs and exosomes are specifically isolated from the same samples and then undergo molecular profiling for comprehensive studies.

Methods: OncoBean devices were fabricated by soft lithography using polydimethylsiloxane (PDMS). The fabricated devices were conjugated with biotinylated MCSP and MCAM, using Neutralavidin-biotin chemistry. Using 6ml blood samples, 3ml of whole blood and 3ml of plasma were used for CTC isolation and melanoma-associated exosome isolation, respectively. In order to remove cellular debris, the plasma sample was filtered using a 200nm filter. After CTC isolation, captured cells were stained with fluorescent antibodies for melanoma specific (Melan-A and S100), leukocyte (CD45), and nucleus (DAPI) markers. The captured and stained CTCs were enumerated with consideration of size, nucleus to cytoplasm ratio and melanoma marker expression. The isolated exosomes were examined by scanning electron microscopy (SEM) to evaluate their abundance and size. The protein concentration and exosomal protein expression were evaluated by protein extraction using RIPA buffer.

Results: All melanoma patient samples showed 1-35 CTCs per 3ml of blood. Healthy donors had no CTCs. The abundance of isolated exosomes was evaluated by SEM and showed sizes ranging from 117-143nm. The average exosomal protein from melanoma was 28.6±19.2μg/ml, considerably higher than that of healthy donor, 11.0±3.1μg/ml. The exosomes isolated by OncoBean tested positively for the common exosomal protein, CD9, implying that the present device isolated melanoma exosomes specifically.

Discussion and conclusion: We showed that melanoma patients have both circulating tumor cells and cancerous exosomes in their blood samples. Even though their absolute CTC number and exosomal protein concentration showed no high correlation, simultaneous isolation using the identical microfluidic devices will be helpful for further verification of disease and cross validation according to their roles in disease.

#1335

Digital droplet PCR (ddPCR)-based detection of androgen receptor splice variant 7 (AR-v7) in non-metastatic castration resistant prostate cancer.

Mel Pilar Espaillat,1 Yashoda Rajpurohit,1 Mike Gormley,1 Denis Smirnov,1 Ian McCaffery,1 Angela Lopez-Gitlitz,2 Deborah Ricci,1 Shibu Thomas1. 1 _Janssen Research & Development, Spring House, PA; _2 _Janssen Research & Development, Los Angeles, CA_.

Background: The androgen receptor (AR) is a clinically important driver in prostate cancer. In metastatic castration resistant prostate cancer (mCRPC), increased expression of the ligand-independent AR variant 7 (AR-v7) is a biomarker of hormonal therapy resistance. However, the prevalence and clinical importance of AR-v7 in non-metastatic CRPC (nmCRPC) is not yet known. Low circulating tumor cell (CTC) frequencies in these patients make emergence of AR-v7 during anti-androgen therapy difficult to study. We report blood-based detection of AR-v7 using digital droplet PCR (ddPCR) in nmCRPC patients enrolled in the SPARTAN trial, a randomized phase 3 study testing ADT vs apalutamide (APA)+ADT.

Method: To investigate simultaneous and quantitative expression of AR-v7 and AR, we utilized ddPCR to measure individual mRNA transcripts in blood samples collected in PAXgene tubes. AR-v7 positivity was calculated as the normalized fraction of AR-v7 vs total AR transcripts (AR-v7/AR). Normalized AR-v7/AR frequency in healthy volunteers (HV) and mCRPC was measured to determine an expression cutoff to separate normal and prostate cancer blood samples. The ddPCR AR-v7 biomarker assay was then used to measure AR-v7 expression in ADT (N=47) and APA+ADT (N=53) SPARTAN samples taken at time of study initiation and correlated with clinical outcome.

Results: By setting a cutoff at 0.3 AR-v7/AR normalized fraction we could differentiate HV from mCRPC patients. In nmCRPC, mean AR-v7 and AR-FL expression were calculated as 1.2 and 349.3 transcripts, respectively. The 0.3 AR-v7/AR normalized fraction cutoff could not differentiate AR-v7 expression between HV and nmCRPC. Using this assay, we detected AR-v7 transcripts in 47% of nmCRPC SPARTAN patients analyzed. However, results of AR-v7 expression as a continuous and discretized variable were inconclusive when correlated with clinical outcome.

Conclusion: This study reports ddPCR-based detection of whole blood mRNA as a sensitive assay to detect simultaneously low and high expressing AR transcripts in nmCRPC. Our technical analysis demonstrates that unlike in mCRPC, low level transcript counts of AR-v7 in nmCRPC may not distinguish expression from baseline in healthy patients. Data from this limited cohort suggest that while AR-v7 is detected in 47% of patients, a higher threshold of expression may be biologically important for driving treatment resistance. Further analysis of this assay in mCRPC and APA refractory samples sequenced with other therapies are needed to confirm the clinical and biological utility of AR-v7 detection by ddPCR assay and inform disease continuum management.

#1336

An assay for the detection and isolation of single live circulating tumor cells using Accucyte/Rarecyte platform.

Mohamed Kamal, Shahin Saremi, Remi V. Klotz, Oihana Iriondo, Min Yu. _University of Southern California, Los Angeles, CA_.

Introduction: Circulating tumor cells (CTCs) are critical metastatic drivers for the interface between the primary tumor and the target secondary organs. CTCs are heterogeneous, therefore, investigating their phenotypic, molecular and biological properties on a single cell level warrant a better understanding to metastasis and patient's response to therapy. AccuCyte® - Rarecyte® system is a density based CTC isolation platform which allows for automated visual identification and retrieval of fixed rare cells from blood.

Aims: To establish an assay for the detection and isolation of live single CTCs valid for subsequent molecular and functional characterization.

Methods: Normal human whole blood samples were spiked with cells from breast cancer patient-derived CTC lines and were processed using the AccuCyte system. Buffy coats containing spiked CTCs were stained live with a cocktail of PE-594-conjugated antibodies for immune cell markers (CD14, CD16 and CD45) and either a live cell dye or a cocktail of Alexa Fluor 488-conjugated antibodies for cancer cell surface markers (EpCAM, Her2, EGFR and CDH11). Stained cells were then plated on chamber slides in the presence of serum free RPMI media and investigated using RareCyte fluorescence platform. CTCs were identified based on the lack of immune cell marker expression and the presence of the live cell dye (negative selection) or the expression of cancer cell surface markers and the lack of immune cell markers (positive selection). Single CTCs detected by both approaches were retrieved using our optimized interactive picking module. Cells detected by negative selection were tested for their ability to proliferate ex-vivo, whereas those isolated by positive selection were processed for single cell RNA sequencing analyses.

Results: Detection rates of our protocols reached up to 85% (12 out of 14 CTCs). Single CTCs isolated by negative selection showed a slower proliferation rate during the first 6 days of culture (1.1 Fold increase relative to Day 0) compared to control cells (2.5 Fold increase). However their proliferation rates were comparable to control cells at day 13 (2.7 Vs. 3.2) and day 21 (2.2 Vs. 2.5). Currently, other live cell dyes are being tested to improve the proliferation ability of CTCs isolated by negative selection. Moreover, single cells which were retrieved by positive selection are currently being validated for RNA sequencing analyses.

Conclusion: We have developed a novel assay which allows for the detection and isolation of single viable CTCs. Our assay has two modules; one uses a positive detection approach combined with a two-step cell picking protocol to provide ultrapure single CTCs valid for gene expression analyses. The other module provides an unbiased identification of CTCs using the negative selection approach and a single step of cell picking that provides viable single CTCs able to proliferate in culture.

#1337

Circulating angiogenic factors predict metastatic liver progression in rectal cancer patients given curative-intent oxaliplatin-containing neoadjuvant therapy.

Sebastian Meltzer,1 Kathrine Røe Redalen,2 Svein Dueland,3 Annette Torgunrud Kristensen,3 Arne Mide Solbakken,4 Kjersti Flatmark,4 Anne Hansen Ree5. 1 _Akershus University Hospital, Nordbyhagen, Norway;_ 2 _Norwegian University of Science and Technology, Trondheim, Norway;_ 3 _Oslo University Hospital, Oslo, Norway;_ 4 _Oslo University Hospital - The Radium Hospital, Oslo, Norway;_ 5 _University of Oslo, Oslo, Norway_.

Purpose: Systemic failure remains a challenge in rectal cancer. Our recent data from a prospective study of intensified treatment in locally advanced rectal cancer indicated that neoadjuvant chemotherapy (NACT) and sequential long-course chemoradiotherapy (CRT), both modalities containing oxaliplatin, may have eradicated hypoxic tumor components and promoted an immune response that favored survival without metastatic progression. Because a tumor microenvironment with aberrant vasculature and hypoxia provokes immune tolerance, which is frequently connected to systemic inflammation, we hypothesized that circulating angiogenic factors may predict development of metastatic disease in rectal cancer patients given curative-intent treatment. Recognizing that angiopoietin 2 (ANGPT2) disrupts vascular remodeling by inducing endothelial cell apoptosis, we analyzed the soluble ANGPT2 and its endothelial receptor TEK at diagnosis and their linkage to inflammatory factors and progression-free survival (PFS).

Experimental procedures: We used clinical data from 275 rectal cancer patients prospectively enrolled onto three studies. In two of the studies, patients were referred either directly to pelvic surgery or to standard CRT (without oxaliplatin) or short-course radiotherapy before surgery. In the third, introduced above, 80 patients received oxaliplatin-based therapy (two cycles of NACT followed by CRT) before resection. PFS was defined as time from study enrolment until the first metastatic event or up to five years after surgery in the case of no event. Serum ANGPT2 and TEK were measured by immunoassays. The measures (range of 1.60-8.16 ng/ml for ANGPT2 and 1.42-2.97 ng/ml for TEK) were normalized according to each intracohort mean value to enable analysis of pooled samples. Statistical methods were Pearson product correlation and Cox regression.

Results: A correlation was seen between ANGPT2 and TEK levels (r = 0.34, p < 0.001), and each factor correlated with C-reactive protein (for ANGPT2: r = 0.29, p < 0.01; for TEK: r = 0.19, p < 0.01). The higher ANGPT2 or TEK, the higher risk of developing liver metastasis (for ANGPT2: hazard ratio 1.01, p = 0.002; for TEK: hazard ratio 1.01, p = 0.039) in the oxaliplatin-exposed cohort. No such association was found for patients given treatment devoid of oxaliplatin.

Conclusions: High circulating ANGPT2 and TEK at diagnosis were associated with systemic inflammation and metastatic progression to the liver in rectal cancer patients receiving oxaliplatin-containing neoadjuvant therapy. Since we have previously shown that oxaliplatin may have breached the immune tolerance in patients with favorable outcome, there is a rationale for adding an anti-angiogenic agent in patients with hypoxic tumors as reflected by elevated circulating angiogenic factors.

#1338

The application of circulating tumor DNA analysis for detecting minimal residual disease and predicting recurrence in colorectal cancer patients.

Hiu Ting Chan, Satoshi Nagayama, Yoon Ming Chin, Rie Hayashi, Kazuma Kiyotani, Yusuke Nakamura, Siew-Kee Low. _Japanese Foundation of Cancer Research, Tokyo, Japan_.

Background: Although the prognosis of colorectal cancer (CRC) has improved in the past decade, a subset of CRC patients may still suffer from relapse due to the progression from minimal residual disease (MRD) after surgical resection. A sensitive and non-invasive method to efficiently detect MRD is critically essential to further improve the cure rate. In this study, we have evaluated the feasibility of circulating tumor DNA (ctDNA) analysis in detecting MRD in CRC patients. Methods: Plasma samples were prospectively collected from 45 CRC patients, ranged from stage I to IV, who underwent surgical resection. A preoperative blood sample was taken just before surgery and post-operative samples were collected on multiple time-points to monitor the changes of mutation profiles. Cell-free DNA/RNA (cfDNA/RNA) were extracted for library construction using Oncomine Pan-Cancer Cell-Free Assay and were subjected to next-generation sequencing. Results: Sufficient amount of cfDNA/RNA was extracted from 39 of the 45 pre-operatively samples for sequencing. The median input was 19 ng (ranged from 8-20 ng). The median of molecular coverage and barcode tagging efficiency of the sequencing were 4,344x and 83%, respectively. Twenty-seven (69%) of 39 pre-operative patients were found to have at least 2 copies of somatic mutations. Seven samples (ranged from stage I to IV) showed an increase in mutant allele frequency (MAF) postoperatively. The median increase in MAF ranged from 1.2-fold to 7.9-fold with a median of 2.0-fold. One of the 7 patients showed an increase in MAF relapsed 6 months after the primary tumor resection. This stage IIIb patient carried BRAF-V600E with a MAF of 0.067% pre-operatively, showed an 8-fold increase (0.530%) in MAF at the time of diagnosis of recurrence, and showed further increase in MAF to 0.685% at the time of surgical removal of the metastatic lesions. The MAF of BRAF-V600E was reduced to 0.1759% one month after the surgery. The dynamic changes in the MAF detected in ctDNA appears to reflect the recurrence status which might not be detected by imaging tests or currently available biomarkers such as CEA (< 5ng/mL before and after tumor resection in this case). Conclusions: Our current results indicate that ctDNA analysis before and after resection allows the detection of MRD in CRC patients. The integration of ctDNA analysis with current standard monitoring guidelines holds great promise in the early detection of recurrence to allow clinical intervention to be applied promptly.

#1339

High dimension single cell analysis reveals complex immunophenotypes altered in metastatic melanoma patients that are associated with immunotherapy outcomes.

David M. Woods, Andressa S. Laino, Aidan Winters, Jason Alexandre, Jeffrey Weber, Pratip Chattopadhyay. _NYU Health, New York, NY_.

For patients with metastatic melanoma immunotherapies have revolutionized treatment, but many patients fail to respond, and mechanisms of resistance remain unclear. To address this, we evaluated peripheral blood samples from 12 healthy donors (HD) and 41 metastatic melanoma patients treated with sequential nivolumab and ipilimumab by high dimension flow cytometry. Samples were assessed for 68 unique markers across four panels. Data were analyzed by gating on defined lineages (e.g. CD3+CD4+) and evaluation of all possible marker combinations for up to 15 markers, totaling over two billion unique marker combinations. Previously, analysis of datasets of this magnitude were limited due to the computational power required to assess such a large number of combinations in 53 samples each containing over one million cell events (~1x10^18 data points). In lieu of utilizing dimension reduction techniques (e.g. tSNE) which result in a loss of higher order data, we used a novel computational approach named CytoBrute to evaluate the data through semi-comprehensive Boolean gating combinations. In a comparison of HD vs patients a total of 2,895 signatures were identified with a p<0.05 after correcting for multiple comparisons. Significant signatures included both simple (e.g. CD4+CD95+KRLG1+) and complex immunophenotypes (e.g. CD4+CD127-CD45RO-PD1+CD95+CD134-CD25-TBET-HLADR-CCR7+Ki67-). Assessment of shared markers expressed in the significant signatures revealed an overrepresentation of several markers (e.g. CD127, CD39, CD45RO, GARP, GITR and CCR7). We also evaluated significant signatures for differences in patient outcomes. We found that 270 of the signatures that significantly differed between HD and patient samples also significantly differentiated responding and non-responding patients (e.g. CD4+CD95+KLRG1+), and 360 signatures were associated with differences in overall survival. Further, in 205 signatures differentiating patient responses, responding patients more closely resembled HD samples (p<1e-8). Finally, we employed Random Forest machine learning to categorize samples. Using Leave-One-Out cross validation, the model was able to correctly categorize 92% of samples (AUROC = 0.96), demonstrating the ability of high-dimensional data to accurately differentiate melanoma patients' peripheral blood phenotypes from that of healthy individuals, and highlighting the potential for its further development as a diagnostic tool. Collectively, we have demonstrated a novel and powerful approach to interrogating complex immunophenotypes and revealed novel peripheral blood immune signatures associated with metastatic melanoma, patient survival and response to immunotherapy.

#1340

High NK cell number predicts poor overall survival in de novo treatment naive acute myeloid leukemia.

Weiwei Wang, Guang Fan, Andy Kaempf, evan Lind, Tomi Mori, Byung Park, Jennifer Saultz. _Oregon Health Science University, Portland, OR_.

Introduction Acute myeloid leukemia (AML) is a deadly disease associated with high mortality and morbidity. Previous studies report defects in NK cell maturation and function in AML patients leading to innate immune evasion. Considering that the number of NK cells may be an important indicator of innate immune regulation of AML, our group sought to understand the prognostic significance of NK cell number in de novo AML patients.

Methods Patient samples were obtained from the Oregon Health and Science University (OHSU) Pathology Department between September 2010 and March 2016. This cohort study includes 82 newly diagnosed adult AML patients who had a bone marrow biopsy that was subjected to flow cytometry and a targeted-NGS panel within 30 days prior to induction treatment. Patients were excluded if they had antecedent hematological diseases or therapy-related AML. Bone marrow immunophenotyping was performed using the Ion Torrent PGM platform. Absolute numbers of NK cells were estimated from flow cytometric analysis of the bone marrow samples whereby lymphocytes were identified based on CD45 staining and light side scatter and NK cells were defined as CD3-/CD56+.

Results This retrospective cohort of 82 newly diagnosed AML patients were 53.7% male with a median age of 61 years (range 18 - 83). Using 2017 ELN risk stratification guidelines, 36.6% of patients were classified as favorable, 26.8% as intermediate, and 36.6% as adverse. Absolute NK cell numbers in the bone marrow ranged from 1 to 896 cells/μl with a median of 98. When stratifying NK cells by staining intensity to identify subpopulations, the number of CD56 bright NK cells ranged from 0 to 69 cells/μl (median 3) and the number of CD56 dim NK cells ranged from 1 to 585 cells/μl (median 77). Overall survival (OS) was measured from diagnosis to date of death or last contact and varied from 1 day to 5.7 years (median 20.5 months). There were 3 deaths in the first week after diagnosis. Separate Cox proportional hazards regression models were applied to each NK cell group in both the univariable and multivariable settings, with the latter models accounting for patient age and ELN risk. Higher absolute numbers of NK cells (CD3-/CD56+) were significantly correlated with worse OS in both model settings, with a multivariable p-value of 0.001. The number of bright NK cells was a significant predictor of OS in the univariable model but lost significance when controlling for other variables. However, similar to the number of all NK cells and contrary to previous reports, higher counts of dim NK cells were significantly associated with shorter survival times, with a multivariable p-value of 0.001.

Conclusions In this study, we show that higher NK cell numbers at diagnosis are associated with worse OS and thus play an important role in prognostication for AML patients. This association with survival is most evident in patients at least 60 years old or those with intermediate or adverse ELN risk.

#1341

Magnetic isolation and identification of exosomes using Fe/Au nanowires.

Zohreh Nemati, Thomas Gage, Mohammad Reza Zamani Kouhpanji, Joseph Um, Alicia Donnelly, Daniel Shore, Kelly Makielski, Yali Zhang, Rhonda Franklin, Jaime F. Modiano, Bethanie J. Stadler. _University of Minnesota, Minneapolis, MN_.

We are developing segmented magnetic nanowires (MNWs) as a new platform for highly specific biolabels. We found that cancer cells internalize MNWs and package them into exosomes, which are then secreted for several days. These MNWs thus enable magnetic isolation of exosomes, which could prove useful in future diagnosis. Our immediate goals are to study how cells internalize MNWs, to determine the timing and reproducibility of exosome secretion, and to improve the magnetic isolation of exosomes. Following this work, we aim to use ferromagnetic resonance (FMR) to identify specific MNW biolabels, similar to radio frequency identification (RFID).

In this study, we incubated osteosarcoma (OSCA-8) cells with Fe/Au segmented MNWs with and without PEG coating for 48 hours. Internalization of MNWs as a function of concentration (5-40 µg/ml) was followed by fluorescence and transmission electron microscopy (TEM). We obtained quantitative estimates of MNW internalization by magnetic measurements. Our experiments indicated better internalization of the MNWs using PEG as a capping agent. As we increased the concentration of MNWs, both the number of cells with MNWs and the number of MNWs per cell increased. TEM images show that after uptake by cancer cells, MNWs were predominantly located within lysosomes, and they appeared to be fragmented into small segments of similar size as exosomes. These segments were mostly composed of either Fe or Au, suggesting that fragmentation occurred at or near the boundary of segments.

We used two different methods for exosome isolation: non-magnetic isolation (centrifuge and ExoQuick TC) as a control and magnetic isolation. We incubated OSCA-8 cells with different concentrations of MNWs (0 to 35 µg/ml), and exosomes were isolated for up to 11 days. We analyzed the exosome size distribution using a nanoparticle tracking analyzer (NanoSight) and found that exosomes isolated magnetically had similar size distributions as those isolated non-magnetically. A minimum concentration of 25 µg/ml MNWs in 3×105 cells was required for appreciable magnetic isolation. Increasing the concentration of MNWs progressively; however, isolated microvesicles that had wider and more heterogeneous size distributions.

Next, we successfully identified two different types of MNW biolabels by FMR. MNWs were fixed in a polymer that was placed onto an RFID chip. Each type of MNW exhibited a unique RF signature as the sample was exposed to an external magnetic field.

Our preliminary data show that MNWs appear to allow fast, inexpensive magnetic exosome isolation and is our future goal is MNW identification of exosomes. The methodology developed in this study should be transferable to develop comparable approaches to isolate and identify exosomes from virtually any type of cell.

#1342

Polymorphisms in genes involved in mitophagy pathway predict clinical outcome in patients (pts) with metastatic colorectal cancer (mCRC): Data from TRIBE and FIRE3 phase III trials.

Stephanie Zhang,1 Joshua Millstein,2 Shu Cao,2 Francesca Battaglin,2 Ryuma Tokunaga,2 Shivani Soni,2 Jae Ho Lo,2 Tricia Ning,2 Vittorina Zagonel,3 Chiara Cremolini,4 Sebastian Stintzing,5 Fotios Loupakis,3 Alfredo Falcone,4 Volker Heinemann,5 Heinz-Josef Lenz2. 1 _Portola High School, Irvine, CA;_ 2 _USC/Norris Comprehensive Cancer Center, Los Angeles, CA;_ 3 _Veneto Institute of Oncology IOV – IRCCS, Padua, Italy;_ 4 _Unit of Medical Oncology 2, Azienda Ospedaliera Universitaria Pisana, Istituto Toscano Tumori, Pisa, Italy;_ 5 _Ludwig Maximilian University of Munich, Munich, Germany_.

Background: Mitophagy is the selective degradation of mitochondria by autophagy. Recent studies are suggesting that dysregulation of mitophagy pathway contributes to neoplastic progression and drug resistance in various types of tumors, includes colorectal cancer. We hypothesized genetic variants in mitophagy pathway genes may predict clinical outcome in mCRC pts treated with bevacizumab-based chemotherapy.

Methods: Genomic DNA extracted from blood samples of pts enrolled in two independent randomized phase III trials, TRIBE and FIRE3. 16 SNPs from 4 mitopahgy pathway genes (PINK1, BNIP3, NIX and PARK2), which were selected based on frequency and function, were genotyped by PCR-based direct DNA sequencing. The associations between SNPs and clinical outcomes were analyzed using chi-square test for tumor response, and the Kaplan-Meier curves and Cox regression for PFS and OS.

Results: A total of 451 pts were included in the analysis. TRIBE FOLFIRI/bevacizumab (bev) arm served as discovery cohort (n = 215, median PFS/OS: 9.7/26.2 mo), FIRE3 FOLFIRI/bev arm as validation cohort (n = 107, mPFS/OS: 11.5/31.4 mo). In the discovery cohort, pts carrying the BNIP3 rs4450 C/C variant showed an increased tumor response rate compared to pts with any T allele (65.3% vs 50.5%, p = 0.032); this finding was consistent in overall pts in FIRE3 cohort, pts with BNIP3 rs4550 C/C genotype had increased tumor response rate compared to pts with any T allele (75% vs 53.8%, p = 0.024). Additionally, in the discovery cohort, pts with BNIP3 rs11556626 any C allele showed a longer PFS (12.7 vs 9.5 mo) and OS (49.1 vs 25.6 mo) compared to pts with A/A variant in both univariate (PFS: HR = 0.52; 95%CI 0.34-0.81; p = 0.002) (OS: HR = 0.50; 95%CI 0.31-0.81; p = 0.004) and multivariable analysis (PFS:HR =0.46; 95%CI 0.28-0.76; p=0.002) (OS:HR =0.45; 95%CI 0.27-0.76; p=0.003).

Conclusions: Our results demonstrated for the first time that mitophagy pathway gene BNIP3 polymorphism may have a predictive value in mCRC pts treated with bevacizumab based chemotherapy. This finding supports a possible role of mitophagy BNIP3 genes in contributing to resistance to antiVEGF treatment.

#1343

**SNPs in the NFKB alternative pathway genes** CD40, BAFFR **and** LTβR **in CRC.**

Anna Antonacopoulou, Anastasia Kottorou, Angelos Koutras, Foteinos-Ioannis Dimitrakopoulos, Thomas Makatsoris, Haralabos P. Kalofonos. _University of Patras, Patras, Greece_.

We sought to assess the role of single nucleotide polymorphisms (SNPs) in genes of the alternative NF-KB pathway, important in regulating different aspects of immune functions in colorectal cancer (CRC). The study comprised DNA samples from 418 patients with CRC and 299 age-matched healthy individuals. Genotyping was performed with real time PCR followed by high resolution melt curve analysis for the following SNPs: CD40 rs1883832 (C/T), BAFFR rs7290134 (A/G), LTβR rs10849448 (A/G). Genotypes of random samples were confirmed by sequencing. Genotype frequencies differed between CRC and healthy individuals for CD40 rs1883832 and LTβR rs10849448 (p = 0.001 and p = 0.016, respectively) but were similar for BAFFR rs7290134. The LTβR rs10849448 G allele was more frequent in CRC patients compared to healthy controls and increased risk for CRC by 61% (p = 0.024). Moreover, the G allele was found more frequently in stage D patients compared to stages A and C while the A allele was more prevalent in stage B patients (p = 0.007). Additionally, most patients with grade III tumors were G allele carriers whereas the majority of grade I tumors were AA homozygotes (p = 0.019). The CD40 rs1883832 T allele was less frequent in CRC patients and reduced risk for CRC by 71% (p = 0.027). The CD40 rs1883832 SNP proved to be a prognostic factor for PFS with the CC individuals displaying a longer PFS compared to T carriers (p = 0.045). The BAFFR rs7290134 SNP was independent of all clinical parameters evaluated. Primary site was independent of genotypes for all three SNPs. SNPs within genes of receptors of the NF-KB alternative pathway, CD40 rs1883832 and LTβR rs10849448, influence PFS and risk for CRC development. Additionally, particular genotypes correlated with stage and grade.

#1344

IL-6 blood level represents an independent biomarker of melanoma patient prognosis.

Yuling Wang, Dawen Sui, Kejing Xu, Lauren E. Haydu, Shenying Fang, Merrick I. Ross, Jeffrey E. Gershenwald, Jeffrey E. Lee. _UT MD Anderson Cancer Center, Houston, TX_.

IL-6 is an inflammatory cytokine produced by both immune and tumor cells, including melanoma cells. IL-6 has been shown to stimulate melanoma growth and invasiveness, and blood levels of IL-6 have been proposed as a melanoma prognostic marker. We previously provided validated evidence that CRP, downstream of IL-6 in the inflammatory cascade, is an independent blood biomarker of melanoma patient prognosis. In the current study, we performed coordinated evaluation of blood levels of both CRP and IL-6 in a large cohort of melanoma patients evaluated and treated at a comprehensive cancer center for whom consistent evaluation, staging, treatment and follow-up were performed. 473 patients underwent blood draw following melanoma diagnosis. There were 177 female and 296 male patients. Median age at blood draw was 58. Median primary tumor thickness was 1.39 mm and 29% of tumors were ulcerated. At blood draw, 187 patients were stage I/II and 286 were stage III/IV. Median follow-up from blood draw was 25 months. There were 60 deaths during follow-up, including 37 melanoma-related deaths. Plasma IL-6 and CRP levels were determined using Elisa kits (R&D Systems). Outcome measures (overall survival, OS; melanoma-specific survival, MSS) were determined from the date of blood draw. On univariate analysis, an elevated level of IL-6 was associated with elevated age at blood draw (Spearman correlation coefficient (CC) 0.2190, P<0.0001), increased tumor thickness (CC 0.2450, P<0.0001), advanced stage at blood draw (ANOVA P=0.01), decreased OS from blood draw (Cox P=0.0001), and decreased MSS from blood draw (Cox P<0.0001). IL-6 levels were highly correlated with CRP levels (CC 0.680, P<0.0001), and on univariate analysis CRP demonstrated similar patterns to IL-6 with regards to age, stage and outcome measures. In a multivariate model that included age, sex, stage, IL-6 and CRP, elevated IL-6 was an independent predictor of shorter OS (HR 1.72, 95% CI 1.39-2.13; Cox P<0.0001), while CRP was not. Additionally, in a multivariate model of MSS, elevated IL-6 was an independent predictor of shorter OS (HR 2.24, 95% CI 1.73-2.89; Cox P<0.0001), while CRP was not. In summary, in a large cohort of melanoma patients, blood level of IL-6 was a strong and consistent predictor of important outcome measures, including OS and MSS. Furthermore, predictions of patient outcomes remained robust following adjustment for important covariates, including the clinicopathologic predictors of age, sex, and stage, as well as the downstream inflammatory biomarker CRP. These results suggest that IL-6 is a clinically relevant inflammatory biomarker in melanoma patients, and that the previously identified association of CRP with melanoma patient outcome is likely downstream and secondary to the effect of IL-6. Prospective investigations are ongoing, including of the potential predictive role of IL-6 in melanoma patients treated with targeted and immune-based therapies.

#1345

Automated capture and analysis of circulating tumor cells across different types of tumors in pediatric cancer patients.

Izhar S. Batth,1 Wafik Zaky,1 Soumen Khatua,1 Najat C. Daw,1 Kris M. Mahadeo,1 Sajad Khazal,1 Aisha Albert,1 Wilber Huang,2 Diva Chen,2 Jonathan B. Gill,1 Eugenie Kleinerman,1 Richard Gorlick,1 Shulin Li1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Abnova Corporation, Taipei City, Taiwan_.

The clinical utility of blood analysis such as circulating tumor cells (CTCs) has garnered increasing interest in recent years. However very few studies were conducted in pediatric cancer patients especially in brain tumor, osteosarcoma, and leukemia patients due to the lack of an effective tool to isolate/capture CTC from these patients. For example, only three independent studies were published to investigate CTC capture from adult brain tumor patients but zero studies were conducted with pediatric brain cancer patients. Our previous studies showed the high specificity of using cell surface vimentin (CSV) to identify and capture CTCs from various types of adult tumor patients using a manual isolation method. Here we present the first automated system for capturing CTCs across different types of pediatric tumors. This automated system utilizes the Abnova CytoQuest system loaded with the CSV antibody 84-1-coated microfluidic cassette for capturing CTCs from peripheral blood samples. This automated assay was very sensitive because as few as one tumor cell can be captured out from one million normal cells. Based on our preliminary study, our success rate for non-DIPG brain tumor, ALL, and AML patients is 100%. This success provides a potential opportunity for analyzing brain tumor genetic properties using blood samples instead of the invasive tumor biopsy; likewise, this high sensitivity assay opens up clinical opportunities to use this assay for transplant and treatment decision making.

#1346

Panel-based tumor mutational burden (TMB) analysis of matched tumor and plasma specimens using Illumina's TruSight Oncology 500 next-generation sequencing assay.

Stephanie B. Hastings,1 Gunjan Hariani,1 Victor J. Weigman,1 Tingting Jiang,2 Chen Zhao,2 Rajiv Raja,3 Traci Pawlowski,2 Philip Brohawn,3 Patrick Hurban1. 1 _Q Squared Solutions, Morrisville, NC;_ 2 _Illumina, San Diego, CA;_ 3 _MedImmune, Gaithersburg, MD_.

Checkpoint inhibitor (CPI) therapy demonstrates a remarkable clinical benefit in many cancer types. However, the ability to successfully select patients who will benefit from CPIs is still limited. Tumor mutational burden (TMB), a measure of the number of somatic mutations per coding area of tumor genome, is a putative biomarker of response showing great promise in CPI and immunotherapy combination trials. The ability to measure TMB from tumor biopsies or plasma samples will be important for clinical adoption of this biomarker. Herein we report on initial performance evaluation of Illumina's TruSight™ Oncology 500 gene (TSO500) NGS assay for the analysis of TMB in FFPE tissue and plasma cell-free (cf)DNA specimens. Illumina's TSO500 assay employs hybrid-capture based approach for target enrichment coupled with unique molecular indices to enable low frequency variant detection of single nucleotide variants and indels. This comprehensive cancer panel interrogates relevant cancer biomarkers in >500 cancer genes (~2 Mb) from as little as 40 ng of FFPE DNA or 30 ng of cfDNA. In addition to variant calls, Illumina's analysis pipeline reports a TMB score and microsatellite instability (MSI) status. Results obtained with the TSO500 TMB assay were compared to our validated whole exome sequencing (WES) TMB assessment for FFPE tissue specimens, with and without matched normal. As previously reported, the WES TMB tumor-only pipeline uses somatic variant classifications determined using a random forest model to generate TMB scores from analysis of tumor FFPE specimens. TSO500 TMB and MSI statues were evaluated in a set of 50 tumor FFPE specimens obtained from late-stage (III+) colorectal and bladder cancer patients. TSO500 TMB results correlated well with the WES-derived TMB results, although some differences were observed in the magnitude of high TMB scores. TSO500 TMB levels correlated strongly with the MSI status, with 100% of MSI-high tumors demonstrating high TMB scores (> 20), and greater than 85% of MSI-low tumors characterized as TMB-low (<20%). Additionally, correlations between tissue and blood-based TMB assessments in matched FFPE and plasma specimens are reported. Overall, our results demonstrate a strong correlation between TSO500 panel-based and whole exome sequencing-based TMB results in tissue and report comparisons to TMB calling in liquid biopsies. Our data further confirm that tumor mutational load is strongly connected to DNA damage repair deficiency in colorectal and bladder cancer patients.

#1347

Peripheral blood TCRB chain convergence in chronic viral infection and cancer: Emerging trends from a novel immune repertoire biomarker.

Timothy J. Looney. _Thermo Fisher Scientific, Austin, TX_.

Background T cell receptor (TCR) convergence refers to the phenomenon whereby antigen-driven selection enriches for TCRs having shared antigen specificity but different nucleotide sequences. Previous work has demonstrated the potential utility of TCR convergence as a predictive biomarker for response to checkpoint blockade and dendritic cell based immunotherapy for cancer. The extent to which convergence arises owing to chronic viral infection is not yet established. Here we sought to identify features of chronic cytomegalovirus (CMV) infection using TCRB profiling of peripheral blood (PBL) total RNA.

Methods Total RNA from PBL was obtained from 35 blood donors of known CMV status, then used for TCRB sequencing via the Oncomine TCRB-LR assay (amplicon spanning CDR 1, 2 and 3) and the Ion Torrent S5. In parallel, we prepared libraries via the Oncomine TCRB-SR assay (CDR3 only). Data were used to identify TCRB repertoire features correlated with CMV status and compare repertoire features across the two assays. For context, we compare CMV-related convergence to previous reports detailing convergent T cell responses in individuals with cancer.

Results T cell clone evenness was reduced in CMV positive individuals irrespective of age, predictive of CMV status (AUC=.86, p=2E-4, Wilcoxon), and strongly correlated between LR and SR assays (Spearman cor=.96). TCR convergence was elevated in CMV positive individuals and uncorrelated with evenness (Spearman cor = -.03) such that the combination of convergence and evenness improved the performance of a logistic regression classifier (AUC= .93).

Conclusions We identify reduced T cell evenness and elevated TCR convergence as features of chronic CMV infection. CMV infection appears to significantly alter the T cell repertoire, suggesting that CMV status may be required for proper interpretation of T cell expansion in the context of immunotherapy for cancer. TCR convergence may detect T cell responses to a broad range of viral and tumor neoantigens and thus may serve as a particularly useful biomarker for the identification of immunogenic tumors having few genetic alterations.

#1348

Characterization of SLFN11 protein expression in circulating tumor cells (CTCs) of patients with metastatic castration resistant prostate cancer (mCRPC) prior to platinum based chemotherapy.

Luisa Fernandez,1 John Poirier,2 Angel Rodriguez,1 Melanie Hulling,2 Robin Richardson,1 Ramsay Sutton,1 Rhett Jiles,1 Joseph Schonhoft,1 Jerry Lee,1 Nadia Ebrahim,1 Emily Carbone,2 Ethan Barnett,2 Mark Landers,1 Yipeng Wang,1 Ryan Dittamore,1 Charles Rudin,2 Howard Scher2. 1 _Epic Sciences, San Diego, CA;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Background: Clinical benefit in response to chemotherapeutic agents that induce replication stress such as PARP inhibitors and cisplatin has been associated with mutations in DNA damage repair (DDR) genes. However, prediction of benefit is not solely explained by DDR mutations; therefore, additional biomarkers for prediction of clinical benefit of these agents are needed. SLFN11 is recruited to stalled replication forks where it sensitizes cells to DNA damage induced replication stress . Presence of SLFN11 protein expression in tumor cells is related to pre-clinical and clinical benefit of PARPi in small cell lung cancer. Previously, we reported high cellular heterogeneity in progressive mCRPC, and as such, we sought to examine whether SLFN11 protein expression was expressed in patients offered platinum chemotherapy to understand the subclonal heterogeneity of CTCs with and without SLFN11 protein expression.

Material and methods: 30 blood samples from mCRPC patients prior to treatment with a platinum chemotherapy from MSKCC were sent to Epic Sciences for CTC enumeration and SLFN11 protein expression characterization. Nuclear localization of SLFN11 was reviewed by a trained technician. 70 CTCs from patients with and without SLFN11 CTCs were single cell sequenced and analyzed for copy number alterations (CNA) and clonality.

Results: 83% (25/30) of patient samples had detectable CTCs, and 53% (16/30) samples had at least one SLFN11+ CTC. For patients with SLFN11(+) CTCs, 81% (13/16) had only nuclear localized SLFN11 expression; 6% (1/16) had a mix of CTCs with nuclear and non-nuclear localized SLFN11 expression; 13% (2/16) had no nuclear localized SLFN11 expression. In 13 patients with exclusively nuclear localized SLFN11 expression, the % of SLFN11(+) CTCs ranged from 2-100% with median of 11%. Single CTC NGS analysis of patients demonstrated genomic heterogeneity between CTCs. In patients with both SLFN11+ and SLFN11- CTCs, no discernable clonal or CNA difference was observed between SLFN11+ and SLFN11- CTCs.

Conclusions: SLFN11 protein expression was observed subclonally in a majority fraction of patients with progressive mCRPC prior to platinum chemotherapy. SLFN11 expression heterogeneity was not consistent with a genetic mechanism. Studies examining whether SLFN11+ CTCs as a sensitivity marker to PARPi & cisplatin, but not ARSi or taxanes in mCRPC are ongoing.

#1349

Personalized circulating tumor DNA profiling in malignant pleural mesothelioma.

Luke J. Martinson,1 Annabel J. Sharkey,2 Alan G. Dawson,1 Robert K. Hastings,1 Gareth Wilson,3 David Waller,4 Apostolos Nakas,5 Charles Swanton,3 Jacqui A. Shaw,1 Dean A. Fennell1. 1 _University of Leicester, Leicester, United Kingdom;_ 2 _Northern General Hospital, Leicester, United Kingdom;_ 3 _The Francis Crick Institute, London, United Kingdom;_ 4 _Barts Health NHS trust, London, United Kingdom;_ 5 _Glenfield Hospital, Leicester, United Kingdom_.

Background:

Malignant pleural mesothelioma (MPM) is an aggressive tumor associated with exposure to asbestos and is rising in incidence worldwide. A lack of early detection methods and treatment successes have maintained a dismal prognosis of just 12 - 16 months. Circulating tumor DNA (ctDNA) is emerging as an important stratification biomarker in other thoracic malignancies, however research in this area has been lacking in MPM. Homozygous deletion of CDKN2A is one of the few established negatively prognostic molecular markers in MPM.

Methods and Results:

We conducted multiregional whole exome sequencing (m-WES) on tumor tissue (4 or 5 regions) and matched germline DNA from 11 patients with MPM that received surgery (extended pleurectomy decortication) as their primary treatment modality. Using a validated bioinformatics pipeline (developed as part of the TRACERx study), clonal single nucleotide variants (SNVs) and/or indels (present within all tumor regions) were selected from m-WES data for patient-specific assay design. Variants were prioritized for selection based on tumor driver status and higher mean variant allele frequencies (VAFs) amongst tumor regions. A minimum of 10 ng cfDNA isolated from pre-surgical blood was analyzed for ctDNA detection using Droplet Digital PCR (ddPCR), with positivity being defined as ≥ 3 mutant droplets and a plasma VAF ≥ 0.1%. Four out of eleven (36.4%) patients were ctDNA-positive using this patient-specific approach with VAFs ranging between 0.16% and 2.96%. Two additional patients had one or two positive mutant droplets, but these were excluded as potential false positives. In this pilot study, ctDNA-positive patients had significantly shorter survival than ctDNA-negative patients (P = 0.047), although this failed to reach significance by Cox regression analysis, likely due to a small sample size. Furthermore, in this cohort ctDNA status was a stronger prognostic biomarker than CDKN2A deletion status.

Conclusions:

Considering the short survival of patients with MPM despite surgery, we consider ctDNA-based analyses could be used to stratify patients regarding surgery, where risk-benefit would be marginal for those ctDNA-positive. This study provides proof-of-principle that the detection of tumor DNA from minimally invasive sources is possible in MPM and has alluded to how this could potentially impact clinical decision-making.

#1350

Correlation of levels of cell-free DNA in plasma with imaging-based assessment in Stage III/IV Non-Small Cell Lung Cancer.

Robert Kovelman,1 Patrick F. Desmond,1 Natalie J. Kennel,2 Juan P. Hinestrosa,1 Ellen B. Christie,1 Delia Ye,1 Michelle M. Nguyen,1 Alfred D. Kinana,1 Mark S. Schechter,3 David Chu,4 Chirag M. Shah,5 Osama Hlalah,6 John E. Doster,7 Rajaram Krishnan1. 1 _Biological Dynamics, San Diego, CA;_ 2 _NJK & Associates, San Diego, CA; _3 _Imaging Healthcare Specialists, Rancho Santa Fe, CA;_ 4 _New York Cancer and Blood Specialists, East Setauket, NY;_ 5 _Community Medical Center, Toms River, NJ;_ 6 _Bond Clinic, Winter Haven, FL;_ 7 _Anderson Area Cancer Center, Anderson, SC_.

There are significant limitations to the tools currently used to monitor clinical status and treatment response in late-stage non-small cell lung cancer (NSCLC) patients. Standard-of-care imaging is generally performed every few months, and emerging assays such as tumor mutational burden have yet to be clinically validated. Most of these tools are expensive, further limiting their use. We are exploring a promising new biomarker, high molecular weight cell-free DNA (hmw cfDNA, predominantly longer than 300 bp), which we assay using proprietary ACE (Alternating Current Electrokinetics) technology directly in plasma.

In this study, we tested the hypothesis that changes in the level of hmw cfDNA in cancer patients correlate with traditional, imaging-based clinical assessments. We collected longitudinal blood specimens from subjects diagnosed with stage III and stage IV NSCLC and being treated with various therapies (chemo, immuno, targeted) at multiple sites across the US. Specimens were obtained within nine days of routine imaging and were processed to plasma prior to testing. For this analysis, using our hmw cfDNA assay and orthogonal PCR assays, we obtained validated results on samples from 62 subjects which included a total of 117 "deltas", defined as the difference in hmw cfDNA levels between consecutive blood draws. The correlation between these deltas and clinical assessments, which were made independently by the physician responsible for the subject's routine care and by a third-party radiologist, was determined.

In post-hoc analysis of these results, we found significant correlation between deltas and clinical assessments across classes of therapies, according to receiver operator characteristic (ROC) analysis. This dataset produced an area under the ROC curve (AUROC) of 0.696, with a 95% confidence interval of 0.594 - 0.797. The time intervals between imaging dates varied, due to common practice being followed by physicians, and we found that exclusion of longer time intervals resulted in a progressive improvement in the correlation between hmw cfDNA delta and clinical assessment. Analyzing results with time intervals of less than 120 days, similar to NCCN guidelines, resulted in an AUROC of 0.784 (95% CI of 0.673 - 0.880). Analysis of data from a small subset of subjects (N=11) on immune checkpoint inhibitors resulted in an AUROC > 0.8.

These results suggest that hmw cfDNA is a promising biomarker for monitoring treatment response of NSCLC patients, including those on immunotherapy. The ease and lower cost associated with obtaining, processing, and testing samples using this assay would enable it to be employed on a more frequent basis, and it would therefore complement current technologies based on imaging and sequencing. Additional studies to refine the application of this assay and to determine its impact on clinical outcomes will further inform its future use.

#1351

**A novel application of DEPArray** TM **NxT System to isolate circulating tumor cell (CTC)-clusters from patients with metastatic breast cancer (MBC).**

Qiang Zhang,1 Lorenzo Gerratana,2 Ami N. Shah,3 Andrew A. Davis,3 Lisa Flaum,3 Youbin Zhang,3 Amir Behdad,3 Firas Wehbe,3 William Gradishar,1 Leonidas Platanias,1 Massimo Cristofanilli1. 1 _Northwestern University, Lurie Cancer Center, Chicago, IL;_ 2 _Northwestern University, University of Udine (Italy), Chicago, IL;_ 3 _Northwestern University, Chicago, IL_.

Introduction: CTCs play a critical role in MBC, and a portion of CTCs may form clusters that contain two or more CTCs bound together which were reported to have up to 50-fold of potential of forming distant metastasis in MBC as compared to individual CTCs. However, genomic characterization of CTCs-clusters remain largely because the enrichment of CTC-clusters is technically challenging. Herein, we describe a novel isolation workflow to select CTC-clusters expressing specific biomarkers for patients with MBC.

Methods: Whole blood samples (7.5ml/each) were collected from 5 patients with stage III/IV BCa patients. CTC enumeration was performed with the FDA approved CELLTRACKS ANALYZERII® System (Menarini) by using CTC Kit targeting the Epithelial Cell Adhesion Molecule antigen for capturing CTCs, and immunofluorescent staining reagents including Anti-CK-PE (specific for epithelial cells), DAPI (for nucleus), anti-CD45-APC (specific for leukocyte), and anti-HER-2/neu-FLU. Or, using CXC kit includes anti-CCR5-PE. After confirming CTCs-clusters, both CTCs and CTC-clusters were enriched from Celltracks cartridge and were loaded into DEPArrayTM NxT cartridge, and then were isolated and sorted by using fluorescent imaging based DEPArrayTM NxT System.

Results: All these samples were identified having CTCs (≥ 5, between 34 to 208 CTCs) with CTC-clusters (between 1 to 16) by CellSearch analysis. The images of CTCs and CTC-clusters were displayed in CellBrowserTM by the DEPArrayTM NxT System. According to multiple channels assigned for PE, DAPI, APC and FITC, the CTCs and CTC-clusters were classified and then sorted based on morphology and correct phenotype as CK+, EpCAM+, DAPI+ and CD45-, with additional markers of HER2 or CCR5. The targeted CTCs and CTC-clusters were routed, parked and then recovered under the direction of Recovery ManagerTM. By using this technique, we successfully recovered and harvested 27/34 (DEPArray harvest/CellSearch diagnosis), 54/151, 57/115, 75/196, and 115/208 CTCs from each CellSearch cartridge sample respectively. Meanwhile, we collected 1/1 (100%), 6/16 (37.5%), 1/1 (100%), 3/11 (27.3%), and 4/4 (100%) of CTC-clusters respectively, as each CTC-cluster includes average of 2.85 CTCs. CTC-clusters at diameter as much as 45µm could be routed and recovered smoothly. Furthermore, CTC-Clusters with biomarkers of interest such as HER2+ or CCR5\+ expression were separated into different tubes. The white blood cells from the same samples were also selected successfully as the controls.

Conclusions: We first reported a new workflow for CTC-clusters isolation. With further optimization, this feasible and reliable strategy will help streamline isolation of single CTC and CTC-clusters in MBC patients for genomic analysis providing the opportunity to gain new insights on the molecular mechanisms associated with the metastasis process.

#1352

Serum neurofilament light (NfL), glial fibrillary acidic protein (GFAp) and tau protein are possible serum biomarkers for activity of brain metastases and gliomas.

Jason Porter,1 Adriana Hepner,2 Manjari Pandey,1 Philippe Prouet,1 Felicia Hare,1 Syed S. Nasir,1 Madison Boles,3 Henrik Zetterberg,4 Kaj Blennow,4 Michael Martin1. 1 _University of Tennessee Health Science Center and The West Cancer Center, Germantown, TN;_ 2 _The State of Sao Paulo Cancer Institute, Cerqueira César, Sao Paulo, Brazil;_ 3 _University of Tennessee Health Science Center, Memphis, TN;_ 4 _The Sahlgrenska Academy at the University of Gothenburg, Mölndal, Sweden_.

Introduction: While the morbidity and mortality attributable to gliomas and brain metastases is significant, there are currently no approved biomarkers for detecting and monitoring intracranial disease and imaging remains the primary modality. Blood levels of the neurofilament light (NfL) and tau, as well as glial fibrillary acidic protein (GFAp) show promise as biomarkers for brain injury. We therefore hypothesized that these biomarkers would correlate with CNS activity of gliomas and brain metastases.

Methods: Serum concentrtaions of NfL, tau and GFAp were measured using the ultrasensitive single molecule array (Simoa) technology (Quanterix, Lexington, MA). Levels of the three proteins were then correlated with status of gliomas and brain metastases in the patients. Statistical analysis was performed using GraphPad Prism7 and all p-values were 2-tailed with an accepted 2-sided alpha level of 0.05 as statistically significant.

Results: 36 patients were enrolled with varying malignancies. Both serum NfL and GFAp levels were significantly associated with the state of intracranial disease (ANOVA psNfL= 0.0307; ANOVA pGFAp=0.0348). In contrast, serum tau levels were discordant with brain involvement with cancer (ANOVA pTau= 0.1453).

Conclusion: Serum NfL and GFAp both apparently vary closely with presence and activity of gliomas and brain metastases. Our studies are among the first to identify possible candidate serum biomarkers for screening and monitoring cancer patients for CNS involvement with malignancy. Further studies in larger populations are needed to expand these findings.

#1353

The clinical utility of ctDNA in colorectal cancer validated by multiregional sequencing and protein analysis.

Mizunori Yaegashi,1 Takeshi Iwaya,1 Masashi Fujita,2 Zhenlin Ju,3 Doris Siwak,3 Kei Sato,1 Fumitaka Endo,1 Ryo Sugimoto,1 Tamotsu Sugai,1 Lance Liotta,4 Yiling Lu,3 Gordon Mills,5 Hidewaki Nakagawa,2 Satoshi Nishizuka6. 1 _Iwate Medical University School of Medicine, Morioka-shi, Japan;_ 2 _RIKEN Center for Integrative Medical Science, Yokohama, Japan;_ 3 _The University of Texas, MD Anderson Cancer Center, Houston, TX;_ 4 _George Mason University, Fairfax, VA;_ 5 _Oregon Health Science University School of Medicine, Portland, OR;_ 6 _Iwate Medical University Institute of Biomedical Sciences, Morioka-shi, Japan_.

Background : Circulating tumor DNA (ctDNA) is considered to be a new class of tumor-specific personalized biomarkers. Since the variant allele frequency (VAF) of ctDNA is very low (0.01-1%), it is more important to quantify tumor-specific mutations using specific probes than to perform exploratory analyses. A molecular targeting drug is often selected by mutations, and it has not been verified whether the mutations reflect the level of proteins as the drug target molecule.

Purpose: To clarify: (a) the utility of ctDNA from tumors with genetic heterogeneity; (b) the ability of early detection for relapse; and (c) the validity of protein level prediction by gene mutation.

Methods: Tumor DNA was extracted from three regions of 14 Stage III/IV surgically-resected colorectal tumors. Plasma DNA was also extracted from pre-/post-operative as well as follow-up blood samples every three months. The multi-regional sequencing analysis was conducted using a next generation sequencer (NGS) with a panel of 151 genes and phylogenetic trees were subsequently generated. Of the detected non-synonymous mutations, ctDNA was monitored using digital PCR (dPCR) for which primers and probes were designed by the Hypercool Primer & Probe™ technology to facilitate a specific PCR with short (~70bp) DNA fragments. A reverse phase protein array (RPPA) was used to quantitate 294 proteins in cancer tissues for three regions.

Results: The average number of mutations per tumor and per region was nine and six, respectively. The average number of founder mutations (i.e., mutations found in all three regions) and non-founder mutations was three and seven, respectively. Phylogenetic analysis revealed that mutations with high VAF tended to emerge at the trunk of the evolutional tree. Interestingly, when non-founder mutations in the regions that had not exhibited the mutation by NGS were detected using dPCR in 5 of 13 regions. mutations were still detected in 5 of 13 regions (38.5%). The VAFs of founder mutations were significantly higher than those of non-founder mutations. In pre-operative plasma, ctDNA was detected in 10 of 12 patients (83.3%) with low VAF (range; 0.028-13.3%). Founder mutations in ctDNA were detected in 8 of 10 patients with low VAF (range; 0.04-13.3%). In each patient, founder mutations appeared to exhibit higher VAFs in ctDNA than those of non-founder mutations. Importantly, the VAF of ctDNA started to increase 6 months before the relapse was diagnosed by CT scan. Among 42 gene-protein matched pairs, the level of phosphorylated proteins that could be molecular targets did not seem to be predicted by the coding gene mutation.

Conclusions: To select mutations for ctDNA monitoring, founder mutations with high VAF in a tumor are mandatory. Since mutations in colorectal cancer do not seem to predict the protein level, the proper clinical utility of ctDNA is a tumor marker over the entire course of colorectal cancer therapy.

#1354

4-marker positive selection system for improved CTC analysis in metastatic triple-negative breast cancer.

Bradley Hancock,1 Chun-Li Chang,2 Yuan Zhong,2 Yu-Hsiang Chen,1 Jeffrey Solzak,1 Casey Bales,1 Emily Nelson,1 Robin Paul,1 Cagri Savran,2 Milan Radovich1. 1 _Indiana University, Indianapolis, IN;_ 2 _Purdue University, West Lafayette, IN_.

Introduction: Circulating tumor cells (CTCs) represent a potent opportunity to glean important information about in vivo breast cancer biology using a non-invasive technique. However, EpCAM-based techniques are significantly limited by the presence of marker-negative CTCs, a phenotype that is enriched in the CTCs of metastatic triple-negative breast cancers (mTNBCs). This interim analysis is part of an exploratory study to investigate the utility of supplementing EpCAM with additional markers specifically relevant to mTNBC for more complete CTC enumeration.

Methods: Marker candidates used in this analysis were: (1) previously associated with TNBC, (2) exclusively surface markers to avoid permeabilization, (3) not associated with leukocytes or endothelial cells, and (4) targetable with commercially available antibodies. TNBC cell lines were characterized across the selected markers by flow cytometry. Immunomagnetic enrichment (IE) of cell lines from culture medium was conducted using antibodies conjugated to magnetic beads in concert with a rare cell detection system developed by the Savran Research Group at Purdue University. The recruitment goals for the study stand at twenty healthy individuals and twenty individuals with mTNBC. All blood samples were collected in CellSave tubes and were assessed using four-marker IE with a parallel monochromatic cross-stain. Anti-CD45 and Hoechst stain were incorporated into the cross-stain in standard fashion. Samples from individuals with mTNBC were also subjected to standard EpCAM-based CTC detection on the same device, which served as a pairwise baseline comparator.

Results: Surface expression of four markers, including TROP2, N-Cadherin, EGFR, and EpCAM, was assessed across eleven TNBC cell lines. All cell lines were positive for at least one of four markers in the panel. EpCAM-negative cell lines (4/11) were positive for N-Cadherin and/or EGFR. Unique to these cell lines, four-marker IE was significantly more efficient than EpCAM alone (p=0.0006). As of November 2018, ten normal and six mTNBC participants have been recruited. All blood samples were able to be analyzed. Healthy participants yielded 0 CTCs, with the exception of one participant, in which a single positive cell was found. CTCs were detected in 50% and 33% of participants with mTNBC when utilizing the experimental method vs. EpCAM-directed detection. Futher, in 100% (3/3) of cases in which CTCs were detected, the four-marker system detected more CTCs than the EpCAM-based comparator.

Discussion: Compared to EpCAM-based capture, the four-marker experimental system presented here has potential to enhance CTC analysis by more completely representing the heterogeneity of TNBC. This results in better overall capture efficiency and enumeration while still maintaining sufficient specificity. The parent analysis is expected to be completed in the summer of 2019.

#1355

Comparison of five commercial kits on extraction of cfDNA from human plasma containing defined amount of genomic DNA contamination: The usage of DNA-depleted plasma as a control matrix for monitoring ctDNA assay.

Yabin Lu, Alina Polonskaia, Keegan Shi, Thomas Fu. _Anchor Molecular Inc, Pleasanton, CA_.

The use of PCR-based or NGS-based liquid biopsy assays detecting circulating tumor DNA (ctDNA) is rapidly increasing in clinical labs. However, there are still uncertainties around assay performance and consistency among assays from different developers. One lacking area of research is on the understanding of how plasma factors affect the cell-free DNA (cfDNA) extraction. For certain applications, the contamination of large molecular weight genomic DNA (gDNA) from cell lysis can cause interference to the quantitation of ctDNA. The ability to minimize this contamination by commercial extraction kits was examined.

Anchor Molecular Inc. has developed a proprietary technology that specifically removes the DNA from plasma without affecting other composition of the plasma. The effect of plasma factors on extraction and ctDNA quantitation was studied by quantitatively spiking low concentration of DNA into the DNA-depleted plasma. The result indicated that, while the recovery from DNA extraction was comparable for the 170bp DNA fragment, there was 2.3 fold more gDNA extracted from plasma than from buffer (quantitated by calculating the area from Bioanalyzer). This suggested plasma factors affected the large molecular weight gDNA recovery more significantly. The ability of extracting gDNA from plasma sample was compared among five commercial cfDNA extraction kits. The plasma contained a mixture of synthetic 170bp ctDNA fragment and large molecular weight undigested gDNA. After extraction, the number of copies of the recovered ctDNA and gDNA (represented by RNasP) was assayed by qPCR. The calculated copy numbers were represented as a percentage of the originally spiked copy numbers. From Kit1 to Kit5, the recovery was 74, 60, 27, 34 and 68%, respectively for the 170 bp DNA, and 75, 24, 0, 42 and 29%, respectively for the gDNA. Kit3 was able to completely eliminate the gDNA (0%) while others showed varying degree of contamination. The relative capacity to retain gDNA contamination was 101, 40, 0, 124 and 42%, respectively. This variation could cause large differences in Allele Frequency determination if the relative amount of gDNA contamination were significant. The results from plasma-based sample and from buffer-based (or synthetic plasma) sample was also compared.

The data demonstrated that the DNA profile from plasma is different from buffer. Different extraction kits showed different ability in eliminating contaminating gDNA. Quality Controls samples based on DNA-free Plasma are good tools for ctDNA assay validation and extraction monitoring.

#1356

GENOCTC, a highly efficient system for enrichment of circulating tumor cells and its clinical application.

Miso Lee,1 Jiyeon Ryu,1 Hyeon Jin Kim,1 Dohyeong Kim,1 Mi Young Kim,2 Jin-Soo Kim,2 Seok-Woo Shin,3 Young Kee Shin,4 Seokbum Ko,1 Hun Seok Lee1. 1 _GenoBio. Corp., Seoul, Republic of Korea;_ 2 _Seoul National University Boramae Medical Center, Seoul, Republic of Korea;_ 3 _International Medical Center, Seoul, Republic of Korea;_ 4 _Research Institute of Pharmaceutical Science, Seoul National University College of Pharmacy, Seoul, Republic of Korea_.

Background:

Liquid biopsy has been reported as a potential surrogate for detection of cancer biomarkers with minimally invasive procedures. Particularly, numerical, phenotypical and genetical information of circulating tumor cells (CTCs), one of critical liquid biopsy materials, is well known to have clinical significances predicting and monitoring of disease progression, drug response and metastasis. However, a number of technical challenges for isolating CTCs and appropriate downstream analysis are still to be addressed.

Methods:

Here we designed high-throughput CTC enrichment device, GenoCTC, which adopted microfluidic magnetophoresis and CTC isolation chip with optimized ferromagnetic wire patterned on it. CTCs were enriched either by Anti-human EpCAM beads, a marker for mesenchymal-epithelial transition (MET), or Anti-human Vimentin beads, a marker for epithelial-mesenchymal transition (EMT). After immuno-magnetic based separation for EpCAM positive or Vimentin positive cells, CTCs are characterized by immunocytochemistry with Cytokeratin 18 and CD45, and then enumerated. Furthermore, MET amplification was investigated with isolated CTCs by ddPCR.

Results:

GenoCTC performance was optimized using the EpCAM positive cancer cell line, MCF7; 63% of recovery, 88% of separation rate and 93% of purity in whole blood spiking test. Analyzing 16 clinical blood samples from 10 non-small cell lung cancer (NSCLC) patients receiving drug treatment, the number of CTCs ranged from 2 to 112 CTCs per 7.5 mL. In serial CTC assessment of 5 NSCLC patients, transient changes in CTC numbers were observed which was positively correlated with the clinical course of disease progression. Moreover, c-MET amplification was confirmed only in CTCs, but not in ctDNA or tissue biopsy, at the point of progressive disease (PD) status in NSCLC patient with stable disease (SD). We also investigated the different CTCs counts between EpCAM and Vimentin based isolation in breast cancer patients. Interestingly, in patient with triple negative breast cancer, we observed only 1 CTC in case of separation based on EpCAM, but when separated by Vimentin, 26 CTCs were found. This result suggests CTCs may show the MET or EMT status of cancer in each patient.

Conclusion:

A newly developed GenoCTC can isolate CTCs sensitively and precisely and enable to reveal the clinical significance of CTCs. We expect this novel microfluidic device may facilitate the use of CTCs on diagnostic and prognostic criteria.

#1357

Serum levels of programmed cell death molecule-1 (PD-1) as a biomarker in metastatic melanoma patients randomized for treatment with autologous dendritic cell or tumor cell vaccines.

Robert O. Dillman,1 Gabriel I. Nistor,1 Aleksandra J. Poole,1 Andrew N. Cornforth2. 1 _AIVITA Biomedical Inc., Irvine, CA;_ 2 _TCR2, Cambridge, MA_.

The purpose of this study was to analyze PD-1 serum levels, in patients with metastatic melanoma who were enrolled in a randomized phase 2 trial, to determine: [1] if baseline PD-1 serum levels were prognostic for overall survival (OS), [2] if baseline PD-1 levels were predictive for survival benefit in the specific treatment arms, [3] if PD-1 levels changed between week-0 and week-4 by treatment, and [4] if changes in PD-1 levels were predictive of survival benefit overall and by treatment. Patients with metastatic melanoma were enrolled in a randomized phase 2 clinical trial comparing treatment with autologous dendritic cell vaccines (DCV) presenting autologous tumor antigens (ATA) derived from irradiated cancer cells from short-term autologous cell lines, compared to tumor cell vaccines (TCV) consisting of irradiated tumor cells [Dillman et al. J ImmunoTher Cancer 2018]. Vaccines were injected s.c. weekly for three weeks, then at weeks 8, 12, 16, 20, and 24. OS was longer for patients treated with DCV (median 43 vs 20 months; 70% reduction in risk of death). Cryopreserved serum samples from week-0 and week-4 for 39/42 patients were analyzed using a quantitative, multiplex enzyme-linked immunosorbent assay (Raybiotech, Inc., Norcross, GA). The patients for whom PD-1 levels were missing were two TCV-treated subjects who had rapidly progressive disease and had no week-4 samples, and one DCV-treated subject who survived more than 5-years, but rescinded permission to test his blood. Baseline serum PD-1 levels ranged from 0 to 511 ng/ml. After three weekly vaccinations, median serum levels of PD-1 did not change between week-0 and week-4 (1.51 and 1.58 ng/ml), nor did mean serum levels change (52.5 and 52.6 ng/ml). For all 39 patients, baseline PD1 levels were not prognostic for OS. DCV was associated with longer OS than TCV both in patients whose baseline PD1 was <1.2 ng/ml and > 1.2 ng/ml. Among DCV-treated subjects, baseline levels <50 ng/ml were associated with OS > 3.5 years in 9/13 compared to 0/4 for those with levels > 50 ng/ml (p=0.029), but there was no difference in the TCV arm (5/19 vs 1/3, p=1.00). Overall, PD1 levels decreased after 3 DCV injections, but increased after 3 TCV injections. DCV-treated subjects who had a decrease in PD-1 were more likely to survive 4 years (6/9 vs 1/8, p=0.05) but this difference was not seen in patients treated with TCV (0/6 vs 6/16, p=0.289). CONCLUSONS: In these metastatic melanoma patients, baseline serum PD1 levels were not prognostic for survival, but were predictive of survival benefit in patients treated with DCV, as was a decrease in PD1 level after 3 weekly DCV vaccinations. PD1 levels should be prospectively evaluated in larger studies to see if they may be useful as a companion diagnostic and/or surrogate marker for efficacy.

#1358

Tumor-derived extracellular vesicles as kidney cancer biomarkers.

Richard C. Zieren,1 Liang Dong,1 Sarah R. Amend,1 Philip M. Pierorazio,1 Theo M. de Reijke,2 Kenneth J. Pienta1. 1 _The James Buchanan Brady Urological Institute, Johns Hopkins University School of Medicine, Baltimore, MD;_ 2 _Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands_.

Renal cell carcinoma (RCC) is a fairly common and lethal cancer. The wide variety of RCC histological subtypes constitutes a challenge in treatment decision-making. Exosomes are extracellular membrane vesicles that are produced by all cell types in physiological conditions. Extracellular vesicles (EVs) are now accepted as a mode of intercellular communication and transport proteins, RNAs, DNA, and lipids to surrounding and distant cells. The lipid bilayer membrane of the EVs helps to protect these cargos. EVs are involved in many pathological processes, such as cancer, and can be easily obtained through liquid biopsy. Currently, EVs are rarely considered as candidate biomarkers for kidney cancer. However, improvements in the characterization of tumor-derived EVs could lead to the implementation of blood- and urine-derived EVs as biomarkers in the management of oncologic patients. Since all organs, not just the tumor, contribute to EV population, the thoughtful discrimination of tumor-derived EVs remains an unmet need for the clinical application of this kind of liquid biopsy technology. To determine the contribution of the tumor to blood- and urine-derived EVs, we utilize a novel approach to isolate tissue-derived EVs in parallel with liquid biopsy-derived EVs. To our knowledge, only a handful of studies (only one in RCC) have studied EVs directly derived from tissue. We propose the use tissue-derived EVs to screen for candidate EV biomarkers in plasma and/or urine. We hypothesize that using tissue-derived EVs would increase the tumor specificity for the characterization of EVs as liquid biopsy biomarkers. We optimized a protocol in which we used tissue of RCC patients (normal kidney or tumor) to condition media and isolate tumor-derived EVs alone by ultracentrifugation. Additionally, we isolated plasma- and urine EVs by ultracentrifugation using standard protocols. Nanoparticle Tracking Analysis (NTA) showed normalized concentrations of >2 x 109 particles/mL with a size distribution in the small EV-range. Transmission Electron Microscopy (TEM) images showed typical exosome morphology, with the characteristic cup-shaped membrane vesicles. Western Blot (WB) confirmed the presence of exosome markers. We successfully isolated EVs from human RCC and healthy kidney tissue. We will proceed with screening the EV-samples for candidate miRNA-biomarkers by multiplexed gene expression analysis, followed by confirmation of the candidate markers by RT-qPCR.

#1359

Detection of PD-L1 positive circulating tumor cells from patient with bladder cancer.

Yongjoon Suh,1 Seong-Kun Kim,1 Hye Seon Lee,1 Yun-Gyu Jeong,1 Jeong-Mi Moon,1 Myoung Shin Kim,1 Byung Hee Jeon,1 U-Syn Ha2. 1 _CytoGen, Seoul, Republic of Korea;_ 2 _The Catholic University of Korea, Seoul, Republic of Korea_.

Immunotherapy with PD-L1 inhibitors greatly enhanced the clinical outcomes of patients with late stage metastatic bladder cancer. However, a concern has been raised in the front-line clinical use of PD-L1 inhibitors (permbrolizumab and atezolizumab) as a single treatment to PD-L1-low patients, due to a poorer overall survival compared with patients treated with plantinum-based chemotherapy. In accordance, reliable evaluation of PD-L1 expression is required for selecting patients relevant for treatment with PD-L1 inhibitors. However, due to the regional heterogeneity in primary tumor, evaluation of PD-L1 levels in tissue biopsy might be less reliable than the levels in circulating tumor cells (CTCs). Here, we developed the evaluating system of PD-L1 levels in CTCs isolated from the blood of patients with bladder cancer. Initially, CTCs were enriched by Smart Biopsy Cell Isolator (CytoGen), which utilizes the size-based gravity filtration for CTC isolation. The isolated CTCs were then validated with the expression of EpCAM, Vimentin and CK as well as a negative selection with CD45, and were analyzed for PD-L1 expression status by immunocytochemical staining. Using the evaluating system, we show that PD-L1 (+) cancer cell was efficiently recovered from the healthy human blood samples in a spike and recovery experiment. Furthermore, we evaluated PD-L1 expression status in CTCs isolated from ~25 patients with bladder cancer. Collectively, this study implicates the possibility that PD-L1 expression status could be analyzed at the levels of CTCs instead of the matching primary tumor tissue biopsy, probably with a better clinical diagnostic option.

#1360

MMP-degraded vimentin is elevated in serum from patients with non-small cell lung cancer - development and biological validation of a new serum biomarker.

Neel I. Nissen,1 Morten Karsdal,2 Nicholas Willumsen2. 1 _Copenhagen University, Copenhagen, Denmark;_ 2 _Nordic Bioscience, Herlev, Denmark_.

Background: Non-small cell lung cancer (NSCLS) accounts for 85% of lung cancers. The five-year survival rate decreases from 47% in stage I to 1% in stage IV, emphasizing the need for early detection. In lung cancer, the intermediate filament protein vimentin has been shown to be applicable for clinical pathology and associate with epithelial-to-mesenchymal transition. Vimentin has been found extracellularly and may be a target for proteolytic cleavage by matrix metalloproteases (MMPs). In fact, a MMP-degraded and citrullinated vimentin fragment (VICM) has shown biomarker potential in serum from patients with NSCLC. The aim of this study was to determine whether (un-citrullinated) MMP-degraded vimentin (VIM) can serve as a biomarker in patients with NSCLC.

Methods: A monoclonal antibody was raised against a MMP-generated vimentin fragment identified by mass spectrometry. A competitive enzyme-linked immunosorbent assay (ELISA) was developed and technically validated. Levels of VIM were measured in serum from patients with NSCLC (n=40) and healthy controls (n=20) and compared to the equivalent citrullinated vimentin fragment (VICM).

Results: The VIM ELISA assay was highly specific to MMP-degraded vimentin and with no cross-reactivity to VICM and the full-length vimentin. VIM was significantly elevated in serum from patients with NSCLC (p=0.0003) compared to controls whereas VICM was not (p=0.0741). In support, the diagnostic accuracy (AUROC) for VIM was 87.1% (p=<0.0001) vs 64% (p=0.0685) for VICM.

Conclusion: The newly developed VIM assay detected significantly elevated levels of MMP-degraded vimentin in serum from patients with NSCLC compared to controls. Furthermore, this non-citrullinated vimentin fragment (VIM) shows stronger accuracy in separating NSCLC patients from healthy controls than the citrullinated equivalent (VICM), suggesting its biomarker potential and biological relevance in NSCLC. Future studies are needed to validate and expand on these findings.

#1361

Automated flow cytometric analysis of tumor heterogeneity, immunophenotypic and functional profiles, and co-expression of checkpoint receptors in the context of tumor aneuploidy.

Konstantin Salojin,1 Dai Liu,1 Christine Hauther,1 Santosh Putta,2 Norman Purvis,1 Matt Westfall1. 1 _Pierian Biosciences, Franklin, TN;_ 2 _Qognit,Inc., San Carlos, CA_.

Traditional methods of analysis of tumor microenvironment (TME) and tumor-infiltrating leukocyte (TIL) rely on a limited selection of histology & IHC markers and fall short in addressing tumor cell heterogeneity, aneuploidy, TIL functional status, and expression of immune checkpoint receptors (ICP-R) and their ligands (ICP-L).

We developed a flow cytometry platform (ImmunoINTEL) comprised of quantitative multi-parameter flow cytometry panels and automated sample/data processing procedures for HTP immunophenotyping of blood and solid tumor specimens in 96-well plates. The platform is fully integrated into an SMS/LIMS system with automated processing procedures and equipment for: 1) tumor dissociation, 2) isolated sample plating, modulation, and staining, 3) 96-well plate based flow cytometry acquisition, and 4) proprietary data analysis software. These integrated automation systems ensure consistency in sample processing, acquisition, and analysis. Multiplexing of lineage-specific markers permitted quantitative analysis of ICP-R/ICP-L (CD47, CD73, CD112, CD155, CD172a, CD274, CD279, CD366, Galectin-9, LAG3, TIGIT) on TILs and epithelial cells, including rare immune cell subsets such as NK cells, mDCs/pDCs, and CD14+HLADRlo MDSCs, using a limited number of cells isolated from lung, breast, renal, and colorectal carcinomas. The functional capacity of the T cell component of the TME was further examined by measurement of intracellular IFNγ, TNFα, Granzyme B and quantitation of Treg levels. DNA aneuploidy in epithelial cancer cells (a possible surrogate for genomic instability or TMB) was also performed in parallel to the phenotypic and functional TME assessments.

This wholistic analysis approach characterized various aspects of tumor biology, such as heterogeneity of TME and functional/exhaustion status of TILs. Ploidy measurements when combined with phenotypic/functional profiles yielded data indicating a strong relationship between tumor aneuploidy, CD8+ T cell infiltration, granzyme B production, and expression of ICP-L on tumor epithelial cells and myeloid TILs (including expression of CD274 tumor cells). We identified distinct immunophenotypic profiles characterized by differential expression of ICP-R and ICP-L, variations in TIL makeup, and tumor aneuploidy, which correspond to atypical histological profiles and can be used in biomarker development.

In conclusion, the ImmunoINTEL platform facilitates the analysis of functional status of TILs in the context of tumor heterogeneity and tumor aneuploidy. The immunosuppressive signature of tumor (the % of ICP-R/L+ immune and tumor cells) combined with analysis of aneuploidy adds an additional dimension to TIL analysis and patient stratification.

### Current Developments in Non-invasive Biomarkers for Assessment of Cancer 2

#1362

Genome-wide sequencing of cell-free DNA enables detection of copy number alterations in cancer patients where tissue biopsy is not feasible.

Taylor J. Jensen,1 Aaron M. Goodman,2 Shumei Kato,2 Christopher K. Ellison,1 Kimberly Kelly,1 Lisa Kim,2 Gregory A. Daniels,2 Kerry Fitzgerald,1 Erin McCarthy,1 Prachi Nakashe,1 Amin R. Mazloom,1 Graham McLennan,1 Eyad Almasri,1 Daniel S. Grosu,1 Marcia Eisenberg,3 Razelle Kurzrock2. 1 _Sequenom, Inc., San Diego, CA;_ 2 _Moores Cancer Center, San Diego, CA;_ 3 _LabCorp, Burlington, NC_.

In instances where tissue biopsy is not medically prudent or tumor tissue material is insufficient for molecular testing, alternative methods are needed. Since cell-free DNA (cfDNA) has been shown to provide a representative surrogate for tumor tissue, we sought to evaluate its utility in this scenario. Here we present data from 91 patients with known neoplasms whose tumors were not able to be tested using conventional tissue biopsy. The cfDNA from each patient was assayed with low-coverage (~0.3X) genome-wide sequencing and copy number alteration (CNA) events were identified and characterized using analytical methods originally developed for noninvasive prenatal testing (NIPT). To quantify the level of CNAs present in the plasma of cancer patients, we utilized the genomic instability number (GIN). First, the technical variability of the GIN was evaluated in a large sample cohort. We therefore processed genome-wide sequencing results from 27,742 pregnant women who consented to have their samples used for research and whose NIPT results yielded no detected CNAs. Utilizing these data, we established a threshold whereby we could differentiate technical noise from biological signal when CNAs were present down to a 1% ctDNA fraction with 99.7% specificity. Applied broadly, CNAs were detected in 32 of the 91 patients with difficult-to-biopsy cancers evaluated (35%) with detection frequencies tightly linked to cancer type. Importantly, there was a significant reduction in progression-free survival for the first treatment after liquid biopsy for patients with an elevated GIN relative to those without, suggesting a prognostic link between GIN and patient outcome across a wide variety of tumor types. In addition, samples with an elevated GIN also showed a significant concomitant reduction in cfDNA fragment length, consistent with previous reports suggesting that cfDNA derived from tumors is shorter in length than cfDNA from healthy tissue. Finally, we characterized the genomic location of the detected CNAs to potentially enable the utility and interpretation of these results clinically. Collectively, the detected CNAs covered more than 95% of all autosomes, highlighting a benefit of using genome-wide sequencing. Taken together, these data demonstrate a proof-of-concept for using low-coverage, genome-wide sequencing of cfDNA in patients where tissue biopsy is not feasible or medically recommended.

#1363

Identifying blood exosomal miRNAs as biomarkers in the diagnosis and prognosis of pancreatic ductal adenocarcinoma.

Shiwei Guo,1 Huan Wang,1 Hao Qin,2 Dadong Zhang,2 Zhuo Shao,1 Yanan Zhang,2 Bin Song,1 Xiaoya Xu,2 Jing Shen,1 Zishuo Chen,2 Xiaohan Shi,1 Suizhi Gao,1 Yaqi Pan,1 Fugen Li,2 Gang Jin1. 1 _Changhai Hospital, Second Military Medical University, Shanghai, China, Shanghai, China;_ 2 _3D Medicine Inc., Shanghai, China_.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is of high mortality with a shortage of effective non-invasive biomarkers in the diagnosis and prognosis. Without a proper method to identify reasonable target molecules and their references for normalization, robust and accurate blood miRNA biomarkers have not been reported. We aim to explore the exosomal miRNAs as biomarkers for the diagnosis and prognosis of PDAC with sufficient biological insights and robustness.

Materials and Methods: miRNA-seq was performed on miRNAs extracted from blood exosomes. 16 non-metastatic PDAC, 11 metastatic PDAC and 15 pancreatitis patients were enrolled in 2017 as the training set. Another 18 non-metastatic PDAC, 12 metastatic PDAC and 18 pancreatitis patients were enrolled in 2018 as the testing set. Bayesian network was built on the levels of miRNAs to identify candidate biomarkers with biological insights. The area under the curve (AUC) of receiver operating characteristic (ROC) was used to measure the accuracy of each pair of miRNAs.

Results: The exosomal miRNA from the plasma of these patients with PDAC or pancreatitis has been successfully extracted and sequenced. The Bayesian network consisted of 337 nodes and 713 edges. The quotient of miR-95-3p (a "consequence" of "cancer") over miR-26-5p (a "cause" of "pancreatitis") performed 81.5% sensitivity and 93.3% specificity in the training cohort, and 86.7% sensitivity and 100% specificity in the testing cohort respectively. Furthermore, considering either high serum level of CA19-9 or the quotient of miR-95-3p/miR-26-5p as PDAC, the sensitivity increased to 96.5%, and the specificity was maintained at 96.9%. In addition, miR-335-5p level in blood exosome could predict metastasis with a mean accuracy of 77.2%, and showed strong relation with overall survival after surgeries.

Conclusions: MiRNAs biomarkers from blood exosomes in differential diagnostics and prognosis are identified and rationale by Bayesian network. Integration of biomarkers of various molecular types could enhance the diagnostic accuracy beyond single molecular types. Blood exosomal miRNAs could be applied to monitor the development of PDAC.

#1364

A Non-Small Cell Lung Cancer Reference Material.

Farol L. Tomson,1 Catherine Huang,2 Omo Clement,2 Russell Garlick,2 Bharathi Anekella1. 1 _Seracare Life Sciences, Germantown, MD;_ 2 _Seracare Life Sciences, Gaithersburg, MD_.

Lung cancer is the leading cause of cancer death in the United States as well as globally. The major diagnosis (~85%) of lung cancer patients is non-small cell lung carcinoma (NSCLC). About 10-50% of NSCLCs have several activating EGFR driver mutations which have been clinically validated as therapeutic biomarkers for anti-EGFR drugs. Identifying these driver mutations through liquid biopsies is a promising alternative to traditional biopsies; however, the precision of mutation analysis for some of these targets, e.g., T790M, especially in advanced-stage metastatic NSCLC patients remains a challenge. To address this, we developed a platform of single and low complexity, highly patient-like circulating tumor DNA (ctDNA) EGFR reference standards designed for PCR assays that are used for EGFR-based patient testing. We mixed genomic DNA from the highly characterized human cell line (GM24385) with four synthetic EGFR variant-containing DNA sequences: T790M, L858R, G719S, and an ex19 deletion, in both individual and combined format. Using digital PCR we were able to precisely blend the material at the defined variant allele frequencies of 1% and 0.1%. In addition, wild-type reference material without any synthetic EGFR variants was also created as a comparable negative control. The materials were size-selected (160-170bp) after fragmentation for more patient-like ctDNA size characteristics. After size-selection the material is further refined to improve conversion efficiency. In order to make the material comparable to native ctDNA, such as that isolated from cancer patients, we encapsulated the DNA fragments into a synthetic plasma matrix. The process of DNA size selection followed by encapsulation and formulation into a synthetic plasma matrix enhances the commutability of these reference standards and improves the long-term stability of the samples to at least two years. The allele frequencies of the reference materials were confirmed by both dPCR and NGS. The Archer Reveal ctDNA 28 Kit was used for NGS library preparation while an Illumina MiSeq, using v2 (2x150 bp) PE chemistry reagents, was used for sequencing. Additional evaluation of these materials was conducted by laboratories that provide EGFR RT-PCR tests for patient anti-EGFR therapy stratification. The data from the dPCR, NGS and RT-PCR assays were comparable and highlight the broad compatibility of these EGFR ctDNA reference standards on a variety of assay testing platforms for NSCLC.

#1365

Serial circulating tumor DNA analysis in locally advanced esophageal squamous cell cancer treated with neoadjuvant chemoradiotherapy followed by surgery.

Mian Xie,1 Xiaoxiang Li,1 Xin Zhang,2 Chaosheng He,3 Ze Xu4. 1 _Guangzhou Medical University, Guangzhou, China;_ 2 _Sun Yat-sen University Cancer Center, Guangzhou, China;_ 3 _Guangdong General Hospital, Guangzhou, China;_ 4 _Shantou University Medical College, Shantou, China_.

Background Neoadjuvant chemoradiotherapy (nCRT) followed by surgery is the most common approach for patients with locally advanced esophageal squamous cell cancer (ESCC). The aim of our study is to investigate circulating tumor DNA (ctDNA) as a prognostic marker for survival in ESCC patients treated with nCRT and surgery.

Method From September 2010 to December 2014, 165 ESCC patients with clinical stage IIB—III from multicenter institutes participated in a phase III clinical trial were enrolled in this study. Patients were treated with nCRT before surgery. We used targeted next-generation sequencing (NGS)-based assays to identify somatic genomic alterations in tumor tissues and designed personalized assays to quantify circulating tumor DNA in serial plasma samples collected before treatment, after nCRT, and after surgery. ctDNA was classified as detectable (ctDNA positive) or undetectable (ctDNA negative) based on a permutation test that compared the mutation frequency in the sample of interest with the mutation frequencies in controls.

Results ctDNA was detectable in 72.0%, 10.8%, and 7.3% of pretreatment, post-nCRT and post-surgery plasma samples, respectively. Postoperative ctDNA detection was associated with ypT3-4 and ypN1-2 stage. The conversion of ctDNA status from positive at baseline to negative at 4-6 weeks after chemoradiotherapy was significantly associated with pathological complete response (pCR) (pCR vs non-pCR, 96% vs 72%, P = 0.03). Detectable ctDNA at baseline was associated with worse overall survival (OS) (HR 7.8, P < 0.001). Patients with detectable ctDNA after nCRT had worse recurrence-free survival (RFS) (HR 6.7, P < 0.001). Significant worse disease-free survival (DFS) was seen if ctDNA was detectable after surgery resection (HR 10.3, P < 0.001). In multivariate analysis, detectable postoperative ctDNA was significantly associated with DFS (HR 6.5, P < 0.001) after adjusting for other clinicopathological risk factors.

Conclusion ctDNA status before and after surgery was a strong prognostic factor of recurrence and survival in locally advanced ESCC patients. Consequently, ctDNA could potentially improve pre- and post-treatment risk assessment and facilitate individual therapy for locally advanced ESCC patients.

#1366

Significance of circulating tumor cells (CTCs) with fibroblast growth factor 2 expression in gastric cancer patients.

Kenji Kuroda. _Osaka City University Graduate School, Osaka City, Japan_.

[Background] Amplification of the fibroblast growth factor receptor 2 (FGFR2) is reportedly identified in 3-10% of primary gastric cancers (GCs). We found that FGFR2 overexpression was closely associated with poor prognosis of patients with gastric cancer. Recently, FGFR signaling inhibitors have been considered a key drug in the treatment of GCs with FGFR2 overexpression. FGFR2 overexpression can be assessed by immunohistochemical staining of primary tumors; however, this method does not sufficiently reflect FGFR2 expression in recurrent tumors owing to the heterogeneity of tumor cells between primary and recurrent tumors. Circulating tumor cells (CTCs) might overcome this issue of "temporal" and "spatial" heterogeneity. This study aimed to assess the significance of the detection of FGFR2-positive CTCs in patients with gastric cancer.

[Methods] Seventy-eight patients with gastric cancer who underwent gastrectomy in our hospital were enrolled in this study. A total volume of 10 ml of peripheral blood was collected prior to surgery from each patient, and mononuclear cells were enriched by Ficol density gradient centrifugation. These cells were immunostained with PI/CD45/EpCAM/FGFR2 and PI/CD45/EpCAM/CK. The number of cells in each sample was enumerated depending on the positivity of EpCAM, FGFR2, and CK by fluorescence-activated cell sorting (FACS). FGFR2 expression was assessed by immunohistochemical staining of resected specimens. Patients were categorized into four groups as follows: group IHC-0 (n=32), no staining; group IHC-1+ (n=24), cytoplasmic staining in 1-19% or membranous staining in 1-5%; group IHC-2+ (n=16), cytoplasmic staining in 80-100% or membranous staining in 5-19%; group IHC-3+ (n=6), membranous staining in 20-100%.

[Results] FGFR2+ cells were detected in 43 (55%) of 78 cases. Among these cases, CK+ cells or EpCAM+ cells were detected in 35 (81%) of 43 cases. By group, the proportions of FGFR2+ cells were 44% in group IHC-0, 52% in group IHC-1+, 69% in group IHC-2+, and 100% in group IHC-3+. The mean number of FGFR2+ cells from patient samples in the IHC-0, IHC-1+, IHC-2+, and IHC-3+ groups were 12, 11, 16, and 54 (per 10 ml peripheral blood), respectively, with a significant difference between group IHC-0 and group IHC-3+, group IHC-1+ and group IHC-3+, and group IHC-0 and group IHC-2+ (p=0.001, p=0.008 and p=0.034, respectively, by Mann-Whitney U test). Among 68 patients with R0 and pStage I-III, the recurrence-free survival of patients with an FGFR2+ cell count ≧10 cells / 10 ml was significantly poorer than that of patients with an FGFR2+ cell count <10 cells / 10 ml (p = 0.018, by logrank test).

[Conclusion] Detection of CTCs with FGFR2 expression by FACScan might be a useful approach to identify gastric cancer patients who would benefit the most from FGFR inhibitor treatment.

#1367

Comprehensive characterization of cell-free tumor DNA in plasma and urine of patients with renal tumors.

Tina Moser,1 Christopher G. Smith,2 Maximilian Seles,3 Gabriel Wcislo,4 Matthew Eldridge,2 Samantha Perakis,1 Florent Mouliere,5 Isaac Lazzeri,1 Katrin Heider,2 Anne Warren,6 Nitzan Rosenfeld,2 Grant D. Stewart,6 Ellen Heitzer1. 1 _Medical University of Graz, Diagnostic and Research Center for Molecular Biomedicine, Graz, Austria;_ 2 _Cancer Research UK Cambridge Institute, Cambridge, United Kingdom;_ 3 _Medical University of Graz, Graz, Austria;_ 4 _Military Institute of Medicine, Warsaw, Poland;_ 5 _Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam, Netherlands;_ 6 _Cambridge University Hospitals NHS Foundation Trust, Cambridge, United Kingdom_.

Renal cell carcinoma (RCC) represents a heterogenous disease in terms of histologic subtypes, prognosis and treatment response. Genetic heterogeneity offers a particular challenge to direct available targeted therapies that best match the patient. Profiling and monitoring of tumor-specific alterations from body fluids has been demonstrated as a valuable tool for many tumor types. Yet, the utility of circulating tumor DNA (ctDNA) in RCC has not been well established. To characterize the levels and composition of ctDNA in the plasma and urine we employed a broad range of targeted and untargeted methods to two independent cohorts of patients with renal tumors. We applied shallow Whole Genome Sequencing (sWGS) and modified Fast Aneuploidy Screen Test-Sequencing System (mFAST-SeqS) to 43 patients with metastatic RCCs. Using the mFAST-SeqS, ctDNA was detectable in only 2 out of 43 patients (4.7%). However, assessment of tumor fractions based on sWGS using the ichorCNA algorithm revealed 6 further patients with detectable amounts of ctDNA. In silico size selection of fragments < 150bp further improved the detection rate to 27% (12 out of 43 patients). This is consistent with previous reports that tumor-derived fragments are often smaller compared to cell-free DNA of normal cells and as such enrichment of smaller fragment increases the sensitivity. High-resolution mutation analysis of 10 recurrently mutated genes in RCC was performed using a QIASeq custom capture panel, enabling detection of tumor-specific mutations at baseline in 18% (8/43) of patients. Of these five had detectable tumor fractions as observed with ichorCNA. Furthermore, we had access to longitudinally obtained plasma samples for 37 of our 43 (86%) patients with a median follow-up period of 6 months (range, 0.4-19.2). The QIASeq panel was applied to follow-up patients of which mutations were identified at baseline. For most of these patients, ctDNA was elevated at treatment initiation but decreased with response. At the time of progression, or when a response could not be achieved, ctDNA increased or remained elevated. Plasma and urine samples were available for a second cohort (n=47) of patients with a wide range of renal tumors. Detection rates using both broad, untargeted sequencing methods and targeted, sensitive approaches were similarly low with 7/47 (14.9%) and 45.5% (10/22), respectively. Interrogation of those patients with detectable ctDNA revealed, for the first time, that urine ctDNA is capable of overcoming genetic heterogeneity and offers information that is complementary to that provided by plasma. Taken together, our data revealed that ctDNA levels are lower in RCC than other cancers of similar stage. Although, ctDNA can be detected in blood and urine of RCC patients and there is potential for clinical utility, improved isolation and detection methods are needed to achieve a broad patient coverage.

#1368

Simultaneous cell-free RNA PD-L1 expression and MSI from the same single-tube of blood in solid tumors.

Glen J. Weiss,1 Paul Walker,2 Dilek Aktas,3 Andrew Ford,4 Charmaine Brown,4 Chen-Hsiung Yeh,4 Nader Javadi5. 1 _Beth Israel Deaconess Medical Center/Harvard School of Medicine, Boston, MA;_ 2 _Brody School of Medicine at East Carolina University, Greenville, NC;_ 3 _Damegen Genetic Diagnostic Center, Ankara, Turkey;_ 4 _Circulogene, Birmingham, AL;_ 5 _Hope Health Center, Calabasas, CA_.

Background: Liquid biopsy is a minimally invasive technique available in the clinic using next-generation sequencing (NGS) of plasma circulating cell-free DNA (cfDNA) and cfRNA. Here we report liquid biopsy results from solid tumors using the Circulogene Theranostics Personalized Gene Profile (CGP, 50-gene panel), as well as, microsatellite instability (MSI) testing. CGP uses proprietary in situ enrichment requiring minimal input volume of 20 uL plasma (cfDNA), 400 uL (cfRNA), and 100 uL buffy coat per case from the same single-tube of blood.

Methods: cfRNA PD-L1 expression, MSI, and co-occurring cfDNA mutations were retrospectively compiled from CGP ordered at multiple centers. Ct value cutoff for 1 copy PD-L1 (positive) was 42. MSI was performed using paired buffy coat gDNA (normal) and plasma cfDNA (tumor) on 5 microsatellite mononucleotide markers, BAT-25, BAT-26, NR-21, NR-24 and MONO-27 (Promega; current an industry standard that has been used in KEYNOTE pembrolizumab trials) and analyzed by capillary electrophoresis genetic analyzer. Based on batched processing, Circulogene's turnaround time (TAT) is 5 business days on average. Data was summarized and the correlation between PD-L1 and MSI was measured.

Results: 397 patients (median age 67 years, range 27-96; 210 men: 187 women) underwent CGP testing for both PD-L1 and MSI (11/2017-10/2018). The majority of cancer types were lung (n=187), colorectal (n=43), breast (n=40), pancreatic (n=32), and prostate (n=30). 59/397 (14.9%) were PD-L1 positive, in the range of expected PD-L1 positivity across solid tumors. 110/397 (27.7%) were MSI-High (MSI-H). There was no correlation between PD-L1 and MSI results (r^2 <0.01). There was a median of 3 mutations/patient (range 0-19), with a 0% sample failure rate. Overall, 23 cases had no detectable mutations (5.8%), but 11 of these 23 (47.8%) had either PD-L1 positivity or MSI-H.

Conclusions: Simultaneous cfRNA PD-L1 and MSI testing from the same single-tube blood is feasible in a liquid biopsy and their positivity has frequency in the expected range for solid tumors (Xang et al. Onco Targets Ther 2016, Zhang et al. AMP Conference 2018, Abstract ST009). Despite the low plasma sample input, there were no sample failures, suggesting CGP in situ enrichment is robust and has a rapid TAT. Assay results with linkage to clinical outcomes is warranted.

#1369

Establishment of a workflow for analysis of mRNA and gDNA from circulating tumor cells, extracellular vesicles and cell-free DNA from the same blood sample to mirror the genomic and transcriptomic complexity in metastatic breast cancer patients.

Corinna Keup,1 Markus Storbeck,2 Peter Hahn,2 Siegfried Hauch,2 Markus Sprenger-Haussels,2 Ann-Kathrin Bittner,1 Oliver Hoffmann,1 Mitra Tewes,1 Rainer Kimmig,1 Sabine Kasimir-Bauer1. 1 _University Hospital of Essen, Essen, Germany;_ 2 _QIAGEN GmbH, Hilden, Germany_.

Background: Blood analytes derived from liquid biopsies are discussed as useful tools for therapy stratification and for monitoring of clonal evolution. To gain comprehensive insights into the genomic and transcriptomic complexity in metastatic breast cancer (MBC) useful for therapy management, we aimed to isolate and analyze mRNA and gDNA from circulating tumor cells (CTCs), mRNA from extracellular vesicles (EVs) and cell-free DNA (cfDNA) from the same blood sample with minimized volume in a condensed workflow.

Patients and Methods: EDTA blood (2x 9 ml) was drawn from 35 MBC patients with hormone receptor positive and HER2 negative primary tumor at time of disease progression and at two further consecutive staging time points. CTCs were isolated in duplicate from 5ml blood by immunomagnetic selection (AdnaTest EMT2/StemCell Select, QIAGEN). Plasma (4 ml) of the CTC-depleted blood was used for cfDNA isolation (QIAamp MinElute ccfDNA Kit, QIAGEN), plasma (4 ml) from the blood not used for CTC/cfDNA isolation was applied for EV isolation (exoRNeasy, QIAGEN). The mRNA purified from CTCs and EVs was analyzed by qPCR panel (AdnaPanel TNBC prototype, QIAGEN), while cfDNA was analyzed with a customized QIAseq Targeted DNA Panel for Illumina (QIAGEN) with unique molecular indices. We are working on a workflow to isolate gDNA from CTCs, starting from mRNA-depleted CTC lysates and analyzing the gDNA with a customized QIAseq Targeted DNA Panel. Isolation and mutation analysis of CTC gDNA was shown to be feasible in spiking experiments.

Results: Isolation of mRNA and gDNA from CTCs, mRNA from EVs and cfDNA was successfully established in a parallel workflow. CTC and EV mRNA profiles showed substantial differences synergizing with regard to their clinical relevance. Whereas overexpression of mTOR was related to therapy responsiveness in CTCs, mTOR signals in EVs related to therapy failure. ERBB2 overexpressing CTCs were found in one third of all MBC patients enabling new therapeutic options. Matched cfDNA revealed the appearance of pathogenic mutations (e.g. PIK3CA H1047R) across treatment indicating underlying resistance mechanisms. Moreover, we identified significant correlations of therapy outcome with the overexpression of transcripts/ presence of mutations in each of the isolated liquid biopsy analytes. Comparison of CTC gDNA and cfDNA is further conducted.

Conclusions: We were able to describe a complete workflow for parallel CTC mRNA, CTC gDNA, EV mRNA and cfDNA isolation from a minimized blood volume. In this research study each analyte showed synergistic potential for therapy management, thus the comprehensive picture of the genomic and transcriptomic complexity might in future enable to identify the most suitable therapy regiment in each individual patient.

#1370

Germinal immunogenetics predicts treatment outcome for PD1 PD-L1 checkpoint inhibitors.

Sadal REFAE,1 Jocelyn GAL,1 Nathalie EBRAN,1 Josiane OTTO,1 Delphine BORCHIELLINI,1 Frederic Peyrade,1 Emmanuel CHAMOREY,1 Patrick Brest,2 Gerard Alain Milano,1 Esma SAADA-BOUZID1. 1 _Ctr. Antoine Lacassagne, Nice, France;_ 2 _Université Cote d'Azur, CNRS, INSERM, Institute of Research on Cancer and Ageing of Nice (IRCAN) , FHU-OncoAge, Nice, France_.

Background Checkpoint inhibitors (CPIs) benefit only a proportion of patients and may be associated with severe adverse events (AEs) which cannot be predicted. We hypothesized that the host genetics could be used as predictive biomarkers for CPI response and AE prediction. Therefore, we conducted a study based on single nucleotide polymorphisms (SNPs) from genes affiliated with immune response and tumor-microenvironment interaction.

Methods Germinal DNA was obtained from advanced cancer patients treated with anti PD-1/PD-L1 CPIs in the Centre Antoine Lacassagne (Nice, France) from July 2012 to January 2018. DNA was genotyped on the MassARRAY system (Agena Bioscience®) using a custom panel of 166 SNPs covering 86 preselected immunogenetic-related genes (Minor allele frequency MAF>0.05 in Caucasians). All tested SNPs were in Hardy-Weinberg equilibrium. Univariate analysis was performed to select the significant SNPs (p<0.05) by either Fisher or Ki2 tests. Treatment outcome prediction was based on an elastic-net penalized logistic regression with 5-fold cross validation. The predictive ability model was performed using a concordance (c)-index (c-index > 0.5 being considered as good prediction). Computational analysis using a GTEX portal was used to determine potential eQTL (expression Quantitative Trait Loci) in tissues.

Results 94 patients were identified, with median age 68 (32-85), 67% male, with a majority (51%) having advanced non-small cell lung cancer. Median follow-up was 16.3 months (95% CI: 12.5-18.3). Overall response rate (ORR) was observed in 49/94 (54%) of patients, with adverse events (grade 3-4) observed in 15/94 (16%) of patients. ORR was significantly predicted by tumor microenvironment related gene polymorphisms (CCL2, NOS3, IL1RN, IL12B, CXCR3, IL6R). In contrast, grade 3-4 AEs were linked to target-related gene SNPs (UNG, IFNW1, CTLA-4, PD-L1, IFNL4). The predictive (c)-index was 0.81 (95% CI: 0.72-0.9) for response and 0.89 (95% CI: 0.76-1.00) for toxicity. In silico functionality exploring (GTEX portal) pointed IL6R (rs4845618) and CTLA4 (rs3087243) as impacting gene expression.

Conclusion Our data strongly support the role of distinct SNPs in immunogenetic related genes to predict efficacy and safety of anti PD1/PD-L1 therapies. These data support the notion that patient-specific, germinal biomarkers may supplement tumor-specific biomarkers in predicting response to CPI therapy, and that additional germinal biomarkers may predict grade 3-4 AEs.

#1371

Assessing the utility of cell-free DNA in identifying prostate cancer and characterizing tumor heterogeneity via whole exome and whole genome, multi-region sequencing.

Emmalyn Chen,1 Clinton Cario,1 Lancelote Leong,1 Karen Lopez,1 Patricia Li,1 Erica Oropeza,1 Imelda Tenggara,1 Janet Cowan,1 Jeffry Simko,1 Daniel Wells,2 Robin Kageyama,2 June Chan,1 Terence Friedlander,1 Pamela Paris,1 Peter Carroll,1 John Witte1. 1 _University of California - San Francisco, San Francisco, CA;_ 2 _Parker Institute for Cancer Immunotherapy, San Francisco, CA_.

Early cancer diagnosis, especially while the disease is still localized and before symptoms appear, results in significantly higher survival rates compared to late-stage diagnosis. At the time of diagnosis, it is also common to find multiple foci within a single prostate gland in men with localized disease. Cell-free DNA (cfDNA) may not only reflect underlying disease biology, but also simultaneously allow for the identification of genetically distinct tumor subclones. The objectives of this study are to determine if 1) cfDNA levels are able to distinguish between healthy individuals from patients with localized or metastatic castration-resistant prostate cancer (mCRPC), and 2) if somatic mutations identified in tumor tissue are detectable in cfDNA and representative of the distribution observed in tumor tissue.

This study includes samples from 130 individuals at UCSF: 21 healthy donors, 100 patients with localized prostate cancer who underwent radical prostatectomy (RP), and 9 mCRPC patients. Blood samples and matched tissue from adjacent normal seminal vesicles and multiple tumor regions (1-9 samples per patient) were collected from patients undergoing radical prostatectomy. CfDNA was extracted from plasma, and the concentration and fragment length distribution were measured with a Bioanalyzer 2100. Comparisons of cfDNA levels between groups were assessed with a Welch's t-test due to the potential for unequal variances. Fifty-seven samples from nine patients have been subjected to whole exome sequencing, and 22 samples from five patients have been subjected to whole genome sequencing at ~40x coverage. Somatic variant calling was performed with Broad Institute's Firecloud platform (GATK4/MuTect2) for tumor tissue and with the Curio platform for cfDNA to build consensus sequences leveraging unique molecular tags.

CfDNA levels are able to distinguish between healthy and localized (p = 0.005), as well as healthy and metastatic groups (p = 0.043). Tumor foci within a patient's prostate gland are genetically heterogeneous, with the majority of somatic mutations private to tumor regions and a subset of mutations at the intersection of these regions. Preliminary analyses result in an average of 72 somatic SNVs and indels per tumor tissue region, with ~10% overlap between regions in the same patient. Additional mCRPC and follow-up blood samples are being collected from all patients. The association between cfDNA levels prior to RP surgery and biochemical recurrence will be investigated when follow-up collection concludes. Further analysis of mutational concordance, clonality, and copy number variation between tumor tissue DNA and cfDNA, along with clinical data, will be performed.

#1372

Detection of early stage pancreatic cancer using 5&#8211;hydroxymethylcytosine signatures in circulating cell free DNA.

Francois Collin,1 Yuhong Ning,1 Gulfem D. Guler,1 Tierney Phillips,1 Erin McCarthy,1 Aaron Scott,1 Chris Ellison,1 Chin-Jen Ku,1 Kim Chau,1 Alan Ashworth,2 Stephen R. Quake,3 Samuel Levy1. 1 _Bluestar Genomics, CA;_ 2 _University of California San Francisco, San Francisco, CA;_ 3 _Stanford University, CA_.

Pancreatic cancers are typically diagnosed at late stage where disease prognosis is poor as exemplified by a 5-year survival rate of 8.2%. Earlier diagnosis would be beneficial by enabling surgical resection or earlier application of therapeutic regimens. We investigated the detection of pancreatic ductal adenocarcinoma (PDAC) in a non-invasive manner by interrogating changes in 5-hydroxymethylated cytosines (5hmC) in circulating cell free DNA in the plasma of a PDAC cohort (n=51) in comparison with a non-cancer cohort (n=41). 5hmC profiles from PDAC and non-cancer samples were generated using a previously published modified hMe-Seal protocol that utilizes chemical labeling of 5hmC by β-glucosyltransferase and allows detection of cell free 5hmC from small amounts of cfDNA (1). We found that 5hmC sites are enriched in a disease and stage specific manner in exons, 3'UTRs and transcription termination sites. Our data show that 5hmC density is reduced in promoters and histone H3K4me3 associated sites with progressive disease suggesting increased transcriptional activity. 5hmC density is differentially represented in thousands of genes, and a stringently filtered set of the most significant genes points to biology related to pancreas (GATA4, GATA6, PROX1, ONECUT1) and/or cancer development (YAP1, TEAD1, PROX1, ONECUT1, ONECUT2, IGF1 and IGF2). Regularized regression models were built using 5hmC densities in a comprehensive set of genes with the most variable 5hmC counts and performed with an AUC = 0.94 - 0.96 on training data. We tested the ability to classify PDAC and non-cancer samples with the Elastic net and Lasso models on three independent pancreatic cancer 5hmC data sets (n = 26, 23 and 7) compared with corresponding independent non-cancer cohorts (n =103, 53 and 10), and found validation performance to be AUC = 0.74 - 0.97. The findings suggest that 5hmC changes enable classification of PDAC patients with high fidelity and are worthy of further investigation on larger cohorts of patient samples. Reference: 1. Song, C. - X. et al. 5 - Hydroxymethylcytosine signatures in cell-free DNA provide information about tumor types and stages. Cell Res 27, 1231 (2017).

#1373

High throughput isolation and expansion of circulating tumor cells (CTCs) from Non-small cell lung cancer (NSCLC) patients for personalized treatments.

Mina Zeinali,1 Maggie Lee,1 Arthi Nadhan,1 Anvya Mathur,1 Wei Huang,1 Eric Lin,1 Ramdane Harouaka,1 Max S. Wicha,1 Nallasivam Palanisamy,2 Mathias Hafner,3 Rishindra Reddy,1 Gregory P. Kalemkerian,1 Bryan J. Schneider,1 Khaled A. Hassan,1 Nithya Ramnath,1 Sunitha Nagrath1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _Henry Ford Health System, Detroit, MI;_ 3 _Institute for Medical Technology of Heidelberg University & University of Applied Sciences Mannheim, Mannheim, Germany_.

Background: Circulating tumor cells (CTCs) have emerged as important blood-based surrogate markers of primary tumors. Current methods for isolation of lung CTCs mostly rely on biomarker dependent antibody-based capture, missing populations that may be stem-like in nature.

Results: We have applied the microfluidic Labyrinth device for high throughput, label-free, size-based isolation of CTCs from non-small cell lung cancer patients (NSCLC). The Labyrinth device was optimized and tested for inertial separation of cancer cells using the human lung cancer cell line H1650. The recovery and purity were >82% and >78%, respectively, operating at a flow rate of 2.5 mL/min. Using the biomarker-independent Labyrinth separation device, heterogeneous CTC populations were isolated from metastatic NSCLC patients (n=21). Heterogeneous CTC populations were detected, including CTCs (PanCK+ and CD45-), CTCs expressing EpCAM or Vimentin, and CTCs expressing both markers representing an EMT-like population of CTCs. Using Labyrinth, we were able to isolate CTCs from 100% of patients with an average yield of 180±168 CTCs/mL. Among the captured CTCs, EpCAM- CTCs were significantly more common than EpCAM+ CTCs (115.7 vs. 39.1 CTCs/mL respectively). Cell clusters of 2 or more CTCs were also observed in 95% of patients; 79% of these clusters were negative for EpCAM expression, whereas 35% expressed Vimentin, suggestive of an EMT phenotype. Recovered CTCs from patients with RET, ROS1 and ALK rearranged tumors showed aberrations matching with the primary tumor for each gene using FISH analysis. We have successfully expanded the recovered CTCs from 2 patients and screened for therapeutic targeting. We have found that TPX-0005 might be effective in these patients and would direct them to a clinical trial using this compound.

Conclusion: The label-free Labyrinth device demonstrated the capability of collecting recovered CTCs from the device using a continuous processing technique while in a suspension state. This advantage opens the opportunities not only for CTC expansion off-chip, but also for ex-vivo drug testing to direct patient-specific therapies.

#1374

Building an effective concordance study: Plasma Next Generation Sequencing (NGS) for oncogenic fusion detection in non-small cell lung carcinoma (NSCLC).

Julianna G. Supplee,1 Marina S. Milan,1 Kristy T. Potts,2 Lynette M. Sholl,3 Tristan S. Shaffer,2 Lee P. Lim,2 Pasi A. Janne,1 Geoffrey R. Oxnard,1 Cloud P. Paweletz1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Resolution Bioscience, Inc., Bellevue, WA;_ 3 _Brigham and Women's Hospital, Boston, MA_.

Background: Non-invasive genotyping of cell-free DNA (cfDNA) is increasingly used in cancer care as tumor biopsies may be inadequate or unavailable. As clinical adaptation of liquid biopsies is driven in large part by commercial vendors that offer proprietary PCR or NGS-based tests, rigorous validation of these assays is essential to ensure maximum clinical benefit. Recent efforts to compare cfDNA diagnostics have not focused on clinically actionable mutations or used tumor to verify plasma results. The present study analyzes the concordance in reports of actionable gene fusions in NSCLC from two independent, commercial plasma NGS tests, compares the breakpoint characteristics between tissue and plasma, and highlights how differences in sequencing and bioinformatic analysis can be a source of false negatives.

Methods/Results: We studied a cohort of 169 NSCLC patients (pts) that had plasma analyzed by Guardant 360 between April 2016 and July 2018. In those with tumor positive ALK, ROS1, or RET fusions (n=16), banked cfDNA from a separate tube of plasma (69% collected within 2 weeks) underwent local testing and sequencing at DFCI using Resolution Bioscience's pre-production ctDx-Lung kit with remote bioinformatic variant calling performed by Resolution Bioscience; all parties involved were blinded to tumor genotype and Guardant results. Locked results were unblinded for post hoc analysis. The ctDx-lung kit detected 13 out 16 fusions (AF% range 80-0.3%), while Guardant360 detected 7 (AF% range 10-0.3%). Guardant360 tended to report lower AFs than ctDx-lung, though corroborating TP53 alterations were of similar AFs. Of the cases where both assays did not detect any fusions, no other shared SNVs were detected, possibly due to low shed of tumor DNA. Of the 13 cases where a fusion was detected in plasma by ctDx-lung, 4 rearrangements could be characterized as 'non-productive' due to opposite transcriptional orientations and comprised 50% (3/6) of the fusions not detected by Guardant360. Only one case detected by Guardant360 was 'non-productive'. Of the 13 cases where a fusion was detected in plasma, 89% (8/9) of pts with productive fusions and 75% of pts (3/4) with 'non-productive' fusions responded to TKI therapies. Additional unblinding is ongoing to better understand false negative cases.

Conclusions: Here, we demonstrate that a rigorous approach to benchmarking plasma genotyping assays should 1) focus on actionable mutations, 2) use tumor as a gold standard for establishing true and false positives and negatives, and 3) include orthogonal validation to address assay design and bioinformatic analyses as sources of discordance. Our study further highlights the challenges of fusion detection and interpretation and the need for platform cross-comparisons to realize the potential of liquid biopsy to increase access to personalized cancer care.

#1375

Multiplex gene expression using the HyCEAD assay in CTCs isolated with the Parsortix™ system.

David F. Englert,1 Mariya Kolesnikova,1 Nishat Zaman,1 Arianna Hustler,2 Gabrielle Wishart,2 Daniel J. O'Shannessy2. 1 _ANGLE Biosciences, Toronto, Ontario, Canada;_ 2 _ANGLE Europe Limited, Guildford, United Kingdom_.

Background: Discrimination between benign and malignant pelvic masses by gene expression profiling of circulating tumor cells (CTCs) may provide information to assist in treatment decisions and improve outcomes. CTCs can be isolated and harvested from blood based on cell size and deformability using the Parsortix™ system. Because the number of CTCs in the blood of pelvic mass patients suspected of having ovarian malignancies is likely very low, gene expression profiling of these CTCs requires a highly sensitive detection system that is tolerant of the presence of normal nucleated blood cells.

Objective: To assess the suitability of the HyCEAD assay for gene expression profiling of cells in Parsortix harvests. HyCEAD (Hybrid Capture Enrichment Amplification and Detection) is a fast and simple method for the simultaneous analysis of 100 or more mRNA species captured from cell lysates. The products of the multiplex amplification with HyCEAD can be quantified by hybridization on a flow-through chip.

Methods: Multiple HyCEAD primer/probe sets were designed for 125 relevant genes, and purified total RNA or lysates from Parsortix harvests were processed with the HyCEAD assay. Primer/probe sets were screened using total RNA from cell lines and ovarian cancer tissues. Genes with significant expression in white blood cells were culled from the gene set. A set of non-human poly-adenylated mRNA molecules (separately quantified using digital droplet PCR) were used as spike-in standards to assess absolute sensitivity. CaOV3 cells spiked into blood from normal healthy volunteers (NHVs) were used as a model system for sensitivity analyses.

Results: Many genes were identified that yielded substantially greater signal intensities in harvests from HNV blood spiked with CaOV3 cells relative to the unspiked blood samples. The expression levels could be quantified over a signal intensity range of 2.5 log10 units. Small numbers of non-human standard molecules could be detected; stochastic detection failures were observed when five or fewer molecules of the standards were assayed (dropout level). CVs in repeatability experiments were typically less than 20% with numbers of standard molecules greater than the dropout level. CVs in repeatability measurements of human genes related to ovarian cancer in spiked Parsortix harvests averaged about 25%. CVs were greater between assays of Parsortix harvests of different HNVs, reflecting in part biological variability and variation of the number of cells actually spiked into the blood and the number of cells recovered in the individual harvests.

Conclusion: The HyCEAD assay provides adequate sensitivity to simultaneously profile expression levels of more than 100 genes in small numbers of CTCs directly from the lysates of Parsortix harvests. Expression profiling is limited to genes expressed at much higher levels in CTCs than in normal nucleated blood cells.

#1376

A simple blood-based serologic assay for early detection of multiple cancers.

Yisrael Katz, Jian Zhang, Luhui Shen, Stephen A. Johnston. _Arizona Biodesign Institute, Tempe, AZ_.

BACKGROUND:Liquid biopsies for tumor DNA have limited sensitivity for early-stage disease due to paucity of circulating molecules. Wwe have focused on discovery of frameshift peptides (FSPs) produced by RNA transcription errors, not DNA mutations, as they may represent a much larger pool of neoantigens to be used as diagnostic and therapeutic targets. Our hypothesis builds on the notion that RNA level errors are more frequent than DNA, and yet more prevalent in cancer cells, resulting in larger numbers of RNA-sourced neoantigens - namely, FSPs. These are generated by insertions and deletions (INDELs) in microsatellites (MS) or by exon mis-splicing.

FSPs have become recognized as a key source of neoantigens. It has been recently shown that FSPs from INDELs of tumor coding region MSs correlate with immunotherapy response. Additionally, FSP antigens in cancer vaccine trials elicited stronger immune responses than did neoantigens from DNA point mutations. We hypothesized that FSPs may induce an immune response in cancer patients with distinct reactivity signatures, which could then be analyzed as a diagnostic biomarker.

METHODS: The human genome has ~220,000 possible FSPs resulting from exon mis-splicing and ~14,000 from INDELs in MSs, totaling ~234,000 potential FSP neoantigens. Our group invented a micropeptide array containing all possible FSPs inferred by informatics, represented by 400,000 15-mer peptides on an array, designed for detecting high-affinity, cognate antibody-peptide binding. Sera from human patients were analyzed for fluorescent intensity, and samples with normalized values larger than standardized cutoffs were counted as positive. Archived blood samples were obtained from prospective screening trials, enabling analysis of blood obtained prior to clinical presentation and diagnosis.RESULTS: Pre-Stage 1 Melanoma vs. Controls: 20 female patients with pre-stage 1 melanoma (blood collected 0-6 months prior to diagnosis) were compared with 20 age-matched female controls, yielding 100% diagnostic accuracy. Stage 1 Breast Cancer vs. Controls: 42 patients with confirmed stage I breast cancer were compared with 65 non-cancer age-matched controls, yielding 95% diagnostic accuracy and 99% specificity. Pre-Stage 1 Breast Cancer vs. Controls: 64 patients with pre-stage I breast cancer (blood collected 0-6 months prior to diagnosis) and 40 age-matched controls were compared, yielding 83% sensitivity and 95% specificity, with 87.5% accuracy.

CONCLUSIONS:Harnessing the natural signal amplification of antibody synthesis, we have created a simple blood-based serologic assay screen for anti-FSP reactivity in stage I and pre-stage disease, with high accuracy and sensitivity for pre-clinical disease. This represents a novel platform for early diagnosis, as well as identification of neoantigens for cancer vaccines.

#1377

Improved ctDNA detection in early stage non-small-cell lung cancer.

Katrin Heider,1 Jonathan C. Wan,1 Davina Gale,1 Florent C. Mouliere,2 Wendi Qian,3 Angels Kateb,3 Gail Doughton,3 Nicola Ramenatte,3 Ruth Tysoe,3 Christopher G. Smith,1 Doris M. Rassl,4 Susan Harden,5 Robert C. Rintoul,6 Charles Massie,1 Nitzan Rosenfeld1. 1 _CRUK Cambridge Institute, Cambridge, United Kingdom;_ 2 _Amsterdam UMC, Amsterdam, Netherlands;_ 3 _Cambridge Clinical Trials Unit – Cancer Theme, Cambridge, United Kingdom;_ 4 _Royal Papworth Hospital NHS Foundation Trust, Cambridge, United Kingdom;_ 5 _Addenbrooke's Hospital, Cambridge, United Kingdom;_ 6 _Department of Oncology, University of Cambridge Hutchison–MRC Research Centre, Cambridge, United Kingdom_.

Overall survival of non-small-cell lung cancer (NSCLC) patients remains poor as patients are frequently diagnosed at a late stage. The evaluation of circulating tumour DNA (ctDNA) has been shown to offer a non-invasive method for detection of cancer. However, detection rates of ctDNA in patients with early stage cancers, including NSCLC, have been limited due to sampling and sensitivity issues. We developed a novel algorithm for INtegration of VAriant Reads (INVAR), which uses sequencing data across hundreds to thousands of tumour-mutated loci to detect ctDNA in plasma samples at high sensitivity. We applied this to a cohort of stage I-III NSCLC patients recruited in the LUCID study. LUCID is a prospective and observational study of 100 stage I-IIIB NSCLC who are planning to undergo radical treatment (surgery or radiotherapy +/- chemotherapy) with curative intent. Plasma samples were collected before and after treatment with curative intent. We analysed a total of 50 patients using patient specific-sequencing panels and detected ctDNA in 78% of cases before treatment, at ctDNA fractions as low as 1.7x10-5. For 17 of those patients staging information was available. Here, we detected ctDNA in 50% of stage I patients (split evenly between stages IA and B) and 100% of stage II and III patients. We also applied INVAR to whole exome and shallow whole genome sequencing data from plasma samples, and showed that this algorithm can be used to detect low ctDNA fractions in such data. Our findings highlight an opportunity to improve ctDNA detection in early stage NSCLC by using patient specific sequencing information. Additionally, our algorithm has the potential to aid in longitudinal cancer monitoring and is applicable to a variety of sequencing data types. We aim to apply this approach to serial samples obtained through the LUCID study to investigate its application in the treatment management.

#1378

**Digital PCR probe library for** TP53 **mutations in early relapse prediction.**

Satoshi S. Nishizuka,1 Mizunori Yaegashi,2 Noriyuki Sasaki,2 Takeshi Iwaya2. 1 _Iwate Medical University Institute of Biomedical Sciences, Morioka, Japan;_ 2 _Iwate Medical University School of Medicine, Morioka, Japan_.

Precise relapse prediction in cancer patients is urgently needed as conventional serum tumor markers do not predict relapse in a timely manner. Circulating tumor DNA (ctDNA) is a new class of quantitative biomarkers that reflect tumor burden. The ctDNA is usually found as mutated DNA fragments in the blood, which are generally consistent with the mutations found in the concomitant tumor in the body. Variant allele frequency (VAF) of ctDNA is usually very low and ranges from 0.001 to 1% even in advanced cases. To quantify absolute copy number of ctDNA at the very low range, digital PCR (dPCR) is one of the most practical methods. However, specific primers/probes for dPCR must be designed individually as tumor mutation varies between individual patients. The TP53 gene is the most frequently mutated in human cancer and 90% of the mutations occur in its DNA binding domain coding region. According to the COSMIC database, there are 1,284 unique mutations from a total of 25,376 recorded mutations in the DNA binding domain. The majority (77%) of the 1,284 unique mutations are not recurrent and thus 42 unique mutations cover 50%, 132 unique mutations cover 70%, and 426 unique mutations cover 90% of the total mutations. Here we have designed a dPCR probe library for the set of 132 unique mutations to cover 70% of TP53 mutations in the DNA binding domain using Hypercool Primer & ProbeTM technology. With this technology, modified bases, 2-Amino-dA and 5-Methyl-dC, were substituted in adenine and cytosine positions, respectively. This modification increases the Tm, thus allowing for highly specific amplification despite short PCR products of 70-90 bp. The dPCR library allows "off-the-shelf", highly sensitive ctDNA tracking once the TP53 mutation is identified in the target tumor (e.g., surgical specimens). In our pilot cases, we found TP53 mutations from 97% (34/35) of esophagus, 70% (7/10) of stomach, and 93% (13/14) of colorectal primary tumors using next generation sequencer (NGS). There were 40 unique mutations for which all mutations were covered with our TP53 dPCR probe library. We then validated the dPCR library for ctDNA in terms of: (a) the ability to detect the target mutation; (b) tumor genomic heterogeneity; and (c) early detection of relapse. Of >500 DNA samples from primary cancers and patient plasma, 98% of probes tested were confirmed to detect the target mutation properly. Multiregion sequencing of stomach and colon cancers by NGS revealed that 85% (17/20) of tumors exhibited TP53 founder mutations, offering a high chance that the corresponding ctDNA was detected in plasma. Among cases who experienced relapse confirmed by CT scan, ctDNA elevation was seen approximately 6 month prior to the confirmation by CT scan. Overall, the sensitivity of VAF using dPCR with the probes was as low as 0.001%. These results suggest that the TP53 dPCR probe library can potentially be used for a wide range of human cancers as a new class of biomarkers for early relapse prediction.

#1379

Insights into analytical factors impacting variant concordance of plasma-tumor NGS testing.

Daniel Stetson. _AstraZeneca R &D Boston, Waltham, MA_.

Next generation sequencing (NGS) of circulating free DNA (cfDNA) is transforming the treatment paradigm in oncology. Sensitive non-invasive cfDNA assays could revolutionize clinical trial design with improved patient safety. However, there are substantial challenges to working with cfDNA, particularly in an early-stage setting. It has been shown that solid tumors from early stage disease shed less DNA into the bloodstream making the detection of somatic variants difficult. Because of this, the current landscape of commercial cfDNA assays are focused on advanced stage patients and may lack the sensitivity and specificity to interrogate early stage samples. To understand the limitations of the currently available cfDNA assays on early-stage oncology samples, we have designed several studies to examine the factors that impact the concordance of plasma-tumor testing and hinder the ability to detect low frequency somatic variants. Three comparison studies have been completed examining both the inter- and intra- variability of commercially available plasma-based NGS assays. Two inter-vendor studies utilized matched sets of 24 tumor-plasma samples purchased from a commercial bio-banking company. The plasmas were aliquoted into 2 ml volumes and sent to the commercial vendor for sequencing with the currently available cfDNA assay, while the tumor-normal tissues were sequenced by an external tumor sequencing vendor. The unique study design allowed for confident determination of true positive, false positive and importantly, false negative variants from the reported plasma variants. The third comparison study also used commercially purchased early-stage patient and normal samples to examine the intra-vendor assay reproducibility. Summarizing the results from all three studies, we found that the majority of discordant variants were from technical rather than biological factors. The technical factors identified include mutational bias, bioinformatic filtering, and variant annotation, while the biological factors found were clonal hematopoiesis and tumor heterogeneity. Through a detailed examination of the discordant variants from the first inter-vendor comparison, we believe that less than 15% of discordance was from biological sources. Overall these findings differ from the current literature which suggests that the sub-clonal nature of a tumor is the cause of most discordance in plasma testing. We have begun to establish guidelines to identify these technical factors in our current and past datasets, with the hope of liquid biopsy testing improvement for early-stage cancer patients.

#1380

Clinical relevance of cell-free DNA analysis for bile juice assessed through amplicon-based deep sequencing in bile duct and pancreatic cancer patients with obstructive jaundice.

Marie Muramatsu, Marie Muramatsu, Hiromichi Ito, Hitoshi Zembutsu. _Japanese Foundation For Cancer Research, Ariake,Koto, Japan_.

Personalized treatment for patients with pancreatic or bile duct cancer is limited because cancer cells could not be detected in some patient. Cell-free DNA (cfDNA) sequencing is expected to enable a precision oncology approach for these patients. The development of gene panel for cfDNA covering hundreds of mutation hotspots is important for the establishment of clinically practical cfDNA detection system. We enrolled 40 patients with bile duct or pancreatic cancer which caused obstructive jaundice. Amplicon-based genomic profiling of 14 genes, which are commonly mutated in digestive cancer, in preoperative bile juice by next-generation sequencing (NGS) was carried out to evaluate the feasibility of this assay, and was compared with their clinical parameters. One or more somatic mutation(s) of the 14 genes in bile cfDNA were detected in 25 patients (62.5%) with bile duct or pancreatic cancer. Mutations in TP53, KRAS and APC genes were detected in 19 (47.5%), 11 (27.5%) and 4 (10.0%) patients, respectively. Mutant allele frequencies in plasma were not associated with CEA nor CA19-9. Of the 40 patients with bile duct or pancreatic cancer, the cancer cells could be detected by cytology in 18 patients (45.0%). On the other hand, mutant alleles from cancer cells could be detected in 14 patients who showed negative cytology. Hence, 32 patients (80.0%) could be diagnosed as cancer before surgical operation by using the information from liquid biopsy (cfDNA analysis using bile juice) in addition to that from cytology. Our data confirmed that mutant allele in bile cfDNA could be sensitively detected by amplicon-based NGS system. These results suggest that the detection of cfDNA in bile juice using high-sensitive deep sequencing system could be a novel diagnostic tool, and these technologies could increase the ratio of definitive diagnosis of refractory cancer such as bile duct or pancreatic cancer.

#1381

Technical validation of Roche AVENIO sequencing platform for liquid biopsies.

Daan C. Vessies, Theodora C. Linders, Kalpana L. Ramkisoensing, Petra M. Nederlof, Gerrit A. Meijer, Kim Monkhorst, Daan van den Broek. _Netherlands Cancer Institute, Amsterdam, Netherlands_.

Purpose

Circulating cell-free tumor DNA (ctDNA) is rapidly gaining attention as a valuable biosource for the detection and diagnosis of multiple types of cancer. ctDNA can be easily obtained through a minimally invasive liquid biopsy. Multiple NGS-based methods have been developed specifically for application in liquid biopsies.

Before such methods can be implemented in clinical care, they need to be technically validated through measurement of independent reference standards. In the current project we technically validated the Roche AVENIO ctDNA Assay, which is based on the CAPP-seq technology [Newman AM, et al. Nat Biotechnol. 2016;34:547-55]. Its Targeted Kit interrogates 17 genes for Single Nucleotide Variants (SNVs), Insertions and Deletions (InDels), Copy Number Variations (CNVs) and gene fusions in a single workflow.

Methods

The detection of SNVs, CNVs, Indels and gene fusions was evaluated as follows:

\- SeraCare Seraseq ctDNA Complete was analyzed in four replicates at 2.5%, 0.5% and 0.1% mutant allele frequency (AF), and at 50ng and 10ng input. This material contains SNVs, InDels, CNVs and gene fusions.

\- An in-house characterized reference pool of patient plasma, containing SNVs and InDels at various AF (0.07% - 5.51%), was analyzed in four replicates at 50ng and 10ng input.

\- Plasma and serum samples obtained from patients with tissue confirmed presence of CNV were used to validate the robustness of CNV detection. Twelve samples were selected to represent a range of background DNA (serum had 2-4 times higher wildtype DNA levels compared to plasma), amplification levels (CNV levels 1.4-fold to 4.2-fold by ddPCR) and total input (10-50ng cfDNA input).

\- Plasma obtained from five patients with EML4-ALK gene fusion confirmed on tissue was used to further validate fusion detection.

Sequencing was performed on an Illumina NextSeq on High Output mode with sixteen samples per run (20-33M read pairs per sample).

Results

Sensitivity for detection of SNVs and InDels was 100% for all variants down to 0.18% AF when using 50ng input, and 50% for variants at 0.10% AF. For 10ng input the variants were detected down to 0.60% AF with sensitivity of 92%. For variants with AF between 0.07% and 0.53%, sensitivity was 67%.

Results from twelve plasma and serum samples obtained from patients with EGFR CNV were 100% concordant with ddPCR results. These results were confirmed in SeraCare samples at 10ng and 50ng input.

Sensitivity of fusion detection in SeraCare material was 100% for AF of 0.5% and 50ng input, and 38% for input of 10ng. No fusions were detected at 50ng or 10ng input for AF of 0.1%. We furthermore confirmed 3 out of the 5 fusions found in tissue.

Conclusions

We have determined the lower limits of detection for SNVs, CNVs and gene fusions. Sensitivity was dependent on DNA input with performance optimized at 50ng.

Our results confirm the feasibility of in-house use of the AVENIO ctDNA Targeted Kit for broad molecular profiling in liquid biopsies.

#1382

Megakaryocyte potentiating factor as a predictive biomarker for therapies against malignant mesothelioma.

Liang Cao, Yunkai Yu, Anish. Thomas, Jingli Zhang, Masanori Onda, Paul Meltzer, Raffit Hassan, Ira Pastan. _National Cancer Inst., Bethesda, MD_.

Objectives: Effective biomarkers for malignant mesothelioma (MM) are needed for clinical management and the development of cell surface mesothelin-targeted therapies. We evaluated serum megakaryocyte potentiating factor (MPF) as a biomarker predictive of clinical outcome in MM patients, for patient monitoring, and for developing mesothelin-targeted therapies.

Patients and Methods: Serial serum samples from MM patients in two clinical trials of an anti-mesothelin immunotoxin were tested for serum MPF. For the purpose, we developed a new MPF assay and conducted both analytical and clinical validation. Correlative studies were performed to determine the test effectiveness in treatment monitoring and outcome prediction. MPF was further evaluated for an association with response to an anti-mesothelin therapy and for disease monitoring.

Results: Our data showed that in MM patients, elevated serum MPF is a predictor of poor survival with a hazard ratio (HR) = 2.46 (Log Rank P = 0.003; n = 95). In refractory MM patients, elevated MPF is a strong predictor of poor outcome with a HR = 6.12 (Log Rank P = 0.0007; n = 57). There was a significant reduction of serum MPF in patients with elevated baseline and radiological response, with an average change from -52% to -78% after 1 to 6 cycles. Using a -50% change as the cutoff, MM patients with a positive MPF-response had significantly improved progression-free survival (PFS) (P < 0.0001), with median extended from 1.9 to 11.3 months. These MPF-response patients further exhibited improved overall survival (OS) (P = 0.004), with median extended from 8.8 to 22.3 months. In refractory MM patients, there was an association between elevated pre-treatment serum MPF and radiological response to an experimental anti-mesothelin therapy (P = 0.033). Furthermore, in these response patients, serum MPF was monitored between 32.2 and 63.8 months and found to reflect the response and disease progression.

Conclusion: In MM patients, elevated MPF was observed in 2/3rd of the patients and is a predictor for poor survival, throughout the course of the disease. Following systemic therapies, a > -50% reduction in MPF was associated with improved clinical outcome, both PFS and OS. An elevated baseline serum MPF may be a predictor of response to an experimental anti-mesothelin therapy in refractory MM patients, however, the finding needs to be confirmed in a larger study.

#1383

Isolating circulating tumor cells from a large screening blood volume: A pilot study using diagnostic leukapheresis.

Liang Dong, Zhongyuan Zhang, Changxue Lu, Diane Reyes, Amber de Groot, Sarah R. Amend, Jun Luo, Kenneth J. Pienta. _Johns Hopkins University School of Medicine, Baltimore, MD_.

Purpose: The presence of circulating tumor cells (CTC) in blood is associated with poor prognosis in metastatic prostate cancer (mPCa) patients. For non-metastatic PCa patients and patients with biochemical recurrence (BCR), it is extremely difficult to find CTCs in 5-10 ml of peripheral blood (PB). The purpose of this study is to evaluate the feasibility of using diagnostic leukapheresis (LP) followed by other enrichment technologies to increase the CTC yield by using a large screening blood volume.

Methods: Blood samples were collected from one healthy donor and one mPCa patient. During LP procedure, different blood elements were separated to different layers based on their densities. The mononuclear cell layer (that also contains CTCs) was collected. To assess recovery rate of different CTC isolation and detection methods, the healthy donor LP was spiked with a known number of PCa cell line LNCaP cells. Three CTC isolation and detection methods were used, including FAST (Clinomics, Ulsan, South Korea), a size-selection platform followed by immunofluorescence (IF); the AccuCyte-CyteFinder system (RareCyte, Inc., Seattle, WA), a selection-free method that utilizes IF; the AlereTM CTC AdnaTest (AdnaGen, Langenhagen, Germany), an EpCAM positive selection method followed by RT-qPCR. The criteria for defining a CTC are DAPI+, CK/EpCAM+, and CD45-. The Ct value thresholds for positive CTC signal by RT-qPCR were EpCAM 36.4, PSA 36.3, HOXB13 40, AR 35.4.

Results: For the healthy donor LP with spiked LNCaP cells, the CTC recovery rate from FAST was 53.7%~67.2%. For Adna test, 0, 44, 95 and 1000 LNCaP cells were spiked in 5 ml of LP respectively. The Ct values of EpCAM were 36.28, 29.32, 28.22 and 24.76. For the mPCa patient, two tubes of 7.5 ml of PB were collected for FAST and RareCyte. The FAST found 3 CTCs while the RareCyte found 1 CTC. An LP sample was also collected from the same mPCa patient. The LP sample volume was 162 ml (WBC concentration was 83×109/L). Triplicates of 5 ml of samples were sent through AdnaTest and CTCs were identified in all samples (Ct: EpCAM 34.21±0.77, PSA 33.86±1.81, HOXB13 36.25±0.34, AR 35.29±0.22). Then the remainder of the mPCa LP was subjected to Ficoll-Paque density centrifugation to further concentrate mononuclear cells to a 90 ml volume. 1 ml samples were then applied to FAST in triplicate. The CTC numbers were 22 and 15. The density of cells on one of the filters was too high to be successfully scanned.

Conclusion: Finding CTCs in LP samples by size-based technology or AdnaTest is feasible and offers a promising method to do liquid biopsy for mPCa patients. Screening a larger volume of PB by LP has significantly increased the CTC yield compared to using the same capture technology on 7.5 ml of PB.

#1384

nCounter for detection of clinically relevant alterations in liquid biopsies of solid tumor patients.

Ana A. Giménez-Capitán,1 Chung-Ying Huang,2 Jill Bracht,1 Rich Boykin,2 Clara Mayo-de-las-Casas,1 Joseph M. Beechem,2 Cristina Teixidó,3 Ariadna Balada-Bel,1 Beatriz Garcia-,1 Sergio Villatoro,1 Monica Garzón,1 Nuria Jordana-Ariza,1 Cristina Aguado,1 Santiago Viteri,1 Juan José García,1 Rafael Rosell,4 Jay Gerlach,2 Noemi Reguart,3 Miguel Angel Molina-Vila1. 1 _Quiron Dexeus University Hospital, Barcelona, Spain;_ 2 _Nanostring Technologies, Seattle, WA;_ 3 _Clinic Hospital, Barcelona, Spain;_ 4 _Institut Catalá d'Oncologia, Badalona, Spain_.

Background: With the advent of precision medicine, screening for clinically relevant mutations and gene fusions is mandatory in many tumor types. However, in a significant number of cancer patients, the tumor tissue available is insufficient for genetic analysis. In addition, repeated tissue biopsies for monitoring the course of the disease and the emergence of mechanisms of resistance to targeted therapies are not feasible. Liquid biopsies constitute the only alternative available in these cases. The nCounter technology has been adapted to detect mutations and gene fusions in FFPE biopsies from cancer patients with a minimum requirement of tumor material and sample handling, a short turnaround time and a straightforward data analysis. However, nCounter has not been tested in liquid biopsy samples.

Methods: For mutation analysis, the SNV Solid Tumor Panel was used, which allows for detection of 97 driver mutations in 24 genes. For fusions, a customized panel for ALK, ROS1, RET, and NTRK1 fusion transcripts was used with a 14-cycles preamp step. First, proof-of-concept experiments were run by spiking plasma samples with a mixture of genomic DNAs or RNAs from positive cell lines. Next, 65 circulating-free DNA (cfDNA) samples from advanced cancer patients, previously genotyped by other techniques, were analyzed using the SNV panel. Of those, 60 had been purified from plasma, 4 from ascites and 1 from the pleural effusion. Nineteen were positive for EGFR mutations, 20 for KRAS, 13 for BRAF, 5 for PIK3CA, 2 for NRAS, and 6 were pan-negative. Finally, 8 circulating cell-free RNA samples isolated from plasma were tested with the nCounter Low RNA Input Kit and the lung fusion panel. Of those, 6 corresponded to lung cancer patients harboring ALK or ROS1 rearrangements in tumor tissue, but previous RT-PCR only detected fusions transcripts in 2.

Results: Spiking experiments revealed that the nCounter SNV Panel was able to detect mutations at allelic fractions as low as 0.2% for most of the drivers. When testing liquid biopsies, 63/65 cfDNA samples from cancer patients were evaluable, despite having DNA concentrations lower than 1 ng/µL. The SNV Panel successfully detected EGFR, KRAS, BRAF, PIK3CA and NRAS mutations with a concordance rate of 97.5% with previous genotyping by NGS, Therascreen® or Taqman® with PNA, corresponding to a Cohen's kappa of 0,913. In the case of the lung fusion panel, ALK, ROS1 and RET fusion transcripts were detected in all spiked plasma cfRNA. Two samples from lung cancer patients with positive RT-PCR results were also detected by the nCounter low-input lung fusion panel. Research is ongoing to further improve the performance of the nCounter low-input fusion panel in liquid biopsy samples.

Conclusions: Our results demonstrate the feasibility of mutation analysis in the cfDNA of advanced cancer patients using nCounter. The nCounter technology also shows promise for the detection of gene fusions in cfRNA

#1385

Prognostic and predictive utility of copy number variations (CNVs) in circulating tumor DNA (ctDNA) from metastatic castration-resistant prostate cancer (mCRPC) patients.

Heidi Fettke,1 Edmond M. Kwan,2 Jianjun Yu,3 Amy Wang,3 Carlos Montesinos,3 Calvin Wong,3 Xue Gong,3 Tiantian Zheng,3 Peter Pan Du,3 Shidong Jia,3 Andrew Mant,4 Phillip Parente,4 Carmel Pezaro,5 Arun A. Azad6. 1 _Monash University, Melbourne, Australia;_ 2 _Monash Health, Melbourne, Australia;_ 3 _Predicine Inc., Hayward, CA;_ 4 _Eastern Health Clinical School, Melbourne, Australia;_ 5 _Sheffield Teaching Hospitals NHS Foundation Trust, Sheffield, United Kingdom;_ 6 _Peter MacCallum Cancer Centre, Melbourne, Australia_.

Background: Due to difficulties with routine metastatic tissue biopsy in mCRPC, the identification of prognostic and predictive biomarkers for treatment with androgen-receptor signalling inhibitors (ARSI) and chemotherapy remains an unmet clinical need. Plasma circulating tumor DNA (ctDNA) has emerged as a promising minimally-invasive tool with which to interrogate the cancer genome. However, detection of copy number variations (CNVs) in ctDNA has proved challenging. This poses a major problem in mCRPC, which commonly harbours pathogenic CNVs. Here we use an ultra-sensitive next-generation sequencing (NGS) assay to identify CNVs in ctDNA from mCRPC patients and correlate findings with clinical outcomes in men commencing ARSI (abiraterone or enzalutamide) or chemotherapy (docetaxel or cabazitaxel).

Methods: mCRPC patients commencing ARSI or chemotherapy were prospectively recruited at two Australian centers. Plasma was collected and platelet poor plasma (PPP) fractions were processed uniformly and cell-free DNA (cfDNA) extracted. Plasma samples were analysed using the PredicineLITE NGS assay, which reports genomic alterations in 90 cancer genes. CNVs from this cohort were correlated with PSA response rate (Fisher's exact test), PSA progression-free survival (PSA-PFS), clinical/radiographic progression-free survival (clinical/rPFS), and overall survival (OS).

Results: Median follow-up was 19.85 months (mo) (IQR 12.5-23.0). In total, 32 pre-treatment samples were analyzed (7 chemotherapy, 25 ARSI). The most common CNVs were PTEN loss (n=12, 38% of cohort), RB1 loss (n=5, 16%) and AR gain (n=12, 38%). Notably, OS was decreased in patients with PTEN loss (median 9.7 mo vs. not reached; p=0.03) and RB1 loss (median 7.1 mo vs. 17.1 mo; p=0.1), while PSA response rates were also lower in RB1 loss (1/5, 20% vs. 19/27, 71%; p=0.053). In addition, AR copy number gain was associated with decreased clinical/rPFS (median 3.4 mo vs. 10.7 mo; p=0.05) and inferior OS (median 9.7 mo vs. not reached; p=0.05).

Conclusion: Using an ultra-sensitive NGS assay, we demonstrate the robust detection of CNVs in plasma ctDNA of patients with mCRPC. Copy number losses of PTEN and RB1, and gains of AR were associated with worse clinical outcomes on chemotherapy and ARSI. Validation in a larger cohort is ongoing.

#1386

**Application of PAXgene** ® **Blood ccfDNA Tubes* as a standardized approach for therapy monitoring in late-stage lung cancer.**

Bernd Schmidt,1 Erkan Arslan,2 Dana Reinicke,3 Thorsten Voss,4 Andrea Ullius,4 Stephan Eisenmann,3 Michael Fleischhacker2. 1 _Universitätsklinikum Halle/Saale, Med. Klinik I; DRK Kliniken Berlin, Klinik für Innere Medizin, Germany;_ 2 _DRK Kliniken Berlin, Klinik für Innere Medizin – Schwerpunkt Pneumologie, Germany;_ 3 _Universitätsklinikum Halle/Saale, Med. Klinik I, Schwerpunkt Pneumologie, Germany;_ 4 _QIAGEN GmbH, Hilden, Germany_.

Introduction In June 2016, the FDA approved the first blood-based genetic test to detect gene mutations of epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC). With this move, the genetic characterization of cell-free DNA became a routine application for the care of NSCLC patients. Nevertheless, there are many unresolved preanalytical issues. Particularly important is defining the optimal method for blood draw and sample handling before plasma preparation. The aim of this research study is to answer two questions: - Would the PAXgene Blood ccfDNA Tube* (PAXgene Tubes) be a viable alternative to EDTA blood tubes as the current gold standard? - Could a longitudinal analysis of methylated cell-free plasma DNA allow predicting response to NSCLC treatment?

Experimental procedures To date, 45 advanced-stage lung cancer patients were enrolled in this ongoing research study. Blood samples from 17 were used for the first study part to compare the quantity of plasma mPTGER4 and mSHOX2 DNA in both tubes types. At diagnosis, blood from these therapy-naive subjects was drawn into EDTA and PAXgene tubes. The EDTA tubes were processed immediately (within 4 h after blood draw) and spun twice to separate the plasma. The PAXgene tubes were stored for up to 7 days at room temperature (15-25°C) before processing to mimic transport between clinical and laboratory sites. All plasma samples were stored at --80°C before DNA isolation and quantification of the two methylated sequences by real-time PCR. For the longitudinal study, blood was drawn from 28 subjects into PAXgene tubes only in approximately one week intervals, starting before therapy until conventional re-staging. The quantity of mPTGER4 and mSHOX2 plasma DNA was measured as in the comparative research study.

Summary of data The amount of plasma mPTGER4 and mSHOX2 DNA in EDTA and PAXgene tubes was completely congruent for both markers in all 17 samples of the first study part. The analysis of the two methylation markers in the second study part allowed predicting response/non-response to the given therapy in 25/28 subjects (88%), when compared to standard computer tomography-based re-staging. PCR results show that an assessment of therapy success could already be possible within two to three weeks after therapy start.

Conclusions PAXgene tubes stabilize blood cells and thus prevent a dilution of cell-free DNA by genomic DNA from white blood cells. No change in the amount of tumor-derived cell-free DNA was seen. Therefore, PAXgene Blood ccfDNA Tubes* could be a good alternative to EDTA tubes. Therapy monitoring by longitudinal measurements of mPTGER4 and mSHOX2 plasma DNA could be several weeks faster than re-staging by the currently used digital imaging method. *The PAXgene Blood ccfDNA Tube is for Research Use Only in the US.

#1387

Tracking minimal residual disease in post-operative cell-free DNA using MSK-ACCESS.

Maysun M. Hasan, Juber Patel, Ian Johnson, Fanli Meng, Grittney K. Tam, Xiaohong Jing, Julie L. Yang, A. Rose Brannon, Jayakumaran Gowtham, Dennis P. Stephens, Monica Diosdado, Ryma Benayed, Ahmet Zehir, Chin-Tung Chen, Martin R. Weiser, Dana Tsui, Brian Houck-Loomis, Michael Berger. _Memorial Sloan Kettering, New York, NY_.

A noted clinical application for liquid biopsy is as a non-invasive method of detecting and monitoring of minimal residual disease (MRD) in patients with cancer. The low concentration of circulating tumor DNA in blood, especially in early stage cancers, however, complicates the detection of tumor-derived cell-free DNA. To address this challenge, we have developed MSK-ACCESS (Analysis of Circulating cfDNA to Examine Somatic Status), a custom NGS assay covering selected exons from 129 cancer related genes for high-sensitivity detection of somatic mutations from plasma. Using ultra-high depth sequencing, with duplex unique molecular indexing (UMI), unique dual sample barcodes, and background error suppression, MSK-ACCESS is able to detect low-frequency (0.1%) variants with high confidence. The design of MSK-ACCESS leverages our dataset of more than 30,000 tumors profiled by our institutional tumor sequencing assay, MSK-IMPACT, ensuring that the majority of patients harbor multiple mutations that can be tracked in plasma.

We have validated MSK-ACCESS using plasma samples collected from 40 healthy individuals and 70 cancer patients harboring a range of somatic mutations in 11 genes. Greater than 95% of mutations at allele fractions >0.1% were empirically detected, and we established the performance characteristics of the assay through intra- and inter-assay reproducibility tests and dilution experiments. We have initiated clinical trials in multiple tumor types to evaluate the benefit of early therapeutic intervention in patients where MSK-ACCESS can detect circulating tumor DNA following surgery. Tumor mutations revealed by MSK-IMPACT in surgically resected specimens will be monitored at regular intervals as evidence of MRD. As a proof of concept, we have applied MSK-ACCESS to monitor variants known from tissue tumor sequencing in pre- and post-surgical cfDNA samples from 9 colon adenocarcinoma patients. All samples were sequenced to an average total depth of approximately 20,000X coverage and subsequently collapsed to consensus sequences exhibiting an average noise level less than 0.0006%. Circulating tumor DNA was detected in 66%(6/9) of the pre-surgical samples. Of these samples, ctDNA was also detected in 50% (3/6) of the post-surgical samples. Overall, this study shows that MSK-ACCESS can be used to successfully detect MRD.

#1388

A novel Hyper-Methylation Outlier method for blood biomarker discovery.

Bradley M. Downs, Juanjuan Li, Mary Jo Fackler, Antonio C. Wolff, Saraswati Sukumar, Chris B. Umbricht, Leslie M. Cope. _Johns Hopkins Univ. School of Medicine, Baltimore, MD_.

Introduction: DNA methylation analysis of circulating cell-free DNA in blood enables minimally invasive detection of screening early stage cancer, and monitoring of changes in tumor burden. However, there is no accepted best practice for developing blood cancer markers. In this study, we tested the ability of different methods to discover serum methylation biomarkers from breast tissue methylation arrays. We compare the ability of two previously published biomarker discovery programs, Limma and iEVORA, with a novel Hyper-methylation Outlier method to identify makers from breast tissues that can detect tumor cfDNA in serum.

Experimental Design: The Hyper-Methylation Outlier method approach was designed to be robust to loss of signal associated with dilution from non-tumor DNA in blood. Candidate CpG sites were selected to meet two criteria in training data from primary tissue: A) At least 95% of healthy control samples have methylation beta values <0.10 and, B) at least one tumor sample is a distinct outlier, with beta value >0.30, and prioritized according to the number of outlying tumor samples. A serum sample is called positive if it is an outlier for at least one marker, using a threshold of beta > 0.20 to help account for the expected loss of signal. The three marker discovery methods were used to discover cancer markers using two sets of breast tissue samples. Set 1 included 103 primary breast cancers, 6 normal-adjacent breast tissues, and 15 normal breast organoids, while set 2 included 236 primary breast cancer and 27 normal breast tissue samples. While the Hyper-Methylation Outlier method has a built-in classifier, Limma and iEVORA were paired with logistic regression and random forest, to develop classifiers. Selected markers were tested in a simulation in which 6,400 breast cancer serum samples were generated in silico by mixing tumor DNA methylation profiles with profiles from normal serum at various dilutions. We then compared the performance of the various methods, an independent set of normal serum (n= 67) along with a small number (n=6) of metastatic breast cancer serum samples.

Results: Limma paired with random forest, iEVORA paired with logistic regression and the Hyper-Methylation Outlier method performed similarly in the in silico cancer serum set. All three methods exhibited a sensitivity of over 90%, at dilutions in the range of 80-50% breast cancer to normal serum. In the independent set of normal and metastatic breast cancer serum samples, the Hyper-Methylation Outlier method achieved an AUC of 0.819 and a classifier built by pairing iEVORA paired with logistic regression set achieved an AUC of 0.820.

Conclusions: We have shown that two discovery methods that identify outlier markers, iEVORA and the Hyper-Methylated Outlier method, have the ability to discover blood cancer markers from cancer tissue arrays. However, these markers need further technical validation before their true utility is fully understood.

#1389

Single-cell sequencing to identify genetic variant accelerating shedding of CTCs into peripheral blood in gastric cancer patients.

Yilin Li,1 Xiaotian Zhang,1 Jifang Gong,1 Daisy Dandan Wang,2 Mingxia Qi,3 Yanyan Li,1 Dan Liu,1 Chunxiang Wang,3 Peter Ping Lin,2 Lin Shen1. 1 _Peking Univ. Cancer Hospital & Institute, Bejing, China; _2 _Cytelligen, San Diego, CA;_ 3 _CoWin Bioscience Inc., Boston, MA_.

Circulating tumor cells (CTCs) are shed from primary or metastatic tumors into blood circulation, which may carry specific genetic characteristics related to hematogenous metastasis. Drawbacks of conventional EpCAM or cell size-dependent CTC detection and relevant single-cell sequencing technologies have limited discovery of specific genetic variants in CTCs. In the current study, a novel integrated SE-iFISH strategy, despite cell size or epithelial marker status, was applied to detect CTCs in advanced gastric cancer (GC) patients. Obtained CTCs were classified into diverse subtypes upon different chromosome ploidy. Fifty-three aneuploid CTCs identified by iFISH in 6 of naïve GC patients were isolated by a novel non-laser microscopic single cell manipulator (NMSCM), and subjected to modified Multiple Displacement Amplification (MDA) and subsequent single-cell sequencing. Compared with the conventional PCR, MDA generates 2 to 100-kb products with a lower error rate and achieves high fidelity whole genome amplification. Sequencing targeting 50 tumor genes of single CTC as well as paired primary tumor specimens was performed. Moreover, leukocytes genomic DNA as germline controls were included. Obtained results showed that high frequent SNV in genes such as TP53 were detected in both CTCs and the paired primary tumor mass. Furthermore, CTCs had considerable

genetic heterogeneity compared to the paired primary tumor lesions. In particular, the CDKN2AA68V variant is frequently detected in CTCs but the paired primary GC tumor. We propose that the specific CDKN2AA68V variant carried by GC CTCs with high frequency, but low in the examined whole population of cancer cells in paired primary tumor lesions, may serve as the driver to promote CTCs shed into peripheral blood and to further facilitate hematogenous metastasis. Systematic in vitro and ex vivo studies are in progress to investigate roles of CDKN2A mutation in hematogenous dissemination in GC.

#1390

Molecular characterization of circulating tumor cells in cholangiocarcinoma patients: A new tool for treatment management.

Carolina Reduzzi, Marta Vismara, Marco Silvestri, Monica Niger, Rosita Motta, Giorgia Peverelli, Patrizia Miodini, Luigi Celio, Filippo De Braud, Maria G. Daidone, Vera Cappelletti. _Fondazione IRCCS Istituto Nazionale dei Tumori di Milano, Milano, Italy_.

BACKGROUND: Cholangiocarcinoma (CCA) is a highly fatal disease mainly treated with standard chemotherapy, albeit with limited efficacy. New therapeutic options are greatly needed, but the use of targeted treatments is often prevented by the impossibility to obtain tissue biopsies for molecular characterization. Here, we propose the use of circulating tumor cells (CTCs) as an alternative source of tumor material to perform molecular characterization for the identification of novel therapeutic targets.

MATERIALS AND METHODS: Blood samples (10 ml) from patients with advanced CCA were processed for CTC isolation as follows: -CTC enrichment with Parsortix -identification and single-cell recovery of epithelial CTCs (expressing epithelial markers) and non-conventional CTCs (lacking epithelial and leukocyte markers) using the DEPArray -whole genome amplification and quality check using Ampli1 kit and Ampli1 QC kit -mutational profiling using Ion AmpliSeq Cancer HotSpot Panel v2 and AmpliSeq somatic pipeline for variant calling -copy number alteration (CNA) analysis using Ampli1 LowPass kit, plus unsupervised clustering and frequency alteration analyses.

RESULTS: We analyzed 88 single CTCs isolated from 38 blood samples longitudinally collected from 23 patients (12 with intrahepatic, 9 with extrahepatic CCA and 2 with gallbladder cancer). CNA profiles showed a high level of both inter- and intra-patient heterogeneity, with each CTC displaying a unique profile. Intra-patient heterogeneity was further confirmed by clustering analysis as, in most cases, CTCs from the same patient clustered independently. CTC clustering was also not affected by sampling time (before/during chemotherapy), nor by the anatomical location of primary tumor. Conversely, we observed an enrichment of CTCs derived from patients non-responding to therapy (showing a PD according to RECIST criteria) in 2 of the 4 identified clusters (p=0.00041). By pairwise comparison of CNAs among clusters, we identified 2 regions more frequently altered in one cluster enriched for CTCs from non-responders: 10q22.2 and 3p11.1. The latter encodes, among others, for EPHA3, a targetable gene whose involvement in chemoresistance will be investigated by in vitro studies. Mutational profiling of 19 CTCs (from 6 patients) also confirmed the high intra-patient heterogeneity with most mutations being present in only 1 CTC. This limits the applicability of this approach in patients with few CTCs. Nonetheless, in 1 patient presenting 9 CTCs, we identified 1 mutation in KIT shared by 7/9 CTCs, indicating it as a possible treatment target for this patient.

CONCLUSIONS: Our results support the possibility of using CTC molecular characterization to identify both resistance mechanisms and patient-specific targets, thus opening the way for a shift in treatment management of CCA towards an innovative and personalized therapy.

#1391

**Development of** in vitro **multiplex colorectal cancer associated mutation detection assay powered with xenonucleic acid.**

Michael Y. Sha, Anna J. Lee, Michael J. Powell, Aiguo Zhang. _DiaCarta, Inc., Richmond, CA_.

Precise detection of cancer associated mutations have many advantages including diagnosis and monitoring of the disease and monitoring development of drug resistance. However, unlike germline mutations, somatic mutations in cancer are found at a much lower frequency. For this reason it remains a challenge especially when trying to detect mutations from limited amount of patient tissue biopsy or liquid samples. Currently, detection and genotyping of these mutations is heavily dependent on deep NGS methods. These procedures, however, are costly and time consuming. Here, we developed an alternative high-throughput method that combines DiaCarta's proprietary xenonucleic acid (XNA) technology that allows target enrichment, and Luminex MAGPIX platform technology that enables simultaneous detection of up to 50 mutation targets. Briefly, multiplex PCR amplification of cancer associated genes is conducted in the presence of XNA. In this reaction, XNA specifically suppresses the wild-type DNA from being amplified and thus, only generates amplicons from the genes that carry mutations. The amplicons are then hybridized in a single tube that contains a set of magnetic beads. Each bead is uniquely color-coded for identification, and each corresponds to a single mutation of interest. The beads are analyzed on Luminex platform for genotyping of the mutations. Using this method, we compared conditions with or without XNA for target amplification. Our preliminary data show that when we tested the cancer oncogene KRAS exon 2 mutation variations, we find that 100% correct allele call was made with XNA for all 7 reference mutations genes. However, in conditions without XNA, the PCR product was composed of mostly wild-type sequences, and thus, only two correct allele calls (28.6%) were made. Consequently we have showed a clear advantage of XNA technology for efficient mutant target enrichment, followed by Luminex MAGPIX platform technology for simultaneous detection of multiple genetic targets. Multiplex capacity of both PCR and Luminex MAGPIX platform allows a significant reduction in the total amount of patient samples needed for precise mutation detection from each assay, especially for the mutations with low allelic frequency. Further development on this assay will provide a more reliable, sensitive and quick method for simultaneously detecting mutations associated with cancer in a single reaction. 

### Deep Learning

#1392

Machine Learning Radiomic Biomarkers Non-invasively Assess Genetic Characteristics of Glioma Patients.

saima rathore, Spyridon Bakas, Hamed Akbari, MacLean P. Nasrallah, Stephen Bagley, Christos Davatzikos. _Univ of Pennsylvania, Philadelphia, PA_.

Genetic heterogeneity of gliomas, both across and within patients, is a significant challenge in precision diagnostics and treatment planning. The emerging field of radiogenomics focuses on imaging signatures reflecting underlying genomic characteristics. In this study, we use advanced radiomic analysis of clinically acquired multi-parametric MRI (mpMRI) sequences to non-invasively detect clinically-relevant genetic alterations in gliomas, including isocitrate dehydrogenase (IDH1), epidermal growth factor receptor variant-III (EGFRvIII), and promoter methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) gene. Such non-invasive, in vivo determination of tumor genetic alterations could assist in assessing spatial heterogeneity (currently not captured via single-specimen analyses), as well as prognostic stratification, and treatment planning. We delineated all tumors, from a retrospective cohort of 202 glioma patients with available pre-operative mpMRI (T1, T1-Gd, T2, T2-FLAIR, DTI, DSC-MRI), into sub-regions of enhancement, non-enhancement, and peritumoral edema/invasion. We used CaPTk (www.cbica.upenn.edu/captk) to extract quantitative imaging phenomic (QIP) features across sub-regions from all mpMRI, describing size, morphology, texture, and intensity. Cross-validated (CV) sequential feature selection determined the most discriminative QIP features. The predicted classifications, following a 10-fold CV, were compared with the results of a next-generation sequencing panel performed on specimens from the patients in our cohort, as well as pyrosequencing for MGMT. The CV accuracy of the radiomic assessment was 85% (spec = 86%, sens = 83%, area under the curve [AUC] = 0.85), 87% (spec = 90%, sens = 79%, AUC = 0.86), and 83% (spec = 85%, sens = 83%, AUC = 0.84) for mutations in IDH1, EGFRvIII, and MGMT methylation, respectively. These signatures were consistent with EGFRvIII-mutated, IDH1-wildtype, and MGMT-methylated tumors having increased neovascularization and cell density, as well as a distinctive spatial pattern involving relatively more frontal and parietal regions in EGFRvIII-mutants, more frontal regions in IDH1-mutants, and relatively right spherical regions in MGMT-methylated tumors. These detections were independent of age, gender, and additional genetic changes. By non-invasively capturing the tumor in its entirety, our proposed QIP signatures can assist in evaluating the tumor's spatial heterogeneity, hence overcoming common sampling limitations of tissue-based analyses. These signatures can non-invasively stratify patients for therapies targeting specific genes, and potentially monitor the dynamic mutational changes during treatment. Given the routine use of imaging in clinical practice, our QIP signatures may provide an unprecedented opportunity to improve decision-support in cancer treatment at low cost.

#1393

Automated adeno/squamous-cell NSCLC classification from diagnostic slide images: A deep-learning framework utilizing cell-density maps.

Mustafa I. Jaber,1 Liudmila Beziaeva,2 Christopher W. Szeto,3 John Elshimali,2 Shahrooz Rabizadeh,1 Bing Song1. 1 _NantOmics, Los Angeles, CA;_ 2 _NantHealth, Los Angeles, CA;_ 3 _NantOmics, Santa Cruz, CA_.

Background: The most common form of lung cancer, non-small cell lung cancer (NSCLC), is further categorized into two major histopathological subtypes: ~40% Adenocarcinoma (LUAD), and ~30% squamous cell carcinoma (LUSC). Classifying patients accurately is important for prognosis and therapy decisions, but requires costly pathologist review. Here we present an automated algorithm to differentiate LUAD and LUSC diagnostic whole slide images (WSIs).

Methods: 488 subtyped NSCLC high-resolution diagnostic WSIs were obtained from TCGA sources. Adjacent normal regions were identified and excluded from analysis. Cancer cell-density maps were created based on cell counts within discrete patches. These maps were then binned into ten ranges of cell counts (20-30 cells per patch, 30-40, etc. up to >110 cells per patch). 2D color patches were transformed into 1D descriptive vectors using the inception v3 deep learning framework. Samples were randomly split into 70% training and 30% testing sets. Ten LUAD/LUSC linear SVM classifiers (one for each cell-density bin) were trained on such transformed data. Subtype prediction in unseen testing samples was achieved by averaging subtype predictions from the 10 subsequent models.

Results: 338 TCGA diagnostic WSIs (164 LUAD and 174 LUSC) were used to train, and 150 (71 LUAD and 79 LUSC) were used to test. The proposed system achieved an AUC of 0.9068 in test samples, corresponding to a classification accuracy of 83.33%. The (heretofore excluded) adjacent normal regions were classified correctly almost as accurately as tumor regions (74.7%).

Conclusions: This fully-automated histopathology subtyping method generates maps of regions-of-interest within WSIs, providing novel spatial information on tumoral organization. For example, our results on test data show tumor patches of size 100 square microns with 60 to 100 cells distinguish LUAD from LUSC better than other cell-density ranges. Moreover, adjacent normal tissue may provide additional insights into tumorigenesis mechanisms.

#1394

Diagnosis of thyroid cancer using deep convolutional neural network models applied to sonographic images from clinical ultrasound exams.

Xiangchun Li,1 Sheng Zhang,1 Qiang Zhang,1 Xi Wei,1 Yi Pan,1 Jing Zhao,1 Xiaojie Xin,1 Xiaoqing Wang,1 Fan Yang,1 Jianxin Li,2 Meng Yang,1 Qinghua Wang,1 Xiangqian Zheng,1 Yanhui Zhao,3 Lun Zhang,1 Xudong Wang,1 Zhimin Zheng,4 Christopher T. Whitlow,5 Metin N. Gurcan,5 Boris Pasche,5 Ming Gao,1 Wei Zhang,5 Kexin Chen1. 1 _Tianjin Medical University Cancer Institute & Hospital, Tianjin, China; _2 _Weihai Municipal Hospital, Shandong Province, China;_ 3 _Affiliated Hospital of Chifeng University, Mongolia Province, China;_ 4 _Integrated Traditional Chinese and Western Medicine Hospital Of Jilin city Jilin Province, Tianjin, China;_ 5 _Wake Forest School of Medicine, Winston-Salem, USA, NC_.

Background Incidence rate of thyroid cancer is steadily increasing due to overdiagnosis and overtreatment. Thyroid ultrasound is commonly used to diagnose thyroid cancer. The aim of this study is to examine the accuracy of using deep convolutional neural network (DCNN) models to improve diagnosis of thyroid cancer by analyzing sonographic imaging data from clinical thyroid ultrasound.

Methods A total of 131,731 sonographic images from 17,627 thyroid cancer patients and 180,668 sonographic images from 25,325 controls used as training set were obtained from Tianjin Cancer Hospital. Images from anatomical sites that did not have cancer according to location sign on the image were not included. All thyroid cancer patients and 13·2% of controls (51,255 images) were confirmed by pathological reports. DCNN is a specific type of neural network optimized for image recognition. We trained two DCNN models on the training set and subsequently evaluated the performance on one independent internal (Tianjin, 1,118 individuals) and two external (Jilin,154 individuals; Weihai, 1,420 individuals) validation sets. Individuals in the validation sets all have pathological examinations. We compared the specificity/sensitivity of DCNN models with the performance of six thyroid ultrasound radiologists on these three validation sets.

Findings DCNN model achieved high performance in identifying thyroid cancer patients versus six experience radiologists: for Tianjin validation set, sensitivity was 92·2% versus 96·9% (95% CI 89·7% - 94·3% vs. 93·9% - 98·6%; p = 0·003), and specificity was 85·6% versus 59·4% (95% CI 82·4% - 88·4% vs. 53% - 65·6%; p < 0·0001); for Jilin validation set, sensitivity was 84·3% versus 92·9% (95% CI 73·6% - 91·9% vs. 84·1% - 97·6%; p = 0·05), and specificity was 86·9% versus 57·1% (95% CI 77·8% - 93·3% vs. 45·9% - 67·9%; p < 0·0001); for Weihai validation set, sensitivity was 84·5% versus 89% (95% CI 81·2% - 87·4% vs. 81·9% - 94%; p = 0·2), and specificity was 87·5% versus 68·6% (95% CI 85·1% - 89·6% vs. 60·7% - 75·8%; p < 0·0001).

Interpretation DCNN models exhibited high accuracy, sensitivity, and specificity in identifying thyroid cancer patients at levels comparable to or higher than six experienced radiologists. Conferred by the high specificity of DCNN models, the rate of overdiagnosis and overtreatment of patients with thyroid cancer is expected to decrease. This supports future application of the deep learning models to clinical practice for thyroid cancer diagnosis. However, further validation of these DCNN models in prospective clinical trials is warranted.

Funding The Program for Changjiang Scholars and Innovative Research Team in University in China (IRT_14R40), National Natural Science Foundation of China (31801117).

#1395

Residual convolutional neural network for predicting response of transarterial chemoembolizationin hepatocellular carcinomafrom CT imaging.

Jie Peng. _nan fang hospital, guangzhou, China_.

Purpose: We aimed to develop and validate a model of deep learning for the preoperative prediction of response of patients with intermediate stage hepatocellular carcinoma undergoing transarterial chemoembolization.

Methods: The training and two independent validation CT images were acquired for 540 patients from the Nan Fang Hospital (NFH), 84 patients from Zhu Hai Hospital Affiliated with Jinan University (ZHHAJU) and 136 patients from the Sun Yat-sen University Cancer Center (SYUCC). We built a predictive model from the outputs using transfer learning techniques of a residual convolutional neural network (ResNet50). Then, we revaluated the prediction accuracy for each patch in two independent validation cohorts.

Results: In the training (NFH) set, the deep learning model had a high accuracy of 84.3% and an areas under the receiver operating characteristic curves (AUROC) of 0.97, 0.96, 0.95 and 0.96 for complete response (CR), partial response (PR), stable disease (SD) and progressive disease (PD), respectively. In the validation (ZHHAJU) set, the deep learning model had an accuracy of 85.1% and an AUROC of 0.98, 0.96, 0.95 and 0.94 for CR, PR, SD and PD, respectively. In the other validation (SYUCC) set, the deep learning model had an accuracy of 82.8% and an AUROC of 0.97, 0.96, 0.95 and 0.95 for CR, PR, SD and PD, respectively.

Conclusion: The proposed ResNet model showed excellent performance for the individualized and non-invasive estimation of TACE therapy, which may help clinicians to better identify patients with HCC who can benefit from the interventional treatment.

#1396

Detection and local histological staging of prostate cancer foci in H&E whole slide images using convolutional neural networks.

Carolin Stürenberg,1 Umair Khan,2 Kevin Sandeman,1 Oguzhan Gencoglu,2 Adrian Malen,1 Andrew Erickson,1 Timo Heikkinen,2 Antti Rannikko,1 Tuomas Mirtti1. 1 _Univ. of Helsinki, Helsinki, Finland;_ 2 _Top Data Science Ltd, Helsinki, Finland_.

A pathologist's histological diagnosis is the gold standard of prostate cancer diagnostic measures. In addition to cancer grade, the stage of cancer determines the follow-up and possible adjuvant therapies after surgery. Not only glandular pattern recognition but also the assessment of three-dimensional extent and size of cancer, in relation to the whole organ, are parts of the subjective diagnosis, which may vary among pathologists. Furthermore, evaluation of lymph nodes for any possible metastases is time-consuming and missing cancer in lymph nodes can lead to undertreatment of the patient. For the abovementioned reasons, our team is developing an algorithm that calculates the amount of cancer tissue objectively, assisting the pathologist in the diagnostic procedure. In addition, we are creating an assistant for cancer detection in lymph nodes, which are removed for histological evaluation. All in all, we aim to create a tool that will help pathologists to a better, faster, more secure and more accurate diagnosis. Our material consists of full sets of scanned whole slide images from 302 prostates that were retrieved by radical prostatectomy. After supervised learning procedure, convolutional neural networks were employed for the classification of cancerous and non-cancerous regions in the images. A tiling based approach was used in which a slide was divided into square shaped small tiles. Millions of cancerous and non-cancerous tiles were sampled from the dataset for training and validation. During the convolutional neural network training, several different tile sizes were used, i.e., 256x256, 512x512, 1024x1024 pixels. Four different types of architectures were fine-tuned and trained for the task of tile-wise binary classification, namely InceptionV3, Xception, ResNet50 and a custom convolutional neural network architecture. In our preliminary assays for cancer detection, in both pixel-wise and tile-wise evaluation, InceptionV3 performed outstandingly well with an AUC score of 0.97 and 0.951, respectively. In conclusion, our algorithm has developed very well thus far with an accuracy in cancer detection of 97%. It is not only a versatile assisting tool, aiding pathologists to a more objective, standardized and accurate diagnosis, but also serves as a second opinion in difficult and challenging diagnostic cases.

#1397

Quantitative MR imaging measures predict intratumoral molecular heterogeneity in clear cell renal cell carcinoma.

Durga Udayakumar, Ze Zhang, Durgesh Dwivedi, Yin Xi, Tao Wang, Ananth Madhuranthakam, Payal Kapur, Asghar Hajibeigi, Allison Joyce, Qurratulain Yousuf, Michael Fulkerson, Alberto Diaz de Leon, Matthew Lewis, Jeffrey Cadeddu, Aditya Bagrodia, Vitali Margulis, James Brugarolas, Ivan Pedrosa. _UT Southwestern Medical Center, Dallas, TX_.

Clear cell renal cell carcinoma (ccRCC) is primarily driven by mutation in von Hippel-Lindau (VHL) leading to constitutive hypoxia inducible factors (HIFs) upregulation promoting angiogenesis. ccRCC is the most aggressive and common histological subtype of kidney cancer. It is characterized by high pathologic and molecular intra-tumor heterogeneity (ITH), a reflection of the genetic branched evolution during the tumor development. The overall molecular complexity in ccRCC may be underestimated with limited tissue samples in percutaneous biopsies. Non-invasive imaging methods that can provide quantitative spatial information on those varying features in the whole tumor may be a valuable tool for predicting tumor progression and therapy outcome. In this work we aim to understand the predictive value of quantitative Magnetic Resonance Imaging (MRI) measures of tumor vascularity as a noninvasive tool to identify molecular heterogeneity in ccRCC. In this IRB approved, prospective, HIPAA compliant study, 62 ccRCC patients underwent 3T multi-parametric MRI: T2-weighted (T2W), dynamic contrast-enhanced (DCE), and arterial spin labeled (ASL) MRI. All tumors were manually segmented with a region of interest (ROI) drawn on the central slice of the tumor. A grey-level co-occurrence matrix (GLCM) was constructed for each ROI and Haralick texture features were extracted. After surgery, 182 snap frozen samples from 49 tumors were subjected to RNA extraction, library preparation and mRNA sequencing using established protocols (Admerahealth, NJ). Spearman correlation coefficient between first- and second-order MRI statistics, including Haralick texture features, and gene expression levels were calculated. Gene ontology (GO) analysis was performed to identify the biological pathways associated with imaging features. Entropy, a measure of ITH, was correlated with standard deviation of normalized gene expression levels in multiple samples obtained from the same tumor. False discovery rate (FDR), q-values <0.05 were considered statistically significant. GO analysis of the top positively correlated genes with ASL-MRI and DCE-MRI measures of tumor perfusion indicated enrichment of immune system and cellular metabolic processes (q<0.05). ASL-MRI perfusion levels correlated positively with 81 HIF2 specific target genes (p<0.05). Gene set Enrichment Analysis (GSEA, Broad Institute, MA) indicated that correlated HIF2 target genes overlapped with key hallmark Molecular Signature database (MSigDB) gene sets, including G2M checkpoint, MTORC1 signaling, and glycolysis. Entropy of the DCE-MRI images correlated with heterogeneity in both metabolic processes, and expression of HIF1/2 target genes. Our study has set the initial framework for utilizing quantitative radiomics to assess the association of the imaging phenotype in ccRCC with specific molecular signatures.

#1398

**Modeling of drug-protein interactions to support clinical decision making for therapy-resistant** EGFR **or** ALK **-positive non-small cell lung carcinoma.**

Juliana F. Vilacha Madeira R Santos, Birgitta I. Hiddinga, Leon C. van Kempen, Arja ter Elst, Lucie B. Hijmering, Thijo J. Hiltermann, Ed Schuuring, Matthew R. Groves, Harry J. Groen, Anthonie J. van der Wekken. _University Medical Center Groningen, Groningen, Netherlands_.

Background Activating mutations in epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) rearrangements are drugable targets in respectively 10% and 4% of advanced non-small cell lung cancer (NSCLC) patients. Different EGFR and ALK inhibitors are available for target-dependent and sometimes independent drug resistance. Mutations in the EGFR or ALK kinase domain are major mechanisms of drug-induced resistance. Prediction of protein-drug interactions using protein modeling of these mutated domains in EGFR and ALK could provide valuable information for the selection of drugs for subsequent treatment. Methods Biopsies from advanced NSCLC patients with an activating EGFR mutation or ALK rearrangement that were taken at therapy resistance were analyzed by targeted panel sequencing. In case of unknown or accumulating mutations in the EGFR and ALK kinase domains, docking poses and binding affinities of drug-protein interactions were determined. The outcome of both calculations were used to create a likelihood ranking of currently available ALK and EGFR inhibitors to bind to specific mutated targets. Follow-up data was available for a limited number of patients who were treated based on this prediction. Results Prediction Protein-drug interactions were modeled for 59 EGFR mutations and 24 ALK mutations that were detected at therapy resistance. Prediction of therapeutic activities for gefitinib, afatinib, dacomitinib, for EGFR mutations, and alectinib, ceritinib, entrectinib and lorlatinib for ALK mutations will be shown. Case example Female (35y), never-smoker, cT1bN2M1c NSCLC with metastases in lymph nodes, liver, bones and asymptomatic brain metastases, ALK translocation-positive without known ALK gatekeeper mutations. Treatment with crizotinib was started, but unacceptable liver enzyme toxicity developed after 6 weeks. Switching therapy to alectinib resulted in a complete metabolic response with absence of brain metastases by MRI. After 9 months the patient progressed with liver, brain and leptomeningeal metastases. Treatment with ceritinib had no effect. A liver biopsy revealed the ALK p.(L1196M) gatekeeper mutation. The case was discussed in the molecular tumor board, because this gatekeeper variant was reported to be sensitive to ceritinib. In contrast, ALK-drug interaction modeling indicated a better binding profile of lorlatinib than ceritinib, not only based in binding affinity but also in the ability of lorlatinib to reproduce the bioactive conformation found in literature. The patient was treated with lorlatinib and recovered in 5 days with a persistent partial response at 2 month follow up and significant clinical benefit. Conclusion Modeling of the protein-drug interactions supports clinical decision making for treatment of NSCLC patients in case of therapy resistance to EGFR and ALK inhibitors.

#1399

Ex-vivo targeting of urothelial carcinomas by fluorescent pHLIP imaging agents.

Borivoj Golijanin,1 Anna Moshnikova,2 Donald M. Engelman,3 Oleg A. Andreev,2 Yana K. Reshetnyak,2 Ali Amin,1 Dragan Golijanin1. 1 _The Miriam Hospital and the Warren Alpert Medical School of Brown University, Providence, RI;_ 2 _University of Rhode Island, Kingston, RI;_ 3 _Yale, New Haven, CT_.

Introduction: The detection of urothelial carcinomas of the bladder and upper tract is not satisfactory. Incomplete findings by contemporary imaging modalities provide a challenge in patient care, so there is a definitive need to improve diagnosis and treatment methods for urothelial carcinomas.

Experimental procedures: ICG (indocyanine green) near infrared fluorescent (NIRF) dyes were conjugated with a pH-Low Insertion Peptide (pHLIP®), which senses and targets the acidity at the surfaces of cancer cells. In a few cases IR800 pHLIP® was used. Urinary neoplasm (kidney, ureter and bladder) specimens obtained through radical surgery were irrigated and incubated ex-vivo with ICG pHLIP® followed by fluorescence imaging. Pathology findings were correlated with the ICG pHLIP® NIRF imaging.

Results: In the upper urothelial tract, nine of ten patients with nephroureterectomy had urothelial carcinomas (UTUC). Eighteen (100%) malignant lesions were found in NIRF imaging. Only thirteen (72%) of malignant lesions were found under white light macroscopic examination. ICG pHLIP® NIRF imaging gave a 28% increase in diagnosis of UTUC in ex-vivo specimens. There was no non-specific uptake in the non-tumoral tissue. The ICG pHLIP® imaging agent identified malignant upper tract urothelial lesions with 100% specificity and sensitivity.

In the lower urinary tract, thirty-eight radical cystectomy specimens were incubated with pHLIP® conjugated with one of the NIRF dyes (ICG or IR800). In the thirty-eight bladders, eighty-one lesions were identified using fluorescent pHLIP®(specificity (97%) and sensitivity (100%)). White light cystoscopy did not detect 20.5% of lesions identified under NIRF cystoscopy (p<.001). Normal tissue did not show any uptake by the construct; however, entrapment of the dye in the necrotic tumor bed was noted. All regions showing the NIRF signal of pHLIP® were confirmed by standard pathological analysis.

Conclusions: The ICG pHLIP® NIRF revealed additional lesions not seen with white light ex-vivo cystoscopy or preoperative ureteroscopy. pHLIP® based agents promise to improve diagnostic accuracy for urothelial carcinomas and may enable targeted treatment.

#1400

C1GALT1 regulates malignant phenotypes of cholangiocarcinoma cells.

Po-Da Chen,1 Ai-An Chang,2 Min-Chuan Huang,2 Yao-Ming Wu3. 1 _National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan;_ 2 _National Taiwan University College of Medicine, Taipei, Taiwan;_ 3 _National Taiwan University Hospital, Taipei, Taiwan_.

Identifying molecular targets for cholangiocarcinoma is an urgent need to overcome the treatment failure and death from cancer cell invasion and metastasis. Core 1 β1,3-galactosyltransferase (C1GALT1) has been documented as the key enzyme in morphogenesis and cellular adhesion, for it adds galactose to form Gal-GalNAc-Ser/Thr structure (T antigen); whereas abnormal C1GALT1 expression and aberrant O-glycans are commonly associated with tumor progression. However, functional roles of C1GALT1 in cholangiocarcinoma remains unclear. With clinical correlation, the immunohistochemistry confirmed that higher expression of C1GALT1 in cancer tissue is associated with larger tumor size, advanced cell grade and overall staging. Consistently, stable knockdown of C1GALT1 regarding cholangiocarcinoma cell lines inhibits cell viability, migration, and invasion; whereas the overexpressed C1GALT1 promotes cell viability, migration, and invasion of cholangiocarcinoma cell lines. Additionally, control of C1GALT1 regulates the colony formation, as well as sphere formation of cholangiocarcinoma cells. Moreover, our data suggest that down-regulated C1GALT1 modifies AAL, VVA, PNA, LEL, and MALII lectin, in correlation with the cell malignant behaviors. Given that, C1GALT1 is sufficient to regulate malignant behaviors of cholangiocarcinoma cells with a significant correlation between gene expression and clinical prognosis, implying that C1GALT1 plays a crucial role in cancer progress of cholangiocarcinoma.

#1401

Sensitive detection of integrated HPV in carcinoma cells.

Maurizio Mauro, Richard Delle Bovi, Jack Coleman. _Enzo Life Sciences, Farmingdale, NY_.

It is estimated that 79 million people in the US are currently infected with human papillomavirus (HPV). There are more than 200 different HPV types that are common worldwide. HPV types are categorized as either high risk (HR) or low risk (LR). LR types, such as HPV 6 and HPV 11, can cause genital warts or squamous papillomas of the oral cavity, while persistent infection with HR HPV types can induce cervical, anal, or head and neck cancers. Episomal HPV DNA can be detected in advanced stages of cancer. HPV integration in the host genome drives oncogenesis by the expression of the two major viral oncogenes, E6 and E7, which abrogate cell cycle checkpoints, promote cellular proliferation, and cause progressive genetic instability. Hence, screening for the presence of E6/E7 RNA transcripts expression is considered the gold standard for confirming active HPV infection. Early detection of active HPV is critical for better understanding of prognosis and therapeutics. So far, detection of integrated HPV DNA in tissue specimens has been challenging. A novel, cost-effective, in situ hybridization (ISH) detection assay has been developed using two sets of HPV probes with enhanced signaling, termed hyper probes. The Hyper HPV D probe detects HR HPV DNA and the Hyper HPV DR probe can simultaneously detect DNA and E6/E7 RNA of HR HPV subtypes in cancer cell lines and formalin fixed paraffin embedded (FFPE) biopsy specimens. The probes were tested on cervical carcinoma SiHa cells containing 1-3 copies of integrated DNA of HPV 16 and HPV negative cervical cancer C33A cell line. The Hyper HPV DR probe resulted in 1-3 puncta in more than 90% of SiHa cell nuclei, indicating integrated copies of HPV DNA into the host genome. There was also the additional presence of a diffuse signal which was abolished by pretreating SiHa cells with RNase. These data suggest the diffuse signal corresponds to E6/E7 mRNA expression. No signals were detected in HPV negative C33A cells. When cells were stained with Hyper HPV D probes, only punctate signals in nuclei of SiHa cells was present and no signal was evident in C33A control cells. Additionally, human cervical and anal FFPE biopsies were analyzed. Simultaneous presence of both punctate signals and diffuse signals were detected when using Hyper HPV DR probes in HR HPV infected tissues, while only punctate signal was detected when using HPV D hyper probes. The signal from the hyper probes was significantly stronger when compared to other commercially available HPV detection kits. Moreover, other commercially available kits failed to detect low copy of integrated HPV. In conclusion, these data demonstrate the Hyper HPV probes can reliably detect low copies of HPV DNA integrated in the host genome and the presence of HPV mRNA using ISH in HPV infected cells.

#1402

**Single cell transcriptomic profiling of** ex vivo **tumoroid models reveal therapeutic vulnerabilities of pancreatic ductal adenocarcinoma.**

Vincent Bernard,1 F. Anthony San Lucas,1 Jonathan Huang,1 Reid T. Powell,2 Paola A. Guerrero,1 Alexander Semaan,1 Clifford C. Stephan,2 Peter Davies,2 Gauri R. Varadhachary,1 Matthew H. Katz,1 Cullen M. Taniguchi,1 Hector A. Alvarez,1 Senthil Muthuswamy,3 Anirban Maitra1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Texas A &M University Institute of Biosciences and Technology, Houston, TX; _3 _Beth Israel Deaconess Medical Center, Boston, MA_.

By 2030, pancreatic ductal adenocarcinoma (PDAC) will likely become the second leading cause of cancer-related death. A lack of actionable mutations continues to be a significant challenge in addressing the disease in a precision medicine setting. As a result, current treatment options are often limited to genotype-independent cytotoxic agents. Patient derived tumor organoid (PDOs) models have gained traction as potential tools for therapeutic stratification. We characterized single cell gene expression profiles of PDOs and applied an in silico therapeutic drug prediction model to identify and validate combinatorial strategies targeting heterogeneous subpopulations. Four established PDOs from PDAC were profiled through single cell RNA-Seq analysis in order to identify subpopulations of cancer cells with differing therapeutic response. The PDOs were then subjected to a high-throughput drug screen of 764 agency approved candidates. Top candidates, including combinatorial drug selections, were orthogonally validated through single cell gene expression analysis and evaluated for efficacy and synergistic interactions. Single-cell transcriptomic profiling of PDOs revealed unique subpopulations of cancer cells with differential expression of DNA Damage response, protein and fatty acid metabolism, and inflammatory related pathways. Effective combinatorial therapies were projected based on pharmacogenomic predictions targeting opposite gene expression patterns of each subpopulation and validated through drug library screens. In a PDO, common sensitivity nodes utilizing epigenetic modifiers were detected among all tumor derived subpopulations. A synergy matrix seeded with PDOs and evaluated using Bliss independence revealed that combinatorial treatment with HDAC-inhibitors and PARP1-inhibitors produced the most profound synergistic result. Mechanistically, HDAC inhibitors limit the activity of DNA repair proteins which in conjunction with PARP-inhibition leads to synergistic outcomes. We have demonstrated the utility of single cell transcriptomic profiling from PDOs in identifying viable treatment options using a pharmacogenomic strategy. The sophisticated heterogeneity of PDOs, revealed the necessity of using combinatorial strategies to target all relevant subpopulations. Furthermore, this strategy revealed an exciting synergistic interplay between HDAC and PARP1 inhibitors, an interaction that has never before been shown in a PDAC case. Looking forward, our model not only may be a vehicle used to discover new drug candidates and elucidate novel mechanisms, but may also be the first step towards the establishment of a true precision medicine paradigm for PDAC.

#1403

**Combining oligonucleotide library-based RNA fluorescence** in situ **hybridization and fluorescent immunohistochemistry on the same tissue section.**

Robert A. Ach, May Tom-Moy, Peter Tsang, Kelly Kroeger. _Agilent Technologies, Inc., Santa Clara, CA_.

The ability to visualize both RNA and protein expression in a single formalin-fixed paraffin-embedded (FFPE) tissue section is a powerful technique for studying biological problems such as the regulation of protein expression and RNA/protein interactions, as well as for antibody characterization and validation. We have leveraged our ability to chemically synthesize oligonucleotides in massively parallel reactions to produce DNA libraries that can be used to generate probes for the detection of DNA or RNA by Fluorescence in situ Hybridization (FISH). The sequences of the oligonucleotides in these libraries are selected in silico using empirically determined criteria so as to avoid repetitive elements or regions homologous to other non-targeted nucleic acids. We have used these oligonucleotide library-derived FISH probes in combination with a novel signal amplification technique to detect the localization of a variety of both coding and non-coding RNAs in fixed tissue culture cells and FFPE tissue sections, using both conventional fluorescence and structured illumination microscopy. We report here combining our RNA FISH methodology with antibody-based fluorescent immunohistochemistry (IHC) detection of proteins on the same FFPE slide. We show that our combined RNA FISH/IHC protocol works on a variety of RNA probe/antibody combinations for both nuclear and cytoplasmic proteins. We also show that our fluorescent assay can be converted to a colorimetric one for both the RNA and protein detection.

#1404

Optical metabolic imaging of the effects of acute and intermittent hypoxia in murine breast cancer cells.

Lisa Rebello, Narasimhan Rajaram. _University of Arkansas, Fayetteville, Fayetteville, AR_.

About 90% of the patients diagnosed with breast cancer succumb due to metastases and not from primary tumor. There are currently few methods that can distinguish indolent and aggressive breast tumors. Recent studies have shown differences in the metabolic profiles of primary, circulating, and metastatic breast cancer cells. One of the key regulators of metastases in breast cancer is the transcription factor Twist. The goal of our study was to determine the relationship between Twist expression and cellular metabolism, and the effect of this relationship on metastatic potential. Multiphoton microscopy can provide non-invasive, label-free and reproducible measurements of cellular metabolism. An optical redox ratio of endogenous fluorescence from nicotinamide adenine dicnucleotide (NADH) and flavin adenine dinucleotide (FAD) (calculated as FAD/NADH+FAD) can reflect the balance between mitochondrial oxidative phosphorylation and glucose catabolism in the cell. Because hypoxia is known to promote metastasis, we studied the effect of both intermittent and acute hypoxia on cellular metabolism in 4T1 murine breast cancer cells. We generated a 4T1-Twist- cell line using CRISPR-Cas9 technology. Western blots of Twist protein expression confirmed that Twist expression was significantly lower in the selected cell population compared with the parental 4T1 line. Both cell lines were exposed to 0.5% oxygen for 3 hours and one hour of reoxygenation (acute hypoxia), and 3 cycles of one-hour hypoxia followed by one-hour reoxygenation (intermittent hypoxia). Multiphoton imaging of endogenous fluorescence as well as metabolic flux analysis (Seahorse Biosciences) was performed for both hypoxic exposures in both cell lines.

Our results indicate a significant increase in the optical redox ratio in 4T1 cells when exposed to acute hypoxia, indicating a possible increase in oxidative metabolism. There were no signfiicant differences in the 4T1-Twist- line. On the other hand, we observed a decrease in the optical redox ratio in response to intermittent hypoxia in the 4T1 cells and the opposite effect in the 4T1-Twist- line. In addition to these cell lines, we will also present data from sibling cell lines of the 4T1 with varying Twist levels and hence metastatic potential. These data indicate indicate potentially measurable metabolic differences between indolent and aggressive breast cancer cells when subject to a hypoxia-reoxygenation regimen.

#1405

Identification of high and middle penetrance pathogenic variants in patients with hereditary breast/ovarian cancer by multigene panel analysis.

Jin-Sun Ryu,1 Eun Hae Cho,2 Kyong-Ah Yoon,3 Eun-Gyeong Lee,1 Eun-Sook Lee,1 Sun-Young Kong1. 1 _National Cancer Center, Korea, Goyang, Republic of Korea;_ 2 _Green Cross Genome, Yongin, Republic of Korea;_ 3 _Konkuk University, Seoul, Republic of Korea_.

Background: Genetic testing for breast cancer-related genes can have implications for diagnosis, treatment, and management of breast cancer. The identification of the variants causing the disease would guide the examination and diagnosis of the at-risk relatives. The purpose of this study was to identify the frequency of pathogenic or likely pathogenic variants associated with hereditary breast and ovarian cancer in Korean patients. In this study, we conducted a multigene panel containing 23 genes with high and middle penetrance rates in hereditary breast and ovarian cancer.

Methods: A multigene panel was performed on 382 patients who agreed to this study and visited the genetic counselling clinic at the National Cancer Center of Korea, between October 2016 and 2018 July. For variants classification, we examined Sanger sequencing, family history, and the clinical pathological characteristics for patients according to the American College of Medical Genetics and Genomics (ACMG) standards and guidelines. We also measured the expression of alternative spliced transcripts by conducting quantitative real-time PCR (qRT-PCR) to investigate the splice donor variants interpreted with pathogenic or likely pathogenic.

Results: We confirmed that 42 patients (11.00%) had 23 pathogenic or 10 likely pathogenic variants for the high and middle penetrance genes such as BRCA1, BRCA2, ATM, CHEK2, MLH1, PALB2, TP53, BRIP1 and PMS2, except for heterozygous MUTYH mutation carriers. The number of mutation careers was evaluated as follows: BRCA1 in 9 patients, BRCA2 in 18 patients, ATM in 2 patients, CHEK2 in 4 patients, MLH1 in 1 patient, PALB2 in 4 patients, TP53 in 1 patient and PMS2 in 1 patient. Two patients had 2 pathogenic or likely pathogenic variants for other genes; BRCA1/BRIP1 and BRCA2/CHEK2. All of these variants were validated by Sanger sequencing. The mRNA level of CHEK2 or PALB2 genes was further reduced in the patients with the splice donor variants compared to controls. The patients with a family history were advised to follow targeted screenings according to the National Comprehensive Cancer Network (NCCN) guidelines so that early detection of cancer and reduction of risk factors can be achieved.

Conclusions: Multigene panels including high and middle penetrance rate genes will provide important information for patients with a family history of cancer. However, functional studies are needed to determine potential clinical impact of these variants. Thus, we are conducting the additional functional assays to resolve the questions for these variants (This study was supported by National Cancer Center, Korea, 1710172)

#1406

Multigene signatures based risk estimates in ER+/HER2- breast cancers: The predictive value of inexpensive statistical models and changes in adjuvant chemotherapy use.

Laurence Slembrouck,1 Giuseppe Floris,2 Hans Wildiers,2 Ann Smeets,2 Erik Van Limbergen,2 Philippe Moerman,2 Caroline Weltens,2 Kevin Punie,2 Griet Hoste,3 Els Van Nieuwenhuysen,3 Sileny Han,2 Ines Nevelsteen,2 Lynn Jongen,1 Patrick Neven,2 Isabelle Vanden Bempt2. 1 _KU Leuven - University of Leuven, Leuven, Belgium;_ 2 _KU Leuven - University of Leuven and University Hospitals Leuven, Leuven, Belgium;_ 3 _University Hospitals Leuven, Leuven, Belgium_.

Background Multigene signatures (MGS) select women with estrogen receptor positive human epidermal growth factor receptor 2 negative (ER+/HER2-) breast cancers where adjuvant chemotherapy (aCT) can be avoided. However, MGS are expensive and not always reimbursed. We investigated the predictive value of five inexpensive statistical models in tumors with low or high risk of relapse based on MGS and investigated the change in decision to add chemotherapy following MGS results.

Patients and Methods In this retrospective study, we evaluated patients diagnosed with primary operable ER+/HER2- lymph node negative or positive breast cancer diagnosed at University Hospitals Leuven between 2013 and 2018. Patients were analyzed by MammaPrint® (MP) (n=24), OncotypeDX® (ODX) (n=44) or Prosigna® (n=57) as there was uncertainty about benefit of aCT during multidisciplinary meeting (MDM). Magee equations (ME), Memorial Sloan Kettering simplified score (MSK), Breast Cancer Recurrence Score Estimator (BCRSE), new Adjuvant! Online (nAOL) and PREDICT v2.0 were computed. TAILORx cut-offs were used for ODX. A 5% cut-off was used for 10-year survival benefit with aCT for nAOL and PREDICT.

Results All ME- and BCRSE-high cases were classified by MGS as high or intermediate and not as MGS-low risk, as shown in Table 1. None of the low risk classifications by ME and nAOL resulted in MGS-high risk with ODX. High risk classification with nAOL showed strong concordance with all MGS-high risk results. Chemotherapy switch according to MGS results was observed in 46% (57/125) of patients. Following MGS testing, aCT was given to 56 patients which resulted in 19% relative and 10% absolute reduction.

Conclusion Inexpensive statistical models based on pathologic parameters can be useful to select patients who may need MGS testing. Integration of MGS into MDM decisions, resulted in a substantial decisional switch and reduction in aCT administration.

Table 1 Predictive value of inexpensive statistical models in MGS tested tumors.

---

|  | MGS high risk (n=52) | |  | MGS low risk (n=52)

|

|

ODX (n=17) | MP (n=10) | Prosigna (n=25) | ODX (n=27) | MP (n=14) | Prosigna (n=11)

MSK high | 59% (10/17) | 30% (3/10) | 32% (8/25) | 4% (1/27) | 36% (5/14) | 0% (0/11)

ME high | 24% (4/17) | 10% (1/10) | 4% (1/25) | 0% (0/27) | 0% (0/14) | 0% (0/11)

BCRSE high | 0% (0/17) | 10% (1/10) | 4% (1/25) | 0% (0/27) | 0% (0/14) | 0% (0/11)

nAOL high | 100% (17/17) | 60% (6/10) | 96% (24/25) | 85% (23/27) | 86% (12/14) | 27% (3/11)

PREDICT high | 47% (8/17) | 40% (4/10) | 48% (12/25) | 26% (7/27) | 36% (5/14) | 0% (0/11)

MSK low | 18% (3/17) | 30% (3/10) | 24% (6/25) | 41% (11/27) | 29% (4/14) | 46% (5/11)

ME low | 0% (0/17) | 10% (1/10) | 8% (2/25) | 7% (2/27) | 0% (0/14) | 18% (2/11)

BCRSE low | 18% (3/17) | 30% (3/10) | 40% (10/25) | 26% (7/27) | 43% (6/14) | 64% (7/11)

nAOL low | 0% (0/17) | 40% (4/10) | 4% (1/25) | 15% (4/27) | 14% (2/14) | 73% (8/11)

PREDICT low | 53% (9/17) | 60% (6/10) | 52% (13/25) | 74% (20/27) | 64% (9/14) | 100% (11/11)

#1407

Histone marks interplay at LIFR enhancer determines the response to HDAC inhibitors in breast cancer.

Xiaomin Wang, Jun Xu, Jian Ding, Meiyu Geng, Min Huang. _Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China_.

The dynamic crosstalk of epigenetic modifications is believed to impose a strong impact on epigenetically-targeted therapies, such as histone deacetylase (HDAC) inhibitors, but current understanding in this regard is extremely limited.Our previous work has revealed that the transcriptional feedback activation of leukemia inhibitory factor receptor (LIFR) limits the response to HDAC inhibitors in breast cancer. However, LIFR transcriptional response appears highly heterogeneous in breast cancer and the reason behind is unclear. This study takes LIFR as a representative to dissect how histone marks interplay determines the transcriptional outcome of epigenetically-targeted therapies. We herein discover that the H3K27 methylation specifically at LIFR enhancer restricts the H3K27 acetylation upon HDAC inhibition and prevents the upregulation of LIFR. The intrinsic level of H3K27 methylation at LIFR enhancer stratifies the responsive subset to HDAC-targeted therapies in a panel of patient-derived breast cancer models. This study provides strong evidence showing how existing histone marks and their specific localization together determine the transcriptional output and the outcome of epigenetically-targeted therapies, highlighting the significance of histone marks crosstalk in epigenetic therapeutics.

#1408

Diffuse optical spectroscopic imaging reveals regimen-dependent changes in breast tumors throughout neoadjuvant chemotherapy.

Anup Tank,1 Hannah Peterson,1 Vivian Pera,1 Syeda Tabassum,1 Naomi Ko,2 Anais Leproux,3 Bruce Tromberg,3 Darren Roblyer1. 1 _Boston University, Boston, MA;_ 2 _Boston Medical Center, Boston, MA;_ 3 _University of California, Irvine, Irvine, CA_.

Diffuse Optical Spectroscopic Imaging (DOSI) is a non-invasive near-infrared imaging modality than can provide label-free functional and metabolic information about breast tumors, probing depths up to several centimeters. Previous work has shown that DOSI has prognostic value for pathological response at key timepoints during neoadjuvant (presurgical) chemotherapy (NAC), including baseline, 24 hours after the first infusion, week 1, and at the midpoint of therapy. To date there have been almost no reports on how specific NAC regimens affect the prognostic capability of DOSI. Here we present a large multi-center DOSI dataset consisting of 54 breast tumors and 313 unique measurements taken across three timepoints during NAC: Baseline, week 1, and midpoint of treatment. We will show for the first time that the manifestation of DOSI response parameters (oxyhemoglobin, de-oxyhemoglobin, water, and lipid) is highly dependent on the specific NAC regimen used. Subjects were treated with either a Maximum Tolerated Dose (MTD) regimen, in which a higher dose is administered less frequently, or a Metronomic (MET) regimen, in which a smaller dose is given more frequently. MTD regimen of cytotoxic agents has traditionally been the most effective method to treat breast cancer as it aims to kill the maximum amount of tumor cells before regrowth. MET administration of cytotoxic therapies has shown antiangiogenic properties aiming to limit toxic side effects and drug resistance, while potentially inducing tumor dormancy and immunomodulation. Subjects treated with MTD regimen and also responding to this treatment exhibited an oxyhemoglobin flare in the first day of NAC. In contrast, subjects treated with MET presented only small fluctuations in oxyhemoglobin throughout the first week of NAC. The oxyhemoglobin flare has been theorized to be induced by an inflammatory effect significant for cell death, which could be in response to specifically the MTD regimen and its higher dosing. This study indicates that the specific regimen must be considered when using DOSI for NAC monitoring. It also suggests DOSI may provide biological insight into the regimen-specific mechanisms of action.

#1409

Characterization of the LAR subtype triple negative breast cancer population.

James Crespo, Seth Sahil, Elizabeth Ravenberg, Lei Huo, Kenneth Hess, Lumarie Santiago, Beatriz Adrada, Gaiane Rauch, Damodaran Senthil, Rashmi Murthy, Jennifer Litton, Debu Tripathy, Naoto Ueno, Stacy Moulder, Bora Lim. _MD Anderson Cancer Center, Houston, TX_.

Background: The LAR subtype of triple negative breast cancer (TNBC) was defined using the Vanderbilt genomic signature in cell lines. The characteristics of this population remain unclear.

Methods: We collected the clinicopathological, molecular and imaging characteristics of the LAR population who were enrolled in the ARTEMIS trial, a prospective trial to treat women with TNBC to receive neoadjuvant anthracycline therapy followed by experimental arms based on biomarker versus taxane. The ultrasonography-based response assessment was performed at baseline, after 2 and 4 cycles of anthracycline chemotherapy. The Vanderbilt genomic signature was used to classify the TNBC into the basal-like 1, basal-like 2, immunomodulatory, mesenchymal, mesenchymal stem-like and luminal androgen receptor (LAR) subtypes. The clinical data for this analysis included age, race, menopausal status, TNM stage and BRCA mutation status. Pathological and molecular characteristics included histologic subtype, nuclear grade, Ki67, vimentin expression, androgen receptor (AR) immunohistochemical (IHC) nuclear staining, stromal tumor infiltrating lymphocytes (TIL) percentage and residual cancer burden (RCB) at surgery.

Results: Total 28 patients with the LAR signature were analyzed. The characteristics are showed in the table: | |  | |  | |  | |

|

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

Age | |

Race | no. (%) | Clinical Stage | no. (%) | AR IHC Staining | |

BRCA Mutation  | no. (%)

Median (yr) | 55 | White | 19 (68) | IA | 1 (4) | Median (%) | 52 | No mutation | 16 (57)

Distribution(%) | no. (%) | Asian | 3 (11) | IIA | 6 (21) | Distribution (%) | no(%) | Mutation present | 0

18-40 | 2 (7) | Black | 2 (7) | IIB | 10 (36) | <25 | 9 (32) | VUS | 1 (4)

41-60 | 12 (43) | Hispanic | 3 (11) | IIIA | 6 (21) | 25-50 | 5 (18) | Test not indicated | 6 (21)

>60 | 14 (50) | American Indian | 1 (3) | IIIB | 0 | 51-75 | 1 (3,5) | Unknown | 5 (18)

|  | |  | IIIC | 5 (18) | 76-100 | 12 (43) | |

|  | |  | |  | Not available | 1 (3,5) | |

Menopause | no. (%) | Histologic subtype | no. (%) | Ki67  | |

Stromal TIL | no. (%) | Vimentin expression  | no. (%)

Postmenopausal | 23 (82) | IDC | 24 (87) | Median (%) | 40 | <5% | 10 (36) | < 1% | 24 (85)

Perimenopausal | 2 (7) | ILC | 2 (7) | Distribution (%) | no. (%) | 5% - <50 % | 16 (57) | ≥1% | 3 (11)

Premenopausal | 3 (11) | Apocrine Carcinoma | 1 (3) | <20 | 5 (18) | ≥50% | 1 (3,5) | N/A | 1 (4)

|  | Metaplastic carcinoma | 1 (3) | ≥20 | 13 (46) | N/A | 1 (3,5) | |

|  | |  | N/A | 10 (36) | |  | |

T stage | no. (%) | Lymph node involvement | no. (%) | Nuclear grade  | no. (%) | Tumor reduction (%)  | no. (%) | RCB  | no(%)

T1c | 3 (11) | Yes | 22 (79) | 1 | 1 (4) | ≥ 70 | 12 (43) | pCR-1 | 11 (39)

T2 | 13 (46) | No | 6 (21) | 2 | 6 (21) | < 70 | 14 (50) | 2-3 | 15 (54)

T3 | 11 (39) | |  | 3 | 21 (75) | N/A | 2 (7) | N/A | 2 (7)

T4 | 1 (4) | |  | |  | |  | |

Experimental arm | no. (%) | |  | |  | |  | |

Enzalutamide | 8 (29) | |  | |  | |  | |

Atezolizumab | 4 (14) | |  | |  | |  | |

None | 16 (57) | |  | |  | |  | |

Relevant characteristics are: median age of 55 years, 82% postmenopausal, 89% breast tumor size > 2cm, 79% with lymph node involvement, no BRCA mutation detected, 75% nuclear grade III, 46% with Ki67 ≥ 20%, median AR IHC staining of 52%, 85% vimentin expression of <1%, 93% had stromal TIL <50%, 39% RCB 0-1 and 54% RCB 2-3. We found that 43% had ≥ 70% reduction in size after 4 cycles of AC. Univariate and multivariate analysis using characteristics did not reveal contributing factor to the RCB.

Conclusion: The LAR subgroup harbors unique characteristics that require further confirmation in larger cohorts.

#1410

Clinicopathological characteristics of lung cancer patients with concomitant idiopathic pulmonary fibrosis.

Kota Araki, Kazuhiko Shien, Shunsaku Miyauchi, Akihiro Miura, Yuta Takahashi, Eisuke Kurihara, Yusuke Ogoshi, Kei Namba, Ken Suzawa, Hiromasa Yamamoto, Shuta Tomida, Junichi Soh, Masakiyo Sakaguchi, Shinichi Toyooka. _Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Science, Okayama City, Japan_.

Background: Idiopathic interstitial pneumonias (IIPs) are diffuse parenchymal lung disease of unknown etiology. Among IIPs, idiopathic pulmonary fibrosis (IPF) is the most frequent subtype. IPF is characterized by the accumulation of activated fibroblasts and extracellular matrix within the parenchyma. IPF is associated with pathologic and radiologic pattern of usual interstitial pneumonia (UIP). There are several possible underlying mechanisms linking lung cancer and IPF, however, many of them are still unknown. The purpose of this study is to reveal the clinicopathological characteristics of lung cancers patients with concomitant IPF.

Materials and Methods: The clinical and pathological data of lung cancer patients with IPF who underwent surgical resection of lung cancer between 2001 to 2016 at our institution were analyzed. We defined IPF as IIPs with UIP pattern, based on the chest CT scan and pathological findings. Mutations of EGFR (L858R point mutation and exon19 deletion), AKT1, BRAF, EGFR, KRAS, MEK1, NRAS, PIK3CA, and PTEN were analyzed.

Result: A total of 37 patients' data of lung cancer with concomitant IPF was collected. Two patients had metachronous lung cancers. Thus, we analyzed 39 lung cancers from 37 patients. Based on the radiographic findings, 33 (84.2%) of the available 38 tumors arose from the fibrotic legion, which were mainly observed in peripheral region. The frequency of EGFR mutation was detected in 1 (2.6%) of 39 lung cancers. As control, we had EGFR mutation profile of 583 lung cancer without IPF. For this population, EGFR mutation was found in 164 (28.1%) of 583 cases. There was a significant difference in EGFR mutation between lung cancers with or without IPF (p = 0.0001). Among other examined genes in 16 tumors, one PIK3CA mutation was found.

Conclusion: The lung cancer with IPF mainly arouse from fibrotic lesion. The frequency of EGFR mutation was less common in lung cancers with IPF than those without IPF. Our result suggests that molecular profile of lung cancer with IPF is different from that without IPF.

#1411

Semi-quantitative mapping of oncological therapies with mass spectrometry imaging.

Dodge Baluya,1 Bindesh Shrestha,2 Erik N. Cressman1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Waters Corp., Beverly, MA_.

Hepatocellular carcinoma (HCC) is one of the challenging cancers to treat with occurrence steadily rising in the United States. While surgical resection is considered curative, it is not available to patients who have underlying liver problems, such as chronic liver disease. Liver transplantation may be considered in these cases but recurrence is a possibility, and transplantation occurs for <5% of all HCC cases. Doxorubicin (DXR), a drug derived from a natural product extracted from several wild strains of Streptomyces, is used to treat different types of cancer, particularly liver cancer such as HCC. While DXR use for treatment shows positive efficacy, the side effects such as cardiomyopathy and typhlitis, are easily triggered when the systemic concentration dose reaches a particular threshold. Also, there is no confirmation that the drug reaches the target area with a viable therapeutic concentration. In this work, we use mass spectrometry imaging (MSI) platform on Waters SYNAPT G2-Si MALDI to gather spatial information and quantity of the DXR to determine the delivery efficacy. To generate a calibration curve that is suitable for MSI, a phantom tissue template was created. Livers were extracted from pig and homogenized using a Qiagen Tissuelyzer with a 30% w/v in deionized water, and created DXR tissue homogenate solutions (20, 10, 5, 2, 1µM) using serial dilution. The resulting solutions were pipetted into a gelatin mold before it was frozen using liquid nitrogen and stored at -80 °C until needed. For the 'unknown' samples, the liver was explanted from a pig that was treated with doxorubicin, then flash-frozen using liquid nitrogen and kept at -80 °C until needed. The tissue was sectioned using a Leica cryotome and mounted on glass microscope slides. For each analysis, two adjacent sections were selected, one was submitted for MSI analysis and the other for IHC staining. A good correlation (R>0.99) of the DXR signal from the tissue homogenates showed that the calibration curve could be used to comparably measure the DXR amount in the similar tissue type. MSI of the explanted liver showed the distribution of DXR towards a particular section. While this experiment showed only the response for DXR, this approach can be applied to other drug therapies to show the localization and concentration, thereby helping the effectivity assessment of the therapy complementary with the other evaluation tools for a successful treatment.

#1412

CT texture analysis to predict response to target therapy of hepatic metastases from colorectal cancer.

Simone Mazzetti,1 Valentina Giannini,1 Lorenzo Vassallo,1 Arianna Defeudis,1 Angelo Vanzulli,2 Rita Golfieri,3 Silvia Marsoni,4 Daniele Regge1. 1 _University of Turin and Candiolo Cancer Institute FPO-IRCCS, Candiolo (Turin), Italy;_ 2 _Niguarda Cà Granda Hospital, Milan, Italy;_ 3 _University of Bologna, Policlinico S.Orsola-Malpighi, Bologna, Italy;_ 4 _Precision Oncology, IFOM – The FIRC Institute of Molecular Oncology, Milan, Italy_.

Introduction Colorectal cancer (CRC), the 2nd cause of cancer death worldwide, is an indolent disease with 50% of patients eventually developing liver metastases (mCRC). Repeated cycles of different chemotherapies, combined with surgery in oligo-metastatic cases, are the therapeutic standard in mCRC. However, this strategy is resolutive in less than 15% of cases. Differentiating non- and short-term responders from potentially "cured" patients will spare patients needless toxicity and allow alternative treatments earlier, with conceivable cost and life savings. In this study we aimed to use CT texture analysis (CTTA) to identify specific imaging biomarkers of hepatic metastases, able to predict patient's response to therapy and overall survival.

Methods We exploited the imaging data-set of the HERACLES trial (NCT03225937): 23 patients with amplified Human Epidermal growth factor Receptor 2 (HER2) mCRC were included in the study. All had received anti HER2 treatment, and underwent CT examination every 8 weeks, until disease progression. CT scans were semi-automatically segmented to extract for each patient all liver metastases. Texture analysis was performed on each segmented area, computing for each lesion 34 quantitative parameters. Both mono-parametric and multi-parametric analysis were assessed to identify features most correlated to therapy response. We also performed a correlative survival (OS) analysis, considering subjects with good survival those with OS > 9 months.

Results In 23 patients we found 124 metastases, 55 of which were classified as responding and 69 as non-responding. Nine parameters reached statistical significance in the mono-parametric analysis (best AUC=0.67, p=0.001), while in the multivariate regression ten parameters were used in the model, achieving and AUC equal to 0.82, with sensitivity of 82% and specificity 72%. For OS analysis, 12 patients were "good" and 11 "poor" survivors. In the mono-parametric analysis "cluster prominence" and "sum entropy" predicted OS with AUC equal to 0.78 and 0.83, respectively. The regression model with two variables ("cluster prominence" and "dissimilarity") reached a sensitivity of 83% and a specificity of 82%.

Conclusions Our study demonstrated CTTA as a potential biomarker to predict response of hepatic metastases to chemotherapy treatment, possibly saving patients predicted as non-responder from toxicity. Moreover, CTTA could give indications on patients OS, without the need for additional tests.

Acknowledgments This study was funded by the Italian Association for Cancer Research (AIRC), ref. 21091.

#1413

Using conventional imaging to predict water content and electrical properties at 200 kHz in brain and GBM tumor tissues: a feasibility study in three TTFields patients.

Cornelia Wenger,1 Hadas Sara Hershkovich,2 Catherine Tempel Brami,2 Moshe Giladi,2 Zeev Bomzon2. 1 _Novocure, Lucerne, Switzerland;_ 2 _Novocure, Haifa, Israel_.

Introduction

Tumor Treating Fields (TTFields) are approved for the treatment of Glioblastoma (GBM). The efficacy of TTFields increases with field intensity at the tumor which depends on its highly heterogeneous electrical properties (EPs) distribution. Thus, an imaging technique, preferably with conventional sequences, rapid acquisition and fast processing, that is able to assess the EPs non-invasively might be of high interest for patient-specific TTFields treatment planning.

An approach termed water content electrical properties tomography (wEPT), estimates the EPs of brain tissue at 128 MHz from water content (WC) maps which are created with two spin echo sequences resembling a T1w and a PD image. Recently we performed experiments in tumor-bearing rats suggesting that wEPT could be adapted to map EPs in the brain at 200 kHz. Here we tested the feasibility of applying wEPT to map EPs at 200 kHz in GBM patients.

Methods

Analysis was performed for three patients that participated in the EF-14 trial. The image ratio was calculated as pixel-by-pixel division of T1 and PD images. The WC maps were estimated with a transfer function and two separate equations were used to calculate the maps of the electrical conductivity σ and the relative permittivity εr at 200 kHz. The table summarizes the median values in 5 tissues of normalized T1 and PD signals and the wEPT-estimations of WC, σ, and εr at 200 kHz.

Results  | |  | |  | |

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

median values | T1norm | PDnorm | WC[%] | σ[S/m] | εr[-]

white matter | Patient 1 | 0,23 | 0,58 | 76% | 0,11 | 2575

|

Patient 2 | 0,18 | 0,32 | 76% | 0,11 | 2462

|

Patient 3 | 0,24 | 0,35 | 74% | 0,10 | 2191

gray

matter | Patient 1 | 0,20 | 0,60 | 83% | 0,22 | 2923

|

Patient 2 | 0,16 | 0,33 | 81% | 0,17 | 2799

|

Patient 3 | 0,22 | 0,38 | 81% | 0,17 | 2919

necrosis | Patient 1 | 0,23 | 0,86 | 89% | 0,47 | 2495

|

Patient 2 | 0,12 | 0,32 | 98% | 1,43 | 1381

|

Patient 3 | 0,24 | 0,44 | 84% | 0,24 | 2988

enhancing

tumor | Patient 1 | 0,37 | 0,83 | 71% | 0,09 | 1546

|

Patient 2 | 0,17 | 0,35 | 81% | 0,17 | 2961

|

Patient 3 | 0,23 | 0,42 | 85% | 0,26 | 2932

non-enhancing

tumor | Patient 1 | 0,23 | 0,73 | 85% | 0,26 | 2979

|

Patient 2 | 0,18 | 0,36 | 78% | 0,14 | 2769

Conclusions

We adapted the wEPT approach to map WC and EPs at 200 kHz in three GBM patients. In healthy brain tissues the estimations are consistent with literature and also among patients, contrary to the highly variable tumor. This fast approach only needing conventional MRI holds some promise for patient-specific TTFields treatment planning. Yet the mapping of tumor tissues needs to be validated further, possibly including EP measurements of excised tumor samples and generally a higher number of patients analyzed.

#1414

Durable response to first-line Trastuzumab in HER2 amplified colorectal cancer.

Jeremy Kratz, Nataliya Uboha, Kayla Lemmon, Hannah Houtler, Mark Burkard, Dustin Deming. _University of Wisconsin Madison, Madison, WI_.

Background: Colorectal cancer (CRC) remains the second leading cause of cancer-related mortality with limited precision strategies. The human epidermal growth factor receptor (HER2) is amplified in 5% of patients with metastatic CRC. Therapeutic studies have demonstrated activity of HER2 targeting regimens in the late-line treatment setting though little characterization has been done in the setting of not having prior anti-epidermal growth factor receptor antibody therapy or in the first-line treatment setting.

Methods: Patient with metastatic colorectal cancer underwent next-generation sequencing (NGS) panel testing by Strata Oncology through the University of Wisconsin Precision Medicine Molecular Tumor Board. Patients were treated per multi-disciplinary recommendation across multiple lines of therapy and tracked by prospective registry (IRB#UW15068). HER2 copy numbers >/= 10 were used to determine amplification. A subset of patients with HER2-amplified CRC were treated with single agent trastuzumab.

Results: HER2 amplifications were identified in 4 of 78 cases of metastatic CRC (5.1%). All biopsies were performed prior to exposure to anti-epidermal growth factor receptor inhibitors. Median estimated copy number (CN) was 55.5 [range 13-178]. There was no concurrent alterations in extended RAS, BRAF, PIK3CA, AKT or MTOR. All cases included concurrent mutations in TP53 at R175H (2), C135W, or C135F. Single agent trastuzumab was administered to two patients at standard dosing (trastuzumab 8mg/kg intravenous day 1 cycle 1 followed by 6 mg/kg intravenous day 1 cycle 2+ on a 21 day cycle). One patient was treated in the treatment refractory setting. They had resolution of bowel obstructive symptoms and a minor response on imaging. Additionally, a patient with a HER2 amplification (CN 178) was treated in the first-line setting with single agent trastuzumab. The patient had grade 1 fever with the first cycle and no other treatment related adverse events thereafter. The patient's CEA decreased from 7,506 to 2,525 ng/mL with the first cycle of therapy and overall reduced to 6.5 ng/mL after 12 cycles of therapy. The patient has had a partial response per RECIST v1.1 response criteria with a reduction of cross-sectional diameter of their disease by 60%. The patient currently continues on this therapy without evidence of disease progression.

Conclusions: HER2 amplified colorectal cancers are an important subtype of colorectal cancer. Here we describe the potential benefit of HER2 targeting with single agent trastuzumab and even demonstrate single agent activity in the first-line setting. Further studies should be done to further characterize the benefit of this low toxicity treatment option for patients with metastatic HER2 amplified colorectal cancer.

#1415

Staging with [18F]FDG PET/CT.

Ramsha Iqbal,1 Tuba Aras,1 Lemonitsa Mammatas,2 Wouter V. Vogel,2 Daniela E. Oprea-Lager,1 Hendrik M. Verheul,1 Ronald Boellaard,1 Catharina W. Menke-van der Houven van Oordt1. 1 _Amsterdam University Medical Centers - location VU University Medical Center, Amsterdam, Netherlands;_ 2 _Antoni van Leeuwenhoek Hospital, Amsterdam, Netherlands_.

Background: Accurate staging of patients with primary breast cancer is essential for an optimal treatment plan. The current imaging standard for staging, positron emission tomography/computed tomography with [18F]Fluorodeoxyglucose ([18F]FDG PET/CT), might be insufficient for detection of distant metastases, specifically in low grade, estrogen receptor positive (ER+) breast cancer, due to lower metabolic activity. The aim of this study was to investigate the efficacy of [18F]FDG PET/CT in staging patients with low-intermediate grade, ER+ breast cancer.

Methods: 79 patients diagnosed with grade 1-2, ER+ clinical stage IIB/III or locoregional recurrent breast cancer were retrospectively included. Visual analysis was performed, comparing the lesions detected on conventional imaging modalities (mammography, ultrasound, magnetic resonance imaging, computed tomography, bone scintigraphy) with lesions detected on [18F]FDG PET/CT. Conventional imaging and/or pathology outcomes were considered as the gold standard for this comparison. Tracer uptake in each PET-positive lesion was (semi)quantified: volumes of interest were defined on the PET scan to determine standardized uptake values (SUVmax, SUVmean, SUVpeak), total lesion glycolysis (TLG) and metabolic tumor volume (MTV). These quantitative parameters were correlated with pathological features of tumors (i.e. histological subtype, grade, ER/PR/HER2 expression and mitotic activity index) to assess whether tracer uptake is influenced by these features.

Results: Scans were analyzed visually and (semi)quantitatively. 370 lesions could be identified with all imaging modalities (primary lesions: 80, locoregional lymph nodes: 161, distant lesions: 129). Based on the gold standard, 226/370 (61.1%) lesions were interpreted as "true positive" on [18F]FDG PET, 134/370 (36.2%) as "false positive" and 10/370 (2.7%) as "false negative" on [18F]FDG PET (p = 0.016). "False positive" lesions were mainly located in the axilla region (e.g. reactive lesions), gynecological and gastro-intestinal tract (e.g. adenomas) whereas "false negative" lesions were predominantly located in osseous tissue. The average SUVmax for "true positive" and "false positive" lesions was 4.24 ± 2.97 and 3.96 ± 2.03, respectively. For "true positive" lesions SUVmax, SUVpeak, SUVmean and MTV correlated with histological subtype, showing higher uptake in ductal carcinoma compared to lobular carcinoma (p <0.023). SUVmax and SUVmean also correlated with PR expression (p <0.034). No other correlations could be found between quantitative parameters and pathology outcomes.

Conclusion: These preliminary data indicate that [18F]DGG PET/CT might not correctly identify a substantial amount of lesions and therefore could lead to incorrect staging of patients with low grade, ER+ breast cancer. This suggests that there is a need to improve current staging procedures.

#1416

ChemoINTEL: A multi-parametric assay of chemotherapeutic responses in isolated primary tumor cells.

Kevin J. Polach, Kellye C. Kirkbride, Samantha J. Braxton, Megan D. Hoeksema, Dustin C. Rogers, Durdica Vojnic Zelic, Patricia Ladd-Ward, Santosh Putta, Norman B. Purvis, Matt Westfall. _Pierian Biosciences, Franklin, TN_.

The efficacy of chemotherapeutic cancer treatment is limited by the absence of "personalized" therapies that pair individual patients with therapeutic agents optimized for their specific disease. The ChemoINTEL platform utilizes kinetic measurements of apoptotic and cell death responses in primary tumor cells treated with a panel of cytotoxic agents to score the in vitro response to each drug as a potential measure of drug-induced apoptosis of these malignant cells in vivo. The ChemoINTEL platform provides a semi-automated system for brightfield and fluorescence-based assessments of cell responses utilizing a combination of automated equipment for: tumor dissociation and enrichment, sample and drug delivery to 384-well imaging plates, and kinetic brightfield and fluorescent imaging. The platform is fully supported through integrated systems of data analytics including sample management (SMS), information management (LIMS), and data analysis (integration of multiple software systems), as well as reagent formulation and a quality and document management program under ISO regulatory guidelines. Assay development studies, completed with multiple cell lines demonstrate fluorescent measurements of apoptosis and cell death (delta values between treated and untreated) provide endpoint responses equivalent or better than established drug testing methods by determining both kinetics and magnitude of response. In proof-of-concept studies utilizing primary carcinomas, enriched tumor cells isolated from 62 patient solid-tumor samples were screened through a panel of 6 cytotoxic agents and one PARP inhibitor (either as single treatment agents or in combination) to assess cytotoxic responses. Tumor viability varied significantly across patient samples and tumor types (ovarian, breast, lung, renal, gastric, colon, and pancreatic) with initial median viabilities of 75% (Interquartile Range (IQR) 19%) dropping to a median of 39% (IQR 29%) at 24 hours and a median of 34% (IQR 27%) at 48 hours. Using pre-defined inclusion criteria, 23 of 87 samples from multiple tumor types exhibited drug responses to the limited drug panel tested. Most tumors responded to multiple drugs, including Cyclophosphamide, Cisplatin, and Etoposide as single agents, and Carboplatin in combination with Docetaxel or Paclitaxel. Applications of this technology to research and development may facilitate screening of early stage therapeutic reagents in primary tumor samples, and clinical applications have the potential to improve patient outcomes and minimize toxicities by limiting the use of cytotoxic agents not suited to the patient's disease.

#1417

Multiple gastric cancer and predisposing genes.

Anqiang Wang, Zhongwu Li, Xin Ji, Tao Fu, Xiaojiang Wu, Ji Zhang, Zhaode Bu, Jiafu Ji. _Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking Universi, Beijing, China_.

BACKGROUND: Multiple gastric cancer (MGC) is one special type of gastric cancer with more than two different tumors at various locations of stomach. However, the clonal relationship and carcinogenesis of MGC are still remain unclear. Therefore, we investigated the clonal relationship and role of germline mutations playing in the carcinogenesis of MGC.

METHODS: We collected 16 multiple gastric cancer patients who underwent subtotal and total gastrectomy at Peking Cancer Hospital form January 2016 to December 2017. Thirty-three tumor samples and sixteen normal gastric tissue or blood samples were obtained for experiment. We also conducted analysis for 208 gastric cancer and 49 esophagogastric junction cancer (GC-EGJ) tumors from TCGA. DNA extraction from our samples was conducted for whole exome sequencing. We analyzed nonsynonymous mutations and somatic copy number variations (CNVs) based on exome data. Driver mutations, germline and cancer predisposing genes, significantly mutated genes (SMGs) were also analyzed.

RESULTS: Tumor mutation burden (TMB) was not statically significant within database and our data in GCs-EGJ groups (P=0.0591). And the TMB level was also similar between two databases in GCs groups (P=0.3113). The mutation spectrum and mutation signatures also showed uniform distributions in GCs and GCs-EGJ groups within our data and TCGA database. Among sixteen patients, four were identified as monoclonal, in which 11, 10, 26 and 6 somatic mutations were shared within different tumors of P7, P8, P9 and P16 respectively. However, no common mutation between different tumors of same patient was found from the other 12 patients in spite of thousands of somatic mutations in P2. After identifying predisposing genes, we found that germline MSH2 and NCOR2 mutations were significantly dominant in 8/12 and 10/12 of genetic MGCs patients. And all patients were identified MSH2 mutations in cancer samples of those same patients. Interestingly, the NCOR2 mutations were not detected significantly with non-frameshift INDELs in only three patients. Taking genetic MGCs as a whole, we identified that TP53 were significantly mutated in 14 of 25 tumor samples. And MSH2 mutations were found in all patients. The correlation analysis between clinical information and mutation signatures showed that the signature B was associated with the level of CA72.4.

CONCLUSIONS: Whole exome sequencing analyses are suggestive of monoclonal and polyclonal origin of MGC, which may promote the classification MGC into genetic and metastatic MGC. For genetic MGC patients, germline MSH2 mutations may contribute to the carcinogenesis of them, may thus giving rise to the consideration of more radical surgery and PD-1/PD-L1 therapy.

#1418

Brother of regulator of imprinted sites (BORIS) inhibits cisplatin induced DNA damage in non-small-cell lung cancer.

Yongfei Song, Chao Li, Mengdie Fang, Juan Ren, Jianfei Fang, Xiaoju Wang, Yanmei Zhang. _Center for Molecular Medicine, Zhejiang Academy of Medical Science, Hangzhou, Zhejiang, China_.

Cisplatin (DDP) treatment is primary modality for non-small-cell lung cancer (NSCLC) chemotherapy. However, patients benefited from DDP treatment limitedly due to the DDP resistance. Brother of Regulator of Imprinted Sites (BORIS, CTCFL) is the paralogue of CCCTC-binding factor (CTCF) and expressed abnormally in most type of cancers and is potential tumor target for breast, cervical carcinoma and lung cancer. However, the funciton of BORIS on carcinoma is unrevealed. In our previous study, we found that BORIS suppressed apoptosis and resisted 5-FU (Fluorouracil) treatment in colorectal cancer. As either 5-FU or DDP induces DNA damage and apoptosis of carcinoma cells, BORIS perhaps resists DDP treatment too. Based on the prevalent expression of BORIS and the high incidence of DDP resistance in NSCLC, we proposed that BORIS contributed to DDP resistance by suppressing DNA damage in NSCLC. We collected RNA sequencing data of 156 NSCLC patients who received DDP chemotherapy from The Cancer Genome Atlas and analyzed the expressions of BORIS. We found high BORIS expression correlated with short survival in NSCLC patients who received DDP chemotherapy. BORIS expression declined from prospective to retrospective collected tissue which responded to DDP chemotherapy. It suggested the probability function of BORIS on DNA damage repair. In cell cultured lung cancer cell lines, BORIS was silenced or over-expressed including DDP resistant cell line. It indicated that A549/DDP (DDP resistant A549 cell line) expressed relative higher BORIS than A549 parental cells and BORIS expression levels correlated with cell viability. H1299 and A549/DDP expressed comparative BORIS and growed comparativly. H460 and A549 expressed relative lower BORIS levels and grew slower than H1299 and A549/DDP. BORIS knockdown inhibited the proliferation of NSCLC cells, enhanced the sensitivity of A549/DDP and H1299 to DDP treatment and induced DNA damage. Reversely DDP induced DNA damage was suppressed by BORIS over-expression. The excision repairing gene ERCC1, which is regulated by BORIS, might be the assistant for DDP resistance. Either high expression of BORIS or ERCC1 indicated short survival rates of NSCLC patients. Our data suggested that BORIS suppressed DNA damage and promoted the progression of NSCLC and DDP resistance, which indicated its potential in NSCLC prognosis and therapy.

#1419

Metastasis and cancer stem cell markers identify two colorectal cancer subtypes with different molecular and clinical features.

Burair Alsaihati, Shaying Zhao. _University of Georgia, Athens, GA_.

Introduction Colorectal cancer (CRC) falls among the top prevalent cancer types with lethal outcome both in the United States and worldwide. The heterogeneity of CRC highlights the importance of identifying molecular markers for meaningful classification and prognosis. The recently published Consensus Molecular Subtypes (CMS) represent a widely-used molecular subtyping of CRC. However, our analyses indicate that clear heterogeneity still exists in some CMSs. To better classify CRC, we performed the following study.

Methods We collected from literature 19 gene markers classified as metastasis or cancer stem cell (CSC) markers in CRC. We then examined their expression in 622 CRC samples by The Cancer Genome Atlas (TCGA), and validated the findings using another published dataset, GSE39582, with 566 CRC samples.

Results We found among the 19 gene markers examined, 9 genes cluster into two oppositely co-expressed groups (VEGFC, SMAD4, TNIK, ITGB1, CD44 and ALCAM in group 1 and VEGFB, OTUB1 and DLL4 in group 2). Principal component analysis and hierarchical sample clustering of theses 9 genes identify two subtypes among the samples of each CMS, particularly CMS2 and CMS4. We named the two subtypes S1, which corresponds to increased expression in group 1 genes, and S2, which corresponds to higher expression of group 2 genes. Analysis of cancer hallmarks indicates a strong enrichment of six pathways (androgen response, protein secretion, mitotic spindle, UV response down, PI3K and TGF-β signaling), a common and defining feature of the S1 subtype in all CMSs. Immune activation pathways and most immune cells are strongly enriched in the S1 subtype of CMS4 samples only. Conversely, S2 is the subtype that exhibits immune activation across CMS2 samples. Tumor microenvironment signatures indicate more lymph vessels and stromal invasion in S1 samples within CMS4. However, within CMS2, a higher leukocyte fraction and invasive signature feature the S2 subtype. The epithelial and proliferative signatures are associated with the S1 subtype in general, but more prominently within CMS2. Consistent with the invasive signature of S2 samples in CMS2, the analysis of TCGA clinical data indicates increased lymphatic invasion, metastasized tumors and worse overall survival for them. On the other hand, GSE39582 clinical data show that worse overall survival is associated with the S1 subtype in CMS4.

Conclusion Each of CMS2 and CMS4 can be further divided into two subtypes, S1 and S2. While S2 may define more aggressive CMS2 cancers, S1 can be linked to worse prognosis in CMS4 patients.

## IMMUNOLOGY

### Adoptive Cell Therapy 1

#1420

Novel expansion of CD8+PD1+ spleen cells for therapeutic intent in pancreatic adenocarcinoma cancer.

Kathryn Cole, Quan P. Ly, Jesse L. Cox, Michael A. Hollingsworth, James C. Padussis, Jason M. Foster, Luciano M. Vargas, James E. Talmadge. _Univ. of Nebraska Medical Ctr., Omaha, NE_.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with poor prognosis and overall survival. In 2018 it is estimated that there will be 44,333 deaths making PDAC the 3rd cause of cancer related deaths. A distal pancreatomy for PDAC in the body/tail of the pancreas usually includes a splenectomy, with current practice to discard the spleen post-surgery. Spleens can provide a large number of lymphocytes, including CD8+ T-cells with potential to be utilized for adoptive cellular therapy. Our studies have focused on spleen from this patient population, which we posit (and show herein) provides a large number of CD8+PD1+ cells for expansion and the first clinical strategy to expand activated, tumor specific T-cells for subsequent infusion with therapeutic intent. In our studies we used Milteny magnetic bead isolation of CD8+PD1+ T-cells from the spleens of PDAC patients with disease in the distal pancreas. The purity of the cells was 98% with a yield of 79% providing 9x108 CD8+PD1+ cells from an idealized 160 gm spleen. CD8 expansion was undertaken with various growth factor cocktails incorporating combinations of three interleukins (IL); IL-7, IL-15, and IL-21 at varying concentrations. IL-7 supports T-cell survival and proliferation, IL-15 is a potent T-cell growth factor and promotes naïve and memory T-cell survival, and IL-21 limits T-cell exhaustion in response to chronic stimulation. The optimized cocktail incorporated IL-7, IL-15, and IL-21 at 40 ng/mL, which demonstrated maximum T-cell expansion, based on cellularity and cell viability. The predominant T-cell memory phenotype of the CD8+PD1+ cells following expansion was transitional memory (TTM) T-cells that increased from 35.6 ± 4.3 to 52 ± 3%. This memory T-cell phenotype has a high proliferative potential while retaining effector function. Following six days of culture with the three interleukins we observed a 10 fold increase in CD8+PD1+ cells such that 9x109 cells are obtained from the 160 gm spleen. These cells, based on ELISPOT analysis with autologous tumor lysate pulsed dendritic cells retained tumor specificity. In conclusion, CD8+PD1+ cells can be isolated from resected spleen, expanded with retention of tumor specificity and potentially used for adoptive cellular therapy.

#1421

Low dose gemcitabine increases the sensitivity of human Vgamma9Vdelta2T cell mediated cytotoxicity through NKG2D-NKG2D ligands axis in bladder cancer cells.

Teruki Shimizu,1 Mako Tomogane,2 Yusuke Sano,2 Daiki Shimizu,2 Masatsugu Miyashita,2 Atsuko Fujihara,3 Fumiya Hongo,3 Osamu Ukimura,3 Eishi Ashihara2. 1 _Matsushita Memorial Hospital, Osaka, Japan;_ 2 _Kyoto Pharmaceutical University, Kyoto, Japan;_ 3 _Kyoto Prefectural University of Medicine, Kyoto, Japan_.

INTRODUCTION AND OBJECTIVES: In clinical trials, Zoledronic acid (ZOL) pretreatment in cancer cells is widely used to improve the efficacy of human γδT cell immunotherapy. We herein provide new strategies for the enhancement of γδT cell function by anticancer pretreatment in urinary bladder cancer (UBC) cells. The aim of this study was whether pretreatment of cancer cells with standard anticancer agents would increase the cytotoxicity of γδT cells.

METHODS: The surface expression of NKG2D, TCRVγ9, TCRVδ2 and the intracellular levels of perforin and Granzyme B in γδT cells were examined. In in vitro assays, UBC cell lines T24, TCCSUP, and UMUC3 were used. Flow cytometric analysis of Carboxyfluorescein diacetate succinimidyl ester (CFSE) /propidium iodide (PI) staining was used in in vitro cytotoxicity assays. Imaging of cancer cells lysed by human γδT cell was captured by laser microscopy. UBC cells were treated with various standard anticancer agents at sub-lethal concentrations, including cisplatin (CDDP), gemcitabine (GEM), methotrexate (MTX), vinblastine (VBL), adriamycin (ADR), and mitomycin C (MMC). The expressions of MICA/B, ULBP1, and ULBP2/5/6 in UBC cells were investigated. In in vivo experiments, the efficacy of intravesical administration of ex vivo-expanded γδT cells was examined in an orthotopic xenograft model using In Vivo Imaging System (IVIS).

RESULTS: Ex vivo-expanded γδT cell expressed NKG2D receptor on the cell surface and both TCRVγ9, TCRVδ2 positive lineage were well expanded. Activation of ex vivo-expanded γδT cell was confirmed by intracellular staining of granules, containing perforin and Granzyme B. We examined the synergistic effects of anticancer agents and γδT cells in vitro. We found that these anticancer agents upregulated the expressions of MICA/B and ULBP family in UBC cells, which γδT cells use to recognize cancer cells and resulted in the increased cytotoxicity of γδT cells. The cytotoxicity was abrogated by induction of siRNA against MICA/B. These findings were also confirmed in MICA/B or NKG2D blocking experiments. We also found that intravesical administration of γδT cells showed potent cytotoxicity using an orthotopic xenograft. Finally, we revealed that low dose gemcitabine pretreatment synergistically increased the cytotoxicity of γδT cells in vivo.

CONCLUSIONS: These results indicated that γδT cell adoptive immunotherapy in combination with standard anticancer agents is an effective strategy and may be a promising approach to the treatment of UBC.

#1422

Preclinical development of CD37CAR T-cell therapy for treatment of B-cell lymphoma.

Pierre Dillard, Hakan Köksal, Sarah Josefsson, Solrun Melkorka Maggadottir, Sylvie Pollmann, Anne Fåne, Yngvild Nuvin Blaker, Klaus Beiske, Kanutte Huse, Ane Kolstad, Harald Holte, Gunnar Kvalheim, Erlend Bremertun Smeland, June Myklebust, Else Marit Inderberg, Sebastien Wälchli. _Oslo University Hospital, Oslo, Norway_.

T cells modified to express chimeric antigen receptor (CAR) targeting CD19 have produced remarkable clinical responses in patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). CD19CAR T-cell therapy has also demonstrated prominent effects in B-cell non-Hodgkin lymphoma (B-NHL) patients. However, a subset of patients who relapse after CD19CAR T-cell therapy have outgrowth of CD19-negative tumor cells. Hence, development of alternative CARs targeting other B-cell markers represents an unmet medical need for B-ALL and B-NHL. Here, we confirmed previous data by showing that B-NHL overall have high expression of CD37. A second generation CD37CAR was designed and its efficacy in T cells was compared to that of CD19CAR. In vitro assessment of cytotoxicity and T-cell function upon co-culture of the CAR T cells with different target B-cell lymphoma cell lines demonstrated comparable efficacy between the two CARs. In an aggressive B-cell lymphoma xenograft model, CD37CAR T cells were as potent as CD19CAR T cells in controlling tumor growth. In a second xenograft model, using U2932 lymphoma cells containing a CD19-negative subpopulation, CD37CAR T cells efficiently controlled tumors and cured the mice while CD19CAR T cells had limited effect. We further show that, unlike CD19CAR, CD37CAR was not sensitive to antigen masking. Finally, CD37CAR reactivity was restricted to B-lineage cells. Collectively, our results demonstrated that CD37CAR T cells effectively can eradicate B-cell lymphoma tumors also when CD19 antigen expression is lost, and support further clinical testing for patients with relapsed/refractory B-NHL.

#1423

AIM ACT, a novel nanoparticle-based technology that generates therapeutic numbers of functional tumor-specific CD8+ T cells with T stem cell, central and effector memory phenotype in 14 days.

Sojung Kim, Lauren Suarez, Emily Lu, Celine Walmacq, Daniel Dembrow, Juan Varela, Dan Bednárik, Kenneth Carter, Naimish Pandya, Kristi Jones, Mathias Oelke. _NexImmune, Gaithersburg, MD_.

Background:

Efficient ex-vivo generation of functional tumor-specific T cells with an optimal memory phenotype remains a significant hurdle to the broad application of adoptive cell transfer (ACT) protocols for the treatment of cancer. Genetically engineered T cells represent a currently available option, but the genetic manipulation of T cells presents significant challenges in terms of manufacturing, cost and addressing tumor escape mechanisms. Here, we describe a novel nanoparticle-based approach for generating tumor-specific T cells at clinical grade and scale from the endogenous T cell repertoire using artificial antigen presenting cells (aAPC).

Methods:

aAPC consist of a paramagnetic nanoparticle to which humanized HLA-A2-Ig dimer-molecules and anti-CD28 antibodies are covalently linked. aAPC are loaded with multiple HLA-A2 restricted peptides and used to magnetically enrich and expand tumor-specific CD8+ T cells. Using peptide loaded aAPC, a fully enclosed and automated GMP T cell expansion platform has been developed that consistently generates clinically relevant numbers of tumor-specific, T stem cell, central and effector memory CD8+ T cells in 14 days, providing an alternative to genetically manipulated T cells.

Results:

Starting from a healthy donor leukopak, tumor-specific CD8+ T cells were generated using an aAPC cocktail loaded with 5 HLA-A2 epitopes from AML tumor antigens WT1, PRAME and Cyclin A1. On average, 1-2 x109 T cells were generated that were >90% of the memory phenotype that averaged 18% T stem cells, 40% central memory and 35% effector memory CD8+ T cells. AML-specific T cells were expanded 500 to >5000-fold from low frequency precursor populations. The system has also been used to generate MM specific T cells of the same quality from both from fresh leukopacks and cryopreserved PBL. All generated T cells were fully functional, as demonstrated by intra-cellular cytokine analysis and tumor cell killing.

Conclusion:

AIM ACT is a novel nanoparticle-based T cell platform developed for the rapid, streamlined generation of clinically-relevant numbers of tumor-specific, T stem cell, central and effector memory CD8+ T cells from donor PBMC in 14 days. The platform described here will be used to generate multi-antigen specific CD8+ T cell products that will be evaluated in multi-center P1/2 clinical trials. These trials will enroll r/r MM patients and r/r AML patients pre- and post-allogeneic hematopoietic stem cell transplant. The flexibility of the AIM ACT system, as shown has potential for clinical evaluation in other hematological tumors and can be used for targeting neo-epitopes.

#1424

Preclinical development of TAC01-CD19, a CD19 specific T-cell Antigen Coupler (TAC) therapy for the treatment of CD19-positive B cell malignancies.

Christopher W. Helsen,1 Danielle Hayes,1 Joanne Hammill,2 Criag Aarts,2 Andreas Bader,1 Donna Rill,1 Jonathan Bramson2. 1 _Triumvira Immunologics, Hamilton, Ontario, Canada;_ 2 _McMaster University, Hamilton, Ontario, Canada_.

Background: The T cell antigen coupler (TAC) is a chimeric receptor co-opting the natural TCR receptor signaling complex to target tumor antigens in an MHC-independent manner (Helsen et. al., Nat. Comm. 2018). TAC engineered T cells mediate biological effects that are distinct from conventional chimeric antigen receptors (CARs) and may offer safety advantages due to greater target selectivity and no detectable off-target toxicity. Here, we present preclinical data of TAC01-CD19, an autologous CD19-specific TAC-T cell product candidate for clinical testing in patients with CD19-positive B cell malignancies

Materials and Methods: T cells from healthy donors and lymphoma patients were engineered with a CD19-specific TAC using lentivirus. Anti-tumor activity, T cell expansion and persistence were measured in the CD19-positive NALM-6 and Jeko-1 mouse tumor xenograft models. We used flow cytometry to determine surface expression of CD19-TAC, cytokine production and in vitro T cell proliferation. Cytotoxicity was evaluated in vitro using luciferase-based killing assay.

Results: Cytotoxicity was measured against multiple CD19 positive tumors cells (NALM6/Jeko-1/Raji) showing strong cytotoxicity in all cases. Treatment of established NALM-6 (acute lymphoblastic leukemia) and Jeko-1 xenografts (mantle cell lymphoma) with CD19 TAC-T cells resulted in clearance of tumors within a few weeks of T cell infusion. In vivo efficacy was tested across multiple donors, establishing consistent and reproducible anti-tumor efficacy. Monitoring of TAC T cells post-infusion revealed robust expansion that peaked in the peripheral blood 1-2 weeks post-infusion. Mice that cleared tumors following TAC-T cell treatment were resistant to subsequent challenge with fresh tumor cells demonstrating persistence of TAC-T cells. In contrast, treatment with control TAC-T had no impact on tumor growth. A histological examination of normal mouse tissues failed to detect TAC-T cell induced toxicity.

Conclusion: The pre-clinical evaluation of TAC01-CD19 has demonstrated specific anti-tumor responses without signs of toxicity. Results were reproducible across multiple donors demonstrating the robustness of the CD19 TAC system. TAC01-CD19 is expected to enter clinical testing in patients with CD19-positive hematological malignancies in Q2 2019.

#1425

Chimeric antigen receptors based on T cell receptor-like antibodies.

Ling Wu,1 Joanna Brzostek,1 Shvetha Sankaran,1 Triscilla Tan,1 Conrad Chan,2 Jiawei Yap,1 Junyun Lai,1 Paul MacAry,1 Nicholas Gascoigne1. 1 _National University of Singapore, Singapore, Singapore;_ 2 _Defense Medical and Environmental Research Institute, Singapore, Singapore_.

T cells are essential in adaptive immunity and play an indispensable role to eliminate abnormal or virus infected cells. By merging antibody technology and cell engineering, T cells equipped with a chimeric antigen receptor (CAR) can be redirected to the target in a non-MHC restricted fashion. Within the past few years, clinical trials using CAR T cells engineered to recognize B cell cancers have shown high rates of response (70%-90%) and durability of response that are unprecedented in acute and chronic leukaemia. However, severe toxicity has been observed due to massive, and to some extent nonspecific, T cell activation. Although CARs have been improved and investigated heavily, the extent to which a CAR is similar to its parental TCR extra- or intracellularly, and whether critical elements involved in normal TCR signalling are kept or rewired in CAR signalling, are unclear. A better understanding of these questions would greatly facilitate improvements in CAR technology and its usefulness in clinical practice. A CAR targeting a peptide-MHC complex (using an Fv from an antibody that recognizes MHC-peptide: in other words, a TCR-like Ab) mimics the process of TCR recognition and takes advantage of CAR technologies developed so far. It is thus an excellent study object to compare TCR and CAR signalling. Well-built research methods on TCR can be transferred immediately to TCR-like CAR, while the knowledge and findings generated through the studies of TCR-like CAR can in turn renew our perceptions on TCR.

TCR-like antibodies targeting the EBV epitopes LMP1125-133, LMP2A426-434 or EBNA1562-570, were engineered as CARs. We find that conformation change, if any, upon ligand binding is not enough to activate downstream TCR signalling, but oligomerization is a crucial factor. Although CAR with a TCR-like specificity can recruit CD8 co-receptor to the immunological synapse, it is dispensable for activation of the T cell. Furthermore, the activation kinetics of CAR and TCR are distinctly different. TCR shows a pulse-like activation, whereas CAR shows an activation that gradually plateaus and remains steady. These unique properties of CAR identified in our study demonstrate that CAR signalling properties may be amenable to modifications leading to better specificity and activity in vivo. Our long-term goal is that the TCR-like CAR can be used to combat EBV induced Nasopharyngeal Carcinoma.

#1426

**Expansion of CD8** + **PD-1** + **T cells restored TCR repertoire as a prognostic marker to adoptive T cell immunotherapy of advanced pancreatic cancer.**

Guoliang Qiao. _Capital Medical University, Beijing, China_.

Purpose: We have previously reported the clinical benefits of autologous adoptive T cell immunotherapy in advanced pancreatic cancer (APC). We therefore aimed to further identify the core functional population of CD8+ T cells which exerted on the aberrant TCR repertoire and subsequently contribute to favorable prognosis after adoptive T cell therapy(ACT).

Experimental Design: PBMC from APC patients (n=25) treated on a protocol of S-1 plus adoptive T cell immunotherapy were expanded ex vivo with a cytokine cocktail. The different fractions of CD8+PD-1+ ,CD8+LAG-3+, CD8+TIM-3+ and CD8+4-1BB+ T cells from time dependent segment were quantitated and expression of co-inhibitory molecules and biological functions on tumor killing and comparative analysis TCR repertoire from each patient were measured. The multivariate Cox hazards regression analysis was performed to identify the key T cell subset that affected the TCR repertoire recovery and subsequently associated with clinical outcome.

Results: The CD3+, CD3+CD4+, and CD3+CD8+ T cells within the expanded cell product reached peak levels by day 15. CD8+ T cells exhibited enhanced expression of PD-1, LAG-3, and TIM-3, but not 4-1BB, after ex vivo expansion. Survival analysis showed that patients with a ratio of post/pre CD8+PD-1+ T cells > 2 had significantly favorable progressive-free survival (PFS) (median PFS time 180 days vs 85 days, P=0.002) and overall survival (OS) (median OS time 238 days versus 142 days, P=0.024). The sorted CD8+PD-1+ T cells displayed enhanced anti-tumor activity, and increased IFN-γ secretion after co-culture with autologous tumor cell lines. TCR repertoire diversity simultaneously rose during the ex vivo expansion and was associated with an increase in CD8+ T cells and decline in CD8+/CD28- T cells. Moreover, an increased TCR diversity was associated with significantly favorable PFS (median PFS time 166 days vs 79 days, P=0.043) and OS (median OS time 216 days versus 112 days, P=0.031) and was associated with the extent of expansion of the CD8+PD-1+ T cells (r2=0.464, P=0.001). Cox proportional hazards analysis showed that post/pre CD8+PD-1+ T cells after expansion ex vivo was an independent prognostic marker associated with both OS(P=0.009) and PFS(P=0.012).

Conclusion: CD8+PD-1+ T cells, after ex vivo expansion over 15 days, are tumor-reactive. The extent of CD8+PD-1+ T cell expansion could restore TCR repertoire and was an independent prognostic factor associated with clinical outcomes in APC patients treated with ACT.

#1427

IL6 trans-signaling robustly promotes the expansion and antitumor activity of CAR T cells.

Peng Li,1 Zhiwu Jiang,1 Rui Liao,1 Zhaoyang Tang2. 1 _Guangzhou Insts. of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China;_ 2 _Guangdong Zhaotai Invivo Biomedicine Ltd, Guangzhou, China_.

Chimeric antigen receptor T (CAR T) cell immunotherapy targeting CD19 positive B cells malignancies induces promising clinical remissions. However, the treatments are often accompanied with high levels of IL-6 characterized cytokine release syndrome (CRS). Previous studies had shown that sIL-6R are constitutively present in high concentrations in serum. Therefore, when the level of IL-6 is elevated (above nomogram per ml), sIL-6R combines with IL6 to form the IL-6/sIL-6R complex (trans-signaling). The IL-6 trans-signaling plays important roles in regulating immune responses, cell survival, apoptosis, and proliferation. To our knowledge, comprehensive assessment of the IL-6 signaling in biologic functions of CAR T has not been well addressed. We thus tested whether the IL-6 trans-signaling promotes expansion and anti-tumor activity of CAR T. We found that the levels of serum IL-6 and IL-6/sIL-6R complex were positively correlated with anti-CD19 CAR T cell expansion and anti-leukemia response in patients. To simulate the IL-6 trans-signaling, we next constructed a constitutively expression of Hyper IL-6 (HIL-6) with various CAR T cells. Anti-tumor efficacy of HIL-6-CAR T cells were confirmed in leukemia (anti-CD19) and solid tumors (anti-MUC1 and anit-GPC3 CAR T cells targeting lung cancer and hepatocellular carcinoma, respectively). Our results demonstrate that HIL-6-CAR T cells are more effective for suppressing tumor growth and more persistent in xenograft models. Transcriptomic profiling analysis revealed that the expression of genes that facilitate T-cell migration, early memory differentiation and the IL-6/GP130/STAT3 signaling was upregulated in HIL-6 simulated CAR T cells compared to the ones without HIL-6 overexpression. Because IL-6 trans-signaling activates downstream signaling via GP130, we also generated a novel CAR vector with constituted GP130 activation and demonstrated the CAR T cells were superior in anti-tumor activity with delayed xGVHD in xenograft models of leukemia, lung cancer and liver cancer. Taken together, these results showed that IL-6 trans-signaling can significantly enhanced the expansion anti-tumor activity of CAR T cells via GP130/STAT3 activation and GP130/CAR T may have the potential to promote anti-tumor effects with reduced toxicity in vivo.

Keywords: Chimeric antigen receptor T, cytokine release syndrome, IL-6 trans-signaling, GP130

#1428

Allogeneic CRISPR/Cas9 gene-edited CAR-T cells targeting CD33 show potent preclinical activity against AML cells.

Brigid McEwan, Zinkal Padalia, Ashley Porras, Jason Sagert, Jonathan A. Terrett, Tony Ho, Demetrios Kalaitzidis. _Crispr Therapeutics, Cambridge, MA_.

Acute myelogenous leukemia (AML) has a high mortality rate and remains difficult to treat, making new treatment approaches critical. Chimeric antigen receptor T (CAR-T) cell therapies have shown impressive clinical responses in B-cell neoplasia. However, comparable successes have not been reported to date in myeloid malignancies, potentially due to difficulty in manufacturing efficacious CAR-T cells from AML patients and lack of a suitable tumor antigen for AML. To address these issues, we have developed allogeneic CAR-T cells from healthy donors targeting the CD33/Siglec-3 antigen, a protein expressed on most AML cells and subpopulations in the majority of AML patients at presentation and relapse. Allogeneic anti-CD33 CAR-T cells were produced from healthy donor-derived T cells using CRISPR/Cas9 gene editing. In these cells, the TRAC locus was disrupted to reduce the risk of graft versus host disease (GvHD). At the same time, a CAR construct targeting CD33 was inserted site-specifically into the TRAC locus. In addition, the beta-2-microglobulin locus was disrupted to prevent clearance of the allogeneic CAR-T cells by the host immune system. These allogeneic anti-CD33 CAR-T cells showed potent effector activity in vitro against human AML-derived cell lines, as measured by both tumor cell lysis and effector cytokine secretion. The allogeneic anti-CD33 CAR-T cells also potently reduced AML tumors in vivo in xenograft mouse models.

#1429

Preclinical characterization of a KK-LC-1-specific T cell receptor for the treatment of epithelial cancers.

Bridget Marcinkowski, Carylinda Serna, Benjamin Jin, Scott Norberg, Christian Hinrichs. _National Cancer Institute, Bethesda, MD_.

T cell receptor (TCR) gene therapy can mediate tumor regression in patients; however, successful treatment of epithelial cancers has been limited. We studied the targeting of Kita-Kyushu lung cancer antigen 1 (KK-LC-1), a cancer germline antigen expressed in multiple epithelial cancers, with TCR gene-engineered T cells. We thoroughly vetted KK-LC-1 expression in both cancerous and healthy tissue by pulling data from BioGPS and cBioPortal and analyzing a broad range of patient samples, primary cancer lines, and healthy tissue by both qRT-PCR and fluorescence in situ hybridization. Our findings show that KK-LC-1 is expressed in numerous epithelial cancers including breast, lung, cervical, ovarian, melanoma, and prostate whereas expression in healthy tissue is limited to immune-privileged sites including the epididymis and testis. We identified an HLA-A*01:01-restricted KK-LC-152-60-specific TCR isolated from the tumor infiltrating lymphocytes of a patient with metastatic cervical adenocarcinoma. T cells from two healthy donors were transduced with the KK-LC-1 TCR and tested for in vitro targeting of tumor cell lines. The KK-LC-1 TCR recognized the HLA-A*01:01+, KK-LC-1+ cell lines 4156, EKVX, and PC-3, as determined by IFN-γ release. The HLA-A*01:01- cell lines, HeLA, DU-145, and MDA-MD-468, were initially not recognized by the KK-LC-1 TCR; however, following stable transduction with HLA-A*01:01, these cell lines were targeted. The HLA-A*01:01+, KK-LC-1- cell lines, PC-3 and 3748, were not recognized by the KK-LC-1 TCR. To assess the TCR's potential for off-target activity, we used amino acid scanning to determine the recognition motif, allowing for subsequent targeted in silico searches to identify peptides containing the same recognition motif. Using NCBI Blast and the ScanProsite database, 10 peptides with homologous essential but different non-essential residues were identified. A retroviral vector encoding the KK-LC-152-60 TCR was used to genetically engineer peripheral blood T cells. No peptide recognition by the TCR was observed when dendritic cells were pulsed with the individual peptides and co-cultured with KK-LC-1 TCR-transduced T cells. Finally, we assessed if human genetically engineered T cells expressing the KK-LC-1 TCR could mediate in vivo tumor regression in an NSG mouse model. Genetically engineered T cells expressing the KK-LC-1 TCR induced tumor regression in both a melanoma and cervical cancer line in a dose-dependent manner. Collectively, these findings indicate that KK-LC-1 is a suitable target for TCR gene therapy and they provide preclinical support for testing of the KK-LC-1 TCR in a phase I clinical trial.

#1430

Anti-CD3 x anti-EGFR bispecific antibody armed T cells (EGFR BATs) kills resistant pancreatic cancer cell lines and increases sensitivity to chemotherapy.

Lawrence G. Lum, Johnson Ung, Archana Thakur. _University of Virginia, Charlottesville, VA_.

Chemotherapy (chemoT) responses of pancreatic cancer (PC) are short and PC rapidly develop multidrug resistance (MDR) with median overall survival (OS) of locally advanced PC and metastatic PC patients of 7 months. Several MPC patients developed dramatic responses to the same chemoT after receiving multiple infusions of EGFR BATs. Novel therapeutic strategies are needed to improve clinical results. This study asks whether pre-targeting tumor cells with EGFR BATs can "sensitize" drug resistant tumor cell lines so that subsequent treatment can not only kill the MDR PC lines but also decrease the doses of chemotherapy required to kill tumor cells. Gemcitabine (Gem) and cisplatin (CIS) were used to generate drug-resistant MiaPaCa-2 and L3.6 by exposing step-wise increasing concentrations of Gem and Cis. MiaPaCa-2 and L3.6 survive to become resistant to Gem and CIS. Specific cytotoxicity was measured by 51Cr release. Flow cytometry data showed increased CD44+/CD24+/EpCAM+ cancer stem like cells and increased ABC transporter ABCG2+ cells in resistant cell lines compared to parental lines. EGFR or HER2 BATs from normal donors would kill Gem or CIS resistant MiaPaCa-2 or L3.6 (n = 6-8 normals) in 51Cr release assays and secrete Th1cytokines, IFN-γ and TNF-α, MIP-1b, and RANTES. We assessed whether pretreatment with EGFR BATs modify the effective dose of CIS activity directed at MiaPaCa-2 and PANC-1 cells using the xCelligence system. EGFR BATs at an E:T of 2:1 were added to the target cells for 24 hours, washed out, and CIS was added at concentrations of 3.5, 7, and 14 µM (IC50 by MTT) and MiaPaCa-2 at concentrations of 4.7, 9.3, and 18.6 µM (IC50 by MTT). The survival of the MiaPaCa2-2 cells was monitored continuously to track survival of the tumor cells. The mean cytotoxicity directed at MiaPaCa-2 increased with BATs priming at concentrations lower than the IC50 (4.7 and 9.3 µM). At the 4.7 µM dose, cytotoxicity increased from CIS alone (31±15%) to BATs primed followed by CIS (57±17%), and BATs together with CIS (77±12%). In PANC-1 cells, there was no specific cytotoxicity of with CIS concentrations at 3.5, 7.0, and 14 µM. BATs priming before addition of CIS increased specific cytotoxicity to 51±13%, 60±9%, and 84±5% at CIS concentrations of 3.5, 7.0, and 14 µM, respectively. Similar data have been obtained for 5-fluorouracil and preliminary data show similar trends for CIS resistant MiaPaCa-2 and PANC-1. In summary, we showed that: 1) BATs can kill chemo resistant tumor cells; and 2) pretreatment or sensitization of the tumor with BATs lowers the threshold for effective cytotoxic doses of CIS against MiaPaCa2-2 and PANC-1. This BATs priming strategy may augment clinical responses, decrease chemoT toxicities, and improve overall survival for pancreatic cancer patients.

#1431

In vivo CRISPR/Cas9 screen of adoptively transferred T cells reveals novel mediators of T cell exhaustion and immunosuppression.

Jin K. Park,1 David A. Canner,1 Rebecca Herbst,2 Amy Li,1 Olivia C. Smith,1 Aviv Regev,2 Tyler E. Jacks1. 1 _Koch Institute for Integrative Cancer Research, Cambridge, NY;_ 2 _Broad Institute of MIT and Harvard, Cambridge, NY_.

Adoptive cell therapy (ACT) is a form of cancer immunotherapy in which patients receive an infusion of a polyclonal population of autologous CD8+ T cells that can be genetically modified ex vivo to optimize their function against the tumor. Although clinical responses to ACT have been successful for a very limited number of indications (most notably pediatric leukemias), its efficacy in solid tumors is very limited, likely due to immunosuppressive mechanisms constraining efficacy. To date, there have not been systematic approaches to discover novel T cell-intrinsic mediators of exhaustion in the context of ACT. Here, we describe a systematic and unbiased approach to uncover novel regulators of exhaustion in adoptively transferred CD8+ T cells. We report the results of longitudinal RNA sequencing of adoptively transferred cells at various time points post-transfer, revealing the complex transcriptional changes that transferred cells undergo. In addition, we report the results of an in vivo CRISPR/Cas9-mediated genetic screen. Taken together, this integrated approach of longitudinal RNAseq and CRISPR/Cas9 screening has yielded mediators previously uncharacterized in T cells that can be targeted to drastically improve functionality, cytotoxicity, and persistence in the tumor microenvironment.

#1432

Enhanced effector responses of regenerated CAR-T cells derived from genome edited iPSCs.

Tatsuki Ueda,1 Shoichi Iriguchi,1 Yohei Kawai,1 Atsutaka Minagawa,1 Hiroyuki Miyoshi,2 Seitaro Terakura,3 Yasushi Uemura,4 Knut Woltjen,1 Yuzo Kodama,5 Hiroshi Seno,6 Yasumichi Hitoshi,7 Tetsuya Nakatsura,4 Koji Tamada,8 Shin Kaneko1. 1 _Center for iPS Cell Reserch and Application, Kyoto University, Kyoto, Japan;_ 2 _Keio University, Tokyo, Japan;_ 3 _Nagoya University, Nagoya, Japan;_ 4 _National Cancer Center, Kashiwa, Japan;_ 5 _Kobe University, Kyoto, Japan;_ 6 _Kyoto University, Kyoto, Japan;_ 7 _Thyas Co. Ltd., Kyoto, Japan;_ 8 _Yamaguchi University, Yamaguchi, Japan_.

Chimeric antigen receptor (CAR) is an artificial protein that provides HLA-independent antigen specificity to T cells. CAR-T therapy has shown remarkable clinical responses especially in hematologic malignancies. But this therapy requires cell preparation for each patient and it cause some limitations for applicability of CAR-T therapy. We have reported regeneration of T cells from iPSCs (Cell Stem Cell. 2013). Since this technology can provide unlimited number of T cells, CAR-T therapy using iPSCs is thought to broaden its applicability. To target solid tumors, it is important to avoid immunosuppressive factors from tumor microenvironment and to exert sufficient cytotoxicity. Tumor reactive T cells are known to fall into anergy state by continuous antigen stimulations. To overcome immunosuppression in tumor microenvironment, enhancement of TCR signaling by modification of genes related to TCR signal is a promising strategy. Recent studies revealed that the efficacy of CAR-iPS-T cells are not equivalent to primary CAR-T cells. To enhance the efficacy of CAR-iPS-T cells and to produce resistant CAR-T cells to immunosuppression, we focused on TCR signaling pathway. We found that antigen reactivity of CAR-iPS-T cells was insufficient compared with primary CAR-T cells. To overcome the weakness of TCR signal, we disrupted genes negatively related to TCR signal and successfully enhanced TCR signal. As a result, genome edited CAR-iPS-T cells could persist longer in vivo and displayed enhanced tumor suppressive function comparable with primary CAR-T cells. Genome edited iPSCs can be a unlimited cell source of enhanced CAR-T cells. These findings indicate that regenerated CAR-T cells derived from genome edited iPSCs would be a promising CAR-T therapy which would overcome immunosuppressive tumor microenvironment.

#1433

NeoTCR-P1, a novel neoepitope-specific adoptive cell therapy, consists of T cells with 'younger' phenotypes that rapidly proliferate and kill target cells upon recognition of cognate antigen.

Barbara Sennino,1 Andrew Conroy,1 Bhamini Purandare,1 Adam Litterman,1 Kyle Jacoby,1 Robert Moot,1 William Lu,1 Diana Nguyen,1 Fabrizia Urbinati,1 Susan Foy,1 Theresa Hunter,1 Olivier Dalmas,1 Michael Bethune,1 Tim Park,2 Songming Peng,1 Alex Franzusoff,1 Stefanie Mandl1. 1 _PACT Pharma, South San Francisco, CA;_ 2 _Arcus Biosciences, Hayward, CA_.

Clinical benefit observed with immuno-oncology trials often depends on the unleashing of a pre-existing intrinsic T cell immune response in each cancer patient. The targets of these intrinsic T cells are commonly ascribed to recognition of patient-specific neoantigens that arose from cancer mutations.

PACT Pharma has developed the ability to selectively capture neoantigen-specific CD8+ T cells from peripheral blood of the patient. Leveraging this technology, PACT Pharma is developing personalized, autologous neo-epitope specific TCR-engineered T cell therapies for the eradication of solid tumors.

Briefly, using PACT's proprietary TCR isolation technology neoepitope-specific TCRs are cloned and autologous CD8+ and CD4+ T cells from the same patient with cancer are precision genome engineered (using a DNA-mediated (non-viral) method) to express the neoTCR. NeoTCR expressing T cells are then expanded in a manner that preserves a "younger" T cell phenotypes, resulting in a NeoTCR-P1 product in which the majority of the T cells exhibit T memory stem cell and T central memory phenotypes.

Upon cognate antigen encounter, NeoTCR-P1 rapidly differentiate into potent effector T cells. Engineered NeoTCR-P1 cells rapidly expand, secrete effector molecules such as perforin and granzyme B, and cytokines such as interferon-gamma (IFN-γ), IL-2 and TNF-alpha (TNF-α). Single cell secretome analysis demonstrates that NeoTCR-P1 cells are highly polyfunctional (secretion of two or more cytokines or effector proteins). These results demonstrate that PACT's autologous ex vivo engineered NeoTCR-P1 T cells represent a highly personalized adoptive T cell therapy with potential to provide significant clinical benefit to subjects with solid tumors.

#1434

The Costimulatory Signal Domains 4-1BB and CD3ζ Do Not Improve the Function of CD16A Chimeric Antigen Receptor Transduced NK Cells.

Long Chen, Vicky Li, David Allan, Robert Reger, Elena Cherkasova, Stephanie Pierre, Stefan Barisic, David Granadier, Emily Levy, Giacomo Waller, Susan Doh, Mala Chakraborty, Kate Stringaris, Richard Childs. _NHLBI, Bethesda, MD_.

Chimeric Antigen Receptors (CAR)-T cells are genetically modified to express an extracellular binding domain, transmembrane domain and intracellular signaling domain. The extracellular binding domain is usually derived from antibodies; however, it can be replaced with other ligand-receptor binding systems, such as with an Fc receptor. CD16A has been tested in second-generation 4-1BB-CD3ζ CAR T cells and NK cell lines (NK-92) and enhances tumor killing; however, it is not clear whether these cells provide superior tumor killing compared to cells modified to express CD16A alone.

In this study, we designed three CAR constructs containing the extracellular binding domain of high affinity mutant CD16A (CD16A-V158) and intracellular signal through 4-1 BB-CD3ζ that differed in leader sequences and transmembrane domains. CAR1 contained the CD16A leader and CD16A transmembrane domain; CAR2 had the CD16A leader and CD8α transmembrane domain; CAR3 contained the CD8α leader and CD8α transmembrane domain. When transduced into Jurkat cells via lentivirus, CD16A V158 and all three CARs showed stable high surface expression of CD16A (> 70%). In contrast, NK-92 cells were only successfully transduced to express CD16 by CD16A V158 (29.8%), CAR2 (42.3%), and CAR3 (42.5%) with no expression of CD16A by CAR1. Transduced cells were sorted by FACs based on CD16A expression. All sorted cells showed >90% stable expression of the constructs for up to 42 days post sorting. We observed a much higher CD16 MFI in cells transduced with CAR2 and CAR3 compared with CD16A V158. Western blotting with anti-CD3ζ identified the full assembly of CARs.

We then tested the function of the transduced cells by a Rituximab binding assay. Although there was no difference in binding to Rituximab at 0.01 and 0.1 mg/mL; CAR2 and CAR3 showed better binding at the 1 and 10 mg/mL. In a 4-hour coculture toxicity assay with 721.221 LCLs, CD16A V158 and CAR3 increased the target lysis by two-fold while CAR2 only showed a 50% increase. When cocultured with 721.221 HLA-E+ LCLs, a 721.221 subclone more resistant to killing by NK-92 cells, we observed similar tumor killing with CD16A V158 and CAR3 (killing doubled) in contrast to CAR2(killing increased by only 50%). We next tested against the B-cell line Raji and found that there was only a mild increase of target lysis with transduced NK-92 cells; however, the CD16A V158 and CAR3 still showed higher toxicity (around a 40% increase).

In summary, CAR2 and CAR3 expressed a higher CD16 MFI in NK cells and demonstrated better binding to Rituximab at1 and 10 mg/mL compared to CD16A V158; however, CAR3 and CD16A V158 had similar killing of target cells that was higher than was observed with CAR2. Our data indicate that NK cells transduced to have a CAR structure combining CD16A with 4-1BB-CD3ζ have augmented ADCC, although this augmentation is not superior to NK cells that are simply transduced to express CD16A alone.

#1435

An ultra-sensitive and high-throughput technology (imPACT) for the identification and isolation of intrinsic and emergent neoepitope-specific T cells from the peripheral blood and TILs of cancer patients.

Songming Peng, Boi Quach, Duo An, Salemiz Sandoval, Robert Bao, Zheng Pan, Michael Bethune, Olivier Dalmas, Michael Yi, Corey Meadows, Katherine Heeringa, Linlin Guo, Benjamin yuen, John Sorfleet, Kyle Jacoby, Robert Moot, William Lu, Diana Nguyen, Barbara Sennino, Andrew Conroy, Bhamini Purandare, Adam Litterman, Stefanie Mandl, Alex Franzusoff. _PACT Pharma, Hayward, CA_.

T cells capable of targeting neoepitopes (neoE) from tumor-specific mutations hold the potential to uniquely recognize and kill tumor cells. However, most cancer patients fail to mount a sufficient intrinsic T cell immune response to translate into clinical benefit. PACT Pharma has developed an ultra-sensitive and high-throughput technology (imPACT) for identifying and isolating neoE-specific T cells from peripheral blood. Whole exome sequencing of tumors and computational prediction identify patient-specific neoepitopes resulting from tumor-specific mutations. We then interrogate patient blood for neoE-specific T cells using human leukocyte antigen (HLA) protein-based reagents comprising a spectrum of human HLAs, thus enabling the evaluation of >99% of all individuals with cancer. We have identified and isolated neoE-specific T cells from the peripheral blood of >80% treatment-naive patients with bladder and colorectal cancers, melanoma and other solid tumors. Primary human T cells engineered with T cell receptor sequences (TCRs) cloned from the imPACT-isolated T cells gain the ability to kill cognate neoE-presenting tumor cells, thereby also confirming the specificity of the isolated TCR sequences to bind to the neoE target. This approach is also amenable to the longitudinal analysis of patients undergoing treatment for their cancers, to characterize the neoE-specific T cell populations likely to confer clinical benefit. In summary, the imPACT technology efficiently discovers potentially meaningful intrinsic neoE-specific TCRs from patients, enabling the development of personalized neoTCR-T cell therapies for the eradication of solid tumors.

#1436

Development of a chimeric antigen receptor against glycosylated annexin A2 on ovarian tumors.

Leonard W. Leong, Simeon Cua, Heng Liang Tan, Andre B. Choo. _Bioprocessing Technology Institute, Singapore, Singapore_.

Chimeric antigen receptors (CARs) have been a significant focus of cancer therapeutic investigation in recent years, due to the remarkably effective responses observed in hematological cancers. However, the utility of CAR T cells has yet to be fully exhibited in solid tumours. One of the key challenges facing the treatment of solid tumors with CAR T cells is the dearth of viable antigen targets, which limits the expansion of this therapeutic space.

Here, we describe how the anti-glycan monoclonal antibody 2448, which target annexin A2 on a number of ovarian and breast cancer cell lines, has been successfully reformatted as a CAR T cell construct.

Messenger RNA (mRNA) encoding CAR(2448) was transfected into activated T cells, resulting in cytotoxicity and cytokine release upon co-incubation with target cells expressing annexin A2. While CAR(2448) variants with differing spacer chain lengths all mediated cytotoxicity against target cells compared to untransfected T cells, real-time target cell growth monitoring revealed that the long spacer CAR(2448) mediated higher levels of cytotoxicity, consistent with the membrane-proximal location of the annexin A2 target epitope.

This optimization of CAR spacer length in the in vitro setting, using mRNA transfected T cells and real-time target cell growth monitoring, suggests that several aspects of CAR construction can be optimized in vitro, thereby lowering the threshold for CAR T cell development.

These results suggest the potential for anti-glycan antibodies to be repurposed as CARs, and the development of CAR(2448) is currently being extended to lentiviral vectors for the purpose of developing long-term expressing CAR(2448) T cells, in anticipation of in vivo studies in murine models.

This project was funded by the Agency for Science, Technology, and Research (A*STAR), Singapore and the A*STAR Graduate Academy (A*GA).

#1437

Improving efficacy and accuracy of CAR-T cells for neuroblastoma.

Babak Moghimi, Sakunthala Muthugounder, Shahab Asgharzadeh. _CHLA, Los Angeles, CA_.

Background: Anti-disialoganglioside (GD2) specific CAR T-cell therapy has shown limited efficacy in children with recurrent/refractory neuroblastoma (NBL). A recent pre-clinical study demonstrated significant neurotoxicity in mice treated with CAR T-cells bearing a high-affinity anti-GD2 short chain variable fragments (scFv; Richman S et al. 2017). To devise a safe and more efficacious CAR T-cells, we developed a gated CAR T-cell system utilizing the Syn-Notch technology.

Methods: GD2 and CD19 specific Syn-Notch gated systems were constructed by linking corresponding scFv to the Notch core followed by Gal4Vp64. Upon target recognition, Gal4VP64-dependent UAS element would initiate expression of the B7H3-BBZ CAR. Reporter activity of CD19 or GD2 Syn-Notch systems were monitored using flow cytometry after exposure to CD19+ transduced CHLA255 NBL cell line. In vitro cytotoxicity was assessed using DIMSCAN and xCELLigence impedance assays at varying Effector: Target ratios. NSG mice with established metastatic CHLA255(GD2+CD19+) NBL were treated with 1x10^7 transduced and control T cells and monitored for disease burden.

Results and discussion: Jurkat and human primary T cells were successfully transduced with the Syn-Notch system. GD2-Notch-Gal4VP64 and CD19-Notch-Gal4VP64 T-cells effectively activated downstream reporter in exposure to CHLA255(GD2+CD19+) within 6 hours of exposure but not in the presence of NBL cells lacking GD2 or CD19. The GD2 system exhibited greater leakiness than the CD19 counterpart. GD2- Syn-Notch-B7H3-BBZ or CD19- Syn-Notch-B7H3-BBZ CAR T-cells demonstrated statistically significant cytotoxicity against CHLA255(GD2+CD19+) NBL cell line. Preliminary in vivo data also show the anti-tumor effect of these gated CAR systems compared to mock-transduced T-cells.

Conclusions: We have successfully established a novel GD2-B7H3 gated system using the Syn-Notch technology, and demonstrate successful specificity to both antigen targets. This system increases the specificity of CAR T-cells against GD2 and B7H3 positive neuroblastoma tumors and likely minimize GD2-induced toxicity. These gated CARs show great promise for establishing immunotherapies that maximize tumor specificity while minimizing toxicity.

#1438

Integrated proteogenomics utilizing mass spectrometry to identify MHC-associated neopeptides in EGFR mutant lung adenocarcinoma.

Yue Qi, Tapan K. Maity, Xu Zhang, Shaojian Gao, Nitin Roper, Meriam Bahta, Khoadang Nguyen, Constance M. Cultraro, Udayan Guha. _National Cancer Institute/NIH, Bethesda, MD_.

Introduction: The epidermal growth factor receptor (EGFR) is one of the most frequently mutated oncogenes in human lung cancer. Although immune checkpoint inhibitors have been therapeutically effective in multiple cancers, patients harboring EGFR mutations have not responded well to this immunotherapy. Adoptive T cell therapy (ACT) has resulted in complete and durable regression of metastatic cancers. ACT requires that neoantigens are presented by major histocompatibility complex (MHC) class I molecules on tumor but not normal cells. The MHC binding algorithms, such as IEDB, most commonly used for antigen prediction on MHC molecules is far from perfect; thus, experimental proof of MHC-bound peptides is needed to provide direct evidence of antigen presentation.

Method: We developed a mass spectrometry (MS)-based platform integrated with a whole exome sequencing (WES)-based protein database to identify tumor-specific mutant peptides presented by MHC class I. Experiments were conducted using EGFR mutant PC9 lung adenocarcinoma cell line that harbors the EGFRDel746-750 mutant. MHC class I proteins and their associated peptides were immunoprecipitated, and the peptides separated from MHC proteins. The MHC proteins were subjected to trypsin digestion and HLA typing using MS. Class I-associated peptides underwent C-18 separation and tandem MS analysis. A PC9-specific database was built adding all SNVs and INDELs identified by WES to the normal human database and used to search MS data. Peptides identified by MS were subjected to IEDB algorithm search using PC9 specific HLA types.

Results: The MS HLA typing identified A*02, A*24, and B*39 in PC9 cells. We identified 13,765 MHC bound peptides. 10,711 peptides were 6~15 amino acid residues in length (6~15mer), which are generally considered as class I epitopes. Using the PC9-specific protein database, 11 variant peptides (neo-peptides) were identified. These include plectin isoform 1 (PLEC_H1459R, 9mer), proteasome subunit beta type-4 (PSMB4_I234T, 9mer), prostaglandin reductase 1 (PTGR1_A27S, 8mer), cytochrome b-c1 complex subunit Rieske mitochondrial (UQCRFS1_S6A, 7mer) and obscurin-like protein 1 (OBSL1_E1365D, 10mer). The IEDB results predicted the binding of PLEC_H1459R with HLA-B*39, OBSL1_E1365D with HLA-A*02 and PSMB4_I234T with HLA-A*24; however, 8 identified variant peptides were not predicted by IEDB.

Conclusion: Our MS data provided direct experimental evidence for MHC class I presentation of 11 neopeptides in PC9 cells. Neopeptides discovered by this proteogenomic platform could potentially trigger T cell response, which needs to be validated by immunological assays. Experiments are underway to utilize this workflow to identify variant neopeptides from patient tumors and patient-derived xenografts (PDXs).

#1439

Identification and characterization of neoantigen specific T cells.

Martin S. Naradikian,1 Leslie Montero,1 Samantha Hall,1 Milad Bahmanof,1 Rukman Thota,1 Luise Sternberg,1 Jerome Lane,1 Zeynep Kosaloglu-Yalcin,1 Manasa Lanka,1 Aaron Miller,1 Bjoern Peters,1 Ezra Cohen,2 Stephen Schoenberger1. 1 _La Jolla Institute for Immunology, La Jolla, CA;_ 2 _UCSD, La Jolla, CA_.

Neoantigens (NeoAg) offer attractive therapeutic targets for directing a patient's immune response to the immunogenic subset of mutations expressed exclusively by their cancer cells. Despite the specificity with which NeoAg enable tumor recognition, the majority of approaches for their identification rely on purely predictive methods such as calculating the ability of mutated peptides to bind to a patient's set of HLA molecules, and have met with limited success in revealing natural targets present on tumor cells. We have developed a novel HLA-agnostic functional approach to NeoAg identification which combines genomic sequencing with bioinformatic analysis to nominate mutations for subsequent functional analysis using patient's own T cells in an effort to identify natural responses generated under physiologic conditions. Using this, we identified a missense mutation (V205>I) in the ribosomal protein RPS2 that is recognized by CD8+ T cells from tumor-infiltrating lymphocytes (TIL) of a metastatic HPV-16+ Head and Neck Squamous Cell Carcinoma (HNSCC) lesion. We then performed adoptive cellular therapy (ACT) using either unseparated TIL or those enriched for RPS2 V205>I-specific CD8+ T cells and found the latter to be superior in controlling outgrowth of tumor of a PDX cell line generated from this lesion in NSG mice. Finally, we used single-cell transcriptomics to isolate the genes encoding the RPS2-specific TCR and show that it recognizes the mutated peptide bound to HLA-B*07.02 as well as the tumor cell line expressing the RPS2 protein. These results demonstrate that high-affinity NeoAg-specific T cell responses can be identified in cancer patients, that ACT of these cells can control tumor growth, and that the relevant TCR can be isolated for use in TCR engineering-based immunotherapy.

#1440

Evolution of CD19 expression in pediatric B-cell malignancies in the era of targeted therapy.

Diane Libert,1 Constance M. Yuan,1 Haneen Shalabi,1 Dalia Salem,1 Bonnie Yates,1 Cindy Delbrook,1 Terry Fry,2 John Shern,1 Maryalice Stetler-Stevenson,1 Nirali Shah1. 1 _National institutes of Health (NIH), Bethesda, MD;_ 2 _Children's Hospital of Colorado, Denver, CO_.

Introduction: Ubiquitous expression of CD19 renders it an ideal target for immunotherapy in B-cell neoplasms. As such, loss or decrease in CD19 is emerging as a means of cancer resistance. As a referral center for CD22-based approaches, an alternative target in B-cells, we had the opportunity to serially evaluate CD19 changes following targeted therapy.

Methods: In this retrospective chart review, we assessed for evolution of CD19 expression in patients referred for CD22 immunotherapies. Data collection included demographics, prior therapies, and serial CD19 expression as assessed by flow cytometry (% positivity and site density), response to prior CD19 targeted therapy, and change from baseline.

Results: 56 unique patients were analyzed. The median age was 19 years (range 4-30). At the time of referral (baseline), 23 (41%) subjects were CD19 negative, all following > 1 forms of CD19-targeted therapy. 8 (14%) were CD19 partial positive including 2 patients who were 28% and 81% CD19 positive but CD19-immunotherapy naïve. 25 (43%) were CD19 positive, 2 were CD19 dim following blinatumomab, and 7 were CD19-immunotherapy naïve. In 40 of 56 subjects (71%), there was no change in CD19 expression. 16 (29%) patients had either gain or loss of CD19. In those with CD19 re-expression, the median time for this occurrence was 91 days from baseline.

Conclusions: In this heterogenous population, we document evolution in CD19 expression following CD19-immunotherapy and identified unique patients with inherent CD19 alterations. Some patients retain or regain CD19 expression, conferring new susceptibility to CD19 targeting, while others permanently lose CD19, rendering further CD19-targeted approaches futile. The development of this latter population raises the concern for the outgrowth of subclinical CD19 negative clones. Pairing these findings with genomics will be important to understand the mechanism behind these changes in expression in the search to optimize treatment for B-cell cancers.

Table 1 | |  | |  | |

|

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

Baseline Demographics | First Change from Baseline

|

n=56 (%) | # with CR to prior therapy | |

Change | n (%) | Days to ↑ CD19 expression (Median, IQR)

Age (median, range in years) | 19 (4-30) | |  | |

|

Male  | 38 (68%) | |  | |

|

At study baseline* | CD19 negative | 23 (41%) | |

Baseline CD19 Negative

(n=23) | Remains CD19 neg | 17 (74%) | NA

|

Prior CD19 CAR | 16 | 14 | |  | |

|

Prior blinatumomab | 3 | 2 | |

Becomes CD19 Partial | 4 (17%) | 133 (351)

|

Both | 3 | 3** | |  | |

|

Other CD19 targeted (SGN19a) | 1 | 0 | |

Becomes CD19 Positive | 2 (9%) | 416 (39)

|

No prior targeted therapy | 0 | \-- | |  | |

|

CD19 partial# | 8 (14%) | |

Baseline CD19 Partial

(n=8) | Becomes CD19 neg | 0 | NA

|

Prior CD19 CAR | 1 | 0 | |  | |

|

Prior blinatumomab | 6 | 2 | |

Remains CD19 partial | 4 (50%) | NA

|

Both | 0 | 0 | |

Becomes CD19 positive | 4 (50%) | 53 (66)

|

No prior targeted therapy | 2 | \-- | |  | |

|

CD19 positive | 23 (41%) | |

Baseline CD19 positive

(n=25) | Becomes CD19 neg## | 2 (8%) | NA

|

Prior CD19 CAR | 5 | 5 | |  | |

|

Prior blinatumomab | 7 | 4 | |

Becomes CD19 partial | 3 (12%) | NA

|

Both | 4 | 2** | |  | |

|

No prior targeted therapy | 7 | \-- | |  | |

|

CD19 dim^ (uniformly positive)  | 2 (4%) | |  | CD19 dim to positive | 1 (4%) | 21 (NA)

|

Prior CD19 CAR | 0 | 0 | |  | |

|

Prior blinatumomab | 2 | 1 | |

Remains CD19 positive | 19 (76%) | NA

|

Both | 0 | 0 | |  | |

|

No prior targeted therapy | 0 | 0 | |  | |

CR: Complete remission

IQR: interquartile range

*Baseline represents first sample that subject had at the time of referral to the NIH with detectable disease

^Indicates CD19 expression < 2000 sites/cells

#Partial positivity defined by < 95% antigen positive

**Complete response to CD19 CAR, no response to blinatumomab

##Following interval CD19 targeted-therapy

#1441

A new subtype of MSCs confers antitumor activity by modulating intratumoral immunity.

Yang Zeng, Tao Li, Mark Poznansky, Huabiao Chen. _Massachusetts General Hospital, Charlestown, MA_.

Background and Purpose: Both anti-tumoral and pro-tumoral effects of mesenchymal stem cells (MSCs) in preclinical treatment of ovarian cancer have been previously demonstrated. The purpose of this study is set to gain a better understanding of the potential for MSCs in cancer immunotherapy.

Experimental Procedures: We profiled the phenotypes of mouse compact bone derived MSCs (CB-MSCs) and bone marrow derived MSCs (BM-MSCs) by flow cytometry. We further examined gene expression of immune regulating cytokines of MSCs in 2D and 3D culture. Finally, we tested antitumor effects of MSCs in a syngeneic orthotopic mouse model of ovarian cancer.

Results: Gene expressions of immune activating cytokines IL-12, IL-21, IFN-γ and a pro-inflammatory cytokine CXCL10 in CD90low MSCs were increased whereas anti-inflammatory cytokine IL-10 and CCL5 were decreased in 3D culture compared to that in 2D culture. CD90low MSCs conferred significantly higher antitumor activity than CD90high MSCs in tumor control and animal survival. This was associated with increase of activated anti-tumoral CD4\+ and CD8+ T cells while decrease of Treg cells in the tumor microenvironment.

Conclusion: Our study for the first time identified an anti-tumoral subtype of MSCs and provided new mechanistic insight into CD90low MSC-mediated immunomodulation in the tumor microenvironment. This study highlights the enhanced antitumor efficacy of a cell therapy in mouse ovarian cancer, which may provide a new therapeutic approach for patients with this disease.

#1442

Chimeric antigen receptor T cell therapy targeting ICAM-1 in gastric cancer.

Minkyu Jung,1 Marjan Zaman,2 Ygindra Vedvyas,2 Xianglan Zhang,1 Jaclyn E. McCloskey,2 Yanping Yang,2 Irene M. Min,2 Raza Zamegar,2 Yoon Young Choi,1 Jae-Ho Cheong,1 Sung Hoon Noh,1 Sun Young Rha,1 Hyun Cheol Chung,1 Moonsoo M. Jin2. 1 _Yonsei Univ. College of Medicine, Seoul, Republic of Korea;_ 2 _Weill Cornell Medicine, New York, NY_.

Introduction:

Despite advances in treatment, gastric cancer (GC) remains among the most fatal malignancies. Intercellular adhesion molecule-1 (ICAM-1) is overexpressed and associated with various cancers, including GC. We developed a third-generation chimeric antigen receptor (CAR) targeting ICAM-1 (ICAM-1-CAR) and investigated ICAM-1 as a immunotherapeutic target for CAR T therapy in GC.

Methods:

We investigated ICAM-1 expression in tissues of curatively resected GC patients (n=134) by immunohistochemical staining to determine its prognostic value. Effector to target assays were performed with ICAM-1-CAR T cells co-cultured with 8 GC cell lines with varying levels of ICAM-1 expression to investigate specific target cell death. We created a firefly luciferase-expressing human GC model in mice to measure tumor growth and killing by whole body bioluminescence imaging. To find the best treatment route and optimal dose of T cells in GC mice model, ICAM-1-CAR was injected via tail vein and intraperitoneal administration (IP) at two different doses, i.e., low dose (1 million CAR T) and high dose (10 million live T cells).

Results:

The ICAM-1 expression was higher in advanced stages (22.2% in stage II vs. 48.8% in stage III, p=0.002) and the patients with high ICAM-1 expression showed significantly poor survival (disease free survival, hazard ration [HR], 4.55, p<0.001; overall survival, HR, 3.89, p<0.001). The efficacy of ICAM-1-CAR T cells in vitro showed a strong correlation with the level of ICAM-1 expression in target cells, i.e., faster killing of GC with higher ICAM-1 expression. ICAM-1-CART facilitated regression of tumor in MKN-28 xenograft IP model. Additionally, the efficacy of ICAM-1-CAR was more prominent in mice treated with high dose of T cell and IP route. Compared to no treatment, ICAM-1CAR via IP led to tumor reduction that persisted for over 80 days and significantly improved survival without toxicity (p=0.049).

Conclusion:

ICAM-1 specific CAR T cells demonstrated significant therapeutic efficacy in vitro and in vivo against preclinical GC models. ICAM-1-CAR T cells may be developed into a promising treatment strategy for patients with ICAM-1 positive GC cancers.

#1443

Sensitive detection and quantification of CAR-T cells in the treated patients.

Xiaoyu An,1 Xiaolong Tu,2 Xinhe Feng,2 Jianjian Zhang,2 Dandan Song,2 Haijuan Yu,2 Henry Li1. 1 _Crown Bioscience, San Diego, CA;_ 2 _Crown Bioscience (Taicang) Inc, Taicang, China_.

Background. Cell therapy for cancers has gained great momentum as a promising new cancer treatment approach. In particular, autologous T cells modified with a chimeric antigen receptor (CAR-T) has recently been regulatory-approved for the treatment of the patients of CD19+ acute B-cell leukemia [1][2]. However effective monitoring and characterizing the CAR-T cells after administration, vital to understand the treatment effect and its underneath mechanism, remain to be an urgent unmet need for CAR-T therapy in the clinics. There have been two major methods to monitor the presence of CAR-T in patients at present: a) flow cytometry (a single cell based assay recognizing the tag engineered in CAR molecule), but with extremely poor sensitivity ~1%); and b) Real-Time PCR (amplifying the engineered tag sequences), being more sensitive, but without the ability to identify and characterizing the individual CAR-T cells [3][4]. Thus, a method with high sensitivity and also the ability to characterize individual CAR-T is in high demand.

Methods. We are attempting to establish a novel platform, based on rare cell detection in pathology format, to detect and character rare CAR-T cells in patients treated by CAR-T using Rarecyte technology. First, 1x106 white blood cells (PBMC from C57BL/6 mice), spiked with the serial dilution of 0-250 human breast cancer MCF-7 cells, were smeared on a pathology slide and subjected to the staining by antibodies against human Cytokeratin 18 (Abcam, ab181597) and mouse CD45 (R&D, AF114), followed by either flow cytometry or Rarecyte system, in order to test the detection sensitivity and quantification dynamic ranges. Second, PBMC obtained from the DLBCL patient treated with CD19 CAR-T cells were (100 μL) were subjected to the same two analysis as above with the CAR-T specific staining (CD3-FITC, CD19-CAR-PE, DAPI (nucleus)), as controlled by the PBMC from healthy donors.

Results. Our preliminary data from the spiked study indicated that our method has high sensitivity down to 2 cells (>90% recovery), and good linear range of quantification. The sensitivity (< 1/1-million) is at least 3-orders of magnitude above that of flow cytometry (~1%). Our CAR-T treated clinical data demonstrated the detection and quantification of CAR-T in patients that flow cytometry cannot. In addition, the single CAR-T can be picked up for further characterization, e.g. single cell genomics.

Conclusions. Our method detection, monitoring and characterization of CAR-T offers an great opportunity in the clinical setting to monitor patient treatment and to understand the treatment mechanisms, that otherwise is unavailable. This could become an useful diagnostics to provide guidance on the treatment and prognosis of patients.

#1444

The new therapy strategy for treatment of peripheral T cell lymphomas: CD30-targeted CAR-modified T cell therapy.

Yang Wu, Dan Chen, Rong Ma, Jun-ying Zhang, Yuan Zhang, Hai-xia Cao, Zhuo Wang, Chang-wen Jing, Si-wen Liu, Ji-feng Feng, Jian-Zhong Wu. _Nanjing Medical University Affiliated Cancer Hospital, Nanjing, China_.

Objective: Peripheral T-cell lymphoma (PTCL) is a type of non-hodgkin lymphoma with high metastasis and poor prognosis. The guidelines (NCCN guidelines, 2017) recommended CHOEP or CHOP chemotherapy regimen for treatment of PTCL. Unfortunately, the 5-year survival rate of PTCL is lower than 30% according to the present clinical data. Establishing new effective treatment methods and strategies for PTCL has been an urgent problem. Car-T cell therapy is a promising therapy for individuals with lymphoma. In USA, CD19 Car-T cell therapy has been proved to be effective for acute and chronic B-cell lymphoma treatment in more than 1000 clinic trials. However, there is currently no known Car-T cell therapy for PTCL, one of the most prevalent malignancies in Asia. In this study, to provide a safe and effective treatment for the recurrence and malignant CD30 positive PTCL, we establish the world-first CD30 Car-T cell therapy strategy and test its effect in PTCL.

Method: The extracellular fragment gene sequences of CD30 were obtained from CD30 positive tumor tissues of PTCL patients and cloned into a recombinant plasmid to obtained CD30 protein. Then, the CD30 targeting single-chain antibody fragment (scFv) gene sequences were obtained from CD30 positive monoclonal hybridoma cell. The T cells were stimulated by using OKT3, CD28 and IL-2 from peripheral blood mononuclear cell (PBMC), then transfected with CD30 Lenti-CAR. The numbers of CD30 positive T cells were counted by flow cytometry after 14 days culture. The cytotoxic function of Car-T cells to CD30+ PTCL cells were detected using chromium release assay (CRA) and real-time cell electronic sensing assay (xCelligence station, ACEA biosciences).

Results: The SDS-PAGE data shows that the molecular weight of CD30 protein was 45KDa, and the mass spectrometry data indicate that amino acid sequences were consistent with the prediction. The flow cytometry data shows 35% CD30 positive Car-T cells after transfection with CD30 Lenti-Car at day of 14. The Car-T cells can significantly inhibit the 299 cells proliferation at 20:1 effector-to-target ratios after 12 hours. The real-time cell electronic sensing data shows the Car-T cells can significantly inhibit the 299 cells proliferation at effector-to-target ratios of 5:1, 10:1, 20:1 and 40:1 after 6 hours co-culture.

Conclusions: In summary, we have shown a strategy for treating CD30 positive PTCL with Car-T cell therapy. We obtained a CD30 antibody with high specificity and affinity though immunized of mouse by CD30 antigen which sequences comes from the CD30 positive PTCL tissue. We also established real-time cell electronic sensing method to assessing cytotoxic function of Car-T cells and Car-T cells show high efficiency to cytotoxic of Karpas 299 cells. Therefore, this new CD30 Car-T cells therapy strategy may provide an effective and safe treatment of PTCL.

#1445

Tumor-specific T cell engineering for enhanced effector function via microfluidic delivery of bioactive molecules.

Luke Cassereau, Julie M. Cole, Roslyn Yi, Jacquelyn L. Hanson, Josh Bugge, Tia DiTommaso, Howard Bernstein, Armon Sharei. _SQZ Biotechnologies, Boston, MA_.

Background: Tumor-specific T cells possess unique potential for cancer therapy but are limited by T cell exhaustion and anergy induced in the tumor microenvironment. Ex vivo manipulation of these T cells to maintain their full function is critical to their success clinically. Yet, limitations of existing ex vivo delivery approaches dramatically restrict their function and thus limit their therapeutic use.

Methods: Genome-wide profiling was used to identify the impact of optimized electroporation treatment and the SQZ cell therapy platform on gene expression in human T cells. The profiling was paired with a 42 key T cell cytokine-multiplex analysis comprised of to assess perturbation of cytokine secretion. We then compared the in vivo functionality of immune checkpoint deleted antigen-specific T cells, modified by either electroporation or SQZ delivery of CRISPR/Cas9, and adoptively transferred into tumor bearing mice. Finally, genomic editing of tumor infiltrating leukocyte (TIL) derived T cells was compared using either electroporation or SQZ and subsequent effector response upon re-exposure to tumor cells.

Results: Impactful disruptions in transcript expression after treatment with electroporation (17% of genes mis-regulated, FDR q <0.1) we identified, whereas cells treated with SQZ had similar expression profiles to untreated control cells (0% of genes mis-regulated, FDR q <0.1). These genetic disruptions result in concomitant perturbation of cytokine secretion and effector response. Ultimately, the effects at the transcript and protein level resulted in functional deficiencies in vitro and in vivo with electroporated antigen-specific and TIL derived T cells failing to demonstrate sustained antigen-specific effector responses and tumor control with or without immune checkpoint editing.

Conclusions: This work demonstrates that functional modifications to tumor-specific T cells ex vivo can restore and improve their function upon re-exposure to tumor cells but that the delivery mechanism used is critical to the desired phenotype. The significant differences in outcomes from the two techniques tested here underscores the importance of understanding the impact of intracellular delivery methods on cell function for research and clinical applications. For both research and therapeutic applications with primary T cells, the functional consequences of the selected intracellular delivery technique and its impact on cell phenotype should be carefully evaluated. 

### Cancer Vaccines and Intratumoral Immunomodulation

#1446

Characterization, evaluation and safety studies of the oncolytic Vaccinia virus TG6002 for canine cancer therapy.

Jérémy Béguin,1 Johann Foloppe,2 Eve Laloy,1 Virginie Nourtier,2 Isabelle Farine,2 Murielle Gantzer,2 Christelle Pichon,2 Sandrine Cochin,2 Pascale Cordier,2 Dominique Tierny,3 Jean Marc Balloul,2 Eric Quémeneur,2 Christelle Maurey,1 Bernard Klonjkowski,1 Philippe Erbs2. 1 _Ecole Nationale Vétérinaire d'Alfort, Maisons-Alfort, France;_ 2 _Transgene, Illkirch-Graffenstaden, France;_ 3 _Oncovet Clinical Research, Loos, France_.

Introduction: Oncolytic virotherapy with tumor selective viruses, such as Vaccinia viruses (VV), offers a promising treatment modality for cancer. TG6002 is a recombinant oncolytic VV deleted in two viral genes (thymidine kinase and ribonucleotide reductase) and armed with the suicide gene FCU1 that encodes a bifunctional chimeric protein which efficiently catalyses the direct conversion of the nontoxic 5-fluorocytosine (5-FC) into the toxic metabolite 5-fluorouracil (5-FU). Canine tumors are relevant predictive preclinical surrogates for human oncology. The first objective was to evaluate the susceptibility, the replication rate and the oncolytic potency of VV in canine tumor cell lines. The second objective was to evaluate oncolytic potency of TG6002 in xenograft model and fresh canine tumor biopsies. The third objective was to assess safety and viral shedding of TG6002 in healthy dogs.

Materials and Methods: Transduction efficiency, replication and oncolytic potency of TG6002 were evaluated in vitro in a variety of different canine cancer cell lines. In vivo anti-tumor effect of TG6002 was examined in a canine tumor xenograft model. TG6002 was injected intratumorally or intravenously with or without 5-FC. Three canine mammary adenocarcinoma explants were infected with TG6002 in presence of 5-FC during 6 days. Oncolytic potency was assessed by histological exams. Concentrations of 5-FC and 5-FU were monitored. TG6002 was administered intramuscularly for 7 healthy dogs and intravenously for 4 healthy dogs. Clinical exams, complete blood count and biochemistry analysis were performed. Blood, saliva, urine, feces were collected for virus detection by qPCR and plaque assay.

Results: Canine cell lines were highly susceptible to VV infection. A replication factor of 106 to 107 was determined 4 days after infection and a significant reduction of cell viability was noticed 5 days after infection. In xenograft model, intratumoral or intravenous injections of TG6002 with oral 5-FC induced a significant inhibition of tumor growth compared to control groups. In canine mammary adenocarcinoma biopsies, a lysis of 90% of tubular cells was observed on histological exams. Conversion of more than 50% of 5-FC to 5-FU was noticed. In healthy dogs, a good tolerance of intramuscular and intravenous injections of TG6002 without viral shedding was assessed.

Conclusion: This study demonstrates that TG6002 is able to infect and replicate in canine tumor cell lines and is oncolytic in both cell lines, xenograft model and canine mammary adenocarcinoma samples. This study also confirms that TG6002 can be safely administered in dogs. These promising results support the use of TG6002 in a clinical trial for both human and canine species. This study emphasizes the importance of a One Health approach in oncology.

#1447

Predicting immunogenic neoepitopes with biology-aware machine learning.

Giovanni Mazzocco, Iga Niemiec, Oleksandr Myronov, Edyta Kowalczyk, Jan Kaczmarczyk, Anna Sanecka-Duin, Piotr Stepniak. _Ardigen, Krakow, Poland_.

Introduction: The purpose of this study is to present a novel method for neoepitope prediction, along with extensive benchmarks to existing solutions and identification of the most predictive constituent biological features. The accurate prediction of neoepitope immunogenicity represents an invaluable tool for the design of personalized cancer vaccines with effective treatment outcomes. The effectiveness of the host's adaptive immune response against cancer relies on the correct HLA-mediated neoepitope presentation and the recognition by specific CD8+ clones. Cancer immunotherapies act by boosting the activity of these effector T-cells.

Methods: The proposed AI-driven bioinformatics solution allows to perform an accurate prediction of HLA I-restricted neoepitope immunogenicity by including several analytical modules simulating biological processes leading to the activation of CD8+ T-cells. These modules can be flexibly composed on the basis of the data available and include: (i) selection of potential neoepitope based on cancer NGS data, (ii) expression of neoepitope-associated genes, (iii) similarity to self, (iv) prediction of neoepitope-HLA binding affinity and stability, (v) effect of post-translational modification on neoepitope presentation and TCR recognition, (vi) prediction of neoepitope:TCR recognition probability based on neoepitope-HLA:TCR structural-derived features and TCR CDR3 sequence similarities. The model was trained on a curated dataset joining records of multiple studies containing neoepitopes experimentally validated for their ability to elicit adaptive immune response.

Results: We present the results of a benchmarking study where we compare the performance of widely used methods for the assessment of neoepitope immunogenicity along with our solution. We investigate the predictive power of each analytical module to determine the individual information gain and determine the most informative ones. All the methods included in the benchmarking study were tested using the dataset provided by Chowell et al. 2015. As the metric for performance evaluation we use ROC AUC and the overall percentage of correctly detected immunogenic neoepitopes (precision).

Conclusion: The results present the utility of the compared methods for personalized cancer vaccine design and the importance of modules related to biological processes underlying the neoepitope presentation and immunogenicity.

#1448

Lymphangiogenesis-inducing whole-cell vaccine against melanoma promotes broad tumor-specific T cell immunity and effective tumor control.

Maria Stella Sasso, Sylvie Hauert, Priscilla S. Briquez, Yue Wang, Jun Ishihara, Jeffrey A. Hubbell, Melody A. Swartz. _The University of Chicago, Chicago, IL_.

We recently reported that the growth of lymphatic vessels (lymphangiogenesis) in mouse melanomas dramatically enhances the efficacy of immunotherapy by promoting immune cell infiltration and broadening the anti-tumor T cell repertoire (Fankhauser et al, Sci. Trans. Med. 2017). Correlative evidence supporting such 'lymphangiogenic potentiation' was also seen in human melanoma patients, suggesting a possible immunotherapeutic use of the pro-lymphangiogenic growth factor VEGFC. On the other hand, tumor lymphangiogenesis is known to promote cancer cell dissemination and metastasis, raising concerns about the possibility of manipulating lymphatic vessels directly within the tumor site as a treatment strategy for cancer immunotherapy. Here, we sought to manipulate and exploit the immune-promoting functions of lymphatics remotely from the tumor by developing a lymphangiogenesis-inducing cancer vaccine that mimics the microenvironment of a lymphangiogenic tumor. Whole-cell vaccines were formulated using lethally irradiated B16F10 or B16F10-OVA mouse melanoma cells, either mock-transduced or genetically modified to overexpress VEGFC, and combined with topically retained immune adjuvants (ctrl-vax and VEGFC-vax, respectively). Upon intradermal injection, VEGFC-vax induced extensive local lymphatic growth and stimulated increased lymphatic transport from the vaccine site to the draining lymph nodes. Consistently with our previous study, VEGFC induced CCL21 upregulation and local recruitment of CCR7+ naïve T cells, which could undergo in situ priming in the vaccine site. Compared to ctrl-vax, VEGFC-vax elicited a robust T cell response directed against multiple mouse melanoma antigens, as assessed by ex vivo antigen stimulation and IFNγ ELISPOT. In a prophylactic vaccine setting, VEGFC-vax induced stronger tumor-specific T cell immunity and greater protection from tumor challenge compared to both ctrl-vax as well as a GM-CSF-secreting whole-cell vaccine. Overall this study provides a proof of concept for the use of VEGFC in cancer vaccines and characterizes a whole-cell lymphangiogenesis-inducing vaccine formulation with potential for clinical translation.

#1449

MG1 Maraba boost following adenovirus prime generates tumor antigen-specific T cells which are potentiated by anti-PD-1 antibody combination.

Kyle Stephenson, Kwame Twumasi-Boateng, Amy Patrick, Caroline Breitbach, Michael Burgess, Brian Lichty. _Turnstone Biologics Inc., Ontario, Canada_.

Immune checkpoint inhibitors, such as antibodies blocking PD-1 and PD-L1, have been shown to potentiate pre-existing immune responses and improve patient survival. MG1 Maraba is a novel oncolytic virus that we bioselected and engineered to cause cancer cell death through two distinct and complementary mechanisms-of-action, direct cancer lysis and tumor-antigen specific T cell generation. MG1 Maraba encoding tumor antigens has been demonstrated to boost pre-existing immune responses. We are currently using a non-replicating adenovirus as the priming entity in our ongoing preclinical and clinical studies for our MG1 Maraba product candidates. This therapeutic platform is able to generate a large number of highly-functional antigen-specific T cells, in addition to its oncolytic activity, in mice, non-human primates (NHP) and patients treated with Adenovirus and oncolytic MG1 Maraba, expressing the same tumour associated antigen. This study investigates the ability of αPD-1 to potentiate immune responses generated by Ad/MG1 prime/boosting, and whether the timing of αPD-1 administration impacts the immune responses and therapeutic outcome. The combination of αPD-1 and Ad/MG1 treatment was investigated in a challenging murine model of metastatic melanoma. In three independent experiments, mice bearing advanced B16F10 lung metastases treated with Ad/MG1-hDCT in combination with αPD1 antibody, overall survival was increased to greater than 90%, compared to survival rates of less than 40% in mice treated with Ad/MG1-hDCT alone. The increased efficacy was correlated with improved anti-tumour immune responses in the αPD-1 combination group. The strongest effects of αPD-1 were observed when αPD-1 treatment was initiated immediately following Ad-hDCT immunization leading to significantly increased anti-tumour immune responses at all timepoints analyzed. When delayed until 1 week after MG1 treatment, αPD-1 was unable to improve the anti-tumour immune responses, or therapeutic efficacy, elicited by Ad/MG1 treatment. Similar effects were observed using an αPD-L1 targeted antibody. Therefore, the timing of PD-1/L1 blockade during Ad/MG1 treatment was determined to be a critical parameter for successful therapeutic outcomes. In addition, a non-human primate study is underway to assess the combination of αPD-1 with Ad/MG1-E6E7 (expressing HPV E6 and E7) when delivered concurrent with adenovirus immunization. Moreover, these data highlight the timing of checkpoint inhibitor treatment as a critical parameter for consideration when administering immune checkpoint inhibitors with other agents, including oncolytic viral immunotherapies.

#1450

Anti-EGF antibodies generated by vaccination significantly improve the activity of kinase inhibitors in preclinical models.

Jordi Codony-Servat,1 Silvia Garcia-Roman,1 Miguel Ángel Molina-Vila,1 Jordi Bertran-Alamillo,1 Ana Giménez-Capitán,1 Santiago Viteri,2 Andrés F. Cardona,3 Delvys Rodríguez,4 Manuel Cobo,5 Noemi Reguart,6 Niki Karachaliou,2 Erik d'Hondt,7 Rafael Rosell8. 1 _Pangaea Oncology, Barcelona, Spain;_ 2 _Instituto Oncológico Dr. Rosell (IOR), Barcelona, Spain;_ 3 _Clinical and Translational Oncology Group, Institute of Oncology, Fundación Santa Fe de Bogotá, Bogotá, Bogotá, Colombia;_ 4 _Hospital Universitario Insular de Gran Canaria, Las Palmas de Gran Canaria, Spain;_ 5 _Hospital Universitario Regional de Málaga, Málaga, Spain;_ 6 _Hospital Clínic Barcelona, Barcelona, Spain;_ 7 _Bioven, Aberdeen, United Kingdom;_ 8 _Institut Catalá d'Oncologia i Institut d'Investigació en Ciències de la Salut Germans Trias i Pujol, Badalona, Spain_.

Background: We have previously described that EGF blocks the activity of EGFR tyrosine kinase inhibitors (EGFR-TKIs) in EGFR mutant NSCLC cells, an effect that is reversed by anti-EGF antibodies generated by vaccination (anti-EGF VacAbs) (1). In this study we aimed to determine the effect of EGF and anti-EGF Vac Abs in the activity of different kinase inhibitors in tumor cell lines with different genetic alterations.

Methods: Anti-EGF VacAbs were obtained by immunizing rabbits with recombinant EGF. The cell lines used in this study were H2228 and H3122 (NSCLC, EML4-ALK positive), H23 (NSCLC, KRAS-G12C), DLD1 (colorectal carcinoma, KRAS-G13D), PC9 and PC9-GR4 (NSCLC, EGFR-mut). Tumor cell lines were treated with EGF, anti-EGF VacAbs and combinations with the kinase inhibitors alectinib, crizotinib, brigatinib (ALK inhibitors), trametinib (MEK inhibitor), dacomitinib and osimertinib (EGFR inhibitors). Cell viability was analyzed by MTT, changes of total and phosphorylated proteins were determined by Western blot and emergence of resistance by direct microscopic examination in low density cultures.

Results: EGF significantly decreased the antitumor activity of alectinib, crizotinib and brigatinib in ALK translocated cells (H2228 and H3122), trametinib in KRAS mutant cells (H23 and DLD1) and osimertinib and dacomitinib in EGFR mutant cells (PC9). In combination with these TKIs, the anti-EGF VacAbs reversed the effects of EGF and significantly potentiated the antitumor activity of all the kinase inhibitors, blocking the activation of EGFR, Akt and Erk. Finally, the addition of the anti-EGF VacAbs to the culture medium delayed the appearance of resistant clones to kinase inhibitors.

Conclusions: Anti-EGF VacAbs potentiate the antitumor effects of ALK, MEK and EGFR kinase inhibitors in tumor cell lines and delay the emergence of resistance in vitro. A clinical trial is currently testing anti-EGF vaccination in combination with afatinib in EGFR-mut advanced NSCLC patients.(1)Anti-Epidermal Growth Factor Vaccine Antibodies Enhance the Efficacy of Tyrosine Kinase Inhibitors and Delay the Emergence of Resistance in EGFR Mutant Lung Cancer Cells" Codony-Servat J, García-Roman S, Molina-Vila MÁ, et al. J Thorac Oncol. 2018.

#1451

HER2 peptide pulsed dendritic cell vaccine induce senescence and eliminates disseminated cancer cells in a preventive model of transgenic HER2/neu breast cancer.

Ganesan Ramamoorthi, Krithika Kodumudi, Colin Synder, William Dominguez-Viqueira, Scott Kidd, Shari Pilon-Thomas, Brian Czerniecki. _Moffitt Cancer Center, Tampa, FL_.

HER-2/neu overexpression plays a critical role in breast cancer development, and its expression in ductal carcinoma in situ (DCIS) is associated with development of invasive breast cancer. The detection of disseminated cancer cells (DCCs) in early-stage DCIS likely to become invasive and manifests prominent role in metastasis and recurrence. The HER2-positive DCCs can easily escape targeted therapies and become a source of tumor recurrence suggesting that targeting these cells may provide eminent benefits in HER2 positive breast cancer patients. Unfortunately, the conventional therapies that target these DCCs are limited. Previous studies from our lab have shown that HER2 peptide pulsed-DC1 (HER2-DC1) vaccine induced 30% complete response (pCR) in DCIS patients. However, little is known about the effect of HER2-DC1 vaccine on DCCs. Here, we investigated the efficacy of HER2-DC1 vaccine on DCCs in a transgenic HER2 mammary cancer model (neuT). The key features of this model are each one of ten mammary glands develops an independent carcinoma that slowly progresses from a microscopic lesion to an invasive tumor and metastasis is mainly driven by DCCs. Since this model mimics some of the most critical features of human disease, we evaluated the efficacy of HER2-DC1 vaccine on DCCs. Mice received six doses of HER2-DC1 vaccine subcutaneously twice a week. The spontaneous tumor development in the mammary glands of NeuT mice was examined once a week by ultrasound until the age of week 16. HER2-DC1 vaccine significantly prevented spontaneous tumor growth in NeuT mice. Remarkably, HER2-DC1 vaccine treatment significantly decreased HER2+CYT8/18+Ki-67+ proliferative DCCs (2%, p<0.002) in the bone marrow (BM-DCCs) compared to untreated mice (6%) by flow cytometry. We also observed increased senescent DCCs (82%, p<0.0001 compared to untreated mice, 37%) by a β-gal assay. BM-DCCs in HER2-DC1 vaccinated mice had reduced expression of Ki-67 compared to untreated mice. Multicolor flow cytometry demonstrated elevated levels of CD4 T cells (2 fold increase) and CD8 T cells (1 fold increase) in the bone marrow after HER2-DC1 vaccination compared to untreated mice. Importantly, accumulation of CD4 T cells (5 fold increase), CD8 T cells (19 fold increase) and B cells (100 fold increase) was observed in the mammary glands of HER2-DC1 vaccinated mice with strong anti-HER2 Th1 immune responses. Notably, HER2-DC1 vaccine led to an increased in the M1 macrophage phenotype (3 fold increase) and decreased M2 type (8 fold decrease) at the tumor site. Treatment of DCCs with Th1 cytokines, IFN-γ or TNF-α significantly induced senescence (76.3%, p<0.0001) in cultured DCCs compared to untreated cells (26%) in vitro. These results describe the novel role of HER2-DC1 vaccine in targeting DCCs and a possible role in prevention of metastasis and recurrence.

#1452

Development of ONCR-148, a miR-attenuated oncolytic HSV-1 designed to potently activate antitumor T cell response.

Agnieszka Denslow, Brian B. Haines, Michael S. Ball, Jacqueline Gursha, Daniel Wambua, Cecilia Kwong, Lingxin Kong, Allison Colthart, Prajna Behera, Peter Grzesik, Jennifer Lee, Terry Farkaly, Caitlin Goshert, Edward M. Kennedy, Lorena Lerner, Christophe Quéva. _Oncorus, Cambridge, MA_.

Oncolytic viruses, through cancer cell-selective lysis and accompanying release of danger signals that promote immune activation, have demonstrated antitumor efficacy in monotherapy of metastatic melanoma and have shown promising activity in combination with checkpoint inhibitors. We present here the activity of ONCR-148, a recombinant oncolytic Herpes Simplex Virus (oHSV) designed to be a safe and efficacious therapy for the treatment of solid tumors. Neuron-specific miR attenuation of ICP4 gene inhibits viral replication in neurons while preserving its potent oncolytic activity in tumor cells. The antitumor potency of the virus is enhanced by incorporating in its genome a transgene expressing fibroblast activation protein (FAP)- and CD3- bispecific antibody that recruits and re-directs the cytolytic activity of T cells toward FAP-expressing stromal cells. FAP is a cell-surface serine protease that is almost exclusively found in fibroblasts within tumor stroma. However, systemic administration of a FAP-CD3 bispecific antibody was not tolerated due to the toxicity toward FAP-expressing bone marrow mesenchymal cells (Tran et al., 2013. J.Exp.Med, 210(6), 1125-1135). Thus, the local intra-tumoral delivery of FAP-CD3 is an attractive approach to promote tumor lysis and T cell activation against cancerous lesions. To test the in vivo activity of ONCR-148, as FAP is poorly expressed in syngeneic mouse tumor models, we developed a bilateral mouse FAP-expressing MC38 syngeneic colon carcinoma model (MC38-FAP), allowing to test local antitumor activity in oHSV-injected tumors and abscopal efficacy mediated by immune cells in the non-injected tumors. Intra-tumoral administration of ONCR-148 in the MC38-FAP model resulted in tumor growth inhibition of 60% (p<0.03) and 46% (p<0.01) on the injected (ipsilateral) and non-injected (contralateral) tumor, respectively. We then investigated additional transgenes that could potentiate the antitumor activity of ONCR-148. We combined in vivo ONCR-148 with ONCR-153, an oHSV armed with the NK and T cell activating cytokine IL-12 and the chemokines CXCL10 and CCL4, allowing for expansion and recruitment of T cells and antigen presenting cells in the tumor milieu. Combined intra-tumoral administration of ONCR-148 and ONCR-153 increased the antitumor response to 90% on the injected tumor (p<0.0001), leading to complete tumor remission in 5 out of 9 treated mice and to 80% on distant tumors (p<0.0001, with no complete responses observed).These data indicate that oHSV expressing bispecific antibodies, while being well tolerated, is efficacious in injected tumors and mediate systemic antitumor immune response. The efficacy can be potentiated by a local co-expression of immunostimulatory cytokines. Such data support the further development of oHSV-1 armed with CD3 bispecific antibodies for the therapy of patients with metastatic cancer.

#1453

Development of a novel PD-1 vaccine and in combination with two Chimeric HER-2 peptide vaccine provides synergistic inhibition of tumor growth in a syngeneic Balb/c model challenged with CT26/HER-2 carcinoma cell line.

Tanios Bekaii-Saab,1 Jay Overholser,2 Yuhong Yang,2 Manuel Penichet,3 Pravin Kaumaya2. 1 _Mayo Clinic Cancer Center, Phoenix, AZ;_ 2 _Ohio State University, Columbus, OH;_ 3 _UCLA, Los Angeles, CA_.

Background: Therapeutic blockade of the signaling axis between PD-1 and its ligand programmed cell death ligand-1 (PD-L1) with monoclonal antibodies has shown remarkable clinical success in the treatment of cancer and demonstrated impressive activity across a broad set of cancer subtypes, even at advanced and metastatic stages of disease. Contrary to treatment with monoclonal antibodies, chimeric B-cell cancer vaccines have the advantage of producing a specific immune response that can potentially induce memory B & T cell responses, while reducing immune evasion and suppression.

Methods: We have translated two HER-2 combination peptide vaccines (B-Vaxx) to the clinic in a Phase 1/2b trial to safely deliver curative and transformative cancer immunotherapies to advanced cancer patients. We have created and established the development of a novel B-cell peptide vaccine (Key-Vaxx) with high immunogenicity that binds to human PD-1 and produces tumor inhibition in vivo in an animal model of colon cancer. Here, we describe the transferable CT-26-HER2 neu tumor model in Balb/c was used to test for synergistic effects of anti-PD1 immunization therapy in combination with anti-HER2 immunization therapy to determine whether this combination could increase immunogenicity, enhance anti-tumor responses and provide synergistic benefit in inhibiting tumor growth.

Results: We show robust HER-2 and PD-1 antibody responses in all vaccinated mice indicating that the combined vaccination is effective in reducing tumor growth in a Balb/c syngeneic model of colon carcinoma versus either the PD-1 vaccine or more importantly the positive control gold standard that is anti-mouse PD-1 monoclonal antibody. The vaccine combination was found to be safe and did not appear to exhibit toxicity or autoimmunity.

Conclusion: Development of a novel PD-1 vaccine in combination with two Chimeric HER-2 peptide vaccine provides synergistic inhibition of tumor growth with no evidence of toxicity or autoimmunity. A clinical trial with the combination is under planning.

#1454

Long-term immunogenicity results from a first-in-human study evaluating the anti-ASPH cancer vaccine, SNS-301.

Michael S. Lebowitz, Hong Dai, Kanam Malhotra, Ildiko Csiki, Steven Fuller, Hossein A. Ghanbari. _Sensei Biotherapeutics, Gaithersburg, MD_.

SNS-301 is a first-in-class immunotherapeutic cancer vaccine candidate targeting human aspartate β-hydroxylase (ASPH). ASPH is an oncofetal antigen expressed in multiple human cancers but not in healthy adult tissue. As a biologic modifier of the NOTCH pathway, ASPH is associated with tumor cell growth, motility and invasiveness. SNS-301 is a vaccine which delivers over 400 copies of an extracellular domain of the ASPH protein displayed on a bacteriophage vector. This phase 1 first-in-human study of SNS-301 was primarily designed to evaluate the safety and tolerability of the vaccine and to establish the recommended phase 2 dose. Vaccine was administered intradermally every 21 days in a 3+3 dose-escalation trial in patients with biochemically (rising PSA) relapsed prostate cancer with evidence of ASPH expression. The safety, tolerability and immunogenicity of SNS-301 have been presented previously (Nordquist, et al. Annals of Oncol., 29, suppl_8, 1 October 2018, mdy279.404, Lebowitz, et al., JITC 2018, 6(Suppl 1):P167). Per protocol, all subjects were required to receive at least three doses of SNS-301, but were given the option to continue on-study receiving further vaccinations every 3 weeks. Furthermore, once the safety of higher dose levels of SNS-301 was established, subjects that had initially received lower doses were escalated to the higher dose levels. All patients tolerated the vaccine well with minimal numbers of adverse events and opted to remain on-study, some receiving >20 doses over a period of more than 420 days. Immune responses in these patients were continuously followed at three-week intervals. Specifically, both anti-ASPH and anti-phage antibody levels in serum as well as percentages of ASPH-specific B-cells were monitored. Levels of anti-ASPH antibody and of ASPH-specific B-cells were well correlated. An initial increase in the levels was seen within the first six treatment cycles in a dose dependent fashion with higher doses achieving more rapid and more robust levels of response. As subjects continued on-study, ASPH-specific immune responses reached a peak level, then subsequently declined. Over the longer-term, these responses re-emerged and declined over multiple cycles. This pattern of response may be indicative of immune fatigue and is suggestive that a more refined dosing regimen in which later vaccinations are further spaced in time may be beneficial. Anti-phage antibody responses were also observed and also occurred in a dose-dependent fashion. Interestingly, subjects who received lower doses of vaccine and were subsequently switched to higher doses did not see a concomitant increase in anti-phage antibodies, suggesting that they had achieved a level of tolerance to the phage vector. Phase II trials in heme and solid tumors are planned and will incorporate a vaccine dosing regimen informed by this phase I study.

#1455

Design of ONCR-177 base vector, a next generation oncolytic herpes simplex virus type-1, optimized for robust oncolysis, transgene expression and tumor-selective replication.

Edward M. Kennedy, Terry Farkaly, Prajna Behera, Allison Colthart, Caitlin Goshert, Judy Jacques, Kyle Grant, Peter Grzesik, Jennifer Lerr, Laura Viggiano Salta, Lorenz Ponce, Caroline Web, Brian Haines, Agnieszka Denslow, Jacqueline Gursha, Mitchell Finer, Christophe Quéva, Lorena R. Lerner. _Oncorus, Cambridge, MA_.

ONCR-177 is a novel oncolytic herpes simplex virus type-1 (oHSV-1) developed for the treatment of metastatic cancers. ONCR-177 base vector, ONCR-159, has been optimized to elicit potent oncolysis, even in presence of host cell antiviral response by keeping the neurovirulence gene ICP34.5. ONCR-159 oncolytic activity and replication in human cancer cell lines is less sensitive to IFNα compared to ICP34.5 deleted oHSV. A dual bidirectional promoter enables the expression of 5 transgenes in ONCR-177: the NK and T cell activating cytokine IL-12, the chemokines CCL4 and FLT3LG (extracellular domain) to allow for expansion and recruitment of classical dendritic cells, and antagonists to the clinically validated immune checkpoints PD-1 and CTLA-4 to counter T cell exhaustion. To ensure selective replication in cancer cells, ONCR-177 utilizes a unique conditional-lethal strategy in which tissue specific microRNA (miRNA)-binding cassettes (miR-T) are inserted into early genes essential for viral replication and in ICP34.5. We leveraged our comprehensive normal and malignant tissue expression analysis to identify highly expressed tissue-specific miRNAs and tailored their selection to ONCR-177 clinical indications of interest. In addition, we performed a siRNA screen to identify essential HSV-1 genes for replication in which miR-T cassettes were introduced. This attenuation strategy dramatically reduced transgene expression and viral replication in healthy tissues expressing the selected miRs without compromising oncolytic activity in cancer cells. ONCR-159 and ONCR-177 demonstrate potent tumor cell killing in human cell lines in vitro and in 3 syngeneic tumor models in vivo. Furthermore, mutations that inhibit axonal retrograde transport have been introduced in ONCR-159 and ONCR-177 (Richards et al., 2017) providing an orthogonal protection of the nervous system and preventing virus of latency.In conclusion, ONCR-177 has been optimized to be a safe and efficacious therapy for the treatment of solid tumors, eliciting direct oncolysis and enhancement of antitumor immune response warranting its evaluation in phase 1 clinical trial.

Richards et al., 2017. PLoS Pathog 13(12): e1006741.

#1456

MEDI5395: A recombinant oncolytic virus with oncolytic and immune modulatory properties.

James A. Harper,1 Shannon Burke,1 Andrew Leinster,1 Nicola Rath,1 Xing Cheng,2 Hong Jin,2 Robert W. Wilkinson,1 Danielle Carroll3. 1 _Medmmune Ltd, Cambridge, United Kingdom;_ 2 _Medmmune LLC, South SanFransisco, CA;_ 3 _AstraZeneca Ltd, Cambridge, United Kingdom_.

MEDI5395 is a genetically modified attenuated Newcastle disease virus (NDV). A reverse genetic system has overcome environmental and regulatory concerns by uncoupling oncolytic potency from avian pathogenicity. MEDI5395 has the intrinsic ability to infect and kill tumor cells and has been inserted with a GM-CSF transgene to potentiate a stronger adaptive immune response. Described here is an extensive in vitro and in vivo pharmacology package that reveals the broad oncolytic activity and immune-modulatory properties of MEDI5395 in a variety of pre-clinical models.

In vitro, MEDI5395 lytic activity in human tumor cells is associated with elevated levels of tumor selective viral replication and expression of the GM-CSF transgene. MEDI5395 infection of immune cells indicated preferential uptake of virus and subsequent self-limiting replication in myeloid cells. Infected myeloid cells expressed cell surface activation markers (e.g. PD-L1) after 24 hours and were effective carriers of virus and mediated the transfer of infectious NDV particles to tumor cells resulting in death through oncolysis. Further mechanistic studies, indicated NDV-killed tumor cells released antigens that were capable of cross-presentation by dendritic cells driving activation of tumor antigen-specific autologous T cells.

In murine models, IV delivery of NDV leads to long lasting tumor selective replication and transgene expression. MEDI5395 administration results in significant anti-tumor activity, observed in patient-derived xenograft models, and in murine syngeneic cancer models. Together, the results suggest that MEDI5395 may act to positively transform the tumor microenvironment. This, coupled to its tumor-selective oncolytic capacity, further underscore NDV as a promising multimodal cancer therapeutic platform and why FTIH studies are planned to start in 2019.

#1457

Sustained persistence of IL-2 signaling enhances the antitumor effect of peptide vaccines .

Takumi Kumai,1 Hussein Sultan,2 Yasuaki Harabuchi,1 Esteban Celis2. 1 _Asahikawa Medical University, Asahikawa-shi, Japan;_ 2 _Georgia Cancer Center, Augusta University, GA_.

Peptide vaccines can be a successful and cost-effective way of generating T-cell responses against defined tumor antigens, especially when combined with immune adjuvants such as poly-IC. However, strong immune adjuvants can induce a collateral increase in numbers of irrelevant, nonspecific T cells, which limits the effectiveness of the peptide vaccines. Here, we report that providing prolonged IL2 signaling in the form of either IL2/ anti-IL2 complexes or pegylated IL2 overcomes the competitive suppressive effect of irrelevant T cells, allowing the preferential expansion of antigen-specific T cells, and increasing the therapeutic effectiveness of the vaccines against established tumors. This vaccination strategy using peptides and sustained IL2 could be taken into the clinic for the treatment of cancer.

#1458

Study on cervical cancer preventive effect of HPV vaccine in Japan.

Masato Nishimura, Tomohiro Kagawa, Ayuka Mineda, Eri Takiguchi, Akiko Abe, Minoru Irahara. _Tokushima University, Tokushima, Japan_.

Objective: In Japan, side effects are emphasized and ingestion of HPV vaccine is not widespread. Meanwhile, the number of people suffering from cervical cancer has increased, and there is concern that deaths due to cervical cancer will increase. The objective of this study is to clarify the differences in the frequency of cytological abnormalities by presence or absence of HPV vaccination, sex hormone intake, and smoking history.

Methods: We examined each item described in medical questionnaire of a consultant who received cervical cancer screening in April 2015 - March 2016 and cytological diagnosis.

Results: There are 23,001 consultation examiners for 1 year. Of these, 3609 people aged 20-29, 6457 people aged 30-39 years old, 4433 people aged 40-49 years old were examined each item. With or without HPV vaccination, the frequency of cytological abnormalities (ASC-US and more) in 20-29 years old was 8/262 (3.1%), 121/3307 (3.7%), and there was no difference. Frequency of LSIL or more was significantly lower in vaccinates as 0/262 (0%) vs 70/3307 (2.3%) p<0.05. There was no difference in the frequency of cytological abnormalities between the groups at 30-39 years old and 40-49 years old. With regard to the presence or absence of sex hormones, there was no difference in the frequency of cytological abnormalities in all ages. With smoking history, the frequency of LSIL or more was significantly higher in a group with smoking history, at the ages of 20-29 years (3.7% vs 1.4%) and 30-39 years (2.3% vs 1.2%).

Conclusion: If there is smoking history, it is important to undergo regular cervical cancer screening. In addition, HPV vaccination reduces the frequency of cytological abnormalities, so HPV vaccine is also important for the prevention of cervical cancer.

#1459

**Construction and assessment of a novel** Listeria monocytogenes **-based** **vaccine targeting squamous cell carcinoma.**

Alyaa F. Hessin,1 Nancy E. Freitag2. 1 _The National Research Centre, Cairo, Egypt;_ 2 _University of Illinois Chicago, Chicago, IL_.

Introduction: Immunotherapy represents a potential therapeutic option for many tumors including squamous cell carcinoma (SCC). L. monocytogenes is a potent delivery vehicle that can elicit cellular immunity specific for the encoded antigens, and overcome the immunosuppressive cancer environment. Here we test the hypothesis that L. monocytogenes strain lacking 2 secreted chaperones (ΔprsA2 ΔhtrA) would be able to induce a specific immune response against SCC and would represent a safe vaccine due to its hyper-attenuation.

Methods: Recombinant L. monocytogenes ΔprsA2 ΔhtrA strains were engineered to express Cyp2e1 squamous carcinoma antigen using standard molecular techniques. All genes were verified by DNA sequencing, and secreted proteins were assessed by western blot. Bacterial growth assays were tested in broth and intracellularly in J774 mouse macrophage cells. The safety of the vaccine strains were tested in vivo by counting the CFUs in target organs and measuring the liver function tests in serum. The efficacy was then assessed in a subcutaneous mouse model of tumor growth together with L. monocytogenes ΔactA prfA* strain and ΔprsA2 ΔhtrA strain with no tumor antigen. Mice were first challenged with KLN205 tumor cells, and then vaccinated with 3 immunizations weekly. The tumors volumes were recorded with a caliper every 3 days and organs weights were measured at the end of the experiment.

Results: The Cyp2e1 antigen was detectable in L. monocytogenes supernatant fractions, and the secretion of tumor antigens did not impact the growth of mutant strains in broth culture. Bacterial replication within infected macrophage cells showed defective intracellular growth for L. monocytogenes ΔprsA ΔhtrA expressing tumor antigen while L. monocytogenes ΔactA prfA strains exhibited modest levels of inhibition in comparison to wild type. Mice infected with ΔprsA2 ΔhtrA strains had no CFUs recovered from liver or spleen while half of the mice infected with ΔactA prfA strains exhibited bacterial burdens within the liver and no bacteria were recovered from the spleen. Consistently, all the infected mice exhibited normal liver function as measured by AST and ALT levels. The results confirm the safety of ΔprsA2 ΔhtrA strains in vivo. Additionally, ΔprsA2 ΔhtrA vaccine strains significantly halted the SCC growth when compared to the control mice. Preliminary data suggests that tumor antigen-specific differences in tumor size and spleens weights could be visualized between the groups of mice immunized with L.monocytogenes recombinant strains expressing Cyp2e1 antigen versus no antigen. These data suggest that the highly attenuated L. monocytogenes ΔprsA2 ΔhtrA strains may indeed be efficient in stimulating SCC regression.

Conclusion: L. monocytogenes ΔprsA2 ΔhtrA strains represent a novel and safe vaccine that altered the immunosuppressive microenvironment of SCC and inhibited tumor growth.

#1460

Generation of a novel, allogeneic cell-based, Gp96-Ig/OX40L cancer vaccine, improves anti-tumor immunity and long-term memory T-cell generation.

Vikas Tahiliani, Jayalakshmi Miriyala, Patrick Dillon, Jason Rose, Louise Giffin, Jeff Hutchins, Matthew M. Seavey. _Heat Biologics, Inc., Durham, NC_.

Our technology is focused on developing a next generation cellular vaccine platform – referred to as ComPACT (COMbination Pan-Antigen Cytotoxic Therapy), that incorporates a tumor antigen chaperone (gp96-Ig) with T-cell costimulation (OX40L-Ig), into a single tumor cell line that secretes both. Viagenpumatucel-L (HS-110; ImPACT), a human lung adenocarcinoma cell line, stably transfected to express gp96-Ig is being tested in a phase 1/2 clinical trial (NCT#02439450) with checkpoint inhibition for NSCLC. A similar line is being generated that will complement HS-110, providing costimulation in the form of secreted OX40-Ig (HS-130). To model how the addition of human HS-130 to HS-110 may impact anti-tumor immune responses, we generated mouse surrogates of these human lines and established an analogous system, that treats tumor-bearing animals with tissue-matched irradiated cancer cell lines (B16F10) expressing gp96-Ig (mHS-110) and OX40L-Ig (mHS-130) both expressing ovalbumin, as our model tumor-associated antigen. Single dose vaccination with mHS-110 identified that 1 to 10 million cells (290 ng to 2,900 ng of secreted gp96-Ig) provided sufficient anti-tumor CD8+ T-cell expansion, in vivo (*p<0.01; 1-way ANOVA), with the greatest expansion observed on day 7, post-immunization. To identify the best ratio of mHS-110 to mHS-130; a dose ratio study was performed. Fixed numbers of mHS-110 (1 million cells, 290 ng of secreted gp96-Ig) were matched with different ratios of mHS-130. Similar to our single dose vaccination study, our results demonstrated that the peak CD8+ T-cell expansion occurred on day 7 post-immunization, and that the addition of mHS-130 further boosted anti-tumor CD8+ T-cell expansion by 3-fold when the ratios of mHS-110 to mHS-130 were at a 1-to-1 ratio (290 ng of secreted gp96-Ig to 290 ng of secreted OX40L-Ig) (*p<0.05, Mann-Whitney). These animals were subsequently boosted 14-days post-immunization, and we similarly found that the 1-to-1 ratio of mHS-110 to mHS-130 gave the maximum expansion of CD8+ T cell responses, peaking on day 21 and contracting thereafter (*p<0.05; Mann-Whitney). Importantly, these ratios led to high frequencies of IFNγ+ antigen-specific CD8+ T cells at both priming and boosting, which enhanced rejection of established B16F10 tumors and increased overall survival.

#1461

mRNA therapeutics against cancers and infectious diseases.

Smriti Arya, Qiubin Lin, Nan Zhou, Jiandong Huang. _The University of Hong Kong, Pokfulam, Hong Kong_.

In vitro transcribed (IVT) mRNA is forming a new basis of drug delivery as it can be synthetically engineered to express the desired protein in lesser time and be more specific than the other 2 types of antigens. Using DNA (coding for proto-oncogenes) for immunization poses a risk for host cell transformation into a malignant one and hence proto-oncogenes would express for a longer time. Therefore, mRNA was considered for cancer immunotherapy as it does not integrate into the host cell's genome, so no question of insertional mutagenesis arises. Our main aim is to develop a stable mRNA system working both in vitro and in vivo and then use this system to develop mRNA vaccines. The stability of IVT mRNA can be increased by changing the 3' and 5' UTRs, adding a 5'cap and 3' poly-A tail which could even increase their half-life to a few days. For creating a stable mRNA working system in vitro, EGFP-2A gene was cloned along with the AdsA (Staphylococcus aureus gene important for infection) in a vector backbone with TMV 3'UTR and 5'UTR. The mRNA created in vitro was with modified uridine and cap 1 structure and transfected to the mammalian cell lines. This transfection was further enhanced by using liposome created in the lab (using some cationic and helper lipids in different ratios) by film rehydration method. The expression of luciferase mRNA was then detected in mice by live imaging indicating that the mRNA system is stable in vivo. Further, to direct the mRNA encoded antigens to MHC1 and MHC2 pathways they were tagged with a htPA secretion tag and MHC1 targeting signal along with a 3XFLAG tag. We tried various mode of injections with AdsA mRNA to have a profile of ELISPOT with differently exposed mRNA (in vivo in mice). Now we are focussing on some neo antigens for tumor to be used as. Avaccine in the form of mRNA.

#1462

Novel oncolytic virus armed with interleukin-21 enhances immune oncotherapy.

Rong Zhang, Tingting Wang, Yun Qing, Shuaijun Sun, Meili Liu, Fang Hu, Jin Fu. _Hangzhou Converd, Hangzhou, China_.

Oncolytic viruses have elucidated the broad anti-tumor properties through selectively destroying tumor cells, promoting tumor antigen release and inducing the activation of immune response. Vaccinia virus (VV) has been engineered for tumor selectivity by the deletion of two essential genes (DDvv), viral growth factor (VGF) and thymidine kinase (TK). However, it still has certain obstacles, such as spread of virus, tumor microenvironment barrier, immune cell infiltration and tumor infiltration lymphocytes (TILs) proliferation. In the present study, we engineered the novel oncolytic mV01 and hV01 viruses, recombinant DDvv viruses carrying human interleukin-21 (hIL-21) or mouse IL-21 (mIL-21), respectively. mV01 and hV01 exhibited potent inhibition of various tumor cells with IC50 < 0.5 MOI. When in combination with hNK cell, hV01 significantly enhanced the inhibition of cell proliferation in SK-HEP-1 cells and HepG2 cells as compared to mock virus. Animal studies showed that mV01 (i.t.) significantly reduced tumor burden in B16 C57/BL6 xenograph mice that were pre-treated with low dose cyclosporine A (i.p), compared to mock virus treatment. Simultaneously, mV01 regimens improved tumoral immune cell infiltration and TILs proliferation. Additionally, in HCT116 NCG xenograph mice, administration of hV01 (i.t.) exhibited significant inhibition of tumor growth (T/C < 40%) when in combination with hNK cells (i.v). Together, these data revealed that V01 is a promising candidate for tumor immunotherapy through viral oncolysis and anti-tumor immunity.

#1463

FAST vaccines based on frameshift neoantigens may have advantages over personal vaccines.

Milene Tavares Batista, Sierra Nicole Murphy, Ji'an Zhang, Luhui Shen, Phillip Stafford, Stephen A. Johnston. _Arizona State University, Tempe, AZ_.

It is widely hoped that personal cancer vaccines will extend the number of patients benefiting from checkpoint and other immunotherapeutics. However, it is clear creating such vaccines will be challenging. It requires obtaining and sequencing tumor DNA/RNA, predicting potentially immunogenic neoepitopes and manufacturing a one-use vaccine. This process takes time and considerable cost. Importantly, most mutations will not produce an immunogenic peptide and many patient's tumors do not contain enough DNA mutations to make a vaccine. We have discovered that frameshift peptides (FSP) produced in tumors though errors in RNA production are a rich source of neoantigens. There are ~220K bioinformatically predictable possible FSP allowing us to make arrays representing them as 15aa peptides. These arrays can then be used to screen cancer patient blood antibodies for reactivity to the arrays. In screening many cancer patients blood on these array, we found both personal and cancer-type specific peptides. This suggests a new type of vaccine consisting of pre-made FSP components for a specific type of cancer. We term these FAST vaccines. Here we use the mouse 4T1 breast cancer model to test the relative effectiveness of a FAST and a PERSONAL vaccine. To create the vaccines, we initially challenged mice subcutaneously with 4T1 tumor cells and, seven days later, sera were collected. Pre-challenge and 7-days sera were assayed on peptide microarrays containing 200 FS neoantigens. For the PERSONAL vax, the top 10 candidates (higher median intensity fluorescence) were select and personal vaccines constructed and administrated to respective mice (n=10). For the FAST vax, we selected the top 10 candidates with higher prevalence among all the mice challenged (n=24), a common Breast cancer FAST vax was constructed (mBC FAST-vax). Mice were challenged with 4T1 cells subcutaneously. Vaccines were then, administrated twice with one-week interval, combined or not with checkpoint inhibitor (CPI) (anti- PD-L1/ CTLA-4). Our results demonstrated that both vaccine approaches, FAST and PERSONAL vax, alone reduced tumor growth as well as increased animal survival. Nonetheless, the FAST vax protected 70 % of mice (7/10 - tumor free) even after re-challenge, 29 days after vaccine regimen. For the Personal vax group, co-administration with CPI resulted in enhancement of tumor control with 57 % of the mice strongly controlling the tumor. The FAST vax performance was not improved by CPI. Both vaccine approaches elicited a robust and homogenous B- and T- cell immune response against both vaccine peptides and tumor cells. Additionally, use of Non-reactive FSPs and a Non-Breast cancer FAST vax were not able to control tumor development. We conclude that the FAST technology may open new opportunities to develop a low cost, feasible and efficacious vaccines against cancer.

#1464

Inhibition of p38 MAPK in dendritic cells enhances their antigen presentation and induces Th17 T cell differentiation.

Yan Luo,1 Shreeder Barath,1 Deborah A. Bahr,1 Navnita Dutta,1 Geraldine Raja,1 Martin J. Cannon,2 Keith L. Knutson1. 1 _Mayo Clinic, Jacksonville, FL;_ 2 _University of Arkansas, Little Rock, AR_.

Dendritic cells (DCs) are the most potent antigen-presenting cells which bridges innate and adaptive immunity, but how DC-derived signals regulate and induce T cell lineage choices remains unclear. The p38 MAPK pathway plays an essential role in regulating cellular processes such as cell differentiation, growth, and death. In this study, SB203580 was employed to selectively inhibit p38 pathway in bone marrow derived DCs (BMDCs) to investigate the effects of this pathway on the characteristic phenotype and function of DCs, especially the induction on T helper cell differentiation. We found that inhibition of p38 increased the proliferation of BMDCs, but did not alter their phagocytic ability. Also, inhibition of p38 enhanced the activation of BMDCs, as evidenced by the up-regulated expression of DCs-related surface markers including CD80, CD86 and MHCII. However, the secretion levels of IL-12 and IL-10 in BMDCs were reduced significantly, whereas the level of IL-6 was not influenced by the p38 inhibitor. In addition, we immunized the mice with BMDCs vaccines and studied the production of antigen-specific Th17 T cell in spleen. Our data demonstrated that p38-inhibited DCs vaccine could induce more antigen-specific Th17 T cells than regular DCs vaccine in vivo. Collectively, these results support the hypothesis that the p38 pathway plays a critical role in the immunologic activation of DCs. Our findings suggest that inhibition p38 pathway in DCs may be a new strategy for generating potent DC vaccines for cancer immunotherapy.

#1465

Priming immunity against SCLC with oncolytic virotherapy.

Patrick Kellish,1 Connor Hartzell,1 Daniil Shabashvili,1 Masmudur M. Rahman,2 Maria V. Guijarro,1 Akbar Nawab,1 Grant McFadden,2 Frederic J. Kaye,1 Maria Zajac-Kaye1. 1 _University of Florida, Gainesville, FL;_ 2 _Arizona State University, Tempe, AZ_.

Small cell lung cancer (SCLC) is an aggressive subtype of lung cancer with few treatment advances over the past 3 decades and poor survival. High levels of tumor infiltrating immune cells are associated with improved SCLC patient survival Independent of tumor stage, patient performance status or treatment. Oncolytic virotherapy presents a new approach where tumor specific infection and replication stimulate host immune responses and increase infiltrating immune cells. Myxoma virus (MYXV) has been widely tested in Australia to control rabbit populations with no toxicity to humans. We have now shown that MYXV selectively infects SCLC cells with no cytotoxicity to normal tissues. In vitro studies were performed using human, murine, and cisplatin-resistant SCLC cell lines. Efficient MYXV infection, replication, and cytotoxicity was observed in all SCLC cell lines with rapid induction of immunogenic cell death (ICD), demonstrating MYXV is effective for SCLC regardless of cisplatin resistance. In contrast, we did not detect productive infection or cytotoxicity in non-tumor cells. Using an optimized conditional genetically engineered mouse (GEM) model (Ad-Cre mediated p53/Rb1/p130 null) we show untreated advanced SCLC tumors are devoid of infiltrating immune cells. Following intrapulmonary MYXV treatment, MYXV localized exclusively within lungs of tumor bearing mice and was cleared by 7 days. Despite clearance within 7 days, a robust immune response was sustained 30 days after MYXV treatment. When intrapulmonary MYXV is administered in combination with anti-PD-1 or anti-CTLA-4, both combinations show greater reduction in tumor burden throughout the lung compared to MYXV alone at the 60 day post treatment timepoint. In addition, the immune cell population localized within SCLC tumors were enhanced following combined MYXV/anti-PD-1 at 60 days post treatment. In our GEM model, MYXV alone results in a statistically significant prolongation of survival compared to both PBS and cisplatin treated animals. To determine the effect of MYXV delivered by intratumoral injection we utilized patient derived xenografts (PDX) and newly developed subcutaneous syngeneic tumor models for SCLC in immunocompetent mice. Following MYXV treatment in PDX tumors we observed extensive necrosis and persisting virus detected 10 days post treatment. In our immunocompetent subcutaneous syngeneic tumor model, MYXV is cleared by 7 days, which is consistent with clearance following intrapulmonary MYXV delivery in our GEM model. This was accompanied by extensive necrosis, reduction in tumor volume, increased infiltrating immune cells, and induction of de novo endogenous tumor specific antibody responses. In conclusion, we demonstrate the potential for MYXV as an oncolytic virotherapy for SCLC, with selective infection leading to enhanced anti-tumor immune responses.

#1466

Combination immunotherapy successfully control tumor growth in a transgenic mouse model.

Kue Peng Lim,1 Syafinaz Zainal,1 Natasha Zulaziz,1 Chai Phei Gan,1 San Jiun Chai,1 Bryan Kit Weng Lye,1 Chuan Wang,2 Ruhcha V. Sutavani,2 Christian Ottensmeier,2 Emma King,2 Gareth Thomas,2 Natalia Savelyeva,2 Sok Ching Cheong1. 1 _Cancer Research Malaysia, Subang Jaya, Malaysia;_ 2 _University of Southampton, Southampton, United Kingdom_.

The use of checkpoint inhibitor anti-PD1 has achieved significant clinical benefit, in recurrent and metastatic head and neck cancer patients, where the overall response rate is between 16-20%. The use of antigen-specific cancer vaccine to increase the level of cytotoxic T cells is believed to work synergistically with anti-PD1. In this study, we evaluated the efficacy of antigen-specific DNA vaccine when it's used in combination with anti-PD1. The B6.Cg-Tg(HLA-A/H2-D)2Enge/J mice bearing established tumours overexpressing the tumour antigen were vaccinated either with anti-PD1, DNA vaccine or anti-PD1 in combination with DNA vaccine. Tumour volume was monitored and antigen-specific immune responses were evaluated at the endpoint. Our data demonstrated DNA vaccine induced antigen-specific immune responses and animals vaccinated with DNA vaccine harboured smaller tumour volume compared to controls. Significantly, animals that received DNA vaccine expressed consistently higher levels IFN-γ and resulted in the upregulation of PD1 compared to control animals. We then demonstrated mice vaccinated with both DNA vaccine and anti-PD1 had near to complete tumour control, indicating a synergistic effect of DNA vaccine with anti-PD1. Animals treated with DNA vaccine in combination with anti-PD1 also have better overall survival. Importantly, these animals showed increased antigen-specific responses by the ELISPOT assay. In summary, our data suggest antigen-specific DNA vaccine works synergistically with anti-PD1 and conferred an excellent tumour control; this opens up a new opportunity of combinatory immunotherapy that might benefit a wider population of patients in an antigen-specific manner.

#1467

Bioengineering AAV6 vector-based vaccine for cancer treatment.

Karina Krotova, Andrew Day, Edward Hinchcliffe, George Aslanidi. _University of Minnesota, Austin, MN_.

Introduction: Adeno-associated virus (AAV) is a small virus of the parvovirus family and is widely used in clinical-stage gene therapy because of its low toxicity and limited side effects. There are a number of naturally occurring serotypes with different tissue tropism and further engineering of the viral capsid can increase the vectors suitability for use as a cancer vaccine. We recently showed that AAV serotype 6 (AAV6) successfully delivers a transgene to dendritic cells (DC) in vitro. In the present study we analyze several rationally designed AAV6 mutants to more efficiently target DC and further increase an antigen-specific immune response.

Methods: AAV6 mutants encoding ovalbumin, tyrosinase, or GFP were developed and produced in the lab by standard procedure. Vaccination was performed as a single intramuscular (i.m.) injection in C57BL/6 mice. The immune response was measured against both ovalbumin (Ova) as a model antigen and tyrosinase (Tyr) as an endogenous antigen. The Ova-specific CD8 T-cells were identified in peripheral blood and splenocytes by binding epitope-specific tetramers. Activity of antigen-specific CD8 cells was estimated by IFNy production measured by ELISpot, and by in vivo and in vitro killing assays. A humoral response was analyzed by the presence of antigen-specific antibodies in mouse serum measured by ELISA or by Western Blotting. Vaccination with GFP was used to show direct antigen expression in DC. Briefly, 3 days after i.m. injection DC from drained lymph nodes were isolated by FACS (CD11c+/MHC II+) and the presence of GFP expression was analyzed by wide-field fluorescence microscopy.

Results: Several significant observations were made: (i) optimized AAV6 directly targets DC in vivo as evidenced by the presence of GFP positive DC in mouse drained lymph nodes after i.m. injection with AAV6-GFP; (ii) AAV6 vectors induced a strong immune response not only against a strong immunogen such as Ova but also against a weak endogenous antigen such as Tyr. (iii) Transgene-specific cytotoxic CD8 T- cells showed superior killing capabilities against cancer cells in vitro and in cytotoxic assay in vivo. (iv) AAV6 based vaccine generates high transgene-specific antibodies titers detectable starting 3 weeks after injection. Our data allows us to choose the AAV6 mutant with superior capacity to induce an immune response against a transgene. As a future direction we will optimize the expression cassette to directly rout an antigen into the MHC class II processing and presentation pathway which is known to dramatically increase vaccine potency. The combination of optimized AAV capsid and expression cassette will be used to confirm an ability of our vaccine approach to reduce tumor growth and extend survival in a murine tumor model. In addition, our technology is highly compatible with, and complementary to current cancer treatments, including other immunotherapy approaches such as checkpoint (PD-1/PD-L1) inhibitors.

#1468

Synergistic cancer immunotherapy combination of MVA-CD40L with tumor targeting antibodies or checkpoint blockade to achieve strong antitumor immune responses against large, established tumors.

Jose Medina-Echeverz,1 Maria Hinterberger,2 Raphael Giessel,1 Barbara Bathke,1 Ronny Kassub,1 Giovanna Fiore,1 Paul Chaplin,1 Hubertus Hochrein,1 Henning Lauterbach1. 1 _Bavarian Nordic, Planegg, Germany;_ 2 _Bavarian Nordic, München, Germany_.

Virus-based vaccines and appropriate costimulation potently enhance antigen-specific T cell immunity against cancer. In the present study, we exploit both innate and adaptive immune responses triggered by recombinant modified vaccinia virus Ankara (rMVA) encoding costimulatory CD40L against large, established tumors in different combinatory settings. Therapeutic treatment with rMVA-CD40L resulted in the control or eradication of solid tumors in several unrelated tumor models. The expansion of non-exhausted, tumor-specific cytotoxic CD8+ T cells was essential for the therapeutic activity and was partially dependent on CD8α+ cross-presenting dendritic cells. Combination of rMVA-CD40L with PD-1 checkpoint blockade further enhanced the therapeutic activity of MVA virotherapy in colorectal carcinoma. In addition, rMVA-CD40L combined with antibodies targeting Tumor-Associated Antigens (TAA) resulted in increased therapeutic antitumor efficacy against two unrelated tumor models. We describe a translationally relevant therapeutic synergy between viral vaccination and CD40L costimulation. We show strengthened antitumor immune responses when both rMVA-CD40L-induced innate and adaptive immune mechanisms are exploited by combining immunotherapeutic regimes, such as checkpoint blockade and TAA targeting antibodies. This novel immunotherapeutic approach could translate into clinical cancer therapies where ADCC competent TAA targeting antibodies PD-1 checkpoint blockade are employed.

#1469

Optimizing the tolerability of intravenous oncolytic viral immunotherapy administration: investigation of a low-high-high dose schedule of enadenotucirev (EnAd) administration.

Hilary McElwaine-Johnn, Sam Illingworth, Rochelle Lear, Richard Brown, Brian Champion. _PsiOxus Therapeutics Ltd, Abingdon, United Kingdom_.

Background:EnAd is a tumor-selective oncolytic adenovirus with highly selective replication and cell killing in a broad range of carcinoma cell lines. Following intravenous dosing in clinical studies, uptake and replication of EnAd, associated with CD8+ cell infiltration, has been shown. It is currently in combination studies with nivolumab and paclitaxel. Intravenous delivery of EnAd stimulates an acute response characterised by an increase in cytokines in peripheral blood. As previously reported (ESMO IO 2017, abstr 203), the MTD of EnAd (3x1012 viral particles (vp) Days 1, 3 and 5, flat dosing schedule) has been determined primarily based on AEs occurring on first dose (C1D1), with the cytokine response, including interleukin-6 (IL-6), to subsequent doses of EnAd appearing attenuated when delivered within a few days of the first dose.

Methods: The effect of the first EnAd dose on subsequent acute particle-mediated cytokine (IL-6) responses in mice was investigated following administration of systemic doses equivalent to between 5.5 x 1012 vp and 5.5 x 1013 vp in humans.

Results: The magnitude of IL-6 response to a low first dose, which itself induces very low or undetectable levels of IL-6 cytokine, is sufficient to attenuate the response to subsequent high doses (10 times more vp). When administered as first dose these high doses induced a significant elevation in systemic IL-6.

Conclusions: Tolerability of EnAd is primarily determined by C1D1, correlating with the cytokine response across the patient cohort. The data from this in vivo pharmacology study support the concept of delivering a lower dose of enadenotucirev on Day 1 and administering higher doses thereafter, with the aim to deliver a higher viral load to tumour tissue and potentially optimise the overall risk:benefit of EnAd administration. This type of dosing regimen (low-high-high) has been successfully implemented in clinical studies of other systemically administered oncolytic viruses (Clin Cancer Res 2006;12:2555-2562) and is under clinical investigation as part of the ongoing combination study with nivolumab. (EudraCT:2013-001276-38).

#1470

Development & characterization of a new oncolytic immunotherapy platform based on herpes simplex virus type 1.

Suzanne Thomas,1 Linta Juncheria,1 Victoria Roulstone,2 Joan N. Kyula,2 Howard L. Kaufman,1 Kevin J. Harrington,2 Robert S. Coffin1. 1 _Replimune, Woburn, MA;_ 2 _The Royal Marsden/The Institute of Cancer Research, National Institute for Health Research Biomedical Centre, London, United Kingdom_.

A number of oncolytic viruses have shown single agent clinical activity and evidence of clinical synergy with immune checkpoint blockade. However, not all patients respond, and as yet most of the promising clinical data have been generated in patients with melanoma. With the objective of maximally activating a patient's immune system against their own cancer which will then provide enhanced synergy with anti-PD1/L1 blockade, we developed a new oncolytic immunotherapy platform based on herpes simplex virus (HSV). This has the dual objectives of killing tumor to provide abundant release of tumor antigens, and potently activating immune responses against these antigens once released. To augment the natural ability of the virus to kill tumors and activate the immune system, these viruses are each armed with multiple therapeutic genes. Initially, we screened 29 new clinical strains of HSV isolated from volunteers who suffer from cold sores across a panel of human tumor cell lines to select a candidate for further development. This strain was then engineered to provide tumor selective replication (deletion of the genes encoding ICP34.5 and ICP47) and to express a potent fusogenic glycoprotein (GALV-GP R-). These modifications greatly enhance both local and systemic antitumor effects, increase immunogenic cell death, potently activate anti-tumor immunity, and are synergistic with PD-1 blockade in pre-clinical models. A virus expressing both GALV-GP R- and human GM-CSF is currently in a Phase 1/2 clinical trial in approximately 150 patients as a single-agent and combined with PD1 blockade in four solid tumor types. Further viruses express additional immune-activating proteins expected to act at the site of immune response initiation in tumors and draining lymph nodes, in particular an anti-CTLA-4 antibody-like molecule and immune co-stimulatory pathway-activating ligands. Each of these demonstrated increased activity in pre-clinical models, particularly an enhanced anenestic effect. The anti-CTLA-4 expressing virus (RP2) is targeted to enter clinical trials in the first half of 2019. Thus, a modular approach to oncolytic immunotherapy development can rapidly generate multiple clinical candidates with multiple mechanisms of action intended to act synergistically with other approaches to cancer therapy. The latest pre-clinical data utilizing this platform will be presented.

#1471

Short hairpin RNA-expressing oncolytic adenovirus-mediated inhibition of PDL1 significantly suppresses human cancer cell growth.

Jipo Sheng, Xue Wang, Xiaoming Dong, Jin Fu, Sanmao Kang, Fang Hu. _Hangzhou Converd Co.,Ltd, Hangzhou, China_.

In cancer immunotherapy, it is known that aberrant expression of immune checkpoint proteins in tumor cells and their resulting immunosuppression in surrounding tumor microenviroment and even in whole immune system represent the crucial limiting factors toward successful cancer treatment. Multiple studies evidenced that oncolytic viruses carrying biological molecule including cytokines, shRNAs and miRNAs relieve or attenuate immunosuppression, and may be a promising way toward curing tumor. In this study we generated an oncolytic adenovirus with expression of shRNA targeting human PDL1. The adenovirus exhibited proliferation activity more than two orders of magnitude higher in human cancer cells than in human normal primary cells. Western blot showed significantly decreased expressing levels of PDL1 protein in human cancer cells upon infection of the PDL1 shRNA expressing-oncolytic virus. FACS assay also showed decreased expression levels of PDL1 on the cell membrane after the infection of same viruses. In vivo experiments in two different genetic backgrounds mouse model <NOD-SCID mice and BALBC nude mice> demonstrated that the PDL1 shRNA expressing adenoviruses lysed the majority of the tumor cells derived from HCT116 colorectal cancer cells that were subcutaneously inoculated. Moreover, the adenoviruses downregulated PDL1 expression levels within the HCT116 colon cell-derived tumor cells, remarkably attenuated immunosuppression surrounding the HCT116 tumor. Consisted with these observations, TIL cell counts in the tumors derived from mice with shRNA expressing oncolytic adenovirus treatment were significantly higher than that from mice in other control groups. The growth of HCT116 colon cell-derived tumor in mice with oncolytic adenovirus treatment was greatly inhibited for a longer period compared with that in mice in other control groups. The oncolytic adenovirus described in this study features highly selective amplification in human tumor cells versus normal cells and inhibition of PDL1 expression in tumor cells, therefore it may be important toward developing novel method for cancer therapy.

#1472

Novel vaccine targeting colonic adenoma: A pre-clinical model.

Toan D. Pham, Sandra Carpinteri, Shienny Sampurno, Lloyd Pereira, Sara Roth, Vignesh Narasimhan, Kasmira Wilson, Phillip Darcy, Jayesh Desai, Alexander G. Heriot, Robert G. Ramsay. _Peter MacCallum Cancer Centre, Melbourne, Australia_.

Background

Colorectal cancer (CRC) is the second leading cause of cancer related mortality. Over 80% of CRC develop from adenomatous polyps. Hence, early treatment and prevention of adenomas would lead to a significant decrease of disease burden for CRC. MYB is a transcription factor that is over-expressed in both adenomatous polyp precursors and colorectal cancer and hence an ideal immunotherapeutic target. We have developed a cancer vaccine, TetMYB, that targets MYB and aim to evaluate its efficacy in the prophylactic and therapeutic management of adenomatous polyps.

Material and Methods

Six to eight-week-old Apcmin/+ (Familial Adenomatous Polyposis model) and Apc580S (sporadic model) C57BL/6 mice were used. The Apcmin/+ mice are carried a germline mutation of one Apc allele whereas the Apc580S model has an inducible silencing of one Apc allele, when exposed to Tamoxifen, via the Cre-Lox recombination enzyme system.

In the prophylactic treatment group, Apcmin/+ and Apc580S C57BL/6 mice were vaccinated and then surveyed for clinical signs of distress according to animal ethical endpoints. Number of adenoma and survival were measured.

In the therapeutic cohort, Apc580S C57BL/6 mice were given Tamoxifen-laced food to activate Cre-Lox recombinase mediated silencing of one Apc allele and thus inducing adenoma development. Following adenoma detection using mouse colonoscopy, mice were vaccinated with TetMYB and treated with anti-PD-1 antibody and were analyzed for adenoma growth rate.

Results

In both the prophylactic and therapeutic setting, mice vaccinated with TetMYB had a significantly improved outcome.

In the prophylactic treatment group, the vaccinated Apcmin/+ mice had a median survival benefit of 70 days (p = 0.008) and the vaccinated Apc580S mice having a mean survival benefit of 134 days (p = 0.01) over the unvaccinated mice.

In the prophylactic cohort, immunofluorescence confirmed a stronger cytotoxic CD8+ T-cell infiltrate in the vaccinated group, implying an anti-tumor immune response. In the therapeutic cohort, vaccinated Apc580S mice showed significantly reduced adenoma growth rate compared to the unvaccinated mice (p = 0.0005).

Conclusion

TetMYB vaccine has shown benefit in a prophylactic and therapeutic setting in the management of colonic adenoma in a murine model. This will form the basis for a future clinical trial to prevent and treat colonic adenomatous polyps, and perhaps colorectal cancer prevention.

#1473

Dual oncolytic viral immunotherapy generates large polyfunctional tumour-specific CD8 + T cell responses that infiltrate immunosuppressive tumors.

David Stojdl. _Turnstone Biologics, Ottawa, Ontario, Canada_.

Generating effective T cell mediated clearance of solid tumors remains an unmet challenge in cancer therapy. Suppressive tumor microenvironments limit the generation of significant numbers of tumor specific T effector cells, their migration to tumor beds, and their subsequent functionality within tumors, thereby blocking the patient's normally potent acquired immune response from contributing to tumor control.

In an effort to meet this challenge, we have developed novel oncolytic viruses that were bioselected and engineered to cause cancer cell death through two distinct and complementary mechanisms-of-action, direct cancer lysis and tumor-antigen specific T cell generation. Specifically, we have selected and engineered MG1 Maraba virus which both infects tumor tissue to reverse immune suppressive programs while simultaneously delivering vector encoded tumor antigens to the spleen to vaccinate against the patient's tumor. The result is a large increase in peripheral and tumor infiltrating CD8+ T effectors cells, strong intratumoral inflammatory signatures and ultimately curative efficacy in preclinical solid tumor models. This first-in-class therapeutic strategy is currently being evaluated in Phase 1 and Phase 2 clinical trials.

In this new study, we describe the development of a novel viral immunotherapy platform based on Farmington virus that is: (1) oncolytic in solid tumor models, (2) a potent inducer of highly-functional antigen-specific T cells (expanding tumor specific CD8+ T effector pools over 1000 fold from pre-existing T central memory) and is (3) immunologically distinct from our clinical MG1 Maraba platform. We show that when used sequentially in a heterologous boosting regimen, T cell responses to encoded tumor antigens, either foreign (HPV E7), self (TRP2) or multi-neoantigen, can exceed greater than 50% of all CD8+ T cells in the periphery. The majority of these CD8+ T effectors show markers of polyfunctionality with little expression of the PD-1 exhaustion marker. We will describe our current data assessing the phenotype, localization and potency of these T cell responses in preclinical models of solid tumors and propose strategies to deploy our novel dual oncolytic viral immunotherapy boosting paradigm to the clinic.

#1474

Repurposing the yellow fever vaccine for intratumoral immunotherapy.

M. Angela Aznar, Carmen Molina, Alvaro Teijeira, Arantza Azpilikueta, Saray Garasa, Alfonso R. Sanchez-Paulete, Iñaki Etxeberria, Maite Alvarez, Pedro Berraondo, Ignacio Melero. _Center for Applied Medical Research University on Navarra (CIMA), Pamplona, Spain_.

Introduction: Yellow Fever attenuated virus vaccine (17D) induces very potent cellular and humoral immunity. Its delivery in situ in the tumor bed may induce an immune response leading to the killing of infected cells.

Experimental procedures: 17D virus was propagated in Vero cells and infectious supernatants were harvested. To evaluate in vitro sensitivity of tumors to 17D infection, a panel of human and mouse tumor cell lines were infected with increasing MOIs and viability was quantified.

To evaluate the antitumor efficacy of intratumor (i.t.) injection of 17D, 4x106pfu/50μl were injected (4 doses) in MC38- and in B16-OVA-tumor bearing C57BL/6 mice. Systemic antitumor effects of i.t 17D were examined in bilateral MC38 models in which one of the tumors was left uninjected. Depletion experiments were performed to assess the contribution of CD4, CD8, Gr1 and NK cells. To determine the role of conventional type-I dendritic cells (cDC1), identical experiments were performed in BATF3-/- mice. Tumor and lymph node immune infiltrates were examined by multiparametric flow cytometry. Combination with intraperitoneal anti-PD1 or anti-CD137 was performed to improve antitumor efficacy.

To evaluate the effects of 17D-preexisting immunity, mice were preimmunized with 17D prior to MC38 bilateral inoculation and were subsequently treated. To assess the individual cell type contribution to the antitumor effects induced upon 17D pre-immunization, CD8, CD4, CD8+CD4 or sera from preimmunized mice were passively transferred 24h prior to first 17D i.t injection to MC38-tumor bearing mice.

Results: 17D live attenuated strain is able to induce cell death of mouse and human tumor cell lines. In MC38 or B16-OVA mice models, repeated i.t. injections of 17D clearly delayed tumor progression. Regarding the systemic effect of i.t. 17D, certain contralateral therapeutic efficacy was induced upon i.t injection in bilateral MC38 models. These local and distant effects were dependent on CD8+ and cDC1 cells. 17D i.t injection increased in CD8+ T cell infiltrates and decreased in Tregs in injected tumors, while in contralateral tumors induced an increase of CD8+ and NK cells expressing key immunomodulatory receptors. I.t. 17D with anti-CD137 monoclonal antibody (mAb) induced a strong synergistic effect, while combination with anti-PD-1 mAb showed mild local additive efficacy compared to 17D monotherapy. Both combinatorial treatments delayed contralateral tumor growth. Notably, 17D preimmunization induced an important improvement of local and distant antitumor immunity. Such beneficial effects of preimmunization were transferred along with CD8+ T cells.

Conclusions: 17D can be safely injected i.t. in preclinical models, giving rise to immune-mediate antitumor effects that can be combined with other immunotherapy agents. The repurposed use of this GMP-grade vaccine in preimmunized patients represents a promising and feasible immunotherapy approach.

#1475

Microbiome derived peptides stimulate strong immune response against tumor associated antigens and trigger in vivo tumor regression after vaccination.

Laurent Chene, Caroline Diveu Sader, Joao Magalhaes, Francesco Strozzi, Lorenzo Tibaldi, Celia Mendez, Simon Baeriswyl, Arnaud Laveissiere, Christophe Bonny. _Enterome, Paris, France_.

Peptide-based vaccination is an immunotherapeutic approach for the treatment of cancer that aims to deliver immunogenic peptides corresponding to specific tumor associated antigens to patients. Despite promising pre-clinical results in animal models, most cancer vaccination approach has not demonstrated unequivocal efficacy in patients. The ability of a peptide cancer vaccine to generate a strong immune response depends on several factors including the avidity of the peptide for the MHC complexes, the capacity of the antigen to be recognized by the immune system as self or non-self and the existence of T cell clones (naïve or memory) that are able to recognize those peptides. Gut colonization is one of the factor that drive the development of T cells and the presence of commensal specific memory T-cell is well reported in the literature and is now currently admitted. Several evidences support that these T cells can be re-activated and can migrate to inflammatory sites where the antigen is expressed. We have developed an innovative, microbiome-based approach for the development of therapeutic peptide cancer vaccines base. Our approach is based on the notion of molecular mimicry, by which a microbial derived peptide vaccine holding molecular similarity with a tumor-associated epitope would trigger a tumor-specific cytotoxic T cell immune response. By developing a dedicated in silico pipeline, we were able to identify bacterial antigens that could elicit strong immune response in WT mice. Furthermore, these bacterial antigens could be selected to generate T cell reactivity against self-peptides that are by themselves not immunogenic. The same pipeline was used to identify bacterial antigens able to bind human HLA-A2 and displaying molecular mimicry with selected tumor associated antigens. While HLA-A2 transgenic mice vaccination with tumor associated antigens doesn't lead to immunization, vaccination with bacterial antigens results in a strong immune response against both bacterial peptides and selected tumor associated antigens. Furthermore, adoptive transfer of T cell from mice immunized with bacterial peptides into tumor engrafted nude mice leads to tumor control in the presence of Check Point Inhibitors. A first vaccine candidate targeting three different tumor drivers overexpressed in Glioblastoma and other solid tumors will enter the clinic this year.

### Cellular Interactions in the Tumor Microenvironment

#1476

Decoding the immunologic landscape of human clear cell renal carcinoma.

Nivedita Chowdhury,1 Aleksandar Obradovic,2 David Aggen,2 Vinson Wang,2 Xinzheng Guo,2 Guarionex J. DeCastro,2 Hongxu Ding,2 Andrea Califano,2 Charles G. Drake2. 1 _Columbia University/Johns Hopkins University, New York, NY;_ 2 _Columbia University, New York, NY_.

Immune cells in the tumor microenvironment (TME) play a critical role in regulating tumor development and response to targeted immunotherapy. While it is well-accepted that clear cell renal carcinoma (ccRCC) is characterized by a T-cell infiltrate, differential activation states within the tumor and the contribution of other immunomodulatory cells remain relatively unexplored. We sequenced approximately 30,000 immune cells at the single-cell level from tumor and matched adjacent normal tissue in ccRCC patients. With a median of 3300 Unique Molecular Identifiers (UMIs)/cell, we were able to capture the expression of 1000 genes per cell on average and 21,000 genes across all cells from each patient. We used Multidimensional Scaling (MDS) -transformation to visualize the various clusters and found that the tumor and adjacent normal tissue were distinct, with each having two major sub-clusters - lymphoid and myeloid cells. The data demonstrated a higher fraction of lymphoid infiltrate(~70-75%) than myeloid in a relatively stable ratio across patients, with tumor containing a slightly higher fraction of lymphoid cells. The normal and tumor cell subtypes were distinguished by multiple iterations of PAM clustering (Partitioning Around Mediods) with feature selection using the iterClust algorithm. Top differentially expressed genes in each cluster were identified and cross-referenced with known immune cell markers along with pathway enrichment analysis for cell typing. Among the tumor lymphoid population, genes specific for T-cells, including markers of functional exhaustion were the most highly expressed. A separate sub-cluster of cells consistent with cytotoxic T and/or NK cells was also identified. We found a distinct population of monocytes within the myeloid cluster that was present exclusively in the tumor tissue. This population expressed high levels of complement factors C1QA, C1QB, C1QC, as well as ApoE. The latter has been implicated as a driver of myeloid cell immunosuppression, indicating a possible regulatory role in the TME of ccRCC that has not previously been described. The phenotype of the immune cell populations in tumor was further validated by spectral flow cytometry. This study demonstrates relative abundance of cell types in the immune microenvironment within ccRCC tumors and also identifies a novel C1Q-high myeloid sub-population specific to the tumor, suggesting its possible role in RCC tumorigenesis or progression.

#1478

Human transgenic IL-15 NOG mice reconstituted with human NK cells as a model for NK cell depletion.

Anna Bunin,1 Ann Marie Rossi,1 Christian Vidal,1 David Trihn,1 Marco Garcia,1 Joseph Woska,1 Lawrence Iben,1 Luca Rastelli,1 Paul Volden,2 Enrique Alvarez1. 1 _Kleo Pharmaceuticals, New Haven, CT;_ 2 _Taconic Biosciences, Rensselaer, NY_.

Natural killer cells (NK cells) are emerging as powerful mediators of anti-tumor effects in many cancer immunotherapy modalities.A multitude of these targeting strategies, however, cannot avoid cross-reactivity and potential NK cell depletion is a result of treatment. Furthermore, some therapies aim to directly engage surface receptors expressed by NK cells, including killer-cell immunoglobulin-like receptors (KIR) or NKG2D, as well as NKp30 and NKp46, to enhance their activity. Thus, NK cell activity modulation is an active area of immune-oncology drug development and there is a need for robust rodent models that can reliably evaluate human NK cell activity without the effort required in non-human primate (NHP) models. In addition, therapeutic intervention can have depletion of NK-like cells as its final goal as in NK- and NK/T lymphomas. To enable monitoring of NK cell response to treatment in vivo, we have established a humanized mouse model in which NK cell depletion can be followed in response to a wide variety of therapeutic interventions. To this end, human IL-15 NOG mice were preconditioned with busulfan, and reconstituted with human NK cells purified from a leukopheresis product. Our model allows for a long-term engraftment of human NK cells. We find that intraperitoneal administration of human NK cells is a route that yields long term robust engraftment of human NK cells in these mice. Peak engraftment of human NK cells in circulation of IL-15 NOG mice occurs at 3 weeks post-engraftment (hu CD45+ cells, 50%±10, SD, n=2). Human NK cells persist in the spleen and bone marrow compartment of IL-15 NOG mice for as long as 6 weeks post-engraftment (huCD45+cells: spleen 47%±3 SD n=3, blood 5%±0.4 SD n=3, bone marrow 0.3%±0.05, n=2). Furthermore, NK cells harvested ex-vivofrom IL-15 NOG mice can mediate a robust ADCC response against RAJI Burkitt's lymphoma cells in the presence of Rituximab (32% cytotoxicity). These harvested cells also maintain their original expression of CD56+ 99.6%, CD16+ 71%, NKG2D+ 89.7%, NKp46+ 96%. To evaluate the in-vivoresponse of these engrafted human NK cells, we administered Daratumumab as a therapeutic agent to these mice. Daratumumab is an antibody against CD38, which is highly expressed by human NK cells. Our results demonstrate specific, complete depletion of NK cells in treated mice 41% ctrl vs 2.5% (Daratumumab) in the blood, and 24% (ctrl) vs 0% (Daratumumab) in the spleen. We therefore have established a humanized mouse system that can be used to monitor the ability of therapeutic agents to deplete NK cells both as a surrogate and as a direct readout of efficacy. This human NK cell fratricide mouse model can be used in a preclinical drug development setting that looks to augment our understanding of human NK cell depletion.

#1479

Bacterial dysbiosis in the mouse model of hemochromatosis: Increased risk of colitis and colitis-associated colon cancer.

Bojana Ristic,1 Sathish Sivaprakasam,1 Rao Kottapalli,2 Abdul Hamood,1 Vadivel Ganapathy1. 1 _Texas Tech University Health Sciences Center, Lubbock, TX;_ 2 _Texas Tech University, Lubbock, TX_.

Hemochromatosis, an inherited iron-overload disease, causes tissue damage due to deposition of iron in toxic levels in most tissues including the liver, heart, kidney, pancreas and colon. Free iron, both in the inorganic form (i.e, non-transferrin-bound and non-ferritin-bound) and organic form (i.e. heme), facilitates Fenton reaction to generate hydroxyl radicals and to induce cellular oxidative damage that is observed in carcinogenesis. The primary site of iron deposition in hemochromatosis patients is liver, and therefore, liver cancer formation in these patients has been studied extensively. However, little is known about the iron-elicited colonic pathologies, mainly colonic inflammation and colon cancer. Here we postulate that iron overload as observed in hemochromatosis disrupts healthy colonic homeostasis and exacerbates the development of colon inflammation and colon cancer. To test our hypothesis, we examined the progression and severity of colitis and colon cancer in Hfe-/- mouse and the wild type control. Hfe-/- mouse is a model for classical hemochromatosis. Besides serum and liver, high concentrations of iron and heme deposits are found in the colon of Hfe-/- mouse. Experimental colitis was induced by administration of Dextran Sodium Sulfate (DSS) in drinking water. Colitis-associated colon cancer was initiated by the intraperitoneal injection of carcinogen, azoxymethane (AOM) and the carcinogenesis was driven by DSS administration. Colonic inflammation was more severe in the Hfe-/- mouse than in control. In addition, hemochromatosis mouse developed more and larger colonic polyps than the control group. We observed that this high susceptibility to colitis and colon cancer in Hfe-/- but not wild type mouse, lies in the differences in strains regarding the bacterial composition and colonic defense machinery. 16S ribosomal RNA sequencing of fecal bacteria revealed that the microbiota composition of Hfe-/- mouse altered to favor the pathogenic bacteria that belong to phyla Proteobacteria and TM7. In addition, the Hfe-/- proinflammatory bacteria adhered to colon and thus increased the colonic bacterial load. This phenomenon, addressed as bacterial dysbiosis, is considered a hallmark of colon inflammation and colon cancer. Furthermore, we observed that the colonic epithelial cells of Hfe-/- mouse had a lower expression of antimicrobial peptides and thus a defective first line defense against the pathogens. Finally, hemochromatosis mouse colon under inflammation released higher concentration of the pro-inflammatory cytokines that contributed to the creation of the hostile colonic environment. In summary, iron overload as seen in hemochromatosis, impairs the colonic defense machinery and it causes bacterial dysbiosis, thus providing the ideal environment for the colitis and colon cancer development.

#1480

A novel mechanism of immunosuppression via nanotube mediated mitochondrial trafficking between cancer cell and immune cell.

Tanmoy Saha, Chinmayee Dash, Sachin Khiste, Shiladitya Sengupta. _Harvard Medical School, Brigham and Women's Hospital, Cambridge, MA_.

Cancer cells deploy multiple mechanisms for immune evasion. Here we report a novel mechanism of cancer cell-induced immune evasion via organelle trafficking between cancer cells and immune cells through physical nanotubes. Using high resolution scanning electron microscopy to analyze a co-culture assay of breast cancer cells and NKT cells revealed that the cancer cells and NKT cells form physical nanotube connections at early time points. Immunofluorescence imaging and quantification using flow cytometry revealed that the cancer cells rapidly hijack mitochondria from immune cells through these nanotubes. The transfer of mitochondria from immune cell to cancer cells imposes a metabolic impairment and results in a reduced population of immune cells. This is not observed in a control coculture using a Boyden chamber, where the two cell types are prevented from forming nanotubes. As the nanotubes are assembled via cytoskeletal rearrangement, we next tested whether the members of Rho-family of GTPases, Cdc42, Ral and Rac are involved in the actin polymerization. Indeed, an inhibitor for Cdc42/Rac1 GTPase inhibitor (ML141) and a geranylgeranyltransferase 1 inhibitor (L-778,123) significantly decreased mitochondrial transfer, and prevented the depletion of NKT cells in co-culture. Furthermore, treating 4T1 tumor-bearing animals with a combination of L-778,123 and a PD1 immune checkpoint inhibitor increased the anti-tumor efficacy compared to PD1 inhibition alone.

Conclusion: Our study sheds insights on a novel mechanism of cancer immune suppression via nanotube-mediated intercellular organelle trafficking. The efficacy of GTPase- or geranylgeranyltransferase-inhibitors, in perturbing this process, and the increased efficacy of the combination with PD1 inhibitor, opens up the possibility of developing these as novel immunotherapy agents for cancer.

#1481

Elucidating the cell type-specific mechanisms of the Ron receptor in the prostate tumor microenvironment.

Camille Sullivan, Nicholas E. Brown, Susan E. Waltz. _University of Cincinnati College of Medicine, Cincinnati, OH_.

The incidence of advanced prostate cancer (PCa) in American men has grown by 72% within the last decade yet current treatments fail to effectively treat advanced disease. We study the Ron receptor tyrosine kinase as a novel target for PCa treatment. Ron is primarily expressed in macrophages and epithelial cells and is found overexpressed in PCa. Ron expression increases with disease severity in humans and mouse models, suggesting Ron drives progression to advanced PCa. Our laboratory has shown that loss of Ron signaling in prostate epithelial cells or in myeloid cells markedly reduces prostate tumor growth and modulates the tumor microenvironment, particularly the functions of macrophages and CD8+ T cells. Thus, we hypothesize that the Ron receptor utilizes cell type-specific mechanisms to suppress the antitumor immune response. However, our previous studies were limited because they were performed separately in different orthotopic models. Therefore, we employed the well-established Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) PCa mouse model to study loss of Ron signaling in either cell type in a single autochthonous model. We used Cre-Lox technology to generate TRAMP mice containing conditional deletion of Ron signaling in prostate epithelial cells or in myeloid cells. At 30 weeks of age, prostate tumors are harvested and analyzed by weight, immunohistochemical staining, quantitative PCR, Western blot, and flow cytometry. To assess the effects of Ron modulation in each cell type and interrogate the interaction between tumor cells and macrophages, we perform in vitro co-culture assays utilizing an established prostate cancer epithelial cell line derived from the TRAMP model and bone marrow-derived macrophages. We observe that Ron signaling in either cell type is critical for prostate tumor growth in TRAMP mice. Furthermore, loss of Ron in either cell type leads to increased macrophage and CD8+ T cell tumor infiltration, and our in vitro data suggest that loss of Ron in either cell type suppresses antitumor M1 macrophage activation and promotes tumor-supporting M2 macrophage activation. Additionally, preliminary data suggest loss of Ron in either cell type leads to increased activation of CD8+ tumor-infiltrating lymphocytes. Overall, our data suggest the Ron receptor coordinates immunosuppressive mechanisms in both the malignant tumor cells and the host macrophages to dampen antitumor immunity and promote prostate cancer. These studies implicate Ron targeting as a powerful therapeutic tool with broad utility for understanding and refining therapeutic strategies to treat advanced PCa.

#1482

Tissue specific splicing program of hMENA: impact on tumor immune microenvironment in node-negative NSCLC.

Francesca Di Modugno, Sheila Spada, Anna Di Carlo, Paola Trono, Isabella Sperduti, Barbara Antoniani, Enzo Gallo, Giulia Campo, Francesco Facciolo, Paolo Visca, Paola Nisticò. _IRCCS Regina Elena National Cancer Institute, Rome, Italy_.

Deciphering the complexity of the tumor microenvironment (TME)is essential to unveil mechanisms of therapy resistance and develop novel microenvironment-related anti-tumor treatment. Actin cytoskeleton dynamics act as platforms for gene regulation and key signaling transduction pathways involved in the cross-talk among tumor cells and cellular and non-cellular components of TME.The actin regulatory protein hMENA undergoes tissue specific splicing, generating two alternatively expressed isoforms hMENA11a and hMENAΔv6 with a crucial role in EMT. We have previously demonstrated that hMENA11a and hMENAΔv6, respectively inhibit or increase cell invasiveness, TGFβ and β1 integrin signaling and the secretion of several key extracellular matrix (ECM) proteins. Early node-negative NSCLC patients show a prolonged disease-free survival (DFS) when expressing high tumor hMENA11a/low stromal FN1. Tertiary Lymphoid Structures (TLS), sites of transient lymphoid neo-genesis and determinants of antitumor immunity, have been associated with a favorable clinical outcome in NSCLC patientsandfound in responding lesions of ICB-treated melanoma patients.

The aim of the present study was to analyzethe pattern ofhMENA isoforms as biomarker of EMT signature in the context of ECM composition and TLS presence and localization.

We evaluated by gain and loss of function experiments the role of hMENA isoforms in TLS neogenesis. hMENA isoforms expression, TLS presence and stromal fibronectin were evaluated in 110primary tumors of node negative NSCLC patients by immunohistochemical analysis using pan-hMENA, hMENA11a, CD3, CD20 and fibronectin (FN) antibodies. The Chi-Square or Fisher Exact tests were used to estimate associations among categorical variables.

We found, by RNA-SEQ analysisand subsequent validation by QRT-PCR, in NSCLC cell lines depleted for the expression of 'epithelial' hMENA11a isoform,that hMENA11asustains the expression of lymphotoxin beta receptor (LTBR), a regulator of TLS formation.

The evaluation of TLS presence and spatial distribution in the primary tumors indicatedthat the presence of TLS within the tumor core is significantly correlatedwith hMENA11a expression in tumor cells, whereas the presence of TLS at the margin oftumor nests correlates with the absence of hMENA11a. When we evaluated also the fibronectin we found a trend of association between low stromal fibronectin and intratumoral TLS, however a low level of stromal FN in concomitance with the expression of hMENA11ain tumor cells,strongly associated with intra-tumoralTLS presence.

Our findings indicate that the alternative splicing of hMENA is crucial in the reciprocal signaling between tumor cells and their immune microenvironment, by participating in tertiary lymphoid structure neo genesis and spatial distribution.

Funded by Airc

#1483

Ex vivo single cell RNA-sequencing of tumor derived organotypic spheroids identifies a unique mesenchymal resistance program to PD-1 blockade.

Andrew J. Portell,1 Jonathan Greene,2 Luke J. Taus,1 Patrick Lizotte,1 Elena Ivanova,1 Kalil O. Menezes,1 Amir R. Aref,1 Paul Kirschmeier,1 Russell W. Jenkins,3 David Barbie,1 Cloud P. Paweletz1. 1 _Dana Farber Cancer Institute, Boston, MA;_ 2 _Acme Informatics, CA;_ 3 _Massachusetts General Hospital Cancer Center, Boston, MA_.

Introduction: Immune checkpoint blockade, including αPD-1 and αPD-L1 form the backbone of personalized medicine for lung cancer and other malignancies. Yet the underlying mechanisms of resistance to therapy are not fully characterized partly because functional models to perform mechanistic studies are lacking. Here we report on single cell RNA sequencing from murine (or patient) derived organotypic tumor spheroids (DOTS-seq) that enables analysis of tumor and immune cell intrinsic changes that occur during αPD-1 treatment ex vivo.

Methods: Murine-derived organotypic spheroids from syngeneic MC38 tumors were grown and treated with αPD-1 and isotype matched IgG in a microfluidic device as previously described1. At day 6, libraries were prepared from individual viable cells using the 10X Genomics platform and sequenced at DFCI. Sequencing data was processed using the Seurat package and corrected for UMI, ribosomal, mitochondrial and cell cycle transcripts. Dimensionality reduction, clustering, and identification of differentially expressed genes were performed on log normalized data. Gene set enrichment analysis was performed using the SetRank package.

Results: Transcripts were obtained for 2,543 IgG treated and 2,626 αPD-1 treated cells that were resistant to ex vivo killing. 60% of αPD-1 treated cells fell into 2 unique clusters which each had downregulated genes associated with apoptosis and interferon-γ response, such as Dap, Cxcl10 and B2m. Within these two, one cluster contained highly upregulated genes known to be E2F targets or important for G2M transition while the other did not. Interestingly, the quiescent subpopulation exhibited a unique epithelial to mesenchymal transition-like state characterized by expression of Snai1, Mmp2, Mmp14, and Vegfa. This subpopulation also upregulated transcripts of immuno-modulatory cytokines from the IL6 family, including Il11, Lif, and Osm. IL-6 extracellular levels are also elevated in treated cultures, suggesting a mesenchymal-like cell subpopulation responsible for this cytokine modulation of the tumor microenvironment.

Conclusion: Here we show that profiling the interplay between tumor and immune cells at the single cell level is possible ex vivo. We identify a previously uncharacterized subpopulation of EMT-like cells that regulate the tumor microenvironment and promote resistance to αPD-1 in MC38 tumors.

Reference: 1. Jenkins RW, et al. Cancer Discov DOI:10.1158/2159-8290

#1484

Microbial sequencing using a single-pool target enrichment of multiple variable regions of the 16S rRNA gene, the nuclear ribosomal internal transcribed spacer (ITS) region, and antimicrobial resistance genes.

Kayla Peck,1 Robert Stedtfeld,1 Jordan RoseFigura,1 Brett Reed,1 Drew McUsic,1 Jon Irish,1 Brett Etchebarne,2 Timothy Johnson,3 Laurie Kurihara,1 Vladimir Makarov1. 1 _Swift Biosciences, Ann Arbor, MI;_ 2 _Michigan State University, East Lansing, MI;_ 3 _Purdue University, West Lafayette, IN_.

The human microbiome has been studied in the context of a number of diseases and has recently been gaining momentum in the field of cancer research. The composition of the gut microbiome, for example, has been shown to affect cancer progression and may act as both a biomarker and therapeutic target. Metagenomic shotgun sequencing has played a large role in characterizing the composition of microbial communities. However, direct sequencing of multiple complex samples in parallel has relatively low sensitivity and struggles to accurately characterize the microbiome. Alternatively, targeted PCR and sequencing can be used to simultaneously enrich for and identify multiple microbial targets of interest in parallel. Here, we present a single-pool multiplex PCR assay to enrich for multiple variable regions of the 16S ribosomal RNA (rRNA) gene, the nuclear ribosomal internal transcribed spacer (ITS) region, and 35 antibiotic resistance genes from multiple bacteria species listed as threats by the CDC. Our two-hour protocol generates target-enriched libraries from genomic DNA (gDNA) that are compatible with Illumina sequencing platforms. We validated our protocol using gDNA from mock microbial communities comprising 20 bacterial strains mixed at varied proportions. The specificity, sensitivity, and quantification of our assay was also evaluated using clinical isolates and extracts from swine manure. We found that our single-tube, target-enriched library workflow accurately and reproducibly identified genomic content from reference strains with as few as six starting genomic copies mixed in complex samples. Further, enrichment of seven variable (V) regions of the 16S rRNA gene facilitated the detection of greater diversity and improved classification of microbial communities compared to the conventionally used primer set, which only targets the V3-V4 regions. Simultaneous analysis of both the 16S rRNA gene and antibiotic resistance genes provided greater differentiation of bacterial pathogens and greater characterization of the overall structure of the community's functional repertoire. This study highlights the power of targeted enrichment via single-pool amplification for sequencing and characterizing microbial communities, including those that have important consequences for human health.

#1485

Hepatic cell cycle-related kinase shapes a metastatic-prone liver microenvironment via crosstalk between myeloid-derived suppressor cell and natural killer T cell.

Xuezhen ZENG,1 Jingying Zhou,1 Zhewen Xiong,1 Hanyong Sun,2 Weiqin Yang,1 Wenshu Tang,1 Yu Feng,1 Alfred Sze-Lok Cheng1. 1 _The Chinese University of Hong Kong, Hong Kong, Hong Kong;_ 2 _Ren Ji Hospital, Shanghai, China_.

Metastasis is a prominent cause of cancer-related death governed by both cancer cell-intrinsic mechanisms and extrinsic microenvironment. Clinical observations demonstrated liver as a common metastatic site for various cancers, which may be due to its immune tolerant environment. Myeloid-derived suppressor cell (MDSC) is a heterogeneous cell population of immature myeloid cells that contribute to the formation of a favorable metastatic environment partially via suppression of immune effector cells. However, the underlying mechanisms in liver tropism of tumor metastasis remain poorly understood. We have previously discovered that cell cycle-related kinase (CCRK) can promote primary hepatocellular carcinoma (HCC) development via MDSCs (Gut 2018; Nat Commun accepted). Here we hypothesize that the accumulation of hepatic MDSCs induced by CCRK may contribute to the formation of a favorable metastatic liver microenvironment. By the construction of a liver-specific CCRK inducible transgenic (TG) mouse model by a Cre/loxP system, induction of CCRK expression by tamoxifen injection could increase CD11b+Gr-1+Ly6G+Ly6Clow polymorphonuclear (PMN)-MDSCs liver accumulation specifically in male mice with upregulated Cxcl1 and Gcsf expression. Intrahepatic injection of a mouse hepatoma cell line Hep1-6 in male TG mice developed larger tumors compared to control and positively associated with increased PMN-MDSCs levels in liver. Moreover, the tumorigenicity was abolished by PMN-MDSC depletion. Notably, intrasplenic injection of a mouse melanoma cell line B16F10 exhibited increased level of liver-infiltrating PMN-MDSCs and enhanced liver metastasis in male TG mice compared to control mice. Moreover, depletion of PMN-MDSCs suppressed metastasis in liver. Mechanistically, anti-tumor NKT cells, rather than NK cells and CD8+T cells, were negatively correlated with tumor weight and MDSC proportion, indicating the potential crosstalk between MDSC and NKT in liver metastasis. Our findings suggest that hepatic CCRK expression create a tumor growth- and metastasis-supportive liver microenvironment via enhancing immunosuppression. Additional colorectal cancer metastasis models will be evaluated in our established TG mice. Moreover, the roles and underlying mechanisms of CCRK-PMN-MDSC interactions in the establishment of liver metastasis await further investigation. This project is supported by Collaborative Research Fund C4017-14G and the Focused Innovations Scheme 1907309.

#1486

The immune atlas and chemokine profile of giant cell tumor of bone.

Binghao Li, Zhan Wang, Zhaoming Ye. _The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China_.

Background and purpose: Giant cell tumor of bone (GCTB) is a locally aggressive tumor with metastatic potential. Given that GCTBs are composed of three major cell populations and the bone-resorption activity is attributed to the multinucleated giant cells which are conjectured to be recruited by "real" tumor cells, it is still elusive that how and what are these immune cells recruited. The purpose of this study is to present the immune landscape of GCTB, as well as to find out responsible chemokines during immune cell recruiting.

Experimental procedures: We collected fresh GCTB tissues from surgical excisions. To identify immune cell composition within the tumor microenvironment, we performed mass cytometry (CyTOF) and conventional multicolor cytometry. Some fresh osteosarcoma and chondrosarcoma samples were also used to compare the immune atlas. For chemokine profiling, chemokine arrays were utilized. Further analysis on potential association between chemokines and immune cells were conducted.

Results: For last updated, 47 GCTB fresh samples were collected, of which 12 cases received CyTOF analysis and the rest 35 samples were analyzed by multicolor cytometry. All GCTB samples were analyzed by chemokine array In-depth interrogation of the immune landscapes showed that compared with osteosarcoma and chondrosarcoma, GCTBs had some unique immune cell subgroups. Some CD163+ CD206+ macrophage clusters which expressed various immune suppressive markers were significantly enriched in GCTBs, while CD4+ CD25+ Treg cells were fewer in GCTB. In addition, some macrophage-like mononuclear cells (CD14- CD68+ CD163- CD206+ HLA-DR+) were only seen in GCTBs.Chemokine array revealed that higher levels of CXCL12, CCL22, and CCL7 might be associated with moremacrophage-like mononuclear cells in GCTBs.

Conclusion: GCTB have very different immune features from other malignant bone tumors. Multiple chemokines may play important roles in recruiting "functional" monocytes. Such in-depth understanding has important implications in appropriate therapeutics for GCTB. Bigger sample size will further help in studying the immune and chemokine features of GCTB.

#1487

Dysregulation of Interleukin-1 receptor-associated kinase 1 promotes prostate cancer-associated chronic inflammation and aggressiveness.

Saheed Oluwasina Oseni,1 Cynthia Nguyen,1 Mirjana Pavlovic,2 James Kumi-Diaka1. 1 _Florida Atlantic Univ., Davie, FL;_ 2 _Florida Atlantic Univ., Boca Raton, FL_.

For decades, chronic inflammation has been linked with cancer development. However, the molecular mechanisms of this association with respect to prostate cancer (PCa) aggressiveness are poorly understood. While previous studies exploiting the contributions of upstream and downstream inflammation signaling proteins to this oncogenic alliance have been clinically unsuccessful, the goal of our study is to elucidate the role of the often-neglected midstream inflammation regulatory proteins, such as the Interleukin-1 receptor-associated kinase 1 (IRAK1). We propose that the dysregulation of IRAK1 protein will promote PCa-associated chronic inflammation and aggressiveness. To explore the importance of IRAK1 in PCa-associated chronic inflammation and aggressiveness, both bioinformatics and experimental approaches were employed. Cutting-edge computational meta-analysis was used to characterize and assess the molecular patterns of gene expression and alteration of IRAK1 in clinical datasets of PCa patients acquired from open resource databases. Inflammation-associated PCa models were established by pre-exposure of aggressive PC3-CRPC cells to low doses (10ng-10µg/ml) of TLR-2, -3, -4 and -7/8 agonists, such as zymosan, poly (I:C), LPS, and resiquimod, respectively, which led to the upregulation of IRAK1 prior to mechanistic studies. Chronic inflammation was confirmed by measuring changes in the levels of IL-6/IL-12 via ELISA. IRAK1 mRNA and protein upregulation were analyzed by RT-PCR and western blot or immunofluorescent staining. Next, we investigated the effects of IRAK1 dysregulation on the aggressiveness of PCa cells by downregulating IRAK1 expression in PC3 cells by either pharmacologic inhibition with thymoquinone (an IRAK1 inhibitor) or via RNAi. MTT and colony forming assays were performed to assess the effects on cell viability and survival. IRAK1 mRNA and protein levels in PC3 cells following treatment with thymoquinone or IRAK1 inhibitor were measured using RT-PCR, western blot or immunocytochemistry. Cell migration was assessed by wound healing assay. Acridine orange/propidium iodide fluorescent staining and flow cytometry were used to analyze the effect on cell cycle and apoptosis. Our computational analysis shows that IRAK1 is commonly upregulated at the mRNA and protein levels and genetically altered/mutated in clinical PCa samples. Downregulation of IRAK1 by thymoquinone or RNAi in PC3 cells result in a decrease in cell viability, cell migration, and proinflammatory cytokines as well as an increase in apoptotic cell death in a dose and time-dependent manner. Upregulation of IRAK1 prior to treatment with PI3K/AKT/mTOR pathway inhibitors abrogates the anticancer effects of these drugs. Our study establishes IRAK1 as a promising therapeutic target to treat PCa-associated chronic inflammation and aggressiveness.

#1488

**IL-35** + **B cells establish immunosuppressive network in pancreatic ductal adenocarcinoma.**

Yuliya Pylayeva-Gupta, Bhalchandra Mirlekar, Daniel Michaud. _UNC Lineberger Comp. Cancer Ctr., Chapel Hill, NC_.

Despite advances in our understanding of the mutational landscape in pancreatic ductal adenocarcinoma (PDAC), this devastating disease is now the third-leading cause of U.S. cancer-related deaths. While recent successes of cancer immunotherapy have generated considerable excitement, this form of treatment has been largely ineffective in patients with pancreatic cancer. A major barrier for immunotherapeutic approaches is marked immunosuppression within the PDAC milieu. We have previously identified a novel role for IL-35 producing B cells in the pathogenesis of pancreatic cancer. However, little is known about the mechanisms behind IL-35 activity in cancer. Here, we set out to elucidate molecular and cellular mechanisms by which IL-35 facilitates the emergence of pancreatic cancer. Our results demonstrate that IL-35, but not IL-10, potentiates PDAC growth. This correlates with induction of regulatory T cells and suppression of effector T cell activity, suggesting that IL-35 controls endogenous anti-tumor immune responses in PDAC. Furthermore, while IL-35 is expressed by several immune cell types in PDAC, we show that its expression specifically in B cells is essential for suppression of anti-tumor T cell responses. Importantly, while PDAC is typically resistant to anti-PD-1 immunotherapy, we demonstrate robust synergistic reduction in tumor growth when IL-35 deficiency is combined with anti-PD-1 treatment. Insights gleaned from these and further mechanistic studies of IL-35 in PDAC may be expeditiously translated into IL-35 targeted combination immunotherapy.

#1489

Increased levels of systemic LPS-positive bacterial extracellular vesicles in cancer patients with chemotherapy-induced intestinal barrier dysfunction.

Joeri Tulkens, Olivier De Wever, An Hendrix. _Ghent Univ. Hospital, Gent, Belgium_.

Bacterial extracellular vesicles (EV) are nanometer-sized membrane particles transporting nucleic acids, metabolites, proteins and endotoxins (lipopolysaccharide (LPS)). As such, bacterial EV that enter the systemic circulation may deliver and elicit a variety of immunological and metabolic responses in different organs including the brain. To date, the systemic presence and activity of bacterial EV in patients with intestinal barrier dysfunction have not been investigated. We combined size exclusion chromatography and density gradient (DG) centrifugation to fractionate plasma LPS in two dimensions to separate bacterial EV-associated LPS from other LPS products. We evaluated plasma from healthy donors asymptomatic of intestinal barrier dysfunction versus cancer patients with radiation- or chemotherapy (with or without gastrointestinal mucositis). We quantitatively measured bacterial EV-associated LPS and other LPS products by performing Limulus Amebocyte Lysate (LAL) and Toll-like receptor 4 (TLR4) reporter assays and qualitatively confirmed the results by immunoelectron microscopy. LAL analysis of DG fractions corresponding to the density of bacterial EV, demonstrated significantly increased LPS activity in patients diagnosed with therapy-induced gastrointestinal mucositis compared to respective controls. In accordance, we detected elevated amounts of microbial pattern recognition receptor (PRR) ligands by determining TLR4 agonistic activity. Immunoelectron microscopy confirmed bacterial EV-associated LPS. In conclusion, LPS-positive bacterial EV are present in plasma, and correlate with impaired barrier integrity in cancer therapy-induced intestinal mucositis. Further studies are needed to understand systemic functions of circulating bacterial EV in cancer patients and cancer therapy-induced conditions.

#1490

Novel microbiome-derived peptides activate the host innate immune system by regulation of TLR signaling.

Dhwani Haria, Helena Kiefel, Yuliya Katlinskaya, Sunit Jain, Thomas Weinmaier, Shoko Iwai, Todd DeSantis, Toshi Takeuchi, Karim Dabbagh, Kareem Graham. _Second Genome, South San Francisco, CA_.

The gut microbiome is a key contributor to the maintenance of host physiology. At the same time, increasing evidence implicates microbiome dysbiosis as a key determinant of numerous metabolic and inflammatory disorders. It is, therefore, of paramount importance to understand the interactions between the host and gut microbiota. Second Genome has developed a proprietary, bioinformatics-driven discovery platform to identify novel microbiome-derived peptides with the potential to modulate host immune responses. We have leveraged this platform to evaluate immunomodulatory functions of novel, secreted peptides derived from gut-resident bacterial species Bifidobacterium longum and Bifidobacterium breve. Previously, the microbial genus Bifidobacterium ('Bifido') has been shown to be associated with enhanced dendritic cell functions and antitumor effects. Here, we identify Bifido-derived peptides that are putative agonists of certain murine and human Toll-like receptors (TLRs). These peptides induce secretion of pro-inflammatory cytokines such as TNF-α and IL-6 by in vitro-generated mouse dendritic cells and peritoneal macrophages in a TLR4 and CD14-dependent manner. One of the peptides, SG-A, also induces secretion of Th1-type cytokines and chemokines such as IL-21, GRO-α, and IP-10 by human monocytes and monocyte-derived dendritic cells. Moreover, in vivo administration of SG-A in mice elicited a systemic pro-inflammatory immune response in the absence of any secondary stimulus. Collectively, our results demonstrate the ability of a novel bacterial-derived peptide to engage the host innate immune system. Moreover, our findings validate the potential of the Second Genome discovery platform for identification of novel microbiome-derived agents that: i) have the ability to modulate host cell functions; and ii) may be developed into therapeutic agents for immune and inflammatory disorders.

#1491

Immune resistance emerges from tumor-initiating stem cells.

Yuxuan Miao. _Rockefeller University, New York, NY_.

The remarkable capacity of the long-lived stem cells-like population to self-renew, initiate and propagate a tumor placed these cancer stem cells (CSC) at the root of tumorigenesis. Many unique molecular features of stem cells endow these key population with specialized properties to drive tumor formation. However, in order to achieve these functions, the neoplastic stem cells must first overcome the immune barrier. Although it has long been postulated that, as the core of a tumor, CSCs must be superior at evading the immune detection, direct evidence is still lacking to demonstrate this stem cell trait. Another important feature of CSCs is that these cells are programed to resist traditional cancer treatments, including chemotherapy and radiation. Yet, it is still unexplored how CSCs react to the immunotherapy, which just start to revolutionize the cancer treatments. In order to address these fascinating questions, we have designed a spontaneous skin squamous cell carcinoma (SCC) mouse model that can be effectively targeted by adoptive cell transfer (ACT)-based immunotherapy, without losing the targeted tumor antigen. Employing this model, we have lineage traced the fate of tumor-initiating stem cells during and after ACT treatment. In this study, we will present data demonstrating that a subset of TGFβ-responding CSCs, residing at the tumor-stromal interface, are always able to survive from the injected CD8+ T cell treatment and give rise to the relapsed tumor. Furthermore, the extensive profiling of ACT surviving stem cells revealed that multiple immune resistant pathways are highly up-regulated in these stem cells. In addition, TGFβ appears to orchestrate these immune resistant processes in the tumor-initiating stem cells. This study highlights CSCs as the core to mediate immune evasion.

#1492

Evaluation of combinatorial chemotherapeutics nano-carriers at different dosage regimens in breast cancer model: Tumor immune signaling, inhibit angiogenesis and tumor growth.

Adel Al Fatease,1 Vidhi Shah,2 Kalei Lopez,2 Brianna Cote,2 Deepa Rao,3 Adam Alani2. 1 _Oregon State University, King Khalid University, Portland, OR;_ 2 _Oregon State University, Portland, OR;_ 3 _Pacific University, Portland, OR_.

The objective of this work is to evaluate the effect of individual & combinatorial delivery of Docetaxel (DTX) and Everolimus (EVR) in polymeric micelles at frequent dosing regimen below their maximum tolerated dose on angiogenesis, efficacy & immune signaling in breast cancer animal model. Based on previous in-vivo work in our lab, the maximum tolerated dose (MTDs) for EVR, DTX & combinatorial micelles is 50, 25 and 20 mg/kg respectively. To achieve our goal, two studies have been conducted. First, in-vivo assessment of anti-angiogenic effects of individual & dual DTX-EVR micelles in murine model at 10%, 30%, and 50% MTD, & MTD dose. Second, in-vivo efficacy evaluation of individual & dual DTX EVR micelles at 10%, 30%, 50% MTD, and MTD dose in orthotropic breast cancer murine model. Relative blood vessel invasion & increase in tumor growth will be evaluated for each dosing treatment. We observed that dual DTX EVR polymeric micelles showed significant inhibition of angiogenesis & tumor growth compared to individual agent. The effect of frequent lower dosing schedule is also currently being investigated for its effect immune signaling.

#1493

Therapeutic efficacy and tolerability of combined immune checkpoint blockade in metastatic melanoma patients is influenced by the gut microbiome.

Vancheswaran Gopalakrishnan,1 Miles Andrews,1 Wei-Shen Chen,2 Christine Spencer,3 Luis Vence,1 Alexandre Reuben,1 Zachary A. Cooper,4 Peter A. Prieto,5 Michael T. Tetzlaff,1 MA Abdul Wadud Khan,1 Alexander Lazar,1 Courtney W. Hudgens,1 Lauren E. Haydu,1 Hussein A. Tawbi,1 Patrick Hwu,1 Wen-Jen Hwu,1 Rodabe N. Amaria,1 Elizabeth M. Burton,1 Scottt E. Woodman,1 Adi Diab,1 Sapna P. Patel,1 Isabella C. Glitza,1 Jianhua Zhang,1 Joseph Petrosino,6 Robert R. Jenq,1 Michael A. Davies,1 Jeffrey E. Gershenwald,1 Padmanee Sharma,1 James P. Allison,1 Andrew Futreal,1 Jennifer A. Wargo1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _University of South Florida Morsani College of Medicine, Tampa, FL;_ 3 _Parker Institute for Cancer Immunotherapy, San Francisco, CA;_ 4 _MedImmune, Gaithersburg, MD;_ 5 _University of Rochester, Rochester, NY;_ 6 _Baylor College of Medicine, Houston, TX_.

Background: The gut microbiome is increasingly being recognized as a strong modulator of anti-PD1 based cancer immunotherapy. Compelling evidence demonstrates differential bacterial enrichment and diversity in responders (R) versus non-responders (NR), mediated by profound influences on systemic and anti-tumor immune infiltrates. However, this has not been studied in the setting of treatment with combined immune checkpoint blockade (CICB), which is associated with superior response rates, but higher rates of potentially debilitating toxicities.

Methods: We assembled a cohort of patients with metastatic melanoma receiving CICB (n=54). All patients were classified as R (n=31, CR + PR) or NR (n=23, SD + PD) based on RECIST v1.1, and as having grade 3 or higher (T; n=29), or less than grade 3 (NT; n=25) immune related adverse event(s) by NCI CTCAE 4.0 criteria. Baseline stool samples were characterized by 16S rRNA sequencing. Correlative analyses of peripheral immune cell populations by flow cytometry (n=12) and circulating T cell repertoire by TCR-sequencing (n=12) were done on matched pre-treatment blood samples.

Results: The overall gut microbial landscape in these patients was varied with high abundance of Bacteroidales and Clostridiales. Ordination of beta-diversity distances revealed a lack of clustering by subtype of primary tumor (uveal, mucosal, cutaneous) consistent with no significant effect of the tumor histology. While no apparent response or toxicity associations were evident based on diversity, notable compositional differences were appreciated. Comparison of relative abundances by LEfSe (LDA>2, p<0.05), and pairwise Mann-Whitney tests revealed an enrichment of Bacteroides stercoris (p=0.03), and Parabacteroides distasonis (p=0.04) in R, and Lactobacillales (p=0.005) in NR. Consistent with our prior findings, the median relative abundance of the order Clostridiales was again higher in R (0.34) versus NR (0.26). On the other hand, Bacteroides intestinalis (p=0.01) and Anaerotignum lactatifermentans (p=0.006) were enriched in T and NT, respectively. Importantly, correlative analyses with circulating immune cell subsets revealed distinct associations by differential bacterial enrichment (including positive correlations between overall CD8+ T-cell abundance and R-taxa), and a clustering effect by high or low T-cell repertoire entropy.

Conclusion: These findings build on our prior work and support the notion of a close link between the gut microbiome and therapeutic outcomes to checkpoint blockade therapy. Extensive studies are underway in both matched human biospecimens and in pre-clinical models to further understand mechanisms of interactions with immune markers, and to establish causality. Taken together, these data support a critical role for the gut microbiome as both a predictive tool and therapeutic target.

#1494

Galectin 9 associates with PDL1 expression and promotes gastric cancer cell invasion.

Charlotte Hill, Camille Cabrolier, Elisa Cumsille, Maximiliano Arce, Mauricio P. Pinto, Maria Loreto Bravo, Matias Muñoz, Marcelo Garrido, Gareth I. Owen. _Pontificia Universidad Catolica de Chile, Santiago, Chile_.

Background: The development of immune checkpoint inhibitors (immunotherapy, such as anti-PD1) that induce a sustained T-cell immune response with anti-tumor activity has been a breakthrough in oncology practice. However, in gastric cancer (GC), sustained responses to anti PD-1 therapy are only observed in approximately 20% of patients. The expression of alternative immune checkpoints such as Galectin 9 (Gal-9) and Tim3 has been associated to anti-PD1 resistance in other cancer types. In search of the factors that render gastric tumors resistant to immunotherapy, we hypothesized that Gal-9 expression in gastric cancer cells may enhance metastasis and immunosuppression.

Methods: Bioinformatic analysis of gastric cancer data of patients with gastric adenocarcinoma from TCGA Database was performed. In vitro experiments with Gal-9 transfected gastric cancer cell line AGS were performed to elucidate Gal-9 gain of function effect on cell invasion and immune-suppressive proteins. Invasion assay using a matrigel coated transwell was performed within 24 hours of transfection. To determine changes in PDL1, E-Cadherin and N-Cadherin expression, cells were harvested 24h after transfection to analyze protein and mRNA levels through Western Blot and RT-PCR respectively.

Results: TCGA database analysis demonstrate that Gal-9 expression is significantly correlated to PDL-1 expression in gastric cancer; furthermore, it was also positively correlated to immune cell markers such as FOXP3. In accordance, in vitro analysis demonstrates that exogenous expression of GAL9 results in an increase in PDL-1 protein and greater invasive potential of the AGS cell line.

Conclusion: Taken together our results suggest that further studies should focus on the role played by Gal-9 is the establishment of an immune suppressive microenvironment in gastric tumors.

FONDECYT 1180241, 1180173. CONICYT FONDAP 15130011, IMII P09/016F

BMS CA209-8F3

#1495

Alternative humanized PD-1 mouse models provide more options for PD-1 antibody efficacy study in vivo.

Qing Lin, Huimin Ma, Xiaoyu Zhang, Hengbin Li, Wenjing Yang, Zhewei Yang, Xingchen Shen, Qunsheng Ji. _WuXi AppTec Co. Ltd., Shanghai, China_.

The Programmed Death-1 (PD-1) binding to its ligands PD-L1 and PD-L2 plays a critical role in suppression of T cell function and tumor immune evasion. Blocking antibodies against PD-1 have been shown to induce durable antitumor responses in multiple cancers in many clinical trials. So a well-characterized mouse model is essential for preclinical studies of antibodies that target human PD-1. Here we generated several humanized PD-1 knock-in mouse models, one expresses a hybrid PD1 protein by replacing the amino acids that involve in binding to PD-L1 (mostly IgV domain) with the human counterpart in both C57BL/6J and Balb/C genetic background, another one have both the IgV domain and signal peptide replaced by human counterpart. These mouse models were confirmed by PCR and southern blotting. And homozygous mouse models from these different strategies were also successfully generated and both of them could survive and the birth rate were very similar to heterozygous and wild type littermates. Staining for human and mouse PD-1 in stimulated CD8+ and CD4+ T cells indicate the up regulated expression of human PD-1 and absence of mouse PD-1 in these mouse model. Homozygous human PD-1 knock-in mice displayed normal development, and no obvious signs of autoimmunity for at least six months, whereas PD-1 knockout mice developed tissue-specific autoimmunity. With these human PD-1 knock-in mice engrafted with MC38 and treated with anti-human PD-1 antibodies, we confirmed the binding of anti-human antibody to chimeric PD-1 protein, and found anti-human PD-1 antibodies could significantly inhibit tumor growth in these models. Head to head comparison of these mice with current commercial available models for in vivo efficacy studies are undergoing and will reveal how the signal peptide effect the protein refolding or signaling transduction pathway for these 'human-mouse' chimeric proteins. In summary, these human PD-1 knock-in mouse models are functional and can be used to evaluate anti-human PD1 antibodies in vivo, we provide more options for antibodies which is targeting PD-1.

#1496

Novel hCD3e mouse models for preclinical pharmacology studies of therapeutic bi-specific antibodies.

Jie Xiang,1 Yanan Li,1 Yuelei Shen,2 Yanan Guo2. 1 _Biocytogen, Wakefield, MA;_ 2 _Biocytogen, Beijing, China_.

Recently it has been demonstrated that T cell-mediated immunotherapy is an attractive treatment strategy for various diseases. For this therapeutic approach, the CD3 complex is one of the key targets to modulate T cell activation; however, in many instances we cannot directly evaluate antibody candidates in mice because the monoclonal antibodies specific to human CD3 cannot cross-react to mouse endogenous CD3. In order to overcome the current model limitation, we have generated CD3e-extracellular domain KI mice for anti-human CD3e bispecific antibody in vivo efficacy evaluation. In this model, the mouse CD3e extracellular domain was replaced by human CD3e. We used conventional mouse ES/HR technology to knock-in human CD3e genomic DNA from the 3' end of exon 2 to the 5' end of exon7 to replace murine exon 2-exon 6. CD3e is one of four subunits of the CD3 co-receptor, which plays an important role in the immune response, associating with the T cell receptor (TCR) to couple antigen recognition to intracellular signal transduction, leading to T cell activation. We have characterized our B-hCD3e mice with the following features:

1.Mouse CD3e+ cells were detected in the wildtype C57BL/6 mice, while human CD3e+ cells were only detected in the homozygous B-hCD3e mice.

2.T cell development was normal in B-hCD3e homozygous mice.

3.T cell subtypes in the spleen, thymus, lymph nodes, and PBMC in B-hCD3e mice were similar and comparable to that in wildtype C57BL/6 mice.

4.Normal in vitro T cell proliferation and cytokine production were found in anti-hCD3e antibody treated humanized B-hCD3e mice

5.Mouse anti-PD-1 antibody can significantly repress the growth of MC38 tumor cells engrafted on B-hCD3e mice, suggesting that T cells in B-hCD3e mice are functionally normal

6.Treatment of B-hCD3e mice with anti-human CD3e antibody OKT leads to T cell depletion, resulting in more aggressive tumor growth.

Furthermore, efficacy and toxicity evaluation of CD3e based bispecific antibodies in B-hCD3e mice are ongoing. Based on our data from extensive phenotypical analyses of B-hCD3e mice, we believe this novel humanized CD3e mouse model will be very useful for in vivo efficacy studies of CD3- mediated therapy, especially anti-CD3e bi-specific antibodies.

#1497

Identification of discrepancy between CTLA4 expression and CTLA4 activation in gastric cancer.

Yingyan Yu. _Shanghai Ruijin Hospital, Shanghai, China_.

Objective: Recently, immune checkpoints blockers showed higher anti-tumor activity for advanced gastric cancer (GC). The purpose of the study is to find out biomarkers related to anti-CTLA4 therapy.

Materials and Methods: Datasets of GEO, TCGA and GESA were extracted. Differential expression of CTLA4 between cancer tissues and normal mucosa, enrichment of WT (wild type) vs CTLA4_KO (knockout) upregulated gene set and clinical significance were analyzed. The expression of CTLA4,CD3 and granzyme A (GZMA) were validated on 30 cases of Chinese GC. MSI marker MLH1 and EBV marker EBER were examined on 30 cases of Chinese GC by immunohistochemistry and in situ hybridization.

Results: CTLA4 was up-regulated in GC tissue relative to normal mucosa in datasets of GSE27342 (fold change=1.586, P<0.001) and GSE63089 (fold change=1.365, P<0.001). Increased CTLA4 expression was positively related to CTLA4 activation. EBV-associated GC (EBVaGC) and microsatellite instability GC (MSIGC) disclosed higher CTLA4 levels than other GCs. Genomic stability GC (GSGC) also showed higher enrichment score of CTLA4 activation. CTLA4 activation resulted in shorter overall survival in GC. The expression level of CTLA4 was well correlated to GZMA (R=0.701, P<0.001) and CD3 (R=0.750, P<0.001).

Conclusions: GSGC was firstly identified as the potential GC subtypes responsive to anti-CTLA4 treatment.

#1498

Methylation landscape of cancers associated with immunogenicity.

Changhee Park,1 Kyeonghun Jeong,2 Joon-Hyeong Park,2 Jeong Mo Bae,1 Kwangsoo Kim,2 Chan-Young Ock,1 Miso Kim,1 Bhumsuk Keam,1 Tae Min Kim,1 Yoon Kyung Jeon,1 Dong-Wan Kim,1 Gyeong Hoon Kang,1 Doo Hyun Chung,1 Dae Seog Heo1. 1 _Seoul National University Hospital, Seoul, Republic of Korea;_ 2 _Biomedical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea_.

Introduction: Determining immunogenicity of tumors is important in predicting response to cancer immunotherapy. Tumor mutation burden, the degree of copy number variation expressed as chromosomal instability score and gene expression profiles (GEP) of tumors, such as cytolytic activity score, interferon-gamma signature and immune signature, are known biomarkers of immunogenicity. However, correlation between methylation burden and signature of tumor immunogenicity is unknown.

Methods: We used The Cancer Genome Atlas (TCGA) pan-cancer database to investigate association of RNA sequencing data and methylation signature of tumors generated by HumanMethylation450K BeadChip. The estimated methylation levels in the CpG sites were expressed in β-scores. To determine immunogenicity in a sample, we used cytolytic activity (CytAct) score defined by the summation of RNA transcript levels of granzyme A (GZMA) and perforin 1 (PRF1). In each CpG site, β-scores of top 5% and bottom 5% were defined as "hypermethylated" and "hypomethylated", respectively. Methylation burden of a sample was defined as number of CpG sites having hypermethylated or hypomethylated β-scores.

Results: In TCGA, total of 7,914 pan-cancer samples had both RNA sequencing data and methylation array data. Methylation burdens of pan-cancer samples were negatively correlated with CytAct scores. (Spearman's correlation rho value -0.37, p < 2.2e-16). This negative trend was consistently observed in most of cancer types and methylation subtypes of individual cancer including breast cancer. Using multivariate linear regression model, methylation burden predicted CytAct score independently along with mutation burden and chromosomal instability score. Hypermethylations in CpG sites of genes related to interferon gamma response(CXCL10, STAT1, IFI6 and IFI27), lymphocyte infiltration(CD8A, CD3, CD79, LCK and CCL5) and tumor antigen recognition were associated with decreased CytAct scores whereas hypomethylation in CpG sites of genes related to TGF-β(CTNNB1, COL1A2, IGF2R, ITGB2, MMP17, SPARC and SMO) and fibroblast response(PLAUR, PLOD2, LOXL2 and MET) were associated with decreased CytAct scores in pan-cancer analysis.

Conclusions: The methylation burden of tumor has negative correlation with the immunogenicity of tumors in general pan-cancer. Correlation of the specific methylation pattern with the response of immunotherapy is warranted in further clinical study.

#1499

Recolonizing microbiota may impact tumor response to PD-1 inhibition following antibiotic and radiotherapy treatment in a bioluminescent orthotopic model of murine lung cancer.

Benjamin G. Cuiffo, Caitlin S. Parello, Chelsea Ritchie, Nicholas Rivelli, Alexandra Kury, Sallyann Vu, Gavin Gagnon, Veronica Ritchie, Kasey Reardon, Catarina Costa, Samantha Rogers, Gregory D. Lyng, Stephen T. Sonis. _Biomodels, Watertown, MA_.

Mounting clinical and preclinical evidence demonstrates an important role for the intestinal microbiome in mediating efficacy of immune checkpoint inhibitor (ICI) antibody therapy across a number of tumor contexts. We sought to determine the effects of microbiome modulation on ICI therapy in a clinically recapitulative orthotopic murine lung cancer model. The microbiome of C57Bl/6NHsd mice was sterilized with antibiotic (ampicillin, streptomycin and colistin) for ten days, resulting in a 99.9% mean decrease in fecal aerobic and anaerobic bacterial load in comparison to naïve mice, handled under specific pathogen free (SPF) conditions. Luciferase-expressing murine Lewis lung carcinoma cells (LL/2-Red-FLuc) were surgically implanted into the left lung parenchyma of all animals. Animals were treated with locoregional radiotherapy (2x 9Gy fractions) targeted to the left lung. The gastrointestinal microbiome was reconstituted via oral gavage Q3D of ~1e09/dose commensal A. muciniphila (A. muc) and E. hirae (E. hir) over five doses or sterile saline as control and animals were randomized within commensal/saline treatment groups to equivalent mean tumor burden as measured by Lumina Series III In-Vivo Imaging System (IVIS). Animals were then administered anti-murine PD-1 or isotype control (0.25 mg) antibody (Ab) treatments Q3D over four doses via intraperitoneal injection. Tumor growth was monitored by IVIS over the course of the study, and ex-vivo IVIS was performed on lungs at endpoint (Day 12 post-tumor implantation). Tumor growth of microbiota non-reconstituted antibiotic-sterilized animals was only slightly inhibited by anti-PD-1 therapy. Animals administered commensal A. muc and E. hir and treatments also displayed slightly inhibited tumor growth kinetics, similar to those observed under saline/anti-PD-1 therapy. Fecal microbial sequencing and immunophenotypic analyses are ongoing. This study demonstrates the utility and ongoing development of a clinically recapitulative contextually accurate preclinical murine lung cancer model to assess the effects of specific microbiota in mediating the efficacy of anti-tumor immunotherapy.

#1500

**Mediators of CD8** \+ **cytotoxic T lymphocyte infiltration in pancreatic cancer.**

Joan Miguel Romero,1 Barbara Grünwald,2 Ashton Connor,3 Gun Ho Jang,3 Prashant Bavi,3 Aaditeya Jhaveri,3 Mehdi Masoomian,4 Sandra Fischer,4 Amy Zhang,3 Robert E. Denroche,3 Tracy McGaha,5 Faiyaz Notta,3 Pamela Ohashi,5 Grainne O'Kane,6 Julie Wilson,3 Jennifer Knox,6 Steven Gallinger3. 1 _Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada;_ 2 _Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada;_ 3 _PanCuRx TRI, Ontario Institute for Cancer Research, Toronto, Ontario, Canada;_ 4 _Department of Pathology, Laboratory Medicine Program, University Health Network, Toronto, Ontario, Canada;_ 5 _Department of Immunology, University of Toronto, Toronto, Ontario, Canada;_ 6 _Wallace McCain Centre for Pancreatic Cancer, University Health Network, Toronto, Ontario, Canada_.

Pancreatic cancer continues to have the highest mortality rate of all solid cancers, with a 5-year overall survival of approximately 8%. Despite recent progress in other malignancies, immunotherapy remains ineffective against pancreatic cancer. Previous work from our group and others has shown that tumors with mismatch repair (MMR) and homologous repair (HR) deficiency have increased immune activity, likely attributable to the inherently increased mutational and neoantigen load in these patients. However, there remains a subset of pancreatic cancer patients with increased CD8+ T cell infiltration, despite lacking these specific mutational signatures. Furthermore, an increasing body of evidence has shown that mutational burden and neoantigen load only partially explain the T cell-inflamed phenotype seen in tumors, implicating the presence of additional mechanisms that drive T cell infiltration in cancer. Several recent studies have highlighted the importance of proper CD8+ T cell priming by antigen presenting cells (APCs), particularly Batf3+ dendritic cells (DCs), and subsequent tumor infiltration via chemokines for immunotherapy to be effective. In this study, we investigated the involvement of chemokines in cytotoxic T lymphocyte infiltration in pancreatic cancer. Using a bioinformatics-driven approach, we analyzed 78 treatment-naïve, primary pancreas cancer resections for associations between CD8+ tumour infiltrating lymphocytes (TILs) and chemokine expression by RNAseq. A panel of chemokines, including CCL4, CCL5, CXCL9, CXCL10, and CXCL11, was highly associated with CD8+ TILs (p < 0.001). Segregating 173 tumour-enriched patient RNAseq samples based on expression of CXCL9 and CXCL10, we found those with higher expression of these chemokines had increased immune activation signatures. Importantly, this included increased MHC I presentation, presence of Batf3+ DCs, and T cell/APC co-stimulation (p < 0.001), while we observed no differences in conventional predictors of CD8+ T cell infiltration such as SNV counts or neoantigens between groups. These results were consistent across ICGC and TCGA data sets. Moreover, these results were also recapitulated in 72 tumor-enriched liver metastases, suggesting an underlying immunobiology that may occur in both primary and metastatic sites. The cellular sources of these chemokines, as determined by immunohistochemical analysis, and the role of tumor-sensing innate immune pathways leading to CD8+ T cell priming, by pathway and differential gene expression analysis, are currently being investigated. Taken together, these results demonstrate a potential role for these chemokines in recruiting CD8+ T cells in pancreatic cancer. Understanding mediators driving cytotoxic T cell infiltration will help identify mechanisms leading to proper CD8+ T cell priming and homing into tumors, to stratify patients amenable for known and novel immunotherapies.

#1501

Clinical significance of tumor immune microenvironment in soft tissue sarcoma.

Naoki Oike,1 Hiroyuki Kawashima,1 Akira Ogose,2 Hiroshi Hatano,3 Takashi Ariizumi,1 Taro Sasaki,3 Tetsuro Yamagishi,1 Hajime Umezu,1 Naoto Endo1. 1 _Niigata University, Niigata City, Japan;_ 2 _Uonumakikan Hospital, Minamiuonuma City, Japan;_ 3 _Niigata Cancer Center Hospital, Niigata City, Japan_.

Background: The prognosis of patients with metastatic soft tissue sarcoma (STS) remain poor. Although immunotherapies have been attempted in patients with STS, the prognostic significance of the immune status within the tumor microenvironment in STS is not well understood. The purpose of this study is to investigate the tumor immune microenvironment and evaluate its prognostic impact for patients with STS.

Material and Methods: We retrospectively evaluated the tumor immune microenvironment in 116 primary untreated STS patients with five subtypes: synovial sarcoma (SS: n=36), myxoid liposarcoma (MLS: n=32), undifferentiated pleomorphic sarcoma (UPS: n=27), leiomyosarcoma (LMS: n=11), and dedifferentiated liposarcoma (DDLS: n=10). The infiltration of CD8+ and FOXP3+ lymphocytes, CD163+ macrophages, and the expression of HLA class I and PD-L1 in the tumor were evaluated by immunohistochemistry. Kaplan-Meier method was used to estimate overall survival (OS) and progression-free survival (PFS).

Results: UPS had a higher infiltration of CD8+ and FOXP3+ lymphocytes than SS and MLS. Infiltration of CD163 + macrophages were higher in UPS, LMS, and DDLS than in SS and MLS. HLA class I expression was detected in all of UPS, LMS, and DDLS. However, HLA class I was negative in 17% of SS and 69% of MLS. Although no PD-L1 expression was observed in SS and MLS, PD-L1 expression was detected in 36% of UPS, 28% of LMS, and 25% of DDLS. Moreover, PD-L1 expression was associated with poor outcome in these subtypes. Increasing number of CD8+ lymphocytes was associated with a better OS in patients with SS. A higher infiltration of FOXP3+ lymphocytes was associated with an unfavorable OS in patients with MLS but favorable outcome in SS. Moreover, a higher infiltration of CD163+ macrophages showed a significantly worse OS and PFS in patients with SS and MLS.

Conclusion: Lymphocytes and macrophages were variably infiltrated across STS subtype. UPS, LMS and DDLS, which have numerous genetic mutations, demonstrated high infiltration of lymphocytes, HLA class I and PD-L1 expression. These suggest that T-cell based immunotherapy such as immune checkpoint inhibitors may be effective in these tumor subtypes. Although the SS and MLS are less mutated and do express cancer-testis antigen NY-ESO-1 in common, expression level of HLA class I is significantly different between SS and MLS. In addition, infiltration of FOXP3+ lymphocytes have opposite effect in these tumors. These suggests different immunotherapeutic strategy might be required in these subtypes. Our results might be useful in the clinical strategy of immunotherapy in STS.

#1502

KRAS RNAi therapy in preclinical models of pancreatic cancer sensitizes tumors to checkpoint blockade by affecting the tumor microenvironment.

Shanthi Ganesh, Serena Shui, Kevin Craig, Weimin Wang, Bob D. Brown, Marc Abrams. _Dicerna Pharmaceuticals, Inc, Cambridge, MA_.

KRAS is one of the most frequently mutated oncogenes in pancreatic cancer. Recent research has revealed that activation of KRAS in cancer cells causes recruitment of immunosuppressive molecules to the tumor microenvironment (TME) and blocks T-cell mediated cytotoxicity. Despite huge progress in our understanding of KRAS and its role in tumorigenesis, direct pharmacological targeting of KRAS has thus far not been achieved. In these studies, we inhibited KRAS directly in tumor-bearing mice, using a potent and specific Dicer substrate siRNA (DsiRNA) formulated in tumor-selective lipid nanoparticles. We demonstrate that intravenously-dosed KRAS DsiRNA treatment results in reduction of multiple immunosuppressive signaling molecules, affecting recruitment of myeloid cells and regulatory T cells (Tregs) to the TME. In addition, effective KRAS inhibition reversed MEK-inhibitor-induced resistance in a model of KRAS mutant human PDAC. These data suggest that KRAS DsiRNA has potential to sensitize treatment-refractory PDAC tumors to both immune checkpoint blockade as well as conventional targeted therapeutics

#1503

Effect of immune cell depletion on the responses to PD1 treatment in syngeneic models.

Ying Jin, Yongli Shan, Annie Xiaoyu An, Henry Qixiang Li, Davy Xuesong Ouyang. _Crown Bioscience, Inc., Taicang, Jiangsu, China_.

Immunological checkpoint inhibitors, i.e., PD1 or PD-L1 antibodies, reverse cancer immunosuppression and promote antitumor immune responses in several cancer types. However, targeting PD-1/PD-L1 axis is only effective in ~20% of cancer patients, while some of the initial responders also develop resistance to the treatment. It is critical to fully understand which lineages of immune cells play major roles mediating PD1 responses in a given tumor microenvironment. While it's well-known that PD1 antibody works through activating cytotoxic T cells, the contributions of other immune cell populations in PD1 treatment are not well defined. To understand how different lineages of immune cells impact PD1 efficacy, we removed various immune cell subpopulations in MC38 and Hepa 1-6 murine models when treated with PD1 antibody. In this study, mice inoculated with MC38 or Hepa1-6 tumors were treated with PD1 antibody or a combination of PD1 with depletion antibodies. The effectiveness of immune cell depletion was assessed by flow cytometry. CD4-, CD8- T cells, NK cells, MDSC and macrophages were depleted with anti-CD4 (GK1.5), anti-CD8 (2.43), anti-NK1.1 (PK136), anti-GR1 (RB6-8C5) antibodies and clodronate liposomes, respectively. As expected, depletion of CD8 T cells completely abrogated the antitumor effect of PD1 treatment, confirmed the crucial roles for CD8 T cells in tumor killing. Surprisingly, depletion of CD4 T cells and macrophages showed quite opposite effects in MC38 and Hepa1-6 lines - while significantly sensitizing MC38 tumors to PD1 treatment, PD1 efficacy is deteriorated in Hepa1-6. Depletion of NK cells enhanced the tumor inhibition effect in Hepa1-6 with 4 tumors showed complete or partial regression, without significant impact on MC38 tumors. Taken together, our studies have shown an unequivocal role of CD8 T cells in PD1 induced tumor growth inhibition. However, other lineages of immune cells may act differently upon PD1 blockade release, presumably depending on specific tumor microenvironment. Additional study is needed to differentiate the effects of Treg depletion vs. non-Treg CD4 T cell depletion.

#1504

Characterization of a MC38 mouse syngeneic tumor model expressing human PD-L1 in the transgenic C57BL/6 mouse system expressing human PD-1 and PD-L1.

Michael Koratich,1 Ted Green,1 Jie Liu,1 Charlotte Hammond,1 LaJuana Durbin,1 Jerry Zhou,2 Anna Chen,2 Murray Stackhouse1. 1 _Southern Research, Birmingham, AL;_ 2 _Nanjing Galaxy Bioparma, China_.

The human checkpoint targets PD-1 and PDL-1 continue to demonstrate great promise in the clinic. With much of the current focus turning toward combination regiments, an animal model using the human clinical antibodies will be beneficial for evaluating new combination strategies. Here we continue characterization of a C57BL/6 transgenic mouse model expressing both the human PD-1 and PD-L1 combined with a modified murine MC38 colon tumor cell line expressing human PD-L1. Both PD-1 and PD-L1 expression of the transgenic mice were verified in ex vivo stimulated splenocytes by flow cytometry analysis. Expression of PD-L1 on the genetically modified MC38 cells was also demonstrated by flow cytometry. For in vivo efficacy evaluation, tumor-implanted mice were treated with the clinical agents nivolumab and pembrolizumab at 100 µg and atezolizumab at 1 mg on Days 3, 7, 10, and 14 post implant. Treatment with nivolumab and pembrolizumab initiated tumor regression by Day 17. Complete tumor regressions were seen at Day 28 in nivolumab, 62.5% complete regression, and pembrolizumab, 71.4% complete regression. Growth inhibition was 83.9%, 69.7%, and 95.0% on Day 28 for nivolumab, atezolizumab, and pembrolizumab, respectively, compared to the control animals. There was no significant body weight loss and no signs of toxicity in any of the treated animals. To compare the human anti-PD-1 and PD-L1 clinical agent specificity, the non-transgenic parent C57BL/6 mouse strain was implanted with the unmodified MC38 colon tumor cells. Treatment with pembrolizumab and atezolizumab was conducted as with the transgenic animals. Through 28 days no growth inhibition, tumor size 105.9% of control, was seen in the pembrolizumab treated group, demonstrating lack of cross reactivity of the human therapeutic in the standard mouse model. Atezolizumab did demonstrate a 56.9% growth inhibition compared to controls and is consistent with the known cross reactivity of atezolizumab between human and mouse. We have shown a genetically modified MC38 colon tumor expressing human PD-L1 in transgenic mice expressing both human PD-1 and PD-L1 to be a suitable model for checkpoint inhibitor evaluation of the human form of the particular checkpoint therapeutic. Further research will involve combining each of the checkpoint antibodies with various chemotherapeutic agents. 

### Suppressive Myeloid Cells

#1505

Tumor associated antigen uptake tracking following immuno-radiotherapy.

Esben Christensen,1 Lars Ringgaard,1 Anja Brus,1 Andreas Kjaer,2 Anders E. Hansen,1 Thomas L. Andresen1. 1 _Technical University of Denmark, Kongens Lyngby, Denmark;_ 2 _Rigshospitalet and University of Copenhagen, Copenhagen, Denmark_.

Immuno-radiotherapy is a promising strategy for generation of immunologic cancer recognition, rejection and induction of anti-cancer memory. Radiotherapy (RT) causes single or double strand DNA breaks in a dose dependent manner leading to immunogenic cell death of cancer cells and induction of type 1 interferons. Furthermore, RT increases MHCI expression on cancer cells and infiltration of cytotoxic T cells. Combined, these effects make RT a potent primer of anti-cancer immune responses. The most promising approaches in anti-cancer immunotherapy is to augment the adaptive immune response through danger associated molecular patterns signaling, including toll-like receptor (TLR) agonists. Optimally, these interventions should be given when tumor-associated antigen (TAA) uptake and presentation peaks. Previous studies on TAA uptake have mostly focused on in vitro or ex vivo assays to mimic the in vivo mechanisms. In the present study, we investigate the in vivo kinetics of TAA uptake and trafficking using stably transfected mCherry-transfected B16F10 melanoma. B16F10 transfected with mCherry, a pH-stable fluorescent protein, allows tracking by e.g. flow cytometry in both cancer cells and phagocytosing populations. In vivo trafficking of mCherry fluorescence was investigated in mice treated with RT alone or in combination with TLR7 agonists given locally or systemically. Following treatment, TAA-uptake, -trafficking, and activation of relevant immune populations was evaluated in tumors or tumor draining lymph nodes (tdLNs). Increased TAA uptake was observed in MHCII high populations compared to MHCII low across all phagocytosing populations in tumors. The highest uptake (MFI of mCherry) was found in patrolling monocytes (CD11b+CD11c-Ly6c-CX3CR1high) and tumor-associated macrophages (CD11b+CD11c+CD64+). Interestingly, we exclusively found increased mCherry signal in resident cDC1s in tdLNs after treatment. RT led to a diminished population of migratory CD103+ cDC1s in tumors and we did not observe any increase in mCherry+ CD103+ cDC1s in tdLNs. We did not observe any mCherry signal in spleens - indicating that no cancer-specific immune response are mounted from the spleen. The results also indicate that TAA antigen is trafficked to tdLNs primarily by monocytes or by lymphatic drainage.

#1506

Lymph node metastasis in clinical breast cancer is significantly promoted by myeloid-derived lymphatic endothelial cell progenitors.

Lisa D. Volk-Draper, Radhika Patel, Nihit Bhattarai, Jie Yang, Sophia Ran. _Southern Illinois University School of Medicine, Springfield, IL_.

Introduction: Lymph node (LN) metastasis, a key prognostic factor for poor survival of breast cancer (BC) patients, directly depends on tumor lymphangiogenesis. We previously showed in orthotopic BC models that lymphangiogenesis is strongly promoted by tumor-mobilized myeloid-derived lymphatic endothelial cell (LEC) progenitors (M-LECP) and that M-LECP density correlates with lymphatic metastasis. The goals of this study were to determine the blood levels of M-LECP, their tumor density and correlation with metastasis in clinical human BC.

Methods: Circulating CD14+ cells from BC patients (N=25) and healthy donors (N=24) were analyzed for expression of lymphatic-specific markers. Specimens from BC patients (N=104) along with normal mammary tissues (N=6) were co-stained with antibodies against myeloid and lymphatic markers including CD68, CD14, CD18 as well as LYVE1, podoplanin, and VEGFR-3. Sections were also double-stained to identify LYVE1+ cells expressing lymphoid, M2-type macrophage, and stem cell markers. Densities of tumor CD68+ macrophages, LYVE1+ vessels, and CD68+/LYVE1+ double-stained M-LECP were quantified and correlated with node status and other parameters. Pro-metastatic potential of experimentally produced mouse M-LECP was determined by injecting in vitro differentiated myeloid-lymphatic cells into mice bearing syngeneic EMT6 breast tumors.

Results: 92% of BC patients had monocytes expressing at least one lymphatic marker compared 2% of healthy donors. More than 70% of breast tumors contained M-LECP with density ranging from 2 to 50 cells per high-power field. Tumor-recruited M-LECP express specific markers of myeloid but not lymphoid lineages as well as M2-type, and stem cell markers. Tumors with high density (>20 cells/field) of M-LECP were significantly more likely to belong to aggressive sub-groups (HER2+ and TNBC) and present with lymph node metastasis (P-value <0.02).

Conclusion: These data show for the first time that human breast tumors recruit significant amount of immature myeloid cells with lymphatic phenotype and vessel-promoting properties. Such cells are largely absent from normal breast tissues. Statistical analyses show that M-LECP can significantly contribute to formation of new lymphatic vessels leading to increased nodal metastasis.

#1507

Transcriptional profiles of CD14+ cells in situ in melanoma tumors reveal plasticity and localization dependent function.

Jan Martinek,1 Kyung In Kim,1 Te-Chia Wu,1 Victor Wang,1 Hannah M. Brookes,1 Lili Sun,2 Ananya Gulati,1 Joshy George,1 Philipp Henrich,3 Florentina Marches,1 Anthony Rongvaux,4 Michael Chiorazzi,5 Jeff Chuang,1 Paul Robson,1 Richard Flavell,5 Jacques Banchereau,1 Karolina Palucka1. 1 _The Jackson Laboratory for Genomic Medicine, Farmington, CT;_ 2 _Dana Farber Cancer Institute, Boston, MA;_ 3 _The Jackson Laboratory, Bar Harbor, ME;_ 4 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 5 _Yale University, New Haven, CT_.

Mechanisms contributing to immunotherapy resistance in patients are the object of intense studies. Myeloid cells play a major role in tumors and include cells with different functions that can be grossly summarized as: (1) Antigen capture for presentation (dendritic cells, DCs) or for degradation (macrophages); (2) Tissue repair (macrophages) and (3) Effector function (mast cells, monocytes, granulocytes). However, the functional states of myeloid cells in human tumors are not completely understood. To study intact tumor microenvironments we have established a comprehensive approach for cellular and molecular analysis. Modular analysis of bulk RNAseq data of whole melanoma tissue sections stratified patient samples based on lymphocytic infiltrate but revealed inflammation and myeloid modules spread across samples. Polychromatic immunofluorescence and histocytometry showed that CD14+ cells represent the majority of the total tumor immune infiltrate. Furthermore, the majority of T cells present in melanoma tissue are in direct contact with CD14+ cells rather than melanoma cells. The distribution of CD14+ cells shows two distinct patterns: CD14+ cells within cancer nests (intratumoral) which are in close proximity to melanoma cells and are loaded with melanoma protein; CD14+ cells in the tissue surrounding cancer nests (stromal) which do not contain melanoma protein cargo and are organized in dense infiltrates. Using customized immunofluorescence-guided laser capture micro-dissection, we harvested CD14+ cells based on their tissue location and melanoma protein load for downstream analysis. We find that unsupervised clustering of transcriptomes groups cells by their tissue localization, i.e., intratumoral vs. stromal. Computational analysis, revealed distinct and replicable gene signatures associated with different pathways, in intratumoral and stromal CD14+ cells. Thus, the transcriptome differentiates the functional status of CD14+ cells related to their localization within tumor.

#1508

Primary tumors induce neutrophil extracellular traps with targetable metastasis promoting effects.

Roni F. Rayes,1 Jack G. Mouhanna,1 Ioana Nicolau,2 Phil Vourtzoumis,1 Carson Wong,1 Jules Eustache,1 France Bourdeau,1 Betty Giannias,1 Aya Siblini,1 Emma Lee,1 Veena Sangwan,1 Simon Rousseau,1 Daniela Quail,3 Logan Walsh,3 Nicholas Bertos,1 Jonathan Cools-Lartigue,1 Lorenzo E. Ferri,1 Jonathan D. Spicer1. 1 _McGill University Health Center, Montreal, Quebec, Canada;_ 2 _University of Toronto, Montreal, Quebec, Canada;_ 3 _McGill University, Montreal, Quebec, Canada_.

Targeting the dynamic tumor immune microenvironment can provide effective therapeutic strategies for cancer. Neutrophils (polymorphonuclear neutrophils, PMNs) are the predominant circulating leukocyte population in humans and are vital to fight infection. Despite mounting evidence that PMNs can promote tumor progression, depleting PMNs is not a viable therapeutic option. Neutrophil extracellular traps (NETs) are networks of extracellular neutrophil DNA fibers that are capable of trapping tumor cells and promoting their growth and their metastasis. Targeting NETs can therefore be a potentially successful therapeutic option to block the tumor promoting functions of PMNs. Here we demonstrate that circulating NET levels are elevated in esophageal, gastric and lung cancer patients compared to healthy controls. This increase correlates with disease stage and NET levels are independent predictors of advanced stage. Using pre-clinical murine models of lung and colon cancer, we observe elevated NET levels in tumor bearing mice compared to non-tumor bearing mice; these levels correlated with tumor size. NET levels significantly decrease following tumor resection or treatment with DNase1, a NET degrader, or neutrophil elastase inhibitor (NEi), a NET inhibitor. NET levels do not rise following tumor inoculation in peptidyl arginine deiminase-IV knock out (PAD4-/-) mice; PAD4 being an enzyme essential for citrullination of histones, a crucial step in NET release. Moreover, PMNs from tumor bearing mice are more primed for NETosis than PMNs from non-tumor bearing mice or NEi-treated or PAD4-/- tumor bearing mice. Finally, elevated in vivo hepatic adhesion and spontaneous liver and lung metastases are observed in tumor bearing mice compared to DNase1- or NEi-treated or PAD4-/- tumor bearing mice. Therefore, inhibiting NETs represents a promising strategy to impede metastatic dissemination in several types of cancer patients.

#1509

Identification of immunotherapy resistance mechanisms within tumor microenvironment in a mouse model of oral squamous cell carcinoma.

Liye Zhou,1 Yasutaka Nakahori,2 Fei Guo,2 Joshua Keegan,2 Rachel Riley,1 James A. Lederer,2 Ravindra Uppaluri3. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Brigham and Women's Hospital, Boston, MA;_ 3 _Dana-Farber Cancer/Brigham and Women's Cancer Center, Boston, MA_.

The response rate of 15-20% with anti-PD1 in head and neck squamous cell carcinoma (HNSCC) highlights the urgent need for strategies to overcome resistance. Our lab has previously developed a carcinogen-induced immunocompetent murine oral carcinoma (MOC) model to study HNSCC immunobiology. Specifically, MOC1 is an immunogenic cell line that, despite sensitivity to anti-PD-1, exhibits occasional development of escape tumors (MOC1esc). MOC1esc escape tumors display a resistance phenotype similar to those observed in HNSCC patients undergoing anti-PD1 therapy. When independent escape tumors are harvested and re-transplanted into naïve mice, they grow progressively and are resistant to anti-PD1 therapy. Intriguingly, whileMOC1esc is resistant to anti-PD1, it is completely rejected in tumor bearing mice treated with anti-CTLA4. Therefore, the anti-PD1 responsive MOC1 and resistant MOC1esc mouse model is an isogenic system that provides an excellent opportunity to study mechanism(s) in adaptive resistance to anti-PD1 therapy of HNSCC. To gain a comprehensive insight into the tumor microenvironment (TME) as a contributor to adaptive resistance, we analyzed tumor infiltrating lymphocytes (TIL) in naïve MOC1 and MOC1esc tumors using mass cytometry time-of-flight (CyTOF) with a 38-cell marker panel.MOC1esc tumors were highly infiltrated by CD103+ effector/memory regulatory T cells (Tregs) and M2-like tumor associated macrophages (TAMs), while MOC1 tumors have more M1-like TAMs and neutrophils. Furthermore, we observed that both anti-PD1 and anti-CTLA4 treatment dramatically expanded CD8+ T cells and decreased neutrophils in MOC1esc tumors. In responding MOC1esc tumors, anti-CTLA4 treatment resulted in depleted Tregs, decreased M2-like TAMs and neutrophils, as well as a striking increase in M1-like TAMs compared with isotype control treated tumors. In contrast, anti-PD1 treated resistant MOC1esc tumors showed decreased M1-like TAMs, while M2-like TAMs were increased compared with controls. Tregs were not affected by anti-PD1 treatment. Therefore, the comparison between the TME of anti-PD1 treated resistant tumors and anti-CTLA4 treated responding tumors suggeststhat Tregs, neutrophils, and TAMs may contribute to the sensitivity (or resistance) to checkpoint blockade therapy. Ongoing studies will test the functional contribution of these distinct TME components in antitumor immunity including cytokine production, proliferative capacity, and their roles in immunotherapy resistance. In summary, this study identified immune modulators within TME involved in adaptive immunotherapy resistance of HNSCC. Findings from this study have advanced our understanding of HNSCC immunotherapy resistance and will accelerate the discovery of new therapeutic targets and biomarkers for adaptive resistance.

#1510

Androgen regulated IL-8 expression in prostate cancer: Insights into tumor cell mediated immunosuppression.

Zoila A. Lopez Bujanda,1 Michael C. Haffner,1 Matthew G. Chaimowitz,2 Nivedita Chowdhury,1 Paula J. Hurley,1 Angela M. Christiano,2 Charles G. Drake2. 1 _Johns Hopkins, Baltimore, MD;_ 2 _Columbia University, New York, NY_.

Androgen deprivation therapy (ADT) results in castration-resistant prostate cancer (CRPC) in a significant fraction of patients. We have previously reported that the protein levels of interleukin-8 (IL-8) were inversely correlated with disease progression in men with biochemical recurrent prostate cancer treated with Lenalidomide. Recently, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) were implicated as potential drivers of CRPC. Here we show that IL-8 expression is upregulated as a consequence of ADT and mediates the recruitment of PMN-MDSCs to the tumor microenvironment. We found that IL-8 expression is regulated by both an inflammatory stimulus (NF-kβ mediated) and loss of androgen receptor (AR) signaling following ADT. We confirmed direct binding of both the p65 subunit of NF-kβ and AR to the IL-8 promoter, and their respective effects on promoter activity. The suppressive activity of AR was further supported by a reduction in active transcription markers at the chromatin level surrounding the IL-8 promoter. Accordingly, intratumoral infiltration of PMN-MDSCs correlated with IL-8 expression, and was reduced in IL-8 knockouts. Taken together, these results suggest an innate inflammatory response, loss of AR suppressive activity, and subsequent chemokine upregulation as a potential mechanism that regulates the infiltration of PMN-MDSCs to the tumor microenvironment of CRPC after ADT. These findings open a window of opportunity for therapeutic interventions aiming to improve responses to checkpoint blockade in prostate cancer.

#1511

MEK activation modulates immunosuppressive MDSCs and metabolic phenotypes in TNBC.

Derek A. Franklin,1 Joe T. Sharick,1 Paula I. Ericsson-Gonzalez,1 Violeta Sanchez,1 Phillip T. Dean,1 Susan R. Opalenik,1 Stefano Cairo,2 Jean-Gabriel Judde,2 Michael T. Lewis,3 Jenny C. Chang,4 Melinda E. Sanders,1 Rebecca S. Cook,1 Melissa C. Skala,5 Jennifer Bordeaux,6 Jehovana Orozco Bender,6 Christine Vaupel,6 Gary Geiss,7 Douglas Hinerfeld,7 Justin M. Balko1. 1 _Vanderbilt University Medical Center, Nashville, TN;_ 2 _XenTech, Paris, France;_ 3 _Baylor College of Medicine, Houston, TX;_ 4 _Houston Methodist Research Institute, Houston, TX;_ 5 _Morgridge Institute for Research, University of Wisconsin, Madison, WI;_ 6 _Navigate Biopharma Services, Inc. a Novartis subsidiary, Carlsbad, CA;_ 7 _nanoString Technologies, Seattle, WA_.

Triple-negative breast cancers (TNBCs) are highly heterogeneous and aggressive, with high mortality rates. Although TNBC is typically more responsive to chemotherapy than other breast cancer subtypes, many patients develop chemo-resistance. The molecular processes between tumor and stromal cells involved in developing chemo-resistance are under-explored. Here we report studies of paired TNBC patient-derived xenografts (PDX) established before and after chemo-resistance. Despite significant genetic similarities, the chemo-resistant PDX model harbored a rare constitutively-active KRASQ61R mutation which was not present in the chemo-naive PDX. Further analysis demonstrated that the chemo-resistant KRAS-mutant model exhibited altered gene expression changes including increased expression of CXCR2-ligands CXCL1 and CXCL2, which are responsible for recruiting immune cells to tumors. These expression patterns were largely inhibited in vivo by MEK inhibitor (MEKi) treatment. Moreover, in breast cancer cell lines, CXCL1, CXCL2, and granulocyte macrophage-colony stimulating factor (CSF2, stimulates granulocyte and macrophage differentiation from hematopoietic precursor cells, including immunosuppressive myeloid cells) transcripts were also downregulated by MEKi. Notably, chemo-resistant KRAS-mutant tumors harbored increased Gr1+ and Arginase-1+ cells, consistent with recruitment of immunosuppressive M2-like macrophages and/or myeloid-derived suppressor cells (MDSCs), which was inhibited by MEKi. Further experiments demonstrate that CD45+CD11b+Ly6G+ MDSC accumulation in tumors can be inhibited by MEKi treatment alone, or by CXCR2 inhibition, suggesting that the effects of MEK inhibition on MDSC recruitment are CXCL1/2-dependent. Confirming the translational relevance of these findings, in >200 murine and >1000 human breast tumors, Ras/MAPK transcriptional activity correlated with myeloid-recruiting CXCL1/2 expression and negatively with T-cell recruiting chemokines (CXCL9/10/11), even in the absence of activating KRAS mutations. The association with Ras/MAPK activity was also confirmed using immunofluorescence to quantify MHC-II-low myeloid cells in 80 post-chemotherapy TNBC tumors. Importantly, MEKi and chemotherapy combination treatment reversed immunosuppressive cell accumulation and metabolic phenotypes exemplified by altered optical redox ratios (NAD(P)H/FAD) in the chemo-resistant KRAS mutant tumors, resulting in tumor growth suppression in mice. MEKi treatment also reduced redox ratios in 3D cultures of breast cancer cell lines further suggesting that MEK inhibition targets multiple oncogenic processes in breast cancer. These results suggest that Ras/MAPK pathway inhibitors may be effective in some breast cancer patients to reverse Ras/MAPK-driven tumor metabolism and immunosuppression, particularly in the setting of chemo-resistant disease.

#1512

Nicotine promotes brain metastasis of lung cancer by inducing M1/M2 conversion and suppressing the innate immune response of microglia.

Shih-Ying Wu, Fei Xing, Sambad Sharma, Abhishek Tyagi, Kerui Wu, Yin Liu, Dan Zhao, Ravindra Deshpande, Yusuke Shiozawa, Kounosuke Watabe. _Wake Forest University, Winston Salem, NC_.

Approximately 50% of patients with metastatic lung cancer develop brain metastasis which is almost always fatal, and the median survival time of these patients is only a few months. Smoking is the major risk factor for lung cancer, and tobacco smoke contains a toxic mix of more than 7,000 chemical. Among those chemicals, nicotine has been shown to promote cancer progression and metastasis. However, the exact mechanism by which nicotine derives this process remain poorly understood. The communications between neoplastic cells and microglia, especially M2 type pro-tumor microglia, profoundly affect the brain metastasis progression. Therefore, we hypothesize that nicotine skews microglial polarization to M2 phenotype which promotes brain metastasis of lung cancer. We found that the brain metastasis-free survival rate was significantly decreased in current smokers compared to non-smokers. Abundant CD206\+ M2 microglia were observed in brain metastatic lesions in such patients. Consistent with these results, when we transplanted the H2030 brain metastasis of lung cancer cell line (H2030BrM) into nude mice by intra-cardiac injection followed by with or without nicotine treatment, the brain metastasis-free survival was decreased in nicotine treatment group. The number of CD206+ cells were also increased in the mouse brain after nicotine treatment. To test the effect of nicotine-driven microglia on tumor progression, H2030BrM cells were treated with conditioned medium (CM) generated from human microglia that were treated with or without nicotine. We found that the CM significantly increased the population of ESA+/CD44+ cancer stem cells and promoted their sphere forming abilities. The expression of SOX2 and Nanog were up-regulated when the H2030BrM cells were cultured with the CM of nicotine-treated microglia. Blocking nicotine-driven M2 microglial polarization by a STAT3 inhibitor suppressed the M2 polarization, resulting in inhibition of tumor growth. To examine the mechanism by which nicotine suppresses anti-tumor function of microglia, we measured the expression of "do not eat me" signal, SIRP on microglia and CD47 on tumor cells. H2030BrM and human microglial cells were treated with nicotine. We found that the mRNA and protein expression of SIRP were up-regulated on human microglia, while the expression of CD47 on H2030BrM were elevated by nicotine treatment. We used FACS to detect the phagocytic ability of microglial by co-culturing H2030BrM with microglial cells. We found that the anti-tumor phagocytic activities of microglia cells were significantly decreased upon nicotine treatment. In conclusion, our results suggest that nicotine promotes lung cancer brain metastasis by enhancing both tumor progression and stemness by skewing microglial polarization and inhibiting its anti-tumor phagocytic ability.

#1513

PI3Kγ inhibition activates T cell memory and relieves T cell exhaustion through the reprogramming of tumor-associated macrophages.

Hideyuki Takahashi, Paulina Pathria, Ann Shih, Ryan M. Shepard, Marc A. Paradise, Judith A. Varner. _Moores Cancer Center, University of California, San Diego, La Jolla, CA_.

We previously reported that macrophage PI3-kinase γ (PI3Kγ) controls a critical switch between immune stimulation and suppression during inflammation and cancer. PI3Kγ inhibition repolarizes tumor-associated macrophages, leading to downregulation of immune suppressive factors such as Arginase and IL10 and upregulation of IL12 and other pro-inflammatory cytokines. This results in recruitment and activation of intratumoral CD8+ T cells, as well as induction of immunological memory (Kaneda et al 2016). In the present study, we investigated the effect of PI3Kγ inhibition on T cell immune responses, including T cell memory induction and T cell exhaustion, in mouse models of head and neck squamous cell carcinomas (HNSCC). We found that Pik3cg−/− mice cleared implanted HPV+ HNSCC tumors; when re-challenged with tumor cells, these mice rapidly cleared secondary tumors and remained cancer-free. The proportion of T cells, especially CD8+ T cells, significantly increased in primary tumors from Pik3cg−/− mice. These CD8+ T cells expressed significantly more granzyme B and interferon and less T cell exhaustion markers than T cells from WT animals, indicating that PI3Kγ inhibition in macrophages results in T cell activation. We found that anti-tumor activity was transferable, as adoptive transfer of splenic T cells from Pik3cg−/− mice to naive WT mice suppressed tumor growth. Accordingly, Pik3cg−/− mice with primary or secondary tumors exhibited more splenic CD62L-CD44+ CD8+ effector memory T cells than WT mice. In summary, Pik3cg−/− mice exhibit a more activated T cell immune response and T cell memory than WT mice, resulting significant suppression of tumor growth. These results suggest that PI3Kγ-targeted therapy may activate durable T cell immune responses in patients with HNSCC.

#1514

Myeloid cell derived myeloperoxidase links cellular innate immunity to inhibition of NF-κB signaling in melanoma tumor cells.

Tracy W. Liu, Seth T. Gammon, Ping Yang, David T. Fuentes, David Piwnica-Worms. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Myeloid-derived cells are considered the primary mediators of the cellular innate immune system where myeloperoxidase (MPO) is the major enzyme present in these cells. MPO is essential for fighting infections but little is known about the role of MPO in cancer. Using the murine melanoma cell line, B16F10, in an immune-competent orthotopic tumor model, tumor growth was compared in wild type (MPO+/+) and syngeneic MPO-deficient host (MPO-/-) mice. Survival studies demonstrated that B16F10 tumors grew slower in MPO+/+ animals (mean survival 27.4 days ± 4.5 days) compared to MPO-/- animals (mean survival 23.4 days ± 1.6 days; p ≤ 0.005). Using a specific and potent inhibitor of MPO, 4-aminobenzoic acid hydrazide (4ABAH), we pharmacologically mimicked the MPO-/- phenotype by continuously dosing MPO+/+ animals. B16F10 tumors grew faster in MPO+/+ animals treated with 4ABAH (mean survival 23.8 days ± 2.5 days; p ≤ 0.017). Utilizing intravital imaging with skinfold window chamber animal models, we evaluated in real-time the recruitment of MPO-expressing myeloid-derived cells during melanoma progression in MPO+/+ and MPO-/- animals. B16F10 tumor NF-κB signaling, and MPO-mediated activation from immune infiltrates were imaged simultaneously using a multi-spectral, multi-modal imaging strategy. Because NF-κB signaling is a central coordinator of the immune system and cancer development, the dynamics of NF-κB signaling in B16F10 tumor in vivo were assessed using a transcriptionally-activated NF-κB-promoter-driven Firefly luciferase reporter enabling real-time bioluminescent imaging and quantitative monitoring of NF-κB transcriptional activation. Mean NF-κB transcriptional activation within the tumor compartment in MPO-/- mice was 42.2% ± 1.6% greater compared to MPO+/+ animals. Intravital microscopy demonstrated heterogeneous activation levels of NF-κB within tumor cells in MPO+/+ animals. To explore whether the spatial heterogeneity was a consequence of myeloid-derived cell distribution within the microenvironment, myeloid-derived cells were labeled in vivo using an i.v. injection of fluorophore-labeled dextran and fluorophore-labeled αGr1 antibody. Remarkably, tumor cells in contact with myeloid cells in vivo demonstrated decreased NF-κB transcriptional activation compared to tumor cells not in contact with myeloid cells. These in vivo studies demonstrate that MPO-expressing myeloid-derived cells suppress NF-κB transcriptional activation within B16F10 tumors in a tight spatially-localized and proximity-dependent manner where MPO, expressed broadly by myeloid-derived cells, contributes to host protection and decreased tumor progression. We demonstrate that MPO-expressing myeloid-derived cells function as an anti-tumor component of the cellular innate immune response during early melanoma progression in a NF-κB-dependent manner.

#1515

Removal of innate suppressors facilitates tumor-immune surveillance.

Yao Zhang,1 Christina Lee,1 Na Diao,2 Liwu Li1. 1 _Virginia Tech, Blacksburg, VA;_ 2 _Southern Medical University, Guangzhou, China_.

Although tumor immune environment is increasingly recognized to be highly important during the modulation of tumorigenesis and tumor regression, the role and regulation of innate leukocytes such as neutrophils during the modulation of tumor immune environment remain controversial and less defined. In this study, we tested the hypothesis that removal of innate signaling suppressors may boost the anti-tumor immune function of innate neutrophils in vitro and in vivo. To this regard, we examined two key innate signaling suppressors Tollip and IRAK-M and their roles in modulating neutrophil anti-tumor immune functions. We observed that selective deletion of Tollip enhanced tumor immune surveillance in the AOM-DSS chemically induced colon cancer model. Tollip deficiency released the neutrophil suppression on T cell proliferation and activation. The adoptive transfer of Tollip deficient neutrophils were sufficient to transfer enhanced tumor immune surveillance and reduce tumor burden. Likewise, the study of another innate suppressor IRAK-M revealed that IRAK-M expression was up-regulated in the human patients with colorectal cancer. We also demonstrated that IRAK-M deficient mice exhibited reduced tumor burden following AOM-DSS challenge. Together, our data reveal a novel anti-tumor immune-enhancement strategy through utilizing reprogrammed neutrophils with targeted removal of innate signaling suppressors.

#1516

**Building a translational pathway using human** **myeloid cell assays to enable development of cancer immune therapies.**

Louise S. Brackenbury, S Rhiannon Jenkinson, Robert D. Nunan, Neil A. Williams. _Charles River (Portishead), Bristol, United Kingdom_.

Myeloid-derived cells are a key immune population in the tumor microenvironment (TME) and as such represent a tractable therapeutic target. Myeloid precursors can differentiate into multiple cell types, one of which is macrophage (Mɸ). Mɸ exhibit multiple phenotypes, which are referred to as ranging from an inflammatory (M1) Mɸ to an anti-inflammatory (M2) Mɸ. In the TME a broader set of tumor-associated macrophage (TAM) can be found. TAM and myeloid-derived suppressor cells (MDSC) exhibit overlapping features and play a key role in promoting tumor growth and metastasis. We have developed a range of in vitro Mɸ assays to model the role of myeloid-derived cells in the TME. These assays provide an understanding of whether the suppressive mechanisms of this cell type can be overcome by immuno-modulators. Briefly, CD14+ monocytes were isolated from healthy donors and differentiated into TAM, with a range of characteristics, following exposure to human tumor cell supernatants, or to MDSC-like cells, following exposure to GM-CSF and IL-6. Monocytes were also differentiated into classical M1 or M2(a) or M2(b) phenotypes for comparison. The cell phenotype was assessed by flow cytometry and cytokine/chemokine production by using Luminex multiplexing. Functional ability was assessed by use of phagocytosis and antigen-presentation assays. The ability of MDSC to suppress T cell responses was also assessed. TAM exhibited a resting phenotype; CD25lo, CD127lo, CD184hi, CD80lo, CD163hi, CD68lo and MHCIIlo whereas M1 Mɸ were CD25hi, CD127+, CD80hi, CD163lo CD68hi and MHCIIhi. M2a Mɸ were CD25lo, CD184int, CD80lo, CD163lo, CD68lo and MHCIIint whereas M2b Mɸ expressed high levels of CD127, CD184 and CD80 and little CD163 and CD68. TAM produced low levels of IL-12p70 compared to Mɸ differentiated in the absence of TCM and raised IL-10 and VEGF. M1 Mɸ produced IL-6, IL-12p70, TNFα and IL-23 whereas M2a Mɸ produced little detectable cytokine. M2b produced IL-6, IL-10 and TNFα. Various Mɸ populations were also shown to express checkpoint inhibitors such as PD1 and Tim-3. Phagocytosis assays were performed using tumor targets in the presence or absence of anti-CD47. Enhanced uptake in the presence of anti-CD47 was seen with most Mɸ. Their ability to act as antigen presenting cells was also determined, examining downstream effects of modulating Mɸ activation. TAM and MDSC were tested for their ability to suppress T cells and the control therapeutics, sorafenib and sunitinib were tested. Mɸ were also screened for wider gene expression analysis following treatment with benchmark therapeutics using an nCounter® Myeloid Innate Immunity Panel (Nanostring). Our myeloid/macrophage-based assay platform can be used to test novel myeloid-targeted therapies, and help elucidate the MOA. This platform has potential for broad use in immune oncology screening programs and is likely to accelerate progress into the clinic.

#1517

Targeting M-MDSC intrinsic enhancer reprogramming enhances the efficacy of immune checkpoint therapy in hepatocellular carcinoma.

Man Liu, Jingying Zhou, Alfred Sze Lok Cheng. _The Chinese University of Hong Kong, Hong Kong_.

Fibrous liver promotes an immunosuppressive environment to facilitate hepatocellular carcinoma (HCC) initiation. However, the underlying mechanisms of liver fibrosis-elicited immune evasion are poorly elucidated. Myeloid derived suppressor cells (MDSCs) play a key role in immunosuppression, leading to low responsiveness of immune checkpoint blockade (ICB). Emerging evidence highlights the pivotal role of enhancer remodeling in cell identity establishment and tumor immunity. Therefore, we aimed to determine whether enhancer remodeling participates in MDSC generation and aggressive HCC development in fibrous liver, and further explore the potential of enhancer inhibition in targeting MDSCs and improving efficacy of ICB in fibrotic HCC treatment.

By a mouse hepatoma cell Hepa1-6 orthotopic fibrotic HCC mouse model, we found that monocytic MDSC (M-MDSC) subset was also dramatically expanded in murine fibrous liver and positively correlated with increased hepatic tumorigenicity. Hepatic stellate cell (HSC), as key profibrogenic stromal cell, stimulated immunosuppressive M-MDSC generation from human peripheral blood mononuclear cells (PBMCs). Mechanistically, activated HSCs initiated p38 MAPK-mediated enhancer remodeling and triggered monocyte-to-M-MDSC identity shifting. Enhancer inhibition by a clinically-trialed drug iBET-762 disrupted HSC-M-MDSC crosstalk and abrogated M-MDSC generation and immunosuppressive functions. Notably, combination of iBET-762 with anti-PD-L1 antibody significantly induced tumor-infiltrating T lymphocytes, leading to tumor eradication and prolonged host survival in fibrosis-associated HCC model. As we also showed profound suppression of HCC patient-derived M-MDSCs by enhancer inhibition, our results delineate an enhancer deregulation mechanism for M-MDSC generation in fibrous liver environment, highlighting a promising therapeutic strategy of combined enhancer and immune-checkpoint targeting for desmoplastic tumors.

This project is supported by Collaborative Research Fund C4017-14G and the Focused Innovations Scheme 1907309.

#1518

Gene expression profile and gender differences related with immune processes in solid tumors.

Jhajaira M. Araujo,1 Luis E. Raez,2 Christian D. Rolfo,3 Luis J. Schwarz,1 Ulises Infante-Huaytalla,4 Kevin J. Paez,4 Luis R. García,4 Hober Alvarado,4 Fany P. Ramos,4 Sheyla S. Delgado-Espinoza,4 Jhon B. Cardenas-Farfan,4 Joshuan J. Barboza-Meca,5 Jose Caballero-Alvarado,5 Joseph A. Pinto1. 1 _Oncosalud-AUNA, Lima, Peru;_ 2 _Memorial Cancer Institute/Memorial Health Care System, Florida International University (FIU), FL;_ 3 _University of Maryland Marlene and Stewart Greenebaum Comprehensive Cancer Center, MD;_ 4 _Universidad Nacional San Luis Gonzaga de Ica, Ica, Peru;_ 5 _Universidad Privada Antenor Orrego, Trujillo, Peru_.

Background: We have described before differences in gene expression profile (GEP) of immune gene-sets between genders in non-small cell lung cancers (NSCLC). On the other hand, there are reports mentioning the benefit of immune checkpoint inhibitors (ICI) could be influenced by gender, suggesting differences in immune processes (IP). We decided to evaluate if there are gender-specific differences in solid tumors including melanoma, gastric adenocarcinomas (GAC), head and neck cancer (HNC) and clear renal cell carcinoma (CCRCC).

Methods: GEO (https://www.ncbi.nlm.nih.gov/geo/) datasets were retrieved and analyzed for melanoma, GAC, HNC and CCRCC that contained GEP across Affymetrix and Illumina Microarrays of primary tumors. Datasets were manually verified and cases without gender information or non-tumoral tissue were excluded. Cell lines were not considered in our analysis. Data was pre-processed and gene-set enrichment analysis (GSEA) was conducted individually in the datasets GSE6791, GSE30784, GSE78060, GSE34105, GSE65858 (HNC); GSE73731, GSE36895, GSE11904, GSE40435 (CCRCC); GSE26899, GSE26901 (GAC) and GSE15605 (melanoma). Immune process (IP) grouped by gene ontology and identified with a p-value<0.05 and a FDR<25% were considered enriched.

Results: The number of IP enriched in women was higher than men in HNC (306 IP), GAC (297 IP) followed by melanoma (128 IP) and CCRCC (30 IP). Pathways significantly enriched in women in the four evaluated malignancies were: activation of immune response, activation of innate immune response, antigen receptor mediated signaling pathway, immune response regulating cell surface receptor signaling pathway, positive regulation of innate immune response, regulation of innate immune response and T cell receptor signaling pathway. In contrast to other cancers, only CCRCC showed enrichment of immune gene sets in males.

Conclusions: We find strong patterns of GEP of immune genes in tumors from female patients, suggesting gender-specific patterns of response to ICI. This pool of immune genes differentially expressed could be used as candidate biomarkers for prediction of response to immunotherapy.

#1519

The impact of ICOS+ Tregs on prognosis of gastric cancer patients.

Shinya Urakawa,1 Hisashi Wada,1 Masaki Mori,2 Yuichiro Doki1. 1 _Osaka University, Suita, Japan;_ 2 _Kyushu University, Japan_.

Background and Aim: Foxp3\+ CD4+ regulatory T cells (Tregs) have a suppressive role in antitumor immunity. Among Foxp3\+ CD4+ population, effector Tregs (eTregs) with highly suppressive functions are located in a subpopulation of Tregs. We have reported that ICOS has the potential as a novel marker for eTregs in gastric cancer (GC) and ICOS+ Tregs were abundant in H. pylori (HP) Ab-positive GC patients. In the present study, we investigate whether HP induce ICOS+ Tregs in GC.

Materials and Methods: Tumor-infiltrating lymphocytes (TILs), normal mucosa-infiltrating lymphocytes (NILs) and peripheral blood mononuclear cells (PBMCs) were obtained from 117 gastric cancer patients. Tregs, ICOS+ Tregs, linage\- CD11c\- CD123\+ HLA-DR\+ plasmacytoid dendritic cells (pDCs), TLR9+pDCs, ICOS-L+ pDCs were analyzed by flow cytometry and multicolor immunohistochemistry. HP Ab was analyzed by ELISA. For induction assay of ICOS+ Tregs, naïve CD4 T cells were cultured with pDCs under stimulation with TGF-β, CD3/CD28 mAbs and frozen-and-thawed HP (NCTC11637).

Results: In GC tissues, %ICOS-L in pDCs was positively correlated with %ICOS in Tregs (r2=0.52, p<0.001). %TLR9 in pDCs and %ICOS-L in pDCs were significantly higher in HP Ab-positive patients than others (p=0.001 and p=0.042, respectively). %ICOS\+ in Tregs was significantly higher in HP-Ab positive patients than others (p=0.020). By multicolor immunohistochemistry, a large number of ICOS/Foxp3 double positive cells were observed. In induction assay, addition of HP (NCTC11637) increased the expression of ICOS-L in pDC and induction of ICOS\+ Tregs, which were suppressed by TLR9 antagonist. %ICOS\+ in Tregs were significantly lower in HP-eradicated patients than others (p<0.001). Interestingly, %ICOS\+ in Tregs were also significantly lower in patients eradicated within four weeks before their surgical treatments (p<0.001).

Conclusion: Our results indicate the induction pathway from HP to ICOS+ Tregs mediated by TLR9 and ICOS-L in pDCs. Preoperative eradication for HP may have a potential as an indirect immune therapy for gastric cancer patients.

#1520

IL-6 up-regulates pd-l1 and facilitates lung cancer escape from NK cell immune function through its downstream MEK-ERK signaling.

Soo Ok Lee,1 Rongying Zhu,1 Xiang Xue,1 Donglai Cchen,2 Shanzhou Duan,2 Mingjing Shen,2 Feng Chen,1 Ying Tsai,1 Peter Keng,1 Yongbing Chen,2 Yuhchyau chen1. 1 _Univ. of Rochester Medical Ctr., Rochester, NY;_ 2 _Soochow Hospital, Suzhou, China_.

Background: We previously reported that IL-6 was associated with therapy resistance to radiotherapy and cisplatin chemotherapy, the two most common treatments for non-small cell lung cancer (NSCLC). Emerging in vivo evidence shows that natural killer (NK) cells have an important role in immune defense. In this study, we examined the role of IL-6 modulation in lung cancer PD-L1 expression and the impact on NK cell cytotoxicity. We also revealed the key molecular signaling pathway involved in this process, which serves as the potential therapeutic target to improve clinical lung cancer treatments.

Methods: We examined the prevalence of IL-6 in tumors of NSCLC in immunohistochemical staining. The susceptibility of IL-6 expressing scramble control (A549sc and H157sc) cells and IL-6 knocked down (A549siIL-6 and H157siIL-6) cells were investigated in in vitro and in vivo human tumor xenograft studies in mice. In in vivo studies, tumors were developed by luciferase tagged A549siIL-6 vs. A549sc cell injection, and primary human NK cells were injected intravenously at the early stage of tumor development. IL-6 regulation of PD-L1 at the transcriptional level was investigated and NK cell binding to tumor cells was also examined.

Results: We found the ubiquitous presence of IL-6 in NSCLC. We found IL-6 expressing lung cancer cells were more resistant to NK cell cytotoxicity than IL-6 knocked-down cells. In addition, we found higher expressions of programmed death receptor 1 ligand (PD-L1) in IL-6 expressing lung cancer cells than IL-6 knocked-down cells. In excised human tumor xenografts and in human NSCLC tumors, co-localization of IL-6 signaling and PD-L1 expression was observed. Furthermore, we discovered that IL-6 promoted PD-L1 expression at the transcriptional level via the regulation of its downstream MEK/Erk signaling pathway. In addition, we found that IL-6-MEK/Erk signaling also contributed to diverting NK cells away from binding to tumor cells.

Conclusions and Impact: In conclusion, we found that IL-6 develops the resistance to NK cell actions by inducing PD-L1 in NSCLC cells and diverts NK cells away from tumor cells via its downstream MEK/Erk signaling. NK cell-mediated immune function plays an important role in cancer surveillance, invasion, and metastasis, but its role in lung cancer is not clear. In in vivo studies, we proved the importance of NK cell-mediated immune reaction. Our discovery of revealing the IL-6 downstream signaling, MEK/Erk, in triggering the PD-L1 up-regulation and in blocking NK cell binding with lung cancer cells is novel. Data from this investigation support that blocking MEK/Erk signaling pathway has the potential to enhance NK cell immune function to NSCLC by overcoming IL-6 mediated NK cell resistance.

#1521

Tumor-produced hyaluronan contributes to the formation tolerogenic immunosuppressive microenvironment.

Paul R. Dominguez-Gutierrez, Paul Crispen, Sergei A. Kusmartsev. _Univ. of Florida College of Medicine, Gainesville, FL_.

Immunosuppressive ligand PD-L1 considered as a major player in mechanism of negative regulation of anti-tumor immune response and its expression is highly up-regulated in human and experimental tumors, including bladder cancer. Bladder cancer is characterized by aberrant hyaluronan metabolism resulting in increased HA production in tumor tissue. Hyaluronan or hyaluronic acid (HA) is a prominent component of tumor stroma/microenvironment. Membrane-bound or free extracellular HA favors tumor progression by inducing tumor cell motility, invasive properties, proliferation, production of growth factors and epithelial-mesenchymal transition. Here, we provide evidence that tumor-produced hyaluronan, one of the key tumor stroma components, contributes to the formation of tolerogenic/immunosuppressive tumor microenvironment and stimulated PD-L1 expression. We found that tumor-derived HA specifically binds to CD44 receptor expressed by myeloid precursors, such as myeloid-derived suppressor cells (MDSCs) and promotes its expansion with differentiation toward PD-L1-expressing F4/80+ macrophages. Inhibition of HA synthesis in tumor cells with pharmacologic inhibitor 4-MU, or blockade of CD44 signaling in myeloid cells with antagonistic anti-CD44 antibody, prevented the macrophage differentiation and tumor-induced up-regulation of PD-L1 expression. We also found that tumor-derived HA stimulated production of immunosuppressive and inflammatory factors IL-6, IL-10, TNF-alpha, IL-1beta and PGE2 by myeloid cells in CD44-dependent manner. Moreover, tumor-produced HA stimulated development of PD-L1-expressing macrophages in both murine and human myeloid cells. Our study reveals that tumors may evade the immune system by creating a protective tolerogenic "shield" in the form of tumor-produced HA, which binds to the CD44-expressing tumor-recruited MDSCs, stimulating production of immunosuppressive factors and promoting development of the PD-L1+ macrophages. Inhibiting of hyaluronan synthesis by tumors or targeting HA-CD44 signaling could provide an attractive approach to break tumor-induced immune tolerance and unleash the anti-tumor immune response.

#1522

**NovelNOD-** scid IL2rgnull **(NSG)mice for preclinical evaluation of TLR agonists in cancer immunotherapy.**

Ken-Edwin Aryee,1 Lisa Burzenski,2 Dale Greiner,1 Giles F. Whalen,1 Leonard Shultz,2 James Keck,2 Michael Brehm1. 1 _Univ. of Massachusetts, Worcester, MA;_ 2 _Jackson Laboratory, Bar Harbor, ME_.

TLR agonists that induce inflammation have been used since the 18thcentury for the treatment of cancer. The inflammation induced by TLR agonists is thought to stimulate tumor-specific immunity in patients and augment control of tumor burden. Although only two forms of TLR agonists are currently FDA approved for cancer treatment, Bacillus Calmette-Guerin and monophosphoryl lipid A, several clinical trials are ongoing with new TLR agonists, including trials targeting TLR3 and TLR4 pathways. Currently there is a paucity of preclinical models to evaluate the efficacy of TLR agonists in activating human immune responses and to assess the impact on tumor growth.Humanized mice are emerging as an exciting translationalplatform to study human immuno-oncology and provide tools to test new immunotherapies. Our laboratory uses NSG mice that have been humanized with hematopoietic stem cells (HSC) to study human immunity and to evaluate therapeutics. While NSG mice lack murine adaptive immunity (T and B cells), these mice maintain a residual innate immune system with the potential to respond to TLR agonists. Challenge of unengrafted NSG mice with either LPS or poly(I:C) stimulates production of mouse cytokines and maturation of murine innate immune cells. Moreover, LPS or poly(I:C) challenge of NSG bearing a PDX melanoma significantly delays tumor growth kinetics in the absence of an engrafted human immune system. Thus differentiating between human and mouse responses to TLR agonist is difficult in currently available NSG mice. To address this issue, we have created NSG mice lacking TLR4 and Type 1 IFNR1 that fail to respond to LPS and poly(I:C), respectively. Challenge of unengrafted NSG-TLR4nulland NSG-IFNR1nullmice bearing a PDX melanoma with LPS and poly(I:C), respectively, had minimal impact on tumor growth kinetics, confirming the utility of these novel NSG strains to study specifically human immune responses to TLR agonists. To validate the NSG-TLR4nullmouse, we humanized these mice with HSC and evaluated human immune system development and function as compared to HSC-engrafted NSG mice. Our results show that human immune system development in HSC-engrafted NSG-TLR4nullmice is comparable to HSC-engrafted NSG mice with similar levels of human CD45+ cells, CD3+ T cells, CD20+ B cells, and CD33+ myeloid cells detectable in blood and spleen. Moreover, both HSC-engrafted NSG-TLR4nulland NSG mice show similar activation profiles of human innate immune cells and human cytokine production following challenge with LPS. Lastly, LPS challenge of HSC-engrafted NSG-TLR4nullmice bearing a PDX melanoma significantly reduced tumor growth kinetics. These findings demonstrate that NSG-TLR4nullmice are an effective tool to test TLR4 agonists for the ability to activate human immunity and to assess impacts on tumor growth in the absence of the confounding effects of the murine innate immune system.

#1523

Inhibition of SUMOylation by TAK-981 induces antitumor innate immune responses by modulating macrophage and NK cell function through Type I IFN pathway activation.

Akito Nakamura, Stephen Grossman, Keli Song, Neeraja Idamakanti, Gary Shapiro, Dennis Huszar. _Takeda Pharmaceuticals International, Co., MA_.

TAK-981 is a novel and selective small molecule inhibitor of the small ubiquitin-like modifier (SUMO) ylation enzymatic cascade in Phase 1 clinical trials. SUMOylation is a reversible post-translational modification that regulates protein function by covalent attachment of a SUMO protein to protein substrates. We have found that in vivo inhibition with TAK-981 promotes an antitumor immune response characterized by induction of a Type I interferon (IFN) response in immune cells. Interestingly, others have also reported a role for SUMOylation in restraining Type I IFN responses, upon exposure to pathogenic stimuli, using genetic approaches to inhibit SUMOylation. In this study, we demonstrate that inhibition of SUMOylation with TAK-981 enhances antitumor innate immunity by modulating macrophage and NK cell function. In vitro studies revealed that TAK-981 treatment increased expression of pro-inflammatory markers and prevented expression of anti-inflammatory markers on macrophages. TAK-981 treatment of macrophages further resulted in potent phagocytic activity against cancer cells. TAK-981 also induced expression of activation markers on NK cells and enhanced cell killing activity against cancer cells. Enhancement of macrophage phagocytosis and NK cell cytotoxicity by TAK-981 were shown to be dependent on Type I IFN receptor signaling, and both activities against CD20-positive cancer cells were further enhanced in the presence of the anti-CD20 therapeutic monoclonal antibody rituximab. Evaluation of combination activity in vivo in CD20-positive lymphoma xenograft models demonstrated synergistic antitumor activity in multiple models when TAK-981 was combined with rituximab. Inhibiting SUMOylation with TAK-981 appears to represent a novel and mechanistically unique means to the end of leveraging the potential of Type I IFNs to promote antitumor immune responses.

#1524

CD137 (4-1BB) engagement fine-tunes the synergistic IL-15- and IL-21-driven NK cell proliferation.

Laurent Vidard,1 Christine Dureuil-Sizaire,2 Jérémy Baudhuin,1 Lionel Vescovi,1 Laurence Durand,1 Véronique Sierra,1 Eric Parmantier1. 1 _Sanofi, Vitry sur Seine, France;_ 2 _Sanofi, Vitry-sur-Seine, France_.

To understand and dissect the mechanisms driving NK cell proliferation, we took advantage of the methodology used in cell therapy to numerically expand NK cells in the presence of K562-derived artificial Antigen Presenting Cells (aAPCs) and cytokines. For 9 consecutive weeks, high expression of CD137L by a K562-derived aAPC cell line was able to sustain NK cell expansion by 100 million-fold whereas low expression of CD137L by the parental K562 cell line supported the expansion by only 40,000-fold. The level of expression of CD137L, however, did not modulate the sensitivity of the K562 cell line to the intrinsic cytotoxicity of NK cells. Similarly, the low NK cell proliferation in the presence of the parental K562 cell line and cytokines was increased by adding agonistic anti-CD137 antibodies to levels similar to CD137L-expressing K562-derived aAPCs. Finally, the synergy between IL-15 and IL-21 was observed only upon CD137 engagement and the presence of aAPCs. Therefore, we conclude that NK cell proliferation requires cell-to-cell contact, the activation of CD137 axis, the presence of IL-15 (or its membranous form) and IL-21. By analogy with the three signals model required to activate T cells, we speculate, that the cell-to-cell contact represents "signal 1", CD137 would be "signal 2" and cytokines would be "signal 3". The precise nature of signal 1 remains to be defined.

#1525

Kaiso influences immune signaling of breast cancer exosomes.

Md Shakir U. Ahmed,1 Brittany Jenkins,2 Benjamin Adu-Addai,1 Balasubramanyam Karanam,1 Melissa B. Davis,2 William E. Grizzle,3 Honghe Wang,1 Clayton C. Yates1. 1 _Tuskegee University, Tuskegee, AL;_ 2 _Henry Ford Health System, Detroit, MI;_ 3 _Universityof Alabama at Birmingham, Birmingham, AL_.

Introduction: Exosomes are communication vesicles between tumor cells and immune cells. However, the mechanism underlining this cell-cell communication is not well understanding, particularly in African American (AA) breast cancer patients. Kaiso, a bi-modal transcription factor is highly expressed in AA patients and high Kaiso expression correlates with aggressiveness and the disparity in survival outcomes compared to European American (EA) patients. However, the biological consequences of Kaiso in immune signaling of breast cancer exosomes has not been studied. Herein we demonstrate the biological role of Kaiso in immune signaling in breast cancer exosomes.

Methods: We utilized Nanostring immune profiling technology along with multiple in vitro and in vivo assays to study the role of Kaiso in breast cancer immune escape.

Results: Nanostring pan cancer immune profiling showed that EA breast cancer exosomes exhibited higher expression of TILs markers, T cell activation markers and CD8+T Cells markers compared to AA, while we observed an increase in the expression of anti-phagocytic molecule CD47 in breast cancer patient exosomes of AA compared to EA. In addition to that CD47 and SIRP-α (Signal Regulatory Protein) are highly expressed in Kaiso-scrambled MDA-MB-231 cells (sh-SCR) and exosomes, whereas THBS1, which is a regulator of CD47 expression and is regarded as angiogenesis inhibitor is significantly increased in sh-Kaiso MDA-231 cells and exosomes. Additionally, we observed that Kaiso directly binds methylated sequences in the promoter region of CD47 and THBS1 by ChIP assay. Furthermore, in vivo sh-Kaiso cells injected into athymic mice exhibited delayed tumor formation after four weeks with smaller tumor size as compared to sh-SCR cells, and we observed higher expression of THBS1 with lower expression of CD47 and SIRP-α molecules by IHC and exosomes isolated from invivo tumors, indicating that Kaiso is associated with macrophage mediated immune escape.

Conclusion: Our findings demonstrate the role of kaiso in immune signaling through exosomes which may be related with more aggressive cancer phenotype in breast cancer specially in African Americans.

#1526

Colitis promotes intrahepatic cholangiocarcinoma via gut microbiome dependent, CXCL1/CXCR2 mediated MDSC accumulation.

Qianfei Zhang, Chi Ma, Umberto Rosato, Bernd Heinrich, Qiong Fu,Laurence Diggs, Tim Greten. _NCI/NIH, Bethesda, MD_.

Inflammatory bowel disease (IBD) is a known risk factor for cholangiocarcinoma. Gut microbiome dysregulation is closely associate with IBD, however, its role in IBD-promoted cholangiocarcinoma is unknown. Here, we established a preclinical mouse model recapitulating the tumor-promoting effect of IBD on cholangiocarcinoma. Increased translocation of gut bacteria to liver is observed in IBD. Oral antibiotic-mediated bacterial depleting shows that neomycin- but not vancomycin-sensitive bacteria mediate the IBD-accelerated tumor progression without affecting IBD disease status. Gut microbiome regulates anti-tumor immunity. A robust increase of hepatic CXCR2+ granulocytic myeloid-derived suppressor cells (G-MDSC) was observed, which is caused by IBD and can be blocked by oral neomycin treatment, but not dependent on tumor-bearing condition, gender or mouse strain. Depleting the increased hepatic G-MDSC abolished the IBD-enhanced tumor progression. The liver accumulation of CXCR2+ G-MDSC was regulated by CXCL1 expression of hepatocytes, which was controlled by gut microbiome-mediated LPS/TLR4 axis. The increase of liver granulocytes can also be found in patients with active IBD. Our study provides novel knowledge that bacterial dysregulation in IBD fosters a tumor-promoting environment in liver and contributes to cholangiocarcinoma disease progression.

#1527

Intraperitoneal myeloid-derived suppressor cells in peritoneal dissemination mouse model.

Yutaka Sugita,1 Kimihiro Yamashita,1 Rishu Takimoto,2 Tomoko Tanaka,3 Akira Arimoto,1 Eiji Fukuoka,1 Tetsu Nakamura,1 Satoshi Suzuki,1 Yoshihiro Kakeji1. 1 _Kobe University Graduate School of Medicine, Kobe, Japan;_ 2 _Sapporo Medical University School of Medicine, Sapporo, Japan;_ 3 _Osaka saiseikai nakatsu hospital, Osaka, Japan_.

Background: Myeroid-derived suppressor cells (MDSCs) are heterogeneous cell populations derived from bone marrow, and two subsets of M-MDSC and PMN-MDSC are known in mice. They accumulate in the tumor-bearing host, and suppresse cell-mediated immunity mainly on T cells, and inhibit anti-tumor immune responses. We have previously reported that (1) the increase of MDSCs in lung, peripheral blood, and spleen reflects the cancer progression, (2) MDSCs in peripheral blood would be useful as a marker of recurrence as it increases in recurrent cases after surgical resection. On the other hand, no detailed report on the dynamics of intraperitoneal MDSCs has been made. In this study, we made peritoneal dissemination mouse models and monitored intraperitoneal MDSCs, and investigated whether MDSCs are involved in the progression of peritoneal dissemination.

Methods: MC38 colon cancer cell line was intraperitoneally injected to C57BL / 6J mice and the change in the number and ratio of immune cells (MDSC, Macrophage, T-cell, B-cell) in the spleen, peripheral blood and peritoneal cavity was monitored over time. We examined the correlation between transition of immune cells and cancer progression.

Results: MDSCs increased with cancer progression in all spleen, peripheral blood, and intraperitoneal cavity. The proportion of PMN-MDSCs in MDSCs increased in peripheral blood. In contrast, the proportion of M-MDSCs increased initially, then the PMN-MDSCs increased in the abdominal cavity.

Conclusion: Intraperitoneal MDSCs were thought to reflect the progression of peritoneal dissemination as well as spleen and peripheral blood. The proportion of M-MDSCs reflects the disease state in the microperitoneal dissemination with serous ascitic fluid, and ascites became bloody and the proportion of PMN-MDSCs increased with the progression of seeding. We are examining treatment for peritoneal dissemination with this feature in mind.

#1528

Characterization of the tumor microenvironment of key suppressive myeloid populations in 11 commonly used preclinical syngeneic cancer models.

Lars Ringgaard,1 Esben Christensen,1 Lotte K. Kristensen,2 Camilla Stavnsbjerg,1 Andreas Kjaer,3 Anders E. Hansen,1 Thomas L. Andresen1. 1 _Technical University of Denmark, Kongens Lyngby, Denmark;_ 2 _Minerva Imaging, Copenhagen, Denmark;_ 3 _Rigshospitalet and University of Copenhagen, Copenhagen, Denmark_.

The tumor microenvironment (TME) composition is a critical parameter for therapeutic outcome in cancer patients. Understanding the TME composition and influence on anti-cancer therapies is a valuable tool for developing new treatments. High TME infiltration of activated tumor specific cytotoxic T-cells (cT) and central memory T-cells is crucial for improved prognosis. Conversely, interpreting infiltration of innate myeloid populations is more complex due to their plasticity and overlapping phenotypes. Myeloid derived suppressor cells (MDSC) potently inhibit cT function and proliferation in the tumor. Especially monocytic MDSCs (Mo-MDSCs) are associated with poor prognosis due to high secretion of arginase, suppressive cytokine production, and ROS-induction. Patrolling monocytes (pMos) have recently gained interest due to their regulatory function and high phagocytic capacity. Both Mo-MDSCs and pMos are progenitors to tumor-associated macrophages (TAMs), which either activate or suppress cT function. Myeloid subsets are the most prevalent infiltrating populations in both human and murine tumors and additional research is needed to provide rational selection when choosing preclinical models. In the present study, we characterized the infiltration of key myeloid subsets and activation markers in 11 commonly used syngeneic tumor models with subcutaneous tumors between 100-500 mm3. Populations of interest included; polymorphonuclear-MDSCs (PMN-MDSCs/G-MDSCs), Mo-MDSCs, TAMs, pMos, and classical dendritic cells type 1. Furthermore, we characterized key activation/regulatory markers CD80, CD86, CD163, CD206, MHCII, arginase-1, PD1, and PD-L1 to elucidate how the expression of these varies across cancer models and populations. The presented work provides a valuable tool to researcher across all categories of anti-cancer therapies by providing optimal grounds for model selection in the preclinical setting.

Cell line | Origin | Dominant infiltrating immune population (% of viable cells)

---|---|---

ASB-XIV | Pulmonary squamous cell carcinoma | cT (5.3%)

CT26 | Colorectal carcinoma | pMo (7.1%)

A20 | B cell lymphoma | Mo-MDSC (3.7%)

MC38 | Colorectal adenocarcinoma | TAM (31.4%)

EG-7.OVA | Thymoma | Mo-MDSC (7.4%)

SA1N | Fibrosacroma | pMo (17.6%)

B16F10 | Melanoma | Mo-MDSC (3.1%)

LL/2 | lung carcinoma | Mo-MDSC (17.9%)

RENCA | Renal adenocarcinoma | pMo (9.2%)

4T1 | Ductal breast carcinoma | PMN-MDSC (17%)

J558 | Myeloma | Very low immune infiltration

#1529

STAT3 mediates epigenetic regulation of MDSCs in tumors.

Brian J. Christmas, Christine I. Rafie, Elizabeth M. Jaffee, Evanthia T. Roussos Torres. _Johns Hopkins University School of Medicine, Baltimore, MD_.

This project aims to test the hypothesis that the epigenetic modulatory drug (EMD), entinostat, is capable of inhibiting the immunosuppressive capabilities of MDSCs by altering STAT3 activity. Our previous work has shown that the HDACi entinostat is capable of sensitizing the inflammatory environment in both pancreatic ductal adenocarcinoma (PDAC) and HER2+ breast cancer mouse models to the immune checkpoint inhibitors anti-PD1 and anti-CTLA4 by inhibiting the immunosuppressive function of intratumoral MDSCs. These MDSCs not only produced significantly less arginase-1 protein, but also demonstrated reduced suppression of T cell proliferation when co-cultured with activated CD8+ T cells. The MDSC dysfunction induced by these combinatorial therapies resulted in significantly improved survival in both models as well as significantly improved infiltration of cytotoxic effector CD8+ T cells. Western blot analysis of isolated intratumoral G-MDSCs from treated animals showed a decrease in levels of phospho-STAT3 (pSTAT3), an important regulator of MDSC immunosuppressive genes. We hypothesize that the reduced levels of pSTAT3 alters the promoter binding of STAT3, thus preventing the translation of genes key to the immunosuppressive activity of MDSCs. To test this hypothesis, we are conducting mechanistic studies in ex vivo and in vitro MDSC models. Our ex vivo system involves isolating intratumoral MDSCs and subsequently culturing the MDSCs using tumor conditioned media. Our in vitro model uses the previously described "MDSC-like" J774M cell line, which was derived by sorting CD11b+ Gr1+ cells from the ATCC cell line J774A. We have shown that this cell line expresses the surface markers Ly6G and Ly6C in a similar manner to what is observed in intratumoral MDSCs. We have also performed immunosuppression assays and determined this cell line is capable of inhibiting T cell proliferation in a dose dependent manner. In addition to establishing and characterizing these models, our work has demonstrated that entinostat treatment results in a significant decrease in the production of functional arginase-1 protein in both ex vivo G-MDSCs and J774M cells. Western blot analysis of the J774M cells showed that this decrease in arginase-1 production correlated with a decrease in pSTAT3. Our current studies aim to confirm a decrease in pSTAT3 in ex vivo MDSCs as well as determine the effect of entinostat on the ability of J774M cells to suppress T cell proliferation. Additionally, we are performing ChIP-Seq on entinostat treated and untreated ex vivo MDSCs and J774M cells to determine how entinostat alters the binding of STAT3 to the promoters of key immunosuppressive genes. These results will not only help elucidate the molecular mechanism of action involved in entinostat's reprogramming of MDSCs, but it will also help identify new, more specific targets to manipulate MDSC function, and improve efficacy of checkpoint inhibition in non-immunogenic cancers.

#1530

Polymorphonuclear myeloid derived suppressor cells secrete IL6 to promote colon tumorigenesis in an inflammatory vicious cycle.

Mohammed L. Ibrahim, John D. Klement, Priscilla S. Redd, Chunwan Lu, Kebin Liu. _Georgia Cancer Center, Augusta University, Augusta, GA_.

It is widely accepted now that chronic inflammation can create a favorable environment for neoplastic transformation and tumor progression. Tumor Microenvironment consists of various innate and adaptive immune cells, which secrete numerous inflammatory mediating and immunoregulatory cytokines. Among all immune cells infiltrated to the tumor site, myeloid cells have been considered as major players in the tumor associated inflammation. Recently, a subpopulation named myeloid-derived suppressor cells (MDSCs) have gain a great attention as a potential link between inflammation and tumor progression. MDSCs are heterogeneous population of immature myeloid cells that is subdivided into monocytic (M-MDSC) and polymorphonuclear (PMN-MDSC) subsets depending on different markers expressed differentially on their surface. Both PMN-MDSC and M-MDSC have been shown to be immune-suppressive and to exert an immunoregulatory role in the tumor microenvironment. However, a clear distinction in their functional and molecular properties has remained a matter of debate. Here we report that PMN-MDSC but not M-MDSC is the major secreting immune subset of the proinflammatory and tumor-promoting cytokine Interleukin 6 (IL6) in colon cancer. IL6 has been shown in literature to function as a tumor promoter through enhancing tumor growth, invasion and chemoresistance. We observed that IL6 upregulates DNMT1 and DNMT3b in colon carcinoma cells to boost their growth and survival in a STAT3 dependent mechanism. We also observed that tumor-expressed IL6 acts as a key modulator of the immune profile in colon tumor microenvironment. We generated an IL6-overexpressing colon carcinoma cell lines. We observed that IL6-overexpressing tumor triggers a significant increment in PMN-MDSCs but not M-MDSCs infiltration and accumulation in mouse colon carcinoma xenograft model. This remarkable PMN-MDSC tumor enrichment is associated with less effector T lymphocytes infiltration and more progressive and proliferative tumor. Moreover, DSS-induced colitis murine model is characterized with higher infiltration of PMN-MDSC but not M-MDSC, which secrete significant amount of IL6. Therefore, inflammation-derived IL6 induces neoplastic transformation of colonic epithelial cells through upregulation of DNMT1 and DNMT3b. Collectively; our data indicate that PMN-MDSC is the primary IL6 producer among all tumor-infiltrated leukocytes. PMN-MDSC-derived IL6 has a dual tumor favoring function through nurturing colon tumor cells in DNA-methylation dependent mechanism, and raising an immunosuppressive context enriched with more PMN-MDSCs, which secrete more IL6 in an endless vicious cycle. Thus, targeting this circuit, may have a promising implication in colon cancer immunotherapy.

#1531

The Polycomb repressor complex 1 promotes recruitment of myeloid-derived suppressor cells and immune evasion during bone colonization in castration-resistant prostate cancer.

Wenjing Su,1 Boyu Zhang,2 Hyunho Han,2 Goutam Chakraborty,3 Bing Zhou,4 Jie Su,1 Guangli Yang,1 Ouathek Ouerfelli,1 Neal Rosen,1 Howard I. Scher,5 Filippo G. Giancotti2. 1 _Sloan Kettering Institute for Cancer Research, Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _The University of Texas M.D. Anderson Cancer Center, Houston, TX;_ 3 _Memorial Hospital, Memorial Sloan Kettering Cancer Center, Houston, TX;_ 4 _The University of California, San Diego, La Jolla, CA;_ 5 _Memorial Sloan Kettering Cancer Center; Weill Cornell Medical College, New York, NY_.

The mechanisms that enable immune evasion at metastatic sites are poorly understood. We show that the Polycomb Repressor Complex-1 (PRC1) drives colonization of the bones and visceral organs in Double Negative (AR- NE-) Prostate Cancer (DNPC). In vivo genetic screening identifies the cytokine CCL2 as the top pro-metastatic gene induced by PRC1. Mechanistic studies show that CCL2 not only controls self-renewal and metastatic initiation but also governs the recruitment of M2-like TAMs and Tregs, thus creating a profoundly immune suppressive and proangiogenic microenvironment. Inhibition of PRC1 reverses these processes and cooperates with checkpoint immunotherapy to suppress multi-organ site metastasis. These results reveal a link between epigenetic regulation of cancer stem cells and molding of the tumor microenvironment and identify PRC1 as a potential therapeutic target in M-CRPC.

### Therapeutic Antibodies 2

#1532

**A novel, monovalent tri-specific antibody-based molecule that simultaneously modulates PD-L1 and 4-1BB exhibits potent anti-tumoral activity** in vivo **.**

Tea Gunde,1 Matthias Brock,1 Stefan Warmuth,1 Alexandre Simonin,1 Christian Hess,1 Eva Oswald,2 Julia Tietz,1 Julia Zeberer,1 Dana Mahler,1 Simone Muntwiler,1 Benjamin Küttner,1 Belinda Wickihalder,1 Antonia Pölderl,1 Dania Diem,1 Teddy Beltrametti,1 Robin Heiz,1 Sebastian Meyer,1 Timothy Egan,3 David Urech4. 1 _Numab Innovation AG, Wädenswil, Switzerland;_ 2 _Charles River Discovery Service Germany GmbH, Freiburg, Germany;_ 3 _Numab US, LLC, Chevy Chase, MD;_ 4 _Numab Therapeutics AG, Pfäffikon, Switzerland_.

The combined immunomodulation of PD-L1/PD-1 and 4-1BB is considered a promising strategy to increase response rates among cancer patients who are eligible to receive PD-L1/PD-1 inhibitors. Unfortunately, encouraging pre-clinical results achieved with such regimens have not yet translated into durable clinical success, due to addition of 4-1BB-agonistic antibodies being either intolerable at effective doses or ineffective, despite tolerability, at all doses. To eliminate this safety/efficacy tradeoff, we engineered a novel, tri-specific immunomodulatory drug candidate. The molecule consists of three monovalent antibody Fvs - specific for PD-L1, serum albumin (SA) and 4-1BB - fused in a single chain (a PD-L1/4-1BB/SA tri-specific scDb-scFv). The monovalent and Fc-less structure of the molecule ensures that 4-1BB agonism is conditional upon drug-mediated formation of an immunological synapse between PD-L1+ cells and 4-1BB+ cells, thereby restricting costimulation of 4-1BB+ cells to the tumor microenvironment (TME). Therefore, the scDb-scFv molecule avoids the extratumoral costimulation of immune cells that is believed to cause the dose-limiting toxicities that arise from therapeutic 4-1BB agonism. Meanwhile, the αSA domain extends the molecule's serum half-life and is expected to promote delivery to the TME. In the present study, we demonstrate that a novel PD-L1/4-1BB/SA tri-specific scDb-scFv potently blocks PD-L1/PD-1 signaling and elicits T cell costimulation solely in the presence of PD-L1+ cells. In in vitro experiments, the scDb-scFv molecule exhibits a greater capacity to costimulate T cells than combinations of clinical stage α4-1BB and αPD-L1/PD-1 IgGs. Moreover, in contrast to ADCC-enabled αPD-L1 IgGs, the Fc-less scDb-scFv spared CD11c+ monocytes from depletion. We also demonstrate that the carefully balanced relative affinity between the molecule's component αPD-L1 and α4-1BB Fvs maximizes its pharmacological activity and avoids bell-shaped dose-response curves. Next, in vivo efficacy was demonstrated in two xenograft models - HCC827 NCSLC and HCC1954 breast carcinoma - using humanized mice. While equally effective at slowing tumor progression in vivo, the PD-L1/4-1BB/SA tri-specific scDb-scFv was better tolerated, and was more pro-proliferative vis-à-vis intratumoral CD8+ T cells, than combined αPD-L1 and α4-1BB IgGs. In PK/PD studies in cynomologus monkeys, we confirmed the tolerability, pharmacological activity and extended serum half-life of the scDb-scFv molecule. Finally, the molecule was produced from stable CHO cells at high titers (comparable to IgGs) and exhibits outstanding stability characteristics. In conclusion, our data confirm the successful development of a novel therapeutic that is designed to unlock the full potential of combined immunomodulation and overcome its present limitations.

#1533

IMGC936, a first-in-class ADAM9-targeting antibody-drug conjugate, demonstrates promising anti-tumor activity.

Stuart Hicks,1 Deryk Loo,2 Kerstin Sinkevicius,1 Juniper Scribner,2 Bhaswati Barat,3 Nicholas Yoder,1 Christopher Espelin,1 Marian Themeles,1 Francine Chen,2 Jacquelynn Lucas,1 Jennifer Brown,3 Bahar Matin,1 Megan Fuller,1 Jenny Lee,1 Paulin Salomon,1 Juliet Costoplus,1 Sadiqa Yancey,1 Gundo Diedrich,3 Sergey Gorlatov,3 Thomas Son,2 Michael Chiechi,2 Pam Li,2 Michael Spliedt,3 Valentina Ciccarone,3 Jeff Hooley,2 Nadia Gantt,3 James Tamura,3 Kerry Donahue,1 Paul Moore,3 Syd Johnson,3 Thomas Chittenden,1 Richard Gregory,1 Ezio Bonvini3. 1 _Immunogen, Inc., Waltham, MA;_ 2 _MacroGenics, Brisbane, CA;_ 3 _MacroGenics, Rockville, MD_.

Dysregulation of ADAM9, a member of the ADAM (a disintegrin and metalloproteinase) family of proteases, has been implicated in tumor progression and metastasis, as well as pathological neovascularization. ADAM9 overexpression correlates with poor prognosis in multiple cancers. We have shown that ADAM9 is overexpressed in multiple solid tumor indications and that anti-ADAM9 antibodies are efficiently internalized and degraded by tumor cell lines making ADAM9 an attractive target for antibody-drug conjugate (ADC) development. Here, we describe IMGC936, the first ADAM9-targeting ADC to enter preclinical development. IMGC936 is comprised of a high-affinity humanized antibody site-specifically conjugated to DM21, a next-generation linker-payload that combines a maytansinoid microtubule-disrupting payload with a stable peptide linker at a drug-antibody ratio of two. To maximize the potential for IMGC936 activity, the M252Y/S254T/T256E (YTE) mutation was introduced into the CH2 domain of the antibody to increase in vivo plasma half-life and exposure. In vitro studies demonstrated targeted cytotoxicity of IMGC936 across a panel of ADAM9-positve tumor cell lines with activity at least 2 logs greater than a non-targeting conjugate. Consistent with the in vitro activity, an anti-ADAM9-DM21 conjugate displayed compelling anti-tumor activity in multiple xenograft models representing non-small cell lung, gastric and colorectal cancers. For example, in the EBC-1 non-small cell lung cancer subcutaneous xenograft model with only moderate ADAM9 expression (H-score of 130), anti-ADAM9-DM21 not only induced tumor growth delay but produced complete and durable remissions in 6/6 mice following a single intravenous dose of 8.6 mg Ab/kg (100 ug DM21/kg). IMGC936 demonstrated a favorable pharmacokinetic profile with good conjugate stability in non-human primates. Importantly, IMGC936 was well-tolerated following repeat dosing in cynomolgus monkeys with no ADAM9 target-related toxicities identified at doses exceeding the levels required for anti-tumor activity in murine xenograft models. Based on the totality of the preclinical data, IMGC936 represents a promising therapeutic candidate to target a wide range of ADAM9-expressing tumors.

#1534

MEDI5083, a novel CD40L-Fc fusion protein, activates the CD40 pathway on antigen presenting cells and promotes a robust anti-tumor immune response in a B16F10 murine tumor model.

Sean Turman, Kelly McGlinchey, YaYa Wang, Deepali Malhotra, Ronald Herbst, Yue Wang. _Medimmune, Gaithersburg, MD_.

Targeting CD40 with its natural ligand (CD40L) enhances the immune-stimulatory functions of dendritic cells, B cells, monocytes and macrophages, resulting in T cell priming, expansion and differentiation. Thus, activation of the CD40 pathway is an attractive strategy to boost anti-tumor immune responses. MEDI5083 is a novel fusion protein consisting of a hexameric recombinant human CD40L structure covalently linked to human IgG4p Fc. In this study, we present the functional characterization of MEDI5083. MEDI5083 specifically bound to human CD40 and triggered surface CD40 internalization. As a result, MEDI5083 exerted robust CD40 agonist activities as measured by NF-κb activation in Ramos and THP1 cell lines. In assays using primary human B cells and myeloid cells, MEDI5083 potently upregulated co-stimulation molecules including MHCII, CD80 and CD86, and induced secretion of pro-inflammatory cytokines, such as TNFα and IL6. To examine the anti-tumor activity in mice, we constructed a murine surrogate of MEDI5083 (mCD40L-Fc) which has similar physical and target binding properties to those of MEDI5083. In the B16F10 syngeneic mouse model, repeated dosing of mCD40L-Fc significantly decreased tumor volume and/or delayed tumor growth, compared with isotype control dosed animals. Combination treatment of mCD40L-Fc with anti-PD-L1 and/or anti-CTLA-4 further enhanced the anti-tumor immune response. Moreover, treatment with mCD40L-Fc alone or in combination with anti-PD-L1 or anti-CTLA-4 induced elevated levels of TH1 cytokines IFNγ and IL12 in serum, and increased numbers of intratumoral CD8+ T cells, compared with isotype control treated animals. Thus, mCD40L-Fc elicits robust immune activation and significant anti-tumor activity against B16F10 tumors, which have low responsiveness to checkpoint inhibitors. The insights obtained from the preclinical studies support the development of MEDI5083, which is currently in clinical development for a broad spectrum of malignancies.

#1535

CBA-1205, a novel glycoengineered humanized antibody targeting DLK-1 exhibits potent anti-tumor activity in DLK-1 expressing tumor xenograft models.

Koji Nakamura, Kota Takahashi, Izumi Sakaguchi, Zhang Lingyi, Hiroyuki Yanai, Toru Kanke. _Chiome Bioscience, Inc., Kanagawa, Japan_.

Delta-like 1 homolog (DLK-1) is a type I transmembrane protein with 6 tandem EGF-like motifs in extracellular region. In fetal development, DLK-1 is expressed at high levels in undifferentiated, highly proliferative tissues, such as, liver, kidney, skeletal muscle and brain. In the fetal liver, it has reported that DLK-1 positive hepatoblasts have a differentiation capability toward hepatocyte and cholangiocyte in vitro. In adult tissues, its expression is restricted in tissues such as adrenal grand and hypophysis. On the other hand, overexpression of DLK-1 in cancers have been reported in several cancer types, such as hepatocellular carcinoma (HCC), neuroblastoma, rhabdomyosarcoma, small cell lung carcinoma, myelodysplastic syndrome. Interestingly, the expression of DLK-1 is overlapped with other hepatic progenitor cell markers such as EpCAM in HCC and DLK-1 has been suggested to be a marker of cancer stem cells in HCC. According to these selective expressions of DLK-1 in cancer tissues as well as cancer stem cells in HCC, DLK-1 is supposed to be a potential target for antibody-based therapy. CBA-1205 is a novel humanized antibody (IgG1/κ) targeting DLK-1 which was glycol-engineered by GlymaxX® to potentiate antibody-dependent cellular cytotoxicity (ADCC) activity. The purpose of this study is to evaluate the anti-tumor activity of CBA-1205 in multiple human cancer cell lines in vitro and xenograft models in vivo. In vitro, CBA-1205 showed potent ADCC activity in several DLK-1 positive human cancer cell lines for different cancer types. In vivo, CBA-1205 exhibited potent anti-tumor activity in several human cancer xenograft mouse models. In a Hep3B HCC xenograft model, we evaluated dose-dependent efficacy of CBA-1205 at a dose of 0.1, 0.3, 1, 3 and 10 mg/kg by the intraperitoneally injection. The tumor growth was inhibited in a dose-dependent manner and the inhibition rate at the dose of ≥ 1 mg/kg was more than 90% compared to isotype human IgG1 control. At 10 mg/kg, CBA-1205 treatment resulted in tumor regression in all mice and 4 complete tumor elimination out of 8 mice was observed. Single injection of 10 mg/kg CBA-1205 induced tumor apoptosis and reduced the CD31 positive tumor micro vessel at 24 and 48 h after the treatment. Similar anti-tumor activity of CBA-1205 was exhibited in other xenograft model of HCC-derived HepG2 and neuroblastoma-derived SK-N-FI, BE2-C and Kelly. In conclusion, we demonstrated that a novel glycoengineered humanized anti-DLK-1 antibody, CBA-1205, exerts potent anti-tumor activity in vitro and in vivo in multiple models and could be a novel treatment option for DLK-1 expressing cancer such as HCC. First-in-human Phase I study of CBA-1205 is planned to be initiated in Q1 2020.

#1536

Tumor heterogeneity and primary versus metastatic evaluation of PD-L1.

Lorenzo Colarossi,1 Eleonora Aiello,1 Marie Cumberbatch,2 Cristina Colarossi,1 Chris Womack,2 Lorenzo Memeo1. 1 _Mediterranean Institute of Oncology, Viagrande, Italy;_ 2 _Tristar Technology Group LLC, Washington, DC_.

Immunotherapy with checkpoint inhibitors allowing recovery of effector cell function, has been demonstrated to be highly effective in many tumor types and represents a true revolution in oncology. However, current assays for the prognostic and/or predictive role of tumor PD-L1 expression are not fully standardized with respect to either quantity or distribution of expression, or regarding the concordance of expression in primary tumor versus metastasis. To assess tumor heterogeneity, we evaluated PD-L1 expression using the Ventana SP-263 assay in paired formalin fixed paraffin embedded (FFPE) blocks from the same primary tumor for each of 22 lung squamous cell carcinoma (SCC), 6 lung adenocarcinoma (ADC), 53 gastric ADC, 55 colorectal ADC, 60 urothelial bladder carcinoma, 3 pancreatic ductal ADC and 4 head and neck SCC. In addition, PD-L1 expression in primary tumor and synchronous metastasis was evaluated in 32 cases of lung neoplasm (10 ADC +22 SCC), 30 cases of gastric adenocarcinoma, 55 cases of colorectal adenocarcinoma, and 16 head and neck SCC, each with corresponding synchronous metastases. PD-L1 expression was determined by pathologist evaluation of the percentage of positive tumor cells and cases were grouped into categories representing <1%, 2-9%, 10-25%, 26-49%, 50-75% and 76-100%, with those samples expressing more than 25% tumor positivity considered as expressing "high" PD-L1 levels. In lung carcinoma, just 1 case of SCC exhibited a change in PD-L1 classification when two blocks from the same tumor were evaluated (0% vs 5%), and also for 1 case of ADC (2% vs 0%) . In gastric ADC we recorded a change in classification in 1 out of 53 cases (5% vs 0%), and for CRC ADC we found a difference between the two blocks evaluated in just 1 of the 55 cases (0% vs 50%). For the remaining tumor cohorts (60 bladder carcinoma, 4 head and neck SCC and 3 pancreatic ADC) 100% concordance was observed between two blocks from the same tumor. Evaluation of primary tumors and synchronous metastasis revealed no differences in PD-L1 scores for the 32 cases of lung carcinoma, or for the 16 head and neck SCC cases. In the 30 gastric ADC cases, there was a discordant PD-L1 expression score for just one pair of tumors (primary tumor 3% and metastasis 0%). Similarly, 1 of 55 CRC ADC examined revealed a primary tumor PD-L1 score of 50% while the metastatic tissue was negative. Taken together, these data demonstrate that PD-L1 expression between two sites in the same tumour, and between primary versus synchronous metastases is remarkably consistent. The conclusions drawn are that a representative PD-L1 score may be achieved from a single FFPE tumour block, and that for cases where obtaining tissue from a primary tumor may be challenging, PD-L1 could be evaluated in metastatic tissue.

#1537

Polatuzumab Vedotin alone or in-combination with anti-CD20 antibody significantly enhanced overall survival in xenografted NSG mice against rituximab sensitive and resistant Burkitt Lymphoma (BL) and Primary Mediastinal B-cell Lymphoma (PMBL).

Aradhana Awasthi Tiwari,1 Dina Edani,1 Janet Ayello,1 Christian Klein,2 Mitchell S. Cairo1. 1 _New York Medical College, Valhalla, NY;_ 2 _Roche Pharmaceutical Research & Early Development, Zurich, Switzerland_.

Background: Patients with relapsed or refractory B-cell Non-Hodgkin Lymphoma (B-NHL) have an unfavorable prognosis with few treatment options (Goldman/Cairo et al. Leukemia, 2013). It is therefore critical to investigate and to develop targeted translational strategies in BL/PMBL in order to reduce acute morbidities, decrease late effects, and provide new options for those with recurrent disease. Polatuzumab Vedotin (PV) has been demonstrated to possess significant preclinical activity against indolent CD79b+NHL (Polson et. al. Can. Res. 2009). We previously observed that obinutuzumab vs. rituximab (anti-CD20 mAb) significantly increased overall survival against xenografted NSG mice in BL (Awasthi/Cairo et al., BJH 2015). However, synergistic effects of PV with obinutuzumab against mature PMBL/BL are unknown.

Objective: To determine the efficacy of the PV alone or in-combination with rituximab/obinutuzumab against rituximab-sensitive/resistant BL (Raji & Raji4RH) and PMBL (Karpas1106P) cell lines.

Methods: Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ), were xenografted with intravenous injections of Luc+ BL and PMBL cells. Mice were divided into following groups: PBS, isotype control, PV, anti-CD79B and MMAE (5mg/kg) and or/with obinutuzumab/RTX (20mg/kg) for 8 weeks with 106 ex.PBNK cells. Animals were monitored for tumor burden progression/regression and survival for up to 12-30 weeks via BLI using the IVIS spectrum system as previously described (Awasthi/Cairo et al BJH, 2015).

Results: The probability of OS in B-NHL xenografts NSG mice receiving PV alone was significantly increased compared to anti-CD79b or isotype control in Raji (35.5 vs.17 vs.19.5 days, p=0.0001, 0.0003), Raji4RH (50 vs.18 vs.18.5 days, p=0.0001, 0.0001) and Karpas1106P (150 vs 89 vs 64 days, p=0.03, 0.003), respectively. Furthermore, The probability of OS of B-NHL xenogrfats NSG mice receiving PV+obinutuzumab+NK significantly increased compared to PV+rituximab+NK in Raji (95.5 vs. 50.4 days, p=0.03 ) and Raji4RH (185 vs.47 days, p=0.05). Moreover, obinutuzumab+NK vs. rituximab+NK also significantly increased over all survival both in Raji (92.5 vs. 52 days, p=0.05) and Raji4RH (80.5 vs. 40.0 days, p=0.04) respectively.

Conclusion: Our preliminary data indicates that PV significantly increased overall survival in BL and PMBL NSG xenografts compared to anti-CD79b Ab alone. Furthermore, PV in combination with obinutuzumab+NK-cells significantly enhance improved OS in BL and PMBL NSG xenografts compared to obinutuzumab or PV alone.

#1538

Margetuximab mediates greater Fc-dependent anti-tumor activities than trastuzumab or pertuzumab in vitro.

Liqin Liu,1 Yinhua Yang,1 Robert Burns,1 Jonathan Li,2 Haiquan Li,2 Sergey Gorlatov,1 Paul Moore,1 Jeffrey Nordstrom1. 1 _Macrogenics, Inc., Rockville, MD;_ 2 _Macrogenics, Inc., San Francisco, CA_.

Margetuximab (M) is an investigational Fc-engineered anti-HER2 monoclonal antibody (mAb) with potential for greater immune-mediated anti-tumor activity than trastuzumab (T). M and T bind the same subdomain IV epitope, but M binds with higher affinity to activating Fc receptor, CD16A (FcγRIIIA), and lower affinity to inhibitory Fc receptor, CD32B (FcγRIIB). Pertuzumab (P) binds to a subdomain II epitope and has the same wild type Fc domain as T. We compared in vitro properties of M, T and P and combinations of M+P and T+P to evaluate Fc domain contributions to anti-HER2 mAb mediated activities. By surface plasmon resonance, M, T and P bound to the HER2 extracellular domain with similar high affinity. Binding affinities of M and T were unchanged if P was pre-bound, indicating lack of interaction. M, T and P exhibited comparable binding to HER2-expressing cell lines. Anti-proliferative activities of M and T toward HER23+ cells (N87, SKBR3 or BT474) in the absence or presence of ligands (EGF or HRG1β) were similar, whereas P exhibited weaker activity. In the absence of ligands, M+P or T+P were comparably as active as M or T alone. In presence of ligands, M+P and T+P were generally more active than M or T alone. Antibody-dependent cell-mediated cytotoxicity (ADCC) was evaluated with N87 and SKBR3 target cells (HER23+, expressing a reporter gene to measure viability) and NK effector cells from donors with differing CD16A158 genotypes. M was 7- to 84-fold more potent than T, as well as 69- to 744-fold more potent than P. Greater differences generally were seen with effector NK cells bearing the more common CD16A158 FF and VF genotypes than with the less common, high affinity CD16A158 VV genotype. While addition of P enhanced mean potency of T by 2- to 3-fold, no such effect was seen with M; however, M or M+P was 7- to 25-fold more potent than T+P. Results with JIMT-1 cells (HER22+) were similar, albeit with lower differential than with HER23+ target cells. NK cells were monitored by flow cytometry following incubation of peripheral blood mononuclear cells with HER2+ target cells and mAbs. M induced greater expression of markers of activation (CD137), cytolytic capability (granzyme B, perforin) and proliferation (Ki67) than T. An anti-HER2 mAb with an inactivated Fc domain was ineffective, indicating that induced changes in NK cells were Fc-dependent. In conclusion, M and T bind HER2 with similar affinity and exhibit comparable anti-proliferative activity that is enhanced by addition of P if ligands (EGF or HRG1β) are present. Relative to T, M mediates superior ADCC with effector cells of all CD16A genotypes and promotes greater NK cell activation and expansion. The M+P combination maintains superior ADCC compared to the T+P combination.

#1539

Mechanisms of action of bispecific T cell engager-mediated immune response in a lymphoma tumor model.

Tiffany A. Coupet, Claire Godbersen-Palmer, Charles L. Sentman. _Geisel School of Medicine at Dartmouth, Lebanon, NH_.

Bispecific T cell Engagers are molecules composed of two single chain variable fragments where one arm binds CD3 on T cells and redirects them to a tumor antigen with the other arm. This causes immune synapse formation between the T cell and tumor cell resulting in T cell activation and destruction of tumor cells. Bispecific T cell engagers have been used successfully in the clinic, however, it is unclear which activities of the host immune system are required for efficacy. This study investigates the mechanisms of action of a Bispecific protein specific for the B7H6 tumor antigen in an immune intact tumor model of lymphoma. In this lymphoma model the median survival for the B7H6xCD3 bispecific treated group was greater than 80 days while the median survival of the control bispecific treated group was 20 days. Monitoring of cellular changes in the bone marrow, blood, spleen and lymph nodes by flow cytometry 24h after bispecific protein treatment revealed that most cellular changes occurred in the inguinal lymph nodes were increases in CD8+ T cell and NK cells while CD4+ T cells decreased. Additionally, intracellular staining of cytokines IFNγ, Mip1α and TNFα in mouse T cells cultured in vitro with target tumor cells and bispecific protein showed that CD8+ T cells had greater polyfunctionality than CD4+ T cells. We have shown that IFNγ is essential for efficacy against lymphoma in vivo. To study the relative contribution of CD4 and CD8 T cells in vivo, lymphoma bearing mice that were depleted of CD4+ T cells or were deficient in CD8+ T cells were monitored for survival after bispecific protein treatment. While both CD4 or CD8 deficient mice had a greater survival when treated with B7H6xCD3 bispecific compared to control bispecific protein, the loss of either cell type resulted in much lower survival compared to WT mice treated with B7H6xCD3. All WT mice receiving B7H6xCD3 bispecific protein survived at least 65 days compared to CD4-depleted mice (median survival 20 days) and CD8-deficient mice (median survival 30 days) receiving the same treatment. In conclusion, both CD4+ and CD8+ T cells play a critical role in bispecific therapy. Understanding the mechanisms of action of bispecific proteins in vivo will help to develop more effective ways to use these powerful immune activating proteins to treat cancer.

#1540

**FS222 mAb** 2 **, a bispecific conditional agonist antibody targeting CD137 and PD-L1, induces potent lymphocyte activation and has a favorable safety profile.**

Matthew A. Lakins, Alexander Koers, Jose Munoz Olaya, Raffaella Giambalvo, Daniel Jones, Sarka Pechouckova, Emma Goodman, Sylwia Marshall, Mateusz Wydro, Cristian Gradinaru, Francisca Wollerton, Sarah Batey, Daniel Gliddon, Michael Davies, Michelle Morrow, Mihriban Tuna, Neil Brewis. _F-star Biotechnology Ltd, Cambridge, United Kingdom_.

PD-1/L1 immune checkpoint blockade shows durable responses and extends overall survival in a subset of cancer patients. Tumour Necrosis Factor Receptor (TNFR) activation is being tested clinically to improve patient responses. However, low affinity FcΓR-mediated crosslinking often limits monoclonal antibody (mAb) clinical efficacy, which is further restricted by adverse safety effects. The generation of a bispecific agonist of CD137, where potent agonist activity is conditional upon PD-L1 crosslinking, allows a greater therapeutic window. FS222, an anti-CD137/PD-L1 mAb2, was generated by introducing a CD137-binding specificity into the Fc-region of a human IgG1 mAb targeting PD-L1. A LALA mutation significantly reduces FcΓR binding. Binding characterisation was assessed by surface plasmon resonance (SPR) and cell binding, and in vitro activity measured in human primary T cell assays. A dose-range finding study in cynomolgus monkey was performed to investigate toxicity, pharmacokinetics (PK) and pharmacodynamics (PD). A murine surrogate molecule was generated and its anti-tumor activity and PK/PD was tested in multiple syngeneic mouse tumor models. FS222 binds to human PD-L1 with subnanomolar affinity. This PD-L1 binding is a prerequisite to subsequently enable highly potent CD137 agonism (low nM EC50 values in primary cell in vitro assays). We term this mechanism conditional agonism. In cynomolgus monkey FS222 has a half-life of ~150h and single and repeat dosing resulted in observable PD changes in lymphocytes and soluble receptor levels at low doses. Furthermore, no hepatotoxicity as defined by changes in clinical chemistry and histopathology was detected. A surrogate anti-mouse-mAb2 significantly reduced tumor growth in multiple syngeneic mouse tumor models. The observed dose-dependent tumor growth inhibition resulted in a significant survival benefit and was concomitant with increases in tumor and peripheral activated CD8+ T cells. FS222 is a conditional CD137/PD-L1 bispecific agonist antibody and has superior activity to control mAbs and relevant combinations in vitro assays. A favorable safety profile and immunopharmacology was observed in a cynomolgus dose-range finding study and with the surrogate molecule in multiple syngeneic mouse tumor models. Anti-CD137 agonistic mAbs in clinical development have so far shown limitations due to dose limiting toxicity or poor clinical activity. In preclinical tumor models anti-CD137/PD-L1 surrogate mAb2 treatment resulted in intra-tumoral and peripheral PD changes leading to an increase in CD8+ T cell proliferation. These changes were dose dependent and coincident with tumor growth inhibition. FS222 is a highly active bispecific molecule with a favorable safety profile with the potential to serve a significant unmet need in the immunotherapy of solid tumors.

#1541

Evaluation of ROR1 targeted antibody drug conjugate in ROR1 positive leukemia.

Eileen Hu,1 Priscilla Do,2 Rajeswaran Mani,1 Frank Frissora,1 Rebecca Pearson,1 Gerard Lozanski,1 Haiyong Peng,3 Lorenz Waldmeier,4 Roger Beerli,4 Christoph Rader,3 Ulf Grawunder,4 John Byrd,1 Natarajan Muthusamy1. 1 _The Ohio State University College of Medicine, Columbus, OH;_ 2 _Emory University, Atlanta, GA;_ 3 _The Scripps Research Institute, Jupiter, FL;_ 4 _NBE-Therapeutics Ltd, Basel, Switzerland_.

Introduction: Receptor tyrosine kinase like orphan receptor 1 (ROR1) is expressed on the surface of multiple blood and solid tumors. There have been increased interest in ROR1 as a potential therapeutic target in ROR1 positive leukemia and lymphomas. In this study, we present a novel first-in-class anti-ROR1 monoclonal antibody drug conjugate (ADC) based on chimeric rabbit/human mAb "XBR1-402" or its humanized version "huXBR1-402". Conjugated to a highly potent anthracycline derived toxin, PNU-159682 (PNU) via a non-cleavable peptide/amide linker, it shows both in vitro and in vivo efficacy at eliminating ROR1 positive chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and pre B cell acute lymphocytic leukemia (pre B cell ALL) cells.

Results: We saw a decrease in viability for all ADC treated ROR1 positive leukemic cell lines. We saw a significant decrease in viability of the ROR1 positive pre B cell ALL cell lines 697 and Kasumi-2 when treated with XBR1-402-PNU (EC50 = 77 and 1 ng/mL) when compared to treatment with isotype-matched control Trastuzumab-PNU (Tras-PNU) (EC50 = 2987 and 5330 ng/mL). In in vitro models of MCL, we saw decreased viability for huXBR1-402-PNU treated ROR1 positive Jeko and Mino cell lines but not ROR1 negative Mec-1 cell line when compared to Tras-PNU. While we were unable to observe significant direct cytotoxicity in primary CLL cells, we show that PNU ADCs mediates antibody dependent cellular cytotoxicity and phagocytosis in vitro. We also show that PNU ADCs mediates a G2/M cell cycle arrest in affected cell lines and hypothesize that this is necessary for direct cytotoxicity. As ex vivo primary CLL cells are non-proliferative, we postulate that PNU would target actively dividing cells in proliferation centers (e.g. secondary lymphoid organs). To test this hypothesis, we set up in vivo studies to test with XBR1-402-PNU and huXBR1-402-PNU in disseminated and aggressive ROR1+ ALL and CLL models, respectively. In vivo studies on a murine 697 ALL model suggested XBR1-402-PNU treatment increased overall survival when compared to treatment with Tras-PNU control (Median survival of 32 and 23 days post implantation, p=0.021, n=6). In vivo CLL studies with engrafted murine human-ROR1 expressing TCL1 leukemic cells showed that huXBR1-402-PNU treatment (3 times/week for 1 week) both suppressed leukemia burden and increased overall survival when compared with Tras-PNU control (Median survival of 62 and 41 days post implantation, p=0.0028, n=4). Ongoing proliferation and cell cycling studies will confirm the mechanism of the in vivo cytotoxicity of huXBR1-402-PNU.

Conclusion: Our results suggest that anti-ROR1 ADC huXBR1-402-PNU is an effective and promising targeted cytotoxic therapy for ROR1 positive leukemic cells of CLL, MCL, and pre B ALL and warrants further evaluation for clinical consideration as either a single agent or combination therapy in patients with ROR1 positive leukemia and lymphomas.

#1542

Bispecific T cell engager-induced toxicity in an immunocompetent solid tumor model.

Claire Godbersen-Palmer,1 Tiffany Coupet,1 Zakaria Grada,2 Charles Sentman1. 1 _Geisel School of Medicine at Dartmouth at Dartmouth College, Lebanon, NH;_ 2 _Dartmouth Hitchcock Medical Center, Lebanon, NH_.

Chimeric antigen receptor (CAR) T cells and bispecific T cell engagers have demonstrated clinical efficacy, however are often accompanied by severe toxicity including cytokine release syndrome (CRS), neurotoxicity or macrophage activation syndrome (MAS). Mechanistic understanding of the development of these toxicities and testing of therapeutic interventions is limited by a lack of suitable immunocompetent pre-clinical models. We have developed both human and murine versions of a bispecific molecule with specific activity against target expressing tumor cells in vitro and in vivo. These bispecific proteins are constructed from the extracellular portion of either mouse or human NKG2D receptor and target cells expressing the cognate ligands by redirecting T cells through their CD3 binding arm. Using the murine bispecific protein, mNKG2DxCD3, we have developed an immunocompetent mouse tumor model which exhibits treatment induced toxicity and recapitulates key features similar to those observed in human CRS. Toxicity was observed between the 2nd and 4th bispecific protein injections but was not observed following subsequent injections. Monitoring of toxicity kinetics showed transient symptoms peaking 3-4 hours following bispecific protein injection with complete resolution after 8 hours. Weight loss and elevated plasma cytokines including IFNγ, IL6, TNFα, IL2 and IL10 were also associated with toxicity. Pathology analysis showed a reduction in white pulp in the mouse spleens, however, other organs evaluated (brain, kidney, lung and heart) were normal. Using mouse genetic knockout models, we were able to show that T cells, IFNγ and Perforin were required for development of this toxicity. Additionally, In vivo depletion of either CD4+ or CD8+ T cells was sufficient to prevent the onset of toxicity. These findings highlight the importance of T cell activation and function and shed light on the mechanistic underpinnings of bispecific T cell engager protein induced toxicity.

#1543

Discovery of antibodies for ADC therapy from cancer patients who show durable clinical response to checkpoint inhibitors.

Alexander Scholz,1 Jerald Aurellano,1 Michael Harbell,1 Danhui Zhang,1 Samantha O'Connor,1 Beatriz Millare,1 Felix Chu,1 Sheila Fernandez,1 Cathrin J. Czupalla,1 Iraz Aydin,1 Amy Manning-Bog,1 Yvonne Leung,1 Kevin Williamson,1 Chantia Carroll,1 Dongkyoon Kim,1 Xiaomu Chen,1 Sean M. Carroll,1 Ish Dhawan,1 Ngan Nguyen,1 Shweta Thyagarajan,1 Mark Whidden,1 Gregg Espiritu Santo,1 Nicole Haaser,1 Guy Cavet,1 Lawrence Steinman,2 Tito A. Serafini,1 Wayne Volkmuth,1 Jonathan Benjamin,1 William H. Robinson,3 Norman M. Greenberg,1 Daniel Emerling,1 Jeff DeFalco1. 1 _Atreca, Inc., Redwood City, CA;_ 2 _Stanford University School of Medicine Department of Neurology and Neurological Sciences, Palo Alto, CA;_ 3 _Stanford University School of Medicine Division of Immunology and Rheumatology, Palo Alto, CA_.

Successful anti-tumor therapy using antibody-drug conjugates (ADCs) depends, in part, on antibodies that bind tumor selective targets that are subsequently internalized at a high rate into tumor cells. Only a select number of tumor-selective internalizing antibodies have been identified, and this has limited the scope of ADC therapeutic strategies in the clinic. Here we demonstrate, using Atreca's Immune Repertoire Capture (IRC™) technology, that we can identify tumor selective antibodies that have internalization activity suitable for ADC therapeutics directly from cancer patients showing durable clinical response to checkpoint inhibitors. To identify anti-tumor antibodies with hallmarks suitable for ADC applications, we used IRC™ to analyze blood plasmablasts, a type of antibody secreting activated B cell collected from patients with non‐progressing metastatic cancer. Antibodies representing expanded clonal families were expressed recombinantly and analyzed for binding to human tumor and non-tumor tissues, as well as for internalization activity using a pH-sensitive dye and cancer cell lines. Those antibodies with a high internalization rate were subsequently tested for in vitro ADC activity using a secondary antibody conjugated with the auristatin MMAE.

We identified multiple patient-derived antibodies, from several cancer types, that bound to human tumor tissue, but not adjacent normal tissue. Of these, several also showed internalization into human A549 lung tumor cells when measured through the detection of a pH-sensitive dye. When used in combination with a secondary antibody conjugated to the MMAE cytotoxic agent, internalizing antibodies were able to induce target cell death in vitro.

In this study, we used Atreca's IRC™ platform to identify patient-derived antibodies that bind to tumor-selective antigens and a subset that also internalize into cancer cells. Furthermore, we demonstrate that the internalizing antibodies were able to deliver a cytotoxic payload to induce tumor cell death. Overall, we show that the antibody repertoires of cancer patients contain tumor-selective antibodies with potent internalization properties and strong ADC therapeutic potential.

#1544

Cell penetrating single domain antibody (sdAb) SBT-100 binds KRAS and inhibits growth of human cancers with KRAS activating mutations.

Sunanda Singh,1 Genoveva Murillo,2 Avani Singh,1 Samara Singh,1 Meenakshi Parihar,1 Anjali Singh,1 Rajendra Mehta,2 Ashutosh Parihar1. 1 _Singh Biotechnology, Tampa, FL;_ 2 _IIT Research Institute, Chicago, IL_.

Background: Despite nearly fourth years of research, scientists have failed to develop a clinically viable therapy against KRAS, one of the deadliest families of cancer-causing proteins. Mutations in KRAS are prevalent amongst the top three most deadly cancer types in the United States: pancreatic (95%), colorectal (45%), and lung (35%). KRAS has been thought to be undruggable due to: 1) its intracellular location and lack of binding pockets for small molecules; 2) the high (pM) affinity of RAS for GTP precludes direct targeting of the nucleotide binding pocket; 3) high intracellular concentrations of GTP (uM) inhibits competition for the nucleotide-binding pocket by small molecules; and 4) possible toxicity. Mutations of KRAS result in it being perpetually turned on to propagate signal down the MAPK pathway. This results in constant production of P-ERK and plays an important role in malignant development. To overcome these challenges, Singh Biotechnology has developed SBT-100 a first in class & best in class novel sdAb that penetrates the cell membrane to bind KRAS to inhibit its GTPase activity.

Methods: Human cancer cell lines were purchased from ATCC. BIAcore affinity studies were conducted by Precision Antibody. KRAS GTPase assay was purchased from Promega. Levels of P-ERK were determined using western blots. In vitro cell growth suppression was tested with MTT assay. Athymic nude mice for xenograft studies were purchased from Envigo.

Results: SBT-100 binds KRAS with KD=10-9 and KRAS(G12D) with KD=10-7 as demonstrated by BIAcore affinity assay. Both SBT-100 and SBT-102 significantly inhibit KRAS GTPase activity in vitro and inhibition is comparable to polyclonal antibody to KRAS. Growth of MDA-MB-231 cells with KRAS(G13D) mutation and PANC-1 cells with KRAS(G12D) mutation are significantly decreased in the MTT assay when incubated with SBT-100. Additionally, same cell lines have significantly decreased P-ERK expression when cultured with SBT-100. Xenograft studies demonstrate significant growth suppression of MDA-MB-231 and PANC-1 when treated with SBT-100 in vivo.

Conclusion: SBT-100, crosses the cell membrane, binds to KRAS intracellularly and its most common mutant with nanomolar affinity, inhibits KRAS GTPase activity, downregulates P-ERK signaling, and suppresses the growth of cancers cells in vitro and in vivo without showing any toxic effects.

#1545

Altered myeloid and lymphoid composition of tumor microenvironment following anti-SEMA4D and immune checkpoint combination therapies.

Elizabeth E. Evans,1 Terrence L. Fisher,1 Holm Bussler,1 Crystal Mallow,1 Christine Reilly,1 Sebold Torno,1 Desa Rae Pastore,1 Maria Scrivens,1 Alan Howell,1 Leslie Balch,1 John E. Leonard,1 Clint Allen,2 Paul E. Clavijo,2 Gregory Lesinski,3 Christina Wu,3 Conor Steuer,3 Nabil F. Saba,3 Brian Olson,3 Siwen Hu-Lieskovan,4 Antoni Ribas,4 Emily G. Greengard,5 Ernest S. Smith,1 Maurice Zauderer1. 1 _Vaccinex, Inc., Rochester, NY;_ 2 _National Institute of Health, Bethesda, MD;_ 3 _Winship Cancer Institute of Emory University, Atlanta, GA;_ 4 _UCLA, Los Angeles, CA;_ 5 _University of Minnesota Masonic Children's Hospital, Minneapolis, MN_.

Anti-semaphorin 4D (SEMA4D, CD100) blocking antibody promotes immune infiltration, reduces immunosuppressive myeloid cells, and enhances T cell activity and tumor growth inhibition in combination with various immunotherapies in preclinical animal models. Clinical trials of immune checkpoint inhibitors (ICI) in combination with pepinemab (VX15/2503), humanized anti-SEMA4D antibody, are currently underway in several cancer indications. SEMA4D exerts multi-faceted effects within the tumor microenvironment by creating a barrier at the tumor-stroma margin to restrict immune cell infiltration and promote immunosuppressive activity of myeloid-derived cells. Blocking antibody to SEMA4D directly enhanced M1/M2 ratio and reduced both expression of chemokines that recruit MDSC and the ability of MDSC to suppress T cell activity. Antibody blockade simultaneously restored the ability of dendritic cells and cytotoxic T cells to infiltrate the TME, increasing ratio of Teffector to Tregulatory cells, in syngeneic tumor models. Importantly, anti-SEMA4D MAb enhanced the activity of co-administered immunotherapies, including antibodies to PD1, CTLA-4, and LAG3, epigenetic modulators, and additional novel immunomodulatory combinations in murine breast, colon, head and neck, and melanoma tumor models. New data will describe development and application of methods to assess immune phenotype and biomarkers in translational and clinical studies. These include flow cytometry and whole slide scans of multiplex IHC panels to examine MDSC, M1/M2 macrophage, monocytes, activated DC, B cells, exhausted, activated and stem-like populations of T cells in clinical samples. Expression of SEMA4D and its receptor PlexinB1 were also evaluated in spatial context and cellular co-localization. In summary, SEMA4D represents a novel target to regulate immune infiltration and mesenchymal suppression, sources of resistance to current immunotherapies. Pepinemab treatment was well tolerated in a Phase I oncology trial (NCT01313065) and is currently being evaluated as single agent or in ICI combinations in: (i) a Phase 1b/2a combination trial of pepinemab with avelumab in ICI naïve or ICI refractory NSCLC (CLASSICAL-Lung) (NCT03268057); (ii) a phase 1 combination trial of pepinemab with nivolumab or ipilimumab in melanoma patients who have progressed on any anti-PD-1/PD-L1 (NCT03425461); (iii) a neoadjuvant integrated biomarker trial in patients with metastatic colorectal, pancreatic (NCT03373188) and head and neck (NCT03690986) cancers treated with pepinemab in combination with nivolumab or ipilimumab; and (iv) a Phase 1/2 trial of pepinemab in children with solid tumors and children and young adults with osteosarcoma (NCT03320330). Clinical trials will evaluate safety, tolerability, efficacy, and biological endpoints, including immunophenotyping tumors and blood.

#1546

The CD30/CD16A bispecific innate immune cell engager AFM13 elicits heterogeneous single-cell NK cell responses and effectively triggers memory-like (ML) NK cells.

Nancy Marin,1 Michelle Becker-Hapak,1 Joachim Koch,2 Melissa M. Berrien-Elliott,1 Mark Foster,1 Carly Neal,1 Ethan McClain,1 Sweta Desai,1 Julia A. Wagner,1 Timothy Schappe,1 Lynne Marsala,1 Pamela Wong,1 Martin Treder,2 Todd A. Fehniger1. 1 _Washington University School of Medicine, Saint Louis, MO;_ 2 _Affimed GmbH, Heidelberg, Germany_.

Natural killer (NK) cells are crucial effector cells of the innate immune system capable of rapidly recognizing and eliminating infected, stressed and malignant cells. NK cells discriminate tumor targets from healthy cells via integration of activating and inhibitory receptor signals. One barrier to the broad application of NK cells across many cancer types is inconsistent cancer cell recognition, which may be overcome by immune cell engagers. AFM13 is a tetravalent bispecific antibody based on the ROCK platform characterized by bivalent binding to CD30 and CD16A with clinical efficacy in CD30+ malignancies. However, our understanding of NK cell functional responses triggered via AFM13 remain incomplete. Moreover, adoptively transferred memory-like (ML) NK cells have demonstrated enhanced anti-tumor activity (Romee R et al, Sci Transl Med, 2016) that may be receptive to AFM13-based targeting to enhance target cell recognition. To address these questions, we analyzed single-cell conventional (cNK) and ML (IL-12/15/18-induced) NK cell functional responses to NK-resistant CD30+ lymphoma cells +/- AFM13. Primary cNK cells co-incubated with AFM13-treated Hut-78 cells demonstrated increased IFN-γ, TNF, and degranulation, compared to Hut-78 cells or Raji (CD30-) targets + AFM13 as a negative control (p<0.05). To define the single-cell specificity of NK cell responses to AFM13, similar assays were performed using mass cytometry analysis of 39 lineage, maturation, activating and inhibitory receptors, and function-relevant NK cell markers. tSNE-based multidimensional analyses revealed marked distinctions between Hut-78 and AFM13-Hut-78 stimulated cNK cells, due in part to IFN-γ, MIP-1α, CD107a, and CD16. To define the impact of specific NK receptors, SPADE was used to define highly activated IFN-γ+ NK cell sub-populations. In a KIR3DL1+ donor, activation was primarily within the KIR3DL1+ subset, consistent with the lack of its inhibitory ligand (HLA-Bw4). In KIR3DL1 negative donors, responding NK cells were enriched in mature KIR2DL2/L3+CD57+ NK cells that lacked NKG2A. Additional experiments revealed that both control and ML NK cells exhibited increased IFN-γ, degranulation, and cytotoxicity with AFM13 (P<0.01), and AFM13-stimulated ML NK cells exhibited the highest IFN-γ response and killing. Collectively, these data indicate that target cell recognition of NK cells can be significantly enhanced by AFM13, yet influenced by inhibitory receptor expression, maturation state, and memory-like differentiation. Thus, these data suggest that the status and repertoire of NK cells in a patient may offer diagnostic potential for therapeutic response, and the combination of ML NK cells with AFM13 appears to be a promising therapeutic approach.

#1547

DNA-encoded bispecific T-cell engagers and antibodies present long-term antitumor activity.

Alfredo Perales-Puchalt, Elizabeth K. Duperret, Kar Muthumani, David B. Weiner, Xue Yang, Xizhou Zhu, Krzysztof Wojtak, Edgar Tello-Ruiz, Megan C. Wise. _The Wistar Institute, Philadelphia, PA_.

Specific antibody therapy, including monoclonal antibodies and bispecific T cell engagers (BiTEs), are important new tools for cancer immunotherapy. However, these approaches are slow to develop and may be limited in their production thus restricting the patients who can access these treatments. BiTEs exhibit a particularly short half-life and difficult production. The development of an approach allowing simplified development, delivery and in vivo production would be an important advance. Here we describe development of a designed synthetic DNA plasmid, which we optimized to permit high expression of an anti-HER2 antibody (HER2DMAb) and delivered it into animals through adaptive electroporation. HER2DMAb was efficiently expressed in vitro and in vivo, reaching levels of 50ug/ml in mouse sera. Mechanistically, HER2DMAb blocked HER2 signaling and induced antibody-dependent cytotoxicity. HER2DMAb delayed tumor progression for HER2-expressing ovarian and breast cancer models. We next used the HER2DMAb scFv portion to engineer a DNA-encoded BiTE. This HER2DBiTE was expressed in vivo for approximately 4 months after a single administration. The HER2DBiTE was highly cytolytic and delayed cancer progression in mice. These studies illustrate a novel approach to generate DBiTEs in vivo which represent promising immunotherapies for HER2+ tumors including ovarian and potentially other cancers.

#1548

Acidic pH selective binding of VISTA to PSGL-1 and anti-tumor activity of combined VISTA and PD-1 blockade.

Robert J. Johnston,1 Linhui Julie Su,1 Jason Pinckney,1 David Critton,1 Arathi Krishnakumar,1 Martin Corbett,1 Andrew Rankin,2 Rose A. DiBella,1 Lynne Campbell,1 Xiaodi Deng,1 Haibin Chen,1 Alexander Kozhich,1 Jim Holloway,1 Zheng Yang,1 Ginger Rakestraw,1 Michael Quigley,1 Alan J. Korman1. 1 _Bristol-Myers Squibb, Princeton, NJ;_ 2 _Five Prime Therapeutics, South San Francisco, CA_.

Background: Therapeutic blockade of the immune checkpoints CTLA-4 and PD-1/PD-L1 has provided durable survival benefits in multiple malignancies. However, additional treatment options are often required to maximally reverse immune dysfunction. V-domain immunoglobulin suppressor of T-cell activation (VISTA) is an orphan B7 family ligand that is highly expressed on immunosuppressive myeloid cells and has been shown to inhibit T-cell responses in vitro and in preclinical models of cancer. Here we report that VISTA, a ligand for the receptor P-selectin glycoprotein ligand-1 (PSGL-1), uses a histidine-rich interface to engage PSGL-1 and suppress immune responses selectively in acidic environments, such as tumor beds.

Methods: Recombinant VISTA multimers were used to assess binding to cells and recombinant PSGL-1 over a range of pH values (6.0-7.4). Antibodies against human and mouse VISTA were used to map binding and functional epitopes. Acidic pH receptor-based ligand capture was used to identify PSGL-1 as a VISTA receptor. X-ray crystallography was used to resolve the VISTA structure in complex with an anti-VISTA antigen-binding fragment. The MC38 mouse tumor model was used to assess the effects of VISTA deficiency and the effects of VISTA antibody blockade alone and combined with anti-PD-1 in vivo.

Results: Recombinant VISTA bound leukocytes at pH 6.0 but was not detectable at pH 7.4. Antibodies in a single epitope bin blocked VISTA binding and reversed VISTA suppression of T cells. VISTA-mediated inhibition of T cells was detectable at pH 7.4 but was more pronounced below pH 7.0, suggesting that VISTA functions selectively in acidic conditions. VISTA's structure was resolved at 1.6 Å and characterized by a histidine-rich extension of the immunoglobulin V domain central β-sheet. VISTA blocking antibodies, but not nonblocking antibodies, bound this β-sheet region. Engineered antibodies could distinguish this epitope in its active and inactive states at acidic and neutral pH, respectively. Receptor capture on T cells at acidic pH identified PSGL-1 as a VISTA receptor. T-cell PSGL-1 CRISPR ablated VISTA binding, whereas PSGL-1 expression on CHO cells conferred VISTA binding at acidic pH. Thus, an antibody that blocks mouse VISTA binding to mouse T cells at acidic pH combined with a PD-1 blocking antibody was shown to enhance anti-tumor T-cell responses and drive MC38 tumor rejection in vivo.

Conclusions: VISTA is a highly pH-selective ligand for PSGL-1. VISTA antibody blockade reverses immune suppression in vitro and in vivo, especially when combined with PD-1 antibody blockade. Our results identify acidic pH as a direct regulator of VISTA engagement with PSGL-1 and suggest new strategies to enhance anti-tumor T-cell responses.

#1549

Undesirable metastasis-stimulating effects of anti-TGF-β therapy in breast cancer models involve interference with tumor cell-autonomous tumor suppressive activity.

Yu-an Yang, Howard Yang, Binwu Tang, Alex Wu, Nellie Moshkovich, Maxwell Lee, Lalage Wakefield. _National Cancer Institute, Bethesda, MD_.

TGF-βs are central regulators of normal tissue homeostasis and response to stress and injury. Dysregulation of this pathway occurs frequently in cancer, and overexpression of TGF-βs in many advanced human tumors correlates with metastasis and poor prognosis. Preclinical studies have shown that TGF-β antagonists can slow tumor progression, largely by reactivating anti-tumor immunity, and there are now >45 ongoing or completed early phase clinical oncology trials using TGF-β antagonists. However, TGF-βs also have tumor suppressive activities, although these are conventionally considered to be restricted to the early stages of tumorigenesis. Most preclinical studies have focused on a very small number of widely used tumor models distributed across many different disease sites. Here we have assembled a panel of twelve immunocompetent mouse allograft models of metastatic breast cancer to capture some of the heterogeneity of the human disease within one disease histology. We find heterogeneous responses of these models to treatment with a neutralizing anti-TGF-β antibody, using lung metastatic burden as the clinically relevant endpoint. Anti-TGF-β antibody treatment inhibited metastasis in 5 models (InhibMet), had no effect in 4 models (NoEff) and showed an undesirable stimulatory effect on metastasis in 3 models (StimMet). As seen previously, the therapeutic effect in the InhibMet models was dependent on an intact immune system. However, the metastasis stimulatory effect in the StimMet models was immune-independent. Transcriptomic analysis of untreated primary tumors showed that InhibMet models had highest expression of a cytotoxic T-cell signature, and higher levels of infiltrating CD8 T-cells were detected by immunohistochemistry, suggesting that a therapeutic response to TGF-β antagonism may depend on some level of pre-existing immune response. In the MVT1 StimMet model, blockade of TGF-β signaling in the tumor cell using a dominant negative TGF-β receptor increased metastasis, suggesting that TGF-β antagonists can interfere with metastasis-suppressing effects of TGF-β on the tumor parenchyma. Consistent with this finding, the StimMet models showed higher expression of an independently-derived TGF-β gene signature that specifically reflects tumor suppressive responses to TGF-β in the tumor cell. In vitro, TGF-β suppressed clonogenicity and tumorsphere formation in all StimMet models but not InhibMet models, suggesting that suppressive effects of TGF-β may target the cancer stem cell population in the StimMet models. Thus our data suggest that contrary to dogma, tumor suppressive effects of TGF-β may still be active in some cases of advanced metastatic disease. Good predictive biomarkers will be essential to exclude patients who might experience deleterious responses to anti-TGF-β therapy.

#1550

Bruteforcing immune oncology discovery with computational immuno-engineering.

Sarah Ives,1 David Maurer,1 Christina Pettus,1 Raymond Newland,1 Valerie Chiou,1 Shahrad Daraeikia,1 Chelsea Jones,1 Kieran Hervold,1 Giles Day,1 Chris Smith,1 Ian D. Waddell,2 Sawsan Youssef,1 Jacob Glanville1. 1 _Distributed Bio, San Francisco, CA;_ 2 _Charles Rivers Laboratories, Saffron Walden, United Kingdom_.

Recent Nobel awards in immune checkpoint inhibitor biology, phage display discovery and protein evolution highlight our current golden age of immune oncology research. While it took 15 years to bring anti-CTLA4 and anti-PD1 therapeutics from conception to global patient populations, new advances in synthetic and data driven immune engineering are enabling new therapeutics to be discovered and engineered in weeks rather than years. Here we review ten years of progress in computational optimization of antibody discovery libraries through the lens of high-throughput data collection technologies, with specific case studies of modern repertoire design principles applied to a panel of immune checkpoint targets. We emphasize the case study of PD1, where a computationally optimized library generated over 6500 unique anti-PD1 antibodies in two weeks, including picomolar binders, mouse/cyno/human triple cross-reactive epitopes, antagonists, agonists, and saturated epitope coverage of the target.

#1551

First in class immunotherapy targeting Galectin-9 promotes T-cell activation and anti-tumor response against pancreatic cancer and other solid tumors.

Linxiao Chen,1 Wei Wang,2 Akiko Koide,1 Joseph Bolen,3 George Miller,2 Shohei Koide1. 1 _NYU Langone Health, New York, NY;_ 2 _NYU School of Medicine, New York, NY;_ 3 _Puretech Health, Boston, MA_.

Background: Pancreatic adenocarcinoma (PDA) is one of the deadliest forms of cancer, with a five-year survival rate at or below seven percent. Current immunotherapies show promise in treating other cancers, but success remains elusive for pancreatic cancer. Poor prognosis in pancreatic cancer is invariably linked to immune dysfunction. We have shown that in PDA, Galectin-9 (Gal-9) plays a significant role in facilitating a tumor-permissive immune microenvironment. Specifically, Gal-9 modulates immunosuppression via binding to multiple partner molecules (TIM-3, Dectin-1, CD44, CD206 etc). In light of these findings, we have developed and characterized first-in-class, therapeutic monoclonal antibodies to target Gal-9 and impede tumor growth.

Methods: A proprietary synthetic, human antibody library was screened via phage display to identify high-affinity antibodies cross-reactive with human, cynomolgus and rodent Gal-9, to enable efficacy and toxicity studies. We used surface plasmon resonance and bead-based assays to measure antibody binding affinity. ELISA and cell-based approaches were used to demonstrate antibody blockade of Gal-9 binding to known ligands, inferring potential therapeutic efficacy. Efficacy in multiple IgG formats were assayed using KPC (Pdx1Cre; KrasG12D; Tp53R172H)-derived cancer cells implanted orthotopically into the pancreas of C57BL/6 mice. Patient-derived organotypic tumor spheroids (PDOTs) derived from PDA and other GI tumors, which recapitulate complex tumor architecture, were treated with anti-Gal-9 antibodies and resulting immune profile was analyzed via flow cytometry.

Results: Our lead clinical candidate Gal-9 antibodies exhibit specific binding towards mouse, cynomolgus and human Gal-9 with KDvalues in the sub nanomolar range. These antibodies potently block Gal-9 interactions with known ligands such as CD206 and Dectin-1. These antibodies also inhibit Gal-9 mediated apoptosis of Jurkat cells. Mice bearing orthotopic pancreatic tumors treated with Gal-9 antibodies induced significant reduction in tumor size (p<0.005) compared with relevant controls. Immune profiling showed increased T cell activation and modulation of the monocytic compartment towards an immune permissive state. PDOTs showed similar immune profiles including an increase in type 1 inflammatory cytokines and effector T cell activation.

Conclusions: Our Gal-9 lead therapeutic antibody has been identified, characterized and is being further assessed in expanding pre-clinical efficacy, molecular and safety studies in preparation for an IND filing. Our data demonstrate novel cancer immune biology with therapeutic implications of a first-in-class monoclonal antibody against Gal-9 to reverse immunosuppressive mechanisms contributing to tumor growth immune escape in pancreatic cancer and other solid tumors.

#1552

**A novel PD1-IL2v immunocytokine for preferential** cis **-activation of IL-2R signaling on PD-1 expressing T cell subsets strongly potentiates anti-tumor T cell activity of PD-1 checkpoint inhibition and IL-2R-beta-gamma agonism.**

Christian Klein,1 Laura Codarri-Deak,1 Valeria Nicolini,1 Stefan Seeber,2 Laura Lauener,1 Marine Richard,1 Esther Bommer,1 Maria Karagianni,1 Johannes Sam,1 Ramona Schlenker,2 Marisa Mariani,1 Petra Petra Schwalie,3 Sylvia Herter,1 Marina Bacac,1 Inja Waldhauer,1 Anne Freimoser-Grundschober,1 Volker Teichgraeber,3 Pablo Umana1. 1 _Roche Pharma Research & Early Development, Schlieren, Switzerland; _2 _Roche Pharma Research & Early Development, Penzberg, Germany; _3 _Roche Pharma Research & Early Development, Basel, Switzerland_.

High-dose IL-2 is approved for patients with metastatic melanoma and renal cell cancer, but is associated with significant toxicity. We have described tumor-targeted CEA-IL2v and FAP-IL2v immunocytokines that are based on an engineered IL2v moiety with abolished binding to IL-2Ra (CD25) to avoid undesired CD25-mediated toxicities and Treg expansion. Their antibodies bind with high affinity to either CEA or FAP, which are broadly expressed in various tumors, and mediate retention/accumulation in malignant lesions. In an effort to further maximize the potency of IL-2R activation of T cells (aka signal 3) without the side effects of wildtype IL-2, we have generated PD1-IL2v that binds to the checkpoint inhibitor PD-1 and delivers IL2v preferentially in cis to PD-1+ T cells substituting binding to CD25. This enables high affinity IL2R signaling selectively in recently primed antigen specific TILs and stem-cell like subsets. Binding/competition experiments demonstrated that PD1-IL2v is 50-fold more potent than FAP-IL2v in inducing p-STAT5 in PD-1 positive T cells, whereas it has equivalent potency on PD-1 negative T cells. Notably, although Tregs can express low constitutive levels of PD-1, PD1-IL2v shows preferential binding to Teff vs. Tregs. Furthermore, PD1-IL2v can overcome Treg suppression of Tconv effector cells to a greater extent than PD-1, FAP-IL2v or their combination. For use in syngeneic mouse models muPD1-IL2v was generated. In an orthotopic pancreatic Panc02-Luciferase model administration of muPD1-IL2v (1 mg/kg IV, qw) resulted in rapid and complete elimination of tumor cells (loss of luciferase signal) and in long term survival (> 140 days) in 7/7 treated animals with protection from tumor cell re-challenge, whereas only 1/7 animals in the muPD-1 + muFAP-IL2v combination group (10 mg/kg + 2.5 mg/kg IV, qw) and none of the animals treated with the respective monotherapies showed long term survival. IHC showed that the improved outcome with muPD1-IL2v was related to a strong intra-tumoral increase of CD3+ CD8+ T cells expressing PD-1 and GrzB. In an immuno-PD study with s.c. Panc02 tumors muPD1-IL2v strongly increased the number of IFNg+ TNFa+ multifunctional and cytotoxic GrzB+ PD-1+ T cells in the tumor accompanied with a 20-fold increase in CD8/CD4 ratio and a strong increase in the CD8+ TEM population resulting in subsequent tumor control or remission in 50% of the animals, respectively. In summary, our data demonstrate that preferential cis-targeting of IL2v to PD-1+ antigen specific T cells with PD1-IL2v results in a strong potentiation of T cell response and anti-tumor efficacy as compared to the combination of PD-1 checkpoint inhibition with FAP-IL2v. These preclinical data establish PD1-IL2v as a promising next generation IL-2 for cancer immunotherapy.

#1553

Collagen affinity improves safety and efficacy of antibody and cytokine cancer immunotherapies.

Jun Ishihara, Ako Ishihara, Aslan Mansurov, Koichi Sasaki, Steve S. Lee, John-Michael Williford, Lambert Potin, Peyman Hosseinchi, Laura T. Gray, Kiyomitsu Katsumata, Stephen J. Kron, Melody A. Swartz, Jeffrey A. Hubbell. _University of Chicago, Chicago, IL_.

Cancer immunotherapy with immune checkpoint inhibitors (CPI), interleukin (IL)-2, and IL-12 have demonstrated clinical antitumor efficacy but is frequently accompanied with severe side-effects caused by excessive and systemic immune system activation. Here, we addressed this need by targeting both the CPI antibodies anti-CTLA4 (αCTLA4) + anti-PD-L1 (αPD-L1) and the cytokines IL-2 and IL-12 to tumors via conjugation (for the antibodies) or recombinant fusion (for the cytokines) to a collagen-binding domain (CBD) derived from the blood protein von Willebrand factor (VWF) A3 domain. This approach harnesses the exposure of tumor stroma collagen to blood components due to the leakiness of the tumor vasculature. We show that intravenously (i.v.) administered CBD protein accumulated mainly in tumors, with 57% of total injected dose depositing in an orthotopic MMTV-PyMT breast tumor model. CBD conjugation or fusion decreased the systemic toxicity of both αCTLA4+αPD-L1 combination therapy and IL-2/IL-12. CBD-conjugation to CPI abolished T cell infiltration into the liver and eliminated hepatotoxicity as well as decreased systemic cytokine release in the blood serum. CBD-fusion to IL-2 ameliorated capillary leak syndrome and pulmonary edema. CBD-fusion to IL-12 significantly decreased hepatotoxicity and systemic IFNg concentration. These side-effects have been reported in the clinic. CBD-CPI and CBD-IL-2 significantly suppressed tumor growth compared to their unmodified forms in B16F10 melanoma, CT26 colon carcinoma and MMTV-PyMT slow growing breast cancer models, and both CBD-CPI and CBD-IL-2 increased tumor-infiltrating CD8+ T cells; increases in the ratio of effector CD8+ T cells to T regulatory cells were observed. In an orthotopic breast tumor model, combination treatment with CPI and IL-2 eradicated tumors in 9/13 animals with the CBD-modified drugs, whereas it did so in only 1/13 animals with the unmodified drugs. CBD-IL-12 therapy induced remission of B16F10 melanoma and EMT6 breast cancer, along with increased intratumoral IFNg concentration compared to normal IL-12 therapy. Our data suggest that the A3 domain of VWF can be used to engineer immunotherapies (e.g. CPI and cytokines) with high translational promise as systemically-administered tumor targeting drugs with improved safety and efficacy compared to their native forms. Unlike other active tumor-targeting approaches, CBD-based targeting is novel in a sense that it exploits abnormal tumor vessel structure; this characteristic affords binding to the ubiquitous extracellular matrix protein in the tumor, while limiting exposure to other tissues. CBD-immunotherapeutics may efficiently activate tumor-antigen-specific T cells and increase intratumoral downstream cytokines while maintaining systemic immune homeostasis by avoiding influencing non-tumor antigen specific T cells and systemic cytokine release.

#1554

Uncoupling tumor-cell cytotoxicity from cytokine release with novel T-cell engaging bispecific antibodies.

Kyle Lorentsen,1 Preethi Sankaran,1 Nathan D. Trinklein,1 Duy Pham,1 Ute Schellenberger,1 Ben Buelow,1 Andrew Boudreau,1 Priya Choudhry,2 Starlynn C. Clarke,1 Kevin Dang,1 Katherine E. Harris,1 Suhasini Iyer,1 Brett Jorgensen,1 Payal Pratap,1 Udaya S. Rangaswamy,1 Harshad S. Ugamraj,1 Omid Vafa,1 Arun P. Wiita,2 Wim van Schooten,1 Roland Buelow,1 Shelley Force Aldred1. 1 _TeneoBio, Menlo Park, CA;_ 2 _UCSF, San Francisco, CA_.

T-cell recruiting bispecific antibody (T-BsAb) treatment is an emerging cancer immunotherapy notable for its highly-specific tumor killing activity in humans. However, the success of T-BsAbs in the clinic has been limited due to cytokine-release related toxicities. In order for this technology to reach its potential, the next generation of T-BsAbs must avoid this unintended toxicity by uncoupling cytokine-release from tumor-cell killing. To this end, we used a sequence-based discovery method on CD3-immunized OmniFlic rats, which express human fixed light-chain antibodies, to identify several novel anti-CD3 antibodies. These antibodies bind to multiple CD3 epitopes, each with varying affinities and degrees of T-cell activation. Furthermore, when placed in the context of a T-BsAb, these novel anti-CD3 antibodies achieve similar levels of T-cell mediated tumor-specific lysis, but with a thousand-fold range in potency. Among these antibodies, we identified one lead with particularly favorable properties, in that it stimulates very low levels of cytokine production without sacrificing its potent antigen-specific tumor lysis in vitro, in mouse xenograft assays, as well as ex vivo patient sample studies. This unique behavior gives our novel anti-CD3 antibody the potential to widen the therapeutic window when treating patients and ultimately usher in the next generation of T-BsAbs.

#1555

Monoclonal antibodies targeting the TAM family of receptor tyrosine kinases.

Diego Alvarado,1 Laura Vitale,2 Mike Murphy,1 Thomas O'Neill,2 Andrew Proffitt,1 Jay Lillquist,1 Gwenda Ligon,1 Komal Patel,1 Anna Wasiuk,2 Jeff Weidlick,1 Jenifer Widger,2 Laura Mills-Chen,1 Andrea Crocker,2 Colleen Patterson,2 Russell A. Hammond,3 Li-Zhen He,2 Joel Goldstein,2 Lawrence J. Thomas,3 Henry C. Marsh,3 Tibor Keler,2 Richard Gedrich1. 1 _Celldex Therapeutics, New Haven, CT;_ 2 _Celldex Therapeutics, Hampton, NJ;_ 3 _Celldex Therapeutics, Needham, MA_.

The TAM receptors (Tyro3/Axl/MerTK) family of receptor tyrosine kinases (RTKs) are important negative regulators of innate immunity. TAM receptor activation in myeloid cells by its ligands Gas6 or Protein S (PROS) promotes phosphatidylserine-dependent efferocytosis of apoptotic cells, inducing a tolerogenic state and mediating resolution of inflammation. TAM-deficient mice exhibit phenotypes consistent with systemic inflammation and autoimmunity. Importantly, individual ablation of TAM receptors can confer tumor immunity, increased pro-inflammatory cytokines and tumor lymphocyte infiltration, leading to the proposal that TAM receptors act as checkpoints of innate immunity. We hypothesize that pharmacological targeting of this family of receptors with monoclonal antibodies (mAbs) may lead to a similar pro-inflammatory response and recapitulate the antitumor effects observed in TAM-deficient mice. From a panel of human anti-MerTK, Axl or Tyro3 mAbs derived from phage-display libraries or human IgG expressing mice we identified unique mAbs that markedly enhanced cytokine and chemokine release from primary human immune cells, alone or in the presence of inflammatory stimuli. Interestingly, the qualitative and quantitative pattern of cytokine response from dendritic cells was very similar using antibodies targeting the individual TAM receptors, and also similar to activation of dendritic cells using a CD40 agonist mAb, suggesting that TAM-targeting mAbs can promote immune activation. We identified surrogate mAbs targeting mouse TAM receptors that elicited similar responses in vivo, and demonstrated antitumor activity when dosed alone, or in combination with PD-1/L1 blockade in syngeneic tumor models. In addition, human MerTK transgenic, and TAM knockout mice have been generated and characterized in order to establish in vivo proof-of-concept with human TAM mAbs. Overall, pharmacological modulation of TAM receptors with mAbs enhances cytokine production in human and murine model systems consistent with the published role of TAMs as negative regulators of innate immunity. Mechanistic and proof-of-concept studies support further development of these mAbs as novel approaches to overcome these checkpoints of the innate immune response.

#1556

Preclinical development of antibody drug conjugates targetting EGFRvIII.

Maria Luz Jaramillo,1 Anne Marcil,1 Alma Robert,1 Normand Jolicoeur,1 Cunle Wu,1 Yves Fortin,1 Yuneivy Cepero Donates,1 Mauro Acchione,1 Jacqueline Slinn,2 Binbing Ling,2 Maria Moreno2. 1 _National Research Council, Montréal, Quebec, Canada;_ 2 _National Research Council, Ottawa, Ontario, Canada_.

EGFRvIII is a naturally occurring variant of epidermal growth factor receptor (EGFR) that results from an in-frame deletion that removes 267 amino acids from the extracellular domain. EGFRvIII is a tumor-specific receptor that is amplified and present in 25-64% of glioblastoma multiforme and in 20-36% of breast cancers. Numerous studies show that normal tissues are devoid of EGFRvIII, thus making this target ideal for development as an antibody drug conjugate (ADC) for the treatment of cancer,including glioblastoma.

At the National Research Council (NRC) of Canada, we have selected mouse monoclonal mAbs that are selective for the extracellular domain of EGFRvIII and which have been shown to be internalized into EGFRvIII expressing cells for ADC development . A panel of EGFRvIII internalizing antibodies was directly conjugated to maytansine, the microtubule disrupting agent through a noncleavable linker. EGFRvIII -DM1 ADCs were shown to efficiently induce growth inhibition in vitro, in EGFRvIII expressing U87MG and DKMG glioblastoma cells. When tested in vivo, EGFRvIII -DM1 ADCs were shown to have potent anti-tumor activity in the U87MG-EGFRIII expressing tumor xenograft model.

#1557

Characterization of AB154, a humanized anti-TIGIT antibody, for use in combination studies.

Amy E. Anderson, Daniel DiRenzo, Susan Lee, Akshata Udyavar, Kimberline Gerrick, Hema Singh, Xiaoning Zhao, Lixia Jin, Lisa Seitz, Nigel P. Walker, Matthew J. Walters, Joanne B. Tan. _Arcus Biosciences, Inc., Hayward, CA_.

BACKGROUND: AB154 is a humanized antibody that blocks human TIGIT (T-cell immunoreceptor with Ig and ITIM domains), an inhibitory receptor expressed on natural killer (NK) cells, CD8+ T cells, CD4+ T cells and regulatory T cells (Treg). DNAM-1 (DNAX Accessory Molecule-1) is an activating receptor found on NK cells, monocytes and a subset of T cells that competes with TIGIT for shared ligands CD155 (PVR) and CD112 (Nectin-2), expressed by cancer and antigen-presenting cells. TIGIT blockade prevents binding to its ligands and shifts the immune balance towards a more favorable DNAM-1 interaction. AB154 has the potential to promote sustained immune activation and tumor clearance, particularly in combination with other immunotherapies such as AB122 (α-PD1).

METHODS: Binding affinity of AB154 was determined in CHO cells over-expressing human TIGIT and in human T cells. TIGIT blockade was quantified using a TIGIT-expressing reporter gene cell line. TIGIT and PD-1 expression in cancer patient PBMCs and dissociated tumor cells (DTCs) were assessed by flow cytometry. Gene expression of these markers were also derived from TCGA (The Cancer Genome Atlas), GTEX (Genotype-Tissue Expression Project), RNAseq, and by immunohistochemistry in various tumor types and normal tissues. A receptor occupancy (RO) assay was developed using a competing α-TIGIT antibody and validated ex vivo in whole blood leukocytes from healthy donors and cancer patients.

RESULTS: AB154 binds to and blocks human TIGIT with sub-nanomolar affinity. Data assembled from TCGA identified tumor types in which expression of TIGIT is greater than PD-1, equivalent to PD-1, or less than PD-1. Expression of TIGIT and CD155 at the protein level was confirmed by IHC. Immunophenotyping performed on dissociated human tumor cells demonstrated strong correlation between TIGIT and PD-1 expression on immune cells. The intensity of TIGIT staining was lowest on conventional CD4+ T cells while its intensity in Treg and CD8+ T cells was 1.5 to 3-fold higher on average. Using flow cytometry, we profiled lymphocyte populations in peripheral whole blood and demonstrated target engagement by AB154 on T cells and NK cells in the low nanomolar range in both healthy and cancer patients (ex vivo). This receptor occupancy assay is being used to monitor target engagement in AB154 dosed patient samples in an ongoing Phase 1 dose escalation study in oncology patients.

CONCLUSION: Blockade of multiple immune checkpoint proteins can confer effective and durable responses in the treatment of cancer. The data presented will provide: 1) the basis for selection of tumor types by TIGIT RNA and protein expression profiles, 2) rationale for combining AB154 with AB122 (α-PD-1) in upcoming clinical trials, 3) methodology to evaluate TIGIT receptor occupancy and 4) preliminary PK/PD data from the AB154 Phase 1 dose escalation study in oncology subjects.

#1558

Therapeutic targeting of cell surface plectin induces anti-cancer immune response and pancreatic cancer regression.

Julien Dimastromatteo,1 Amanda Poisonnier,2 Samantha Perez,1 Lisa Coussens,2 Kimberly Kelly1. 1 _University of Virginia, Charlottesville, VA;_ 2 _Oregon Health & Science University, Portland, OR_.

Background: Pancreatic ductal adenocarcinoma (PDA) is the 3rd deadliest cancer, diagnosed typically in advanced stages, with only an 8% 5-year survival rate, thus demonstrating the need for novel therapeutic approaches that significantly enhance chemo- and/or immune-therapy. Our team previously identified a promising functional target for cancer therapy in PDA, cell surface plectin 1 (CSP1) that is aberrantly expressed on PDA cells and thus a cell surface-associated biomarker of cancer. CSP1 expression first becomes apparent in high grade dysplasias, remaining high in early and advanced cancers and in metastases. Our first-in-human imaging trial in PDA patients using a CSP1-targeted imaging agent revealed that CSP is an available target and accessible for binding, a potentially a target for cancer therapy. We hypothesized that a monoclonal antibody (mAb) against CSP1 could lead to novel pancreatic cancer treatment options, thus, we developed a therapeutic mAb, e.g., ZB131, representing a first-in-class antibody selectively targeting CSP1.

Methods: ZB131 is a humanized mAb targeted against human plectin 1 (rhSec8) that also binds murine CSP1. ZB131 affinity and its effect on cancer cells including proliferation, cytotoxicity, and migration were tested in vitro using saturation binding, SRB-based survival assays, flow cytometry, and migration assays on various pancreatic cell types and homeostatic "normal" controls. In vivo validation was performed using two nu/nu mouse models bearing subcutaneous MiaPACA2 or Yapc PDA cells, and a syngeneic KPC-derived tumor model to also evaluate immune responses to tumors treated with ZB131 or IgG control.

Results: ZB131 exhibits high specificity and high affinity (0.4±0.1nM) to CSP1, and functionally induces G0 growth arrest followed by necrotic cell death of PDA cells in culture, and is synergistic with gemcitabine resulting in a 50-fold decrease in IC50. In vivo, in subcutaneous xenograft models, ZB131 monotherapy decreased PDA tumor volume 5-fold as compared to controls, and in combination with cisplatin resulted in sustained tumor reduction with greater than 85% tumor necrosis. In subcutaneous syngeneic PDA models, ZB131 induced complete tumor regression within 35 days mediated by an anti-tumor immune response as upon tumor rechallenge, full tumor regression was again achieved without additional ZB131 therapy. Leukocyte complexity analysis of regressing PDA tumors versus controls revealed an ~3-fold increase in effector and central memory T cells.

Conclusion: CSP1 is a first in class anti-cancer target expressed on the cell surface of PDA, as well as other cancers including ovarian, esophageal and head neck. ZB131, an anti-CSP1 mAb, induces tumor cell intrinsic cell death, as well as a robust anti-tumor T cell response leading to complete tumor regression indicating the potential therapeutic efficacy of ZB131 in late-stage cancers.

#1559

TLR7/8 immune-stimulating antibody conjugates elicit robust myeloid activation leading to enhanced effector function and anti-tumor immunity in pre-clinical models.

Shelley E. Ackerman,1 Joseph C. Gonzalez,1 Josh D. Gregorio,1 Jason C. Paik,2 Felix J. Hartmann,2 Justin A. Kenkel,1 Arthur Lee,1 Angela Luo,1 Cecelia I. Pearson,1 Murray L. Nguyen,1 Benjamin Ackerman,3 Lauren Y. Sheu,2 Richard P. Laura,1 Steven J. Chapin,1 Brian S. Safina,1 Sean C. Bendall,2 David Dornan,1 Edgar G. Engleman,2 Michael N. Alonso1. 1 _Bolt Biotherapeutics, Redwood City, CA;_ 2 _Stanford University, Stanford, CA;_ 3 _Johns Hopkins Bloomberg School of Public Health, Baltimore, MD_.

Deficient anti-tumor immunity often results from an immunosuppressive tumor microenvironment (TME) that renders antigen presenting cells (APCs) unable to effectively stimulate T cells. Recent studies indicate that local delivery of immunostimulatory adjuvants can activate tumor resident APCs, driving uptake, processing and presentation of tumor neoantigens to T cells that mediate anti-tumor immunity. To overcome challenges associated with intratumoral delivery of such adjuvants, we developed a novel class of TLR immune-stimulating antibody conjugates (TAC) that comprise a TLR7/8 agonist conjugated to tumor-targeting monoclonal antibodies. In vitro co-cultures with human cancer cell lines and leukocytes revealed that TACs potently activate primary APCs, leading to increased co-stimulatory molecule expression (e.g. CD40, CD86) and secretion of pro-inflammatory cytokines (e.g. TNFα). The TACs also enhanced antibody-mediated effector functions such as ADCC and ADCP. Surprisingly, these constructs also induced dendritic cell (DC) differentiation from monocytes, as measured by changes in cellular morphology and DC surface markers (e.g. CD14 downregulation). CyTOF-based analysis of intracellular signaling in human leukocytes revealed a unique signaling signature of the conjugate compared to a mixture of its components, suggesting a novel biological mechanism by which the conjugate stimulates APCs. Finally, we demonstrated in vivo efficacy in syngeneic tumor models in which TAC treatment led to tumor clearance and development of immunologic memory. These results provide a strong rationale for this technology as a platform for cancer immunotherapy.

#1560

Selection of a bispecific trivalent HER2 x CD137 TRIDENT format providing optimal tumor-anchored immune co-stimulation.

Liqin Liu,1 Chia-Ying K. Lam,2 Ralph Alderson,1 Vatana Long,1 Yinhua Yang,1 Robert Burns,1 Lusiana Widjaja,1 Jonathan Li,2 Christina Wolf,1 Valentina Ciccarone,1 James Tamura,1 Gundo Diedrich,1 Ezio Bonvini,1 Syd Johnson,1 Paul A. Moore1. 1 _MacroGenics, Inc., Rockville, MD;_ 2 _MacroGenics, Inc., Brisbane, CA_.

Introduction: CD137 (4-1BB) signaling provides co-stimulation of CD8 or NK cells following antigen or FcγR engagement, respectively. Efforts to leverage CD137 co-stimulation via agonistic monoclonal antibodies (mAbs) have been thwarted by limited clinical efficacy or unacceptable toxicity. Bispecific targeting strategies linking CD137 activation to a tumor-targeting moiety provides an approach to localize CD137 activation to the tumor microenvironment. Here we evaluate a panel of Fc-bearing HER2 x CD137 bispecific molecules incorporating different valency and geometry to define the format providing optimal CD137 co-stimulation in a tumor-cell anchor-dependent manner.

Methods: An anti-HER2 mAb specificity that does not cross compete with margetuximab, trastuzumab or pertuzumab and a proprietary anti-CD137 mAb were utilized to assemble a set of HER2 x CD137 bispecific molecules in bivalent and tetravalent DART® or trivalent TRIDENTTM configurations. The resulting molecules were compared in binding, signaling and co-stimulation assays in the presence or absence of tumor cells expressing HER2. Combination studies were performed in vitro and in immune deficient mice reconstituted with human PBMCs.

Results: TRIDENT molecules bearing bivalent CD137 and monovalent HER2 binding achieve optimal HER2-dependent tumor-cell anchored CD137 immune cell co-stimulation. CD137 co-stimulation increases proportionally with the level of HER2 expression as observed with HER2 1+ (MCF7 breast), HER2 2+ (JIMT1 breast) and 3+ (N87 gastric) tumor cells and was paralleled with increased HER2+/CD137+ cell association. No CD137 activation is observed in the absence of HER2-expressing tumor cells. HER2 x CD137 bispecifics enhance NK-cell proliferation and IFN-γ release induced by margetuximab, an Fc-optimized anti-HER2 mAb that up-regulates CD137 expression on NK-cells concomitant with enhanced ADCC against HER2-positive cells. Similarly, HER2 x CD137 bispecific molecules enhanced the in vitro activity of orlotamab, a B7-H3 x CD3 bispecific DART molecule that up-regulates CD137 during T-cell redirected killing. Finally, in vivo mouse studies demonstrate the ability of HER2 x CD137 molecules to expand tumor-associated CD8 cells when co-administered with a tumor targeted CD3 bispecific molecule and support enhance anti-tumor activity.

Conclusion: An optimal HER2 x CD137 bispecific format providing maximal CD137 activation in a HER2- dependent manner was identified as a trivalent TRIDENT molecule bearing bivalent CD137 and monovalent HER2 binding. Combinatorial activity with HER2 x CD137 bispecifics was observed with both a HER2-directed therapeutic mAb and a CD3-engaging tumor-targeted bispecific molecule. HER2 x CD137 TRIDENT molecules have therapeutic potential and provide a structural template for incorporating alternate tumor- and/or co-stimulatory-targeting arms.

#1561

Evaluation of mesothelin BiTE® antibody constructs in models of pancreatic ductal adenocarcinoma.

Noelle R. Jurcak,1 MacKenzie Zarecki,1 Fei Lee,2 Noah Rozich,1 Stephen Muth,1 Elizabeth Jaffee,1 Sabine Stienen,3 Julie Bailis,2 Lei Zheng1. 1 _Johns Hopkins School of Medicine, Baltimore, MD;_ 2 _Amgen Inc., South San Francisco, CA;_ 3 _Amgen Research (Munich) GmbH, Munich, Germany_.

Bispecific T cell engager (BiTE®) antibody constructs are designed to redirect T cells to induce lysis of tumor cells through simultaneous binding to CD3 on T cells and to a tumor associated antigen. BiTE® antibody constructs have previously been shown to be effective at depleting blood and tissue targets in B cell malignancies, suggesting a therapeutic potential for BiTE® antibody constructs in solid tumors. Pancreatic ductal adenocarcinoma (PDA) is a devastating malignant disease with a dismal prognosis even with currently available therapies. The tumor differentiation antigen mesothelin (MSLN) is highly expressed in over 80% of pancreatic tumors. Expression of MSLN is restricted in normal tissue implicating MSLN as an attractive target for BiTE® targeted therapy in PDA. In this study, we report the preclinical characterization of an anti-MSLN/CD3 BiTE® antibody construct with half-life extension (MSLN HLE BiTE®) in xenograft models of PDA. Previously the MSLN HLE BiTE® was found to bind CD3 and MSLN with low nanomolar affinity and have low picomolar cytotoxic activity against MSLN-positive cells in vitro. Moreover, the MSLN HLE BiTE® can mediate redirected lysis of cancer cell lines resistant to chemotherapy. Our study examines the efficacy of the highly potent and specific MSLN HLE BiTE® antibody construct in vivo by utilizing the orthotopic implantation of human AsPC-1 luciferase expressing PDA cells in immunocompromised NOD-Prkdcscid IL2rgnull (NSG) mice. We found that mice receiving the MSLN HLE BiTE® antibody construct survived significantly longer and had significantly reduced tumor burden comparing to control mice. Moreover, tumor bearing mice were given fluorescently labeled human T cells to measure T cell localization. Using live animal fluorescent imaging, we found mice treated with the MLSN HLE BiTE® antibody construct had significantly increased T cell localization and retention to the PDA tumor area compared to control. Mice receiving orthotopic implantation of MSLN knock out human AsPC-1 tumors treated with the MSLN HLE BiTE® did not show increased survival, reduction of tumor burden or increased T cell localization, confirming the BiTE® specificity for the tumor target MSLN. Furthermore, using the KPC mouse model of PDA, which develops cancer spontaneously upon conditional pancreatic expression of KRASG12D and TP53R172H mutations, we tested if a mouse-specific surrogate MSLN HLE BiTE® antibody construct could localize to a naturally formed dense tumor microenvironment. We found that mice treated with a murine MSLN HLE BiTE® labeled with CW800 demonstrated strong localization of the MSLN HLE BiTE® through the dense microenvironment to the PDA tumor. Taken together, these results suggest that a MSLN HLE BiTE® may be a unique targeted immunotherapy for PDA patients. 

## EPIDEMIOLOGY

### Genetics and Genomics 1: Outcomes

#1562

Germline polymorphisms in cancer stem cell genes associate with survival among women with high-grade serous ovarian cancer.

Brett M. Reid, Jennifer B. Permuth, Zhihua Chen, Ann Chen, Thomas A. Sellers, on behalf of the Ovarian Cancer Association Consortium (OCAC). _Moffitt Cancer Center, Tampa, FL_.

Background: Cancer stem cells (CSCs) are a rare population of tumor initiating cells associated with chemoresistance, recurrence and poor survival. Our group previously correlated germline variants in ovarian CSC-related genes with chemoresponse in 362 high-grade serous ovarian cancer (HGSOC) patients. Here, we evaluate CSC variants in a large, independent population to determine their association with overall (OS) and progression-free (PFS) survival.

Methods: Over 2,500 women of European ancestry diagnosed with HGSOC who underwent cytoreductive surgery have been followed for clinical outcome and genotyped on the OncoArray. We identified 4,209 common (MAF>0.01) short polymorphisms (SNPs) that were genotyped or well-imputed (r2>0.70) within 26 CSC-related genes. Associations with OS and PFS were estimated with additive genetic cox proportional hazards models adjusted for ancestral principal components, age at diagnosis, and study. Residual disease after surgery (≤1cm vs. >1cm), and stage at diagnosis (advanced vs. local) were assessed for confounding (covariate), modification (interaction), and mediational (logistic regression) effects. For each SNP, the best fit model was adopted and p-values were adjusted for multiple testing using a false discovery rate (FDR) < 15% threshold. Primary analyses evaluated 1,254 patients who received standard chemotherapy; secondary analysis included 2,659 patients, regardless of chemotherapy regimen.

Results: Two genes that were previously associated with response to chemotherapy (ESRRB, ALDH1A1) and two new genes (NOTCH2, CXCR4) were associated with OS and PFS. Eight variants within ESRRB were significantly associated with improved OS and comprised four independent (rsq<0.2) intronic variants. The top hit rs7160216 (HR=0.61, P=8.4x10-7, FDR=0.004) was modified by residual disease with the association attenuated in optimally debulked patients (HR=0.88, P interaction=0.05). One independent variant in ALDH1A1 was associated with an almost 2-fold lower OS (rs17563633 HR=1.80, P=7.2x10-5, FDR=0.03) and a ~6-fold lower OS in suboptimally debulked patients (HR=5.8, P interaction=9.2x10-4). Analysis of all patients, irrespective of chemotherapy, identified a significant association for rs17848385 in CXCR4 with reduced OS (HR=1.36, P=1.3x10-5, FDR=0.05). Aforementioned variants also displayed similar, significant associations with PFS though without modification. No significant SNP effects on survival were mediated through residual disease or stage; however, uncorrelated SNP blocks in ESRRB and ALDH1A1 associated with these factors proposing possible genomic interaction.

Conclusion: Polymorphisms in CSC genes are associated with survival of HGSOC and more research is needed to clarify the prognostic value of these genes, particularly as carriers may benefit from emerging therapies targeting CSC pathways.

#1563

**The impact of heterogeneity of** AK **T1/2 copy number alterations between primary tumors and lymph node metastases in non-small cell lung cancer.**

Kazuo Tsuchiya,1 Katsuhiro Yoshimura,1 Yusuke Inoue,1 Yuji Iwashita,1 Takashi Yamashita,1 Akikazu Kawase,1 Masayuki Tanahashi,2 Hiroshi Ogawa,2 Kazuhito Funai,1 Kazuya Shinmura,1 Hiroshi Niwa,2 Takafumi Suda,1 Haruhiko Sugimura1. 1 _Hamamatsu University School Medicine, Hamamatsu, Japan;_ 2 _Seirei Mikatahara General Hostal, Hamamatsu, Japan_.

Background: Copy number alterations (CNAs) arise during the evolution of tumors as a consequence of its genomic instability. It is reported that the increase of gene copy number of AKT1/2 correlated with clinicopathological profiles in lung cancer. On the other hand, tumor heterogeneity was also reported to associate with clinical outcomes of the patients in several cancers. However, the clinical significance of AKT1/2 CNAs in patients with non-small cell lung cancer (NSCLC) has been still unclear, especially its contribution to patients' prognoses. We here explored the relationships between intertumoral heterogeneity of CNAs of AKT1/2 and clinicopathological features using the resected tissue pairs of primary sites and corresponding lymph node metastases (LNM).

Method: AKT1/2 CNAs in 130 tissue pairs of NSCLC were assessed by fluorescent in situ hybridization (FISH), which were classified into three categories: amplification, polysomy, and disomy. The associations between CNAs and clinical features were retrospectively analyzed. In addition, we investigated the effect of the increased AKT1/2 copy number and intertumoral heterogeneity of AKT1/2 CNAs between the resected tissue pairs on the patient prognoses.

Result: The median age of patients was 66.0 years old (33-84 years old) and 98 (75.4%) patients were male. 100 (76.9%) patients had smoking history and 69 (53.1%) patients had adenocarcinoma. AKT1 CNAs including amplification and polysomy were observed in 27.3% of primary sites (2.3% for amplification and 25% for polysomy), and in 43.8% of LNM (9.4% for amplification and 34.4% for polysomy). AKT2 CNAs were observed in 33.1% of primary sites (5.5% for amplification and 27.6% for polysomy), and in 36.3% of LNM (9.7% for amplification and 26.6% for polysomy). AKT1 CNAs were significantly associated with sex (p=0.004 for primary sites, p=0.034 for LNM) and smoking history (p=0.004 for primary sites, p=0.038 for LNM). EGFR mutations were more frequently observed in the patients with AKT1/2 CNAs in primary sites (p=0.009 for AKT1, and p=0.037 for AKT2). The mean copy number significantly increased in the LNM compared to the primary sites in AKT1 (2.1 for primary sites vs 3.4 for LNM, p<0.001), whereas there were no significant differences but an increasing tendency in AKT2 (2.9 for primary sites vs 3.5 for LNM, p=0.147). There were no significant differences for recurrence-free survival (RFS) according to AKT1/2 CNA categories. However, cases with the increased AKT1/2 copy number in the corresponding LNM showed significantly worse survivals for RFS (Log-rank, p=0.02 for AKT1, and p=0.04 for AKT2).

Conclusion: AKT1/2 copy number increased in LNM compared to primary sited, and which also associated with worse prognosis. The intertumoral heterogeneity of AKT1/2 CNAs may possess distinct clinical significance in NSCLC.

#1564

Polymorphisms in nucleotide excision repair genes were associated with the survival of post-operative oral squamous cell carcinoma patients treated with adjuvant concurrent chemoradiotherapy.

Chih-Ching Yeh,1 Thomas Senghore,1 Huei-Tzu Chien,2 Wen-Chang Wang,1 You-Xin Chen,1 Chi-Kuan Young,3 Shiang-Fu Huang4. 1 _Taipei Medical University, Taipei, Taiwan;_ 2 _Chang Gung University, Tao-Yuan, Taiwan;_ 3 _Chang Gung Memorial Hospital, Keelung, Taiwan;_ 4 _Chang Gung Memorial Hospital, Linkou, Taiwan_.

Background: The nucleotide excision repair (NER) pathway plays a major role in the repair of DNA damaged by exogenous agents including chemo-and radiotherapeutic agents. Thus, we investigate the association between key potentially functional single nucleotide polymorphisms (SNPs) in the NER pathway and clinical outcomes in oral squamous cell carcinoma (OSCC) patients treated with concurrent chemoradiotherapy (CCRT).

Methods: Thirteen SNPs in five key NER genes were genotypes in 319 OSCC patients using iPLEX MassARRAY. Multivariate Cox proportional hazards models and Kaplan-Meier survival curves were used to estimate risk of death or recurrence.

Results: Carriers of the XPC rs2228000 TT genotype showed a borderline significant increased risk for poor overall survival under the recessive model (hazard ratio [HR]=1.81, 95% confidence interval [CI]=0.99–3.29). The CC genotypes of ERCC5 rs17655 (HR=1.54, 95% CI=1.03–2.29) and ERCC1 rs735482 (HR=1.65, 95% CI=1.06–2.58) were associated with an increased risk for worse disease-free survival under the recessive model. In addition, participants carrying both CC genotype of ERCC5 rs17655 and ERCC1 rs735482 exhibited an enhanced susceptibility for recurrence (HR=2.60, 95% CI=1.11–6.09). However, no statistically significant interaction was observed between them.

Conclusion: Our findings show that the ERCC5 rs17655 CC and ERCC1 rs735482 CC genotypes were associated with an increased risk for recurrence in OSCC patients treated with CCRT. Therefore, CCRT may not be beneficial and alternative treatments are required for them.

#1565

A novel therapeutic target in lethal prostate cancer in African American males.

Salma Kaochar,1 Michael Ittmann,1 Matthew Robertson,1 Jin Wang,1 Darlene Skapura,1 Christel Davis,2 Erik Ehli,2 Matthew Freedman,3 Cristian Coarfa,1 Bert O'Malley,1 Nicholas Mitsiades1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Avera Institute for Human Genetics, Sioux Falls, SD;_ 3 _Dana-Farber Cancer Institute, Boston, MA_.

Background: The largest US cancer health disparity exists in prostate cancer (PC), with African American (AA) men having a 1.6-fold increased risk of developing PC; younger age and more advanced stage at diagnosis; increased risk for recurrence; and a 2.5-fold increased mortality rate relative to Caucasian American (CA) men. In addition to important socioeconomic and environmental factors, this disparity is linked to incompletely understood genetic and other intrinsic biological factors. Consequently, there is an unmet need to identify actionable targets in AA PC in order to develop more effective therapeutics and, ultimately, improve clinical outcomes for AA PC patients.

Rationale/Hypothesis: The p160 Steroid Receptor Coactivator (SRC) family (SRC-1, SRC-2, and SRC-3), are master regulators of transcriptional activity. They cooperate with several oncogenic transcription factors to mediate their gene expression programs, thus representing a critical connecting signaling node in PC. We have hypothesized that the p160 SRCs underlie the higher aggressiveness of AA PC and proposed that the inhibition of the p160 SRCs can target the oncogenic transcriptional programs of AA PC, suppress AA PC cell proliferation, survival, and metastasis, and improve outcomes for AA PC patients. Data from the three p160 SRC knockout mouse models (Src1, Src2, or Src3 knockout mice) show no negative effect on mouse lifespan, suggesting the possibility of a therapeutic window.

Experimental approach: We utilized state-of-the-art in vitro assays and multi-Omics datasets to examine these hypotheses and shed light onto the role of the p160 SRCs in the intrinsic biological aggressiveness of AA PC. Recently, our BCM group synthesized an innovative panel of small molecule inhibitors (SMIs) of the p160 SRCs. We examined the efficacy of our novel p160 SRC SMIs against AA PC cell lines and patient-derived xenografts (PDXs).

Results: The p160 SRCs exhibit increased activity in AA PC compared to CA PC. They are important regulators of the transcriptional programs that drive AA PC cell proliferation, metabolism, invasion, and metastasis. Importantly, our novel p160 SRC SMIs potently suppress multiple oncogenic signaling programs in AA PC cells and inhibit cell proliferation. They also potently suppress key AA PC driver transcription factors, including AR, SREBP1, and MNX1. The latter two are - so far - undruggable oncogenic transcription factors that our extended BCM group recently discovered to be specifically overexpressed in AA PC. Furthermore, our lead SMI is well tolerated in mice and can inhibit PC cell proliferation and tumor growth in vivo.

Conclusions: We propose pharmacologic inhibition of the p160 SRC as an innovative treatment opportunity that will target several signaling pathways simultaneously and improve clinical outcomes in AA PC.

#1566

Guideline-concordant care among women with HER2 negative metastatic breast cancer: A SEER-Medicare analysis.

Ami M. Vyas, Tyler Mantaian, Stephen J. Kogut. _University of Rhode Island College of Pharmacy, Kingston, RI_.

Objectives: Limited data exist regarding the rates of guideline-concordant care among women with HER2 negative metastatic breast cancer (MBC). The primary objective of the study was to determine the proportion of women with HER2 negative MBC who received guideline-concordant care and also its predictors using a multivariable framework.

Methods: A retrospective observational cohort study was performed using the Surveillance, Epidemiology, End Results-Medicare linked database. The study cohort consisted of women age ≥ 66 years diagnosed with HER2 negative MBC in 2010-2013. Guideline-concordant initial care within six months of cancer diagnosis was determined as per The National Comprehensive Cancer Network (NCCN) treatment guidelines. The Andersen behavioral model of healthcare services utilization was utilized to identify the significant factors that affect guideline-concordant cancer care. A multivariate logistic regression was performed to identify the significant predictors of guideline-concordant care in the study cohort, after controlling for predisposing, enabling, and need-related factors, healthcare use, and external environmental healthcare factors.

Results: Among 1,089 older women with HER2 negative MBC, 72.3% received guideline-concordant care, while 27.7% did not receive guideline-concordant care. Marital status and education among enabling factors, hormone status among need-related factors, and the number of oncology visits among healthcare use were significant predictors of guideline-concordant care for women with HER2 negative MBC. Compared to women who did not receive guideline-concordant care, women who received guideline-concordant care were significantly more like to have had positive hormone receptor status, be married, resided in census areas with the higher proportion of people age ≥ 25 years with at least years of education, and had the higher number of oncology visits. The adjusted odds ratios were 4.004 (95% confidence interval (CI)=2.726-5.881, 1.543 (95% CI=1.087-2.191), 2.180 (95% CI=1.111-4.278), and 4.908 (95% CI=3.313-7.270). Among predisposing factors, age at cancer diagnosis was a significant predictor of guideline-concordant care. The likelihood of receiving guideline-concordant care decreased for women who were at least 70 years of age at cancer diagnosis. The adjusted odds ratios were 0.573 for women in 70-74 years age group, 0.592 for women in 75-79 years age group, and 0.403 for women at least 80 years old (p<0.0001).

Conclusion: Approximately 28% of older women with HER2 negative MBC were not treated as per the NCCN breast cancer treatment guidelines. These study findings have identified the opportunities to improve cancer care for older women at least 70 years of age at cancer diagnosis, who are single or divorced, and those with negative hormone receptor status.

#1567

Race, RNA-based recurrence scores, and survival of early hormone receptor-positive breast cancer: A population-based study.

Yunan Han, Graham A. Colditz, Ying Liu. _Washington University in St. Louis, St. Louis, MO_.

Introduction: Compared with white women, black American women are less likely to develop hormone receptor-positive breast cancer, but experience higher mortality of the disease. The 21-gene recurrence scores (RS) predict individualized benefit of chemotherapy for early hormone receptor-positive breast cancer. We hypothesize that the RS scores in early hormone receptor-positive breast cancer are higher in black women than in white women and this contributes to the worse outcomes among black women with hormone receptor-positive breast cancer.

Methods: Utilizing the Surveillance, Epidemiology, and End Results database, we identified 75,861 women (75.7% non-Hispanic white, 7.9% non-Hispanic black, 8.0% Asian/Pacific Islander, and 8.5% Hispanic) with stage I-III hormone receptor-positive breast cancer diagnosed between 2004 and 2014. RS scores were categorized into low, medium, and high groups using traditional cutoffs (18 and 30) and the cutoffs used in the TAILORx clinical trial (11 and 25). We performed multivariable logistic regression to evaluate odds ratios (ORs) of RS categories, and Cox proportional hazard regression to estimate hazard ratios (HRs) for breast cancer-specific mortality and overall mortality.

Results: The multivariable adjusted least square mean of RS was significantly higher in black (23.2) than other racial/ethnic groups (white 22.9, Asian/PI 22.5, Hispanic 22.4; p<0.0001). Compared with white women, black women were more frequently in the high RS category defined by a cutoff of 30 (case-only OR=1.24, 95% CI: 1.11-1.39), Asian/PI (OR=0.88, 95% CI: 0.79-0.98) and Hispanic (OR=0.80, 95% CI: 0.72-0.89) were less frequently in the high RS category defined by a cutoff of 25. Compared with white women, the HR of breast cancer-specific mortality was 1.37 (95% CI: 1.10-1.69) in black women, and 0.65 (95% CI: 0.48-0.90) in Asian/PI women, after adjustment for demographic factors, RS categories and other tumor characteristics, and treatment. This association did not significantly vary by RS categories. The multivariable-adjusted associations between race/ethnicity and overall mortality were similar to those for breast cancer-specific mortality.

Conclusions: Racial/ethnic differences in early hormone receptor-positive breast cancer outcomes could not be explained by RS scores. Potential differences in tumor biology that are not related to the genes in the 21-gene assay may contribute to the worse prognosis of black women with hormone receptor-positive breast cancer.

#1568

Clinical implications of tmprss2-erg fusion and tumor location in prostate cancer.

Kosj Yamoah,1 Shivanshu Awasthi,1 Priti Lal,2 Arash O. Naghavi,1 Shelly Mahajan,2 Jong Y. Park,1 Rob Rounbehler,1 Travis Gerke,1 Anders Berglund,1 Julio PowSang,1 Jasreman Dhillon,1 Timothy R. Rebbeck3. 1 _Moffitt Cancer Ctr & Research Institute, Tampa, FL; _2 _2) The Abramson Cancer Center, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA;_ 3 _Dana Farber Cancer Institute and Harvard TH Chan School of Public Health, Boston, MA_.

Introduction: There is limited data on the impact of race on tumor location and TMPRSS2-ERG (ERG) gene fusion status in Prostate Cancer (PCa). African American men (AAM) often lack the ERG fusion and are likely to experience poor outcomes. Nonetheless, the extent to which ERG status, race, and tumor location impacts PCa outcomes remains unclear.

Methods: This multi-institutional study included a retrospective cohort of 495 AAM and 422 European American men (EAM) who received prostatectomy at Moffitt Cancer Center (MCC) and University of Pennsylvania Health system (UPHS). To assess the race-dependent variation in ERG protein expression and its association with tumor location and PCa pathology, ERG antibody staining on 4 micron sections of FFPE blocks were carried out on all samples. ERG expression was categorized as ERGpos and ERGneg by two pathologists. Additionally, the staining pattern of ERG was analyzed within ERGpos tumors. A review of anatomic location of the dominant tumor lesions was performed and analyzed using methods of categorical analysis and logistic regression. Alpha value of < 0.05 was considered as statistically significant.

Results: In all samples, analysis of ERG immunohistochemistry (IHC) yielded 39.7% ERGpos and 60.6% ERGneg. Variability in histopathologic expression of ERG was significant between races with AAM being more likely to lack TMPRSS2-ERG fusion (ERGneg) compared to EAM, (62% vs 38%, p < 0.001). Lack of ERG fusion was also associated with anatomically distinct tumor location with 72.1% of anteriorly located tumors having ERGneg staining, while only 27.9% were ERGpos, (p < 0.001). Among ERGpos patients, AAM (65.2%) showed significant heterogeneous expression of ERG compared to EAM (31.8%). Furthermore, ERGneg status was associated with higher pretreatment PSA. In a multivariate logistic regression model adjusted for tumor location and race, ERGneg tumors were more likely to have Gleason Score 4+3=7 (Grade Group 3) or higher compared with ERGpos tumors, Odds ratio 2.07, 95% CI 1.20 - 3.56, p = 0.008.

Conclusions: The absence of TMPRSS2-ERG fusion is associated with higher grade disease and is a strong predictor for anatomic tumor location within the prostate gland. The clinical implications of ERGneg tumors among AAM and its association with anteriorly located tumors can be vital in addressing disparities in PCa.

#1569

Racial differences in incidence and impact of TP53 deletion on outcome in African American and Caucasian veterans with multiple myeloma.

Diana Cirstea*,1 Nathanael Fillmore*,2 Hassan Yameen,3 Sarvari Yellapragada,4 Chizoba Ifeorah,4 Nhan Do,1 Mary Brophy,1 Nikhil Munshi5. 1 _Boston Medical Center, VA Boston Healthcare System, Boston, MA;_ 2 _VA Boston Healthcare System, Boston, MA;_ 3 _Boston Medical Center, Boston, MA;_ 4 _Baylor College of Medicine, Michael E Debakey VA Medical Center, Houston, TX;_ 5 _Dana-Farber Cancer Institute, VA Boston Healthcare System, Boston, MA_.

BACKGROUND: In multiple myeloma (MM), the presence of TP53 deletion is associated with poor prognosis. It is found in roughly 10% of newly diagnosed patients. Lower incidence of TP53 deletion is reported in African American (AA) compared to Caucasian (CA), suggesting a possible contribution of disease biology to clinical disparity in AA. Our recent report of a significantly superior age-adjusted risk of death in AA compared to CA patients in the Veteran population also suggests possible racial differences in disease biology. Here we investigated the incidence of TP53 deletion among younger (<65) versus older AA and CA patients with MM at the VA.

METHODS: We identified 15717 patients with MM from 1999 to 2017 using the VA's nationwide Corporate Data Warehouse. We extracted data on patients' age, race, and ISS stage, as well as data on their therapy at induction and stem-cell transplant (SCT). Additionally, we extracted data on TP53 deletion testing and results using a natural language processing algorithm and focused our analysis on patients with these data.

RESULTS: We identified 5,744 MM patients evaluated for TP53 deletion by conventional cytogenetics and/or FISH, including 32.5% AA (1,864) and 67.5% CA (3,880) patients. Greater proportion of AA patients (53%) were younger (<65 years), compared to CA (39%; p<0.001). Overall, among those tested, TP53 deletion was reported in 9.6% of patients, but the incidence was significantly lower in AA (7.2%) compared to CA (10.7%; p<0.001). Among patients <65 years of age, difference in incidence of TP53 deletion between AA and CA was 3.2% (8.1% vs 11.3%; p=0.01), while in those ≥65 years age, it was 4.1% (6.4% vs 10.5%; p=0.0005), suggesting increased difference in the incidence of TP53 deletion with age. The rates of TP53 deletion across different ISS stages were not significantly different, nor did ISS stage differ across race. The majority of patients with TP53 deletion (95.2%) received either proteasome inhibitor or immunomodulator at induction, but only 24.7% were treated with the combination, and only 21.4% proceeded to SCT. Median survival among younger patients with TP53 deletion appeared substantially lower in AA than in CA (5.1 vs 7.0 years; p=0.74), though without reaching significance, perhaps due to sample size. In contrast, younger AA without TP53 deletion had a significantly longer median survival than CA (8.0 vs 6.2 years; p<0.01). Among older patients, AA had longer median survival regardless of TP53 deletion status.

CONCLUSIONS: This large cohort study identified significantly lower incidence of TP53 deletion in AA compared to CA. Yet, when present in younger AA patients, TP53 deletion correlated with worse survival. These results identify racial disparity in both occurrence and impact of TP53 deletion and now suggests the need for a more comprehensive genetic assessment to develop risk stratification in AA myeloma patients.

#1570

Genome-wide association study identifies variants at 3p26.1 linked to survival in ovarian cancer patients among Chinese women.

Hongji Dai,1 Xinlei Chu,1 Wei Wang,1 Huijun Yang,1 Qiong Wang,1 Mulin Jun Li,2 Qingyi Wei,3 Kexin Chen1. 1 _Tianjin Medical Univ. Cancer Inst. & Hosp., Tianjin, China; _2 _Tianjin Medical University, Tianjin, China;_ 3 _Duke University Medical Center, Durham, NC_.

Survival rates for ovarian cancer varies considerably among patients, and some of this variation could be explained by germline genetic variation. In order to identify genetic markers associated with ovarian cancer-specific survival, we performed a genome-wide association study (GWAS) of ovarian cancer survival among Chinese women. In a meta-analysis of 2,130 ovarian cancer patients from the southeast and north part of China, we found a significant association between SNPs at the 3p26.1 and overall survival (lead SNP rs3804994; combined P=9.8×10-9). Patients with the minor allele are at increased risk for mortality (HR: 1.55; 95% CI: 1.33-1.80) relative to patients with the major allele. Statistical fine-mapping and functional prioritization recommend a potential casual SNP rs9311399 that locates in the active chromatin region of ovarian tissue. Using EMSA, luciferase assay and ATAC-seq, we demonstrated that risk allele of rs9311399 could alter the regulatory activity of DNA sequence. Our findings suggest that genetic variants at the 3p26.1 may be promising prognostic biomarkers for ovarian cancer patients.

#1571

Telomere maintenance variants and survival after colorectal cancer: Smoking- and sex-specific associations.

Sheetal Hardikar,1 Hang Yin,2 Sara Lindstroem,2 Li Hsu,3 Kristin Anderson,4 Barbara Banbury,3 Sonja I. Berndt,5 Andrew T. Chan,6 Edward L. Giovanucci,6 Tabitha A. Harrison,3 Amit D. Joshi,6 Nan Hongmei,7 John D. Potter,3 Lori C. Sakoda,8 Martha L. Slattery,9 Robert E. Schoen,10 Emily White,3 Ulrike Peters,3 Polly A. Newcomb3. 1 _Huntsman Cancer Institute, Salt Lake City, UT;_ 2 _University of Washington, Seattle, WA;_ 3 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 4 _University of Minnesota, Minneapolis, MN;_ 5 _National Cancer Institute, Bethesda, MD;_ 6 _Harvard University, Cambridge, MA;_ 7 _Indiana University, Indianapolis, IN;_ 8 _Kaiser Permanente, Oakland, CA;_ 9 _University of Utah, Salt Lake City, UT;_ 10 _University of Pittsburgh, Pittsburgh, PA_.

Background: Telomeres play an important role in determining colorectal cancer (CRC) prognosis; telomere length varies by smoking status and sex. The association between variation in telomere maintenance genes and survival after CRC remains uncertain; evidence on the interaction between these genes and prognostic factors, such as smoking and sex, is also lacking.

Methods: We selected candidates from a subset of the large genome-wide International Survival Analysis in Colorectal Cancer Consortium (ISACC). Common variants (MAF >5%) in 13 telomere maintenance genes (TERT, TERC, TERF1, TERF2, TINF2, TERF2IP, ACD, POT1, TNKS, TNKS2, TNKS1BP1, TEP1, PINX1) were examined. Cox proportional hazard models adjusted for age, sex, study, and genetic ancestry were used to analyze associations of these variants with overall and CRC-specific survival, with additional stratification analyses by prognostic factors, including smoking status and sex. Likelihood ratio tests evaluated the significance of interaction terms. P-values were adjusted for multiple comparisons by the Bonferroni method within each gene.

Results: Eight SNPs within TERT, TERF1, TNKS, TNKS1BP1, TEP1 and TERF2 were associated with overall and CRC-specific survival at the P-value threshold of 0.05. The association between rs73202875 (TNKS) and CRC-specific survival varied significantly by smoking pack-years (adjusted Pinteraction =0.024, never smokers vs. ≥45 smoking pack-years). Significant interactions by sex were observed such that the minor allele at rs2975843 (TERF1) was associated with poorer overall and CRC-specific survival in women, but not in men (adjusted Pinteraction =0.002 and 0.019, respectively). The minor allele at rs74429678 (POT1) was associated with increased CRC-specific mortality in women but not in men [HR (95% CI) =1.33(1.07-1.65) and 0.75(0.52-0.97), respectively; adjusted Pinteraction =0.002]. A significant interaction was also observed for overall survival between sex and rs75676021 (POT1, adjusted Pinteraction =0.023).

Conclusion: This study showed statistically significant associations between candidate genes in the telomere maintenance pathway with overall and CRC-specific survival. Additionally, we observed a statistically significant interaction for telomere maintenance genes with smoking pack-years (TNKS) and sex (POT1, TERF1), respectively. Validation of these findings in other large studies is warranted; further functional annotation of these SNPs should be conducted.

#1572

**Inherited truncating** RAD52 **variant discovered using integrated germline- somatic analysis predicts clinical outcome in patients with lung cancers.**

Semanti Mukherjee,1 Preethi Srinivasan,1 Chaitanya Bandlamudi,1 Matthew D. Hellmann,1 Vignesh Ravichandran,1 Myvizhi Esai Selvan,2 Yelena Kemel,1 Diana Mandelker,1 Marjorie Zauderer,1 Michael Walsh,1 Ahmet Zehir,1 Maria I Carlo,1 Karen Cadoo,1 Steven M Lipkin,3 Marc Ladanyi,1 David Solit,1 Mark Robson,1 Liying Zhang,1 David Jones,1 Charles Rudin,1 Robert Klein,1 Zsofia Stadler,1 Joseph Vijai,1 Zeynep Gumus,1 Barry Taylor,1 Michael Berger,1 Kenneth Kenneth1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 3 _Weill Cornell Medicine, New York, NY_.

Most of the heritability of lung cancer, estimated at 18% in population studies, remains unexplained. We employed a framework for variant interpretation utilizing integrated germline and somatic data.The lung cancer patients(pts) enrolled between 01/2014 and 05/2016 underwent sequencing analysis for 468 genes using MSK IMPACT with pathogenicity assessment performed using ACMG guidelines. Loss of heterozygosity (LOH) in tumor was inferred using the FACETS algorithm. A poorly characterized truncating variant was classified as "potential pathogenic variant" if we observed LOH of the relevant allele in the tumor and the gene was previously reported co-segregating in family studies or significant in genome wide association studies (GWAS). Frequencies of pathogenic germline variants were compared to population frequencies in the genome aggregation database (gnomAD).As per IRB protocol, germline results were anonymized for all cases. 2686 pts consented to tumor-normal sequencing of whom, 80% had lung adenocarcinoma, 10% squamous cell carcinoma and 5% small cell lung cancer diagnosis. We discovered a new truncating RAD52 p.Ser346Ter variant in 89 (3.3%) lung cancer patients; 47(53%) patients had LOH in tumor (23 pts with wild type allele loss and 24 patients with mutant allele loss). Previous lung cancer GWAS had reported common variants at the RAD52 locus as lung cancer susceptibility loci. The identified RAD52 truncating mutation may represent the functional variant within the risk haplotype. Clinico-pathological association with the RAD52 variant suggested that the mutation was more common in patients who had <5 pack years of smoking history (5% in non-smokers versus 2.8% in smokers; OR=1.8, 95%CI=1-3.2,p-value=0.03), more commonly associated with EGFR oncogenic mutations (5.4% vs 2.8%; p-value=0.01) or MYC amplification (8.3% vs 3%; p=0.001) and had a poorer prognosis compared to patients without the variant (adjusted for smoking status; HR=1.5, 95%CI=1-2.4,p-value=0.04). We also identified 237 (8.8%) pts harboring pathogen germline variants in 28 cancer predisposition genes based on ACMG criteria, including 3 DNA repair genes significantly enriched in lung cancer cases compared to gnomAD controls; BRCA2 (OR=2, 95% CI= 1.3- 4.9), TP53 (OR=10.7, 95% CI=2.9-32.7)and NBN (OR= 9.6; 95% CI= 3-26.1) . Notably, 4/5 (80%) TP53variants and 9 /19 (47%) BRCA2 variants showed loss of heterozygosity (LOH) of the wild-type allele in tumor. In conclusion, we discovered a novel truncating RAD52 variant associated with clinical characteristics and poor prognosis, supporting the biological role of pathogenic mutations in DNA repair genes, including BRCA2, in the pathogenesis of a subset of hereditary lung tumors. These data warrant follow-up to refine the scope of germline genetic testing for lung cancer patients.

#1573

Non-canonical NF-kappaB signaling in the tumor microenvironment in relation to ovarian cancer survival.

Demetra Hufnagel,1 Andrew J. Wilson,2 Jamie Saxon,3 Timothy Blackwell,2 Dineo Khabele,4 Marta A. Crispens,2 Alicia Beeghly-Fadiel,2 Fiona Yull5. 1 _Vanderbilt University School of Medicine, Nashville, TN;_ 2 _Vanderbilt University Medical Center, Nashville, TN;_ 3 _Inari, Cambridge, MA;_ 4 _The University of Kansas Medical Center, Kansas City, KS;_ 5 _Vanderbilt-Ingram Cancer Center, Nashville, TN_.

Introduction: Despite advances in treatment, ovarian cancer associated mortality has not substantially improved over the last 50 years. Understanding mechanisms that underlie disease progression is crucial to the development of new treatments for ovarian cancer. Mediated by a family of transcription factors, the canonical and non-canonical nuclear factor-kappaB signaling pathways have known roles in cell proliferation, as well as immune, inflammatory, and neoplastic processes. Non-canonical NF-kappaB signaling requires processing of a cytoplasmic p100/RelB dimer to a mature p52/RelB complex, capable of nuclear translocation to activate transcription of target genes. Prior studies on canonical NF-kappaB expression and ovarian cancer survival have been inconsistent, but no studies to date have evaluated the role of non-canonical NF-kappaB transcription factors.

Methods: We conducted immunohistochemical staining for p52, a component of the non-canonical NF-kappaB signaling pathway, on a clinically annotated tissue microarray of ovarian tumors collected from 1994-2004 at the Vanderbilt University Medical Center. Nuclear and cytoplasmic p52 staining in tumors was semi-quantified by H-score and expression was dichotomized at median values. Associations with progression-free survival (PFS) and overall survival (OS) were quantified by hazard ratios (HR) and 95% confidence intervals (CI) using Cox proportional-hazards regression.

Results: Among 194 ovarian cancer cases, p52 expression was predominantly cytoplasmic, with a relatively smaller proportion of tumor cells showing nuclear staining (median H-scores: 135.6 vs. 1.0). Cytoplasmic and nuclear p52 H-scores were significantly higher among high grade, advanced stage, and serous tumors (all Wilcoxon rank sum p<0.01). Kaplan-Meier survival analysis showed that cases with high nuclear p52 expression had significantly shorter time to disease progression (5-year PFS high vs. low: 72% vs. 17%, log-rank p<0.001). In regression models that included adjustment for age, stage, grade, and histologic subtype, high nuclear p52 was associated with a more than two-fold shorter PFS (HR 2.30, 95% CI 1.44-4.67). Significance of this finding was unaltered by additional adjustment for race, debulking, and platinum sensitivity (HR 2.26, 95% CI 1.15-4.45). In contrast, significant associations between high nuclear p52 and worse OS, and all associations for cytoplasmic p52 staining were attenuated after adjustment for clinical covariates.

Conclusions: This is the first study to demonstrate that nuclear p52, representing active non-canonical NF-kappaB signaling, may be an independent prognostic factor for epithelial ovarian cancer. Thus, interventions that inhibit non-canonical NF-kappaB signaling should be explored as novel therapies to limit ovarian cancer progression.

#1574

Circadian clock gene expression and lethal prostate cancer outcomes.

Sarah C. Markt,1 Ericka Ebot,2 Iona Cheng,3 Lynne Wilkens,4 Ayesha Shafi,5 Karen Knudsen,5 Kathryn Penney,6 Lorelei Mucci,2 Travis Gerke7. 1 _Case Western Reserve University, Cleveland, OH;_ 2 _Harvard T.H. Chan School of Public Health, Boston, MA;_ 3 _UCSF, San Francisco, CA;_ 4 _University of Hawaii, Honolulu, HI;_ 5 _Thomas Jefferson University, Philadelphia, PA;_ 6 _Channing Division of Network Medicine, Boston, MA;_ 7 _Moffitt Cancer Center, Tampa, FL_.

Background: Converging evidence points to a potential role of disruption of the circadian rhythm in prostate cancer progression. There is limited human data in prostate tissue examining the biological consequences of alterations in genes involved in circadian rhythm on outcomes among prostate cancer patients.

Methods: To test the hypothesis that tissue expression of circadian-related genes was associated with prostate cancer outcomes, we leveraged data from the Physicians' Health Study (PHS) and the Health Professionals Follow-Up Study (HPFS) prostate tumor cohorts. Whole transcriptome expression profiling was performed on 404 prostate cancer cases, including 113 lethal cases (metastatic disease or prostate cancer death) and 291 indolent cases (>8 years from diagnosis without evidence of metastasis). Using logistic regression models, we assessed whether mRNA expression levels in tumor (N=404) or paired normal prostate tissue (N=202) of eleven circadian-related genes (AANAT, CLOCK, CRY1, CRY2, CSNK1E, MTNR1B, NPAS2, OPN4, PER1, PER2, PER3) were associated with lethal prostate cancer, stage (T2 vs. T3+), Gleason grade at diagnosis (<8 vs. ≥8), and biomarkers of angiogenesis and apoptosis. We calculated odds ratios and 95% confidence intervals (95% CIs) per standard deviation increase in gene expression.

Results: Pathway analyses showed a statistically significant association between the eleven circadian genes and lethal disease (global test p-value = 5.1e-05). On an individual gene level, men with higher tumor expression of Period Circadian Regulator 1 (PER1) gene had a reduced risk of lethal disease, independent of grade and stage (OR: 0.75, 95% CI: 0.59-0.97). High tumor expression of PER1 (OR: 0.72, 95% CI: 0.57-0.91) and Cryptochrome Circadian Regulator 2 (CRY2) (OR: 0.79, 95% CI: 0.63-1.00) were associated with lower Gleason grade tumors. High tumor expression of PER1 was associated with lower levels of cell proliferation and lower levels of angiogenesis markers; Neuronal PAS Domain Protein 2 (NPAS2) was associated with markers of apoptosis. For none of the genes was expression in normal prostate tissue associated with lethal prostate cancer (global test p-value = 0.06).

Discussion: We found higher expression of several of the circadian related genes were associated with less aggressive prostate cancer features. In line with these findings, PER1 has been suggested to be a tumor suppressor in previous studies. This supports the idea that maintenance of circadian clock function may protect tumor progression.

#1575

DNA methylation-derived neutrophil-to-lymphocyte ratio and survival among pediatric medulloblastoma patients.

Vidal M. Arroyo,1 Philip J. Lupo,1 Michael E. Scheurer,1 Surya P. Rednam,2 Jeffrey C. Murray,3 M F. Okcu,2 Murali M. Chintagumpala,2 Austin L. Brown1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Texas Children's Hospital, Houston, TX;_ 3 _Cook Children's Hospital, Houston, TX_.

Background/Objectives: Medulloblastoma is the most common malignant brain tumor diagnosed in children. Current risk-stratification methods of pediatric medulloblastoma do not fully explain the observed variability in clinical outcomes. Methylation-derived neutrophil-to-lymphocyte ratio (mdNLR) captures immune-specific information and has been identified as a potential prognostic biomarker of outcomes in various cancers, including adult brain tumors. Therefore, we evaluated the association between blood-derived mdNLR and overall survival in a cohort of patients with pediatric medulloblastoma.

Design/Methods: We identified pediatric patients diagnosed and treated for medulloblastoma at Texas Children's Cancer Center between 1995 and 2015. Peripheral blood DNA methylation was measured using the Infinium Human Methylation 450K Beadchip. Methylation data underwent quality control, including beta-mixture quantile normalization and batch correction. Immune cell proportions (CD4+ T-Cells, CD8+ T-Cells, B-cells, natural killer cells, monocytes, and granulocytes) were estimated using cellular deconvolution methods and used to estimate mdNLR, which was then log-transformed to improve normality. Cox regression models were estimated to evaluate the association between mdNLR and overall survival.

Results: Of the 78 eligible patients include in this analysis, 83% (n=65) were alive at last follow-up (median follow-up=7.8 years). Deceased patients (n=13; median follow-up=2.6 years) had a higher mean mdNLR than patients who were alive at last contact (12.3 vs. 4.0, P = 0.046). Elevated log-transformed mdNLR was associated with an increased risk of death in both unadjusted models (HR=1.68, 95%CI: 1.11-2.55) and models accounting for age, sex, race, and clinical risk group (HR=1.97, 95%CI: 1.12-3.45).

Conclusion: We identified a significant association between peripheral blood mdNLR and survival in pediatric medulloblastoma. As a promising prognostic biomarker of outcomes, mdNLR captures immune-specific information and is a potential avenue of research in settings where cytologic determination of NLR may not be possible. Future work should investigate the relationship between elevated mdNLR and specific pediatric medulloblastoma molecular subtypes.

#1576

Identification of six novel candidate miRNAs associated with early relapse in B-cell pediatric acute lymphoblastic leukemia.

Ernest K. Amankwah,1 Kelly W. Maloney,2 Eric C. Larsen,3 Meenakshi Devidas,4 David T. Teachey,5 Karen R. Rabin,6 Mignon L. Loh,7 Patrick A. Brown8. 1 _Johns Hopkins University, Tampa, FL;_ 2 _Children's Hospital of Colorado, Aurora, CO;_ 3 _Maine Children's Cancer Program, Scarborough, ME;_ 4 _Children's Oncology Group, FL;_ 5 _Children's Hospital of Philadelphia, PA;_ 6 _Baylor College of Medicine, Houston, TX;_ 7 _UCSF Medical Center, CA;_ 8 _Johns Hopkins University, Baltimore, MD_.

Background: Aberrantly expressed miRNAs may contribute to treatment resistance in pediatric acute lymphoblastic leukemia relapse (ALL) and could serve as biomarkers for risk of relapse. However, few studies have evaluated the role of miRNAs in ALL relapse and a consensus clinically significant miRNA signature is yet to be identified. In this study, we identified miRNAs associated with pediatric B-ALL relapse, by profiling ~2,500 miRNAs in two independent sample sets.

Methods: We performed global miRNA profiling using Affymetrix miRNA v 4.0 on two independent samples. Profiling was initially performed on diagnostic bone marrow specimens from 6 early relapse (≤3 years after diagnosis) and 6 age and cytogenetics matched prolonged remission (≥4 years) patients treated at the Johns Hopkins School of Medicine (first set). We then performed profiling on 14 early relapse and 14 matched prolonged remission specimens from patients treated on the Children's Oncology Group trials AALL0331 and AALL0232 (second set). Global miRNA expression was compared between early relapse and remission samples in each set. Differential expression was determined based on fold change (>1) and false discovery rate q-value (<0.05).

Results: Twelve and 39 top differentially expressed miRNAs were observed in the first (fold change=1.48-4.45, q<0.05) and second (fold change=1.12-2.19, q<0.05) sets, respectively. None of the top differentially expressed miRNAs in the two datasets overlapped. However, six miRNAs (miR-101-3p, miR-4774-5p, miR-1324, miR-631, miR-4699-5p and miR-922) were consistently upregulated in early relapse compared to remission specimens in both the first (fold change=1.13-2.19, q<0.38) and second (fold change=1.48-4.78, all q<0.05) sets; albeit these miRNAs did not meet the q-value threshold in the first set. Four (miR-631, mir-101-3p, miR-922 and miR-1324) out of the six overlapping miRNAs have been previously implicated in key functional oncogenic pathways in adult cancers, including multiple myeloma, prostate, lung, liver, breast and head and neck cancers.

Conclusion: This study provides evidence for a set of six miRNAs that are associated with early relapse in pediatric B-ALL. Although some of the differentially expressed miRNAs have been implicated in adult cancers, none of them have been previously implicated in pediatric B-ALL. Therefore, validation of these miRNAs in a larger cohort and investigation of their mechanistic role are warranted.

#1577

**Telomerase reverse transcriptase (** TERT **) promoter mutations in cancers derived from multiple organ sites among Middle Eastern population.**

Rong Bu, Abdul K. Siraj, Kaleem Iqbal, Sandeep Parvathareddy, Mark Diaz, Dionne Rala, Ingrid G. Victoria, Khadija Al-Obaisi, Wael Al-Haqawi, Nabil Siraj, Allianah Benito, Khawla S. Al-Kuraya. _King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia_.

Activation of telomerase induced by telomerase reverse transcriptase (TERT) promoter mutations has been implicated in human carcinogenesis. The two mutations termed C228T and C250T in TERT promoter region have been reported in various human tumor entities with different frequencies in Western populations. However, the contribution of these mutations to cancer progression in the Middle Eastern region remains poorly understood. In this study we investigated the frequency of TERT promoter mutations and TERT protein expression with their association to clinicopathological characteristics in 2113 samples from 13 different types of cancers. The TERT promoter mutations were most frequently present in bladder cancers (68.6%), followed by anaplastic thyroid cancers (50%), Hurthle cell carcinomas (40%), central nervous system (CNS) tumors (28.7%) and follicular thyroid cancers (19%). The low frequency of these mutations was found in medullary thyroid cancers (10%), prostate cancers (9.3%), endometrial carcinomas (3.7%), rhabdomyosarcomas (1.4%), colorectal cancers (CRC) (1%), epithelial ovarian carcinomas (EOC) (0.7%) and breast cancers (0.7%). No mutations were observed in gastric cancers, lung cancers, diffuse large B-cell lymphomas (DLBCL) and acute lymphoblastic leukemias (ALL). In CNS tumors the TERT promoter mutations were significantly associated with older age (p<0.0001), histological subtype (p<0.0001), high grade (p<0.0001) and poor 5 years overall survival (p<0.0001). In gliomas, TERT promoter mutations were more frequently present in cases with old age (p=0.003), high grade (p<0.0001) and poor 5 years overall survival (p<0.0001). Furthermore it was also revealed that co-occurrence of TERT promoter mutations and IDH1 wildtype predicted worst survival (p=0.0001) in gliomas. Our results demonstrate that mutations in TERT promoter is a frequently occurring event in several types of cancers and may be utilized for prognostic prediction in CNS tumors from Middle Eastern Region.

#1578

Cohesin complex-related genes in colorectal cancer.

Masaru Sasaki,1 Norikatsu Miyoshi,1 Kazuhiro Saso,1 Shiki Fujino,1 Hidekazu Takahashi,1 Naotsugu Haraguchi,1 Chu Matsuda,1 Taishi Hata,1 Hirofumi Yamamoto,1 Tsunekazu Mizushima,1 Masaki Mori,2 Yuichiro Doki1. 1 _Osaka University, Osaka, Japan;_ 2 _Kyushu University, Fukuoka, Japan_.

Backgrounds: One of the hallmarks of cancer is chromosomal instability and it leads to aneuploidy. Defective sister chromatid cohesion represents a major cause of chromosome instability in human cancers. Stromal antigen (STAG) encodes a subunit of the cohesin complex, which regulates cohesion and segregation of sister chromatids. STAG contains STAG1, STAG2 related to somatic division and STAG3 related to meiosis. We evaluated the relationship between STAG and colorectal cancer.

Materials and methods: We examined STAG mRNA expression in tumor tissues by real-time qPCR. The samples were obtained from 172 colorectal cancer patients who underwent curative resection from November 2009 to December 2013 at the Osaka International Cancer Institute, Japan. STAG mRNA expression was evaluated by comparing with clinicopathological features and patients' survival. Patients were classified into a high STAG expression and low STAG expression groups. Kaplan-Meier survival curves were evaluated using the log-rank test. The association with STAG expression and prognosis was analyzed by the Cox proportional regression model. Then, we utilized siRNA to knock down the expression of STAG3 and examined whether this altered proliferation in colorectal cancer cell lines (HCT116, RKO, HT29 and SW480).

Results: STAG1 and STAG2 did not show no statistically significant differences in disease-free survival (DFS) and overall survival (OS). In contrast, DFS rate was significantly lower in the high STAG3 expression group than the low STAG3 expression group (P = 0.032). A multivariate analysis showed that the high STAG3 mRNA expression was a significant independent risk factor for the recurrence (P = 0.006). In patients with adjuvant chemotherapy, the high STAG3 expression group was significantly worse for DFS (P = 0.043) and OS (P = 0.020), compared to the low STAG3 expression group. However, the proliferation assays evaluating the knockdown of STAG3 did not show any significant differences.

Conclusions: Our study showed that STAG3 can be a novel prognostic biomarker and the high STAG3 mRNA expression was an independent risk factor for the recurrence, suggesting that it relates to the chemotherapy resistance in colorectal cancer. We perform some other assays to evaluate the biology (focusing on the cancer invasiveness, the drug sensitivity, etc.) which reveal the mechanism of STAG3 in colorectal cancer.

### Genetics and Genomics 2: Risk

#1579

Thyroid hormone pathway genetic variation and risk of prostate cancer in the PRACTICAL Consortium.

Jiaqi Huang,1 Esteban Castelao,2 Han Zhang,1 Maria Elena Martinez,3 Stephanie J. Weinstein,1 The PRACTICAL Consortium, Kai Yu,1 Stephen J. Chanock,1 Manuela Gago-Dominguez,4 Demetrius Albanes1. 1 _NCI-DCEG, Rockville, MD;_ 2 _Complejo Hospitalario Universitario de Vigo, CHUVI, Fundacion de Investigación Galicia-Sur, Vigo, Spain;_ 3 _University California San Diego Department of Population Sciences, Moores Cancer Center, San Diego, CA;_ 4 _Complejo Hospitalario Universitario de Santiago, Instituto de Investigación Sanitaria Santiago IDIS, Santiago de Compostela, Spain; UCSD Moores Cancer Center, San Diego, CA_.

Thyroid hormones play important roles in metabolism and development in most human tissues. Although a hypothyroid state has been linked to lower prostate cancer risk, how variation in thyroid hormone-related genotypes may be related to prostate carcinogenesis has been sparsely studied. We comprehensively examined associations of genetic variation in nineteen key thyroid hormone genes with prostate cancer risk using data from 41,319 men with prostate cancer and 26,315 controls from 36 studies within the PRACTICAL Consortium (Prostate Cancer Association Group to Investigate Cancer Associated Alterations in the Genome). Based on the summary-based Adaptive Rank Truncated Product (sARTP) method, we performed gene- and pathway-based analyses using summary statistics for the associations with overall, low-, intermediate- and highly aggressive prostate cancer with SNPs within the candidate gene regions. We found that accumulated genetic variation in the thyroid hormone pathway is statistically significantly associated with overall prostate cancer risk (ppathway=0.0093). The two most significant genes below the threshold of Bonferroni correction (pgene<2.8×10-3) were THRA and POU1F1. Two SNPs in THRA and one in POU1F1 (rs1568400, rs1879265 and rs1825899, respectively) were most strongly associated, with the latter two being significant below the Bonferroni correction threshold (p<9.7×10-6). Our findings provide evidence to support the hypothesis that genetic variation in thyroid hormone-related genes, and possibly biochemical status, is related to the development of prostate cancer.

#1580

**Genetic variability of H3K27me modifier genes** EZH2 **,** KDM6A **and** KDM6B **in association with pancreatic cancer risk.**

Ye Lu, Ofure Obazee, Federico Canzian. _Germen Cancer Research Center, Heidelberg, Germany_.

Background:

Several literature reports explain the critical role of epigenetic regulation in relation to different types of cancers, however this is still an emerging research area in pancreatic cancer. Methylation of lysine 27 on histone H3 (H3K27me) plays a critical role in regulating gene expression and this modification has been reported to have oncogenic effects. Specifically, in pancreatic cancer this mechanism has only been demonstrated in relation to patient outcome.

The demethylase KDM6A (UTX) or KDM6B (JMJD3, a paralog of KDM6A on a different chromosome), and methyltransferase EZH2 (KMT6A) specifically regulate the alteration level of H3K27me. Based on the hypothesis that genetic variants may influence overall gene function, it is plausible that single nucleotide polymorphisms (SNPs) in these genes may similarly affect the roles of these genes in H3K27me deregulation.

Materials and methods:

Using the SNPinfo tool, a total of 21 tag SNPs in EZH2, KDM6A and KDM6B were selected from European populations based on 1000 Genome project. SNPs below 80% linkage disequilibrium threshold or less than 5% frequency among Europeans were excluded.

These SNPs were analysed in relation to pancreatic cancer susceptibility using logistic regression among up to 8,741 pancreatic cancer cases and 6,957 controls from publically-available GWAS data (Pancreatic Cancer Cohort Consortium (PanScan) and Pancreatic Cancer Case-Control Consortium (PanC4)). Imputation of these data sets were performed according to standard protocol and used for subsequent analyses.

Results:

Following adjustment for age and gender, we observed promising associations between KDM6B rs12453250 C>A and EZH2 rs2888566 T>C ((OR=0.92 95% CI (0.85-0.99), P=0.018) and (OR=1.09 95% CI (1.03-1.17), P=0.007) respectively) and pancreatic cancer risk. Interestingly, rs12453250 results in an amino acid change from Phenylalanine (F) to Leucine (L) which is predicted to have a probably-damaging effect on the KDM6B protein.

Conclusions:

The extent to which epigenetic markers can be incorporated into pancreatic cancer risk prediction along with genetic information is unknown. Our novel findings suggest a plausible role of EZH2 and KDM6A SNPs in relation to pancreatic cancer risk among carriers of their minor alleles. These results warrant further validation in independent studies and may enhance systematic risk assessment among Europeans.

#1581

Gut barrier function-related genes and colorectal cancer risk in Western European populations.

Hannah Mandle,1 Mazda Jenab,2 David Hughes,3 Marc Gunter,2 Elio Riboli,4 Veronika Fedirko5. 1 _Rollins School of Public Health, Emory University, Atlanta, GA;_ 2 _International Agency for Research on Cancer, Lyon, France;_ 3 _Conway Institute, University College Dublin,, Dublin, Ireland;_ 4 _School of Public Health, Imperial College London, London, United Kingdom;_ 5 _Rollins School of Public Health, Winship Cancer Institute, Emory University, Atlanta, GA_.

The gut barrier is a multi-later system consisting of two main components: 1) a physical barrier comprised of a thick mucus layer and the epithelium providing defense against microbes and foreign antigens, and 2) a mucosal immune system differentiating between pathogenic and commensal microorganisms, and responsible for the immune response to pathogens and pathogen-associated molecular patterns such as lipopolysaccharide (LPS). Gut barrier dysfunction and related inflammation are known to be associated with and contribute to the development and progression of colorectal cancer (CRC). Whether this association is modified by genetic variation in the genes related to intestinal mucosal and immune function has not been well studied in humans. Therefore, we investigated 292 functional and tagging single-nucleotide polymorphisms (SNPs) in 27 genes encoding proteins in the pathways related to endotoxins/LPS sensing and tolerance, inflammation, Crohn's disease, and colon mucus synthesis in 1,420 incident CRC cases matched 1:1 to control participants from the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. Previously measured serum flagellin- and LPS-specific IgA and IgG levels, considered as biomarkers of exposure to bacterial products and intestinal permeability, were available for a subset of matched case-control pairs. Multivariable odds ratios and 95% confidence intervals were calculated using unconditional logistic regression, with Benjamini-Hochberg (BH) adjustment for multiple comparison testing. The adaptive rank-truncated product (ARTP) method implemented in R-package PIGE was used for gene- and pathway-level analyses. Thirty one SNPs in 16 genes related to LPS response and tolerance (TLR4, TNFRSF1B, LBP, CD38, CD14), colon mucosal function (ABCB1, MUC6/MUC2, CAMP/ZNF589), inflammation (ALOX5, IL10, IL12B, IL2/IL21, IL6, NFKB1), and Crohn's disease (rs3197999, rs762421) were statistically significantly associated with CRC risk (raw P-values < 0.05), but lost significance after correction for multiple testing. Among controls (n = 692), 10 SNPs in 4 genes (ABCB1, MUC6/MUC2, NFKB1, IL1A/IL1B) were statistically significantly associated with biomarkers of intestinal permeability (raw P-values < 0.05; all non-significant after multiple testing correction). Pathway analyses showed no statistically significant effects on CRC risk for either individual genes or genes grouped into distinct pathways. However, the data suggested possible associations between CRC risk and the genes in the LPS pathway (P = 0.18) and genetic variants previously associated with Crohn's disease (P = 0.08). This large and comprehensive study has identified gut barrier function-related genes and pathways possibly contributing to CRC risk in European populations. Additional studies in large populations and consortia are needed to confirm our findings.

#1582

Catechol-O-methyltransferase (COMT) genotypes modify the effects of beta-carotene supplementation on cancer incidence in a controlled trial: findings from the ATBC Study.

Stephanie J. Weinstein,1 Jiaqi Huang,1 Kathryn Tayo Hall,2 Howard D. Sesso,2 Demetrius Albanes1. 1 _NCI-DCEG, Bethesda, MD;_ 2 _Brigham and Women's Hospital, Harvard Medical School, Boston, MA_.

Large cancer prevention trials of vitamin supplementation have yielded mixed results over the past three decades. Whether and how genetic variation that influences oxidative stress or vitamin absorption, transport, and metabolism can modify responses to supplementation remains to be elucidated. Catechol-O-methyltransferase (COMT) genotypes have been shown to impact the effects of vitamin E (i.e., alpha-tocopherol) supplementation on cancer, and the present pharmacogenetic analysis examines interactions among COMT, beta-carotene supplements, and risk of cancer. In a case-control study nested within the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study of 29,133 Finnish men, we examined overall cancer risk based on the randomized assignment to beta-carotene supplementation (BC; 20 mg daily for 5-8 years) and two common genetic variants in COMT, rs4680 and rs4818 (genotyped by Illumina scans). Based on 2396 cases and 2235 non-cases, Cox proportional hazards models that adjusted for age, smoking intensity and duration, and randomized assignment to the trial vitamin E supplement were conducted to estimate overall cancer risk for the trial and 10-year post-trial periods combined. For rs4680, BC supplementation (versus no BC) was associated with higher cancer risk only in men with the AA (met/met) genotype (HR 1.29, 95% CI 1.08-1.53, P=0.004) as compared with the AG (met/val) and GG (val/val) genotypes (respectively, HR 1.04, 95% CI 0.93-1.16, P=0.52; and, HR 0.99, 95% CI 0.86-1.15, P=0.94). We observed a somewhat similar pattern for rs4818, with the BC supplement being associated with higher cancer risk in men with the GG and GC genotypes (respectively, HR 1.21, 95% CI 0.94-1.55, P=0.14; and, HR 1.15, 95% CI 1.01-1.30, P=0.03), but not the CC genotype (HR 0.99, 95% CI 0.88-1.11, P=0.80). Our findings indicate that the AA (met/met) genotype of COMT rs4680 and the G allele of rs4818 potentiate the increased cancer risk resulting from BC supplementation.

#1583

Genetic interaction analysis among oncogenesis-related genes revealed novel genes and networks in lung cancer development.

Yafang Li,1 Xiangjun Xiao,1 Ivan Gorlov,2 Younghun Han,1 Rayjean Hung,3 Christopher Amos1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Dartmouth College, Hanover, NH;_ 3 _University of Toronto, Toronto, Ontario, Canada_.

The development of cancer is driven by the accumulation of many oncogenesis-related genetic alterations and tumorigenesis is triggered by complex networks of involved genes rather than independent actions. To explore the epistasis existing among oncogenesis-related genes in lung cancer development, we conducted pairwise genetic interaction analyses among 35,031 SNPs from 2027 oncogenesis-related genes. The genotypes from three independent genome-wide association studies including a total of 24,037 lung cancer patients and 20,401 healthy controls with Caucasian ancestry were analyzed in the study. Using a two-stage study design including discovery and replication studies, and stringent Bonferroni correction for multiple statistical analysis, we identified significant genetic interactions between SNPs in RGL1:RAD51B (OR=0.44, p value=3.27x10-11 in overall lung cancer and OR=0.41, p value=9.71x10-11 in non-small cell lung cancer), SYNE1:RNF43 (OR=0.73, p value=1.01x10-12 in adenocarcinoma) and FHIT:TSPAN8 (OR=1.82, p value=7.62x10-11 in squamous cell carcinoma) in our analysis. None of these genes have been identified from previous main effect association studies in lung cancer. We further explored the impact of identified genetic interactions on the expression of involved cancer-related genes using the lung eQTL dataset including a total of 409 subjects with both genotyping and gene expression data. In the study between TSPAN8 gene and rs1882898:rs1705235 SNP pair, we identified decreased gene expression in individuals with reduced lung cancer risk which supports the oncogene function of TSPAN8. In the gene expression analysis between SYNE1 and rs10515157:rs1554783 SNP pair, we also identified consistent evidence between gene expression and lung cancer risk. Finally, we conducted gene set enrichment analysis to reveal potential pathways and gene networks underlying molecular mechanisms in overall lung cancer as well as histology subtypes development. Our results provide evidence that genetic interactions between oncogenesis-related genes play important roles in lung tumorigenesis. Epistasis analysis, combined with functional annotation, provides a valuable tool for uncovering functional novel susceptibility genes that contribute to lung cancer development by interacting with other modifier genes.

#1584

Transcriptome analysis links immune genes allelic expression imbalances to lung cancer.

Yanhong Liu, Spiridon Tsavachidis, Farrah Kheradmand, Margaret R. Spitz, Chris Amos. _Baylor College of Medicine, Houston, TX_.

Background: Genome-wide association study (GWAS) have identified over 45 susceptibility loci for lung cancer; many studies including our own group, have focused on low-frequency and rare coding variants using fine mapping and exome sequencing. This strategy, however, has met with limited success as about 90% of GWAS hits are noncoding and act primarily through altering transcriptional regulation in an allele-specific manner. The RNA-Seq based allele-specific expression (ASE) analysis affords an innovative approach to study preferential expression of an allele in direct relationship to its genotype, providing information on cis-regulatory effects for the expression of putative genes. However currently, there are no lung cancer studies that have rigorously evaluated the ASE variation in lung tumor and adjacent tissues.

Methods: Leveraging The Cancer Genome Atlas (TCGA) resource, we performed transcriptomic-wide ASE analysis using existing RNA-Seq datasets of paired tumor and adjacent tissues from 54 lung adenocarcinoma patients. We first quantified the RNA read counts of Referent and Alternate alleles of heterozygous variants, then evaluated the allelic imbalance on a per-sample basis using Beta-binomial test, and explored the differential ASE between tumor and adjacent tissues using paired Wilcoxon test. Functional regulatory consequences were generated from Ensembl Variant Effect Predictor.

Results: We identified total 208 significant ASEs, including 35 tissue-specific (only in tumor or only in adjacent), 28 sharing, and 145 differential variants. Of the 208 candidates, 41 were from the human leukocyte antigen (HLA) locus (primary DQA2, DQB1, DRB1, H and J), 26 were from the immunoglobulin (IG) superfamily (primary IGH, IGL, IGK and F11R). About 80% candidates were noncoding (mostly in 5' and 3' untranslated regions) and with regulatory features (21 promoter, seven enhancer, seven open chromatin region, two induce nonsense-mediated mRNA decay, one CTCF-binding site, and one transcription factor binding site). Other top genes included MDM2, APOL1, and CTSB. Pathway analyses revealed 27 genes involved in immune response pathway, and 12 genes involved in HLA antigen processing and presentation pathway.

Conclusion: This study is the first transcriptomics ASE analysis in lung adenocarcinoma. The key somatic cis-regulatory ASE variants identified from this study, especially immunogenic allelic variations from HLA and IG genes, could be used for identifying high-risk individuals for targeted lung cancer checkpoint blockade and related immunotherapies.

#1585

Genetic variations in progesterone receptor (PGR)-related genes and mammographic density in premenopausal women: Are the associations mediated by circulating progesterone.

Favour A. Akinjiyan, Yunan Han, Jingqin R. Luo, Aldi Kraja, Catherine M. Appleton, Graham A. Colditz, Adetunji T. Toriola. _Washington University in Saint Louis, Saint Louis, MO_.

BACKGROUND: Progesterone is a proliferative hormone in the breast. Combined estrogen plus progestin use in postmenopausal women increases mammographic density; a very strong risk for breast cancer. Circulating progesterone and genetic variations in progesterone receptor (PGR)-related genes are positively associated with mammographic density in postmenopausal women, but there are limited data in premenopausal women, with conflicting results. Notably, besides hormone therapy, genetic variation in PGR genes are known to modulate circulating progesterone levels. We, therefore, investigated the associations of genetic variations in PGR-related genes (PGR and Progesterone Receptor Membrane Component 1 (PGRMC1) with mammographic density in premenopausal women and determine, for the first time, whether these associations are mediated via circulating progesterone.

METHODS: We genotyped 179 PGR-related single nucleotide polymorphisms (SNPs) (155 for PGR and 24 for PGRMC1 - 17 of which were synonymous variants and were filtered) in 364 cancer-free premenopausal women who had screening mammography at the Joanne Knight Breast Health Center at Washington University School of Medicine, St. Louis in 2016. We used Volpara to determine volumetric percent density, and dense volume. Variants were coded in dominant mode of inheritance. For causal mediation analysis, multivaraite linear regression models were applied to both the mediator (circulating PGR in logarithm scale) with variants and volumetric percent density, and dense volume with variant, PGR in logarithm scale and their interaction, both adjusting for age, body mass index, family history of breast cancer, and parity. The causal mediation effect, direct effect and total effect, by gentoype and averaged across genotypes were estimated with 95% confidence intervals and p values. Significance was claimed with p-value≤0.05.

RESULTS: The mean age of the study participants was 47.5 years. Most participants (42.1%) had volumetric percent density between 3.5-7.5%, equivalent to BI-RADS® category b. Rs2020875 (PGR11) had a positive average direct effect (estimate=0.22, 95% CI, 0.016~0.44, p-value=0.024) and rs645213 (PGR11) had a borderline inverse direct effect (-0.25, -0.48~0.0004, p-value=0.052) on volumetric percent density. Further, rs11224565 (PGR11): 0.19, 0.04~0.34, p-value=0.028 had positive average direct effects on dense volume. None of the mediation effects were statistically signficiant.

CONCLUSIONS: SNPs in PGR-related genes are associated with mammographic density in premenopausal women. Circulating progesterone levels did not mediate the associations between variants in PGR-related genes and mammographic density. Larger studies are needed to confirm our findings.

#1586

ADAM32 is amplified in a wide range of malignancy.

Aditya Rajan, Vijay Tonk, Kishore Bhende, Sharda Singh, Sahil Tonk, Sanjay Awasthi. _Texas Tech University Health Sciences Center, Lubbock, TX_.

Cytogenetic analysis using karyotyping and fluorescence in-situ hybridization (FISH) has been the method of choice for identifying chromosomal abnormalities in cancer and other diseases. FISH is a powerful tool enabling detection the amplification or deletion of specific genes of interest using specific fluorescent labelled DNA probes. It has high sensitivity and specificity and enables detection of chromosomal abnormalities present in many genetic disorders. Unfortunately, FISH is labor intensive, gives information only regarding chromosomal changes specifically designated by the nucleotide sequence of the probes. Recently, DNA microarray techniques have allowed the simultaneous detection of vast number of chromosomal abnormalities including deletions, amplifications and translocations. These assays are suitable for large scale screening to discovery previously unknown associations of chromosomal lesions with many types of cancer. In present studies, we performed a retrospective analyses on anonymized data from the Constitutional v3 Array from the Texas Tech University Cytogenetics Lab (Children's Oncology Group reference lab) on 198 sequential patients to determine the frequency of chromosomal aberrations and their disease associations. The patient population included children and adults. The majority of patients had malignancy, but some had pancytopenia or immune deficiency. We found chromosomal rearrangements, amplifications or deletions at 794 loci. The most commonly affected loci were 8p11.21-8p11.22, 15q11.1-15q11.2, 14q11.2, 14q32.33, 9p24 and 9p13. The 8p11.2 locus was amplified in 81 cases and deleted in 17 cases, an amplification/deletion ratio greater than any other locus. At this locus, the KAT6A, ADAM32 and TACC1 genes are known to associate with cancer, while no associations of GPAT4 and TM2D2 have been reported. The ADAM32 gene amplification, previously identified only in breast cancer, was also found in hepatocellular carcinoma, T-cell Lymphoma, acute lymphoblastic leukemia, acute myelogenous leukemia, Wilm's Tumor, neuroblastoma, rhabdomyosarcoma, fibromatosis, chondroblastoma, enchondroma, and giant cell tumor of the bone. An oncogenic role of ADAM32 should be investigated in a broad spectrum of pediatric and adult malignancy.

#1587

Integrated renal cancer post-GWAS functional studies implicates the SWI/SNF DPF3 gene on 14q24 risk locus.

Leandro Machado Colli, Lea Jessop, Sabrina Y. Camp, Timothy Myers, Mitchell J. Machiela, Jiyeon Choi, Kai Yu, Mark Purdue, Kevin Brown, Stephen J. Chanock. _NCI-NIH, Gaithesburg, MD_.

We investigated regulatory elements involved in20 renal cancer (RCC) susceptibility regions identified by Genome-wide Association Study (GWAS) by an integrated approach. In parallel, we conducted Massively Parallel Reporter Assay (MPRA), Assay for Transposase-Accessible Chromatin (ATAC-seq), RCC eQTL analysis, and Capture-HiC. We identified at least one functional variant in 18 of 20 RCC regions. Cis-eQTL linked 16 RCC regions to as many as 198 genes. The integrated approach underscores the spectrum of effects of genetic variation on regulation of genes critical for RCC. We further pursued the region on 14q24 and confirmed the tag SNP, rs4903064, by luciferase assay. Using an EMSA, the rs4903064 T-allele, associated with lower risk, showed higher binding than the C-risk allele. The T-allele creates a binding site for IRX2/IRX5, which can act as a transcriptional repressor. On the other hand, the C-allele creates a HIF1a binding site. The C-risk allele was associated with higher DPF3 expression, a 14q24 region gene, in the TCGA RCC and in normal kidney eQTL. We created four stable renal cell lines that included a DOX-dependency for overexpression of DPF3 isoforms (DPF3a or DPF3b) and performed RNA-seq analysis; we observed over 500 genes deregulated when either isoform is overexpressed. To investigate chromatin accessibility changes due to DPF3 overexpression, we used ATAC-seq and found 86 regions with altered accessibility and 15% of the RNA-seq deregulated genes are located within 2 MB of one of these regions, including the promoter of the top deregulated gene (CEMIP). In growth curve assays, DPF3 increased the growth rate of renal cell lines. When we knocked down the top two DPF3-deregulated genes (CEMIP and IL23R) using siRNA, DPF3's growth effect was reduced, suggesting that CEMIP and IL23R are important cofactors for growth along with DPF3. Since CEMIP is important for apoptosis, we evaluated apoptosis in our dox-inducible-DPF3 cell lines. DPF3 overexpression decreased the percentage of apoptotic cells, measured by flow cytometry, and reduced Caspase-3 and PARP cleavage, measured by Western blot (WB). The second deregulated gene, IL23R, is connected to STAT3, an important RCC oncogene. By WB, DPF3 overexpression leads to increased STAT3 phosphorylation. Furthermore, we saw evidence of increased activation of the STAT3 signaling pathway by RNA-seq pathway analysis. Lastly, SWI/SNF complex genes have been shown to influence response to immunotherapy. Using co-culture of tumor cells and stimulated PBMCs, we found that DPF3 overexpression in RRC cells leads to an increased PBMC-cytotoxicity, due to an increased proportion of T CD8+ cells. In summary, we integrated RCC post-GWAS functional studies to pinpoint notable variants on 14q24 and suggest that altering DPF3 regulation can perturb the SWI/SNF complex by reduction of apoptosis and STAT3 pathway activation.

#1588

Germline variation in DNA repair genes and risk of Barrett's esophagus and esophageal adenocarcinoma.

Matthew F. Buas,1 Li Yan,1 Xuan Peng,1 Qianya Qi,1 Jianhong Chen,1 Aaron Thrift,2 Qianchuan He,3 Lynn Onstad,3 Puya Gharahkhani,4 Stuart MacGregor,4 Thomas L. Vaughan,3 Margaret M. Madeleine3. 1 _Roswell Park Comprehensive Cancer Center, Buffalo, NY;_ 2 _Baylor College of Medicine, Houston, TX;_ 3 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 4 _QIMR Berghofer Medical Research Institute, Brisbane, Australia_.

Incidence of esophageal adenocarcinoma (EA) has risen substantially in Western countries over recent decades. Established risk factors for EA and its precursor lesion, Barrett's esophagus (BE), include reflux, obesity, and tobacco smoking. Inherited genetic variation also influences disease risk, although only a limited number of susceptibility loci have been identified. Genomic analyses of EA tumors have revealed a distinctive mutational signature, high mutational burden, and extensive somatic chromosome alterations, features also observed in high-risk BE tissue. To explore whether germline variation in DNA repair-related genes may be associated with altered disease susceptibility, we analyzed data from a recent meta-analysis of genome-wide association studies (GWAS) encompassing 4,112 EA cases, 6,167 BE cases, and 17,159 controls, representing the largest sample size assembled for these conditions. Using a gene-based testing approach (VEGAS2), we assessed 263 DNA repair-related genes and found that variation in NEIL2, a mediator of base excision repair (BER), was significantly associated with risk of BE (P=1.4×10-5, q<0.05). No other gene-level associations with BE or EA survived correction for multiple comparisons. SNP-level analysis of 239 polymorphisms at the NEIL2 locus revealed six variants strongly associated with altered risk of BE (P<5×10-5, q<0.01), with the index SNP classified as an intronic eQTL for NEIL2 in esophageal tissue. Four of these SNPs were also associated with risk of EA (P<0.05), with odds ratios in the same direction and of similar magnitude. Our results provide evidence that germline genetic variation in a DNA glycosylase enzyme (NEIL2) may influence risk of BE/EA, and suggest a potential novel biological role for altered BER in BE/EA pathogenesis.

#1589

Breast cancer subtype GWAS in Peruvian breast cancer patients.

Katie M. Marker,1 Tatiana Vidaurre,2 Jeannie Navarro Vasquez,2 Valentina Zavala,3 Silvia Serrano Gomez,4 Lizeth Tamayo,5 Renzo Meza Florez,2 S Casavilca,2 M Calderon,2 JE Abugattas,2 Henry Gómez,2 Hugo Fuentes,2 CL Monge Pimentel,2 Sikai Song,3 Daniel Cherry,6 Scott Huntsman,3 Donglei Hu,3 Elad Ziv,3 Laura Fejerman3. 1 _University of California Berkeley, Berkeley, CA;_ 2 _Instituto Nacional de Enfermedades Neoplasicas, Lima, Peru;_ 3 _University of California San Francisco, San Francisco, CA;_ 4 _Instituto Nacional de Cancerología, Bogota, Colombia;_ 5 _University of Chicago, Chicago, IL;_ 6 _University of California San Diego, San Diego, CA_.

Background: We previously investigated genetic ancestry associations with breast cancer subtypes in Peruvian women. We found that women with human epidermal growth factor receptor 2 (HER2) over-expressing tumors had higher Indigenous American ancestry than women with other subtypes. We found a suggestive association between African ancestry and triple negative breast cancer (TNBC). We hypothesized that these associations could be due to the presence of germline variants in the Indigenous American genome or the African genome predisposing women to HER2 over-expression or TNBC respectively. We conducted genome wide association analyses to explore this hypothesis.

Methods: Blood samples and clinical data were collected from 1195 women with breast cancer at the Instituto Nacional de Enfermedades Neoplasicas in Lima, Peru. Genotypic profiles for 821 women were generated using the Affymetrix Precision Medicine Research Array. Four major breast cancer subtypes were identified using immunohistochemical markers (luminal A, luminal B, triple negative, and HER2 over-expressing). Genetic ancestry was determined using ADMIXTURE. Genome wide association analyses were conducted in PLINK.

Results: Three polymorphisms with the lowest p values in the TNBC GWAS are of interest. A variant, rs16910137, at 10q26, is located in an intron of the Glutaredoxin 3 gene (GLRX3 or TXNL2, OR = 2.45, P = 8.60*10-07). The protein encoded by this gene may inhibit apoptosis and play a role in cellular growth. It has also been described as a biomarker for TNBC. Our own RNA-seq data for TXNL2 in 52 Colombian women with breast cancer showed that the expression level of this gene is higher in women with triple negative (N=9) compared to luminal tumors (N=43) (P=0.06). The other two variants, rs12327440 and rs13381183, are located in an intron of the Mitogen-Activated Protein Kinase 4 (MAPK4) gene at 18q21 (OR = 0.44 and 1.94 respectively, P = 4.92*10-07 and 7.230*10-06). RNA-seq data for MAPK4 suggestively showed that the expression level is higher in women with TNBC compared to luminal tumors (P=0.18). The variant in the HER2 over-expressing GWAS with the lowest p value, rs11594103, at 10p11 is located upstream of the Enhancer Of Polycomb Homolog 1 (EPC1) gene (OR = 2.38, P = 8.03*10-07). The encoded protein has been linked to apoptosis and DNA repair among other functions and suggested as involved in the activation of metastasis.

Conclusions: Our data suggests that a germline variant within the GLRX3/TXNL2 gene might be associated with the risk of developing TNBC compared to other subtypes. This is particularly interesting in the light that previous work suggested that TXNL2 autoantibodies could be biomarker of TNBC. The MAPK4 and EPC1 genes have been previously implicated in multiple types of cancer and our results indicate that they may play a role in the etiology of specific breast cancer subtypes. This supports the importance of conducting genetic association studies in diverse samples.

#1590

Robust evolutionary conservation and pair-wise co-mapping of polygenic colon and lung cancer susceptibility loci.

Lei Quan,1 Alan Hutson,2 Peter Demant2. 1 _Tianjin University of SportSchool of Health and Exrcise Sciences, Tianjin, China;_ 2 _Roswell Park Cancer Institute, Buffalo, NY_.

Comparing chromosomal locations of statistically significant colon and lung cancer susceptibility loci detected by linkage in mouse and rat and by GWAS in humans revealed conservation of position of most of them in spite of the 70 million years of separate rodent and primate evolution.

1. When projected onto their corresponding homologous positions in the mouse genome, 79% of human, and rat loci as well as mouse loci mapped together in 31 distinct short clusters, which indicate presence of at least 31 evolutionary conserved colon/lung cancer susceptibility genes or gene pairs.

2. These co-localizations indicate also an evolutionary conservation of the hierarchy of strength of colon and lung cancer susceptibility genes. Generally, both in linkage and in GWAS studies a subset of genes with the largest effect will reach the significance threshold early, while many other genes will remain beneath it and may reach significance only in later studies, especially meta-analyzes. As we analyzed only significant genes, the co-mapping of their majority into the 31 clusters has been possible only if the subsets of genes with the largest effects are very similar in all three species.

3. In addition, the majority of colon and lung cancer susceptibility QTLs formed linked pairs. Consequently, all 31 clusters contained both colon and lung cancer susceptibility QTLs.

4. This pair-wise genomic location of colon and lung cancer QTLs seemed to be associated with their similar quantitative effects on tumorigenesis, as tests of congenic mouse strains each carrying 87% of BALB/c genome and differing from each other in a small subset of colon cancer QTLs revealed that the two strains with very high colon cancer susceptibility and the two strains with very low colon cancer susceptibility exhibited concordantly very high and very low susceptibility to lung cancer, respectively.

5. It has to be established whether the co-mapping of colon and lung cancer susceptibility QTLs and concordant magnitude of their effect on colon and lung cancer indicate that a single gene or two paired organ-specific genes control development of colon and lung cancer.

6. In summary, genetic location and relative strength of polygenic colon and lung cancer susceptibility QTLs have been surprisingly conserved during separate evolution of rodents and primates. These QTLs exhibit frequent pairwise linkage of colon and lung cancer susceptibility loci and directionally correlated effects on both cancers.

7. The presented findings indicate that a majority of colon and lung cancer susceptibility QTLs are robust genetic and biological entities, whose individual functions in colon and lung carcinogenesis may be effectively studied. As the combined mortality of colon and lung cancers exceeds that of combined five other major cancer sources of deaths, their study can have a significant translational impact.

#1591

Large-scale transcriptome-wide association study (TWAS) identifies novel candidate susceptibility genes for pancreatic cancer.

Jun Zhong,1 Ashley Jermusyk,1 Lang Wu,2 Jason W. Hoskins,1 Irene Collins,1 Mingfeng Zhang,3 Song Lei,1 Charles C. Chung,1 Tongwu Zhang,1 Wenming Xiao,4 PanScan, PanC4, Rachael Stolzenberg-Solomon,1 Alison P. Klein,5 Brian M. Wolpin,6 Xiao-Ou Shu,2 Stephen J. Chanock,1 Sara Olson,7 Nilanjan Chatterjee,8 Jill Smith,9 Jianxin Shi,1 Peter Kraft,10 Gloria M. Petersen,11 Wei Zheng,2 Laufey T. Amundadottir1. 1 _National Cancer Institute, Gaithersburg, MD;_ 2 _Vanderbilt University Medical Center, Nashville, TN;_ 3 _US FDA, Silver Spring, MD;_ 4 _US FDA, Jefferson, AZ;_ 5 _Johns Hopkins School of Medicine, Baltimore, MD;_ 6 _Dana-Farber Cancer Institute, Boston, MA;_ 7 _MSKCC, New York, NY;_ 8 _Johns Hopkins University, Baltimore, MD;_ 9 _Georgetown University, Washington, DC;_ 10 _Harvard T.H. Chan School of Public Health, Boston, MA;_ 11 _Mayo Clinic College of Medicine, Rochester, MN_.

Pancreatic Ductal Adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths in United States with a 5-year survival rate of only 8%. Inherited predisposition plays an important role in PDAC risk. Rare, moderately to highly penetrant mutations in hereditary cancer and pancreatitis genes, identified in families with a high incidence of disease, account for a small fraction of cases. At the other end of the spectrum, common risk variants of low penetrance have been discovered through genome-wide association studies (GWAS). Currently, over 20 common PDAC risk signals have been identified through GWAS in populations of European ancestry. However, these loci explain only a small fraction of genetic heritability for PDAC, and the causal genes for the large majority of these are unknown. Most susceptibility alleles discovered through GWAS are located in noncoding regions of the genome and are likely to function through allele specific regulation of gene expression. Transcriptome-wide association studies integrate expression quantitative trait loci (eQTL) with GWAS data to discover genes whose cis-regulated expression is associated with complex traits. To identify novel susceptibility loci and likely causal genes, we applied this approach to PDAC. Firstly, we used two reference datasets generated from histologically normal pancreatic tissue samples from individuals of European ancestry, the DCEG/LTG (n=95) and Genotype-Tissue Expression (GTEx v7, n=174), to build robust predictors for gene expression (n=2,872-5,906; r2>0.01), using 5 different models (Top1, Lasso, Enet, Blup and Bslmm) in FUSION and PrediXcan. Gene expression predictors (n=2,043-21,422) were also generated by Enet in 48 different tissues (n=74-421 samples) from 608 individuals of European ancestry (GTEx). We then applied these predictor models to PDAC GWAS summary statistics from 9,040 PC cases and 12,496 controls. We discovered 24 genes whose genetically predicted expression is significantly associated with PDAC risk (FDR < 0.05), including 13 candidate genes at 10 novel loci (CELA3B, SMC2, SMC2-AS1, RP11-80H5.9, SMUG1, BTBD6, RCCD1, PNMT, CDK12, PGAP3, SUPT4H1, RP11-888D10.3 and PGPEP1) and 11 at 5 known risk loci (TERT, CLPTM1L, ZDHHC11B, INHBA, ABO, PDX1, KLF5, WDR59, CFDP1, BCAR1 and TMEM170A). Pathway enrichment analysis for genes differentially expressed in the top vs. bottom quartile of CELA3B expression highlighted higher expression of inflammatory and immune response genes in both the DCEG/LTG and GTEx pancreatic transcriptome datasets, indicating that low expression of this gene may be associated with an inflammatory state in the pancreas. Our findings showcase the utility of integrating multiple datasets to generate hypotheses and provide novel clues for the genetic mechanisms underlying pancreatic cancer risk.

#1592

Genome-wide meta-analysis of keratinocytic cancers identifies 26 novel risk loci.

Upekha E. Liyanage,1 Matthew H. Law,1 Xikun Han,1 Jiyuan An,1 Jue-Sheng Ong,1 Puya Gharahkhani,1 Mark M. Iles,2 Rachel E. Neale,1 Catherine Olsen,1 Stuart Macgregor,1 David C. Whiteman1. 1 _QIMR Berghofer Medical Research Institute, 300, Herston Road, Brisbane, QLD 4006, Australia;_ 2 _Leeds Institute for Data Analytics, University of Leeds, Leeds, United Kingdom_.

Background: Basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) (collectively referred to as keratinocytic skin cancers (KC)) are the most common forms of cancer in fair-skinned people. KCs account for the highest expenditure of any cancer type in the Australian healthcare system. Knowledge about the genetic architecture of skin cancer may help elucidate the biology of the disease.

Methods: We conducted a genome-wide meta-analysis of KC, including 28248 cases and 353855 controls from United Kingdom, United States and Australian populations. We performed LD score regression to determine the genetic correlation between KC, BCC, SCC, and melanoma.

Results: We identified 41 independent genome-wide significant SNPs associated with KC risk, 16 of which have not been reported previously. A further 10 loci were implicated using gene-based tests. New loci included common variants in BRCA2 (rare variants increase risk of various cancers) and CTLA4 (immune response; antibodies targeting CTLA-4 are used to treat melanoma). We found a strong genetic correlation between BCC and SCC, 0.93 (95% confidence interval [CI] 0.71 - 1.16). The genetic correlation between melanoma and BCC was 0.66 (95% CI: 0.41 - 0.91). Comparatively lower genetic correlations were identified between melanoma and SCC 0.43 (95% CI: 0.12 - 0.74), and melanoma and KC 0.52 (95% CI: 0.27 - 0.76).

Conclusions: We have identified novel genetic loci associated with KC risk which will lead to a greater understanding of the biology of KC. Future work will leverage the genetic correlations between BCC, SCC and melanoma to further advance our understanding of these cancers.

#1593

Genetic interactions in lung cancer using machine-learning approaches in genome-wide association studies.

Jinyoung Byun, Younghun Han, Christopher I. Amos. _Baylor College of Medicine, Houston, TX_.

Genome-wide association studies (GWAS) consisting of hundreds of thousands or millions of single nucleotide polymorphisms (SNPs) have presented the complex inheritance patterns of disease/trait. Although GWAS have identified thousands of SNPS associated with diseases, few studies have explored interactions among these SNPs that may influence disease risks. Machine learning applications can identify SNPs that influence disease risk through interactions and define how these SNPs jointly influence disease risks. Tree-based machine-learning applications; classification and regression trees (CART) and random forest (RF) methods, have become increasingly popular and convenient tools for understanding interactions influencing disease development. Here we apply these methods to understand the genetic architecture of lung cancer. To elucidate the SNP-SNP interactions that influence lung cancer risk, we applied supervised tree-based approaches using 18,444 cases and 14,027 controls from lung cancer OncoArray GWAS data that is the largest lung cancer GWAS so far. To reduce the space of SNPs to consider in modeling, we first selected the SNPs very significantly (p<0.00001) associated with lung cancer risk. Random Forests, which consists of systematically fitting classification trees, was run 1,000 times to identify the most influential SNPs that jointly influence lung cancer risk. Subsequently we applied a classification tree approach to summarize interactions that predict risk. The final parsimonious tree included effects from genetic variants in rs55781567(CHRNA5): rs452384(CLPTM1L): rs9258608(LOC105375010): rs6154144(DNAJC5): rs116506680(HLA-G): rs9271365(near HLADQA1): rs421629(CLPTM1L). The nodes of this tree had Odds ratios for lung cancer ranging from 0.87 to 1.63. Machine-learning approaches in genomics can provide an important genetic mechanism in lung cancer development.

#1594

Transcriptome-wide association study analysis of nasopharyngeal carcinoma.

Wen-Hui Su, Fu-Chun Chan. _Chang Gung University, Taoyuan, Taiwan_.

Arising from the epithelial cells in nasopharynx, nasopharyngeal carcinoma (NPC) is highly prevalent in Southeastern China, including Hong Kong and Taiwan. Genetic and environmental factors play important role in NPC susceptibility. Genome-wide association studies (GWASs) have identified single-nucleotide polymorphisms (SNPs) located in major histocompatibility complex (MHC) region with strong evidence for association with NPC. Recently, combination of GWAS and expression quantitative trait loci (eQTL) datasets, transcriptome-wide association study (TWAS) was used to identify novel associations in transcriptome level. In this study, we conducted TWAS in NPC using two in house GWAS datasets and GTEx eQTL datasets from public domain. Most of the significant results were located in chromosome six MHC region. Our analysis identified significant TWAS genes that explain the most of the GWAS signals : HLA-A, GABBR1 and HLA-F genes with P values of 5.08E-13, 5.78E-09 and 2.30E-08, respectively. One of the novel genes, ZNRD1 (P = 8.22 E-08), was known to be involved in the tumorigenesis of esophageal cancer and liver cancer. Further analysis should be needed to understand the mechanisms between ZNRD1 association in NPC.

#1595

Functional genomic analysis of 18q21.1 locus identifies potential functional variants and genes for colorectal cancer.

Jifeng Wang,1 Xingyi Guo,1 Shimian Qu,1 Joshua A. Bauer,2 Jirong Long,1 Wei Zheng,1 Qiuyin Cai1. 1 _Division of Epidemiology, Department of Medicine, Vanderbilt Epidemiology Center, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN;_ 2 _Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN_.

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers in the world. Genome-wide association studies have identified approximately 50 genetic risk loci for CRC. We have previously identified SNP rs7229639, located in the second intronic region of the SMAD7 gene at locus 18q21.1, in relation to CRC risk in Asians. In this study, we performed functional genomic analysis to identify potential functional variants and target genes for this association. Functional annotation analysis using epigenetic data from ENCODE and Roadmap projects showed functional evidence of promoter and/or enhancer activities for four highly correlated potential functional SNPs, including rs7229639, rs77544449, rs112540150 and rs57668004. Using in vitro luciferase reporter assays in colon cancer HCT116 and PKO cells, we showed that the risk alleles of rs7229639 and rs77544449 increase promoter or enhancer activities, compared to reference alleles. The risk alleles of both SNPs also affect the ability of interacting with nuclear proteins in the electrophoretic mobility shift assays, compared to reference alleles. To identify candidate target genes for this risk locus, we performed cis-expression quantitative trait loci (eQTL) analysis using transcriptome data from the Genotype-Tissue Expression (GTEx) project, The Cancer Genome Atlas (TCGA) and Colonmics. We identified putative target gene LIPG, located 500 kb downstream of rs7229639, in all three datasets. No associations were observed for SMAD7, or eight other genes in the region, via eQTL analysis. To investigate the biological functions of LIPG gene in colorectal cancer, we knock-downed LIPG gene expression using siRNA in HCT116 and PKO cells, which resulted in a significant decrease in cell proliferation, colony formation, migration and invasion in both HCT116 and PKO cells. These results suggest that LIPG gene plays a significant role in colorectal tumorigenesis via disruption of cell behaviors. Taken together, results from our study suggest that the association between the rs7229639/18q21.1 locus and CRC risk may be mediated through SNPs that regulate expression of the LIPG gene.

#1596

Genome-wide gene-environmental interaction and random survival forest analyses: Insulin resistance and breast cancer risk.

Su Yon Jung,1 Herbert Yu,2 Matteo Pellegrini,1 Jeanette Papp,1 Eric Sobel,1 Zuo-Feng Zhang1. 1 _University of California, Los Angeles, Los Angeles, CA;_ 2 _University of Hawaii Cancer Center, Honolulu, HI_.

Purpose: Obesity–insulin connections have been considered potential risk factors for postmenopausal breast cancer, and the association between insulin resistance (IR) genotypes and phenotypes can be modified by obesity-lifestyle factors, affecting breast cancer risk. We explored the role of IR in those pathways at the genome-wide level. With identified IR genetic factors and selected lifestyles, we further generated risk profiles for breast cancer risk.

Methods: Using large-scale cohort data from postmenopausal women in the Women's Health Initiative Database for Genotypes and Phenotypes Study, our previous genome-wide association gene–behavior interaction study identified 58 loci for associations with IR phenotypes (homeostatic model assessment–IR, hyperglycemia, and hyperinsulinemia). We evaluated those single-nucleotide polymorphisms (SNPs) and additional 31 lifestyles in relation to breast cancer risk by conducting a two-stage multimodal random survival forest analysis.

Results: We identified the most predictive genetic and lifestyle variables in overall and subgroup analyses (stratified by body mass index [BMI], exercise, and dietary fat intake). Two SNPs (LINC00460 rs17254590 and MKLN1 rs117911989), endogenous and exogenous factors related to lifetime cumulative exposure to estrogen, BMI, and dietary alcohol intake were the most common influential factors across the analyses. Individual SNPs did not have significant associations with breast cancer, but the combined SNPs and lifestyles synergistically increased the risk of breast cancer in a dose-dependent manner. In subgroup analysis, we further stratified women by the two strongest variables (durations of oral contraceptive [OC] and opposed estrogen use). Shorter-duration OC and ever-opposed estrogen users with both risks of genotypes and lifestyles had greater risk (2.5 times and 6.3 times, respectively) than their counterparts without risks in both. This indicates the combined effect of SNPs and lifestyles and the joint effect with OC use and opposed estrogen on breast cancer risk.

Conclusions: Our findings may contribute to more accurate prediction of breast cancer and suggest intervention strategies for those women with genetic risk to reduce their breast cancer risk.

#1597

Real-world large-scale study kinase domain duplications across diverse tumor types in Chinese populations.

Chun-wei Xu,1 Wen-xian Wang,2 Xiao-jia Wang,2 You-cai Zhu,3 Qu-xia Zhang,1 Yong Fang,4 Xiu-yu Cai,5 Yu Chen,1 Li Lin,6 Hong Wang,7 Mei-yu Fang,2 Yin-bin Zhang,8 Shi-jie Lan,9 Xin Liu,10 Xing-xiang Pu,11 Zong-yang Yu,12 Bing Wan,13 Jian-ying Li,14 Xian-bin Liang,15 Li-ping Wang,16 Wu Zhuang,1 Ling Lin,17 Gang Chen,1 Tang-feng Lv,18 Yong Song18. 1 _Fujian Cancer Hospital, Fuzhou, China;_ 2 _Zhejiang Cancer Hospital, Hangzhou, China;_ 3 _Zhejiang Rongjun Hospital, Jiaxing, China;_ 4 _Sir Run Run Shaw Hospital, Hangzhou, China;_ 5 _Sun Yat-sen University Cancer Hospital, Guangzhou, China;_ 6 _Peking University International Hospital, Beijing, China;_ 7 _Affiliated Hospital of Academy of Military Medical Sciences, Beijing, China;_ 8 _The Second Affiliated Hospital of Medical College, Xi'an Jiaotong University, Xi'an, China;_ 9 _The First Hospital of Jilin University, Changchun, China;_ 10 _Fudan University Shanghai Cancer Center, Shanghai, China;_ 11 _Hunan Cancer Hospital, Changsha, China;_ 12 _Fuzhou General Hospital, Nanjing Military Area, Fuzhou, China;_ 13 _The Affiliated Jiangning Hospital of Nanjing Medical University, Nanjing, China;_ 14 _Nantong Tumor Hospital, Nantong, China;_ 15 _The Third People's Hospital of Zhengzhou, Zhengzhou, China;_ 16 _Baotou Cancer Hospital, Baotou, China;_ 17 _Taizhou Hospital of Wenzhou Medical University, Taizhou, China;_ 18 _Jinling Hospital, Nanjing, China_.

Background: Recently, kinase domain duplications (KDD) in BRAF, EGFR, MET and FGFR1 were reported, along with responses to tyrosine kinase inhibitors (TKI). However, its frequency and clinical outcomes in advanced cancer patients are larglely uncertain. We assessed the frequency of KDD across 14, 491 advanced cancers to reveal the landscape in a wide variety of subtypes.

Methods: A multicenter study in China was initiated from July 2013, and advanced cancer patients have been enrolled as of September 2018. We analyzed data from 14, 491 clinical advanced cancer cases, each of which had results from next-generation sequencing (NGS)-based 381 genes panel assay, analogous to the index patient.

Results: Of this entire cohort [6837 lung cancer (47.18%), 1894 breast cancer (13.07%), 1325 colorectal cancer (9.14%), 710 urinary system tumor (4.90%), 536 gynecological tumor (3.70%), 592 hepatobiliary cancer (4.09%), 221 gastric cancer (1.53%), 312 soft tissue sarcoma (2.15%), 260 head and neck cancer (1.79%) and 1804 others (12.45%)], 71 patients were identified with KDD, including EGFR (16), BRAF (12), MET (11), FGFR3 (8), RET (3), PDGFRA (3), ROS1 (3), FGFR1 (2), ERBB2 (2), SOX17 (2), ALK (2), KIT (1), NTRK1 (1), BAP1 (1), TMPRSS2 (1), ERBB4 (1), VHL (1), NTRK3 (1). KDD were seen in 0.39% (27/6837) of lung cancer [EGFR (9), BRAF (2), MET (6), FGFR3 (2), RET (1), PDGFRA (1), ROS1 (2), ERBB2 (1), ALK (1), NTRK1 (1), and ERBB4 (1)]; 0.21%(4/1894) of breast cancer [BRAF (1), FGFR3 (2), FGFR1 (1)]; 0.60%(8/1325) of colorectal cancer [EGFR (1), BRAF (3), FGFR3 (2), RET (1), ROS1 (1)]; 0.28%(2/710) of urinary system tumor [TMPRSS2 (1), VHL (1)]; 0.19%(1/536) of gynecological tumor [SOX17 (1)]; 0.17%(1/592) of hepatobiliary cancer [EGFR (1)]; 0.45%(1/221) of gastric cancer [ERBB2 (1)]; 0.96%(3/312) of soft tissue sarcoma [EGFR (1), FGFR3 (1), PDGFRA (1)]; 0.77%(2/260) of head and neck cancer [BRAF (2)]; and 1.22%(22/1804) of others [EGFR (4), BRAF (4), MET (5), FGFR3 (1), RET (1), PDGFRA (1), FGFR1 (1), SOX17 (1), ALK (1), KIT (1), BAP1 (1), and NTRK3 (1)]; KDD possibly related to target resistance were seen in ERBB2 amplification gastric cancer and ALK-related NSCLC.

Conclusion: Diverse KDD are found across diverse tumor types and may underlie acquired resistance, and can benefit from matched targeted treatment. In addition, for short- or long-term responses to targeted treatment, we can use the NGS assay to explore differential gene alter in the future.

#1598

Deep targeted sequencing of colorectal cancer cases to study associations of molecular subtypes with clinical, genetic, and lifestyle risk factors.

Syed H. Zaidi,1 Amanda I. Phipps,2 Tabitha A. Harrison,2 Robert S. Steinfelder,2 Quang Trinh,1 Barbara L. Banbury,2 Adilya Rafikova,1 Megan Van Tassel,1 Emma Reid,1 Stefanie Brezina,3 Marios Giannakis,4 Charles S. Fuchs,5 Li Hsu,2 Andrea Gsur,3 Shuji Ogino,6 Steven Gallinger,1 Polly A. Newcomb,2 Peter T. Campbell,7 Wei Sun,2 Thomas J. Hudson,1 Ulrike Peters2. 1 _Ontario Institute for Cancer Research, Toronto, Ontario, Canada;_ 2 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 3 _Medical University of Vienna, Vienna, Austria;_ 4 _Dana Farber Cancer Institute, Boston, MA;_ 5 _Yale Cancer Center, New Haven, CT;_ 6 _Harvard Medical School, Boston, MA;_ 7 _American Cancer Society, Atlanta, GA_.

Colorectal cancer (CRC) is the 3rd most common cancer in men and women, excluding skin cancer, and the 2nd leading cause of cancer death in the United States. In this biologically heterogeneous disease, a comprehensive molecular characterization is valuable for understanding tumorigenesis and studying associations with clinical, lifestyle, environmental, and germline genetic factors.

The Colon Cancer Family Registry (CCFR) and Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO) collected clinical, germline genetic, and epidemiologic data from over 125,000 CRC cases. In a subset of 2,500 CRC cases, we conducted targeted deep sequencing of tumor and matching normal DNA. The custom AmpliSeq sequencing panel includes 205 genes, which were prioritized from literature and analyses of existing CRC tumor whole exome sequencing data. Next-generation sequencing was conducted on Illumina Hi-Seq 2500, and mean sequence coverages of 748x and 287x were attained for tumors and normal DNA samples, respectively. So far, 1,705 cases have been analyzed. Strelka and MuTect were used to identify somatic single nucleotide variants, and VarsScan2, Verdict, and Strelka were used to call indels. Orthogonal technologies were used for validation and improving mutation calling algorithms. Approximately 18% of tumors were hypermutated, among which 70% exhibited microsatellite instability (MSI). Hypermutated tumors with MSI occurred more frequently in proximal colon compared to distal colon and rectum (OR, 10.10; 95% CI, 6.98-14.63; P<0.0001). Hypermutated tumors carried non-silent mutations in DNA mismatch repair genes (MSH2, MSH6, MLH1, MLH3, and PMS2), POLE, and POLD1. In POLE, in microsatellite stable hypermutated tumors, non-silent mutations were frequent in the exonuclease domain. Approximately 96% of non-hypermutated and 99% of hypermutated tumors contained non-silent mutations in genes in the Wnt/beta-catenin, p53, receptor tyrosine kinases/RAS, transforming growth factor-beta, and phosphatidylinositide 3-kinases pathways. Most tumors carried non-silent mutations in genes in more than one of these signaling pathways. The APC gene was the most significantly mutated gene in non-hypermutated tumors followed by TP53, KRAS, and PIK3CA. In hypermutated tumors, RNF43 was the most significantly mutated gene followed by BMPR2, APC, and BRAF.

CRC-specific survival was significantly more favorable among individuals with hypermutated tumors, regardless of POLE mutation status (HR=0.36, 95% CI: 0.23-0.57). This survival benefit was attenuated in analyses of overall survival (HR=0.82, 95% CI: 0.65-1.02).

This large dataset is being used to study associations with clinical, lifestyle, and environmental factors. This study will provide valuable information to develop better strategies for the prevention, diagnosis, and treatment of CRC.

#1599

Determining patient ancestry based on targeted tumor comprehensive genomic profiling.

Justin Newberg, Caitlin Connelly, Garrett Frampton. _Foundation Medicine Inc, Cambridge, MA_.

Background. Cancer gene mutations exhibit mutation patterns of prevalence that vary across different ancestry groups. For example, EGFR variants are more frequent in non-small cell lung cancer among people with Asian ancestry, and KRAS variants are observed more frequently in colorectal cancer among patients with African American ancestry. Additionally, many histological subtypes of cancers demonstrate differences in prevalence between people of different ancestry. However, many cancer studies lack the statistical power to identify such nuances. The large cohort of patients who have undergone comprehensive cancer genomic profiling at Foundation Medicine may provide a useful starting point for characterizing these mutation patterns across ancestry groups.

Methods. To establish the ancestry on de-identified samples, we superimposed SNPs targeted by each of our comprehensive genomic profiling tests (FoundationOne, FoundationOne CDx, FoundationOne Heme) with Phase 3 1000 Genomes data. Using an established approach, we projected the SNPs down to the top five principal components and used random forest ensemble learning to train a classifier on each bait set. 10-fold cross-validation indicates this approach performs with 98-99% precision and recall for the different genomic profiling tests.

Results. Ancestry calls were made on over 170,000 de-identified samples consented for research. Initial analyses indicated that classification of American samples was not as robust as other groups. To address this, we trained classifiers on a per-chromosome basis, and re-assigned samples which exhibited less than 80% consensus across chromosomes to an admixture group. Overall prevalence of patient ancestry in the dataset is 75.9% European, 8.3% African, 4.7% East Asian, 0.8% South Asian, and 0.8% American, and 9.5% admixed. From the resulting data, we summarize cancer types that are well-represented across populations, identifying at least 28 tumor types for which we likely have power to identify ancestry-dependent somatic mutations.

Discussion. The dataset described contains a previously unavailable set of cancer types to be mined for ancestry-dependent cancer-driving alterations. Those results will be presented. The ancestry classification approach described in this work can be applied to a range of genomic profiling tests, and refinements on this approach can be integrated into clinical trials and ultimately clinical care to better elucidate varied biologic behavior across advanced cancer.

#1600

A literature review of whole exome and genome sequencing population studies for identifying novel germline mutations in relation to cancer risk.

Rolando Barajas, Elise Hoover, Mindy Clyne, Tram Lam, Leah Mechanic, Alisa Goldstein, Elizabeth Gillanders, Melissa Rotunno. _National Cancer Institute, Bethesda, MD_.

The application of Next Generation Sequencing (NGS) technologies in cancer research has accelerated the discovery of somatic mutations; however, it has not mirrored the same progress in the identification of cancer associated germline variation. With the goal to inform future studies on commonly used methods and their degree of success and limitations in identifying germline variants associated with cancer, we performed a review of the English literature for genome-wide germline sequencing studies aimed at identifying new cancer susceptibility genes or variants. A key words search in 2005-April 2017 PubMed yielded 7,349 articles which were checked against the following exclusion criteria: non-cancer (1852), somatic mutations (1764), methods (905), reviews (872), non-human (735), expression (272), targeted sequencing (224), commentaries (170), epigenetic (135), non-English (89), functional (85), clinical studies (51), others (44). The remaining 151 publications were examined in-depth and coded in an Access database according to ~60 fields, capturing information on: publication (journal, year, author, title, abstract), study design (goal; cancer type; population and ethnicity; discovery, technical validation, and replication phases; numbers and characteristics of cases, controls, families used in each phase), sequencing technique (center, technology, coverage, reads aligner, variants caller, QCs), data analysis (variants annotation, candidate genes, filtering strategy, association test), in silico and experimental functional assessment, and key conclusions (main results, challenges, next steps). The discovery phase of the examined papers was largely based on familial cases (77%), exome/genome sequencing of one single family (31%), and restricted to the analysis of candidate genes lists. The distribution of ethnicity for the sequenced cancer patients was 38% European, 11% Asian, 10% African, 5% multi-ethnic, 4% African American, 3% Latino/Hispanic, 3% Middle Eastern, 1% Jewish, and 25% unreported. Breast, prostate, colorectal, and lung cancer represented ~40% of the assessed articles, and the remaining 60% a diverse group of rare cancer types. Most studies (~80%) included a technical validation phase, but without clear success rate. Only 60% of studies included a replication phase in an independent population. Although most studies (~80%) described the identification of multiple germline variants and genes thought to have a role in cancer susceptibility, the supporting evidence was often disparate or not clearly stated. Our findings underscore the importance of establishing standards for reporting filtering strategies and rational for variant identification. We identified untapped potentials in the utilization of more/larger families, the harmonization of results across studies, and the expansion beyond candidate genes.

#1601

Genetic and epigenetic intratumor heterogeneity of lung adenocarcinoma.

Wei Zhao,1 Xing Hua,1 Tongwu Zhang,1 Dario Consonni,2 Angela Cecilia Pesatori,2 Jianxin Shi,1 Maria Teresa Landi1. 1 _National Cancer Institute, Rockville, MD;_ 2 _University of Milan, Milan, Italy_.

Genomic studies have revealed remarkable intratumoral heterogeneity (ITH) in lung adenocarcinoma (LUAD) and increasing evidence suggests its impact in cancer treatment. However, to what extent epigenomic abnormalities and somatic copy number alterations (SCNAs) are involved in shaping genetic clones in lung tumors and are associated with clinical outcome is under-appreciated. Here we applied Infinium HumanMethylation450K BeadChips, Infinium Global Screening Arrays and ultra-deep (>500X) targeted sequencing of 35 LUAD driver genes to 369 samples (including 77 normal tissues) from 84 LUAD patients to investigate the genetic and epigenetic architecture of subclonal diversification. SCNAs were mostly clonal events. For patients with more than three tumor regions assayed, 66% SCNAs were located on the trunk of the evolutionary trees. Similar evolutionary trajectories were inferred from SCNAs and DNA methylation. The driver genes' sequencing depth allowed to identify subclonality even in TP53, EGFR, and BRAF mutations, which were largely reported as clonal events in previous studies. Elevated copy number ITH was associated with higher risk of death, while no significant association was found between clinical outcome and DNA methylation ITH or heterogeneous CpG island methylator phenotype (CIMP), in contrast with the observed prognostic capability of DNA methylation ITH in hematological malignancies. These findings expand our understanding of the coevolution of genetic and epigenetic aberrations in tumorigenesis, the subclonal complexity for therapeutic targets and the implication of ITH in cancer prognosis.

#1602

Genomic profiling of lung cancer associated with idiopathic pulmonary fibrosis.

Rintaro Noro, Akihiko Miyanaga, Aya Fukuizumi, Shinobu Kunugi, Teppei Sugano, Miwako Omori, Yuji Minegish, Jitsuo Usuda, Masahiro Seike, Kaoru Kubota, Mamiko Hirao, Kuniko Matsuda, Akihiko Gemma. _Japan, Tokyo, Japan_.

Idiopathic pulmonary fibrosis (IPF) is reportedly associated with an increased risk of lung cancer. However, optimal diagnosis and treatment for lung cancer associated with IPF remains unclear. In this study, we aimed to clarify the mechanism of carcinogenesis and progression in lung cancer with IPF. We used tumor tissues and adjacent non-tumor lung tissues from 29 lung cancer patients who had undergone complete surgical resection at Nippon Medical School Hospital between 2013 and 2017. Whole-Exon Sequencing was performed on 29 paired samples, consisted of 19 lung cancers with IPF (IPF group) and 10 lung cancers with normal lung (non-IPF group). The candidate gene alterations discriminating between the IPF and non-IPF group, were identified using the random-forest method, RelifF method along with the data from Catalogue Of Somatic Mutations In Cancer (COSMIC). Twenty-five gene alterations were specifically found in IPF tissues and/or tumor tissues with IPF. Whenever 5 of 25 genes including MUC2 and TTF1, were altered in both IPF tissues and tumor tissues, 20 of 25 genes including CADM1, were altered in only tumor tissues. We are undergoing the validation study using other tissues in independent cohorts. Among the candidate genes, Cell adhesion molecule genes (CADM1) gene alterations were frequently found in 15 out of 19 in only tumor tissues with IPF. The CADM1 protein expression was decreased in tumors than in non-tumor fibrotic tissues by western blotting. The CADM1 induced the epithelial-mesenchymal transition (EMT) on the multifunctional cell process involved in the pathogenesis of fibrosis in vitro study. Whenever the down-regulation of CADM1 was frequently detected in various human cancers through its allelic loss as well as hypermethylation within promoter region in addition to inactivate gene mutation, the up-regulation of CADM1 was frequently detected in paired IPF tissues. The CADM1 may be a target candidate in lung cancer associated with IPF. 

## PREVENTION RESEARCH

### Nutrition, Screening, and Early Detection for Cancer Prevention

#1603

**Valproic acid and hydralazine reverse increased mammary cancer risk and upregulate Cdkn2a/p16 in mice exposed to a high fat diet** in utero **.**

Leena A. Hilakivi-Clarke, Fabia de Oliveira Andrade, Nguyen Nguyen, Kerrie Bouker. _Georgetown University Medical Center, Washington, DC_.

Maternal or paternal high fat (HF) diet that modifies the epigenome in germ cells and fetal somatic cells can program increased susceptibility to breast cancer among offspring. Resulting epigenetic changes include transgenerational upregulation of DNA methyltransferases (DNMTs). We studied here if this increased breast cancer risk can be reversed by treating HF offspring with a well-tolerated drug that inhibits DNA methyl transferase (DNMT; hydralazine), combined with a histone deacetylase (HDAC) inhibitor valproic acid (VPA). Adult C57BL/6NTac mouse offspring of dams fed either corn oil -based HF or control diet during pregnancy were treated with 5 g/kg/day VPA and 5 mg/kg/day hydralazine in drinking water, starting after mice received 7,12-dimethylbenz[a]anthracene (DMBA) to initiate mammary cancer. Treatment with VPA/hydralazine prevented the increase in mammary cancer risk associated with in utero HF diet exposure: it reduced mammary tumor multiplicity and lengthened tumor latency, compared with non-treated HF offspring. These drugs downregulated DNMT3a protein levels, and reversed down-regulation of Cdkn2a/p16 mRNA in the mammary tumors of HF offspring. p16 is a tumor suppressor that is often epigenetically silenced in human breast cancers. However, in control offspring not exposed to HF diet in utero, VPA/hydralazine increased mammary tumor incidence and burden. Further, this treatment upregulated genes involved in the unfolded protein response pathway in in utero control diet exposed animals that were already upregulated in non-VPA/hydralazine treated mammary tumors from in utero HF diet exposed animals, including HIF-1α, NFkB, PERK and SQSTM1 p62. Thus, VPA and hydralazine had opposing effects on breast cancer risk in HF and control offspring, suggesting the importance of individually tailoring breast cancer prevention strategies.

#1604

**Modulating effects of genistein in a mouse model of conditional** BRCA1 **deletion and triple negative breast cancer cells.**

Micah G. Donovan, Ornella I. Selmin, Thomas C. Doetschman, Donato F. Romagnolo. _University of Arizona, Tucson, AZ_.

Germline mutations in BRCA1 (BRCA1+/-) confer ~72% risk of breast cancer, the majority of which are triple negative breast cancer (TNBC). Hypermethylation of BRCA1 is observed at high frequency in sporadic TNBC and may play a role in tumor development in BRCA1 mutation carriers. The aryl hydrocarbon receptor (AhR) coordinates epigenetic silencing of BRCA1 and is overexpressed in some TNBC. The purpose of our study is to investigate the effect of dietary genistein on BRCA1 epigenetic regulation, the AhR pathway and mammary tumorigenesis in BRCA1+/- and BRCA1+/+ mice. We used Cre-lox recombination to derive a mouse model of conditional BRCA1+/-. We used BRCA1F22/24 (The Jackson Laboratory, stock no. 017835) mice, which harbor loxP sites flanking exons 22-24 of the BRCA1 gene. We crossed dams that were heterozygous for BRCA1F22/24 (BRCA1+/FL) and transgenic for Cre recombinase (TG) under control of the mouse mammary tumor virus (MMTV) promoter with wildtype NJ males to derive our colony. Breeding pairs were administered either control or genistein-enriched (4 ppm) diets upon set up, which were continued throughout the life of the offspring. Changes in mRNA expression and protein levels were determined, respectively, by RT-PCR and Western blot. Cre was not expressed in mammary glands of non-TG offspring and Cre expression had no effect on BRCA1 mRNA levels in BRCA1+/+ mice, which do not harbor BRCA1 loxP sites. BRCA1 expression was ~40% decreased in mammary glands of TG, BRCA1+/FL mice compared with TG, BRCA1+/+ mice. Hepatic BRCA1 expression was not changed, confirming conditional BRCA1 knockdown. Preliminary data indicate BRCA1 expression in mammary glands of genistein-fed TG, BRCA1+/+ mice was elevated (~1.3 fold) compared with control-fed mice. Moreover, the genistein diet induced a compensatory increase in BRCA1 expression in mammary glands of TG, BRCA1+/FL mice. We found genistein rescued BRCA1 levels in TNBC HCC38 cells that have constitutively active AhR and hypermethylated BRCA1. These data suggest dietary genistein may have protective effects against tumorigenesis in these mice. To that end, we are currently monitoring tumor development, BRCA1 methylation, and changes in the AhR pathway in BRCA1+/+ and BRCA1+/FL mice fed either the genistein-enriched or control diet.

#1605

Time-dependent maternal soybean genistein exposure leads to later-life breast cancer chemoprevention in mice.

Yuanyuan Li, Min Chen, Shizhao Li, Trygve Tollefsbol. _Univ. of Alabama at Birmingham, Birmingham, AL_.

Breast cancer is the most common type of cancer and the second leading cause of death among women in the United States. Studies have shown that both environmental and genetic factors contribute to breast cancer risk over women's lifespans. Early-life nutritional status has a significant impact on the uterine environment and subsequent fetal development, predisposing the developing fetus to certain diseases such as breast cancer in adult life. During these windows of susceptibility including maternal pre-pregnancy, pregnancy and lactation periods, certain dietary exposure during these critical time periods may change breast cancer risk in an individual's later life. Alterations of the maternal diet have been shown to induce modifications in the fetal epigenome. Nevertheless, the vulnerability to maternal nutrition status during the critical developmental stage provides an excellent opportunity to re-program epigenetic profiles that may lead to a beneficial outcome such as cancer prevention in the offspring. The bioactive dietary component, genistein (GE) enriched in soybean products, is an important epigenetic modulator that can prevent various cancers including breast cancer. Our study focuses on how maternal dietary soybean GE exposure during the specific time periods may prevent breast cancer in later-life and the potential mechanisms. In this study, we included multiple exposure time points of maternal GE diet (250 mg/kg) including prior-pregnancy (PP-GE), long-term (LT-GE) and short-term (ST-GE) exposures to further determine the efficacy of these strategies on transplacental chemoprevention of breast cancer in the progenies of two spontaneous breast cancer transgenic mouse models. Our pilot study indicates a time-dependent effect of maternal GE exposure on early-life breast cancer development in spontaneous breast cancer mouse models. We also integrated transcriptome and methylome data from mouse breast tumors and identified several candidate genes such as Trp63 and Tprg in response to maternal GE treatment, and considered to play important roles in regulation of development and tumorigenesis. These results suggest that an appropriate exposure window to soybean dietary GE could be a key factor for prevention of human breast cancer and comprehensive mechanisms may be involved in dietary GE-induced in utero breast cancer intervention.

#1606

The ethyl ester forms of the n-3 fatty acids eicosapentaenoic acid and docosahexaenoic acid decrease the proliferation, invasion, and matrixmetalloproteinase activity of colorectal cancer cells.

Eric Pfister, Michelle A. Lane. _Texas State University, San Marcos, TX_.

More than half of individuals diagnosed with colorectal cancer (CRC) will die from their cancer. The 5-year survival rate for patients diagnosed metastatic CRC is only 14%. Effective and well-tolerated therapies, such as dietary supplements, that prevent metastasis may increase the survival of these patients. The ability of the n-3 fatty acids (n-3FAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) to decrease the proliferation of a variety of cancers in vitro, including CRC, is well established; however, the efficacy of n-3FAs with respect to inhibition of CRC metastasis has received little attention. EPA- and DHA ethyl esters (EE) are alternative forms of n-3FAs that may be more efficacious than free, non-esterified n-3FAs due to their ability to resist degradation allowing them to exert their effects for longer durations. Our objectives were to (1) compare the efficacy of free vs EE-n-3FAs with respect to inhibition of CRC cell proliferation, (2) to determine if EE-n-3FAs decreased CRC cell invasion, and (3) to begin to elucidate the mechanism through which EE-n-3FAs decrease cell invasion. We used two human invasive CRC cell lines, HCT-116 and SW620. To examine the effects of each n-3FA on cellular proliferation, cells were treated with 12.5, 25, 50 and 100 µM free or EE- EPA or DHA for 72 h [CAS numbers: free EPA (10417-94-4), free DHA (6217-54-5), EE-EPA (86227-47-6), EE-DHA (81926-94-5)]. All cells received equal volumes of ethanol vehicle. Data shown are mean +/- n=3 unless indicated. As n-3FA concentration increased, cellular proliferation decreased, regardless of n-3 FA type. Importantly, the IC50 (inhibitory concentration) for EE-EPA (7.7 ± 1.6 µM) was lower than that of free EPA (24.5 ±4.2 µM). The ability of EE-EPA and -DHA to decrease the invasion of CRC cells through Matrigel coated Boyden chambers was assessed following treatment with 12.5 µM EE-EPA or -DHA for 24 h. EE-EPA and EE-DHA inhibited the invasion of HCT-116 cells to 37.1 ± 13.5% and 50.6 ± 17.5% of control, respectively (n=4). Similarly, SW620 cell invasion was decreased to 27.5 ± 9.7% of control by EE-EPA and 24.2 ± 6.7% of control by EE–DHA. The decrease in metalloproteinase-2 (MMP-2) activity, assessed via zymography following EE-n-3FA treatment, reflected the decrease in CRC cell invasion. Finally, EE-n-3FAs failed to inhibit the invasion of HCT-116 cells expressing two alleles of constitutively active phosphatidylinositol 3-kinase, suggesting their ability to decrease cell invasion is conferred by inhibition of PI3K activity. In conclusion, EE-n-3FAs are likely more effective inhibitors of CRC proliferation than free n-3FAs, potentially because they are more stable. Additionally, EE-n-3FAs also inhibit CRC invasion and decrease MMP-2 activity through a mechanism that may involve PI3K.

#1607

Saffron restricts MACC1-dependent cell proliferation and motility of colorectal cancer cells, and alters the microbiome structure.

Nazli Güllü,1 Hassan Brim,2 Marjorie Gondre-Lewis,2 Silvia Bisti,2 Hamid Shoraka,2 Dennis Kobelt,1 Akbar Soleimani,2 Lena Timm,1 Janice Smith,1 Hassan Ashktorab,2 Ulrike Stein3. 1 _Experimental and Clinical Research Center, Charité - Universitätsmedizin Berlin, and Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Straße 10, 13125 Berlin, Germany, WA;_ 2 _Howard Univ., Washington, DC;_ 3 _Experimental and Clinical Research Center, Charité - Universitätsmedizin Berlin, and Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Straße 10, 13125 Berlin, Germany, Washington, DC_.

Saffron has proven a beneficial effect as a supplement in the treatment of many conditions through its perceived anti-oxidative and anti-inflammatory properties. We previously identified the gene MACC1 and demonstrated its importance as metastasis inducer, prognostic and predictive biomarker for colorectal cancer (CRC). It is confirmed as decisive driver for tumorigenesis and metastasis for a broad range of solid cancers. Therefore, we explored the inhibitory impact of saffron extracts on MACC1-induced cancer cell growth and motility and its effects on the gut microbiome. First, we assessed cell proliferation of MACC1-high and MACC1-low expressing cells using MTT and the IncuCyte real-time cell imaging system. We employed the endogenously MACC1-low expressing colon cancer cell line SW480 and clones with ectopic MACC1 overexpression. In addition, SW620 cells with endogenous high MACC1 expression and CRISPR-Cas9 mediated MACC1 knock-out were used. Cells were treated with increasing amounts of extracts from 4 different saffron species and concentration-dependent proliferation was analyzed by MTT assay. Changes in cell cycle were assessed by FACS. In vitro migration was measured by Boyden chamber assays. Activation of caspases was analyzed by Western blot. In addition, rats treated with saffron had their gut microbiome analyzed through 16S rDNA sequencing of fecal DNA samples. Upon addition of saffron, cells with high MACC1 expression showed a growth delay compared to cells with reduced MACC1 expression. Further, MACC1-dependent migration was reduced when cells were treated with saffron extracts. We did not find activation of caspases, but we showed a cell cycle arrest of MACC1 positive cells treated with saffron extracts. Using real-time measurement of cell proliferation we identified crocin as most active compound, in our system. Furthermore, rats fed with saffron showed major changes at the phylum level of their gut microbiome. A dramatic reduction/depletion of Cyanobacteria and Proteobacteria and a decrease of Bacteroidetes/Firmicutes ratio was found within the saffron treated rats. These reductions were accompanied by enrichment in Spirochaetes, Tenericutes and Candidatus saccharribacteria phyla in these rats. This is the first identification of saffron-based compounds restricting cancer cells proliferation and motility progression via the novel target MACC1. In addition, change of microbiome composition by saffron favors short chain fatty acid synthesis. The use of saffron or some of its extracts might be of therapeutic value to CRC patients.

#1608

Time-restricted feeding attenuates pulmonary metastasis of Lewis lung carcinoma in mice fed a high-fat diet.

Lin Yan, Sneha Sundaram. _Grand Forks Human Nutrition Research Center, ARS, USDA, Grand Forks, ND_.

Circadian rhythms, which cycle approximately every 24 hours, control patterns of daily functions, e.g. feeding and fasting, rest and activity, and sleeping and waking. Altering the finely tuned circadian rhythms by an erratic lifestyle (e.g. disruption of the daily feeding and fasting pattern) leads to metabolic disorders, including obesity. Obesity is a risk factor for cancer. Obese cancer patients are at a greater risk of recurrence and metastasis than patients with normal body weight. We hypothesized that time-restricted feeding (TRF) attenuates high-fat diet-enhanced metastasis. In a metastasis model of Lewis lung carcinoma (LLC), male C57BL/6 mice were fed the standard AIN93G diet (16% of energy from soybean oil) or a high-fat diet (HFD) containing 45% of energy from soybean oil with or without dark phase restricted feeding (12 hours per day) for 10 weeks. Pulmonary metastases from a primary tumor, established by subcutaneous injection of LLC cells, were quantified. The percent fat body mass of the TRF group was lower than that of the HFD group and remained similar to that of the AIN93G group. There was no difference in energy intake between the TRF and HFD groups. The number and size of lung metastases were significantly higher in the HFD group than in the AIN93G group; however, there were no differences in these measurements between the TRF and the AIN93G groups. Time-restricted feeding prevented HFD-induced increases in plasma concentrations of glucose, insulin, proinflammatory cytokines (leptin, monocyte chemotactic protein-1, and plasminogen activator inhibitor-1), and angiogenic factors (angiopoietin-2, hepatic growth factor, and vascular endothelial growth factor). We conclude that TRF attenuates the HFD-enhanced pulmonary metastasis of LLC. This protection may be through the entrainment of metabolic regulators to the fixed feeding time that results in reductions in body adiposity and associated cancer-promoting inflammatory and angiogenic factors.

#1609

Association between antioxidant levels and inhibition of lung cancer cell proliferation by various garlic extracts.

Zeinab Farhat,1 Pamela Hershberger,2 Diana Aga,1 Tyler Scheving,1 Ajay Myneni,1 Lina Mu1. 1 _University at Buffalo, Buffalo, NY;_ 2 _Roswell Park Comprehensive Cancer Center, Buffalo, NY_.

Introduction:

Garlic is rich in organosulfur compounds that have potential chemopreventive properties. It contains a wide range of potent antioxidant compounds, which contribute by both radical scavenging activity and enhancing activity of enzymes. We aimed to investigate the antioxidant and antiproliferative activity of different garlic forms.

Methods:

Different types of garlic were obtained from a local Wegmans market in Buffalo, NY. Aqueous and methanol extracts of seven garlic forms including: raw garlic, raw garlic set aside for 10 minutes, garlic powder, cooked garlic, black garlic, and two garlic supplements (Garlicin and Kyolic) were investigated for their antioxidant and antiproliferative activity. Antioxidant activity was evaluated utilizing the 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) assay. The antioxidant activity of each sample was calculated in terms of gram equivalents of Trolox (TE) per gram of sample. Sulforhodamine B (SRB) assays were conducted to assess the antiproliferative effects after 48h of treatment with the extracts in cell lines selected to represent common cancer histologic and molecular lung cancer subtypes. Two-fold dilution concentrations of garlic extract were prepared ranging from 0.08% to 2.5% and the 50% inhibitory concentration (IC50) was calculated from dose-response curves. Each measurement of one sample was conducted in triplicate in order to average the antioxidant and antiproliferative measure for each garlic sample.

Results:

Among the different garlic forms, Garlicin supplement presented the highest antioxidant activity in both water and methanol extracts, 2.58 ± 0.43 and 3.45 ± 0.60 μmol trolox/g dry weight, respectively. Black garlic showed the next highest antioxidant activity in water and methanol extracts, 1.36 ± 0.53 and 0.19 ± 0.019 μmol trolox/g dry weight, respectively. Among lung cancer cells, different forms of garlic and different extraction methods resulted in varied percent inhibition. Garlicin alcohol extracts and fresh garlic water extracts resulted in a significant decrease in cell growth in H1975 cells and H520 cells, while other garlic forms showed lower levels of inhibition. Garlicin alcohol extracts and fresh garlic water extracts had the lowest IC50 values in H1975 EGFR mutant cells, 0.54% and 0.17% respectively. The same extracts were also potent in H520 squamous cells, 0.55% and 0.32%, respectively.

Conclusions:

In conclusion, Garlicin supplements and black garlic showed promising antioxidant activity, while Garlicin supplements and fresh garlic showed potent antiproliferative effects among lung cancer cells. Our results also indicate that the antioxidant and antiproliferative activity of different garlic forms are affected by different preparation and extraction methods. Further studies are needed to better understand the bioactive compounds present in garlic.

#1610

Getting under the skin: Fat quality in esophageal cancer prognosis.

Maelle Anciaux,1 Lieveke Ameye,1 Thomas Guiot,1 Patrick Flamen,1 Serge Goldman,2 Pieter Demetter,2 Amélie Deleporte,1 André Van Gossum,2 Marianne Paesmans,1 Vincent Donckier,1 Alain Hendlisz,1 Caroline Vandeputte1. 1 _Institut Jules Bordet, Bruxelles, Belgium;_ 2 _Erasme Hospital, Bruxelles, Belgium_.

INTRODUCTION

The last few years have seen an increased interest in the role of body mass composition parameters in cancer survival. this study sought to assess the impact of the body mass composition (BMC) on prognosis of locally advanced esophageal cancer (EC) patients beyond the usual quantitative weight loss associated with this condition.

METHODS

Diagnostic CT scans were assessed for BMC in 155 all-stage EC patients at diagnosis. The index (area/height2) of skeletal muscle (SMI), subcutaneous (SFI) and visceral fat (VFI) were delineated on two adjacent slides at the third lumbar vertebra level by two independent investigators using PLANET ONCO® software (DOSIsoft, France). Mean attenuation (or density) of fat tissue was measuring to assess the quality of adipose compartments. Survival and relapse free survival (RFS) were calculated from date of baseline CT-scan.

RESULTS

Interobserver correlations were excellent for all BMC parameters measured (r = 0.94 to 0.99). Remarkably, low subcutaneous fat density (SFD) was associated to better outcome, as were low disease stages. Low C-reactive protein (CRP) levels were also associated with better overall survival (OS). In contrast, low BMI did not affect the patients' outcome. Relapse free survival (RFS) analysis showed that only high disease stages and SFD remained associated with poor RFS. Stepwise regression showed that the combination of SFD, stage and CRP was an effective model for OS prediction. No parameter was retained for RFS in the multivariate analysis. Detailed results are shown in Table 1.

CONCLUSION

SFD, stages and CRP appeared as robust prognostic factors of OS in EC patients, in contrast with BMI. While SFD and stages were significant in RFS univariate analysis, none of these two parameters were retained in multivariate analyses. These results confirm the validity of BMC assessment for evaluating patient prognosis and show for the first time that adipose tissues and inflammation may have a preponderant impact on cancer prognosis. | |  | |

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

Parameters | Threshold | OS

|

male | female | p value | HR (95 CI)

Low SFD | < -95,7 | < -99,4 | 0,00028 | 2,2 (1,4-3,5)

Low disease stages | 1+2A vs 2B+3+4 | 0,0047 | 2,2 (1,2-3,7)

Low CRP | < 3,20 | 0,0053 | 2,2 (1,2-3,8)

Low BMI | <25 | 0,13 | 0,71 (0,46-1,1)

Parameters | Threshold | RFS

|

male | female | p value | HR (95 CI)

Low SFD | < -95,7 | < -99,4 | 0,04 | 1,6 (1-2,6)

Low disease stages | 1+2A vs 2B+3+4 | 0,044 | 1,8 (1-3,1)

N.B. Thresholds for SFD are the sex-specific medians. Thresholds for CRP is the median

#1611

Caloric restriction potentiates the therapeutic benefit of androgen deprivation therapy and alters macrophage polarization when combined with PD1 inhibition in murine models of prostate cancer.

May Elbanna,1 Justin Budka,1 Paige Dausinas,1 Remi Adelaiye-Ogala,2 Nur Damayanti,1 Ashley Orillion,3 Eri Banno,1 Luigi Fontana,4 Roberto Pili1. 1 _Indiana University School of medicine, Indianapolis, IN;_ 2 _NCI, Bethesda, MD;_ 3 _The Janssen Pharmaceutical Company, Springhouse, PA;_ 4 _Washington University, St. Louis, MO_.

Background: Data from epidemiological studies have linked dietary intake to the development of prostate cancer (CaP). We have previously shown that dietary protein restriction inhibits tumor growth by modulating PI3K/mTOR signaling in vivo. Additionally we have shown that dietary methionine restriction influence macrophage polarization which enhances the ability of the immune system to fight cancer. In this study we hypothesize that caloric restriction is capable of restricting tumor growth and potentiating the antitumor effect of androgen deprivation therapy (ADT) and PD1 inhibition.

Methods: We utilized two prostate cancer models for our in vivo studies; castrate resistant LuCaP 23.1 AI prostate cancer model and MYC-Driven prostate cancer model in C57BL/6 mice. Caloric restriction was carried out by exposing mice to alternating fasting either as a single intervention (in LuCaP 23.1 model) or in combination with ADT (enzalutamide or surgical castration) or PD-1 inhibition in Myc-CaP model. Tumor sizes and weights were blindly assessed during the study and upon study termination respectively. IHC staining for both Ki-67 and mTOR phosphorylation was done to assess the impact of fasting+/- ADT on tumor growth and mTOR signaling. Macrophage polarization/distribution in tumors was assessed using immunofluorescence. Blood was collected at the end of the study for future comprehensive analysis of PBMCS.

Results: In both models, alternating fasting was associated with significant decrease in tumor weight at the end of study in comparison to control group (*p<0.05). In Myc-CaP model, tumor weight was significantly decreased in combined fasting and enzalutamide/castration group in comparison to the control condition (**p<0.001) but was not significantly better than either intervention alone. Similarly, fasting potentiated the therapeutic benefit of PD-1 inhibition yet it was not significant. Combined fasting and ADT was associated with significant reduction in Ki-67 nuclear expression (****p<0.0001), however it was not significantly less than either agent alone. Combined fasting and ADT was associated with significant decrease in P-mTOR expression in comparison to control mice (****p<0.0001). Although we did not observe significant change in tumor weight with combined fasting and PD-1 inhibition; fasting potentiated the influence of PD-1 on macrophage polarization. M2 macrophages were significantly less in combined treatment compared to either single intervention alone (**p<0.01).

Conclusions: Caloric restriction can hinder prostate cancer growth and potentiate the therapeutic effect of ADT. Our results provide basis for the translational use of dietary modification both as preventive measure as well as a therapeutic intervention that can improve the benefit of current standard treatments.

#1612

The effect of docosahexaenoic acid on androgen-dependent and androgen-independent prostate tumors in combination with abiraterone and enzalutamide.

Andrew R. Cooper,1 Irvin V. Ma,1 Andy Shao,1 Prem Kumar,1 Ronald S. Pardini2. 1 _University of Nevada, Reno School of Medicine, Reno, NV;_ 2 _University of Nevada, Reno, Reno, NV_.

Standard treatments for prostate cancer typically involve androgen deprivation therapy (ADT). In later stages of prostate cancer, the tumor typically evolves to become insensitive to ADT, otherwise known as castration-resistant prostate cancer (CRPC). Despite being hormone-insensitive, CRPC has been shown to respond to both abiraterone and enzalutamide. Although both treatments deprive the tumor of androgens, they both display different mechanisms of action. Abiraterone inhibits androgen production, and enzalutamide acts by interrupting the binding of androgens to the androgen receptor. Studies in our laboratory have shown that docosahexaenoic acid (DHA; C22:6, n-3), a long chain omega-3 polyunsaturated fatty acid (PUFA), inhibits tumorigenesis in both androgen-dependent (LNCaP) and androgen-independent (PC-3, DU145) prostate cancer cell lines in culture. Moreover, in vivo studies with n-3 supplementation have been shown to significantly reduce PC-3 and DU145 tumor growth compared to corn oil diets rich in linoleic acid (C18:2, n-6). With evidence suggesting that n-3 PUFA dietary supplementation can inhibit tumorigenesis in CRPC, this study investigated the efficacy of DHA in combination with both abiraterone and enzalutamide in a subset of prostate tumor phenotypes in vitro. PC-3 and LNCaP cell viability were reduced with DHA in combination with either enzalutamide or abiraterone compared to primary treatments of each. These results suggest that DHA enrichment can augment abiraterone and/or enzalutamide therapy with androgen-dependent and androgen-independent prostate tumors in vitro. The results of this study provide preliminary evidence of the effectiveness of nutritional supplementation in conjunction with cancer therapeutics, and it will serve as a prelude to a pilot clinical trial evaluating the effects of nutritional supplementation with high levels of omega-3 fatty acids on enzalutamide inhibition of CRPC.

#1613

**Dietary isoflavone decreases prostate cancer progression and improves survival in conditional** Pten/Trp53 **-deficient mice.**

Yasunori Mori,1 Marco A. De Velasco,1 Yurie Kura,1 Eri Banno,1 Naomi Ando,1 Noriko Sato,1 Masahiro Nozawa,1 Kazuhiro Yoshimura,1 Kazuko Sakai,1 Kazuhiro Yoshikawa,2 Kazuto Nishio,1 Hirotsugu Uemura1. 1 _Kindai University Faculty of Medicine, Osaka-Sayama, Japan;_ 2 _Aichi Medical University, Japan_.

Epidemiological data has shown that dietary practices can greatly influence cancer rates. Men in East Asian countries, men have significantly lower prostate cancer rates compared to their counterparts the US and Europe. Soybeans are a versatile and rich source of protein and its products constitute a rich portion of Asian diets. Recent interest in healthy eating has expanded the consumption of soy products which also provide a rich source of naturally occurring isoflavones and 17β-estradiol. In this study, we used roasted soybean flour (kinako), which contains high levels isoflavones glycosides and estradiol, as dietary soy source to determine the influence of isoflavones rich diets on prostate cancer. Six-week old conditional Pten/Trp53 double knockout mice were randomized and fed plain AIN-93M (Control) diets or a diets supplemented with kinako ad libitum. Concentrations of kinako were adjusted to for daily intakes of aglycone isoflavones (genistein, daidzein, and glycetein) of 400 (LDI) and 800 (HDI) mg. Mice were sacrificed at 16 and 20 weeks (n=6 mice/group) or maintained for survival assessment (n=8 mice/group). Dietary intake of kinako-supplemented diets did not influence the onset of prostatic intraepithelial neoplasia or tumor burden at the early stages. However, tumors from mice fed the HDI diet experienced reduced tumor proliferation rates. Moreover, mice fed LDI and HDI diets showed reduced androgen receptor (AR) protein expression levels as well as mRNA levels for the AR target genes Fkbp5, Nk3x3.1 and Timp4. Interestingly, mice on the LDI diet, but not the HDI, experienced longer times to disease progression (median times 264, 299 and 250 days for Control, LDI and HDI, respectively, P=0.663), tumor doubling (median times 14, 27 and 14 days for Control, LDI and HDI, respectively, P=0.083), cumulative survival (median times 292, 348 and 320 days for Control, LDI and HDI, respectively, P=0.199), and overall survival times (median times 28, 43 and 35 days for Control, LDI and HDI, respectively, P=0.324). The metastatic incidence was 33%, 14% and 14% for Control, LDI and HDI groups, respectively, P=0.631. We also investigated whether dietary intervention with kinako would impact previously stablished tumors. For this we fed kinako supplemented diets to conditional Pten-knockout mice with established tumors but no changes were observed in tumor burden, proliferation, apoptosis and AR activity. Together our data shows that long-term continuous ingestion of a diet rich in isoflavones may be necessary in order suppress tumor growth. Interestingly, this protective effect appears to be lost with high-doses of the dietary isoflavones. Further studies will need to be performed in order to decipher complex dynamic interplay between survival pathways isoflavones chemoprevention.

#1614

Coconut kernel extract as a novel chemopreventive agent that target cancer stemness.

Sandhya Sorra,1 Joyeeta Talukdar,1 Gayatri Gogoi,2 Hong Li,3 Debabrat Baishya,4 Bikul Das1. 1 _KaviKrishna Laboratory, IIT-Guwahati Campus, Guwahati, India;_ 2 _Assam Medical College, Dibrugarh, India;_ 3 _Forsyth Institute, Cambridge, MA;_ 4 _Gauhati University, Guwahati, India_.

Cancer stemness can be defined as integrated molecular programs that confer stem cell phenotype to cancer cells. Cancer stemness is associated with drug resistance, immune evasion and metastasis. Our ongoing work indicates that oncogene MYC may enhance cancer stemness. We are interested to identify novel dietary agents that can target MYC mediated cancer stemness pathway. Coconut (Cocos nucifera L.) kernel extract (CKE) is traditionally used in tradition Indian medicine as anti-inflammatory diet. Here, we have investigated the potential CKE mediated targeting of MYC mediated skin carcinogenesis.

Methods: To determine tumor-suppressing activity of isolated CKE in vivo, we used a DMBA/TPA-mediated skin cancer in Balb/c mice. C-MYC expression in skin tumor was studied by immuno-histochemistry. The bioactive component of CKE was studied by performing histopathology and immunohistochemistry.

Results: CKE treatment decreased DMBA/TPA induced tumorigenicity by 200-fold (p<0.001, n=10). Histological and Immuno histo-chemistry studies suggested that CKE treatment significantly inhibited over expression of c-Myc protein on skin tumorigenesis induced by DMBA/TPA. Additionally, CKE treatment also shows significant inhibitory effect on lipid peroxidase and stimulatory effect on glutathione, catalase and superoxide dismutase activities in tissues treated with DMBA/TPA. Additionally, in vitro study showed that CKE reduces DMBA/TPA induced stemness in skin keratinocytes. CKE contains lauric acid as per analysis performed by high performance liquid chromatography and Nuclear magnetic resonance. However, purified lauric acid alone did not exhibit potent anti-stemness activity in vitro as compared to CKE.

Conclusions: These findings demonstrate that CKE is a tumor suppressing agent for chemically induced skin cancer. CKE prevent DMBA/TPA induced oxidative stress and potentially act as an inhibitor of MYC-induced cancer stemness.

#1615

Correlation between demographic factors, dietary habits and urinary micronutrient content among minority groups in New York City.

Cristina Zambrano,1 Maayan Beeber,1 April Panitz,1 Katarzyna Wyka,2 Safa Ibrahim,1 Yin Tan,3 Grace Ma,3 Khursheed Navder,1 Ming-Chin Yeh,1 Olorunseun Ogunwobi1. 1 _Hunter College of the City University of New York, New York, NY;_ 2 _Graduate School of Public Health and Health Policy, New York, NY;_ 3 _Temple University, Philadelphia, PA_.

Cancer is the second leading cause of death in the United States. A diet low in fruits and vegetables increases the risk of certain cancer types. Moreover, socioeconomic factors contribute to limited access to fresh and healthy foods and limited opportunities for safe physical activity, leading to poor physical health. In this multidisciplinary, IRB approved study, we explored relationships between demographic factors, self-reported dietary behaviors and gallic acid, a polyphenolic micronutrient that correlates well with fruit and vegetable intake. We recruited participants at a senior center in East Harlem, New York City, a racially diverse and underserved community. The participants completed a NIH-validated survey through which we assessed their dietary habits and collected standardized demographic data and history of cancer. Urine samples from participants were analyzed for gallic acid. So far, 33 participants completed the survey and 25 of them provided urine samples for gallic acid analysis. We found an association between demographic information (Race/ethnicity and age) and intake of certain foods. Specifically, age was negatively associated with french fries/fried potatoes, cooked dried beans and tomato soup intake (p<0.05) and Black/African American race was associated with increased intake of juice (p<0.05). A positive association between self-reported food intake and gallic acid concentration was only observed with french fries/fried potatoes (R = 0.641, p<0.001). Overall, the results suggest a significant association between dietary habits and demographic factors. Ongoing work include further recruitment of participants, and multivariate analysis of the data. This will be followed by an intervention consisting of active nutritional education, and urinary gallic acid analysis to assess the impact of nutritional education on dietary habits.

#1616

Adjuvant supplementation of docosahexaenoic acid with 2-deoxyglucose, metformin, and aspirin on cellular metabolism in breast, lung, and prostate tumor cells.

Irvin V. Ma,1 Michael Mouradian,2 Andrew R. Cooper,1 Erika D. Vicente,1 Ronald S. Pardini2. 1 _University of Nevada, Reno School of Medicine, Reno, NV;_ 2 _University of Nevada, Reno, Reno, NV_.

A standard treatment for cancer often involves the use of adjuvant supplementation with targeted therapy to maximize tumor suppression. Recent studies have shown that docosahexaenoic acid (DHA; C22:6 n-3), a long chain omega-3 polyunsaturated fatty acid, acts on an array of molecular oncogenes that are often targeted by anti-tumorigenic drugs. After a careful review of DHA's mechanistic properties, 2-deoxyglucose (2DG), metformin, and aspirin were identified as drugs that share similar molecular targets as DHA. 2DG and metformin both target cellular metabolism by inhibiting intracellular ATP, albeit different mechanisms. 2DG inhibits glycolysis, while metformin targets the mitochondria. Aspirin inhibits inflammation via irreversible acetylation of platelet COX. The current study investigates the effects of DHA in combination with each drug on numerous cancer subtypes to determine if concurrent treatments can synergistically inhibit tumor proliferation. In BT-474 human breast ductal carcinoma, MDA-MB-231 human breast adenocarcinoma, and A549 human lung adenocarcinoma, DHA was shown to enhance the tumor-inhibiting effects of 2DG via intracellular ATP reduction. In contrast, DHA showed no effect with metformin on the same tumor cell lines. With limited studies localizing DHA's metabolic effects, these results suggest that DHA may have potential selectivity for ATP inhibition via glycolysis. In response to DHA + aspirin, the PC-3 prostate adenocarcinoma and MDA-MB-231 displayed synergistic inhibition of cell viability, while LNCaP prostate carcinoma showed an additive effect. With studies showing DHA's ability to inhibit endothelial COX, further studies will be done to determine whether the synergistic effects of DHA + aspirin expand to COX expression as well. Moreover, downstream targets of 2DG and metformin will be studied to determine the extent of DHA enhancement on oncogenic targets, such as mTOR, 4EB-P1, and GRB10. The results of this study provide preliminary evidence for the efficacy of adjuvant supplementation with DHA in combination with anti-tumorigenic drugs. With ongoing analysis, the potential for adjuvant nutritional supplementation will allow for additional treatment strategies for future patients.

#1617

**Genetic polymorphisms in DNA repair genes** XRCC1 **and** 3 **are associated with increased risk of breast cancer in Bangladeshi population.**

Md. Mustafizur Rahman,1 Nupur Rani Hawlader,1 Md. Mostafizur Rahman,1 Md. Amir Hossain2. 1 _Khulna University, Khulna, Bangladesh;_ 2 _Asa University of Bangladesh, Dhaka, Bangladesh_.

Background: Breast cancer is the most commonly occurring cancer in women worldwide and its prevalence vary among different ethnic groups. Genetic polymorphisms in DNA repair genes may affect individual variation in DNA repair capacity which may contribute to individual susceptibility for developing various cancers. This study aimed to investigate the contribution of polymorphisms in two DNA repair genes, namely XRCC1 (Arg399Gln, rs25487) and XRCC3 (Thr241Met, rs861539) to breast cancer risk in Bangladeshi females and their role as susceptibility markers.

Methods: A case-control study comprising 121 breast cancer patients and 133 healthy volunteers was conducted taking the age, breast feeding status, menopausal status, education etc into account. Genomic DNA isolated from peripheral blood was used to genotype the target polymorphisms using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Risk-stratified subgroup analyses were conducted to find out the association between the genotypes and age-, menopause-, lactating status-related characteristics of breast cancer patients.

Results: A significant positive association was found between XRCC1 (Arg399Gln,) and XRCC3 (Thr241Met) polymorphisms and breast cancer risk in the studied population. In case of XRCC1 Arg399Gln polymorphism, the heterozygous Arg/Gln (G/A) and homozygous Gln/Gln (A/A) genotypes showed 1.78-fold (OR=1.78, 1.0084 to 3.1442, p=0.0467) and 2.41-fold (OR=2.41, 95% CI= 1.0354 to 5.5914, p=0.0413) increased risk of breast cancer, respectively when compared with homozygous Arg/Arg genotype. The presence of any XRCC1 Gln (Arg/Gln or Gln/Gln) was associated with 1.93-fold increased risk (OR=1.93, 95% CI= 1.1574 to 3.2290, p=0.0118). The variant Gln (A) allele also was associated with increased risk of breast cancer (OR=1.78, 95% CI= 1.1885 to 2.6805, p=0.0052). The Gln/Gln genotype was more prevalent in breast cancer cases at ages 55 or above and in menopausal state. In case of XRCC3 (Thr241Met) polymorphism, Thr/Met (C/T) heterozygote, and combined Thr/Met+Met /Met (C/T+T/T) genotypes were significantly associated with 1.85- and 1.89- fold higher risk of breast cancer, respectively (OR=1.85, 95% CI=1.0815 to3.1834, p=0.0248; OR=1.89, 95% CI=1.1199 to 3.1908, p=0.0171, respectively). The variant Met (T) allele showed significant association with increased susceptibility (1.70-fold) to breast cancer (OR=1.70, 95% CI=1.0849 to 2.6172, p= 0.0206). Among breast cancer cases significant differences in genotype frequency were evident in patients with age 55 years or above and with menopause.

Conclusion: Our results suggest that XRCC1 (Arg399Gln) and XRCC3 (Thr241Met) polymorphisms are associated with increased risk of breast cancer in Bangladeshi females. In addition, this association was significantly related to age and lactating status.

#1618

Breast cancer screening in low and lower - middle income country : A program in Vietnam.

Voc Tai Dang,1 Tu Van Dao,1 Thuan Van Tran,1 Huong Thi Tran2. 1 _Vietnam National Cancer Hospital, Hanoi, Viet Nam;_ 2 _Vietnam National Institute for Cancer Control, Hanoi, Viet Nam_.

Background: From 1980 to 2010, new breast cancer cases increased by more than 50% worldwide. Disease burden increased even more rapidly in low- and middle-income countries (LMICs), where over half of breast cancer cases now occur. In Vietnam, approximately 15229 new cases of breast cancer were diagnosed with 6103 resulted deaths in 2018. Breast cancer can be detected early by screening; however, it is not covered by medical insurance. Mammogram is currently one of the most reliable screening tools for breast cancer. Regular mammograms can help detect breast cancer early, thus allowing for early treatment Purposes: To raise public awareness on breast cancer prevention and early detection, while promoting a habitual routine of breast self - examination and breast cancer screening participation; to call on companies to include breast cancer screening on the periodic health examination for female employees. Methods: We collected data on risk factors of breast cancer. Breast Imaging-Reporting and Data System (BI-RADS fifth edition) scores from the radiology reports were utilized. The information collected from women who completed the questionnaires included: age, BMI, family history of breast cancer, personal history of breast or ovarian cancer, number of pregnancies, number of babies born, number of abortion, number of miscarriage, stillbirth, hormonal medication use (hormone replacement therapy and daily oral contraceptive), age of menarche, menopause, smoking habit, and breast self-examination results. Results: There were 10,000 Vietnamese women who were over 40 years old (over 35 years old if their mother or their sister had breast cancer) participated in this program. Rate for regular breast self-examination was 19.1%. In the 40 to 55 years-old age group, the rate was 70%, 2.2% for women aged 35-39 years (with a family history of breast cancer). Eight women were diagnosed with breast cancer, the incidence rate was 80 per 100,000 population, respectively. Conclusions: The total fee for a breast cancer screening in Viet Nam is approximately $25 ($20 for a mammogram and $5 for consultation fee). This remains one of the best solutions to detect breast cancer early and reduce the overall burden of cancer treatment costs on the already strained Vietnamese health system.

#1619

Cervical cancer screening knowledge and behavior among female undergraduates.

Joseph Agboeze,1 Matthew Igwe Nwali,1 Nkiru Ezeani2. 1 _Federal Teaching Hospital,Abakaliki, Abakaliki, Nigeria;_ 2 _Ebonyi State University, Abakaliki, Nigeria_.

Background; Cervical cancer is a highly preventable disease that disproportionally affects women in developing countries with an estimated incidence rate of 33 cases per 100,000 women in Nigeria.. Over the year's awareness and uptake of cervical cancer screening services have remained poor in developing countries. Lack of knowledge and poor attitude towards the disease and risk factors can affect screening practice and development of preventive behavior for cervical cancer

Objective; This study assessed the level of knowledge and barriers towards cervical cancer screening among female university students.

Method; We conducted a cross-sectional study of 234 female students at Ebonyi State University Abakaliki Nigeria from March to July 2017 using multistage sampling technique. Apretested questionnaire was used to collect data on socio-demographic characteristics, knowledge, and screening history. Data were analysed with Epi info version 7.1.4.

Results; Participants' mean age was 22 years (sd=3).Of the respondents 79.5% were not aware of cervical cancer screening while 58.9% and 78.5% had poor knowledge on cervical cancer and Human papilomavirus vaccination respectively. Major sources of information were health workers 70% and 35% Radio while only 4.0% have had Pap smear test. Common barriers include lack of centres where such services are obtainable 88% and fear of cancer being discovered 9%. Reported risk factors were multiple sexual partners 24.2% and early onset of sexual intercourse 19.8%. A significant association was found between institutional and personal barriers and having a Pap smear test.

Conclusion Comprehensive education on cervical cancer screening in universities is critical in reducing the morbidity and mortality associated with cervical cancer. Few screening centres contributed to poor cervical screening uptake.

#1620

Validation of a proteomic signature of lung cancer risk from bronchial brushings.

SM Jamshedur Rahman,1 Yuqian Gao,2 Athena A. Schepmoes,2 Yi-Ting Wang,2 Tujin Shi,2 Sheau-Chiann Chen,1 Heidi Chen,1 Karin D. Rodland,2 Tao Liu,2 Pierre P. Massion1. 1 _Vanderbilt University Medical Center, Nashville, TN;_ 2 _Pacific Northwest National Laboratory, Richland, WA_.

A major challenge in lung cancer prevention and cure hinges on identifying the at-risk population who ultimately develops lung cancer. Previously we reported proteomic alterations in the cytologically normal bronchial epithelial cells collected from the bronchial brushings of individuals at-risk for lung cancer. Proteins were identified by shotgun proteomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and selected candidate biomarkers of risk were validated in an independent sample cohort by parallel reaction monitoring. The purpose of the current study is to validate, in independent cohorts from Vanderbilt University Medical Center, a selected list of 54 candidate proteins associated with risk for lung cancer with sensitive targeted proteomics using selected reaction monitoring (SRM) at the Pacific Northwest National Laboratory. We tested proteins from bronchial brushings and bronchial biopsies collected from individuals at low risk (n=10) and high risk (n=10) based on the Tammemagi risk stratification model as well as from patients with lung cancer (n=10). Multiplexed LC-SRM assays were developed for all 54 proteins and the protein concentrations were determined by LC-SRM using heavy isotope-labeled peptides for 44 and 49 proteins in the brushings and biopsy samples, respectively. Pairwise comparisons by Wilcoxon rank sum test demonstrated significant overexpression, in the high-risk group compared to the low-risk group, of ALDH3A1 and AKR1B10 in both brushings and biopsies, and of SFN/YWHAS only in the biopsies. Significant overexpression of ALDH1A1, ALDH3A1, AKR1B10, ANXA1, CTNNB1, G6PD, LGALS7B, ALOX15, PGK1, SFN/YWHAS, and UBA1 were observed in the lung cancer group compared to the low-risk group in the brushings only. LGALS7B was also overexpressed in the brushings of high risk compared to lung cancer group. In conclusion, these SRM results revealed promise of selected candidate proteins to stratify the at-risk population for lung cancer. Next, our validation efforts will test the promising candidates in second, larger independent cohort; methods with increased sensitivity such as PRISM (high-pressure, high-resolution separations with intelligent selection and multiplexing)-SRM for analysis of the previously undetected low abundant protein candidates and carrier-assisted SRM approaches for analysis of very small-sized samples. Ultimately, we hope to deliver a signature of risk that may provide the basis for lung cancer risk assessment and possibly novel future prevention strategies. This work is supported by UO1CA152662.

#1621

Multiplex analysis of proteins for cancer diagnosis: A case study using ActivSignal technology.

Ilya Alexandrov, Malcolm Mackenzie, Irina Brandina. _ActivSignal, Natick, MA_.

Pancreatic cancer is one of the deadliest types of cancer, killing over 50,000 each year in the US, and with a five-year survival rate below 8%. However, currently there are no reliable diagnostic tests for pancreatic cancer, and the great majority of cases are detected at a late stage, with bleak mortality rates as a result. The ActivSignal IPAD platform monitors activity of 26 cancer signaling pathways in multiplex, by examining phosphorylation status or expression of 70 protein targets. The technology utilizes pairs of antibodies per each protein target to ensure high specificity. These antibody pairs are carefully tested and selected. Detection will only take place if both antibodies in a pair bind to their specific target molecule during the analysis phase - avoiding false hits. The ActivSignal IPAD platform can be used to profile cell culture samples, tissues and different blood fractions. The IPAD assay requires a very small amount of sample. All these characteristics make it potentially adaptable for diagnostics, simultaneously profiling multiple proteins targets. We have tested the assay in a preliminary analysis of human pancreatic cancer patients. Several control and pancreatic ductal adenocarcinoma (PDAC) blood samples were used for analysis. The assay can be performed on a very small volume of material, and 100 microliters of each sample was obtained from a collaborating academic medical center. The profiling was performed on 40 previously identified, relevant proteins. The comparison of Normal controls and PDAC patient samples gave a clear pattern that discriminated these two population groups with a great degree of certainty. Thus, giving a proof of concept that the assay can be used for diagnostic development for pancreatic cancer detection, an urgent unmet need.

#1622

Role of 3d volumetric and perfusion imaging for detecting early changes in pancreatic adenocarcinoma.

Syed Rahmanuddin,1 Erkut Borazanci,2 Vincent Chung,1 Derek Cridebring,3 Ronald Korn,2 Joyce Ho,1 William Boswell,1 Daniel Von Hoff4. 1 _City of Hope Comprehensive Cancer Center, Duarte, CA;_ 2 _Honor Health, Phoenix, AZ;_ 3 _Translational Genomics Research Institute (TGen), Phoenix, AZ;_ 4 _Translational Genomics Research Institute (TGEN), Phoenix, AZ_.

Introduction:

The aggressive nature of pancreatic cancer makes it one of the leading causes of cancer-related deaths worldwide. Survival rates remain low with standard therapy. Pancreatic adenocarcinoma accounts for nearly 90% of pancreatic malignancies, and early detection could have a major impact on disease prevention and early treatment. This study assesses the utility of CT perfusion imaging and 3D modeling for the early detection of pancreatic adenocarcinoma, as well as its role in monitoring treatment response.

Methods:

This study was a collaboration approved by the City of Hope and Honor Health institutional review boards. Thirty patients diagnosed with pancreatic adenocarcinoma who initiated treatment between 2015- 2018 were selected. Six patients treated with Neoadjuvant and Standard of care (SOC) therapy at Honor Health & 24 patients received SOC therapy alone at City of Hope. Multiphase CT imaging was performed with 2.5mm slices according to standard imaging protocol, and reconstructed into 0.625 x 1.25mm slices for 3D post-processing. Images from the picture archiving and communication system (PACS) were uploaded to advanced imaging software (GE Advantage Workstation 3.2) for volumetric analysis. 3D tumor volume and perfusion studies before and during treatment were used to detect early changes in pancreatic cancer and assess response to treatment.

Results:

Measured 3D tumor volume correlated with disease burden, and decreased with clinical response to treatment. Change in arterial vs venous phase perfusion indicates the aggressivity of tumor biologics and potential early detection imaging biomarker in pancreatic adenocarcinoma. Serial imaging in patients receiving combination neoadjuvant and SOC therapy showed decreases in tumor volume and venous vasculature, while imaging in patients receiving standard therapy showed variable responses during treatment.

Conclusion:

3D volumetric imaging biomarker is one of the clinical outcome variables which define the treatment response on follow-up imaging in both neoadjuvant therapies & SOC alone. Results of our pilot study suggest that change in 3D volume and perfusion are important imaging markers that correlate with tumor progression vs regression & define the aggressivity of the tumor. Larger studies are needed to validate the utility of imaging-based volume and perfusion analysis for detecting early changes in pancreatic cancer.

Special thanks to:

1. made possible by the Kemper and Ethel Marley Foundation

2. Stand Up To Cancer-care Research UK-Lustgarten Foundation Pancreatic Cancer Dream Team Research Grant ( Grant number SU2C-AACR-DT-20-16)

#1623

Autoantibodies to human endogenous retrovirus K envelope protein (HERV-K ENV) are associated with features of prostate cancer aggressiveness in African American men.

Tino W. Sanchez,1 Shannalee R. Martinez,1 Greisha Ortiz-Hernandez,1 Evelyn Sanchez-Hernandez,1 Christopher Montgomery,1 Benjamin Becerra,1 Tiffany Dorsey,2 Feng Wang-Johanning,3 Stefan Ambs,2 Carlos A. Casiano1. 1 _Loma Linda University, Loma Linda, CA;_ 2 _National Cancer Institute, Bethesda, MD;_ 3 _SRI International, Menlo Park, CA_.

Prostate cancer (PCa) is the most commonly diagnosed cancer and the second leading cause of cancer-related deaths in American men. African-American (AA) men are more likely to develop PCa at a younger age and are twice as likely to die from the disease compared to men from other ethnic/racial backgrounds. Even when normalized with equal access to healthcare, socioeconomic status and familial PCa history, AA men still die disproportionately from PCa. Thus, it is paramount to improve the early detection of aggressive PCa. A cancer patient's humoral immune response to a tumor offers us a minimally invasive opportunity to detect aggressive cancers before a symptomatic tumor emerges. Previous studies indicate that genes associated with immunity and inflammation are significantly upregulated in prostate tumors from AA men compared to European-American (EA) men, suggesting that there are racial differences in the anti-tumor immune response in patients with PCa. This has prompted our laboratory to evaluate the autoantibody repertoire to tumor-associated antigens (TAA) in ethnically diverse patients. Previous studies showed a higher frequency of autoantibodies to the human endogenous retrovirus K (HERV-K) GAG protein in men with late stage PCa. In addition, investigators in this team reported previously an elevated expression of the envelope (ENV) protein in PCa tumors from AA men compared to PCa tumors from EA men. Given the emerging role of HERV-K in human cancers and the potential use of autoantibodies to the ENV and GAG viral proteins as cancer serum biomarkers and immunotherapeutic tools, we investigated the antibody response to the HERV-K ENV in AA and EA men with and without PCa. Using an enzyme-linked immunosorbent assay (ELISA), we detected a significant increase in the frequency of autoantibodies to HERV-K ENV in AA men with PCa compared to EA men with PCa (N=100, p<0.05) and AA men without PCa (N=100, p<0.0001). The frequency of autoantibodies to ENV was also significantly higher in AA men with stage IV (29%, p<0.0001) and AA men with PCa metastasis (40%, p<0.0001) compared to AA men without PCa or EA men with and without PCa. The immunoreactivity of the sera against ENV was confirmed by immunoblotting. To our knowledge this is the first report of an increased autoantibody frequency to HERV-K ENV in PCa patients and the first report of autoantibodies associated with racial differences in PCa. The observation that these autoantibodies are significantly linked to a more aggressive PCa in AA men is also consistent with previous observation of increased upregulation of HERV-K ENV in prostate tumors from AA men. The biological and clinical significance of anti-HERV-K ENV and GAG autoantibodies in PCa, and the racial differences in their frequency and clinical associations, warrant further investigation.

#1624

**Antigen-independent** de novo **prediction of cancer-associated TCR repertoire.**

Bo Li. _UT Southwestern Medical Center, Dallas, TX_.

Cancer-associated T cells play a critical role in mediating immune responses in the anti-tumor immunity. However, due to the complex nature of cancer antigens, and the limited experimental approaches for collecting antigen-specific T cells, it remains a difficult task in cancer immunology to detect cancer-associated T cells. In this project, we have developed an antigen-independent machine learning approach to perform de novo prediction of cancer-associated T cells. Specifically, we have discovered a signature in the CDR3 sequence that can distinguish cancer-associated T cells from others. We applied this signature to immune repertoire data to define a "cancer score", which is related to the probability of a repertoire being cancer-associated. We applied this approach to investigate 15 sample cohorts from public domain. These cohorts cover healthy donors, viral infected individuals and cancer patients from both early and late stages. Surprisingly, we observed consistently and significantly higher cancer scores using the cancer patients' immune repertoire data, while none of the non-cancer repertoire was significant compared to healthy donors. We therefore used repertoire cancer score as a single predictor for cancer status to distinguish cancer patients from healthy donors, and observed high prediction power measured by area under the ROC (AUROC) curves. The AUROC reached 0.90 for early breast cancer patients, which is better than a number of current early prediction methods based on cancer biomarkers, such as PSA, CA-125, CEA, etc. In addition, cancer scores derived from certain late-stage cancers are associated with patient response to checkpoint blockade therapies, suggesting that it may also be used in combination with existing biomarkers, such as PD-L1 expression or cancer neoantigen load, to improve the prediction of clinical outcome of these cancer types. We anticipate broad utilities of cancer scores in cancer diagnosis and immunotherapy prognosis with the rapidly accumulating TCR repertoire sequencing data in clinical studies.

#1625

Challenges of screening applications for early detection of cancer using liquid biopsy.

Bahram G. Kermani. _Crystal Genetics, Inc., Los Altos, CA_.

In the recent years, early detection of cancer using next-generation sequencing (NGS) has been en vogue, due to its potential to drastically improve the survival rate of cancer patients. Liquid biopsy is often viewed as the modus operandi for early detection, as taking biopsies from the actual organs is not practical for widespread screening tests. Among others, two main approaches attempted for early detection are based on cell-free tumor DNA (ctDNA) and circulating tumor cells (CTCs). The rarity of ctDNA and CTC is often debated as a major potential challenge for detection of cancer at early stages. In this work, we will discuss several other challenges that are necessary to address for developing a practical and high-quality screening application for early-stage detection of cancer. We will also provide a framework in which much of these challenges can be addressed. While a reasonable sensitivity is needed for such test to be of value, the most prominent matter regarding screening for cancer is specificity. Due to low prevalence of cancer, what may appear as a high specificity (e.g., 99%) could actually be insufficient, as it translates to a low positive predictive value (PPV) (e.g., in the 10% range). A low PPV would result in a large number of false positives in the test. These false positives would cause follow-up tests that are not only costly, but also could trigger unnecessary anxiety for the patients. A comprehensive validation process is another critical element in early detection. While it is tempting to perform validation based, solely, on groups of Healthy and Cancer patients, it can be shown that some signals caused by Benign conditions could highly resemble those of Cancer patients, and therefore result in false positives. We will show that distinguishing Benign from Cancer necessitates detection of more complex patterns, as opposed to simplistic measures such as genome-wide copy number variation (CNV). Yet another challenge in a liquid biopsy test is identification of the tissue-of-origin. Due to the mixed-signal problem inherent in ctDNA-based technologies, we argue that they face a higher challenge, as compared to CTC-based counterparts, for obtaining this goal. In conclusion, we will show how a system based on CTCs, whole genome sequencing (WGS), and artificial intelligence (AI) could provide an appropriate framework for addressing much of the challenges in early detection of cancer for a true screening application with the specificity in the 99.99% range.

#1626

Comparison of two flow cytometry detection methods of circulating tumor cells: Direct identification and EpCAM affinity based enrichment.

Li Zhao,1 Lauren Jachimowicz,1 Ming Lei,2 Peifang Ye,2 Garret Guenther,1 Nancy LI1. 1 _ACEA Biosciences, Inc., San Diego, CA;_ 2 _ACEA Bio, Hangzhao, China_.

The dissemination of cancer and development of metastases is the cause of nearly all cancer deaths. Circulating tumor cells (CTC) are cells that detach from the primary solid tumor or metastases and circulates through the peripheral blood. Accumulating evidence shows the importance of CTC detection for cancer prognosis also referred to as a "liquid biopsy", biomarker for metastatic cancer, as well as therapeutic response monitoring. However, detection of CTC in peripheral blood is made difficult because they are present at extremely low concentrations; in some patients as little as a few CTC per million blood cells. Flow cytometry a standard equipment available in many laboratories is capable of analyzing thousands of cells per second, allowing rare cell detection and has been demonstrated to be useful for CTC studies. CTC detection can be further aided by affinity based enrichment methods that eliminate other blood cells from the sample before analysis. In these experiments, we analyzed a colon cancer cell line (SW620) by 2 methods; either direct flow cytometry identification or following EpCAM affinity based enrichment. First, SW620 cells were spiked into blood cells and analyzed of the NovoCyte Quanteon and other comparison cytometers to determine the rare event detection sensitivity. The results show a good linear relationship between the number of cells detected and the predicted number added with a sensitivity of 0.00001% or 1 CTC per million cells. Colon cancer CTC and many other CTC have high expression of epithelial markers, such as epithelial cell surface marker, EpCAM. Affinity based enrichment methods utilize distinct antigens expressed either by CTCs but not blood cells such as EpCAM to separate CTC from blood cells and increase the detection capacity of CTC in the blood. In the next experiments, SW620 cells were spiked into blood samples to simulate CTC in cancer patients. CTC were then enriched by EpCAM affinity based magnetic bead enrichment. The recovery rate of the spiked tumor cells was then detected by flow cytometry to determine the accuracy and specificity of the enrichment process. The results show that a greater number of CTC present in the spiked blood sample increased the recovery rate of CTC through the enrichment process. A high precision syringe pump allowed the direct enumeration of CTC without the need of reference beads. These data demonstrate the high specificity and accuracy of CTC detection by flow cytometry by both direct identification and the use bead based enrichment methods.

#1627

Using internet-platform technology to train community-based researchers in developing countries: Lessons learned from a Caribbean study.

Kamilah B. Thomas-Purcell,1 Marva Primus Primus-Jospeh,2 Christine Richards,3 Lydia S. Atkins,4 Julie Blaze,5 Patsy Wylie,6 Ann Marie Burnie James,7 Kimlin Ashing8. 1 _Nova Southeastern University, Ft. Lauderdale, FL;_ 2 _Turks and Caicos Islands Community College, Cockburn, Grand Turk, Turks and Caicos Islands;_ 3 _St. George's University, St. George's, Grenada;_ 4 _Spartan Health Sciences University, Vieux Fort, Saint Lucia;_ 5 _Dominica State College, Grand Bay, Dominica;_ 6 _Ministry of Healtlh, Wellness and the Environment, Kingstown, Saint Vincent and the Grenadines;_ 7 _Grenada Ministry of Health, St. George's, Grenada;_ 8 _City of Hope Medical Center, Dueate, CA_.

Cancer is increasing as a health crisis in the Caribbean region. In the U.S. advocate engagement and Community Based Participatory Research (CBPR) have been used successfully to partner with community members in each stage of the research process thereby utilizing the knowledge and experience of those individuals for improved community responsive research and greater societal benefit. We utilized advocate engagement and CBPR towards building a cancer prevention program of research between U.S. and Caribbean researchers. In 2016-2017, a two-phased CBPR research study was initiated to address breast and cervical cancer screening in the four countries of the English-speaking Windward Islands of the Caribbean. The aims were to determine 1) community member's perceptions of breast and cervical cancer screening and 2) gatekeepers' perceptions of the quality and availability of breast and cervical cancer screening and treatment services. Mobile communication and internet-platform technologies were used to successfully train Caribbean community researchers on qualitative data collection and analysis methods. Our results showed that the incorporation of internet-based technology facilitated training, communication, social interactions, and alleviated transportation challenges. In particular, mobile platforms including GoToTraining, WhatsApp, Dedoose, Google Drive, and email were to used to deliver interactive training modules to community researchers and facilitate the collaborative development of recruitment materials, data collection instruments, data management, reports, manuscripts and results dissemination. Participatory research studies of this nature not only shed light on the cancer burden in developing nations but also builds community trust and capacity to engage in future international research.

## BIOINFORMATICS AND SYSTEMS BIOLOGY

### Convergence Science for Biomarkers, Early Detection, and Diagnosis

#1628

Modularity of the metabolic gene network as a prognostic biomarker for hepatocellular carcinoma .

Fengdan Ye,1 Dongya Jia,1 Mingyang Lu,2 Herbert Levine,1 Michael Deem1. 1 _Rice University, Houston, TX;_ 2 _The Jackson Laboratory, Bar Harbor, ME_.

Abnormal metabolism is an emerging hallmark of cancer. Cancer cells utilize both aerobic glycolysis and oxidative phosphorylation (OXPHOS) for energy production and biomass synthesis. Understanding the metabolic reprogramming in cancer can help design therapies to target metabolism and thereby to improve prognosis. We have previously argued that more malignant tumors are usually characterized by a more modular expression pattern of cancer-associated genes. In this work, we analyzed the expression patterns of metabolism genes in terms of modularity for 371 hepatocellular carcinoma (HCC) samples from the Cancer Genome Atlas (TCGA). We found that higher modularity significantly correlated with glycolytic phenotype, later tumor stages, higher metastatic potential, and cancer recurrence, all of which contributed to poorer prognosis. Among patients with recurred tumors, we found the correlation of higher modularity with worse prognosis during early to mid-progression. Furthermore, we developed metrics to calculate individual modularity, which was shown to be predictive of cancer recurrence and patients' survival and therefore may serve as a prognostic biomarker. Our overall conclusion is that more aggressive HCC tumors, as judged by decreased host survival probability, had more modular expression patterns of metabolic genes. These results may be used to identify cancer driver genes and for drug design.

#1629

Classification of cancer histology images using deep learning.

Weidong Xie. _The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China_.

Pathological evaluation of tumor tissue images stained with hematoxylin and eosin (H&E) is pivotal and specialists often disagree on the final diagnosis. Meanwhile, automated image analysis approaches have great potential to increase precision of diagnosis, help reduce human error and cut cost. In this study, we utilize various computational methods based on deep learning framework and build a stand-alone diagnostic tool to effectively classify different histopathology images across different types. The dataset of tissue slides from eligible patients with breast cancer/ thyroid cancer consists of 45,000 samples in our hospital. Available tissue samples above were split into a training set, for learning the CNN parameters, and test set, for evaluating its performance. An accuracy of 94% was obtained for non-cancer (i. e. normal or benign) vs malignant (i. e. invasive carcinoma or papillary thyroid carcinoma). In addition, we also demonstrated the utility of our model to discriminate between two subtypes of lung cancer. It provides pathologists or medical technicians a straightforward platform to use without requiring sophisticated computational knowledge, and cancerization would be identified which is not visible under a single microscope.

#1630

The utility of the online pathologist network for cancer research.

Ke Cheng, Christoper Gibson. _HistoWiz, Brooklyn, NY_.

Introduction: Pathologic review of tumors is critical to understand what type of cancer is present, changes in the immune microenvironment, and assess for the presence of necrosis. Simultaneously, in animals treated with immunotherapeutic agents, the assessment of the presence of abnormal inflammation and corresponding tissue damage in normal organs is of the utmost importance to measure a medication's potential side effect profile. The current problem is that most researchers do not have the skill in interpreting histology that pathologists have, barring them from the above histologic observations. This is compounded by the fact that most pathologists have busy clinical schedules and even if they wanted to help a research study, they do not have access to researchers. Another issue is that, even if researchers and pathologists could be connected, there is the issue of compensation. If an online platform could be constructed where researchers can connect to pathologists and pathologists can connect to researchers to help them interpret their slides with equitable compensation, the quality of histologic interpretation of research materials would increase. With improved slide interpretation quality, researchers would more rapidly know which experimental paths have potential and which should be avoided based on unfavorable histology.

Methods: To build this platform we used our histology business where wet tissue is converted to digital scanned slides and offered the pathologist consultation service to our customers. Researcher requests for pathology assistance were first screened by a board-certified pathologist (AB) for addressability by pathologists. When histology questions were deemed answerable by pathology, pathologists on Histowiz's remote pathologist network reviewed the slides and generated reports in 3 days that were then sent to the researchers.

Results: Between February and September 2018 there were 60 online consultations performed at HistoWiz. Nearly half (25/60) of the studies involved measuring inflammation in mouse organs. Only one study asked for tumor grading, one study asked for tumor percentage estimation, and two studies asked for tumor type identification. The remaining studies were for miscellaneous requests such as mast cell counting, GVHD measurement, coronary atherosclerosis measurement, organ damage assessment (skin: 6, small intestine: 1, lung: 3, brain: 2, cartilage: 2, liver: 2), IHC suitability assay, differences between WT and KO mice, and toxicology after chemical treatment of mice.

Conclusions: It is possible to build a platform whereby pathologists assist researchers through remote review of their scanned slides. In 7 months of this service, nearly half of the researcher requests were for assessment of inappropriate inflammation. Less requested services included tumor grading, tumor percentage estimation, and tumor type identification.

#1631

Molecular and clinical associations of histopathological image features.

yu fu, moritz gerstung. _EMBL-EBI, cambridge, United Kingdom_.

Haematoxylin and eosin stained (H&E) histopathological images are widely used as a clinical routine for many cancers for diagnosis, but their interpretation is laborious and has a subjective element. Recent advances in computer vision using deep neural network offers an unprecedented opportunity to quantify patterns on H&E images typically used to classify tumour and normal. It is however possible to associate these traits with molecular alterations to obtain a precise understanding of the molecular underpinnings of histological changes. Here we demonstrate wide-spread associations of quantitative image traits with underlying genetic mutations including TP53, whole genome duplications (WDG) and molecular expression profiles ranging from cell morphology to T-cell infiltration. These traits are then on par with molecular data to predict patient survival. We use Inception-V4, a deep neural network, to extract 1536 image features from 9,754 tumour or normal H&E images of 28 cancers from TCGA (split into 7.9 million tiles of 512 by 512 pixels). Using these features, tissue-specific tumour/normal classification accuracy was high with an average of 0.95, ranging from 0.99 for Thyroid Carcinoma to 0.64 for Head and Neck Squamous Cell Carcinoma. We then used these features to predict molecular alterations using regression models. Out of 104 driver gene mutations, 30 showed an association with histology in at least one cancer (AUC > 0.5). Interestingly, TP53 the most frequent mutated gene in cancer, was found in measurably distinct histology in 10 of 28 cancers. WGD were associated with histology in 24 out of 27 cancers (AUC > 0.5), where 4 cancers showed an AUC as high as 0.8. As expected, presence of WGD also correlated with increased cell nucleus volume. For gene expression, 7.7% showed a positive correlation with image features (FDR < 0.1); 2.7% showed a correlation above 0.6. Interestingly, we detected significant associations for genes indicative of immune cell infiltration. Finally, we used image and clinical data to predict patient overall survival. We obtained a C-index range in [0.64, 1] for 25 cancers, which were comparable to models used expression and clinical data ([0.61, 1]). For 13 cancers, models with image data outperformed those with expression data with an improvement of C-index from 0.05% for Kidney Chromophope to 20% for Esophageal Adenocarcinoma. In this study, we used an established deep learning architecture to quantify tumour histology. These analyses showed a high accuracy of classifying tumour/normal images. We obtained a high accuracy in predicting molecular alterations with image features, indicating that these alterations shape the resulting tumour histology. We showed that image features can be used to predict clinical outcomes for cancer patients with a comparable to sometime higher accuracy compared to expression data, offering an easy and inexpensive method to complement the patients' prognosis.

#1632

Deep learning functional associations using histopathology images.

Javad Noorbakhsh,1 Saman Farahmand,2 Mohammad Soltanieh Ha,3 Sandeep Namburi,1 Kourosh Zarringhalam,2 Jeff Chuang1. 1 _The Jackson Laboratory for Genomic Medicine, Farmington, CT;_ 2 _University of Massachusetts Boston, Boston, MA;_ 3 _Boston University, Boston, MA_.

Histopathological images have critical value for understanding cancer subtype, local structure, and malignancy. Manual interpretation of such images can be time-consuming and error-prone, while also failing to make use of the power of image aggregation for coordinated data mining. Large-scale patient cohorts such as TCGA provide high-resolution H&E images that complement other -omics data, with the important advantage that images provide spatial information about heterogeneity and stromal cell interactions. We implemented a convolutional neural network (CNN) to classify H&E slides according to tissue type, cancer subtype, and genomic mutations. We modified the CNN architecture of Inception v3 according to our problem and trained it with tiled whole slide images from 31 solid cancers in TCGA, and evaluated our algorithm on holdout data. We first classified tumor/normal status from the images and determined AUC values for each cancer type. We then applied our method for tissue classification in a multilabel classification scenario. Furthermore, in few cancers (e.g. gastric cancer) we determined cancer subtypes using supervised learning. Finally we evaluated our model for classifying mutational status of cancer-related genes. To improve classification accuracy, we modified Inception architecture for multi-task classification by replacing the final fully-connected layer by a shared layer which provides the input to individual task layers corresponding to each gene mutation. Furthermore, we adopted image segmentation approaches (e.g.. auto-thresholding) to improve sample background removal, and used several approaches (e.g. resampling, cross entropy weighting) to rebalance the data. In some cases we achieved high classification results (e.g. AUC=0.7 for TP53 in lung adenocarcinoma). Our study suggests that histological images can be used to classify tumors according to their mutational status, and can provide biomarkers for molecular subtyping.

#1633

Pretreatment prediction of recurrence-free survival in hepatocellular carcinoma after surgical resection by machine-learning based imaging analysis.

Jingwei Wei, Yuqi Han, Xia Wu, Yushen Jin, Jie Tian. _Chinese Academy of Sciences, Beijing, China_.

In hepatocellular carcinoma (HCC), patients at early stages are recommended to accept surgical resection as primary treatment by internationally endorsed guidelines. Nevertheless, post-surgical recurrence is one of the main threats that lead to death, with 5-year recurrence rate of over 70%. How to select patients at high risk of recurrence is essential to guide systematic follow-up and provide basis for alternative treatment strategy making, thus to prolong overall survival. In this study, we aimed to predict recurrence free survival (RFS) for HCC patients undergoing surgical resection using MR-based radiomics analysis. A cohort of 176 patients diagnosed with HCC was enrolled with complete recurrence follow-ups. A total of 4404 radiomic features including shape and size, intensity, textural, and wavelet features were extracted on the segmented tumor lesion. Mutual correlation among the features were initially assessed to reduce redundant features by Pearson's correlation analysis. Lasso-cox regression modeling was further applied to select recurrence-related key features and generate the radiomics signature with 10-fold cross validation. In addition, we explored the predictive ability of conventional clinical factors as comparable predictors with radiomics signature by univariate analysis and constructed clinical model by cox proportional hazard regression modeling. The result turned out that 4 T2WI-based radiomic features were identified as most correlated predictors for the recurrence prediction modeling, which were correspondingly gabor10_glszm_SZLGE, gabor12_glcm45_cluster_prominence, W5L5_fos_kurtosis, and W5E5_fos_skewness. The radiomics signature could successfully stratify patients into high-risk and low-risk groups with p-value < 0.001 and of 0.019 in the training and validation cohorts, respectively, by log-rank test. The radiomics-based model yielded C-indexes of 0.761 and 0.719 in the training and validation cohorts, respectively for RFS prediction. Venous invasion and AFP were selected as effective clinical predictors. The clinical model presented with C-index of 0.551 and 0.583 in the training and validation cohorts, respectively for RFS prediction. When adding clinical predictors into radiomics model, it did not show significant improvement for the prediction with Delong-test p-value > 0.05 in both training and validation cohorts. Our study revealed T2WI-based archetypal radiomic features related with RFS and highlighted the radiomics model as an effective noninvasive tool for RFS pretreatment prediction in HCC after surgical resection, which would beyond doubt provide reliable basis for individualized treatment decision making in HCC management.

#1634

A topology based data analysis method to predict survival time.

Cheng Huang,1 Hui-Chuan Sun,1 Junda Chen,2 Lin Shi,3 Fengqing Li,3 Yihui Lin,3 Hanyan Yang3. 1 _Department of Liver Surgery and Transplantation, Liver Cancer Institute and Zhongshan Hospital,, Shanghai, China;_ 2 _Department of Mathematics, Shanghai, China;_ 3 _3D Medicines Inc., Shanghai, China_.

There are different methods to predict survival time, including traditional statistical regression, and modern neural network. Still, precise prediction of survival time remains as a challenge. In recent years algebraic topology was introduced as a novel method to infer information from high dimensional data set, where a mapper uses an algorithm to obtain geometric or topological features of the data set, and associate the features with the predicted variables. In this study, the data set includes gender, age, AFP, GGT, PPT, TB, ALT, tumor size, tumor number, BCLC stage, number of liver nodules, type of surgical approach etc., total 21 diagnostic variables. We use these variables to predicted OS and DFS, and the data set contains 329 records. In this study, the topology mapper is executed to acquire the topological features of all the data (including training and test sets), since this task is unsupervised, so the target values are not considered. Our method is to infer three new features of each data record, including the location of the record on the main branch of the topology, whether it is a branch node (i.e. has more than 2 branches), and whether it is an endpoint. A simple neural network with one hidden layer is designed and implemented with Tensorflow, and the three features will be added to the data set and provide valuable information for prediction of the target values. Without adding the new features from topology mapper, we have 72% accuracy (in predicting the survival time is greater or less than the mean value) on test set data when predicting OS, and we have 75% accuracy on test set data when predicting the DFS. With the new features from topology mapper, we have 83% accuracy on test set data when predicting OS, and 84% accuracy on test set data when predicting the DFS. The three new features inferred from the topology mapper have a significant effect on the accuracy of prediction and provide better accuracy. The topology based data analysis method in this study could be applied to other classification problems related to clinical trial data, and will need further verification.

#1635

A scalable, cloud-based, unsupervised deep learning system for identification, extraction, and summarization of potentially imperceptible patterns in whole-slide images of breast cancer tissue.

Jacob L. Evans,1 William Seo,1 Mary Macheski-Preston,1 Michelle Fritz,2 Samantha Puvanesarajah,3 James Hodge,3 Ted Gansler,3 Susan Gapstur,3 Mia M. Gaudet,3 Michelle Yi1. 1 _Slalom Consulting, LLC, Atlanta, GA;_ 2 _Slalom Consulting, LLC, Philadelphia, PA;_ 3 _American Cancer Society, Atlanta, GA_.

Digital pathology images potentially contain novel patterns that may be perceived by modern deep learning models, but not humans. Prior unsupervised pattern recognition approaches have been used to reveal prognostically-relevant subtypes of glioblastoma (PMID: 28984190) and breast density segmentation (PMID: 26915120), and may complement supervised machine learning models trained using labeled data.

In the Cancer Prevention Study II (CPS-II) cohort (PMID: 12015775), high-resolution, digitized hemotoxylin and eosin diagnostic slides are available for approximately 1,700 breast cancer cases providing an opportunity to perform unsupervised pattern recognition image analysis for epidemiologic breast cancer studies. Given the size of the dataset and complexity of the models, we constructed an end-to-end analytical pipeline, including preprocessing, feature engineering, and clustering, using cloud-based technologies that enable analysis at scale.

Prior to training the unsupervised models, we faced issues converting raw images with open-source software. Specifically, OpenSlides could not open the Leica Versa SCN files due to their proprietary format while BioFormats inverted colors. To fix these issues, we altered the BioFormats library to successfully convert the files into a TIFF format. Since this issue likely affects other researchers, we are in discussions to provide the fix under a public license.

TIFF formatted images were then denoised through color normalization to reduce hue variance and artifact detection to remove unwanted features such as pathologist annotations. Due to the computational complexity of analyzing the full image, images were padded with white space to ensure divisibility and broken into nine tiles of a predefined size. To further reduce computation time, uninformative tiles were filtered based on a predetermined threshold of artifact and white space composition.

The remaining tiles were input to the unsupervised models. We used convolutional autoencoders, specifically a modified VGG-16 model without pretrained weights and a deep embedded clustering algorithm. These models learn representations of the images called 'feature vectors' and encode the images' salient patterns. The final model was chosen based on iterative testing on a subsample of 100 images (N=21,472 tiles) and performance comparison of various VGG-inspired autoencoders. The feature vectors were clustered by K-means to summarize the information in a format suitable for statistical analyses.

Our initial results show that the system captures macro-scale tissue patterns at lower magnifications (1x and 5x) and produces clusters that can be integrated into epidemiological studies of breast cancer etiology and prognosis.

#1636

Development of a cell phone-based electrochemiluminescence biosensor to detect breast cancer biomarkers.

Hyun Kwon, Padma P. Tadi Uppala, Elmer Ccopa Rivera, Rodney Summerscales. _Andrews University, Berrien Springs, MI_.

Introduction: Studies conducted in our laboratory on proteomic profiling of breast cancer serum among women by shotgun LC-MS methodology have identified several biomarkers for early detection of breast cancer. However, not all of the identified biomarkers have been validated due to costs involved with validation tests. The development of a reliable, cost-effective highly sensitive portable system, the Electrochemiluminescence (ECL) biosensor in our laboratory will enable us to validate identified biomarkers that have valuable diagnostic applications. Electrochemical immunosensors have significantly improved the sensitivity of detecting low molecular weight biomarkers present in early stages of cancer. There is a great need for such easily accessible systems for early detection of cancer because of disease prevalence high rates of mortality from breast cancer.

Purpose: of this project is to develop the ECL biosensor using ubiquitous mobile technologies to detect biomarkers of breast cancer. ECL offers great advantages over fluorescence based measurement: ECL is a highly localized and time-triggered detection method. Instrumentation can be minimized as it requires only voltage to trigger the reaction. Cell phone-ubiquity and connectivity creates opportunities to simplify healthcare instrumentation, brings closer to end users, and enable instant data sharing.

Methods: A highly compact and affordable instrumentation was designed and constructed using the mobile technology replacing the traditional ECL detector, computer interface, and data analyzer in an all-in-one mode. Ru (bpy)32+ was embedded in silicon nanoparticles and sandwich immunoassay was conducted. Antibody is conjugated to the Silicon nanoparticles where Ru (bpy)32+ is embedded and the other antibody is attached to magnetic bead so that the bound complex can be attracted to the electrode surface separating the false positive signals. Coreactant tri-n-propylamine (TPrA) or 2-(dibutylamino)ethanol (DBAE) was tested for their ability to enhance the signal.

Results: TPrA provided stronger signals for carbon electrodes and optimal concentration was determined for low signal measurements. The sensing conditions were optimized by experimenting with variables of buffer conditions, trigger voltage, and electrode conditions. The target sensing molecule, vitamin-D binding protein (DBP) was detected at the hundreds of nano-molar level with the optimized conditions.

Conclusion: The sensor demonstrated that a new instrumentation with mobile technology in the point-of-care diagnostics could provide reliability and sensitivity of a high-end equipment. This project is funded by the NSF CBET division.

#1637

Biologically informed deep neural network for genomic discovery and clinical classification in prostate cancer.

Haitham Elmarakeby, David Liu, Saud H. Aldubayan, Eliezer M. Van Allen. _Dana Farber Cancer Institute, Boston, MA_.

Background: Dissection of the molecular properties that distinguish primary and metastatic cancers may reveal new underlying biological drivers of aggressive disease and inform clinical stratification. The rapid increase in the size of molecularly profiled patient cohorts creates an opportunity to develop machine learning algorithms that interrogate these data for discovery and clinical application. However, the superior performance of deep learning models typically comes with the downside of reduced interpretability mainly due to using arbitrary architectures with dense connections. We hypothesized that a biologically informed deep neural network could effectively classify localized or advanced disease (here, in prostate cancer (PrCa)) using molecular features while maintaining interpretability for genomic discovery.

Methods: We introduce P-NET, an artificial neural network with biologically informed, parsimonious architecture that accurately predicts metastasis in PrCa patients based on their genomic profiles. In P-NET, each node encodes some biological entity and each edge represents a known relationship between the corresponding entities. P-NET can be used to simultaneously rank features, genes, and biological pathways based on their importance to the clinical classification. We applied P-NET to whole-exome sequencing data from 1012 primary and metastatic prostate cancers and validated our model on two independent validation sets of 130 primary samples and 95 metastatic samples. We compared P-NET performance to other models and visualized ranked features to generate novel biological hypotheses.

Results: The trained P-NET outperforms other models including Support Vector Machine, Logistic Regression, and Decision Trees (average area under curve AUC=0.93, the area under precision-recall curve AUPRC= 0.91 for 5-fold cross-validation). In general, copy number variation (CNV) was more informative compared to mutations. Highly-ranked genes in P-NET include AR, PTEN, and TP53, which are known PrCa drivers. In addition, less expected genes such as I-kappa-B kinases and proteasome-related genes are suggested to play a role in predicting the outcome. P-NET selected a hierarchy of 17 pathways (out of 3047 pathways on which P-NET was trained) as significantly relevant to classification including post-translational modification pathways such as ubiquitination and SUMoylation. The model accuracy was 0.77 for the primary validation set and 0.83 for the metastatic set.

Conclusion: P-NET, a biologically informed deep neural network, accurately classifies metastatic vs. primary prostate cancers. Visualizing the trained model generates novel hypotheses of mechanisms of metastasis. This represents a novel approach to integrating biology with machine-learning by building mechanistic predictive models, providing a platform for biological discovery.

#1638

Whole-exome sequencing and protein interaction networks to prioritize candidate genes for cutaneous melanoma susceptibility.

Sally Yepes, Margaret Tucker, Hela Koka, Kristine Jones, Aurelie Vogt, Laurie Burdette, Wen Luo, Bin Zhu, Meredith Yeager, Belynda Hicks, Neal D. Freedman, Stephen J. Chanock, Alisa M. Goldstein, Xiaohong R. Yang. _NIH/NCI, Bethesda, MD_.

Background: Known cutaneous malignant melanoma (CMM) genes account for melanoma risk in less than 40% of melanoma-prone families, suggesting the existence of additional risk genes or other modifiers. Whole exome sequencing (WES) of high-risk families usually results in the identification of a large number of potential disease-causing genes. However, prioritizing these genes and finding the true disease-causing ones have been challenging. We hypothesize that physically interacting proteins might have a similar effect on cell function and therefore, might produce a shared phenotype or increase the risk when mutated. To test this hypothesis, we applied a network approach integrating WES and protein-protein interaction (PPI) data to detect and prioritize rare variants in melanoma-prone families.

Methods: We conducted WES analysis on germline DNA of 212 patients from 45 CMM families (2-5 affected people in a family) without known mutations. Variants that were rare (<1% in public and our in-house control datasets), loss-of-function or missense predicted to be deleterious, and showed complete disease co-segregation (present in all affected members within a family) were included in the network analysis. For this analysis, we only focused on genes that interact with known CMM genes (gene list in Goldstein AM, et al. Hum Mol Genet, 2017) which we used as seed proteins. We then applied a degree-aware algorithm (DADA) to rank the set of candidate genes from WES with respect to the set of known CMM genes using human PPI networks. The goal of DADA is to prioritize a candidate set of genes based on their association level with genes known to be related to the disease. Network proximity calculations of the candidates with respect to the seed set were based on Random Walk with Restarts method. We used high quality and scored human proteins interactions collected from InWeb_IM. In the primary analysis, the network contains 625,641 interactions, aggregated from 8 source databases and spanning 87% of reviewed human proteins. We also conducted additional analyses with CMM PPI subnetworks generated using high confidence interaction data from different sources to compare results.

Results: The WES analysis described above identified 546 genes that harbored rare variants, likely affect protein function and co-segregated in affected members in CMM families. After applying DADA across all networks, the rankings of these genes were fairly consistent. Top genes included both known CMM genes (such as MC1R, CDKN2B, KITLG, and TINF2) and genes that have not yet been associated with CMM susceptibility (such as FOXK1, AR, LEF1, RGL4, PABPC1, CHEK2, CTBP2, and BLM). These results highlight the importance of known CMM genes and their networks in CMM susceptibility, and demonstrate the potential value of network approaches in gene prioritization. Further evaluation of novel genes is in progress.

#1639

Predictive modeling of cancer-type in Li-Fraumeni syndrome.

Vallijah Subasri, Nicholas Light, Benjamin Brew, Nathaniel Anderson, Adam Shlien, Anna Goldenberg, David Malkin. _The Hospital for Sick Children, Toronto, Ontario, Canada_.

Objective: Li-Fraumeni syndrome (LFS) is an autosomal dominant cancer predisposition syndrome associated with a germline TP53 mutation. Individuals with LFS are prone to developing a wide spectrum of tumors. This heterogeneity in tumor type makes it difficult to provide patient specific surveillance protocols. As a result, there is an immediate need to develop robust, evidence-based stratification strategies to tailor surveillance protocols to an individual patient's risk of cancer. Germline TP53 mutations themselves do not explain specific cancer phenotypes in LFS, nor the collective clinical heterogeneity. Hence, it is important to consider genomic level data in the development of predictive algorithms in these patients. The proposed study is two-fold—to identify germline modifiers predictive of tumor type in LFS, and to use these modifiers to develop a predictive model for the early detection of cancer type.

Methods: This study consists of blood-derived DNA methylation and whole genome sequencing (WGS) from a cohort of LFS patients (n=134); a subset of this cohort was held out as a test set. The first objective was to identify germline modifiers: single nucleotide variants (SNVs), insertions and deletions (indels), structural variants (SVs), copy number variation (CNV) and differentially methylated regions (DMRs) predictive of cancer type. Blood DNA methylation was generated using Illumina HumanMethylation450 BeadChip array. The top statistically significant DMRs were identified between cancer types by performing pairwise comparisons using a linear model. CNV, SNVs, indels, and SVs were detected from WGS using benchmarked tools: CNVnator, ERDS, GATK, and Delly. Custom filtering pipelines, and a curated list of cancer genes were established to determine high quality, biologically relevant modifiers. The second objective was to develop a model using the identified modifiers to predict cancer type in LFS patients. A generalized linear model was implemented using elastic net regularization to estimate the probability of getting a particular cancer type.

Results: The classifier can accurately identify the cancer type of all the individuals in the test set. The model can determine with > 90% accuracy whether an individual has cancer and can differentiate between cancer types among affected individuals with 42%-92% probability. Pathway analysis of the predictors highlight the TGF-β signaling pathway as a possible therapeutic target for personalized treatment in LFS.

Conclusion: This project is the first comprehensive molecular analysis of LFS and uses the largest LFS cohort to-date. It illustrates the contribution of genetic changes in LFS beyond TP53 and highlights the existence of molecular differences within LFS patients that contribute to phenotypic differences. Ultimately, this study will allow for the early detection of tumor-onset in LFS patients to assist clinicians in developing personalized surveillance protocols.

#1640

Deep learning classification of neuro-oncology medical documents.

Michael Wells, Thais Sabedot, Tathiane Malta, James Snyder, Laila Poisson, Houtan Noushmehr. _Henry Ford Health System, Detroit, MI_.

Introduction

Precision medicine and big data for cancer discovery requires well curated indexed critical health care data, however to date limited resources exist that successfully parse out unstructured clinical data in neuro-oncology. Current practice relies on time consuming manual extraction by researchers or clinicians resulting in data inconsistency and limitation in data set volume. Rule-based natural language processing algorithms could be used for simple consistent text, but medical documents are created longitudinally by multiple people across long periods of time resulting in inconsistencies and semantic heterogeneity that render rule-based techniques insufficient.

Methods

We applied a deep learning text classification method to multiple clinical document categories including clinical pathology reports and a text based clinical database spanning 17 years of clinical narratives with approximately 4000 unique cases. For this study we identified clinically relevant molecular criteria for glioma outlined in the WHO 2016 CNS classification of tumors including IDH mutation, MGMT methylation, and 1p19q co-deletion status. Using a convolutional neural network with two densely connected layers of 30 rectified linear nodes we were able to classify patients into their respected molecular cohort with an accuracy of 98%.

Conclusion

Parsing of unstructured text based clinical narratives and pathology reports using convolutional neural networks is a promising method to extract heterogeneous molecular data in neuro-oncology for large scale data analysis.

#1641

Generation and evaluation of medical synthetic data.

Andre R. Goncalves,1 Priyadip Ray,1 Braden Soper,1 Madhumita Myneni,2 Jennifer L. Stevens,3 Linda M. Coyle,3 Ana Paula Sales1. 1 _Lawrence Livermore National Laboratory, Livermore, CA;_ 2 _National Cancer Institute, Rockville, MD;_ 3 _Information Management Systems, Washington, DC_.

While machine learning (ML) has shown some promise in medical research, its actual impact has been limited relative to other application domains. One reason for this disparity is the lack of high-quality, patient-level data available to the broader ML research community. Such datasets are often not made available due to protections around patient privacy. To overcome these obstacles, high-quality, synthetic datasets could be leveraged to accelerate methodological developments in the application of ML to biomedical research. Clinical data in the form of electronic health records present a rich data source to be used for synthetic data generation. Such data can be high dimensional and predominantly categorical, which poses multiple challenges from a modeling perspective. In this paper, we evaluate four classes of synthetic data generation techniques, as well as several metrics for evaluating the quality of the synthetic data. While the results and discussions are broadly applicable to medical data, for demonstration purposes we generate synthetic datasets from the publicly available Surveillance Epidemiology and End Results (SEER) program. Specifically, our cohort consists of breast cancer cases diagnosed in the year of 2010, which includes over 26000 individual cases. Finally, we discuss the trade-offs of the different methods and metrics, providing guidance on considerations for the generation and usage of synthetic medical data.

#1642

Spatial proteomics with hyperplexed fluorescence imaging predicts risk of colorectal cancer recurrence and infers recurrence-specific protein-protein networks.

Shikhar Uttam,1 Andrew M. Stern,1 Samantha A. Furman,1 Filippo Pullara,1 Fiona Ginty,2 D. Lansing Taylor,1 S. Chakra Chennubhotla1. 1 _University of Pittsburgh, Pittsburgh, PA;_ 2 _General Electric Global Research, Niskayuna, NY_.

Introduction: Colorectal cancer (CRC) is the third leading cause of cancer related deaths in United States with recurrence after resection-with-a-curative-intent being frequently implicated in these deaths. The basis for CRC recurrence is not completely understood, is multifactorial, and involves dysregulation of heterocellular signaling among tumor cells and their microenvironment. Based on hyperplexed immunofluorescence imaging and novel computational analyses, we have developed a recurrence-risk prediction method that samples these signaling networks within the epithelial and stromal domains of the tumor microenvironment and provides improved performance over current state-of-the-art recurrence-risk prediction assays.

Data: In the retrospective study presented here, we used 52 hyperplexed immunofluorescence biomarkers associated with either canonical oncogenic pathways, immune response, or colon cancer per se to spatially profile tissue microarrays obtained from resected tissue samples from 432 chemo-naïve CRC patients.

Results: Using epithelial- and stromal-domain expression and co-expression diversity of the biomarkers, our preliminary results predicted the risk of CRC recurrence with a concordance index of 0.91. We also generated training and validation sets from the CRC patient cohort and demonstrated that the area under the curve (AUC) of the prediction receiver operating characteristic (ROC) was 0.90. We utilized stratified bootstrapping to show that the AUC was stable with a standard deviation of 0.02. Significantly, the penalized model selection used within our method allowed us to infer epithelial and stromal-domain protein networks specific to the risk-of-recurrence from the underlying signaling networks. Despite the limited sampling intrinsic to tissue microarrays we were able to capture immune cell infiltration and the differential modulation of these outcome specific protein-protein networks.

Conclusions: Our CRC recurrence-risk prediction method exploits our spatial proteomics computational pathology platform involving hyperplexed immunofluorescence imaging. This study demonstrates the potential of this paradigm to not only accurately predict risk of CRC recurrence but also to reveal the underlying systems pathophysiology. Inferring outcome- and domain-specific CRC protein networks will enable biomarkers mechanistically linked to disease progression to be determined and their causality corroborated. In turn, this knowledge can be used to inform optimal therapeutic strategies for individual patients.

#1643

Discovery pipeline of cancer transcriptomic biomarkers using machine learning.

Eileen Kim. _Medical College of Georgia (Augusta University), Augusta, GA_.

Introduction: Cancer affects millions of people and causes nearly 1 in 6 deaths worldwide. Precision oncology holds potential in guiding cancer treatment on a molecular basis and elucidating targetable cancer pathways. This study aims to create a discovery pipeline that uses cancer patients' transcriptomes and machine learning algorithms to predict cancer patient survival.

Methods: Transcriptomic and phenotypic data from cancer patients were downloaded from The Cancer Genome Atlas (TCGA). Initial genes were selected using univariate cox proportional hazard models to predict overall survival across all cancer types. Co-expressed selected genes were clustered, creating gene sets per cancer subtype. Then, an amalgam of LASSO and elastic-net regularized generalized linear models (GLMNET), gene bootstrapping, random forest without/with shadow features (Boruta), and recursive feature elimination were used. Monte Carlo iterations were performed for GLMNET to assess model stability and over-/underfitting.

Results: The highest hazard ratios (HRs) and lowest p-values resulted from the univariate cox proportional hazards model (e.g. for the RASGEF1A gene in a uterine serous carcinoma model, HR=2.8e8, p=0.005) and subsequent GLMNET models (e.g. 392 genes for uterine endometrioid carcinoma, HR=2.89e8, p=0.00873). Boruta before GLMNET yielded lower HRs and higher p-values (e.g. 11 genes for uterine serous carcinoma, HR=2, p=0.0517). Bootstrapping genes before GLMNET generally yielded much lower HRs and higher p-values.

Conclusions: We were able to model survival risk in cancer patients with a variety of methods. The best method so far is univariate gene selection and GLMNET, although random forest is promising. Next steps include exploring random forest models further, investigating the biology of prognostic models, using disease-specific survival, analyzing possible confounders, using Breslow-Wilcoxon or accelerated failure time models, replacing histological with molecular-signature-based cancer subtypes, and exploring alternative methods for prognosis prediction. Supported by the Medical Scholars Program, Medical College of Georgia, Augusta, GA.

#1644

Pancreatic cancer survival prediction using machine learning and comparing its performance with TNM staging system and prognostic nomograms.

Mohamed H. Osman. _Faculty of Medicine, Zagazig University, Egypt, 10th of Ramadan City, Egypt_.

Background:

Pancreatic cancer is one of the deadliest cancers with a 5-year survival rate of only 6%. Patients with cancer often want to know how long they have left to live and it's the first question patients ask after diagnosis. Reliable predictions are very important for achieving more personalized care and better management. However, the accuracy of physicians' clinical predictions of survival is poor. Predicting pancreatic cancer survival is challenging due to different tumor characteristics, treatments and patient populations. In this study, we evaluate the performance of Machine Learning (ML) in survival prediction compared to TNM system and published nomograms.

Methods:

Pancreatic cancer patients were identified through the Surveillance, Epidemiology and End Results database (SEER). Clinical data of the patients were extracted including: age, sex, race, marital status, tumor site, tumor histology, grade, tumor size, extension, Lymph Node (L.N) involvement, metastasis, cancer sequence, TNM stage, surgery, radiotherapy, chemotherapy, examined (L.N), positive (L.N) and survival months. Patients' records were randomly divided into a training set (80%) and a validation set (20%). ML algorithms were tested to predict survival at 6, 12 and 24 months as well as TNM staging system and two nomograms.

Results:

A total number of 22,700 patients were identified through SEER from 2004 – 2013 with median survival of 12 months. The most common primary sites were head of pancreas (62.8%), tail (13.2%), and body (10.7%). Random Forests algorithm achieved better results compared to other models. The trained model yielded Area under the Receiver Operating Characteristic Curve (AUCs) of 86.6% at 6 months, 83.4% at 12 months and 82.2% at 24 months. Compared to TNM staging system which achieved AUCs of 66.6% at 6 months, 65.5% at 12 months and 57% at 24 months. The nomograms achieved AUCs of 71.1% and 55.2% in predicting 1-year-survival.

Sensitivity of the trained model were 80%, 75% and 79% at 6, 12 and 24 months, respectively. The most important characteristics which influenced the model prediction performance were: age at diagnosis, surgery and tumor size.

Conclusion:

Machine learning achieved a much better performance compared to TNM staging system and prognostic nomograms. Improved survival prediction can help in in making better treatment decisions and planning social and care needs.

#1645

Data science for cancer research.

W. Jim Zheng. _University of Texas Health Science Center at Houston, Houston, TX_.

Cancer has been studied at all levels, ranging from molecules, to cells, to individuals, to populations. However, cancer research faces significant challenges such as enormous volume and heterogeneity of the data. Furthermore, new technologies applied to cancer research can generate data at extremely high speeds. These "big data" pose a significant challenge to conventional data analysis methodologies. We established a Data Science and Informatics Core Facility for Cancer Research (DSICCR) to analyze big data and provides integrative solutions to analyze cancer data at all levels. DSICCR supports the cancer research community by establishing a powerful "big data" infrastructure using data science-oriented high-performance computing, deployed with analytic and mining algorithms and software built upon DSICCR faculty's cutting-edge data science and informatics research. This infrastructure will support DSICCR's data science and informatics services to the cancer research community through collaborative research, data analysis services, and outreach and education programs. DSICCR is uniquely positioned to leverage existing research networks and make this vast amount of information more available to the cancer research community - an unprecedented opportunity to support multi-scale cancer research statewide.

#1646

Beyond BRCA: Discovery of novel drivers of homologous recombination deficiencies in cancer.

Nidhi Sahni,1 Daniel McGrail,1 Yongsheng Li,2 Gordon Mills,3 Song (Stephen) Yi2. 1 _UT MD Anderson Cancer Ctr., TX;_ 2 _Univ of Texas at Austin, TX;_ 3 _Oregon Health & Science University, Portland, OR_.

Since the discovery of BRCA1 and BRCA2 mutations as cancer risk factors, we have gained much understanding of their role in maintaining genomic stability through homologous recombination (HR) DNA repair. However, mutations in BRCA1/2 and other classical HR proteins such as RAD51 and PALB2 only identify 10-20% of TCGA patients who display HR deficiencies, indicating that we do not understand the vast majority of HR defects. Here, we leveraged the abundance of molecular characterization from TCGA patients for network analysis to fill this knowledge void. We discovered that over half of HR deficiencies originate outside of canonical DNA damage response genes, with particular enrichment for RNA binding proteins (RBPs). Experimental techniques validated over 90% of our predictions in a panel of 50 genes tested by siRNA, as well as 30/31 additional engineered mutations identified in TCGA patients. We further cross-validated these findings in independent patient cohorts, finding that the identified RNA processing mutations again enriched for HR deficient patients to an equal or greater degree than mutations in DNA damage genes. Using a series of experimental approaches, including protein interactome screening, RNA sequencing, and quantitative imaging cytometry, we probed how loss of RBP function induced global DNA damage response rewiring, including changes in RNA splicing, protein-protein interactions, and recruitment of repair factors to DNA damage sites. Clinically, defects in HR are known to promote cancer initiation, but also sensitize cells to targeted therapies such as PARP inhibition. We found that depletion RBPs from PARP-resistant triple-negative breast cancer cells induced sensitivity to multiple PARP inhibitors, indicating that this could identify new cohorts of patients who may benefit from PARP inhibition, beyond the small number of BRCA1/2-mutant patients. Moreover, we find that the identified RBP genes are significantly enriched for genes associated with cancer risk identified through GWAS. In patients, HR deficiencies were not equally distributed across all demographics, so preferential screening of those most at risk could further heighten this benefit. Thus, these novel drivers may show clinical relevance both for treatment stratification and for identifying individuals at high risk for cancer development to improve patient outcomes. Taken together, this exhaustive study greatly expands our repertoire of known drivers of HR deficiencies and mechanisms of damage repair, which may impact research from the most basic biology studies to clinical screening and stratification.

#1647

Integrative subtype analysis of muscle-invasive bladder cancer reveals subtypes that have different overall survival and respond differently to neoadjuvant chemotherapy.

QIANXING MO,1 Roger Li,1 Keith Chan2. 1 _H. Lee Moffitt Cancer Center & Research Institute, TAMPA, FL; _2 _Baylor College of Medicine, Houston, TX_.

Introduction: Muscle-invasive bladder cancer (MIBC) is a heterogeneous disease driven by accumulative alterations on DNA, epigenomics, genomics and protein expression levels. There is a clinical need to classify MIBC samples into molecular subtypes and to identify driver molecular alterations that could be targeted by precision medicine.

Methods: We performed an integrative clustering analysis of 388 TCGA MIBC samples. Each of the samples has somatic mutation, DNA copy number, methylation and gene expression data. We used Bayesian iClusterPlus method to identify integrative MIBC subtypes and subtype-specific genes and genomic features. Pathways and gene sets altered in the subtypes were identified by gene set and gene functional term enrichment analyses. To investigate the prognostic power of the subtype-specific gene expression signature, we compiled 5 more publicly available MIBC data sets and classified the samples into different subtypes using 1-nearest neighbor method. Subtype-specific overall survival was estimated by Kaplan-Meier method. Log-rank test and Cox model were used to compare the subtype-specific survival functions.

Results: We identified two integrative MIBC subtypes with distinct genomic landscapes. The top driver mutated genes included TP53, KDM6A, FGFR3, ELF3, RB1 and PIK3C2A. DNA regions containing MTAP, CDKN2A/2B, and several INFA genes were the driver altered regions. The integrative subtypes had similar gene expression pattern as the basal and differentiated subtypes reported by Mo et al., 2018 (JNCI 110(5):448-459), where a group of basal and differentiated markers were differentially expressed between the two subtypes. For this reason, we named them as basal and differentiated integrative subtypes. The basal subtypes were characterized by up-regulation of immune signaling pathways including CTLA-4, cytokine and inflammatory, NKT and JAK_STAT signaling pathways. A significant amount of the driver genes had negative correlation between methylation and gene expression. Furthermore, the subtype-specific gene expression signature was prognostic. For chemotherapy-naïve patients, the basal subtype was associated with worse overall survival. However, for patients undergone neoadjuvant chemotherapy, the basal subtype of patients had a better overall survival.

Conclusion: We identified basal and differentiated integrative subtypes of MIBC, which were characterized by distinct alterations on the DNA, epigenomics and gene expression levels. The two subtypes were associated with differentially altered pathways such as immune signaling pathways and responded differently to neoadjuvant chemotherapy. This integrative analysis has provided us a comprehensive picture of MIBC biology and revealed targetable molecular pathways for future precision therapeutics.

#1648

Deep learning tissue "fingerprints" to identify patients and predict clinical subtypes of breast cancer from digital pathology images.

Rishi Rawat,1 Fei Sha,2 Darryl Shibata,1 Daniel Ruderman,1 David Agus1. 1 _USC Keck School of Medicine, Los Angeles, CA;_ 2 _USC Viterbi School of Engineering, Los Angeles, CA_.

Inspired by facial recognition, we define an analogous problem in digital pathology called tissue matching. The objective is to match histology image patches to the patient. Unlike many classification problems in pathology, tissue matching comes with its own ground truth, and does not require subjective annotation. Using tissue-matching via deep neural networks, we developed strategies to overcome common sources of noise in histology data, including H&E and slide scanner variability. We demonstrate that the typical approach of increasing the training set size, in the current study up to 12,000 tissue cores, yields only moderate gains in test accuracy, while including a loss term promoting style-invariance cooperatively improves the impact of scaling training set size. Our best neural network learned features that matched patients from the test set of H&E image patches with 93% accuracy (n=104 patients, baseline accuracy <1%). Leveraging this network's identification ability, we applied its internal representation as a feature vector (tissue fingerprint) to apply to other problems in pathology. We found that a fingerprint-based classifier could predict estrogen receptor status from breast cancer whole-slides on two separate cohorts with high accuracy (AUCROC=0.89), a sizeable improvement over previous approaches. This work presents a novel, scalable, and transparent approach to train neural networks to identify distinctive features of pathology images. These features have demonstrated utility in classifying biomarkers from morphology and have the potential to assist in a number of difficult tasks in pathology, including tissue subtype classification and patient tumor similarity assessments based on morphometry.

#1649

Integrating genome, transcriptome and methylome data to identify novel genes for lung cancer: Data from over 50,000 European participants.

Yaohua Yang,1 Qiuyin Cai,1 Lang Wu,1 Xiang Shu,1 Xingyi Guo,1 Ran Tao,1 Bingshan Li,2 Xiao-Ou Shu,1 Wei Zheng,1 Jirong Long1. 1 _Vanderbilt University Medical Center, Nashville, TN;_ 2 _Vanderbilt University, Nashville, TN_.

Genetic factors contribute significantly to lung cancer risk. Genome-wide association studies (GWAS) have identified 18 susceptibility loci for lung cancer. However, these loci explain only approximately 12% of its familial relative risk. In addition, the causal genes remain largely unknown. The present study aimed at identifying novel risk loci and potential functional genes for lung cancer by integrating multi-omics data.

Genome-wide genotyping and gene expression data of 6,124 samples from 369 participants of European ancestry included in the Genotype-Tissue Expression were used to build cross-tissue models to predict gene expression. Genome-wide DNA methylation and genetic data of white blood cell samples from 1,595 individuals of European ancestry in the Framingham Heart Study (FHS) were used to build models to predict DNA methylation. The gene expression and DNA methylation prediction models were then applied to a GWAS dataset of lung cancer, including 27,162 cases and 23,619 controls of European ancestry to investigate predicted levels of gene expression and methylation in association with lung cancer risk. For CpG sites (CpGs) showing an association with lung cancer risk, we further estimated the methylation levels in correlation with the expressions of their proximal genes using data from the FHS (N=1,367).

Of the 15,810 genes investigated, significant associations with lung cancer risk were observed for 22 genes at P<3.16×10-6 (Bonferroni-corrected threshold). Among them, fourteen genes were in GWAS-identified loci and the remaining eight genes were in five novel genomic regions not yet reported as risk loci for lung cancer, including ZFP57, HLA-J, PAIP1P1, HCG18, CCL24, RNASEH2B, RP11-958N24.2, and ZNF565. Among 62,938 CpGs investigated, 177 were significantly associated with lung cancer risk at a Bonferroni-corrected threshold of P<7.94×10-7. Among them, 86 CPGs were located at GWAS-identified loci and 91 CpGs were in five novel genomic regions not yet reported as risk loci for lung cancer, including 85 CpGs at 6p22.1, cg11395890 at 1p36.21, cg09461792 at 7p21.1, cg05712524 at 7q22.1, and three CpGs,

cg09414264, cg16129132 and cg12851049, at 11p15.4. Of these 177 CpGs, significant correlations were observed between 35 CpGs and 14 genes at a false discovery rate-corrected P<0.05. Taking these results together, we proposed a putative genetic variant-DNA methylation-gene expression-lung cancer risk pathway for 22 CpGs and eight potential functional genes including FUBP1, MOG, HLA-F, ZNRD1, FLOT1, DDR1, C4B and UCKL1. Among them, three genes, MOG, HLA-F and ZNRD1 and 15 CpGs were in 6p22.1, a locus not previously reported for lung cancer risk.

Our findings highlight the power of integrating multi-omics data to identify novel risk loci and prioritize putative causal genes at known loci and provide new insights into the etiology of this malignancy. 

### Laboratory and Computational Methods for Cancer Analysis

#1650

Deconvolution of copy number alterations combining bulk and single-cell genomic data.

Haoyun Lei,1 Bochuan Lyu,2 E. Michael Gertz,3 Alejandro A. Schaeffer,3 Xulian Shi,4 Kui Wu,4 Guibo Li,4 Liqin Xu,4 Yong Hou,4 Michael Dean,3 Russell Schwartz1. 1 _Carnegie Mellon Univ., Pittsburgh, PA;_ 2 _Rose-Hulman Institute of Technology, Terre-Haute, IN;_ 3 _U.S. National Institutes of Health, Bethesda, MD;_ 4 _Beijing Genomics Institute - Shenzhen, Shenzhen, China_.

Characterizing the evolution of clonal cell populations in tumor progression is a challenging task given pervasive intratumor heterogeneity (ITH). Bulk DNA sequencing remains the dominant technology for large cohorts, but requires computationally deconvolving clonal substructure from bulk data, an error-prone process with limited resolution. Single-cell sequencing (SCS) is a promising alternative but is not yet practical at the scales needed for large cohorts. To address limitations of both bulk and SCS approaches, we developed strategies for deconvolving ITH by combining bulk sequencing with limited SCS data with specific focus on copy number alterations (CNAs), which are amenable to low-depth SCS and particularly challenging for deconvolution. We introduce two methods based on non-negative matrix factorization (NMF) of bulk data. One method extends the NMF optimization objective with a penalty for deviation of inferred clones from small numbers of observed SCS samples. The other combines clonal deconvolution with tumor phylogeny inference, balancing deconvolution quality against a minimum evolution cost for incorporating inferred and observed single cells into a reconstruction of the history of clonal evolution. We validated the methods on a set of semi-synthetic data derived from true low-depth SCS data consisting of 393 single cells derived from three regions each of two human glioblastoma cases. These data were used to call mean copy numbers at 9934 genomic loci. We artificially mixed the data to generate synthetic bulk samples of known clonal composition and applied each method to correctly reconstruct unobserved single cells and their clonal structures from three, six, or nine semi-synthetic bulk samples plus six single cells each per trial. For three/six/nine bulk samples, we achieved mean RMSD of copy number inference of 1.507/1.483/1.406 for pure NMF without SCS, 0.657/0.641/0.588 for the phylogeny-free SCS method, and 0.479/0.497/0.462 for the phylogeny-based SCS method. RMSD of mixture fractions describing the clonal composition were 0.245/0.242/0.243 for pure NMF without SCS, 0.215/0.198/0.206 for the phylogeny-free SCS method, and 0.215/0.215/0.224 for the phylogeny-based SCS method. The results show that we can substantially improve on bulk CNA deconvolution using limited SCS data, providing a way to balance advantages of pure bulk and pure SCS. The work also supports the value of a principled evolutionary model in inferring accurate clonal structure.

#1651

Utilization of an ensemble approach for identification of driver fusions in pediatric cancer.

Stephanie LaHaye, Kyle Voytovich, James Fitch, Sean McGrath, Anthony Miller, Amy Wetzel, Vincent Magrini, Elaine R. Mardis, Richard K. Wilson, Peter White, Catherine E. Cottrell. _Nationwide Children's Hospital, Columbus, OH_.

Pediatric cancers, which make up ~1% of cancer diagnoses each year, comprise distinct genomic landscapes compared to adult cancers. Pediatric cancers typically exhibit a "quiet" genome with a reduced number of somatic mutations compared to adult tumors, which tend to carry a high mutational burden. In addition, pediatric tumors are often associated with fusion events, including fusions that involve potentially targetable kinases, as well as transcription factors which can often be targeted downstream. Fusion events can occur through chromosomal rearrangements, large deletions, or insertions. These events can lead to dysregulated gene expression, and can often become a driving event in pediatric cancer. Sensitive and specific detection of these fusion events through the utilization of RNA-seq data has proven to be a difficult task, given the complexities in tumor cellularity and clonality, and the numerous false positive identifications that are typically output from single fusion callers. Additionally, there are confounding issues surrounding memory usage and compute time required for certain fusion calling algorithms. The Institute for Genomic Medicine at Nationwide Children's Hospital has implemented an ensemble approach, utilizing 7 fusion callers: FusionMap, JAFFA, STAR-Fusion, SOAPfuse, FusionCatcher, MapSplice, and TopHat-Fusion. This approach allows us to identify fusions that have been called by at least 2 of the above algorithms, and also allows for a prioritization approach based on the number of callers that have identified a specific fusion. We have employed this approach on 67 pediatric cancer cases that we have analyzed in a collaborative Institutional Review Board approved protocol which encompasses scientists, genetic counselors, oncologists, and pathologists at Nationwide Children's Hospital. These cases are comprised of 52 central nervous system (CNS) tumors, 13 solid tumors, and 1 hematologic tumor, to date. To analyze these cases for fusion events, total RNA-seq with ribodepletion was performed on RNA, extracted from either flash frozen or Formalin-Fixed Paraffin-Embedded (FFPE) tissue samples, to generate libraries that were sequenced using 150bp paired-end reads. Through the employment of our ensemble fusion calling approach and a manual knowledge based filtering strategy, we have currently identified 18 clinically meaningful fusions, all of which we have confirmed in a CAP/CLIA laboratory. We have identified both known and novel fusion events, of which several have provided diagnostic value and/or provided targeted treatment options for patients. In conclusion, this method has the potential to offer a streamlined approach to uncover driver fusions in cancer, while also providing additional diagnostics and an opportunity to identify targeted treatment options for patients in a clinical setting.

#1652

Evaluating cell lines andorganoidsas models for metastatic cancer through integrative analysis of open genomic data.

Ke Liu,1 Patrick Newbury,1 Benjamin Glicksberg,2 William Zeng,2 Eran Andrechek,1 Bin Chen1. 1 _Michigan State University, Grand Rapids, MI;_ 2 _UCSF, San Francisco, CA_.

Metastasis is the most common cause of cancer-related death and, as such, there is an urgent need to discover new therapies to treat metastasized cancers. Cancer cell lines are widely-used models to study cancer biology and test drug candidates. However, it is still unknown to what extent they adequately resemble the disease in patients. The recent accumulation of large-scale genomic data in cell lines, mouse models, and patient tissue samples provides an unprecedented opportunity to evaluate the suitability of cell lines for metastatic cancer research. In this work, we used breast cancer as a case study. The comprehensive comparison of the genetic profiles of 57 breast cancer cell lines with those of metastatic breast cancer samples revealed substantial genetic differences. In addition, we identified cell lines that more closely resemble different subtypes of metastatic breast cancer. Surprisingly, a combined analysis of mutation, copy number variation and gene expression data suggested that MDA-MB-231, the most commonly used triple negative cell line for metastatic breast cancer research, had little genomic similarity with Basal-like metastatic breast cancer samples. We further compared cell lines with organoids, a new type of preclinical model which are becoming more popular in recent years. We found that organoids outperformed cell lines in resembling the transcriptome of metastatic breast cancer samples. However, additional differential expression analysis suggested that both types of models could not mimic the effects of tumor microenvironment and meanwhile had their own bias towards modeling specific biological processes. Our work provides a guide of cell line selection in metastasis-related study and sheds light on the potential of organoids in translational research.

#1653

Application of high-density tiling CRISPR screen technology to identify chromatin remodeler complex targets for drug discovery.

Luis Soares,1 Michael Bocker,1 Qianhe Zhou,1 Cigall Kadoch,2 Ho Man Chan1. 1 _Foghorn Therapeutics, Cambridge, MA;_ 2 _Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA_.

The mammalian SWI/SNF (mSWI/SNF or BAF) complexes are widely mutated in different human pathologies, presenting new therapeutic opportunities in a range of genetically well-defined cancers, including lung, endometrial, malignant rhabdoid tumor, synovial sarcoma, and others. In each of these settings, disruption of different SWI/SNF subunits have been shown to contribute to the tumorigenic potential via distinct mechanisms. This observation suggests the potential for a range of therapeutic targeting strategies to effectively inhibit cancer cell proliferation. Using high-density tiling sgRNA CRISPR/Cas9 screens, we have developed large-scale efforts to systematically identify critical protein functional domains within chromatin remodeling complex components to inform drug discovery strategies across several different cancer indications. Using this approach, we have successfully identified and validated critical functional domains in mSWI/SNF complex components with promising drug target potential in indications such as BRG1-mutated non-small cell lung adenocarcinoma and synovial sarcoma.

#1654

Detection of structural variations in a cancer reference sample with multiple NGS platforms.

Yongmei Zhao,1 Wenming Xiao,2 on behalf of Somatic Mutation Working Group of theSEQC2 Consortium. 1 _Frederick National Laboratory for Cancer Research, Frederick, MD;_ 2 _Food and Drug Administration, MD_.

Structural Variations (SVs) are genetic variations or rearrangements in the structure of chromosome. SVs study plays an important role in cancer research as cancer genome is commonly altered with thousands of structural rearrangements such as insertions, deletions, translocation, inversions, duplications, copy number variations (CNVs). Next-generation sequencing (NGS) technology has provided a cost-effective approach for the comprehensive detection of SVs in human cancer cells.

In this study, we systematically investigated the somatic structural variations in a pair of breast cancer cell lines with multiple NGS platforms including Illumina short-read, 10x genomics linked-read, PacBio long-read, and high-throughput chromosome conformation capture HiC. We systematically evaluated the factors that impact the SVs detection accuracy. We measured the reproductivity of somatic SV detections across platforms and benchmarked the different software tools' performance. We performed SV integration from different platforms and established a high-confident SV call set for the reference samples. To independently evaluate the accuracy of our SV call set, we used orthogonal methods such as cytogenetic array as well as fusion gene detected from RNA-seq for cross validation. From this study, we identified the unique strengths of each NGS technologies and provide a practical guide to integrate multiple platform data to improve SV detection sensitivity and accuracy in cancer genome.

#1655

A structural approach for prioritizing variants in personalized Oncology.

Siao-Han Wong, Brors Benedikt. _DKFZ, Heidelberg, Germany_.

In personalized oncology, treatment strategies take individualized genomic aberrations into consideration and relies heavily on how the cancer genomes are interpreted. With immense efforts put into cohort studies, knowledge of pathogenicity at the gene level had been accumulated. On the other hand, the increasing amount of personalized clinical trials and the accompanying need of interpreting pathogenecity at the sub-gene level emphasize the importance of resolving heterogeneous significance within genes.

There had been some initiatives where people identified mutation hotspots within genes, looking at either highly recurrent single residues or mutation clusters. The hotspot clusters can be inferred from protein sequences as well as from protein structures, where residues distant in sequence may be brought together through protein folding. Nonetheless, these information are not well utilized in routine clinical settings yet.

In this study, we aim to build a consensus list of prevalent mutational events for molecular stratification of patients. This is achieved by collecting sequence and structural mutation hotspots from different algorithms, and further combine structural information to identify their neighboring residues in protein structures. Specifically, around 1400 residue hotspots are collected from CancerHotspots and DominoEffect. An additional of around 250 structural hotspots are identified by three structural algorithms, namely 3DHotSpots, HotMAPS and HotSpot3D. These results are mostly derived from TCGA PanCancer cohorts, with CancerHotspots included more other studies in the analysis. With this consensus information in place, we further set up the mechanism to map from genomic variants to protein sequence and protein structures. This enables us to annotate the variants found in patient genome and see whether they co-localize with hotspot residues, in their structural neighborhood, or in structural informative domains like interaction interfaces.

In summary, a higher prevalence of variants might imply higher significance and more well-studied roles than other low prevalent mutant alleles. Transferring this knowledge from the public cohorts helps researchers quickly prioritize variants found in patient genomes. Annotating structural significance of variants further provides us with more confidence in identifying driving events. With these procedures automated, the goal is to help facilitate clinical evaluations to meet the growing number of patients recruited to personalized oncology trials.

#1656

Systematic identification of cancer therapeutic liabilities through adaptive functional proteomics.

Jun Li,1 Wei Zhao,1 Yiling Lu,1 Gordon B. Mills,2 Han Liang1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _OHSU Knight Cancer Institute, Portland, OR_.

Reverse-phase protein arrays (RPPAs) offer a powerful functional proteomic approach to evaluate biomarkers and mechanisms underlying sensitivity and resistance to cancer therapy. This rapidly maturing quantitative antibody-based assay can assess a large number of protein markers in many samples in a cost-effective, sensitive and high-throughput manner. MD Anderson Cancer Center has been a leader in the technology, and our platform currently contains ~300 protein markers, covering all major signaling pathways. We have generated and compiled the RPPA data of cell lines that were treated with >150 compounds under multi-dosage, replicates or triplicates up to 8 time points in 2D/3D cell culture. In total, we have >25,000 perturbed samples profiled using our RPPA platform. From these adaptive RPPA data, we developed computational algorithms to infer therapeutic liabilities including synthetic lethality and related combined therapies in a systematic way. Representative examples include PARP and MEK inhibitor combination showing synergistic activities in RAS mutant tumors. We further built an integrated data portal that contains user-friendly analytic and visualization tools for a broad biomedical community to utilize these data.

#1657

Prediction of pathologic complete response to neoadjuvant therapy for esophageal adenocarcinoma using copy number alterations.

David C. Qian,1 Joel A. Lefferts,2 Bassem I. Zaki,2 Pavlo Mishyn,2 Elizabeth B. Brickley,3 Yue Xue,4 Mikhail Lisovsky2. 1 _Winship Cancer Institute of Emory University, Atlanta, GA;_ 2 _Dartmouth-Hitchcock Medical Center, Lebanon, NH;_ 3 _London School of Hygiene & Tropical Medicine, London, United Kingdom; _4 _Emory University Hospital, Atlanta, GA_.

Background: Standard of care for locally advanced (stage II-III) esophageal adenocarcinoma is neoadjuvant chemoradiation followed by esophagectomy. Pathologic complete response (pCR) to neoadjuvant therapy occurs in 25-30% of patients. Three-year overall and progression-free survival of patients with pCR are nearly double those of incomplete responders (86% and 80% versus 48% and 39%, respectively). Therefore accurate prediction of pCR prior to esophagectomy can potentially spare patients the intraoperative risks and postoperative changes in quality of life associated with surgery. However, no well-established biomarkers of pCR currently exist.

Methods: Paired pre-treatment biopsies and post-chemoradiation resections from patients with stage II-III esophageal adenocarcinoma between 2003 and 2017 were retrieved at a single institution. pCR was defined as absence of cancer cells in the treated specimen, while pathologic poor response (pPR) was defined as greater than 10% of tumor cells remaining in the specimen. In total, 27 pCR and 25 pPR resections were identified along with 20 and 12 of their corresponding DNA samples from pre-treatment biopsies that passed quality control on the OncoScan platform to call copy number alteration events. Within every cytogenetic band in the human genome, the quantity of bases gained by each sample was computed as the sum of gained genomic segment lengths weighted by the surplus copy number of each segment. These cytogenetic band gains were compared between pCR and pPR samples. An independent validation dataset of 47 patients with stage II-III esophageal adenocarcinoma was obtained from The Cancer Genome Atlas (TCGA).

Results: In 3-fold cross-validation, genomic gains in chromosome 14q11 and chromosome 19p13 displayed the most significant difference between pCR and pPR samples in all 3 training sets. Across the test sets, average AUC for correctly predicting pCR/pPR status was 0.829. Selecting 1.1 megabases as the cutoff that optimizes tradeoff between sensitivity and specificity in the discovery dataset also stratified survival in TCGA. Among TCGA patients, those possessing genomic gains in chr14q11 and chr19p13 that exceed 1.1 megabases had superior 5-year overall survival (44% versus 0%, HR = 0.324, P = 0.0249) with median survival of 46 months versus 20 months.

Conclusion: Genomic gains in 14q11 and 19p13 may be a valuable biomarker for inference of pCR/pPR among patients with esophageal adenocarcinoma. To our knowledge, it is the first such biomarker with demonstrated utility in an independent set of patients. Further prospective validation is warranted.

#1658

Performance evaluation of assembly based structural variation discovery in paired tumor normal samples.

Chunlin Xiao. _NIH, Bethesda, MD_.

Structural variations (SVs) are well known to contribute to genetic diversity of human populations, affect biological functions, and cause various human disorders. However, accurately identifying SVs with correct sizes and locations in the human genome, particularly in cancer samples, remains a challenge due to the complexities of the human genome, limitations of sequencing technologies, and drawbacks of analysis methods. Recent advancement of next-generation sequencing technologies has significantly reduced the sequencing cost, while substantially increased the lengths of the sequencing reads. Therefore, using de novo assembly based approaches for discovering a full spectrum of SVs in human genome becomes tempting. While various assembly methods have been developed and proposed for general use by the community, the relative efficiency and predictive accuracy of SVs calling based on these assembly methods have not been fully evaluated. In this study, we applied several popular de novo assembly tools to the sequencing read data that were generated using multiple sequencing technologies with technical replicates for a paired tumor-normal samples. Assemblies and SVs callsets were generated, and repeatability in the SVs of the technical replicates and reproducibility across sequencing sites were evaluated. Results showed that there were substantial variabilities between SVs callsets based on various assemblies within sequencing platform and cross sequencing sites. We also observed considerable variabilities between assembly based SVs callsets and alignment based SVs callsets. The sources of these variabilities will be further discussed. These results allow better understanding of the impacts of de novo assembly methods on SVs calling, thus providing a better insight to precision medicine.

#1659

Overcorrection of batch effects by ComBat can be avoided by using an equal medians method.

John C. Obenauer,1 Thomas P. Stockfisch,2 Marcia V. Fournier3. 1 _Rancho BioSciences LLC, Southaven, MS;_ 2 _Rancho BioSciences LLC, San Diego, CA;_ 3 _Bioarray Genetics, Farmington, CT_.

Combining multiple data sets from the Gene Expression Omnibus (GEO) or other data repositories for an integrated analysis requires appropriate batch correction. ComBat, an empirical Bayesian method for batch correction of microarray data, is widely used and has been reported to be the best correction method. We combined cancer data from 16 public studies representing 8 tissue types and a total of 3,563 samples, used the R "sva" package and ComBat for batch correction, and examined 6 gene sets representing positive and negative controls. As positive controls, we extracted 4 gene sets from the Human Protein Atlas that were found to be expressed at least 5-fold higher in one tissue than in any of 35 other tissues, and we matched these genes to their Affymetrix U133A probesets. This resulted in 16 probesets specific for stomach, 18 for lung, 37 for pancreas, and 27 for prostate. A fifth positive control is a group of 85 genes called BA80 that we have found to be expressed much lower in blood than in solid tissues. As a negative control that we do not expect to change much between tissues, we used a list of 3,804 housekeeping (HK) genes that were reported to show less than a four-fold expression change across 16 tissue types. We compared the ComBat results to a new method we call equal medians. The equal medians method assumes that the 22,277 genes measured on the Affymetrix U133A microarrays can vary widely between tissues and batches, but that the median of the 22,277 genes is the same for every sample. We created boxplots of each gene set across the 16 studies before and after each method of batch correction. The reduction in batch effects was scored using the change in standard deviation of the HK genes. The preservation of biological variability was scored using the fold change of the positive controls, comparing the target tissue's median to the nearest alternate tissue's median. We used two GEO studies as independent representatives of each tissue type, so the two fold changes were averaged to create a single measure.

The results using the HK genes showed that ComBat removed 99.90% of the batch effects visible in the raw data, while equal medians removed 61.58%. However, equal medians did the best at preserving biological variability, with a fold change of 4.8 for stomach, 13.1 for lung, 42.3 for pancreas, 12.0 for prostate, and 3.9 for blood. The corresponding fold changes for ComBat were 1.4, 1.1, 2.2, 1.0, and 1.0.

We conclude that ComBat was best at removing batch effects, but at the undesirable cost of minimizing biological variation. We believe this is due to known and unknown sources of variability that are confounded with batches, which is one of ComBat's known risks. Equal medians showed the opposite performance, preserving biological variation better while partially removing batch effects. We offer the equal medians method as an alternative batch correction method in cases where ComBat shows evidence of overcorrection.

#1660

Identification of allelic imbalance utilizing heterozygous genotype allele frequencies and intensities.

Kyle Chang, Francis A. San Lucas, Zuhal Ozcan, Smruthy Sivakumar, Yasminka A. Jakubek, Richard G. Fowler, Paul Scheet. _UT MD Anderson Cancer Center, Houston, TX_.

Allelic imbalance (AI) events, such as amplification, deletion or copy-neutral loss-of-heterozygosity (cn-LOH) can result in the activations of oncogenes or inactivations of tumor suppressor genes that are critical to the process of carcinogenesis and metastasis. However, tumor samples often have low tumor-cellularity and small subclones that require sensitive and robust algorithm for AI detections. To overcome this challenge, our lab has developed an original software application hapLOH (Vattathil and Scheet, 2013) that identifies AI with high sensitivity in SNP array data by utilizing heterozygous genotype allele frequencies in SNP array data using a Hidden Markov Model. Motivated by the plethora of next-generation sequencing (NGS) data, we carried this logic and functionality and developed hapLOHseq (San Lucas et al, 2016) for detection of subtle AI in NGS data using reference and alternate allele read depths and a set of haplotype estimates based on 1000 genome data. Recently, we sought to improve AI detection and provide support for different data types by developing haploh-cn. Haploh-cn extends our previous tools by supporting both SNP array and NGS data, and by incorporating intensity data and Log R ratios (LRR) into the underlying Hidden Markov Model for SNP array data, and incorporating sequencing depth for NGS data. In order to evaluate haploh-cn's performances, we have downloaded The Cancer Genome Atlas Lung Adenocarcinoma whole exome sequencing data and corresponding Affymetrix SNP6 array data. Then, we simulated tumor cellularity by performing in-silico dilution of NGS and SNP array data. For NGS data, we down sampled tumor sequencing BAM files and mixed in matched-normal sequencing reads. For SNP6 array data, we combined the adjusted B allele frequencies of heterozygous sites in the tumor sample and matched-normal sample. Using the AI events detected in the undiluted tumor samples as the gold standard, we assessed the performance of haploh-cn in the computational diluted samples. Our results showed that haploh-cn had high sensitivity without sacrificing specificity in diluted tumor samples. In summary, haploh-cn is a robust and powerful method for profiling subtle AI in NGS and SNP array data.

#1661

Molecular modeling of an emerging chemopreventive agent wedelolactone against multiple targets in oncogenetic pathway.

sadia sarwar,1 Tauqeer Ahmed,1 Jun Q. Yu,2 Fazlul Huq2. 1 _Riphah International University, Islamabad, Pakistan;_ 2 _The University of Sydney, Sydney, Australia_.

Targeting multiple pathways involved either in drug resistance or/and in intact cancer cell survival are promising strategies. Epigenetics is being immensely recognized as exciting frontier in cancer research. Kinases have direct and indirect epigenetic impacts. Kinases are among the popular discovery targets and kinase inhibitors represent one of the key classes of drugs in the last two decades of oncology research. Braf kinases and cyclin dependent protein kinases exert their role as key mediators of signal transduction, apoptosis and overall intercellular communications. Topoisomerase II inhibitors including etoposide, tinoposide are important drugs used in breast cancer, sarcomas and lymphomas while inhibitors of dihydrofolatereductase, a key enzyme in the synthesis of DNA, are also clinically useful antagonists/inhibitors of ERα and aromatases have the potential to be therapeutically significant towards estrogen mediated breast, ovarian and uterine (endometrium) cancers. Wedelolactone is emerging as a small molecule with cytotoxic potential. Auto Dock Vina program was used for docking study .Affinity of best docked pose of ligand and protein target complex was determined by E-value (Kcal/mol).3D-structures of target proteins were taken from http://www.rcsb.org/pdb/home/home.do. The target proteins include dihydrofolatereductase, braf kinase , cyclin dependent protein kinase, nuclear factor kappa b, topoisomerase II, aromatase, estrogen receptor alpha and alkaline phosphatase (PDB ID: 2C2T, PDB ID: 3Q4C, PDB ID: 1HCL, PDB ID: 1NFK, PDB ID: 5B58, PDB ID: 3S7S, PDB ID: 3ERT, PDB ID: 1ANI). The structures (wedelolactone and standard drugs) were downloaded in.xml format and converted to PDB format via Open Babel JUI software. PDB form of wedelolactone, EGCG, reference drugs and target proteins were converted to PDBQT via AutoDock Tools (Version1.5.6 Sep_17_14). Discovery studio visualizer was used for post docking interactions for number of Hydrogen bonds (classical and non-classical), π-π bonds and other hydrophobic interactions by binding amino acid residues. The cell survival fraction was determined using the MTT reduction assay. The order of binding affinity of wedelolactone was found as: estrogen receptor alpha >dihydrofolate reductase>braf kinase > topoisomerase II > CDPK > aromatase > alkaline phosphatase >Nfkb. We identified wedelolactone as a cytotoxic agent in breast and ovarian tumor models with the potential to inhibit multiple targets in oncogenic as depicted through its In-silico study. Further research should be encouraged to investigate different aspects of wedelolactone.

#1662

Detection of chromosomal allelic imbalances from RNAseq data using hapLOHseq.

Zuhal Ozcan, F. Anthony San Lucas, Yasminka Yakubek, Yihua Liu, Richard G. Fowler, Paul A. Scheet. _The University of Texas M.D. Anderson Cancer Center, Houston, TX_.

RNA-seq is commonly used to profile tumors through transcript quantification and detection of alternative transcripts and gene fusions. Recent advancements enabled assessment of allele-specific gene expression. In this study, we characterize chromosomal allelic-imbalances (AI) through the identification of haplotype-specific patterns in RNA-seq data.

We processed SNP-array, whole-exome sequencing (WES), and RNA-seq data for two cancer sites from The Cancer Genome Atlas (TCGA): colon adenocarcinoma (COAD) and lung adenocarcinoma (LUAD). For the WES and RNA-seq data, we phased variants in the 1000 Genomes Project database and then used the hapLOHseq algorithm to detect AI. AI events detected in the Affymetrix SNP array 6.0 data were used as a gold standard, as it queries a much larger proportion of heterozygous sites relative to WES and RNA-seq.

A comparison of AI calls derived from the TCGA COAD RNA-seq and WES data to the gold standard events inferred from SNP-array yielded similar specificity estimates of 92% and 96% in WES and RNA-seq, respectively. However, with a sensitivity of 43%, RNA-seq derived AI calls had decreased overlap with gold standard events, compared to WES derived AI calls with 86% sensitivity. The TCGA LUAD analysis resulted in specificity estimates of 88% and 95% in WES and RNA-seq, respectively, and sensitivity estimates of 84% and 35%, respectively, for identifying gold standard events. Events detected exclusively in RNA-seq are classified as false positives in our sensitivity/specificity calculations. Possible validity of those events was assessed by investigating clinical correlations between RNA-seq only events and clinical phenotypes such as cancer stage, overall survival, and mutations in 21 bona fide tumor suppressor genes (TSG) involved in chromatin modification. We found a marginally significant correlation between the number of RNA-seq only events and the number of mutations in this set of TSG genes (p value = 0.07, corr. = 0.19) for the COAD study; however, we did not observe such a correlation in the LUAD study. This may be explained by a potential larger influence of extrinsic environmental factors, smoking, leading to epigenetic changes in the LUAD. In the LUAD, we observed a potential clinical association between the existence of RNA-specific events and worse overall survival (p-value = 0.15).

Although at lower sensitivity levels, our results suggest that RNA-seq may be used to detect AI in the absence of available DNA profiles. Clinically, our results may indicate that higher RNA-specific AI load may have a worse overall prognosis and we are exploring this further in additional TCGA datasets. The AI we detect in the RNA-seq samples may reflect long-range epigenetic dysregulation. RNA-exclusive AI may be caused by mutations in cancer driver and lineage genes may lead to selective cis-epigenetic chromatin alterations affecting large genomic regions.

#1663

Analytical methods to identify tumor heterogeneity and rare subclones in single cell DNA sequencing data from targeted panels.

Manimozhi Manivannan, Sombeet Sahu, Shu Wang, Dong Kim, Niranjan Vissa, Jordan Wilheim, Adam Sciambi, Nianzhen Li, Robert Durruthy-Durruthy, Anup Parikh, Keith Jones. _Mission Bio, South San Francisco, CA_.

Current bulk sequencing paradigms are inadequate to characterize complicated biological systems where somatic variation is buried in the landscape of populations. Single-cell DNA sequencing can simultaneously identify zygosity, rare alleles and determine whether mutations co-occur within the same cell. While high-throughput single-cell DNA analysis has been a recent innovation, it is essential to develop new capabilities for assessing genetic variation present in rare cells and to better understand the role that these cells play in the evolution of tumor progression. To address these challenges and enable the characterization of genetic diversity in cancer cell populations, we developed analytical methods to identify mutation signatures which define subclones present in a tumor population. Here we present a workflow for subclone identification using data generated on the Tapestri single-cell DNA platform. The analytical pipeline steps include obtaining raw reads from the sequencer, removing adapters, aligning and mapping the reads, calling individual cells, error correction of reads assigned to cells and identifying genetic variants within each cell. After filtering for high quality variants, we then filter for data completeness to ensure high quality data is used in downstream processing. The variant-cell matrix is then subjected to clustering to identify subclones. Top variants that define the signature of each subclone are also identified. To validate our methodology, we used data from two different targeted panels on different model systems with known truth mutations. Our pipeline shows the distinct clusters correlating with titration and cell line ratios. Cluster associated signature mutations were also identified. The pipeline can be used for multi sample analysis with time-series data from diagnosis to relapse or from primary site to metastasis to understand clonal diversity. These data demonstrate the utility of the Tapestri platform, the analytical pipeline, and associated data visualization capability. Our approach addresses key issues of identifying rare subpopulations of cells down to 0.1%, and transforms the ability to accurately characterize clonal heterogeneity in tumor samples. This high throughput method advances research efforts to improve patient stratification and therapy selection for various cancer indications.

#1664

Comparison of RNA sequencing data generated from formalin-fixed, paraffin-embedded (FFPE) papillary thyroid carcinoma samples using different library preparation methods.

Julie Dragon, Ramiro Barrantes, Jessica Hoffman, Scott Tighe. _UVM, Burlington, VT_.

Comparison of RNA sequencing data generated from formalin-fixed, paraffin-embedded (FFPE) papillary thyroid carcinoma samples using different library preparation methods. Julie Dragon, Ramiro Barrantes, Jessica Hoffman, and Scott Tighe.

Genomics research requires a significant sample size to provide robust biological signal, leaving researchers clamoring for access to large sample sets. As this research continues to expand into the clinical arena, the demand to sequence RNA from banked tissue samples, such as formalin-fixed paraffin-embedded blocks, is unavoidable. Readily available, these samples represent a wealth of both patients and disease variation. However, recovering DNA and RNA from such samples can be challenging depending on age of the sample block and fixture protocols, which influence nucleic acid degradation and ultimately our ability to synthesize RNA-Seq libraries. To fulfill the need for increased recovery of quality reads, several manufacturers have developed solutions to address these challenges, including FFPE-specific extraction kits, as well as library synthesis and quality control reagents.

In this study, we analyzed the quality and outcome of RNA-Seq data generated from RNA from two library synthesis kits applied to FFPE-derived human thyroid tumors with storage times from 4-12 years. The data processing pipeline included adaptor and poor quality base trimming (trimommatic), removal of rRNA reads (sortMeRNA), genomic alignment to hg19 (HiSat2), and read distribution and alignment quality assessment (RNASeqQC) (Adiconis and Levin, 2013). Differential expression was evaluated using DESeq2 (Love et al, 2014).

Results indicated that of the reagents tested, it is possible to improve capture of the usable RNA data, and biological signal, with improved control of rRNA depletion and read duplication with newly developed library preparation kits specifically designed for FFPE samples. Moreover, our analysis illustrates some features of the tested synthesis kits that enable one to perform better for FFPE samples with highly varying quality, and quality metrics to consider when evaluating FFPE samples for sequencing.

#1665

Development of standardized assay and software systems to deliver concordant results across sequencing platforms.

Sudhir Chowbina, Gustavo Cerqueira, Deacon Sweeney, Shantanu Shewale, James Hernandez, Eniko Papp, Mark Sausen, John Thompson, David Riley, Sam Angiuoli. _Personal Genome Diagnostics, Baltimore, MD_.

Introduction The role of NGS testing to inform clinical decisions has greatly expanded, now including biomarkers such as microsatellite instability (MSI) and Tumor Mutational Burden (TMB) for immune therapies. As oncology adopts widespread clinical use of NGS, standardization becomes essential. PGDx is developing IVD NGS assays optimized for these complex and comprehensive tumor profiling applications. Here, we describe the analytical performance for detection of various analytes using the PGDx elioTM tissue hybrid capture-based assay on the Ion Torrent S5 NGS platform. Notably, standardized and systematic design of wet chemistry and bioinformatics algorithms in combination yields high-performing results, regardless of sequencing platforms.

Method The comprehensive PGDx assay covers the coding regions of >500 genes, complex structural alterations, and genomic signatures associated with solid cancers. Specifically, single-base substitutions (SBS), amplifications, fusions, MSI and TMB are analyzed. A custom bioinformatics pipeline was developed to accommodate single-end Ion Torrent reads with analyte-specific algorithms. PGDx's VariantDx software was used to detect variants with allele frequencies > 2%. MSI, amplifications and rearrangements were identified using proprietary methods and algorithms. TMB was calculated from non-synonymous somatic variants across the panel, excluding common germline alterations. TMB was normalized to the targeted region to achieve a mutation density of mutations per megabase. To determine analytical accuracy, we compared TMB values from the PGDx assay against a validated whole-exome sequencing (WES) method for 93 lung cancer patients.

Results TMB results demonstrated a Spearman correlation coefficient of >0.90 compared to whole-exome TMB. MSI and amplifications were detected with 100% analytical accuracy, compared to orthogonal Illumina-based methods (n=19). SBS achieved >94% agreement compared to samples sequenced with Illumina-based methods, covering the same >500 gene panel. ROS1 and RET rearrangements were detected in 100% of 20 cell-line replicates obtained from Horizon®.

Conclusion Collectively, these data indicate the systematic co-development of a hybrid capture-based NGS assay with bioinformatics software is practical on the Ion Torrent S5 NGS platform. PGDx elio comprehensive tissue assays may be utilized for detection of SBSs, amplifications, fusions, MSI and TMB on the Ion Torrent S5 NGS platform with robust correlation to Illumina-based results and gold-standard WES.

#1666

Developing healthcare data scientists using Veterans Health Administration (VHA) data resources: The NCI/VA Big Data Scientist Training Enhancement Program (BD-STEP).

Sarah G. Elder, Michelle Berny-Lang. _NCI, Bethesda, MD_.

Due to advances in molecular profiling and medical imaging and the increased adoption of electronic health records (EHRs), the growth in oncology-related data has surpassed the development of a workforce capable of analyzing these data. This marks a missed opportunity in cancer research to gain insights toward better prediction of disease risk and therapeutic response. The Big Data Scientist Training Enhancement Program (BD-STEP), jointly managed by the National Cancer Institute and the Veterans Health Administration, addresses this gap by training the next generation of healthcare data scientists using the VA's healthcare data infrastructure. Post-doctoral fellows from science and engineering backgrounds are paired with VA medical centers across the country, where they are immersed in the clinical environment as they pursue high-impact clinical research questions through data science. They enrich their data science skills using VA data resources, including clinical data from the VA healthcare system, genomic data from the Million Veterans Program (MVP), and treatment and diagnosis data from the VA Central Cancer Registry. Prospective fellows, data science experts, and healthcare researchers are encouraged to learn more about the program, its training opportunities, and existing projects using VA healthcare data.

#1667

A robust human immune profiling assay using CyTOF technology and automated data analysis software.

Stephen K. Li,1 Daniel Majonis,1 C. Bruce Bagwell,2 Benjamin C. Hunsberger,2 Vladimir I. Baranov,1 Olga I. Ornatsky1. 1 _Fluidigm Canada Inc., Markham, Ontario, Canada;_ 2 _Verity Software House, Topsham, ME_.

Immune monitoring is an essential method for quantifying changes in immune cell population numbers and states over time in health and disease. A cornerstone in translational and clinical research, it is frequently used in the investigation of chronic inflammation, infectious disease, autoimmune diseases, and cancer. The diversity of immune cell populations demands a highparameter approach to more fully and efficiently quantify these changes. Mass cytometry, which utilizes CyTOF® technology, is a single-cell analysis platform that uses metal-tagged antibodies to resolve over 40 markers in a single tube of sample without the need for compensation. It is an ideal solution for routine enumeration of immune cell populations. However, development of a robust, highly multiplexed assay requires panel optimization as well as standardization of instrument setup and an analysis pipeline.

We have developed a sample-to-answer solution for human immune profiling using mass cytometry. It includes an optimized 30-marker immune profiling panel provided in a lyophilized, single-tube format, validated SOPs for human whole blood and PBMC staining, an instrument data acquisition template, instructions for data acquisition on a Helios™ system, and automated software for data analysis. The software analyzes FCS 3.0 files generated with the kit; automatically reports cell counts, percentage calculations, and staining intensity; and produces graphical elements such as histograms, dot plots, and a Cen-se′™ (t-SNE variant) graph for 36 immune cell populations.

Here we present assay analytical validation data on repeatability, reproducibility, software precision, and software accuracy. We also present a performance comparison of the lyophilized material and a liquid formulation of the same antibodies and clones used in the lyophilized format. This assay provides a robust, complete solution for broad immune profiling using mass cytometry that reduces sources of variability and subjectivity in sample preparation and data analysis.

#1668

A comparative study of curated contents by knowledge-based curation system in cancer clinical sequencing.

Masayuki Takeda, Kazuko Sakai, Shigeki Shimizu, Takayuki Takahama, Takeshi Yoshida, Satomi Watanabe, Tsutomu Iwasa, Kimio Yonesaka, Shinichiro Suzuki, Hidetoshi Hayashi, Hisato Kawakami, Yoshikane Nonagase, Kaoru Tanaka, Junji Tsurutani, Kazumasa Saigo, Akihiko Ito, Tetsuya Mitsudomi, Kazuhiko Nakagawa, Kazuto Nishio. _KINDAI UNIVERSITY, Osaka, Japan_.

Background: Medical oncologists are challenged to personalize medicine with

scientific evidence, drug approvals, and treatment guidelines based on sequencing of clinical samples using next generation sequencer (NGS). Knowledge-based curation systems have the potential to help address this challenge. We report here the results of examining the level of evidence regarding treatment approval and clinical trials between recommendations made by Watson for Genomics (WfG), QIAGEN Clinical Insight Interpret (QCII), and Oncomine knowledge-based reporter (OKR).

Patients and methods: The tumor samples obtained from the solid cancer patients between May to June 2018 at Kindai University Hospital. The formalin-fixed paraffin-embedded tumor samples (n=31) were sequenced using Oncomine Comprehensive Assay v3. Variants including copy number alteration and gene fusions identified by the Ion reporter software were used commonly on three curation systems. Curation process of data were provided for 25 solid cancers using three curation systems independently. Concordance and distribution of curated evidence levels of variants were analyzed.

Results: As a result of sequencing analysis, nonsynonymous mutation (n=58), gene fusion (n=2) or copy number variants (n=12) were detected in 25 cases, and subsequently subjected to knowledge-based curation systems (WfG, OKR, and QCII). The number of curated information in any systems was 51/72 variants. Concordance of evidence levels was 65.3% between WfG and OKR, 56.9% between WFG and QCII, and 66.7% between OKR and QCII. WfG provided great number of clinical trials for the variants. The annotation of resistance information was also observed. The larger difference was observed in the clinical trials information. It was due to the different filtering process among three curation systems possibly.

Conclusion: This study demonstrates knowledge-based curation systems (WfG, OKR, and QCII) could be helpful tool for solid cancer treatment decision making. Difference in non-concordant evidence levels was observed between three curation systems, especially in the information of clinical trials. This point will be improved by standardized filtering procedure and enriched database of clinical trials in Japan.

#1669

Full length transcriptome sequencing of melanoma cell line complements long read sequencing assessment of genomic rearrangements.

Elizabeth Tseng,1 Brendan Galvin,1 Ting Hon,1 Wigard P. Kloosterman,2 Meredith Ashby1. 1 _Pacific Biosciences, Menlo Park, CA;_ 2 _UMC Utrecht, Utrecht, Netherlands_.

Transcriptome sequencing has proven to be an important tool for understanding the biological changes in cancer genomes including the consequences of structural rearrangements. Short read sequencing has been the method of choice, as the high throughput at low cost allows for transcript quantitation and the detection of even rare transcripts. However, the reads are generally too short to reconstruct complete isoforms. Conversely, long-read approaches can provide unambiguous full-length isoforms, but lower throughput has complicated quantitation and high RNA input requirements has made working with cancer samples challenging.

Recently, the COLO 829 cell line was sequenced to 50-fold coverage with PacBio SMRT Sequencing. To validate and extend the findings from this effort, we have generated long-read transcriptome data using an updated PacBio Iso-Seq method, the results of which will be shared at the AACR 2019 General Meeting. With this complimentary transcriptome data, we demonstrate how recent innovations in the PacBio Iso-Seq method sample preparation and sequencing chemistry have made long-read sequencing of cancer transcriptomes more practical. In particular, library preparation has been simplified and throughput has increased. The improved protocol has reduced sample prep time from several days to one day while reducing the sample input requirements ten-fold. In addition, the incorporation of unique molecular identifier (UMI) tags into the workflow has improved the bioinformatics analysis. Yield has also increased, with v3 sequencing chemistry typically delivering > 30 Gb per SMRT Cell 1M. By integrating long and short read data, we demonstrate that the Iso-Seq method is a practical tool for annotating cancer genomes with high-quality transcript information.

#1670

A hidden markov modeling approach for identifying tumor subclones in next-generation sequencing studies.

Bin Zhu, Hyoyoung Choo-Wosoba, Paul Albert. _NCI, Bethesda, MD_.

Allele-specific copy number alteration (ASCNA) analysis is for identifying copy number abnormalities in tumor cells. Unlike normal cells, tumor cells are heterogeneous with a combination of dominant and minor subclones with distinct copy number profiles. Estimating the clonal proportion and identifying main and subclone genotypes across the genome is important for understanding tumor progression. Several ASCNA tools have recently been developed, but they have been limited to the identification of subclone regions, and not the genotype of the subclones. In this paper, we propose subHMM, a hidden Markov model-based approach that estimates both subclone region as well as region-specific subclone genotype and clonal proportion. We specify a hidden state variable representing the conglomeration of clonal genotypes and subclone status. We propose a two-step algorithm for parameter estimation, where in the first step, a standard hidden Markov model with this conglomerated state space is fit. Then, in the second step, region-specific estimates of the clonal proportions are obtained by maximizing region-specific pseudo-likelihoods. We apply subHMM to a renal cell carcinoma dataset from The Cancer Genome Atlas. In addition, we conduct simulation studies that show the good performance of the proposed approach.

#1671

Characterization of a gene signature predictive of cancer patient survival prognosis and chemo-response.

Paul Tran, Shuchun Li, Hai-Tao Liu, Lynn Tran, Sharad Purohit, Boying Dun, Jin-Xiong She. _Augusta University, Augusta, GA_.

Precision oncology aims to match cancer patients with drugs based on their unique molecular profiles. The MATCH trial results indicate ~80% of cancer patients do not benefit from screening cancer patients for actionable genomic aberrations. We aim to identify potentially druggable gene signatures that are molecular drivers of poor survival outcome for these cancer patients. We analyze data from The Cancer Genome Atlas (TCGA) to identify patient groups with differential survival characteristics using gene expression data. We applied a univariate Cox regression model to identify genes associated with patient survival and identified a 67 gene signature of highly correlated genes significant for cell cycle related gene ontologies on over-representation analysis. Patients from the TCGA dataset whose tumors have a higher gene expression score are more likely to have worse survival prognosis compared to tumors with a lower gene expression score, demonstrated through Kaplan-Meier survival analysis and univariate and multivariate Cox proportional hazard models. This suggests we have identified a co-regulated gene expression network associated with cellular proliferation which predicts overall and recurrence-free survival in many of the cancers in the TCGA dataset. We further analyzed data from the NCI-60 Human Tumor Cell Lines Screen and found the cellular proliferation gene signature expression is correlated with response to cell cycle inhibitors. We then validated this finding using resistant cell lines and gene knockdown models in vitro. This gene signature may provide an important tool for selecting patients for chemotherapy administration.

#1672

Clinical validation of a genomics-based classifier to predict tissue of origin from targeted tumor sequencing.

Youyun Zheng, Alexander V. Penson, Niedzica Camacho, Evan Biederstedt, Ahmet Zehir, Cyriac Kandoth, Aijazuddin Syed, Anna M. Varghese, Hikmat A. Al-Ahmadie, Nikolaus Schultz, Marc Ladanyi, David B. Solit, David S. Klimstra, David Hyman, Barry Taylor, Michael F. Berger. _Memorial Sloan Kettering Cancer Center, New York, NY_.

Introduction

Various classes of genomic alterations can now be robustly detected from targeted Next Generation Sequencing (NGS)-based tumor profiling assays. Despite recent advances in genome-directed cancer therapies and lineage-agnostic basket clinical trials, tissue of origin remains a critical determinant of tumor biology and therapeutic sensitivity. Therefore, efficiently harnessing the mutational information to predict tissue of origin from NGS data can further aid diagnosis and treatment.

Methods

We have developed a novel random forest machine learning classifier that infers tissue of origin from the mutational features of each tumor NGS profile. We trained and validated the model using prospective sequencing data from >30,000 patients profiled by MSK-IMPACT, a custom FDA-authorized clinical sequencing assay. An initial version of this classifier trained on a smaller cohort, presented previously, has been further optimized to incorporate additional tumor types, genomic features, mutational signatures, and accessibility features for clinicians. Altogether, the model evaluates a comprehensive range of mutational features to generate predictions. In addition, we developed a framework for the prospective clinical implementation of our method that allows for extension to the expanding MSK-IMPACT cohort and utilization at the point of care.

Results

Overall, we predicted the correct cancer type in 74% of cases, with nearly half of cases predicted with high confidence (>95%). In order to make this tool accessible to pathologists for real-time diagnostic and treatment decisions, we also have implemented APIs that transmit our classifier predictions to the cBioPortal for Cancer Genomics and the MPath console from MSK Molecular Diagnostics Service. With the additional samples and genomic features, our model predictions have been improved and are considered during clinical review and sign-out. This practice has brought about critical diagnostic changes and orthogonal validation in several cases. In one case, a patient referred to MSK with a misdiagnosis of metastatic breast cancer to the bladder underwent MSK-IMPACT sequencing for her primary breast tumor and bladder lesion. The model predictions, confirmed by orthogonal testing, classified two lesions as independent primaries of breast and bladder origins, leading to significant changes in treatment regimens.

Conclusion

Our work delineates the framework of utilizing and optimizing machine learning models on NGS-based sequencing data to aid diagnosis and treatment. The same framework can be applied to cell-free DNA sequencing, which will capture tumor spatial heterogeneity and improve classifier performance. These results indicate that leveraging machine learning to

predict tissue of origin complements conventional histologic review to provide integrated diagnoses, often with critical therapeutic implications.

#1673

In silico modeling of RNA binding domains (RBDs) of nucleolin and miRNA interactions.

Avdar San,1 Anjana D. Saxena,2 Shaneen Singh2. 1 _Biochemistry PhD Program, the Graduate Center, CUNY, New York, NY;_ 2 _Brooklyn College and Biology/Biochem PhD Programs at the Graduate Center, CUNY, New York, NY_.

In eukaryotes, the stress-responsive RNA-binding phosphoprotein nucleolin (NCL) plays defined role/s in many critical cellular processes, including transcriptional and translational regulation of various non-coding and coding RNAs. The RNA binding domains (RBDs) in NCL varies in numbers (1 to 4) depending on the organism, and individual RBDs are more conserved in divergent species than within the same protein. NCL targets a diverse RNA species e.g. long-noncoding-RNA, rRNA, mRNA to control gene expression and is also implicated in dysregulation of miRNAs in a variety of cancers; NCL is often highly upregulated in tumors. Analyses of mRNA and miRNA transcriptomics suggest that NCL plays a direct role in biogenesis of certain oncogenic miRNA species; targeting NCL by aptamers reduces the expression of these miRNA as well as in vivo tumor load. However, there is a large gap in knowledge in understanding miRNA-NCL interactions. Full length NCL protein structure still remains unresolved limiting structural predictions. Available partial NCL-RBDs structures are inadequate in providing cues to the plethora of NCL functions, especially its ability to interact with many different species of RNA. Molecular phylogeny for NCL-RBDs remains elusive as does target specificity of NCL. The number of RBDs, which structural combinations of RBDs and the cellular conditions that trigger specific NCL-RNA interactions remain to be understood. We have used in silico tools to model NCL-RBDs to assess their interactions with different RNA species focusing on a subset of miRNAs whose biosynthesis is positively correlated with NCL levels in a variety of aggressive cancer types e.g. breast, lung, pancreas and prostate cancers. Here we present a combination of template-based and ab initio approaches to develop robust theoretical models of all 4 RBDs in tandem as well as 2 consecutive RBDs in different combinations, for human NCL. Development of such theoretical constructs is essential to address hypotheses pertaining to target specificity of NCL for different RNA species in the absence of complete structural information. Further, these refined models were used to probe binding affinities and specificities of various miRNAs using different docking algorithms that analyze protein-RNA interactions. Our results provide an initial glimpse into possible binding modes and RBD specificity of human NCL for different miRNA molecules. Our study is further enhanced by modeling and comparative analysis of RBDs from NCL homologs in different organisms that house a variable number of RBDs. The results from this molecular modeling study lay the groundwork for elaborating the detailed mechanism of NCL-miRNA interactions, the evolutionary basis of RNA-binding and need for the expansion of RBD units in higher organisms.

#1674

Prediction of survival based on tumor size dynamics and new lesions in EGFR mutation-positive non-small cell lung cancer patients treated with gefitinib or carboplatin and paclitaxel.

James Dunyak,1 Jacqueline Buros-Novik,2 Eric Novik,2 Hong Yan,1 Nidal Al-Huniti1. 1 _AstraZeneca, Waltham, MA;_ 2 _Generable, New York, NY_.

Objectives: Dynamics of tumor size have long been used for the clinical diagnosis, staging, prognosis and treatment of non-small cell lung cancer (NSCLC). The aim of this work was to develop a joint statistical framework to interrogate the longitudinal tumor burden, the appearance of new lesions, and changes in therapy following progression in a population of NSCLC patients treated with an EGFR inhibitor gefinitib to predict, given early results, time to progression (PFS) and overall survival at both population and individual levels.

Methods: The model builds on a previously developed joint model for tumor size and survival. The tumor size model was extended to estimate the probability of a new lesion. For the survival submodel, two additional biomarkers were evaluated: the derivative of the sum of longest diameters (SLD) and the probability of new lesions. The model was fitted in a Bayesian framework using STAN. Model was developed from 434 EGFR+, NSCLC patients treated with gefitinib or carboplatin+paclitaxel ('IPASS', NCT00322452). Predictive performance was evaluated on a cohort of 102 EGFR+ NSCLC patients ('IFUM', NCT01203917).

Results: Overall, the model fit well to the observed data and the predictive performance was strong in both within- and out-of-sample evaluations. Surprisingly, SLD derivative and new-lesion association parameters appear to negatively impact model performance, indicating further improvement of these submodels might be necessary. The model recapitulates response evaluation criteria in solid tumors (RECIST) outcomes with nearly 90% accuracy. When comparing progression-specific hazards, it appears the drug effect is mediated by tumor size dynamics rather than new lesion incidence.

Conclusion: This Bayesian joint model of tumor size and survival accurately recapitulates the RECIST-based outcomes and generates well calibrated predictions of survival. It offers insights regarding the relative predictive value of the components of RESICT in NSCLC.

#1675

Analytical validation of MSI High detection with GuardantOMNI.

Jing Zhao,1 Alexander Artyomenko,1 Carlo Artieri,1 John Latham,1 Stephen R. Fairclough,1 Catalin Barbacioru,1 Elena Helman,1 John Strickler,2 Darya Chudova,1 Richard Lanman,1 AmirAli Talasaz1. 1 _Guardant Health, Redwood City, CA;_ 2 _Duke University, Durham, NC_.

Background: Late stage therapy selection is advancing beyond the detection of classical driver alterations (SNV, Indel, CNV, Fusion) to the use of aggregate biomarkers comprising combinations of SNVs, Indels, and fusions, that reflect the underlying mutational process, indicate the cellular dysfunction, and suggest the neoantigen signature. GuardantOMNI (OMNI), is a highly sensitive 500-gene cfDNA sequencing test designed to detect primary somatic mutations as well as aggregate biomarkers including TMB and DDR pathway defects. The newest version of OMNI introduces MSI biomarker detection, which is an indication for the first FDA-approved pan-cancer therapy. Here we present the first analytical validation study of OMNI MSI-H detection.

Methods: Probabilistic models for MSI detection were designed to interrogate instability across >1300 loci, differentiating PCR and sequencing induced artifacts from true biological signals using molecular barcoding as implemented in the Guardant digital sequencing platform. Cut-offs were selected based on the analysis of the training healthy donor cohort and locked prior to validation. Analytical performance was assessed using pre-characterized cell lines, cancer-free donor-samples, and clinical patient samples. Qualitative and quantitative orthogonal confirmation was provided by next-generation sequencing (NGS) and published information.

Results: 350 samples were processed for this study, using both 5ng and 30ng cfDNA input levels, and all samples passed sequencing QC metrics established prior to testing. Cell line-based dilution studies performed in triplicate spanning 5ng and 30ng input range as well as in silico simulation demonstrated 95% limit of detection (LoD) at 0.1% Analysis of 121 healthy donor and 16 MSS advanced cancer patient samples identified no false positives (FP). In 240 patient samples with orthogonal confirmation of MSI status from tissue or panel NGS, GuardantOMNI demonstrated 100% analytic sensitivity (3/3) and 100% analytic specificity (237/237) with a PPV of 1 and and NPV 1. Retrospective analysis of 2000+ clinical samples detected MSI high in 16% of colorectal cancers, 4% of prostate cancers, and 0.6% of non-small cell lung cancers.

Conclusions: Here we have demonstrated the performance of MSI-H detection with GuardantOMNI (OMNI), which has the potential to accelerate clinical trial enrollment, research and discovery with a single, non-invasive blood sample.

#1676

A hybrid ontology and graph system enabling precision oncology applications through real-time incorporation of omics biomarkers into a scalable real-world data platform.

James E. Shima, James L. Chen, Matthew J. Glick, Ryan A. Warrier, Eric C. Abruzzese, Walter C. Mankowski, Jonathan Hirsch. _Syapse, San Francisco, CA_.

Background: The ability to produce timely, clinically actionable recommendations is a cornerstone of a robust precision oncology program. This requires the assimilation of rapidly changing precision oncology data that flows asynchronously from various EMR and lab sources. A key challenge is harmonization of omics biomarkers from hospital-based and commercial testing labs, rendering these omics data interpretable and comparable between patients, labs and reports. To address this challenge, we describe an automated system leveraging a hybrid ontology and graph structure to permit the rapid harmonization and interpretation of molecular diagnostic results across multiple labs.

Methods: A two-part, fully automated system to ingest and harmonize omics biomarker data from hospital-based and commercial testing labs was developed. First, an ontology-based omics lexicon provides annotation of discrete laboratory values provided electronically. The ontology model is Manchester language-based with data stored in a relational document store. Second, a graph structure serves as the knowledge model and relates the annotated lab values to 12 types of clinically informative biomarkers, including sequence and copy number variants, tumor mutational burden, MSI, gene rearrangements, wild type, and expression biomarkers. The graph structure leverages the ontology to fully describe hierarchical and categorical data associated with each biomarker. Access to the omics knowledge model uses a RESTful API and relational database architecture. Acquisition of molecular data uses custom JSON schemas and a RESTful API.

Results: Feeds from 7 commercial and hospital-based labs resulted in the receipt of >12,000 molecular diagnostic reports containing >2 million individual biomarker observations, with integration of each report achieved within 2 minutes of electronic data receipt. As of Nov. 2018, 81.2% of reports had at least one biomarker observation, while the remaining reports lacked results. Within these biomarker observations, 95.8% were represented in the ontology/graph structure, resulting in successful linkage to ~22,000 distinct harmonized biomarkers out of more than 12 million in the knowledge base. Of the linked, harmonized variant biomarkers, 4% are class-level (e.g. BRAF mutation) with the remainder reflecting more specificity (e.g. BRAF V600E). Within the set of more specific biomarkers, 54% represent a protein altering variant biomarker.

Conclusions: We have built an automated, scalable system using a novel ontology/graph structure to reliably and automatically harmonize and integrate omics data into a real-world oncology precision medicine platform. Proof of concept validation has been demonstrated in over 12,000 patient reports, with the results being used to enable clinical trials matching and outcomes research.

#1677

Understanding the TME: Advanced analysis and visualization of multiplexed fluorescence images.

Douglas Wood, Courtney Hebert, Aditi Sharma, Jamie Buell, Sean Downing. _Ultivue, Cambridge, MA_.

Background: Multiplexed immunofluorescence (mIF) has the potential to revolutionize immuno-oncology and pathology research as it enables the identification of complex cell phenotypes and their potential interactions in the tumor microenvironment (TME). But for a whole slide image with millions of cells, as we increase the number of biomarkers imaged for every cell, the complexity of the data analysis and visualization task increases exponentially. For n markers, a total of 2n phenotypes are possible (e.g. 10 markers have 1024 potential phenotypes). To address this problem and reveal the biologically relevant information embedded in the data, we have developed software tools to reduce the complexity, visualize, and quantify spatial distributions of cells across the full spectrum of possible phenotypes.

Methods: Here we present results using two different methods. The first is an image processing technique called Phenotypic Surface Density Mapping (PSDM), that produces not only true surface density images of each phenotype (cells/µm2), but also surface density images that quantify a variety of other statistics such as the level of expression (intensity) of key markers, inter-phenotype nearest neighbor distance maps, and maps of cell size/morphology. Some important features of these surface density maps are that they are quantitatively robust, have real physical units (e.g. cells/µm2 or intensity/µm2), and they are generated in an unbiased fashion to reveal information about every possible phenotype. The second analysis method, dimensionality reduction, exploits a new technique called Uniform Manifold Approximation and Projection (UMAP), reducing dozens or even hundreds of dimensions for millions of cells to a simple 2D scatter plot. We have developed interactive software that displays the UMAP for a slide and allows the user to select a given cell or region of cells to view closeup images of each cell and statistics about the collection.

Results: Examples of the surface density maps provide insights into mapping the complexity of the TME. We assess the results on deidentified samples by comparison with both human generated labels (pathology review) for individual cells and with automatically generated labels (software review). We show how these tools can be used to both identify tumors and quantify the level of activity in different tumor regions. We demonstrate how increasing the level of multiplexing allows one to differentiate subtle variability and separate subclasses of cells from each other.

Conclusions: Multiplexed data brings valuable information about the TME but much of this information is inaccessible by simply viewing the captured images or performing simple cell counting alone. To address this problem, we demonstrate two new software tools, PSDM and UMAP that preserve and quantify the spatial information of the underlying biology and provide this analysis for all possible phenotypes.

#1678

Accurate detection of expressed variation in RNA-seq.

Lisle E. Mose, David Marron, Joel S. Parker. _UNC - Lineberger Cancer Center, Chapel Hill, NC_.

Somatic variant detection has long been an area of great interest and critical importance in cancer research and treatment. DNA-Seq based somatic variant calling pipelines have been maturing over the last several years and although there is still room for improvement, several methods have been developed capable of accurately detecting somatic variants from matched tumor/normal DNA. RNA-Seq has become a widely used tool for measuring gene and isoform abundance, identifying alternative splicing, and detection of gene fusions. Detection of expressed variation is receiving growing interest, however computational methods to detect this variation have received far less attention than that of DNA-Seq. We have developed an optimized RNA-Seq pipeline based upon the ABRA2 realigner capable of accurately detecting expressed somatic variation in RNA-Seq.

We applied this pipeline to the TCGA Breast Cancer dataset. To assess the impact of identification of expressed variation as an indicator of variant significance, we compared the expression state of the top 20 cohort wide significantly mutated genes (SMG) as identified by The Cancer Genome Atlas Network (2012) with all other genes. At the median for SMGs, 23 somatic variants are expressed with over 90% of variants detected in DNA being expressed. For non-SMGs, the median number of expressed variants is 1 and the median fraction of expressed variants is 50%. Among non-SMGs, a total of six genes exceed the median total number of expressed variants and fraction of expressed variants for SMGs (AHNAK, CHD4, ERBB2, FASN, FOXA1 and MYH9). All of these variants have been previously implicated in breast cancer, however none of these variants were among the cohort wide SMGs identified in the original study and only FOXA1 was identified as being a subtype specific SMG (ER+,ER+/HER2-). Notably, variants in the massive TTN gene are found to be coding 73% of the time, but expressed in only 8% of cases. This is substantially less than the 90% found in SMGs and a likely indicator that TTN mutations are passenger variants. The median and third quartile RNA variant allele frequency (VAF) for SMGs is .41 and .69 versus .32 and .46 for DNA. By comparison, the median and third quartile VAF for non-SMGS was 0 and .27 in RNA and .20 and .30 for DNA. The increased VAF for RNA SMGs is a likely indicator of selection and can potentially further be used to identify variants of significance. We believe these results demonstrate the utility of variant expression as a potentail tool to aid in assessment of variant significance.

#1679

Neoepitope screening in colorectal cancer patients by custom exome panel profiling somatic mutations.

Raghavee Venkatramanan,1 Kerry Deutsch,1 Inah Golez,1 Pallavi Shroff,1 Spencer Seale,1 Brendan Ralph,1 Sally. Dow,1 Evan Anderson,1 Saman Tahir,1 Anjali Malge,1 Corey Braastad,1 Steven Anderson2. 1 _Covance, Redmond, WA;_ 2 _Labcorp, MN_.

Colorectal cancer (CRC) is one of the major causes of global mortality. Recent clinical advances in T cell therapy using antibodies that block inhibitory T cell signals have been transforming cancer therapy. Studies show that somatic mutations in protein coding regions produce tumor-specific antigens, or neoantigens. CRC warrants deeper investigation into the non-synonymous exonic mutations that seem to be drivers in tumor development. Tumor mutation burden (TMB) and the abundance of neoantigens are highly correlated and it has been shown that TMB can be estimated using targeted sequencing panels. The aim of this study is to investigate 10 matched tumor/normal CRC samples using a panel targeting 422 genes covering about 1.7 Mb to screen for potential neoepitope candidates. Although sequencing and bioinformatics pipelines are developed to effectively filter neoantigens, it is still unclear which antigens elicit a T cell response. A review of the tools used in neoepitope prediction shows that it is not only a very complex task, but demands high sequencing depth. Comparing currently available tools will enable us to understand the strengths and shortcomings of each algorithm, and develop a custom pipeline framework.

## MOLECULAR AND CELLULAR BIOLOGY / GENETICS

### Cancer Genomics 2

#1680

RhoGAP and PAP2 domain-containing fusions are recurrent and prognostic in gastric cancer.

Hark K. Kim. _National Cancer Ctr. Korea, Goyang, Republic of Korea_.

We conducted an RNA sequencing study to identify novel in-frame gene fusions in 80 discovery dataset tumors collected from young Korean patients with diffuse gastric cancer. Twenty-five in-frame fusions were associated with diffuse gastric cancer, three of which (CLDN18-ARHGAP26, CTNND1-ARHGAP26, and ANXA2-MYO9A) were recurrent in 384 diffuse gastric cancers based on RT-PCR. All three fusions contained a RhoGAP domain in their 3' partner genes. Patients with one of these three RhoGAP domain fusions had a significantly worse prognosis than those without such fusions. The median survival was 29.1 [95% CI, 5.7‒not reached] and 94.6 [95% CI, 62.0‒not reached] months, respectively (P=0.011, log-rank; HR, 2.8 [95% CI, 1.5‒5.3]). Ectopic expression of CLDN18-ARHGAP26 promoted the migration and invasion capacities of diffuse gastric cancer cells. Parallel targeted RNA sequencing analysis additionally identified PAP2 domain-containing fusions as recurrent and poor prognostic in-frame fusions. Overall, in-frame gene fusions containing either RhoGAP or PAP2 domain clearly defined the aggressive subset (7.5%) of diffuse gastric cancer, and their prognostic impact (HR, 3.4 [95% CI, 2.0‒5.7]) was greater than, and independent of, chromosomal instability and CDH1 mutations. Our study may provide novel genomic insights guiding future strategies for managing diffuse gastric cancer.

#1681

Prospective clinical sequencing distinguishes independent primary cancers from recurrent or metastatic disease.

Alison M. Schram, Chai Bandlamudi, Ahmet Zehir, Marc Ladanyi, Michael F. Berger, Barry S. Taylor, David M. Hyman. _Memorial Sloan Kettering Cancer Center, New York, NY_.

Background: Standard histopathology and immunophenotyping of cancer are sometimes insufficient to distinguish independent primary cancers from recurrent/metastatic disease. Accurate classification is essential when making treatment decisions. We hypothesized that broad NGS, which has increasingly been adopted to inform treatment in patients with advanced cancer, also helps discriminate between multiple primary tumors and recurrent/metastatic disease.

Methods: We analyzed results from prospective NGS ('MSK-IMPACT') performed at Memorial Sloan Kettering between 2014-2018 involving >25,000 samples. Patients where MSK-IMPACT had been performed on more than one sample were classified based on 1) histologic determination of cancer type, and 2) the genomic similarity of the tumors. Genomic similarity was defined as having at least one shared somatic mutation or >20% shared copy number segment alterations. Tumors with purity <20% based on pathologic estimation were excluded. Cases were manually reviewed to determine whether the genomic data was useful in classifying tumors and how these results ultimately influenced patient care.

Results: We identified 41 cases where tumor sequencing was utilized to help determine the clonal relationship between either synchronous or metachronous tumors. In five patients with NSCLC, NGS confirmed that multiple lesions were separate cancers as opposed to metastatic or multifocal disease. This determination changed management in two patients who were recommended surveillance rather than adjuvant chemotherapy based on this finding. In contrast, another patient did not receive NGS results prior to starting treatment and could have avoided chemotherapy. Two patients with stage I NSCLC were thought to have recurrent disease but NGS demonstrated de novo metastases from a second NSCLC. This is notable because first-line treatment of metastatic NSCLC is dictated by tumor genomics. Four patients were thought to have recurrent cancer but genomics revealed two unrelated tumors. Three patients were treated definitively with localized therapy for a presumed new primary cancer but genomics later suggested metastasis from a synchronous (N=1) or prior (N=2) tumor. In several additional cases, molecular profiling was useful in supporting what was suggested to be the same (N=19) or different (N=10) tumors histologically.

Conclusion: Broad NGS can be utilized to distinguish independent malignancies from recurrent/metastatic disease, including cases where standard work-up is inconclusive. We identified multiple instances where clonal relationships were used to guide treatment, including whether to pursue potentially curative versus palliative therapy. These data demonstrate a potential additional use of NGS, which to date has largely been reserved for aiding treatment decisions in patients with advanced incurable cancer.

#1682

Non-V600 BRAF mutants in melanoma: Multiple MAPK mutations and distinctive RNA expression.

Elizabeth Kiernan,1 Paul Bastian,1 Liza Dorfman,1 Ioannis N. Anastopoulos,1 Brad Wubbenhorst,1 Clemens Krepler,2 Vito Rebecca,2 Meenhard Herlyn,2 Katherine Nathanson1. 1 _Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA;_ 2 _WISTAR Institute, Philadelphia, PA_.

Large discovery sequencing analyses conducted by our group and others enabled classification of melanomas into major and minor subtypes, helping to elucidate the role and diversity of activating mutations within the MAPK pathway. We sequenced 482 melanoma PDX, cell lines and patient biopsies, of which 20 (4.1%) carry non-V600 BRAF mutations. Of the 20 mutations, 14 are pathogenic, 5 are likely pathogenic and 1 is a variant of unknown significance. Interestingly, 78.5% of tumors with pathogenic non-V600 mutants and all likely pathogenic or VUS non-V600 mutants carry additional mutations within the MAPK pathway. To explore this pattern, we queried publicly available melanoma sequencing data from six previously published sequencing studies of melanoma. Consistent with our findings, nonV600-BRAF mutations were identified in 0 - 9% of melanoma sequencing studies. Of the tumors carrying non-V600 BRAF mutants, 26 of 42 (62%) carry additional mutations in the MAPK pathway. Given the co-occurrence of MAPK mutations with non-V600 mutants, we considered whether these mutations are found together within the same cell or if mutants are encoded in distinct cell populations and reflect admixture of a heterogenous tumor. We compared the variant allele frequency (VAF) of co-occurring MAPK mutants with their non-V600 BRAF counterparts in 41 available samples. The VAF between mutants was discordant (>0.10 change in VAF between mutants) in 27 of 41 (68%) of the samples. To more precisely assess variants at single-cell resolution, we established a PCR based method for variant calling using single-cell-derived cDNA libraries. Pilot studies supported distinct expression patterns and we are working to generate robust and reproducible sequencing data. We also compared RNA expression data between BRAFV600 melanomas and non-V600 melanomas. This analysis revealed distinct transcriptomes that were easily distinguished based on previously established quaternary structure and kinase activity of various BRAF mutants. Interestingly, MAPK-pathway genes did not account for most of the differential expression, and an AXL-high/MITF-low expression program was highly enriched in non-V600 BRAF tumors relative to their V600 BRAF counterpart (FDR <0.01). Taken together, non-V600 BRAF mutant melanomas represent a subtype characterized by a tendency for multiple MAPK mutations and an AXL-high/MITF-low enriched RNA expression pattern differentiated based on BRAF structure and function. Further studies of this rare sub-type of melanoma are important to demonstrate the diversity and interplay of MAPK dysregulation in cancer and may reveal mechanisms of tumorigenesis.

#1683

Mutational landscape of colorectal cancer with POLE gene mutation.

Yoshikage Inoue,1 Nobuyuki Kakiuchi,1 Kenichi Yoshida,1 Yusuke Shiozawa,1 Yuichi Shiraishi,2 Kenichi Chiba,2 Tetsuichi Yoshizato,1 HIroko Tanaka,3 Satoshi Nagayama,4 Satoru Miyano,3 Yoshiharu Sakai,1 Seishi Ogawa1. 1 _Kyoto University, Kyoto, Japan;_ 2 _National Cancer Center Research Institute, Tokyo, Japan;_ 3 _University of Tokyo, Tokyo, Japan;_ 4 _The Cancer Institute Hospital of Japanese Foundation for Cancer Research, Tokyo, Japan_.

Introduction

POLE-mutated tumor is a rare subtype of colorectal cancer (POLE-CRC) and has been poorly understood because of its rarity. In this study, we have investigated the clinical and genetic features of POLE-CRC in the largest cohort of this unique subtype of CRC.

Methods

We included a total of 3,240 patients who had been treated at the Cancer Institute Hospital of Japanese Foundation for Cancer Research between 2004 and 2017, which were screened for POLE mutations, either by targeted-panel sequencing (n=544) or amplicon-based deep sequencing (n=2,696). POLE mutation-positive samples were further analyzed by whole exome sequencing.

Results

In total, 74 POLE variants were detected in 69 samples, of which 40 showed prominent hypermutation (median; 5,346, range; 837-16,990) with the predominance of COSMIC signature 10 associated with defective POLE functions. Patients with POLE-CRC were significantly younger (median; 50, range; 33-84), compared with those with non-hypermutated CRC (median; 65, p-value<0.01) and MSI-CRC (median; 70, p-value<0.01). They also showed significantly favorable prognosis than patients with non-hypermutated CRC (p-value<0.01). In accordance with previous reports, mutational hotspots in POLE gene were found at codons 411, 286, 459, and 456, while a previously unreported variant was found in 1 case, which affected an active site of the exonuclease domain on codon 277. Significantly mutated genes or driver genes in 40 POLE-CRC cases were interrogated by evaluating non-synonymous vs. synonymous mutations in each gene, using dNdSCV. We found 9 genes showing significantly deviated dN/dS (q-value<0.1), including B2M, APC, TP53, PTEN, PIK3CA, PIK3R1, ARID1A, TAP1, and CD58 of which most frequently observed was APC (39/40; 97.5%). Of interest, two of these genes, B2M and TAP1, are involved in the antigen presentation machinery (APM). When samples having truncating mutations in HLA-A, B, C, and TAP2 are included, 70% (28/40) of POLE-CRC samples are thought to have compromised neoantigen presentation. Positive selection of mutations in APM was also found in our analysis of POLE-mutated endometrial cancer. Because of an extremely heavy burden of somatic mutations in POLE-mutated cancers, this finding suggested an important role of anti-cancer immune evasion in the pathogenesis of these POLE-mutated cancers. Also of note is frequent loss-of-function mutations in CD58, a molecule involved in NK-cell recognition. In 70% (7/10) of CD58 mutated cases, co-occurrence with mutations affecting the APM was detected. Loss of intact CD58 might play a role in the evasion from NK cell surveillance for tumors with defective HLA presentation.

Conclusions

Similar to MSI-CRC, POLE-CRC shows a favorable prognosis, where the mutations affecting APM are positively selected to evade immune surveillance, suggesting a possible role of checkpoint blockade in its therapeutics.

#1684

Multi-omics profiling of breast cancers during neoadjuvant chemotherapy identified distinct molecular changes and biomarkers associated with clinical response.

Samir Lal,1 Ying Ding,1 Jeong Eon Lee,2 Soo-Hyeon Lee,3 Se Kyung Lee,2 Jae-Yong Nam,2 Jong Han Yu,2 Yoon-la Choi,2 Seok Won Kim,2 Seok Jin Nam,2 Ji-Yeon Kim,2 Sripad Ram,1 Eric Powell,1 Keith A. Ching,1 Pablo Tamayo,4 William Kim,4 Huwate Yeerna,4 Soo Youn Cho,2 Vinicius Bonato,1 Shibing Deng,1 Jinho Kim,2 Hyuntae Shin,2 Woong-Yang Park,2 Paul A. Rejto,1 Jadwiga Bienkowska,1 Yeon-Hee Park,2 Zhengyan Kan1. 1 _Pfizer, Inc., San Diego, CA;_ 2 _Samsung Medical Center, Seoul, Republic of Korea;_ 3 _Pfizer, Inc., Seoul, Republic of Korea;_ 4 _University of California San Diego, San Diego, CA_.

The molecular bases underlying neoadjuvant chemotherapy (NAC) response are poorly understood. To elucidate the effects of NAC on breast tumor biology and its association with clinical outcome, we have conducted WES and RNA-Seq profiling of a longitudinal breast cancer (BC) cohort consisting of 146 cases (281 tumors, 109 pairs), including 55 (38%) that achieved pathologic complete responses (pCR) and 91 (62%) that harbored residual diseases at time of surgery. Tumor biopsies were collected for each patient at three time points - pre-treatment, three weeks after the first cycle of anthracycline and cyclophosphamide (AC) and at the time of surgery, after 3 more cycles of AC followed by 4 cycles of taxane. In addition to somatic mutations and copy number alterations, we also derived a comprehensive set of genomic and molecular features for each tumor including chromosomal instability, loss-of-heterozygosity, mutation burden, mutation signatures and expression signatures for oncogenic signaling pathways and immune cell subsets. Virtual microdissection analysis inferred 14 factors that represent distinct tissue compartment including a tumor infiltrating lymphocyte (TIL) factor and revealed that initial NAC treatment increased stromal and adjacent normal tissue fractions while reducing tumor cellularity. NAC also induced dynamic changes in immune gene expressions over time, a pattern that was validated through detecting and quantifying the density of TILs from H&E images. To investigate NAC induced changes in tumor intrinsic biology we classified tumors into five oncogenic cellular states on a reference Onco-GPS map defined by transcriptional signatures from breast cancer cell lines. We observed that, as a result of NAC treatment, tumors often change from one oncogenic state to another, transiently upregulating an EMT program that appears to mediate drug resistance and increase the likelihood of residual disease. Multiple regression and multivariate analyses were performed to identify predictive biomarkers of pCR status while adjusting for BC subtypes and tumor purity. We found that ER+ subtype and estrogen response signature but not Ki-67 were independently associated with NAC response. Within TNBC, the immunomodulatory subtype was enriched in responders while the basal-like subtype had the poorest response. Pretreatment TIL and changes in TIL level over time were independently associated with NAC response, implicating anti-tumor immunity in mediating the efficacy of chemotherapies. Through multi-omics characterization of longitudinally paired tumor biopsies, we have revealed dynamic changes in the tumor molecular states in BC patients undergoing NAC treatment, identified molecular markers of treatment outcome and derived insights into the mechanism of action as well as resistance to an important class of therapy.

#1685

Germline mutations in cancer predisposition genes are prevalent among survivors of childhood acute lymphoblastic leukemia in a Chinese population.

Ru Zhang,1 Yao Xue,2 Liwen Zhu,2 Meiyun Kang,2 Xiangye Xu,1 Jie Huang,2 Liucheng Rong,2 Juncheng Dai,3 Zhibin Hu,3 Yongjun Fang,2 Zhaoming Wang4. 1 _Guangzhou Women and Children's Medical Center, Guangzhou, China;_ 2 _Children's Hospital of Nanjing Medical University, Nanjing, China;_ 3 _Nanjing Medical University, Nanjing, China;_ 4 _St. Jude Children's Research Hospital, Memphis, TN_.

Landscape of pathogenic or likely pathogenic (P/LP) germline mutations in cancer predisposition genes have been well-studied among patients or survivors of childhood cancer in the US. However, little is known for cancer predisposing germline mutations in Chinese survivors of childhood cancer including acute lymphoblastic leukemia (ALL). To characterize the spectrum and prevalence of cancer predisposing germline mutations, and to assess the relationship between the mutation status and cancer family history, whole-exome sequencing (>80-fold) was performed on blood DNA for 91 childhood ALL survivors who were ≥5 years from initial cancer diagnosis. All patients enrolled and consented for the study were previously diagnosed and treated either at Nanjing Children's Hospital of Nanjing Medical University (n=28) or Guangzhou Women and Children's Medical Center (n=63). Germline mutations in 156 cancer predisposition genes were analyzed and classified for their pathogenicity based on the American College of Medical Genetics and Genomics (ACMG) guideline. Clinical information was extracted from medical records and family history were collected based on questionnaire and phone interview. Of 91 survivors of childhood ALL (median age [range], 11 [6-22] years; 58 [63.7%] male), P/LP mutations were identified in 7 (6.4%) survivors. The mutations occurred in well-established ALL predisposition genes such as ATM, BLM, and SBDS, and other cancer-associated genes such as PALB2, for which two P/LP mutations were identified. Notably, PALB2 was also included in "Add-On" gene panel provided by Invitae, a genetic testing company, for leukemia predisposition testing. Most of these mutated genes (ATM, BLM, LIG4 and PALB2) are involved in DNA repair pathways. Using East Asian (EAS) population from Genome Aggregation Consortium (genomAD) database as controls, the cancer predisposing germline mutations were significantly enriched (Odds Ratio [95% CI], 6.3 [2.9-13.7]) among survivors of childhood ALL. Among survivors with cancer family information (n=82), the prevalence of mutation carriers is 18.8% (3/16) for survivors with positive cancer family history, compared to 4.5% (3/66) for survivors with negative family history, but the association between mutation status and cancer family history is not statistically significant (Fisher's exact test P = 0.09). To our knowledge, we're the

first to assess cancer predisposing mutations in Chinese survivors of childhood ALL. Our findings showed slightly higher level of prevalence of cancer predisposing mutation than US survivors, suggesting disparity across different populations. The results highlight the importance of genetic counselling and testing which could inform personalized screening strategy for second cancer for survivors and for cancer risk stratification and early prevention for other family members.

#1686

Comprehensive genomic and transcriptomic profiling of gastrointestinal stromal tumors.

Peter Horak,1 Matea Hajnic,1 Laura Gieldon,2 Mario Hlevnjak,3 Susan Richter,2 Barbara Hutter,3 Johanna Falkenhorst,4 Sebastian Uhrig,3 Gregor Warsow,3 Nagarajan Paramasivam,3 Stefan Gröschel,3 Barbara Klink,5 Simon Kreutzfeldt,1 Christoph Heining,6 Christoph E. Heilig,1 Martina Fröhlich,3 Stephan Richter,2 Christian Brandts,7 Wilko Weichert,8 Philipp Jost,8 Olaf Neumann,9 Marc Zapatka,3 Albrecht Stenzinger,9 Alexander Marx,10 Benedikt Brors,3 Evelin Schröck,2 Sebastian Bauer,4 Peter Hohenberger,10 Hanno Glimm,6 Claudia Scholl,3 Stefan Fröhling1. 1 _NCT Heidelberg, Heidelberg, Germany;_ 2 _Faculty of Medicine Carl Gustav Carus, Dresden, Germany;_ 3 _DKFZ Heidelberg, Heidelberg, Germany;_ 4 _University Hospital Essen, Essen, Germany;_ 5 _Faculty of Medicine Carl Gustav Carus, Heidelberg, Germany;_ 6 _NCT Dresden, Dresden, Germany;_ 7 _UCT Frankfurt, Frankfurt, Germany;_ 8 _Technical University of Munich, Munich, Germany;_ 9 _Heidelberg University Hospital, Heidelberg, Germany;_ 10 _University Medical Centre Mannheim, Mannheim, Germany_.

Most adult gastrointestinal stromal tumors (GIST) are driven by activating KIT or PDGFRA mutations. The remaining 10-15% of cases, often referred to as wildtype (WT) GIST, display either alterations of the succinate dehydrogenase complex (SDH) or RAS pathway mutations. To gain additional insight into the biology of GIST, we performed whole-exome or genome and RNA sequencing in 38 GIST patients (WT, n=15; KIT-mutant, n=21; PDGFRA-mutant, n=2) enrolled in a prospective molecular stratification trial of NCT Heidelberg/Dresden and the German Cancer Consortium (DKTK) designed for younger adults with advanced-stage cancer across histologies and patients with rare tumors (NCT/DKTK MASTER). Of the 15 patients with WT GIST, 3 had pathogenic germline mutations in NF1 and 9 harbored SDH alterations (germline, n=5; somatic, n=4). In the 3 patients with quadruple-negative GIST - defined by the absence of KIT, PDGFRA, SDH, or RAS pathway alterations - we detected novel gene fusions affecting RET, FGFR2, and FGF4, respectively. To delineate biologically relevant subgroups of GIST based on RNA sequencing data from the entire cohort (n=34), we used 3 different clustering methods and 4 different measures of stability and consistency. Despite the underlying clinical and molecular heterogeneity, we identified 3 distinct transcriptional subgroups that were characterized by (i) SDH deficiency, (ii) recurrent somatic RB1 alterations and mutational signatures associated with defective homologous recombination DNA repair, and (iii) elevated PDGFRA expression, respectively. Furthermore, we used random forest analysis to identifiy genes that are significantly (p<0.005) differentially expressed between the 3 subgroups. Interestingly, quadruple-negative cases did not form a separate cluster or clustered within a specific subgroup. Collectively, our data illustrate the molecular heterogeneity of advanced-stage GIST and support comprehensive molecular profiling approaches to capture the entire spectrum of clinically actionable genetic alterations, such as diverse fusion genes affecting kinase signaling pathways in quadruple-negative cases or pathogenic germline mutations in patients with inconspicuous family histories. The finding of two separate transcriptional clusters among patients with SDH-proficient GIST may be reflective of distinct regulatory pathways whose molecular underpinnings and clinical actionability warrant further study.

#1687

Identification of bladder cancer subtypes with different sensitivities to immunotherapy.

In-Sun Chu,1 Bic-Na Song,1 Sun-Hee Leem2. 1 _Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea;_ 2 _Dong-A University, Busan, Republic of Korea_.

Bladder urothelial carcinoma (BLCA) is pathologically categorized as non-muscle-invasive (NMIBC) and muscle-invasive (MIBC) bladder cancers. But its clinical heterogeneity has been suggested that biologically relevant subtypes may exist within and between NMIBC and MIBC. Here we identified that BLCA could be subgrouped into four major classes with distinct molecular characteristics regardless of previously known clinicopathological factors, using a series of unsupervised learning approaches. The predictor to discriminate distinct molecular subgroups of BLCA was constructed in a training cohort and validated in six independent cohorts with a total of 1,992 samples. The predictor showed a prognostic significance of the four subtype of BLCA, especially aggressive clinical behavior of the class 3 subtype. Interestingly, class 3 subtype has distinct biological features associated with potential responsiveness to immunotherapy, such as high somatic mutation rates and alterations of genes involved in DNA damage response and repair (DDR). Analyses of anti-PD-L1-treated BLCA patient tumor samples suggested that class 3 subtype was correlated with response to immune checkpoint inhibitors. Our findings have the potential to lead to the improvement of the current molecular classification system of BLCA and the achievement of better clinical outcomes for high-risk patients with BLCA treated with immunotherapy.

#1688

**Integrative sequencing discovers a novel** PGAP3-SRPK1 **gene fusion in sinonasal undifferentiated carcinoma.**

Erin L. McKean, Andrew C. Birkeland, Molly E. Heft Neal, Aditi Kulkarni, Sue K. Foltin, Brittany M. Jewell, Jonathon B. McHugh, Lawence J. Marentette, John Chad Brenner. _University of Michigan, Ann Arbor, MI_.

Sinonasal Undifferentiated Carcinoma (SNUC), a rare and aggressive tumor arising from the sinonasal cavity, remains a difficult-to-treat disease with poor overall survival. There are no agreed upon treatment algorithms and research efforts have focused on identifying new genetic mutations for novel targeted therapies. To date, targeted sequencing studies have identified IDH2 and SMARCAB1 as potential SNUC driver alterations, however the molecular alterations found in SMARCAB1 wild type tumors are unknown. Here, using whole exome sequencing, we characterize a series of 7 SNUC tumors and a SNUC cell line and discover recurrent aberrations to the SWI/SNF, MAGE and FAT gene families. We also validate a novel ALK mutation, recurrent ERBB2 copy number amplifications and a previously undescribed PGAP3-SRPK1 gene fusion in the SNUC cell line. Further investigation of the PGAP-SRPK1 fusion gene reveals a change in the active site with loss of three of nine active site residues. Functional studies are ongoing to determine the importance of the PGAP3-SRPK1 gene fusion for oncogenic phenotypes. This discovery extends the need for comprehensive characterization of gene family mutation status in SNUC and supports a need to understand the downstream molecular mechanisms in order to improve therapeutic strategies for this disease.

#1689

Comparative whole genome analyses of breast cancer aromatase inhibitor resistance models.

Reiner Hoppe,1 Siarhei Kandabarau,1 Ping Fan,2 V. Craig Jordan,2 Heather Cunliffe,3 Hiltrud B. Brauch4. 1 _Dr. Margarete Fischer-Bosch - Institute of Clinical Pharmacology, Stuttgart, and University of Tübingen, Germany;_ 2 _MD Anderson Cancer Center, University of Texas, Houston, TX;_ 3 _Dunedin School of Medicine, University of Otago, Dunedin, New Zealand;_ 4 _Dr. Margarete Fischer-Bosch - Institute of Clinical Pharmacology, Stuttgart, and University of Tübingen, German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany_.

One third of breast cancer patients treated with Aromatase Inhibitors (AI) or Tamoxifen acquire resistance leading to disease recurrence and death. The clinically observed AI resistance is mimicked by in vitro models MCF-7:5C and MCF-7:2A that are susceptible to apoptosis upon E2-treatment. These models have been instrumental to the current understanding of underlying mechanisms such as the reconfiguration of survival signaling, alterations of stress-related pathways (Ariazi et al. 2011; Fan et al. 2013, 2015), and changes in miRNA expression profiles (Hoppe et al. 2016). How the model phenotypes relate to a specific genomic background is poorly understood. In order to identify somatic variants indicative of AI resistance and potentially useful as novel therapeutic targets we performed NGS analyses of the 5C and 2A clones compared to the non-resistant WS8 and MCF-7 (ATCC). Whole genome/exome libraries were sequenced on Illumina HiSeq/NovaSeq platforms, respectively. Previously we reported first results from a preliminary analysis of gains and losses of variants in both resistant derivatives (Hoppe at al. AACR 2018). Here we report an updated analysis of raw reads trimmed and aligned to GRCh38 reference using BWAkit v 0.7.15. Duplicates were removed by Picard MarkDuplicates. Base quality scores were recalibrated using GATK4 BQSR. WGS and WES alignments were merged and tested for somatic variants using GATK4 Mutect2 (Tumor-only mode) with population variation data from gnomAD as a germline source. Called and filtered variants were uploaded to IVA for functional and clinical annotation. Copy number segments were modeled using both read- and allele-counts from WGS alignments by Gaussian-kernel binary-segmentation algorithm applied in GATK4 ModelSegments tool. To interpret copy number events causative for LOH we recovered germline variants and annotated CNVs that led to loss of the corresponding wild-type allele. In line with the COSMIC database we confirmed 461 mutated loci of which 228 were lost in both resistant derivatives. Examples of 5C-specific coding mutations classified as pathogenic include splice site alterations (i.e. SEMA3A), frameshifts (i.e. ARID1A), and stop gains (i.e. CACNA1S, CDH2, NOTCH4). 2A-specific mutations include for example a stop gain affecting ADGRV1. Copy number variation analyses revealed that both resistance models share prominent genetic defects such as amplified regions at Chr6q25.1 and Chr21q22.2. A 5C-specific aberration is a large Chr18 deletion affecting most of the q-arm, and a 2A-specific aberration is the amplification of most parts of Chr6q23.3-q24.3. We will present the status of this currently ongoing comparative genome analysis of AI resistance models and discuss the putative effects of homozygous mutations at loci with copy number aberrations within the context of AI resistance.

#1690

A tumor deconvolution DREAM Challenge: Inferring immune infiltration from bulk gene expression data.

Brian S. White,1 Andrew J. Gentles,2 Aurélien de Reyniès,3 Aaron M. Newman,2 Andrew Lamb,1 Laura Heiser,4 Joshua J. Waterfall,5 Thomas Yu,1 Justin Guinney1. 1 _Sage University, Seattle, WA;_ 2 _Stanford University, Stanford, CA;_ 3 _Ligue Nationale Contre le Cancer, Paris, France;_ 4 _Oregon Health and Science Institute, Portland, OR;_ 5 _PSL Research University, Paris, France_.

Introduction: Immune cell infiltration in solid tumors correlates with patient outcome and therapeutic response. While specific cell-type infiltration can be elucidated by single-cell transcriptomic techniques, these suffer biases and limitations of scale. To instead leverage existing large repositories of bulk gene expression data with clinical outcomes, computational methods have been developed to deconvolve sample profiles into their stromal (including immune) and malignant cell components. However, their performance has yet to be compared within an unbiased, objective framework. To assess methods and catalyze development of new approaches, we are organizing a community-wide DREAM Challenge.

Methods: The Challenge consists of: (1) an open phase, during which methods are trained on publicly-available transcriptomic profiles of cell populations; (2) a leaderboard phase, during which methods are submitted, assessed, and revised using bulk expression data having ground truth (e.g., ratios from mixing experiments or from flow cytometry); and (3) a validation phase, during which final submissions are assessed using independent expression profiles of known admixtures. The latter are generated in vitro by mixing RNA from multiple types of purified stromal and cancer cells. To assess sensitivity, we provide in silico admixtures from expression profiles of purified populations with a range of tumor "contamination." Models will be submitted as Docker containers, executed in the cloud, and assessed based on their ability to predict levels of an individual cell type across samples. In sub-Challenge 1 models will predict coarsely-defined stromal populations, while in sub-Challenge 2 models will further dissect these into subsets (e.g., of T-cell subtypes).

Results: We have isolated cell populations of interest and performed quality control on datasets for use in the leaderboard phase. We have defined mixing proportions to assess sensitivity and specificity of deconvolution algorithms. The infrastructure for conducting the Challenge is in place. There is robust interest, with 240 participants pre-registered. The active phase will launch in early 2019 for seven weeks. At its completion, we will identify features of best performing models and provide guidelines where improvements are needed.

Discussion: We expect methods to have difficulty deconvolving cell types with closely-correlated expression profiles and in detecting low-frequency populations. An assessment of these limits will enable appropriate use of deconvolution methods in prognostic and predictive models. Our synthetic admixtures model a diverse range of stromal microenvironments that mimic realistic tumors as far as possible. In a subsequent in vivo Challenge, methods will be tested against a future data set in which patient samples are profiled in bulk for deconvolution and at the single-cell level to establish ground truth.

#1691

The impact of genomic mutational status on PD-L1 expression and tumor mutation burden in non-small cell lung cancer.

Qing Zhou,1 Weiguang Gu,2 WenFan Fu,3 Shijie Mai,4 Daren Lin,5 Shiyue Zhang,6 Wenjing Wang,6 Peng Zhang6. 1 _Guangdong General Hospital, and Guangdong Academy of Medical Sciences, Guangzhou, China;_ 2 _The People's Hospital of Nai Hai, Foshan City Chancheng Area Foping Street 40, Foshan, China;_ 3 _Affiliated Cancer Hospital and Institute of Guangzhou Medical University, Guangzhou, China;_ 4 _Southern Medical University Nanfang Hospital, Guangzhou, China;_ 5 _Jiangmen Central Hospital, Affiliated Jiangmen Hospital of Sun Yat-Sen University, Jiangmen, China;_ 6 _OrigiMed, Shanghai, China_.

Background: Immune checkpoint inhibitors have provided remarkable antitumor effects in non-small cell lung cancer (NSCLC), while their efficacy in patients harboring driver mutations is still controversial and the correlation between driver gene mutations and PD-L1 expression remains unclear. In this study, we explored the association of PD-L1 expression, tumor mutation burden (TMB) and genomic mutation in NSCLC patients. Methods: FFPE tumor and matched blood samples of 244 NSCLC patients were collected for NGS-based targeted panel sequencing from July 2017 to July 2018. Genomic alterations including single nucleotide variations, short and long insertions/deletions, copy number variations and gene rearrangements were assessed. PD-L1 expression, TMB and microsatellite instability status (MSI) were assessed in 244 (100%), 188 (77%) and 198 (81%) patients, respectively. Results: There were 145 males (59.4%) and 99 females (40.6%) with a median age of 62 (31-84) years were involved, including 183 adenocarcinomas and 61 squamous carcinomas. PD-L1 expression of negative, 1-49% and ≥50% were 63.5%, 20.9% and 15.6%, respectively. The median value of TMB was 9.2 (0.8-68.9) muts/Mb and TMB≥10 muts/Mb was seen in 30.9% tumors. All the tumors but one were MSS status. The demographic features were comparable among different PD-L1 expression and TMB level (cutoff is 10 muts/Mb) group. We found recurrent mutations (>10%) in 6 genes, including TP53 (67.6%), EGFR (60.7%), CDKN2A (15.2%), KRAS (13.9%), PI3KCA (11.1%) and LRP1B (10.7%). AKT2 (2.5%) amplification was associated with higher TMB (p=0.011). For the relationship between driver genes and immunotherapeutic biomarkers, TP53 were positively associated with higher PD-L1 expression (p<0.001), while BRAF associated with lower PD-L1 (p<0.001); TP53 and BRAF mutations were correlated with higher TMB (p<0.001, p<0.001; respectively), while EGFR, KRAS, ERBB2 and ALK correlated with lower TMB (p<0.001, p<0.001, p<0.001,p=0.046; respectively). Interestingly, in terms of double mutants, NSCLC patients with EGFR and NOTCH1 co-mutations had lower level of TMB than either single mutant alone. EGFR and RBM10 double mutant was associated with lower PD-L1 expression than either single mutant. Conclusion: This study revealed the impact of genomic mutational status on immunotherapeutic biomarkers in NSCLC. TP53 mutation was associated with higher PD-L while TP53, BRAF mutations and AKT2 amplification correlated with higher TMB. EGFR with NOTCH1 or RBM10 co-mutation is associated with lower TMB or PD-L1, which needs further investigation in their impact on immunotherapy. Our study indicated that NGS panel sequencing and co-mutation analysis could provide potentially insights to precise immunotherapy.

#1692

Genomic changes in relapsed neuroblastoma.

Susanne Fransson,1 Angela Martinez Monleon,1 Anna Djos,1 Susanne Reinsbach,1 Rose-Marie Sjoberg,1 Per Sikora,1 Per Kogner,2 Tommy Martinsson1. 1 _University of Gothenburg, Gothenburg, Sweden;_ 2 _Karolinska Institutet, Gothenburg, Sweden_.

Neuroblastoma (NB) is an aggressive pediatric malignancy originating from the sympathetic nervous system. Despite recent improvements in multimodal treatment, survival for children diagnosed with high-risk NB, remains below 50% at 5 years from treatment. Although an initial response to treatment is seen in a majority of patients, a significant portion will subsequently relapse with lesions resistant to standard therapy and for NB patients with relapsed or refractory metastatic disease, survival is below 10%. Genetic risk factors previously identified in primary neuroblastoma include MYCN-amplification, 11q-deletion, 1p-deletion and 17q gain and activating ALK mutations. However, treatment resistant tumors will likely have novel genetic aberrations differing from those present at time of diagnosis. To investigate the genetic mechanisms linked to recurrent disease, alterations between five triplet samples of primary tumor, relapsed/refractory tumor and constitutional DNA was investigated through whole genome sequencing. Sequencing was performed using Illumina instrumentation for an average coverage of at least 60X and 30X for tumor samples and normal tissue respectively. Read trimming, mapping to hg19 and variant calling were performed using the CLC Genomics Workbench software while copy number profiles were prepared through the CANVAS software. Systematic filtering was performed using the Ingenuity variant analysis tool. Variants with allele frequency above 3% in common population based cohorts were discarded as well as excluding all synonymous variants or variants in non-coding regions except those affecting canonical splice sites. A median of 21 and 59 non-silent somatic variants were detected in diagnostic and in relapsed samples respectively, while the median number of segmental alterations were 31 in diagnostic samples and 35 in relapse samples. Genes with recurrent alterations that emerged in relapse samples includes CCND1, CDK5RAP1, POLR2A, LSAMP, C3 and CARNS1. One patient showed only one single alteration common between primary and relapse; a t(11;17) resulting 11q-deletion and 17q-gain. The paired tumors did both have CCND1-gain although with different breakpoints and 6p-gain but of different parental alleles. In addition, in one patient an ARID1B mutation was detected in primary tumor but lost in the relapsed sample where an ARID1A mutation had emerged, which could indicate parallel evolution. Gene set analysis of variants in relapsed samples showed no significant functional enrichment differing from primary tumors where cell projection, Rap1-signaling and neuron part showed enrichment. In this study we investigated the genomes of primary and relapsed NB for five patients. We show that mutation load is higher in relapsed NB and includes alterations of previously linked to NB such as ATRX, TERT and CCND1 but lacked alterations in ALK, RAS or other genes of the RAS/MAPK pathway.

#1693

Comparison of commercially available myeloid next-generation sequencing assays.

Sarah Johnson, Nathan Riccitelli, Reinhold Pollner. _Navigate BioPharma Services, a Novartis subsidiary, Carlsbad, CA_.

Introduction

Myeloid neoplasms are a complex class of disorders characterized by a range of genotypes. Accurate, precise, and rapid identification of these genotypes is crucial in giving the right drug to the right patient at the right time. In this study, we present a comparison of three Next-Generation Sequencing (NGS) panels targeting genes associated with myeloid cancers. We evaluated assay performance as a function of sensitivity and specificity of somatic variant callings, sequencing metrics, and general workflow, for the analytically validated Illumina TruSight Myeloid Sequencing Panel (TSM), the new Illumina AmpliSeq for Illumina Myeloid Panel (IA), and the ThermoFisher Oncomine™ Myeloid Research Assay (TA).

Methods

Thirty-two libraries were prepared across all three panels. Samples included a mix of Horizon Discovery and SeraCare myeloid DNA controls, a well-characterized Hapmap sample, and six clinical DNAs isolated from peripheral blood of patients with myeloid disorders. Libraries were prepared according to manufacturer protocols, and quantified by Qubit Flourometer. For Illumina panels, libraries were manually prepared, normalized, pooled, and sequenced on Illumina's MiSeq instruments. For the TA panel, libraries were generated, pooled, templated, and loaded onto sequencing chips by the IonChef, then sequenced on the S5xL system. Primary data analysis was completed using cloud-based bioinformatic solutions followed by manual annotation.

Results

Hands-on time varied for the assays, with the TA panel requiring just 30 minutes compared to four to eight hours for the TSM and IA protocols. Time to results from DNA dilution to variant calls was comparable, at three days for the TSM panel and four days for the IA and TA protocols. The TSM assay exhibited the highest average coverage, but also the greatest amplicon dropout. Both the IA and TA panels exhibited similar coverage uniformity and read depths. Detection of expected variants in control samples down to 5% allele frequency was robust for all assays, however both the IA and TA panels contained increased regions of poor sequencing quality relative to the TSM panel. No false positives were found in the somatic-variant negative Hapmap sample NA12878, and concordance of variant calls for the clinical samples from the IA and TA assays to the validated TSM assay was greater than 95%.

Conclusions

In the complex landscape of myeloid neoplasms, NGS is an invaluable tool for the diagnosis and management of the diseases. Overall, the three sequencing panels evaluated herein provided accurate genotype information for actionable targets in myeloid cancers. Although results for the assays were similar, the short time-to-data for the TSM assay, increased coverage and detection of low frequency variants for the IA assay, and fully-automated option for the TA assay should all be considered when determining the optimal panel for a given need.

#1694

A multi-institutional, multi-tumor analysis of fine needle aspiration (FNA) cytology smear (FNAC) performance in clinical comprehensive genomic profiling.

Jonathan K. Killian, Dean Pavlick, Lindsay Croshier, John Truesdell, Chris Wright, Tim Brennan, Vera Banning, Lazaro Garcia, Laurie Gay, Gene Stirchak, Jo-Anne Vergilio, Christine Malboeuf, Nhu Ngo, Julia Elvin, Siraj Ali, Vince Miller, Jeffrey S. Ross. _Foundation Medicine, Cambridge, MA_.

Background. FNAC are generated during the FNA procedure - for example, commonly used in conjunction with endobronchial ultrasound (EBUS-FNA) as a minimally invasive tool for the diagnosis and management of non small-cell lung cancer (NSCLC) - as a histologic QC for the presence of malignant cells. Formalin-fixed core biopsies and aspirate cell blocks (FNACB) are then created for comprehensive genomic profiling (CGP). However, the routine pathology workup may render the FNACB unable to generate a CGP result, while the FNAC remain in reserve. In the current study we performed a retrospective multi-cancer, multi-institutional analysis of FNAC performance, including computed tumor purity analysis, using a regulated commercial grade CGP assay.

Experimental procedures. 119 FNAC met minimum specimen cellularity (10K total cellularity) and DNA extraction (50ng) criteria for CGP. Extracted DNA from FNACS underwent hybrid-capture-based CGP for all classes of genomic alterations in 315 genes. Tumor mutational burden (TMB) was determined on 1.1 Mbp of sequenced DNA and microsatellite instability (MSI) was determined on 114 loci. A comparison set of 182 FNACB specimens underwent the same CGP assay. Individual specimen CGPs were annotated as Pass, Fail, or Qualified per computational biology standard procedures that factor in sequencing quality metrics such as mean target coverage and computational tumor purity modeling.

Results. The 119 FNAC, collected at 14 different medical facilities, encompassed 44 distinct tumor disease ontologies (DO) from 17 unique anatomic biopsy sites. 105 of 119 FNACS (88%) received a passing report, 10 (8%) were qualified, and 4 (3%) failed. In the FNACB comparison group, 155 of 182 reports were pass (85%), 22 qualified (12%), and 5 failed (3%). The number of passing reports in FNACS versus FNACB was equivalent (P=0.5). The FNAC had a significantly higher mean target coverage than FNACB (703 vs 665; P= 0.04995). The average computational tumor purity metric was significantly higher in FNACS than in FNACB (57.9% vs. 45.3%; P=0.00001).

Conclusions. This study provides new data from FNAC that further support their utility for clinical testing. FNAC derived from multiple medical facilities, biopsy sites, and tumor types were found to be equivalent or superior to FNACB for CGP. Finally, the relative cellular homogeneity and high tumor cell content of both FNAC and FNACB likely confirms the discohesiveness of malignant cells and enables CGP without required enrichment given that macrodissection would be challenging for these samples in a daily practice setting.

#1695

Linking clinical molecular profiles of tumors to the electronic medical record.

Debajyoti Datta, Atul Butte, Theodore Goldstein. _UCSF, San Francisco, CA_.

By linking "real-word" patient data mined from electronic medical records to the results of clinical genomic testing, we can analyze the impact of the rapid expansion in tumor molecular profiling on the management of cancer patients over the past several years. Theoretically, the widespread adoption of comprehensive molecular characterization of patient tumors using next-generation sequencing panels provides new opportunities for advances in precision oncology. For patients, identification of specific alterations can potentially lead to new therapeutic options from an ever-widening array of targeted agents. For example, the recent FDA approval in 2017 of pembrolizumab for patients with unresectable or metastatic, microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) solid tumors is the first example of an FDA approval agnostic with regards to the tumor site of origin. The availability of this novel treatment option has likely led to more frequent use of tumor molecular profiling in the clinical setting, and in the future, we can expect similar examples of treatment strategies that favor specific genomic perturbations over the tumor site of origin. However, despite their ever-expanding use in the clinical setting, the overall impact of genomic testing remains unclear, and evidence demonstrating improvement in patient outcomes is lacking. In this study, we present the development of a database that combines clinical genomic results from our internal next-generation sequencing panel, as well as commercially available external clinical genomic panels, with a variety of data points from our institution's electronic medical record (EMR). This data set allows us to measure the utility of genomic tumor testing for the management of cancer patients. We analyze the magnitude of positive impact for various tumor types, identify barriers to efficient use of genomic tumor testing, and assess for disparities in the utilization of testing. Patient survival data relative to the clinical action of obtaining tumor molecular profiling can also be measured. We find that a small fraction of patients dies before results return and any action can be taken, especially in cases of pancreatic cancer, suggesting that molecular profiling should be considered earlier in the course of disease for certain cancer types. Combining information on patient outcomes from "real-world" EMR data with clinical genomic testing provides the additional opportunity of identifying exceptional responders or non-responders with greater efficiency, which can lead to the discovery of new therapeutic targets and treatments as well as the identification of novel biomarkers for response or resistance to therapy.

#1696

Structural variant detection with long read sequencing reveals driver and passenger mutationsin a melanoma cell line.

Aaron Wenger,1 Marcel Nelen,2 Meredith Ashby,1 Wigard P. Kloosterman3. 1 _Pacific Biosciences, Menlo Park, CA;_ 2 _Radboud UMC, Nijmegen, Netherlands;_ 3 _UMC Utrecht, Utrecht, Netherlands_.

Past large scale cancer genome sequencing efforts, including The Cancer Genome Atlas and the International Cancer Genome Consortium, have utilized short-read sequencing, which is well-suited for detecting single nucleotide variants (SNVs) but far less reliable for detecting variants larger than 20 base pairs, including insertions, deletions, duplications, inversions and translocations. Recent same-sample comparisons of short- and long-read human reference genome data have revealed that short-read resequencing typically uncovers only ~4,000 structural variants (SVs, ≥50 bp) per genome and is biased towards deletions, whereas sequencing with PacBio long-reads consistently finds ~20,000 SVs, evenly balanced between insertions and deletions. This discovery has important implications for cancer research, as it is clear that SVs are both common and biologically important in many cancer subtypes, including colorectal, breast and ovarian cancer. Without confident and comprehensive detection of structural variants, it is unlikely we have a sufficiently complete picture of all the genomic changes that impact cancer development, disease progression, treatment response, drug resistance, and relapse.

To begin to address this unmet need, we have sequenced the COLO829 tumor and matched normal lymphoblastoid cell lines to 49- and 51-fold coverage, respectively, with PacBio SMRT Sequencing, with the goal of developing a high-confidence structural variant call set that can be used to empirically evaluate cost-effective experimental designs for larger scale studies and develop structural variation calling software suitable for cancer genomics.

Structural variant calling revealed over 21,000 deletions and 19,500 insertions larger than 20 bp, nearly four times the number of events detected with short-read sequencing. The vast majority of events are shared between the tumor and normal, with about 100 putative somatic deletions and 400 insertions, primarily in microsatellites. A further 40 rearrangements were detected, nearly exclusively in the tumor. One rearrangement is shared between the tumor and normal, t(5;X) which disrupts the mismatch repeat gene MSH3, and is likely a driver mutation.

Generating high-confidence call sets that cover the entire size-spectrum of somatic variants from a range of cancer model systems is the first step in determining what will be the best approach for addressing an ongoing blind spot in our current understanding of cancer genomes. Here the application of PacBio sequencing to a colorectal cancer cell line revealed thousands of previously overlooked variants, including a mutation likely involved in tumorogenesis.

#1697

Molecular features of refractory metastatic breast cancer.

Zhanhong Chen,1 Yuzi Zhang,2 Jing Zhao,2 Shangli Cai,2 Yongmei Yin3. 1 _Zhejiang Cancer Hospital, Hangzhou, China;_ 2 _3D Medicines Inc., Shanghai, China;_ 3 _The First Affiliated Hospital of Nanjing Medical University, Nanjing, China_.

Background: Breast cancer is the most frequently diagnosed cancer in women worldwide and metastatic disease remains generally incurable, indicating a need for new drug discovery for metastatic breast cancer. In this study, we investigated the molecular features of refractory metastatic breast cancer.

Methods: 74 metastatic refractory breast cancer patients who received molecular screening using next generation sequencing (NGS) between 2015 and 2017 were included in this analysis. Clinical and pathologic characteristics of these patients were reviewed.

Results: Among the 74 patients, 27%, 51.4%, 10.8%, 10.8% had hormone receptor (HR)-negative HER2-negative, HR-positive HER2-negative, HR-positive HER2-positive, HR-negative HER2 positive. 54.1%, 48.6%, 9.5%, 47.3% had liver, lung, brain, bone metastasis. The most significantly mutated genes in refractory HR-positive breast tumors included TP53 (54.3%), PIK3CA (52.2%), CCND1 (19.6%) and ESR1 (15.2%). The most significantly mutated genes in refractory HR-negative breast tumors included TP53 (82.1%), PIK3CA (50%), MYC (14.3%), NF1 (14.3%) and KRAS (10.7%). Genes in PI3K/AKT/mTOR pathway were significantly mutated in 76.1% of HR-positive and 82.1% of HR-negative refractory breast tumors, respectively. Mutation of genes involved in DNA damage response pathway were observed in 23.9% of HR-positive tumors and 32.1% of HR-negative tumors. 21.7% of HR-positive tumors and only 3.6% of HR-negative tumors harbored at least one gene alteration in FGFR pathway (P = 0.04). 30.4% (17/74) of HR-positive tumors and 10.7% (3/28) of HR-negative tumors carried at least one gene alteration in CDK pathway (P = 0.50). 63.6% of tumors with alterations in FGFR pathway concurrently harbored alterations in CDK pathway. Alterations in FGFR or CDK pathway were significantly correlated with liver metastasis (P = 0.001). In addition, TP53 mutation was significantly correlated with brain metastasis (P = 0.022).

Conclusions: These results provide a better understanding of molecular targets in refractory metastatic breast cancer which may lead to an improvement in the development of novel therapeutic strategies.

#1698

A million years of precision oncology knowledge for the world.

Amber Johns, International Cancer Genome Consortium (ARGO Initiative). _Garvan Institute of Medical Research, Sydney, Australia_.

Introduction: The most valuable information for someone with the diagnosis of cancer is the ability to predict which treatment will be most effective for them, and their prognosis. The most effective way to generate the knowledge required to do this for all people affected by cancer is to amass large datasets of deep molecular and detailed phenotypic data through health systems as part of routine care.

However, the pathways and processes are not in place and require development, with the paramount challenge being the acquisition and application of rich, reliable clinical data including treatment and response parameters.

Although multiple regional platforms have been established to generate cancer genomics data, no platform exists that can capture, aggregate, harmonize and appropriately share data globally to enable analysis of pooled individual participant data.

Method: The International Cancer Genome Consortium1, established in 2007, aimed to define the genomes of 25,000 primary untreated cancers. The ICGC solved numerous data governance, ethical and logistical challenges and provided the international community with comprehensive genomic data for many cancer types. The ICGC's next Initiative -ARGO aims to Accelerate Research in Genomic Oncology to provide a million patient-years of precision oncology knowledge to the world. To achieve this, ICGC-ARGO will acquire detailed treatment and response information mostly from participants in clinical trials along with genomic, transcriptomic and proteomic data from their tumors and healthy tissues.

Results: ICGC membership already spans 28 countries, with new commitments to ARGO from 10 programmes investigating a range cancer types, comprising an estimate of 200,000 patient years of data. Regional Data Processing Centres are in operation in 4 countries to coordinate ICGC-ARGO data and to validate and process sequencing data through a standardized series of data analysis pipelines. A central site will integrate the various ICGC-ARGO data sets and disseminate to the community through the ICGC Data Portal. Policies and guidelines have been established that address the global challenges of clinical cancer genomics such as standardisation of clinical data parameters, data access structures, data composition and management and jurisdictional issues that impact global data sharing.

Conclusion: The data generated by ICGC ARGO member programmes will allow for precise correlation of molecular attributes with clinical features, allowing for the discovery of molecular markers of response, resistance and adverse events, as well as novel therapeutic targets. This knowledge will translate into new approaches to improve outcomes for people affected by cancer. The ICGC ARGO project will also address the challenges of generating and sharing genomic data matched with clinical and patient information on a global scale.

#1699

Accurate detection of low AF variants relevant to AML by Anchored Multiplex PCR and next generation sequencing.

Verity Johnson, Kaitlyn E. Moore, Laura M. Griffin, Aaron Berlin, Abel Licon, Ryan Walters. _ArcherDX, Boulder, CO_.

Introduction: Acute Myeloid Leukemia (AML) is clinically and biologically heterogeneous, requiring the detection of multiple mutations for characterization. For instance, FLT3-ITDs and CEBPA mutations represent important markers in AML, however they are difficult to detect by NGS due to the highly variable nature of ITDs, the high GC content of CEBPA, and the difficulty in mapping repeated sequences to a wild-type reference. Tracking low frequency mutations is also of growing importance. The ability to accurately detect variants at low allele fractions (AFs) using a single test can be used to assess treatment efficacy and potential relapse.

Methods: We developed Archer® VariantPlex® myeloid assays based on Anchored Multiplex PCR (AMP™) to detect important mutations in myeloid malignancies. AMP is a target enrichment strategy that uses molecular-barcoded adapters and gene-specific primers for amplification, permitting open-ended capture of DNA fragments from a single end. This approach enables flexible and strand-specific primer design to provide better coverage of ITD-containing and GC-rich regions. We also developed a method to assess SNV sensitivity taking into account both unique coverage depth and noise for single base substitutions. This strategy enables utilization of position-specific detection thresholds and maximizes sensitivity and specificity. We tested this approach using the VariantPlex® Core Myeloid panel, by titrating reference inputs into background normal samples to examine detection of low AF variants.

Results: Our assay enables calling of a 30bp FLT3-ITD down to sub-0.05% allele frequencies. Using optimized low AF conditions improves coverage depth, consistency of low AF FLT3-ITD detection, and sensitivity (98.5% of bases are powered to call a true variant at an allele frequency of 3.0% with 1M reads and 200ng of input). We show >1000X unique molecule coverage across the coding region of CEBPA and use this challenging region to visualize the minimum detectable AF (MDAF) at which a variant has a >95% probability of being detected above the noise (95MDAF). Finally, we show consistent single nucleotide variant (SNV), insertion and deletion (indel), and ITD calling at sub-0.5% allele frequencies, and demonstrate the utility of reporting variant-specific MDAFs and normal dataset P-values when analyzing low AF variants.

Conclusion: AMP provides NGS-based detection of complex mutation types that are relevant in AML. We demonstrate robust calling of FLT3-ITDs and other variants at low AFs. We also demonstrate full coverage of CEBPA with high depth and low noise such that the 95MDAF predicts confident variant calling at low AFs. This approach is accurate and scalable, enabling simultaneous detection of multiple mutation types across multiple target genes in a single assay.

#1700

Alterations of DNA damage repair genes in Chinese primary liver cancer patients and its association with tumor mutation burden.

Jianzhen Lin,1 Honglin Guo,2 Junping Shi,2 Xu Yang,1 Yan Jiang,2 Yi Bai,1 Junyu Long,1 Dongxu Wang,1 Jin Bian,1 Ming Yao,2 Kai Wang,2 Haitao Zhao1. 1 _Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College (CAMS & PUMC), Beijing, China; _2 _OrigiMed, Shanghai, China_.

Background: The genomic mutation features of primary liver cancer (PLC) varied from various pathology and etiology. Alterations in DNA damage repair (DDR) genes could induce genomic instability and accumulate tumor mutation burden (TMB), which was related to the efficacy of immune-checkpoint blockade treatment. However, the characteristics of DDR gene alterations and its association with TMB in PLC remain largely undefined.

Method: We enrolled 357 Chinese PLC patients as training cohort and used the TCGA-LIHC data of 373 hepatocellular cancer patients as validation cohort. FFPE tumor tissues and matched blood samples were collected from these Chinese patients for next-generation-sequencing (NGS)-based 450 genes panel assay, which contains 35 DDR genes. All histological diagnoses were confirmed by independent pathologists. Correlation of the DDR pathway genes alterations with TMB was assessed by multiple linear regression model using R package "nlme". The receiver operating characteristic (ROC) curve was applied to determine the diagnostic efficiency of DDR pathway genes for TMB high (which was defined as the top quantile) using R package "pROC".

Results: The training cohort of Chinese PLC patients included 282 males and 75 females with the mean age of 56.3 years old. The pathologic subtypes include 214 hepatocellular carcinoma (HCC), 122 intrahepatic cholangiocarcinoma (ICC) and 21 mixed hepato-cholangiocellular carcinoma (H-ChC). There are 25.8% (92/357) patients had at least one DDR gene alteration. The DDR mutation rate showed no significant differences among three subtypes of PLC (HCC vs ICC vs H-Chc: 22.9% (49/214) vs 30.3% (37/122) vs 28.6% (6/21), P=0.311). In addition, 16.3% (15/92) of the patients with DDR gene mutations have germline variations. The TMB in HCC patients was significantly higher than that in ICC patients but not in H-ChC patients (median TMB for HCC, ICC and H-ChC was 5.4, 3.1, and 3.9 Muts/Mb, Kruskal-Wallis test, P<0.001). Importantly, PLC patients with DDR gene mutations had significantly higher TMB compared to patients without DDR gene mutation (6.2 vs 3.9 Muts/Mb, P<0.001), which was further validated in TCGA-LIHC data (95 vs 64 mutations, P<0.001). The patients with germline DDR gene mutations had significant lower TMB compared with patients with somatic DDR gene mutations (3.1 vs 6.3 Muts/Mb, Wilcox test, P=0.041). We further defined 3 subtypes of DDR pathways (BER, FA and MMR) that significantly correlated with TMB in the training cohort, which exhibited diagnostic efficiency in distinguishing TMB-high or TMB-low PLC in TCGA-LIHC dataset (AUC: 0.67).

Conclusion: DDR gene alterations were significantly positively associated with TMB in PLC. Mutations occurred in BER, FA or MMR pathways indicated higher TMB in PLC, which have the potential to serve as markers to predict the TMB level to guide the immune-checkpoint inhibitor therapy.

#1701

Improvement of tumor mutation burden measurement by removal of deaminated bases in FFPE DNA.

Warren Tom,1 Ruchi Chaudhary,1 Vinay Mittal,2 Dinesh Cyanam,2 Iris Casuga,1 Elaine Wong-Ho,1 Rob Bennett,3 Fiona Hyland,1 Seth Sadis,2 Janice Au-Young1. 1 _Thermo Fisher Scientific, South San Francisco, CA;_ 2 _Thermo Fisher Scientific, Ann Arbor, MI;_ 3 _Thermo Fisher Scientific, Carlsbad, CA_.

Tumor mutation burden (TMB) is a positive predictive factor for response to immune-checkpoint inhibitors in certain types of cancer. The Oncomine™ Tumor Mutation Load Assay, a 1.7Mb targeted next generation sequencing (NGS) panel, measures TMB and detects mutations in 409 cancer genes. The TMB values obtained using targeted sequencing are positively correlated with TMB measured by whole exome sequencing in NSCLC, colorectal, endometrial and gastric cancers. TMB from these tumor samples are correlated with other phenotypes associated with genomic instability, specifically microsatellite instability (MSI) and mutations involved in mismatch repair and cancer related genes. Analysis of the Oncomine™ TML Assay results with Torrent Suite and Ion Reporter software uniquely measures the degree of deamination of cytosines to uracils in fixed tissues. FFPE preservation methods can lead to significant cytosine deamination of the isolated DNA, resulting in decreased sequencing quality. In these samples, uracils are propagated as thymines and result in false C>T substitutions. To minimize the influence that excess deamination has on TMB results, we have incorporated a UDG enzyme treatment to eliminate damaged targets and improve usable TMB values of DNA from damaged FFPE tumor tissue. The Oncomine™ TML assay for TMB on Ion GeneStudio™ S5 in conjunction with MSI detection is informative and potentially predictive for the use of checkpoint inhibitors in multiple cancer types.

#1702

Comprehensive genomic analysis of predictive biomarkers for checkpoint inhibitor therapy from a limited amount of FFPE tissue and cfDNA.

Xiaodong Wang,1 Zhenyu Yan,1 Quyen Vu,2 David Smith,2 Chad Galderisi,2 Cynthia S. Spittle,1 Jin Li1. 1 _MolecularMD Corp., Cambridge, MA;_ 2 _MolecularMD Corp., Portland, OR_.

PD-L1 expression and MSI status are useful predictive biomarkers for checkpoint inhibitors (CIs) in some tumor types. However, studies have demonstrated that the identification of all potential patients who may benefit from CIs will require the analysis of both tumor cell and tumor microenvironment (TME) biomarkers. For example, recent data suggests that tumor mutation burden (TMB) and tumor mutation signatures as well as inflammatory signatures and T cell clonality in the tumor microenvironment may serve as independent predictive biomarkers of CI response. The need for evaluation of multiple biomarkers using IHC, PCR and/or NGS using limited amounts of tissue poses a challenge in the clinical setting. Here we conduct a study to evaluate whether a panel of tumor and TME biomarkers can be accurately analyzed using low DNA and RNA input from FFPE specimens or using plasma-derived cfDNA. Multiple methods for assessing TMB, MSI status and mutation signatures were compared. The impact of sample quality on biomarker analysis was also evaluated. A set of late stage CRC and matched plasma samples were used in this study. DNA and RNA were co-extracted from FFPE sections using RecoverAll™ kit. cfDNA was extracted from plasma using the MagMAX™ Kit. DNA and RNA yields from FFPE ranged from 855-3875ng and 477-5200ng, respectively. cfDNA yields ranged from 8-38ng. Based on the KAPA DNA QC results, the FFPE samples were determined to be high quality. TMB and mutation signature analysis of FFPE samples were performed using Oncomine Tumor Mutation Load (TML) Assay, the Oncomine Comprehensive Assay v3 (OCAv3) and WES. MSI status in FFPE tissue and cfDNA was determined using the Promega MSI Analysis System. The mutation spectrum in the matched cfDNA samples was determined using the Oncomine PanCancer cfTNA Assay. Inflammatory (IFNG) signatures were assessed using the targeted RNASeq panel, Oncomine Immune Response Research Assay (OIRRA). The clonality of TILs was analyzed using NGS. TMB results generated from the targeted panels using only 20ng DNA input were comparable to WES results. Differences in TMB counts between the NGS assays are likely due to differences in genomic coverage and data filtering. MSI-H status and/or mutations in critical DNA repair or proofreading genes such as MSH6 and POLE correlated with high TMB. Gene expression profiling using OIRRA was successful for all FFPE RNA samples. In summary, this study demonstrates that comprehensive genomic analysis of predictive biomarkers for CI therapy can be performed from limited FFPE samples using co-extraction methods and a combination of targeted NGS and PCR-based assays. DNA and RNA quality have a significant impact on the accuracy of TMB analysis and gene expression profiling. In cases where no tumor tissue is available, the analysis of biomarkers such as MSI and mutation signatures may be possible using cfDNA.

#1703

New genetic variations discovered in KRAS wild-type cetuximab resistant Chinese colorectal cancer patients.

Changwen Jing,1 Ting Wang,2 Rong Ma,1 Haixia Cao,1 EnShun Xu,3 Zhuo Wang,1 Siwen Liu,1 Dan Chen,1 Junying Zhang,1 Yang Wu,1 Yuan Zhang,1 Ping Ding,3 Michael Y. Sha,4 Jianzhong Wu,1 Jifeng Feng1. 1 _Jiangsu Cancer Hospital & Jiangsu Institute of Cancer Research & The Affiliated Cancer Hospital of Nanjing Medical University, Nanjing, China; _2 _Nanjing Medical University, Nanjing, China;_ 3 _Diacarta Inc, Nanjing, China;_ 4 _DiaCarta Inc, Richmond, CA_.

The efficacy of Cetuximab therapy in colorectal cancer patients is limited by the development of therapeutic resistance. The currently known oncogenic 'driver' mutations do not fully explain the resistance mechanism of Cetuximab. Currently there are no reports of any studies that have explored new driver mutations in Cetuximab resistance by whole exome sequencing (WES).

In this study, we performed WES to identify common genetic factors from twenty-two Cetuximab-sensitive and ten Cetuximab-resistant patients, aiming to discover characteristic genes associated with Cetuximab resistance. For Cetuximab-sensitive patients, the mutation frequency of MUC16, FCGBP, DMBT1 was found to be 100%, 90.91% and 86.36%, respectively. MUC16, PDE4DIP and TACC2 were altered in all 10 Cetuximab-resistant samples. We also discovered there are 151 significantly mutated genes (SMGs) in Cetuximab sensitive group and 37 in the resistant group. CYP4A11 was the most frequently mutated gene in Cetuximab-resistant patients. BCAS1, GOLGA6L1 and WNT16 were found to be the second group of frequently mutated genes which found in to occur at a frequency of 60% in resistant patients. After cosine similarity analysis with 30 known signatures, three mutational signatures (signature a, b and c) were found in all CRC tumors, similar to Signature 1, 5 and 6 in COSMIC, respectively. Gene ontology analysis was performed on SMGs and found twelve enriched GO terms. Four genes are enriched in six specific KEGG pathway groups, such as metabolism of xenobiotics by cytochrome P450, Steroid hormone biosynthesis, retinol metabolism and drug metabolism. In conclusion, we show that the establishment of a WES clinical study of Cetuximab resistance CRC with prospective follow-up of patients can help to identify candidate predictive biomarkers of response.

#1704

Predicting survival and improving treatment for uterine serous carcinoma patients using USC78, a genomic risk score.

Lynn K. Tran,1 Emily K. Myers,1 David P. Mysona,2 Paul M. Tran,1 Wonsok Lee,1 John J. Wallbillich,1 Daniel Kleven,1 Sharad Ghamande,1 Jin-Xiong She1. 1 _Medical College of Georgia at Augusta University, Augusta, GA;_ 2 _University of North Carolina, Augusta, NC_.

Uterine serous carcinoma (USC) is a rare but particularly invasive histological subtype of uterine cancer, the most common gynecologic malignancy in developed countries. Though previous studies with small sample sizes have been conducted to identify transcriptomic and proteomic biomarkers for USC, none have resulted in a clinical assay for patient risk stratification. I have discovered a panel of 78 genes highly upregulated in poor prognosis patients from The Cancer Genome Atlas (TCGA) USC dataset. To create a composite score for our biomarker panel, I applied a machine learning algorithm (elastic net regression) to the TCGA USC gene expression data set to generate a model which outputs a linear predictor score (USC78) based on individual patients' expression of the 78 genes from our signature. I then quantified gene expression from formalin-fixed paraffin embedded (FFPE) tumor tissue in our cohort of Augusta University (AU) patients and calculated their USC78 score to demonstrate the ability of this model to separate good and poor survival prognosis in a single-center, retrospective validation study. In both TCGA and AU, higher USC78 scores are associated with worse overall survival. This score is also able to risk stratify serous ovarian carcinoma patients in the TCGA data set, suggesting that USC78 may be an important prognosis prediction tool in serous gynecological cancers. Additionally, network analysis of these genes reveals increased TGF-B signaling correlates with the poor-prognosis USC78-high tumor expression profile. TGF-B inhibition has demonstrated chemosensitization effects in primary USC tumor cell lines. I aim to use USC78 to better understand the biological mechanisms behind poor survival prognosis in USC patients and identify potential therapeutic targets for USC. One such target is TGF-B inhibition, and further knockdown, overexpression, and inhibitor studies will be conducted to test whether TGF-B inhibition can improve USC patient prognosis.

#1705

Genetic analysis of thymoma and thymic carcinoma.

Hiroyuki Yamaguchi, Hiroshi Gyotoku, Hirokazu Taniguchi, Daisuke Sasaki, Midori Shimada, Yosuke Dotsu, Hiroaki Senju, Norihito Kaku, Takaya Ikeda, Minoru Fukuda, Katsunori Yanagihara, Hiroshi Mukae. _Nagasaki University Hospital, Nagasaki-city, Nagasaki, Japan_.

[Background] Thymic epidermal tumors (thymoma and thymic cancer) are rare, and its genetic profiles are unclear. Molecular targeted therapy of thymic tumors has not been established. Clarifying a molecular alteration and to identify the novel targets for molecular targeted therapy is important. So, we conducted the next generation sequencing (NGS) analysis with surgically resected thymic tumor tissues.

[Methods] From July 2013 to October 2018, thirty patients who were resected thymic epidermal tumors in our hospital were enrolled in this study. We got written informed consent from patients, and NGS analysis of extracted DNA sample was performed in 24 patients to the present time. DNA were extracted from fresh frozen surgically resected tissues (tumors and paired normal tissues) and DNA amplicon sequencing was performed with a custom panel of 53 cancer-related genes based on Ion AmpliSeq™ Cancer Hotspot Panel v2 comprising major oncogenes and tumor suppressor genes (including GTF2I). Sequencing was carried out with an Ion Torrent PGM™. Sequencing data were analyzed using Ion Reporter™. In addition, we collected the patient's information (age, sex, Masaoka-Koga staging, World Health Organization (WHO) histologic classification, tumor size, and complication such as myasthenia gravis and pure red cell aplasia) from medical records. The study was approved by the Institutional Review Board (IRB) of the Nagasaki University Hospital.

[Results] A total of 30 patients were enrolled in this study. The median age was 62 years old (range, 34-84 years old), patients characteristics were 9 male and 21 female, WHO histologic classification (type A, AB, B1, B2, B3, thymic carcinoma) was 1, 11, 4, 10, 2, 2, and Masaoka-Koga staging I, II, III, IV were 12, 6, 9, 3, respectively. Median tumor size was 47mm (range 11-110 mm). Moreover, complications were 7 myasthenia gravis, 4 acetylcholine receptor antibody-positive (do not confirm diagnossis of myasthenia gravis), 1 pure red cell aplasia, and 1 agranulocytosis. In NGS sequencing, the nonsynonymous mutations of HRAS and NRAS (HRAS Q61R, HRAS G13R, and NRAS Q61K) was detected in three patients. These RAS gene mutations were reported to be pathogenic in various malignancies. Low frequently DNMT3A mutation was detected in the other two patients, however this mutation were not validated in other methods. No genetic alterations were detected in the rest 19 patients.

[Conclusion] The frequency of genetic alterations in thymic epidermal tumors was very low in this study, however, active mutations of RAS oncogene were detected in three patients. Although the RAS are still not established as treatment targets, it should be one of the interacting molecules. Further investigation is required to establish new therapeutic strategy according to the genetic alteration in thymic tumors.

#1706

High-quality whole-genome sequencing and analysis of FFPE samples from multiplecancer types.

Shannon T. Bailey, James Lund, Muhammad Ekram, Wanfeng Yu, Richard T. Williams, Hongye Sun, Jeffrey R. Gulcher. _WuXi NextCODE Genomics, Cambridge, MA_.

Next-generation sequencing analysis of formalin-fixed, paraffin-embedded (FFPE) samples has the potential to lead to major advances in cancer treatment and prevention. However, whole-genome sequencing (WGS) of these samples has remained challenging due to the difficult process of isolating high-quality DNA and distinguishing true variant calls from artifacts. As evidence of these challenges, only four WGS studies of FFPE samples have been published to date. Previous work has attempted to overcome these challenges by using whole-exome sequencing or smaller exon panels at high coverage depths with publications reporting success from less than half to most samples. Thus, there has not been widespread adoption of using FFPE samples for next-generation sequencing.

We developed a novel DNA extraction and library prep protocol for whole-genome sequencing of FFPE tissue samples. This method produces larger DNA fragments that can be successfully used for WGS. We previously presented results of a large esophageal cancer cohort where we sequenced over 1,000 samples in tumor-normal pairs with data equivalent to that generated from fresh-frozen tissues. We recently expanded the tumor types, ages, and sources and achieved similar results. We have assayed FFPE tissues derived from breast cancer, colorectal cancer, non-small cell lung cancer, prostate cancer, and melanoma. These samples were sourced from several different labs, which have used variations on the standard fixation protocol. Some of these FFPE samples were over 15 years old. Variant calling provides excellent results without excessive C>T mutations. The coverage uniformity was very good, and CNV calls were made with results comparable to published TCGA data. These samples have been analyzed for MSI and TMB, and the results are comparable to those using fresh tissues.

Our method makes new types of clinical studies possible utilizing FFPE samples that have been generated as part of standard hospital procedures. This method also facilitates performing studies comparing primary and recurrent cancers easier to establish and run as recurrent cancer cases can be enrolled and stored FFPE primary samples used to establish a cancer baseline.

#1707

Enteric-type adenocarcinoma in head and neck: Dissecting oncogenic genes alterations through whole transcriptome analysis.

Diana Bell, Achim H. Bell, Ehab Y. Hanna. _UT MD Anderson Cancer Ctr., Houston, TX_.

IntroductionAdenocarcinomas of nose/paranasal are uncommon tumors; an important one is that which shows enteric differentiation (ETA). ETA is typically seen in adults, many whom have had occupational exposure (mainly wood and leather). The histogenesis of ETA is uncertain, an origin from transdifferentiation of the respiratory epithelium is a possibility. We previously reported evidence for such transformation in similar tumors at other anatomic locations (Eustachian tube, base of tongue, epiglottis). Due to the rarity of these tumors, their molecular profile is not well known. To investigate the molecular profile and identify potential oncogenic drivers we compared the whole transcriptome of ETA at different anatomic location in head and neck.

Materials and Methods10 adenocarcinomas of head and neck formed the material of this study. These samples along with matching normal mucosa were microdisected from FFPE. Pools of RNA were subjected to expression profiling using whole-transcriptome shotgun sequencing.

Results 20 tumors were divided as follows: 10 sinonasal and 10 non-sinonasal adenocarcinomas. The sinonasal adenocarcinomas were classified as 8 ETA and 2 seromucinous type (non-ETA, nasal). The 10 non-sinonasal HN adenocarcinomas divided according to histologies and location: 4 ETA (BOT/mobile-2, submandibular-1, ear-1); 3 mucinous adenocarcinoma (buccal-2; 1 ectopic rests neck L2); 1 adenoid cystic carcinoma of BOT; 1 polymorphous adenocarcinoma of buccal; one larynx metastatic colonic adenocarcinoma. Differential gene expression profiles from adenocarcinomas versus normal mucosa were generated and compared to each other. Comparing all tumors versus all normal mucosal tissues, 3029 genes and noncoding transcripts were detected as differentially expressed with fold change >2 and p-values< 0.05 (1625 upregulated and 1404 downregulated genes). These genes and transcripts were annotated and analyzed using IPA program. Further refined analysis of ETA at sinonasal locations showed 622 specific/ non-overlapping genes (325 upregulated and 297 downregulated). The ETA group at other anatomic location segregated with 3344 specific differentially expressed genes, mostly upregulated (1887 versus 1457 downregulated genes). The highest expressed genes and potential drivers were predominantly development- and differentiation-related genes.

ConclusionWe assume that ETA are mainly defined by overexpressed developmental homeobox genes, which provide the potential for transformation/ plasticity, along with the differentiation and proliferation behavior of neoplastic cells. The data form the basis for understanding cell fate determination and cellular homeostasis in the normal respiratory mucosa at different anatomic sites, and the contribution of different mucosal components to the etiology/ molecular pathology of ETA.

#1708

An integrated framework for genome analysis reveals numerous previously unrecognizable structural variants in leukemia patients' samples.

James R. Broach,1 Jie Xu,1 Emily Schleicher,1 Christopher Pool,2 Max Hennessy,2 Kathryn Sheldon,1 Charyguly Annageldiyev,2 Arati Sharma,1 Yuanyuan Chang,3 Alex Hastie,3 Barbara Miller,2 David Goldenberg,2 David Claxton,2 George-Lucian Moldovan,1 Feng Yue1. 1 _Penn State College of Medicine, Hershey, PA;_ 2 _Hershey Medical Center, Hershey, PA;_ 3 _Bionano Genomics, San Diego, CA_.

We have applied a combination of whole genome sequencing and optical genomic mapping to a number of adult and pediatric leukemia samples, which revealed in each of these samples a large number of structural variants not recognizable by current standard genomic analyses. We developed computational methods to determine which of those variants likely arose as somatic mutations. The method identified 97% of the translocations previously reported by karyotype analysis of these samples and revealed an additional fivefold more such somatic rearrangements. The method identified on average tens of previously unrecognizable inversions and duplications and hundreds of previously unrecognizable insertions and deletions. These structural variants affected a number of leukemia associated genes as well as cancer driver genes not previously associated with leukemia and genes not previously associated with cancer. A number of variants only affected intergenic regions but caused cis-acting alterations in expression of neighboring genes. Analysis of TCGA data indicates that several of the recurrently mutated genes significantly affect survival of AML patients. Our results suggest that current genomic analysis methods fail to identify a majority of structural variants in leukemia samples and this lacunae may hamper diagnostic and prognostic efforts.

#1709

Consistent performance of highly multiplexed RNA fusion reference materials across different NGS assays in a multi-lab study.

Dana J. Ruminski Lowe, Deepika Philkana, Catherine Huang, Omoshile Clement, Andrew Anfora, Dan Brudzewsky, Bharathi Anekella. _SeraCare Life Sciences, Gaithersburg, MD_.

Introduction: Gene fusions, usually the result of chromosomal rearrangements, are frequently associated with many cancer types, and hence clinically actionable, making fusion detection an important part of cancer disease management. We developed a new, optimized version of the Seraseq® Fusion RNA Reference Material, and demonstrate its consistent performance across different NGS enrichment assays, sequencing coverage depths, bioinformatics pipelines, and RNA mass inputs from a multi-lab investigation (Sites A, B, C, D & E).

Methods: Biosynthetic DNA was used for transcription of 18 RNA fusions, including more common fusions of ALK, RET, and ROS1, as well as rare fusion events such as PAX-PPARG and ETV6-NTRK3. The in vitro transcribed RNAs were mixed with total RNA extracted from GM24385 reference cell line (The 1000 Genomes Project, Coriell). Digital PCR with TaqMan® chemistry was used to determine the target fusion RNA concentration and serve as the "truth" data set for comparison to NGS, which can be variable depending on input, assay, and bioinformatics. The fusion-total RNA mix was analyzed by five external laboratories; it was tested using the ArcherDx FusionPlex™ Solid Tumor Panel (Site A, Site B, Site C), the ArcherDx FusionPlex™ CTL Panel (Site C), the ArcherDx FusionPlex™ Lung Panel (Site B), a custom ArcherDx FusionPlex™ Panel (Site D), and the TruSight Tumor 170 Panel (Site E).

Results: All eighteen (18) fusions in the new Seraseq Fusion RNA Mix v4 reference standard were detected as expected on each NGS platform with an average of greater than 85% of on-target reads across all assays. Even at inputs as low as 20 ng, all 18 fusions were typically detected above fusion-calling thresholds. In general, the results for individual fusions among the different NGS panels and among replicates were concordant, with observed variance in reads across some fusion junctions between assays and replicates. Within FusionPlex assay results, the average percent of reads supporting the fusion call across all fusions was about 63%, regardless of input (a range between 20 to 250 ng). Collectively, the multi-lab results confirm that the Seraseq® Fusion RNA Mix v4 reference standard is compatible with both amplicon and hybridization-capture based NGS assays.

Conclusions: Seraseq RNA Fusion Mix v4 has broad NGS assay compatibility and allows for reliable and simultaneous detection of 18 clinically relevant RNA fusions even at low input amounts. The data from a multi-lab study support the use of this reference standard for targeted NGS assay development, assay validation, bioinformatics pipeline optimization, and as positive controls in clinical NGS RNA fusion assays. The biosynthetic manufacturing approach produces reference materials that provide consistent results for a wide variety of common and rare gene fusions. 

### Deregulated Cell Processes and Carcinogenesis

#1710

Role of Nurr1 in cervical cancer pathogenic pathway.

Kok Ting Wan, Thomas Ho Yin Leung, Michelle Kwan Yee Siu, Hextan Yuen Sheung Ngan. _The University of Hong Kong, Hong Kong_.

Aims: To study the oncogenic role of Nurr1 in cervical cancer and elucidate the underlying mechanism.

Background: Orphan nuclear receptor related-1 protein (Nurr1) belonging to the NR4A family is important for carcinogenesis in multiple biological processes such as cell proliferation and apoptosis. It has been reported that the expression of Nurr1 is significantly correlated with various types of cancer, such as prostate and breast cancer. However, the significance of Nurr1 in cervical cancer etiology remains poorly understood.

Methods: Primary cancer cells were established from tumor tissues resected from cervical cancer patients. Six pairs of attached and spheroid cells derived from primary cervical cancer tissues were established namely CP-1A/CP-1S, CP-2A/CP-2S, CP-3A/CP-3S, CP-4A/CP-4S, CP-5A/CP-5S and CP-6A/CP-6S. A microarray was performed to identify differentially expressed genes in two pairs of attached and sphere cells, followed by validation with qPCR. Stable clones of cervical cancer cell lines with Nurr1 overexpression and knock-down were generated. The role of the selected gene was then functionally characterized by XTT assay, migration assay, spheroid formation assay, apoptosis assay, in vitro and in vivo tumorigenicity assay in cervical cancer cell lines. Downstream proteins of Nurr1 were detected by western blot.

Results: Microarray analysis showed that Nurr1 was highly expressed in sphere cells as compared with attached cells. Relative expression of Nurr1 mRNA in sphere cells was approximately 200-fold more than that in attached cells in qPCR validation. Cancer cell lines, in which Nurr1 was transiently knocked down by siRNA demonstrated an attenuation in proliferation rate, migration, spheroid formation and colony formation. By flow cytometric analysis, more cells were detected in early apoptotic stage with Nurr1 siRNA transfection as compared with NTC siRNA transfected cells, suggesting the role of Nurr1 in preventing cervical cancer cells from apoptosis. Western blotting also demonstrated that there was an increased expression of Bim and p21 in Nurr1 silenced cells. This may indicate the involvement of Nurr1 in modulating the transcription of pro-apoptotic and tumor suppressor genes.

Conclusion: Nurr1 is a potential oncogenic target in cervical cancer pathogenic pathway. Suppression of Nurr1 expression attenuated cancer progression and promoted apoptosis.

#1711

Kynurenine 3-monooxygenase (KMO) acts as a novel oncoprotein in triple negative breast cancer.

Chun-Yu Liu,1 Tzu-Ting Huang,1 Ji-Lin Chen,1 Chia-Han Lee,1 Wan-Lun Wang,1 Ka-Yi Lau,1 Chun-Teng Huang,2 Pei-Yi Chu,3 Hsin-Chen Lee,4 Ling-Ming Tseng1. 1 _Taipei Veterans General Hospital, Taipei, Taiwan;_ 2 _Yang-Ming Branch of Taipei City Hospital, Taipei, Taiwan;_ 3 _Show Chwan Memorial Hospital, Changhua, Taiwan;_ 4 _National Yang-Ming University, Taipei, Taiwan_.

Purpose: Tryptophan-kynurenine pathway involves in inflammation, immune response and tumorigenesis, in which kynurenine 3-monooxygenase (KMO), an outer mitochondrial membrane protein, mediating kynurenine metabolism. Previous studies indicated KMO showed increased activity in breast cancer. Triple-negative breast cancer (TNBC) tumors exhibited elevated levels of tryptophan metabolites compared to estrogen receptor positive breast cancers. We aimed to study the role of KMO in human TNBC.

Experimental design: The gene alterations and transcripts of enzymes in kynurenine metabolism were analyzed from The Cancer Genome Atlas (TCGA) database. Immunohistochemical staining for KMO was performed and a H-score was assigned to quantify protein expression. Epithelial-mesenchymal transition (EMT) phenotypes were examined by transwell assay and EMT markers expressions. Stemness properties were assessed by mammosphere assay and pluripotent genes expressions. The molecular events were analyzed by Western blot, quantitative real-time PCR and luciferase reporter assay. Tumor growth and metastasis were conducted in nude mice and NOD-SCID mice by subcutaneous and tail vein injection respectively.

Results: TCGA databases showed KMO but not KYNU and KAT2 was amplified in breast cancer. Both the data from TCGA and our in-house IHC-based tissue-microarray exhibited increased KMO expression in TNBC compared to normal tissue. In vitro, overexpression of KMO in TNBC cells resulted in increased cell growth and colony formation. The abilities migration and invasion as well as EMT markers expressions of TNBC cells were elevated by KMO overexpression. In addition, KMO increased mammosphere formation, pluripotent genes expressions and promoter activities. However, inhibition of KMO enzymatic activity by KMO inhibitors did not affect cancer progression or mitochondrial respiration of TNBC cells. KMO upregulated β-catenin, the upstream regulator of pluripotent genes, CD44 and Nanog expressions. Mechanistically, data showed KMO expressed in both cytosol and nuclear fractions and was associated with β-catenin. KMO enhanced pluripotent genes expressions through β-catenin upregulation. Importantly, KMO knockdown suppressed tumor growth and expressions of β-catenin, CD44 and Nanog in TNBC tumors. Moreover, KMO knockout significantly decreased lung metastasis in vivo.

Conclusion: Our data indicated KMO can play an oncogenic role in TNBC, acting as a novel regulator of pluripotent genes via β-catenin and promoted TNBC progression.

#1712

Estrogen receptor beta signaling sensitizes glioblastoma cells to chemo and radiation therapy.

Uday P. Pratap,1 Gangadhara Reddy Sareddy,1 Mengxing Li,1 Yiliao Luo,1 Mei Zhou,2 Suryavathi Viswanadhapalli,1 Rajeshwar Rao Tekmal,1 Andrew Brenner,1 Ratna K. Vadlamudi1. 1 _UT Health San Antonio, San Antonio, TX;_ 2 _Second Xiangya Hospital and Xiangya School of Medicine, Central South University, Changsha,, China_.

Current treatment options for glioblastoma (GBM) are poor, the mortality rates are very high and therapy resistance is a major clinical problem. Standard treatment consists of surgical resection, external beam radiation therapy, adjuvant chemotherapy with temozolomide (TMZ), and tumor treating fields. Nonetheless, despite a heavy investment in therapy, all patients eventually succumb to their disease. Recent studies suggest that estrogen receptor beta (ERβ) may function as a tumor suppressor in GBM. However, the mechanism(s) by which ERβ contributes to chemo- and radiation therapy response remains unknown. The objective of this study is to examine whether ERβ sensitizes GBM to chemo- and radiation therapy and to understand the mechanistic insights of ERβ mediated tumor suppression in GBM.

Methods: To study the functions of endogenous ERβ in GBM cells, we have utilized multiple ERβ overexpressing GBM model (GBM- ERβ) cells using lentiviral transduction. As a second model we have also generated ERβ knockout (ERβKO) cells using CRISPR/Cas9 system, and as a third model we used lentiviral-ERβshRNA transfected primary GBM cells (ERβKD). We then examined the effect of TMZ and radiation on the expression of ERβ using qRT-PCR. The effect of TMZ and radiation on ERβ overexpression and knockout models was examined using MTT cell viability assays. Mechanistic studies were conducted using RNA-seq, HR reporter gene assays, confocal microscopy, western blot, and qRT-PCR analysis. The in vivo role of ERβ on chemo sensitivity of TMZ was studied using orthotopic models of GBM and mouse survival was determined using Kaplan-Meier survival curves.

Results: Cell viability and survival assays using multiple established and primary GBM cells demonstrated that ERβ sensitizes GBM cells to DNA damaging agents including TMZ and radiation therapy. qRT-PCR analysis demonstrated that ERβ expression was decreased following chemo- and radiation treatment. Combination analysis of RNA-seq studies using ERβ overexpression, and ERKO models, revealed an alteration in the number of genes involved in DNA recombination and repair, ATM signaling, and cell cycle check point control. Mechanistic studies showed that ERβ plays a significant role in homologous recombination (HR) mediated repair; and ERβ reduced expression and activation of ATM upon DNA damage. Generation of optimal γH2AX foci following TMZ treatment is dependent on the status of ERβ. More importantly, GBM cells expressing ERβ had increased survival when compared to control GBM cells in orthotopic GBM models. ERβ overexpression further enhanced the survival of mice to TMZ therapy in both TMZ sensitive and TMZ resistant GBM models.

Conclusion: Our results provided evidence that ERβ is required for optimal chemo- and radiation- induced DNA damage response in GBM cells.

#1713

Molecular dissection of Zc3h8 functional domains and the implications for the oncogenic phenotype.

John A. Schmidt, Tyler Doan, Emanuel Irizarry, Emily Harris, Keith G. Danielson, Janice E. Knepper. _Villanova Univ., Villanova, PA_.

Zc3h8 encodes a zinc finger protein that is amplified in 2-6% of human breast tumors. Higher expression of Zc3h8 also results in lower rates of disease-free survival. Amplification of Zc3h8 expression has been shown to increase cell proliferation, migration, invasion and tumor growth in mice. Conversely, decreased expression alters cell behavior by decreasing cell proliferation, migration, invasion, and tumor growth in mice. Zc3h8 comprises three CCCH non-classical zinc finger domains at the C-terminus of the protein that we hypothesize bind to RNA. The interactions between Zc3h8 and nucleic acids may be important for the localization and/or function of Zc3h8 and therefore may be important for the cellular phenotypes observed when the gene is over or under expressed in cells. We used in vitro and in vivo assays to demonstrate that mutations in any of the three zinc finger domains results in a protein that fails to rescue aggressive cellular phenotypes in cells with reduced expression of Zc3h8, while the wild type protein can rescue these phenotypes. This implies that all three zinc finger domains are required for Zc3h8 function in promoting rapid and aggressive cell proliferation. In addition to zinc finger domains, Zc3h8 also contains a casein kinase-2 (CK2) phosphorylation site that may be important for protein localization and/or activation. We have previously shown that CK2 inhibitors alter Zc3h8 localization to nuclear PML Bodies. Mouse cells overexpressing Zc3h8 with a Thr to Ala (T32A) change that prevents CK2 phosphorylation grow very slowly in culture and cannot grow in more challenging 3D or soft agar growth chambers. However, Zc3h8 with a Thr to Glu (T32E) change that mimics constitutive CK2 phosphorylation results in rapidly growing cells that surpass overexpression of Zc3h8 wild type. Zc3h8 phospho-mutants also have altered localization with Zc3h8 T32E becoming diffuse in the nucleus and T32A localizing to nuclear foci. These data suggest that the phosphorylation state of Zc3h8 and its ability to bind nucleic acids via zinc finger domains are critical for the oncogenic behavior that results from amplified expression. We also hypothesize that Zc3h8 has an important role in establishing or maintaining PML Bodies involved in genome maintenance, transcription, and/or post-transcription regulation.

#1714

Nuclear receptor Nurr1 facilitates pancreatic ductal adenocarcinoma cell survival under metabolic stress.

Sneha Harishchandra, Xi Li, Stephen Safe, Mahsa Zarei. _Texas A &M University, College Station, TX_.

Background: Pancreatic cancer is lethal and the fourth leading cause of cancer deaths in the United States with a 5-year overall survival rate of 6.7%. The orphan nuclear receptor 4A2 (NR4A2, Nurr1) has been implicated in various cellular processes, including inflammation, vascular remodeling, and cancer as a transcriptional facilitator of tumorigenesis, through its regulation of various proliferation, invasion, and metastasis-associated genes. In the pancreatic cancer context, very little is known about NURR1. Here, we explored the expression, prognostic value, and function of NURR1 in human PDA growth and metastasis. We also investigated the role of NURR1 in regulating pancreatic cancer metabolism.

Methods: We analyzed The Cancer Genome Atlas (TCGA) database for PDA and microarray expression data from GEO and examined NURR1 expression in normal pancreatic tissue, primary pancreatic tumors and metastatic tumors by using the R statistical computing environment. Kaplan-Meier analysis were performed to investigate the prognostic significance of NURR1 for PDA. NURR1 levels were depleted with siRNA oligos, and also overexpressed with NURR1 cDNA. Following characterization of NURR1 expression, we performed a cell proliferation (XTT) assay and assessed anchorage-independence over a 3-week period by soft agar colony formation assay under normal condition and metabolic stress. Metastatic potential was measured by Matrigel invasion and transwell migration assays. Experiments were performed in MIA PaCa-2, Panc-1 and L3.6pl cell lines.

Results: Analysis of messenger RNA (mRNA) expression data from 177 PDA samples from TCGA showed that NURR1 was overexpressed in PDA tissues compared with the pancreatic normal samples (P< 0.001). Kaplan-Meier analysis revealed that 5-year median overall survival (OS) and progression free survival (PFS) were significantly poorer in the NURR1-overexpression group (P= 0.002). Moreover, knockdown of NURR1 significantly reduced PDA cell proliferation, migration, and invasion, whereas NURR1 overexpression resulted in a significant increase in proliferation and migration. In addition, mRNA expression of NURR1 was increased after 12 hours of glucose deprivation in 2 cell lines (vs. 25mM glucose). However, NURR1 depletion impaired survival in low glucose media (5mM) and overexpression of NURR1 had a protective effect under glucose deprivation in Panc-1 cells.

Conclusion: NURR1 exhibits a tumor-promoting effect on PDA and NURR1 expression is critical for pancreatic cancer cell survival under glucose deprivation. Our work demonstrates that manipulation of NURR1 expression affects key characteristics of pancreatic tumorigenesis (i.e. proliferation, invasion, and migration). Improved understanding of how NURR1 regulates metabolism in response to stress should uncover novel therapeutic strategies against pancreatic cancer.

#1715

**** Sox2 **is required for tumor initiation and maintenance in small cell lung cancer.**

Ellen Voigt,1 Ethan Thompson,1 Hannah Wollenzien,2 Madeline Vande Kamp,1 Michael S. Kareta1. 1 _Sanford Research, Sioux Falls, SD;_ 2 _University of South Dakota, Vermillion, SD_.

Some cancer prognoses have been radically improved in recent years, but little headway has been made with others. One of these diseases, small cell lung cancer (SCLC,) has a five-year survival rate of less than 7% and a standard of care that has been essentially unchanged for forty years. One promising avenue to improve SCLC outcomes is to understand the cancer's underlying genetic alterations that drive its formation and growth. One primary driver of SCLC, is the loss of Rb gene function, one of the most common occurrences in human cancers. Normally Rb promotes differentiation by regulating lineage specific transcription factors concurrent with repression of the pluripotency factors such as Sox2. However, there is evidence that when certain tissues lose Rb, Sox2 becomes upregulated and promotes oncogenesis. To understand the contribution of Sox2 to SCLC, we have utilized a genetically engineered mouse model for SCLC to determine the consequence of Sox2 on SCLC tumor initiation and growth. We observe that Sox2 is indeed required for SCLC. Further elucidation of the Sox2 network in SCLC could identify candidates for future targeted therapies in this devastating disease.

#1716

Evaluating the functional impacts of the BRCA1-mTORC2 interaction in breast cancer.

Kimiko L. Krieger,1 Wen-Feng Hu,2 Tyler Ripperger,3 Nicholas T. Woods1. 1 _University of Nebraska Medical Center, Omaha, NE;_ 2 _Central South University, Changsha, China;_ 3 _University of Arizona, Tucson, AZ_.

Objective: The BRCA1 C-Terminal (BRCT) domain of BRCA1 has been found to interact with three accessory proteins (PRR5, RICTOR, and SIN1) of mTOR complex 2 (mTORC2). BRCA1 BRCT domain dissociates the members of the mTORC2 complex to inhibit the activity of this kinase and block activation of Akt. Most breast cancers exhibit hyperactivation of the PI3K/Akt/mTOR pathway, and we hypothesize that genetic and epigenetic loss of functional BRCA1 contributes this signaling activation event. Therefore, the goal of this project is to study the potential cancer impacts of BRCA1-deficient breast cancers and their response to mTOR inhibition.

Methods: Yeast two-hybrid and orthogonal mammalian overexpression systems were used to confirm the interactions of PRR5, RICTOR, and SIN1 with the BRCT domain. MCF-10A breast cells were used to examine BRCA1-γH2AX foci formation under DNA damage conditions with cisplatin and mTOR inhibitors. Three-dimensional MCF-10A spheroids were used to study the effects of breast acini formation with shRNA-mediated repression BRCA1 and RICTOR. Breast cancer cell lines MCF-7, MDA-MB-231, and HCC1937 were used to study the effects of BRCA1 deficiency and response to pan-mTOR and pan-PI3K/mTOR inhibitors.

Results: Wild type BRCA1 BRCT domain was found to have stronger interactions with PRR5, RICTOR, and SIN1 than with the M1775R mutated BRCA1 BRCT domain and empty vector control. MCF-10A breast cells treated with rapamycin, which hyperactivates mTORC2, exhibited significantly higher amounts of BRCA1-γH2AX foci formation in comparison to control and pan-mTOR PP242-treated cells. This was comparable to the effect of cisplatin-treated cells. BRCA1-RICTOR double KD rescued the effect of distorted breast cell acini formation displayed by BRCA1 loss that could lead to tumorigenesis. This suggests that inhibition of the mTORC2 complex can be an effective target, especially for cells that lack functional BRCA1. MCF-7 and MDA-MB-231 cells with BRCA1 knockdown were significantly more sensitive to pan-mTOR inhibition than non-targeting scramble control. HCC1937 cells, which have a truncated, non-functional BRCA1 BRCT domain, were more sensitive to pan-PI3K/mTOR inhibition that HCC1937 cells with restored full-length BRCA1. Significance: Breast cancer is the most commonly diagnosed cancer among women in the U.S. BRCA1 is the most characterized gene associated with breast cancer. Genetic alterations in BRCA1 appear in about 5-10% of breast cancer cases, and epigenetic loss of BRCA1 appears in about 10-15% of breast cancer cases. Also, about 70% of breast tumors have hyperactive PI3K/Akt/mTOR pathways. This would suggest that breast cancer is heavily reliant on this oncogenic pathway, and therefore BRCA1-deficient patients may benefit from pan-mTOR cancer therapeutics.

#1717

Variants of uncertain significance of the cancer predisposing genes in 3.5 thousand Japanese whole genome sequencing data.

Jun Yasuda. _Tohoku Medical Megabank Organization, Sendai, Japan_.

Many of the cancer predisposing genes are known to be associated with juvenile cancer syndromes such as Li Fraumeni syndrome. The responsible variants of the juvenile cancer syndromes are usually very deleterious and show strong biological effects on the gene functions. It is not so easy to estimate the clinical significance of the moderately deleterious variants in those genes that may have clinically significant effects on the hosts' cancer predisposition and these moderately deleterious (or hypomorphic) variants are now classified as variants of uncertain significance (VUSs). The insight that VUSs may have moderate but clinically significant effects on cancer susceptibility now attracts cancer geneticists. To elucidate the state of VUSs of more than 150 cancer predisposing genes in general population, we functionally annotate the single nucleotide variants (SNVs) in the genes from a 3,552 whole genome sequence dataset (3.5KJPN) obtained from participants in a genome cohort by Tohoku Medical Megabank Organization. Frequency spectrum of the Combined Annotation-Dependent Depletion (CADD) and deleterious annotation of genetic variants using neural networks (DANN) scores of the more than 150 cancer predisposing genes will be discussed. Finally, the association of the CADD and DANN scores with the past and family histories will be analyzed.

#1718

**Down-regulation of p16** INK4a **by hYSK1 promotes cancer cell migration under hypoxic conditions.**

Mee-Hyun Lee,1 Jung-Hyun Shim,2 Zigang Dong,3 Young-Joon Surh,4 Bu Young Choi5. 1 _China-US (Henan) Hormel Cancer Institute, Zhengzhou, China;_ 2 _Department of Pharmacy, College of Pharmacy and Natural Medicine Research Institute, Mokpo National University, Republic of Korea;_ 3 _The Hormel Cancer Institute, Austin, MN;_ 4 _Tumor Microenvironment Global Core Research Center, College of Pharmacy, Seoul National University, Seoul, Republic of Korea;_ 5 _Department of Pharmaceutical Science & Engineering, Seowon University, Cheongju, Republic of Korea_.

The alteration expression of p16INK4a, a well-known cyclin-dependent kinase inhibitor involved in cancer cell cycle control, is unclear, especially under hypoxic conditions. To evaluate p16INK4a regulation, we performed a protein microarray analysis. Among 1,800 proteins, we identified hYSK1 as a novel protein that interacts with the tumor suppressor p16INK4a. hYSK1, a member of the Ste20 family of serine/threonine protein kinases, promotes cell migration & tumorigenesis and is activated by oxidative stress. However, the molecular mechanisms underlying the oncogenic potential of hYSK1 remain elusive. Here, we report here that hYSK1 interacts with p16INK4a under hypoxic conditions like tumors, where it negatively regulates p16INK4a, enhancing cancer cell migration. Hypoxic stimulation of hYSK1 reduces p16INK4a accumulation through p16 promoter regulation to interact with unphosporylated SP-1 & increases matrix metalloproteinase-2 (MMP-2) expression by activating the MMP-2 promoter associated with cell migration and proliferation.Conversely, knocking down hYSK1 expression activated p16INK4a expression & suppressed MMP-2 expression.Thus, hYSK1 is necessary as a trigger for inactivating p16INK4a & activating MMP-2 during tumor migration, suggesting that hYSK1 is a specific negative regulator of the tumor suppressor p16INK4a may represent a novel molecular target for reactivation of tumor suppressor genes in humans.

#1719

Copper levels and the p53 protein: A delicate balance.

Patricia Muller, Yannick von Grabowiecki, Callum Hall. _CRUK Manchester Institute, Manchester, United Kingdom_.

The tumor suppressor p53 is mutated in 50% of all cancers and in about 90% of small lung cancers. Mutations lead to loss of p53 expression or the expression of mutant p53 proteins. These mutants often lose WT function, but they have also been shown to acquire invasive and metastatic functions. p53 requires a zinc molecule in its core domain to fold properly and to function as a transcription factor and tumor suppressor. Loss of this zinc through competition with copper causes p53 to unfold and to lose its transcription function. Copper is an essential heavy metal required for a large variety of biological processes as it is required for the function of many enzymes. In cancer patients, increased serum levels of copper have been found and people with a predisposition for copper accumulation in the liver (Wilson's disease, mutations in ATP7B) are prone to develop liver cancers. Our preliminary results have indicated that an unfolded WT p53 molecule does not only lose its transcription function, but also gains mutant p53-like functions in promoting invasion. Copper-induced unfolded p53 interacted with the mutant p53 specific interacting proteins Dicer, Ago, p63 and p73 and impaired p63 function. These results raise the question as to why a WT molecule would lose its function and whether it perhaps could act as a redox sensor. Experiments are ongoing to explore p53 mediated transcription in response to copper exposure. In addition, we are setting up mouse models to investigate p53 folding in vivo in response to mild increases in copper serum levels and we are developing methods to better determine p53 folding in patients and in mice. These results will help us assess p53 status in relation to serum metal levels in vivo and determine whether p53 is important in the development of cancers and metastasis.

#1720

Correlations between declines in cystic fibrosis transmembrane conductance regulator (CFTR) and arylsulfatase B (ARSB) in prostate malignancy.

Joanne Kramer Tobacman, Sumit Bhattacharyya, Leo Feferman. _Univ. of Illinois at Chicago, Chicago, IL_.

Prior work has demonstrated decline in expression of arylsulfatase B (ARSB, N-acetylgalactosamine-4-sulfatase) in malignant tissue from prostate, mammary, and colon, and melanoma, suggesting a role for ARSB as a tumor suppressor. ARSB functions to remove the 4-sulfate group from chondroitin 4-sulfate (C4S) or dermatan sulfate, and thereby regulates the interaction of C4S with other mediators, including galectin-3 and the non-receptor tyrosine phosphatase SHP2 (PTPN11). Cystic fibrosis transmembrane conductance regulator (CFTR) has also been identified as a tumor suppressor. Declines in CFTR have been associated with malignancy, and increased incidence of malignancy is emerging in cystic fibrosis (CF) patients who have defective CFTR. Previous findings indicated that 1) ARSB expression and activity were reduced in CF cells and 2) repair of CFTR by gene insertion or by small molecule potentiator restored ARSB expression and activity. To further examine the relationship between CFTR and ARSB, the activity and expression of ARSB and CFTR was determined in prostate cell lines (WPE1-NB14, LNCaP, PC-3, TRAMP-C1; ATCC), the malignant and normal prostate tissues (n=6), and in normal and malignant stromal and epithelial tissues obtained by laser capture microdissection (LCMD) (n=3). Expression of both ARSB and CFTR were reduced in the malignant cell lines and in the malignant prostate tissues. Correlation coefficients between ARSB and CFTR were 0.99 and 0.97. Both CFTR and ARSB expression was reduced in the malignant epithelial tissue obtained by LCMD. However, in the stromal tissues, CFTR expression was absent and ARSB was greater than in the epithelium. In the normal prostate epithelial cells, inhibition of CFTR by siRNA or by CFTR(Inh)-172 led to significant declines in expression of ARSB, with no change in expression of galactose-6-sulfate sulfatase (GALNS). Silencing ARSB did not affect CFTR expression in the normal prostate epithelial cells. GATA-3, EGFR, Cyclin D1, and c-Myc were all increased when ARSB or CFTR was reduced by siRNA. Pending experiments are anticipated to show further associations between effects of ARSB and CFTR and to elucidate the mechanism whereby CFTR influences ARSB expression in the epithelial cells.

#1721

FOXO1, a downstream substrate of AKT, function as tumor suppressor in HCC carcinogenesis.

Shu Zhang, Xin Chen. _University of California, San Francisco, San Francisco, CA_.

Hepatocellular carcinoma (HCC) represents the fifth most common tumor types in the world and the third leading cause of cancer-related death. However, the signaling pathways underlying hepatic pathogenesis are still poorly understood, hence lacking of effective targeted therapy. Activation of PI3K/AKT signaling cascade has been implicated in HCC development. It has been well-established that the mammalian target of rapamycin complex 1 (mTORC1), is a major downstream effector of AKT. The reason why mTORC1 can be consistently activated is partly due to inactive mutations of a negative element regulating mTORC1, tuberous sclerosis 2 (TSC2), found in 10% human HCCs. Beyond TSC2, forkhead Box O transcription factor 1 (FOXO1), is another AKT substrate. It has been proved that FOXO1 plays a crucial role in the insulin signal pathway and liver regeneration relying on AKT isoforms. However, the role of FOXO1 in HCC development has not been well established. Here we report, by using TCGA database the mRNA level of FOXO1 significantly decreases among the FOXO family, comparing tumor tissue to the surrounding tissue. The overall survival rate favors patients with high FOXO1 expression level. The inhibition efficacies of a pan-AKT inhibitor, in both TSC2 wide type cells and TSC2-null ones, indicating TSC2 is not the only target downstream of AKT. Protein analysis shows the activity of mTORC1 can still be inhibited as well as the phosphorylation of FOXO1 in TSC2-null cells. Therefore, we hypothesize that in the presence of TSC2, AKTi can suppress the activation of mTORC1 directly through TSC2. Otherwise, in the absence of TSC2, AKTi can still inhibit mTORC1 via activation of FOXO1. Furthermore, we overexpress the activated FOXO1 (FOXO1 AAA) in TSC2-null HCC cell lines. Cell growth are significantly prohibited, implicating FOXO1 is a tumor suppressor in HCC cells. Over expression of activated FOXO1 can also delay mTORC1 activation based HCC in AKT/Ras FVB mice model.Lesions consist mainly of clear-cell hepatocytes, owing to an increase in glycogen and fat storage. Proliferation is nearly the same within the neoplastic nodules. Activated AKT occurs both in the cytoplasm and on the membrane. Interestingly, FOXO1AAA escapes into the cytoplasm instead of remaining in the nuclei where it should stay. In a conclusion, AKT/mTORC1 can be a classic molecular pathway for HCC pathogenesis. Nevertheless, the parallel substrate of AKT, FOXO1, may also be an important factor in a TSC2 independent way.

#1722

Spectrum of solid malignancies associated with mutations in CYLD gene.

Michelle Ellis, Jeff Swensen, Zoran Gatalica. _Caris Life Sciences, Phoenix, AZ_.

Introduction: The CYLD gene product is a de-ubiqutinating enzyme involved in regulation of NF-kappaB signaling. Inactivating germline mutations in CYLD are the cause of Brooke-Spiegler syndrome characterized by multiple cutaneous tumors (cylindromas, trichoepitheliomas and spiradenomas) and tumors of salivary glands. Little is known about the role of CYLD in the development of solid organ malignancies.

Methods: We retrospectively analyzed CYLD gene mutations detected in a large database of solid tumors genetically profiled (NGS of 592 genes) at a reference laboratory (Caris Life Science, Phoenix, AZ).

Results: CYLD pathogenic mutations were identified in 235 tumor samples (0.4% of the tumors analyzed). The most common primary sites included lung (60), endometrium (15) and anus (10). Histologically, the majority of the tumors were classified as squamous (71) and adenocarcinoma (59). A number of mutations were identified, most commonly affecting codons p.N722fs (27), p.S371* (16) and p.P771S/p.P771L (12). 38 cases had only CYLD mutations detected, while the majority of associated mutations occurred in TP53 (45%). Numerous co-occurring pathogenic mutations were present in oncogenes (e.g. PIK3CA in 41, KRAS in 33, BRAF 17) and/or tumor suppressor genes (e.g. APC in 40, PTEN in 36, BRCA2 in 25, RB1 in 23).

Conclusion: Our study provides the largest database analysis of CYLD gene mutations in solid tumors and shows the distribution in numerous primary tumor sites previously unrecognized in association with CYLD. Additional studies may reveal unexpected germ-line associations and will be pursued in the future.

#1723

**Elucidation of novel therapeutic targets for acute myeloid leukemias with** RUNX1 **-** RUNX1T1 **fusion.**

Jae Won Yun,1 Yoon Kyung Bae,2 So Yeong Cho,2 Hee-Jin Kim,1 Do-Hyun Nam,1 Sun-Hee Kim,1 Sejong Chun,3 Kyeung Min Joo,2 Hye Won Lee,2 Woong-Yang Park1. 1 _Samsung Medical Center, Seoul, Republic of Korea;_ 2 _Sungkyunkwan University, Suwon, Republic of Korea;_ 3 _Chonnam National University, Gwangju, Republic of Korea_.

The RUNX1-RUNX1T1 fusion is a frequent chromosomal alteration in acute myeloid leukemias (AMLs). Although RUNX1-RUNX1T1 fusion protein has pivotal roles in the development of AMLs with the fusion, RUNX1-RUNX1T1 fusion protein is difficult to target since it lacks kinase activities. Here, we used a sophisticated bioinformatic tool to elucidate targetable signaling pathways in AMLs with RUNX1-RUNX1T1 fusion. After analysis of 93 AMLs from the TCGA database, the expressions of 293 genes were significantly correlated with the expression of the RUNX1-RUNX1T1 fusion gene. Based on the 293 genes, the COX, VEGFR, PDGFR, and FGFR pathways are predicted to be specifically activated in AMLs with RUNX1-RUNX1T1 fusion. Compared with the AML cells without the fusion, the specific activations of the AML cell lines with RUNX1-RUNX1T1 fusion were confirmed by the increased phosphorylation of VEGFRs, PDGFRs, and FGFRs. Moreover, the in vitro proliferation of AML cells with RUNX1-RUNX1T1 fusion decreased significantly more than that of AML cells without the fusion when the pathways were inhibited pharmacologically. The results indicate that novel targetable signaling pathways could be identified by the analysis of the gene expression features of AMLs with non-targetable genetic alterations. The elucidation of specific molecular targets for AMLs that have a specific genetic alteration would promote personalized treatment of AMLs and improve treatment outcomes for AML patients in clinic.

#1724

Natural history and clinical characteristics of ERBB2 mutant hormone receptor-positive breast cancers: Results from the AACR Project GENIE Registry.

Michele LeNoue-Newton,1 Sheau-Chiann Chen,2 Thomas Stricker,2 David Hyman,3 Philippe Bedard,4 Funda Meric-Bernstam,5 Rinaa Sujata Punglia,6 Deborah Schrag,6 Eva Lepisto,6 Fabrice Andre,7 Lillian Smyth,3 Semih Dogan,7 Celeste Yu,4 Chetna Wathoo,5 Mia Levy,1 Grace Mann,8 Alshad S. Lalani,8 Fei Ye,2 Christine Micheel,2 Monica Arnedos7. 1 _Vanderbilt-Ingram Cancer Center, TN;_ 2 _Vanderbilt University Medical Center, Nashville, TN;_ 3 _Memorial Sloan Kettering Cancer Center, NY;_ 4 _Princess Margaret Cancer Centre, Ontario, Canada;_ 5 _MD Anderson Cancer Center, TX;_ 6 _Dana-Farber Cancer Institute, Boston, MA;_ 7 _Gustave Roussy Cancer Campus, Villejuif, France;_ 8 _Puma Biotechnology, Inc., CA_.

Background: Potential oncogenic mutations in ERBB2 causing constitutive activation of the HER2 kinase in the absence of amplification occur in around 4% of breast cancers. The natural history of this rare genomically-defined population is needed.

Methods: We used the AACR Project GENIE Registry, an international pan-cancer registry that aggregates clinical and cancer genomic data, to identify a cohort of patients with hormone receptor (HR)-positive, HER2-negative metastatic breast cancer (MBC) with oncogenic ERBB2 mutations. Matched wild-type (WT) controls to birth year, race and age at sequencing were abstracted in a 2:1 ratio. The primary aims were to: i) compare clinicopathological features and concomitant molecular alterations of ERBB2-mutant vs. WT patients, ii) investigate the natural history of ERBB2-mutated BC and iii) analyze their response to different therapies.

Results: We identified 45 HR+/HER2- MBC patients with an oncogenic ERBB2 mutation. Most frequent ERBB2 variants were: L755S (38%), V777L (24%), D769Y (13%), L869R (1%), S310F (1%). Most frequent co-occurring molecular alterations were PIK3CA (46%), CDH1 (28%) and TP53 (18%) mutations. No ESR1 mutations were identifed in the 27 ERBB2-mutant patients with concomitant ESR1 sequencing (vs. 14% (4/29) in WT cases). No significant differences were observed between ERBB2-mutated MBC patients and the 90 controls in clinicopathological features including stage, histological grade and menopausal status. There were differences in site of first metastasis, with more liver (p=0.006) and bone (p= 0.019) metastases in the ERBB2-mutant patients compared with more lung metastases in controls (p=0.028). No difference in overall survival (OS) from time of distant metastatic diagnosis was identified between ERBB2-mutant and WT patients (p=0.37). Multivariable analysis shows that duration from diagnosis to late relapse (>5 years) has a significant impact on OS, after adjusting for mutation status, number of prior lines of chemotherapy and number of lines of metastatic therapy (p<0.0001). In the ERBB2-mutant cohort, 38% (17/45) of patients had received neratinib, an irreversible HER2 inhibitor that has demonstrated clinical activity in ERBB2-mutant MBC. There was a trend to improved OS from 1st distant metastasis in the neratinib-treated group. Neratinib-treated patients also had received more lines of systemic treatment than those neratinib-naïve (P=0.001).

Conclusions: In this large series of ERBB2-mutated BC patients, no significant differences in clinicopathological features, except sites of metastasis, were observed between ERBB2-mutant vs. wild-type ER+/HER2- breast cancer patients. Patients with ERBB2 mutations treated with neratinib trended toward an improvement in OS. Further analysis including multivariate analyses in the ERBB2-mutant cases will be presented.

#1725

**Mutationally activated KRAS** (G12D) **upregulates the expression of axonal guidance cue Semaphorin-5A in pancreatic cancer.**

Sugandha Saxena, Babita Tomar, Lingyun Wu, Mohammad Awaji, Satyanarayan Rachagani, Michel J. Ouellette, Surinder K. Batra, Bhavana J. Dave, Rakesh K. Singh. _Univ. of Nebraska Medical Ctr., Omaha, NE_.

Mutation of KRAS is commonly found in different human cancers and is generally responsible for driving tumorigenesis. Unfortunately, direct inhibitors of KRAS are not available. Thus, scientists are in search of alternative strategies that can repress the oncogenic properties of KRAS and can benefit a large number of cancer patients. Drugs targeting KRAS downstream effectors are under investigation but are facing numerous challenges in their clinical implementation. Accumulating evidence suggests that the genetic status of KRAS can modulate the role of axonal guidance cue family members in tumorigenesis. Previous reports from our laboratory have identified upregulation of one such molecule Semaphorin-5A (SEMA5A) in murine model based on KRAS gene mutation in pancreatic progenitor cells that bear a striking similarity to the human PDAC condition. We utilized PDX-cre-LSL-Kras(G12D) (KC) for studying Sema5A expression and observed no expression of Sema5A in the normal pancreas, derived from the control PDX-cre mice, however, we observed Sema5A expression in PDX-cre-LSL-Kras(G12D) mice beginning at ten weeks of age. Furthermore, we observed an increase in SEMA5A expression in the tumors of mice aged at twenty-five and fifty weeks, representing a fully developed PDAC condition. To investigate the relationship between KRAS status and SEMA5A, we assessed whether KRAS(G12D) alters the expression of SEMA5A using immortalized human pancreatic ductal cells having endogenous wild-type KRAS but an exogenous expression of KRAS(G12D) [HPNE-KRAS]. We detected higher expression of SEMA5A in the KRAS(G12D) bearing HPNE-KRAS cells in comparison with their control counterpart HPNE-Tert. Stable clones of CD18/HPAF cell line knocked-down for KRAS(G12D) demonstrated inhibition of SEMA5A expression in comparison with their respective Control CD18/HPAF cells bearing an endogenous expression of mutant KRAS. We also analyzed SEMA5A expression in the cells lines derived from the serial passage of COLO 357 cells through mice to achieve highly invasive/metastatic variants also leading to the selection of cells that express mutationally activated KRAS. L3.3 cells, with no point mutations in the KRAS gene, expressed lower SEMA5A in comparison with highly metastatic L3.6pl cells bearing a point mutation in codon 12, thereby encoding a mutationally activated KRAS(G12D). Together, our data suggest that KRAS(G12D) mutation directly upregulates the expression of axon guidance cue molecule-SEMA5A in the PDAC cells. Our findings open an avenue of molecules that can serve as secondary targets to repress KRAS activity in pancreatic cancer.

#1726

Structure-function analysis of retinoblastoma tumor suppressor protein in human cone precursors.

Sijia Wang,1 Brendan Grubbs,2 Matthew Thornton,2 David Cobrinik1. 1 _Keck School of Medicine, Los Angeles, CA;_ 2 _Children's Hospital Los Angeles, Los Angeles, CA_.

Background: The retinoblastoma protein, pRB, is a key cell cycle governor that is functionally impaired in most cancers, but its mechanism of suppressing tumorigenesis in a tumor cell-of-origin has not been characterized. This study aims to define pRB structural features and corresponding cellular functions that suppress cone precursor (CP) proliferation, likely the first step in retinoblastomagenesis.

Method: Intact human fetal retinae were co-transduced with lentiviruses to knockdown endogenous pRB and ectopically express wild type or structure-based substitution mutant pRBs. The ectopically expressed pRB mutants had selectively impaired interactions with E2F transactivation domains (ΔG), with the E2F1 "marked box" domain (ΔS), or with the LxCxE protein interaction motif (ΔL). At day 12-75 post-infection, transduced retinae were analyzed by quantitative immunofluorescence staining of Ki67/EdU, pRB and CP marker L/M-opsin.

Results: In intact retina, pRB knockdown induced cell cycle re-entry of L/M-opsin+ postmitotic cone precursors, whereas those reconstituted with wild type pRB did not express Ki67 indicative of a block to cell cycle entry. Unexpectedly, all three mutants (pRB-ΔG, -ΔL, and -ΔS) were able to block cell cycle re-entry when expressed at the high-normal range the of endogenous pRB. However, at the low-normal expression level, ectopic wild type but not mutant pRBs suppressed proliferation.

Conclusion: All three surface targeted mutants (pRB-ΔG, ΔL, and ΔS) retain partial ability to suppress cone precursor cell cycle entry. This may be attributed to the presence of multiple independent cell cycle inhibitory functions. These findings suggest that pRB-mediated regulation of E2F-responsive cell cycle genes, interactions with LxCxE motif-containing proteins, and EZH2 mediated repression of repetitive elements are individually dispensable for suppressing aberrant cone precursor proliferation.

#1727

**Dissecting the impact of** KEAP1 **loss and NRF2 activation on lung adenocarcinoma initiation, metabolism, immune contexture and patient outcomes.**

Georgia Hatzivassiliou,1 Anju Singh,2 Anneleen Daemen,1 Dorothee Nickles,1 Sang-Min Jeon,3 Eun-Ji Choi,3 Oded Foreman,1 Shyam Biswal2. 1 _Genentech, Inc., South San Francisco, CA;_ 2 _Johns Hopkins University, Baltimore, MD;_ 3 _Ajou University, Republic of Korea_.

Non-small-cell lung cancer (NSCLC) is a heterogeneous disease of diverse subtypes, each exhibiting unique biology, immune profile and clinical behavior. While recent advances in targeted therapy and immunotherapy are improving outcomes for patients, response rates vary widely and the prognostic and predictive drivers of response remain poorly defined. In lung adenocarcinoma (LUAD), genomic alterations in the KEAP1 and NFE2L2 loci occur in about 20% of patients, leading to the constitutive stabilization of the transcription factor NRF2, a master regulator of antioxidant defenses and redox balance. The unique contribution of NRF2 activation to lung cancer physiology, immune phenotypes and clinical outcomes remains largely elusive. To address this issue, we have modeled NRF2 pathway activation in vitro and in vivo by targeting KEAP1 in KRAS-driven LUAD tumor models. Our data reveals the contribution of NRF2 in lung cancer initiation, differentiation and progression as well as its impact on the tumor microenvironment and immune contexture, both independently and in the context of combined loss of the LUAD tumor suppressor STK11. We develop a novel, 96-gene expression signature diagnostic of NRF2 activation independently of other frequently co-occurring mutations and valid across both non-squamous and squamous NSCLC. We use this signature to evaluate the impact of functional NRF2 activation on LUAD prognosis and patient clinical responses to immunotherapy and chemotherapy regimens. Our work supports the conclusion that cancer cell intrinsic genetic events shape the tumor microenvironment and immune cell infiltration and reveals that NRF2 activation has a distinct impact relatively to other common driver mutations.

#1728

Overactivation of tumor suppressor P53 in hepatocytes promotes hepatocarcinogenesis.

Yuki Makino. _Osaka University Graduate School of Medicine, Suita, Japan_.

Aim: p53 is a tumor suppressor and its dysfunction promotes carcinogenesis of several organs including the liver. Meanwhile, p53 is reported to be overactivated in non-tumorous liver tissues of patients with chronic liver diseases, presumably due to several stressful stimuli to hepatocytes, including viral infection, ethanol exposure, fat accumulation. The aim of this study is to clarify the impact of p53 overactivation on hepatocarcinogenesis.

Methods: To induce hepatocyte-specific overactivation of p53 in liver cancer models, hepatic Mdm2, a negative regulator of p53, was genetically ablated in diethylnitrosoamine (DEN)-treated mice (Alb-CreMdm2fl/fl) and liver-specific constitutive-active Kras-mutant knock-in mice (Alb-CreKrasLSL-G12DMdm2fl/fl called as KrasG12DΔMdm2 mice). To analyze the significance of p53, hepatic p53 was deleted in Mdm2-wild KrasG12D mice (Alb-CreKrasLSL-G12DMdm2+/+p53fl/fl) and KrasG12DΔMdm2 mice (Alb-CreKrasLSL-G12DMdm2fl/flp53fl/fl).

Results: Mdm2 deletion significantly elevated serum ALT levels and increased tumor penetrance in both DEN-treated mice and Kras-mutant mice, suggesting that p53 overactivation accelerated hepatocarcinogenesis. KrasG12DΔMdm2 mice upregulated hepatic expressions of pro-apoptotic molecules (p16, p21, Bax, Noxa), number of TUNEL positive hepatocytes and serum caspase-3/7 levels, indicating accelerated hepatocyte apoptosis. They also increased the number of β-galactosidase positive hepatocytes and upregulated hepatic inflammatory cytokines (Tnf, Ccl2, Il-1β), suggesting senescence-associated secretory phenotype (SASP). Expressions of hepatic progenitor cell (HPC) markers (Afp, Epcam, Ck7, Ck19, Cd90, Cd133, Dlk1) were up-regulated in their liver. HPC marker staining suggested that liver tumors in KrasG12DΔMdm2 mice may originate from HPCs. All the phenotypes including accelerated hepatocyte apoptosis, SASP and emergence of HPCs in KrasG12DΔMdm2 mice were significantly alleviated by p53 deletion. Most importantly, deletion of p53 significantly decelerated hepatocarcinogenesis in those mice, indicating that p53 overactivation was responsible for the accelerated hepatocarcinogenesis. In sharp contrast, p53 deletion significantly accelerated hepatocarcinogenesis in Mdm2-wild Kras-mutant mice, confirming the tumor suppressive role of normal level of p53 in the liver.

Conclusion: Whereas p53 deletion accelerated hepatocarcinogenesis, p53 overactivation induced liver injury and activation of HPCs, leading to the acceleration of hepatocarcinogenesis. p53 overactivation in hepatocytes might be associated with liver cancer development in patients with chronic liver diseases.

#1729

Investigating deregulated circadian clock machinery in cancer cells.

Pauline J. van der Watt, Kate Davis, Virna Leaner. _University of Cape Town, Cape Town, South Africa_.

The circadian clock plays an integral role in cellular functioning by temporally controlling gene expression, and there is accumulating evidence for a link between circadian clock disruption and cancer progression. In this study we aimed to investigate circadian clock gene expression and oscillation patterns in cervical and oesophageal cancers, in order to determine whether disruptions in circadian clock functioning occur in these cancer types. Microarray gene expression analysis revealed the circadian clock gene, Per2, to be one of the most significantly downregulated genes in cervical cancer patient tissue compared to normal epithelium. In addition, Oncomine data-mining revealed significant downregulation of not only Per2, but multiple members of the circadian clock gene family (Clock, Bmal1, Per1, Per2, Cry1, Rev-erbα and RORα) in cervical cancer tissue compared to normal. Real-time RT-PCR analysis of circadian clock genes in oesophageal cancer patient tissue compared to matched normal epithelium revealed significant downregulation of Clock, Per2, Cry1 and RORα in oesophageal tumour tissue. Significant positive correlations were observed where circadian clock genes were simultaneously dysregulated in tumour tissue specimens. In cell line models, Clock, Bmal1, Per2, Cry1 and RORα circadian clock genes were significantly downregulated in transformed cells, compared to their untransformed counterparts, as well as in most cervical and oesophageal cancer cell lines compared to non-cancer epithelial cells. Patterns of protein expression did not accurately match mRNA expression, likely due to extensive post-translational processing, but Clock and Cry1 protein levels were considerably reduced in the cancer cell lines compared to normal. Overexpression of circadian clock genes in cancer cell lines negatively affected cell proliferation, highlighting the tumour suppressor properties of these genes in cervical and oesophageal cancer cells. Despite downreglated expression of circadian clock genes, cervical and oesophageal cancer cells maintain functional circadian oscillations, after synchronisation with Dexamethasone, as shown using real-time bioluminescence imaging, suggesting that their circadian clock mechanisms are still intact. Together, this study is a first to describe deregulated circadian clock machinery in cervical and oesophageal cancer cells, although the cells maintain a functional circadian rhythm. The study has relevance to the field of chronotherapy, where elucidating differences in circadian clock functioning in normal and cancer cells could yield better insights into the timing of administration of chemotherapy, ultimately ensuring a better patient response.

#1730

DACH1 **gene deletion extends portraits of human prostate cancer.**

Xuanmao Jiao,1 Gabriele Di Sante,1 Zhiping Li,1 Agnese Di Rocco,1 Min Wang,1 Adam Ertel,2 Peter A. McCue,2 Andrew P. South,2 Carlos Cordon-Cardo,3 Matthew P. Stokes,4 Marco Marra,5 Steven J. Jones,5 Andrew Kossenkov,6 Richard G. Pestell1. 1 _Baruch S. Blumberg Institute, Doylestown, PA;_ 2 _Thomas Jefferson University, Philadelphia, PA;_ 3 _Mt. Sinai Hospital, New York, NY;_ 4 _Cell Signaling Technology, Danvers, MA;_ 5 _BC Cancer Agency, Vancouver, British Columbia, Canada;_ 6 _The Wistar Institute, Philadelphia, PA_.

Purpose of the study: This study was conducted to define the role of Dachshund in prostate cancer, through assessing human prostate cancer samples and through genetic deletion in the mouse. Prostate cancer (PCa), the second leading cause of death in American men, is a genetically heterogeneous disease, likely representing distinct genetic drivers, with terminal events caused primarily by metastasis. Substratification of PCa into genetic subtypes, forms the basis of rational therapy for PCa. A better molecular understanding of the disease is necessary in order to develop novel targeted therapies of metastatic PCa. Known genetic drivers to tumor initiation include PTEN and NKX3.1 deletions, rearrangements of the TMPRSS2 gene to the oncogenic ETS transcription factor, ERG, and genetic predisposing factors include germline DNA-repair gene mutations. The DACH1 gene, initially cloned as an inhibitor of Elipse, the hyperactive epidermal growth factor (EGFR) in Drosophila, was found to be reduced in abundance in several malignancies including breast and prostate cancer.

Results: In order to determine whether the DACH1 gene is deleted or mutated in prostate cancer we interrogated the genomic sequencing analysis of over 490 patients from 5 population cohorts. Homozygous deletion of DACH1 was identified in 18% (N=61), 11% (N=136), 10% (N=492), 7% (N=103) and 3% (N=150) of prostate cancer in 5 distinct cohorts. The prevalence of DACH1 gene deep deletions was higher in the metastasis than in the primary tumors. The Transgenic Adenocarcinoma Mouse Prostate (TRAMP) transgenic, Dach1fl/fl, and Probasin-Cre, ROSA26mT/mG transgenic mice were used to generate a prostate epithelial cell specific Dach1 gene knockout mouse (Probasin-Cre-Dach1fl/fl ROSA26mT/mG-TRAMP) lines. Prostate specific deletion of the murine Dach1 gene enhanced progression of prostatic intraepithelial neoplasia (PIN), associated with increased prostate epithelial cell proliferation, epithelial mesenchymal transition (EMT), DNA damage and inflammation.

Conclusions: DACH1 gene deletion may define a distinct subclass of prostate cancer that may benefit from PARP inhibitors, and platinum-based chemotherapy.

#1731

**Functional genomics approach reveals distinct neo-morphic activities and co-driver mutations of different missense mutant** TP53 **that promote breast cancer heterogeneity and progression.**

Anasuya Pal, Anasuya Pal. _Arizona State University, Tempe, AZ_.

Phenotypic and molecular profiling of breast cancer demonstrates a high degree of heterogeneity in the breast tumors. TP53 tumor suppressor is mutated in 30% of all breast tumors and 80% of the basal-like subtype with numerous co-existing somatic mutations in other genes. We hypothesize that tumor heterogeneity is a result of combination of neo-morphic functions of specific TP53 driver mutations and functionally important but distinct co-mutations (or 'co-drivers') for each type of TP53 mutation. We thus ectopically expressed 10 most common missense p53 mutant proteins found in breast cancer patients in normal-like mammary epithelial cells and examined their phenotypes associated with various hallmarks of cancer. Supporting our hypothesis, a wide spectrum of phenotypic changes in cell survival, resistance to apoptosis and anoikis, cell invasion and cell polarity across the 10 mutant cell lines were observed compared to those of WT p53 expressing cells. Integrated analysis of ChIP-seq and RNA-seq showed distinct promoter binding profiles of different p53 mutant proteins, implying that non-canonical transcriptional activity of mutant p53 proteins may contribute to the phenotypic heterogeneity in TP53-mutated tumor. Further, enrichment and model-based pathway analyses revealed the key biological pathways associated with invasion of cells expressing different p53 mutant proteins. As a proof-of-concept for screening co-drivers of mutant p53, when PTEN was deleted in non-invasive cells expressing p53-Y234C mutant using CRISPR-Cas9 system, an increase in cell invasion was observed. A genome-wide CRISPR library-based screen on p53-Y234C and p53-R273C cells identified completely different candidate co-driver mutations that promoted cell invasion. The top candidates included several known mutated genes in breast cancer patients harboring TP53 mutations and were associated with cytoskeletal and apoptosis resistance pathways. Overall, our combined approach of molecular profiling and functional genomics screens highlighted distinct sets of genes and co-driver mutations that can lead to heterogeneous phenotypes and promote aggressive behaviors of cells with different TP53 mutant backgrounds, which can guide development of novel targeted therapies.

#1732

**Switch to MET oncogene dependence as a novel mechanism of drug resistance in** EGFR- **mutant lung cancer.**

Pinar Ö. Eser,1 Raymond M. Paranal,1 Michael J. Poitras,1 Man Xu,1 Stephen Wang,1 Atsuko Ogino,1 Jihyun Choi,2 Pavlos Missios,3 Heidi M. Haikala,1 Jieun Son,1 Mika Lin,1 Masahiko Yanagita,1 Prafulla C. Gokhale,1 George Q. Daley,4 Pasi A. Jänne1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Brandeis University, Waltham, MA;_ 3 _Boston Children's Hospital, Boston, MA;_ 4 _Harvard Medical School, Boston, MA_.

Introduction: Non-small cell lung cancers harboring sensitizing gain-of-function mutations in the epidermal growth factor receptor (EGFR) are treated with small molecule EGFR kinase inhibitors including erlotinib and osimertinib. Unfortunately, the clinical efficacy of these inhibitors is limited by progression to drug resistance. Genomic amplification of the hepatocyte growth factor receptor (MET) is a prevalent mechanism of resistance to clinical EGFR inhibitors. EGFR mutant lung cancers with MET amplification are co-dependent on signaling through EGFR and MET kinases, whereby either oncogene can phosphorylate the EGFR family receptor ERBB3, activating downstream signaling and promoting survival. These EGFR/MET co-dependent cancers are characteristically resistant to treatment with individual drugs, but retain sensitivity to combination treatment with EGFR and MET inhibitors. Here, we describe three patient-derived EGFR mutant, MET amplified lung cancer models that exhibit a switch away from EGFR, to MET oncogene dependence. Methods: We established and characterized patient-derived lung cancer cell lines and xenograft models harboring concurrent EGFR activating mutations (EGFR L858R or EGFR Del19) and genomic MET copy number gain. Three patient-derived models—DFCI81, DFCI161, and DFCI307—were developed from pleural effusion and core biopsy specimens of patients whose tumors progressed on erlotinib. Clinical samples were immediately sorted and stably cultured in vitro or engrafted into immunodeficient mice. Results: DFCI81, DFCI161, and DFCI307 cell lines and xenograft models retained mutant EGFR expression, but exhibited resistance to clinical EGFR inhibitors and sensitivity to single-agent c-MET inhibitors, including crizotinib and savolitinib. Comparing our EGFR-mutant and MET-dependent cell lines to EGFR/MET-codependent models, we observed that EGFR expression was significantly reduced in our models compared to controls. Ectopic overexpression of EGFR Del19 and EGFR L858R in DFCI81 and DFCI161 cell lines, respectively, was sufficient to confer crizotinib resistance and induce EGFR/MET co-dependency. We demonstrated that MET-mediated ERBB3 phosphorylation drives downstream PI3K activation to promote cell proliferation and survival in DFCI81 and DFCI161 cells. In these contexts, ERBB3 reactivation by recombinant ligand treatment was sufficient to induce EGFR-mediated ERBB3 activation, conferring resistance to single-agent crizotinib treatment. Conclusions: We have identified and characterized three patient-derived models of treatment refractory EGFR mutant lung cancer that exhibit a switch to MET oncogene dependency. Clinically, we predict a subset of EGFR mutant, MET-dependent tumors exists, and can be identified de novo by a reduced EGFR to MET expression ratio: a potential biomarker predictive of sensitivity to single-agent MET inhibition.

#1733

Role of the SIX1 oncofetal protein in endometrial basal cell metaplasia and carcinogenesis following neonatal exposure to diethylstilbestrol.

Alisa A. Suen,1 Wendy N. Jefferson,1 Charles E. Wood,2 Anna C. Kenan,1 Carmen J. Williams1. 1 _National Institute of Environmental Health Sciences, Durham, NC;_ 2 _Boehringer Ingelheim, Ridgefield, CT_.

Early-life exposure to estrogenic chemicals can increase cancer risk, likely by disrupting normal patterns of cellular differentiation. In a model of this process, female mice exposed to the potent synthetic estrogen diethylstilbestrol (DES) on postnatal days 1-5 develop endometrial adenobasal carcinoma as adults. Neoplastic glands are comprised of abnormal populations of basal cells (cytokeratin (CK)14+), luminal cells (CK18+), and low numbers of "mixed" basal/luminal cells (CK14+/18+), all of which express the oncofetal protein sine oculis homeobox 1 (SIX1). We hypothesized that DES-induced SIX1 expression is necessary for aberrant endometrial differentiation patterns and carcinogenesis. To test this hypothesis, a conditional knockout model was generated in which floxed Six1 was excised in the uterus using progesterone receptor (Pgr) cre. The most prominent change in DES-exposed SIX1 knockout (DES-Six1d/d) mice was the absence of basal cells in the uterine horns. There was a greater than 10-fold decrease in CK14 labeling in the uterine horns of DES-Six1d/d mice compared to DES-exposed SIX1 wildtype (DES-Six1+/+) mice as determined by quantitative image analysis. However, DES-Six1d/d mice exhibited a 42% increase in cancer incidence compared to DES-Six1+/+ mice at 6 months of age (16/18 DES-Six1d/d vs. 7/15 DES-Six1+/+). Interestingly, mixed cells were still present in DES-Six1d/d mice. These findings demonstrate that SIX1 is a cellular differentiation factor necessary for DES-induced basal cells but not mixed cell development or cancer. Furthermore, these data suggest that DES-induced SIX1 expression decreases endometrial carcinogenesis by facilitating basal cell differentiation. Studies investigating the mixed cell population as a putative cancer progenitor cell population are ongoing. Interestingly, mixed cells were present in 35% (63/181) of malignant human endometrial tissue biopsies and in 0% (0/29) of normal endometrial tissue biopsies, suggesting that the DES mouse model has significant similarities to human endometrial cancer.

#1734

Crispr/Cas9 screening of Brca1 and Brca2 susceptibility genes in breast and ovarian cancer precursor cells can identify phenotypically different mutants with variable penetrance.

Justyna Kanska, Kruttika Dabke, Zachary Schwartz, Norma Rodriquez-Malave, Nikoo Safi, Simon Gayther. _Cedars-Sinai Medical Ctr., Los Angeles, CA_.

BRCA1 and BRCA2 mutations are the most significant risk factors for breast and ovarian cancer identified so far. A recent analysis of breast/ovarian cancer risks in approx. 12,000 BRCA1 carriers and 7,000 BRCA2 carriers found that PTVs (protein truncating variants) in the central region of both genes are more associated with ovarian cancer development (Ovarian Cancer Cluster Regions (OCCRs)), while PTVs in the 5' and 3' regions are more associated with breast cancer development (Breast Cancer Cluster Regions (BCCRs)). We aimed to identify the functional consequences of different BRCA1/2 mutants by creating various PTVs in BCCR and OCCR regions of both genes in precursor cell types for Basal-like Breast Cancer (BLBC) and High-Grade Serous Ovarian Cancer (HGSOC). To this end, we created partially transformed models of breast and fallopian tube epithelial cells (MCF12A and FT282, respectively) via an overexpression of a hot-spot p53 mutation (R175H), frequently observed in BLBC and HGSOC. We then generated PTVs in MCF12Ap53R175H and FT282p53R175H cells in two regions of BRCA1 (exon 11 (OCCR) and 16 (BCCR)) and two regions of BRCA2 (exon 9 (BCCR) and 13 (OCCR)) using CRISPR/Cas9 system. We targeted the olfactory receptor OR10A4 as a negative control. We clonally derived cell lines and genotyped PTVs via molecular cloning and Sanger sequencing. We established models of MCF12Ap53R175H and FT282p53R175H cells carrying PTVs for almost all regions. Mostly heterozygous PTVs survived; except of BRCA1 BCCR PTVs, which were lethal in FT282p53R175H, while MCF12Ap53R175H cells survived homozygous PTVs in this region. Typically, all PTVs led to diminished growth, although FT282p53R175H with BRCA2 OCCR PTVs showed similar proliferation rate as control. Heterozygous BRCA1/2 mutations were sufficient to induce nonsense mediated decay of BRCA1/2 mRNA transcripts. BRCA1 OCCR PTVs also specifically increased processing of delta11q splice isoform, known to decrease PARP inhibitor and Cisplatin sensitivity in BRCA1 mutant cell lines. Consistently, derived BRCA1/2 mutant clones showed increased Cisplatin resistance. Immunofluorescence and flow cytometry confirmed decreased BRCA1 protein levels upon introduction of BRCA1 PTVs. Our studies demonstrate that different BRCA1/2 mutations have different phenotypic effects in breast and ovarian cancer precursor cells. We found that BRCA1 and BRCA2 BCCR PTVs in FT282p53R175H had more severe effect on survival and proliferation than OCCR PTVs. This suggests that OCCR PTVs may have survival advantage in FT282p53R175H cells, compared to BCCR PTVs. Next, we will perform high-throughput CRISPR/Cas9 screen of hundreds of BRCA1 and BRCA2 PTVs to functionally validate the genetic epidemiology data indicating the different disease risks for different germline mutations in these susceptibility genes.

#1735

Cathepsin B knocked down in pancreatic cancer cell lines fail to establish tumor in nude mice and influences the differential expression of circular RNA.

Manu Gnanamony, Liang Ge, Tulika Chatterjee, Christopher S. Gondi. _Univ. of Illinois College of Med. at Peoria, Peoria, IL_.

Most malignant tumors show increased transcriptional activation of cathepsin B. Over expression of cathepsin B has been associated with different malignancies and this has made it an attractive pharmacological target. Tumor progression can be defined in several steps with respect to proteases. The first step requires that surrounding tissue be cleared to create a pathway for growth by modification and degradation of the extracellular matrix and basement membrane. Cathepsin B is one of the cathepsins that has been directly linked to extracellular matrix degradation. The second is a proteolytic cascade where activation of matrix metalloproteinases and urokinase plasminogen activator (uPA), results in further tissue invasion, this activation is known to be mediated by Cathepsin B. The last is cleavage of E-cadherin at adherin junctions, detaching cells to enter circulation and migrate. Cathepsin B plays a central role in these processes.

Methods: In this study, we used two pancreatic cancer cell lines PANC-1 and MIA PaCa-2 that show differential sensitivity to gemcitabine. We knocked down the expression of cathepsin B in both PANC-1 and MIA PaCa-2 cells using CrisperCas9. We determined the tumorogenic potential of these down regulated cells and determined changes in gemcitabine sensitivity. Further, we determined the global expression profiles of circular RNA to predict the post-transcriptional regulation of associated microRNAs associated with chemo-sensitivity.

Results: From the tumorogenic studies, we observed that both PANC-1 and MIA PaCa-2 cells downregulated for Cathepsin B failed to develop tumors in nude mice subcutaneous tumor models. Further, from the gemcitabine sensitivity studies using the MTT assay we observed that Cathepsin B down regulation reduced IC50 in both PANC and MIA PaCa-2 by at least 25%. Global analysis of circular RNA expression showed that Cathepsin B downregulated PANC-1 cells showed a 4.2 fold increase in the expression of hsa_circRNA_081069 and a 3.8 fold decrease in the expression of hsa_circRNA_104169. In case of MIA PaCa-2 cells downregulated for Cathepsin B, we observed that hsa_circRNA_042488 was upregulated by 15.9 fold and hsa_circRNA_101692 was decreased by 20.5 fold.

Conclusions: From our observations, we conclude that Cathepsin B expression is necessary for (1) tumor establishment and development; (2) gemcitabine resistance and (3) modulation of circular RNA expression that regulate oncogenic and metabolic pathways necessary for tumorigenesis.

### DNA Damage and Repair 1

#1736

Targeting Hsp90-Cdc37 complex overcomes drug resistance in glioma cells harboring FGFR3-TACC3 fusion gene.

Tao Li,1 Farideh Mehraein-Ghomi,1 Sanjeev V. Namjoshi,1 Lynette M. Phillips,2 Elizabeth A. Ballard,1 Mary E. Forbes,1 ping-Chieh Chou,1 Xuejun Yang,3 Wei Zhang1. 1 _Wake Forest Baptist Medical Center, Winston-Salem, NC;_ 2 _University of Texas, MD Anderson Cancer Center, Houston, TX;_ 3 _Tianjin Medical University General Hospital, Tianjin, China_.

Glioblastoma is one of the most lethal malignancies with the poorest prognosis among tumors of the central nervous system. Fusion genes are common chromosomal aberrations in many cancers that may give rise to an in-frame fusion protein with oncogenic function. These fusion genes can be used as prognostic markers as well as drug targets in clinical practice. The FGFR3-TACC3 (F3T3) fusion gene was discovered in glioblastoma, with an occurrence rate of up to 8.3% and leading to uncontrolled proliferation and chromosomal instability. Our studies have shown that glioblastoma cells harboring the F3T3 are resistant to the frontline glioblastoma drug Temozolomide (TMZ). Using the Comet assay, we show that TMZ-mediated DNA damage is repaired more rapidly in cells harboring the F3T3 compared to control. Our studies on the mechanism of this drug resistance show that the F3T3 confers an aberrant activation of c-Met and an elevated expression of Bcl3, which is an independent predictor of response to TMZ. To further explore druggable target(s) to overcome resistance caused by F3T3, we co-immunoprecipitated (IP) the proteins that interact with F3T3 in U251 glioblastoma cells overexpressing the F3T3 or wildtype FGFR3, followed by LC-MS/MS analyses. Our quantitative proteomic analysis revealed interactions of the F3T3 fusion protein with CKAP5 (ch-TOG), Hsp90α and Cdc37 proteins. These interactions were further validated by reciprocal IPs followed by Western blotting. CKAP5 is necessary for spindle pole organization and proper chromosomal segregation. Activation of many kinases, such as FGFRs, depends on their interaction with the Hsp90 molecular chaperone system. Hsp90 recruitment is mediated by the co-chaperone adaptor protein Cdc37, which simultaneously binds to both the kinase and Hsp90. To test inhibition of F3T3 association with the Hsp90-Cdc37 complex, we treated U251 cells constitutively expressing F3T3 with the Hsp90 inhibitor Onalespib. We show that Onalespib treatment at 74 nM significantly suppresses the proliferation of F3T3 expressing U251 cells in comparison to micromolar levels of the pan-FGFR inhibitor BGJ398. Our results provide evidence that the F3T3 fusion gene contributes to drug resistance via multiple chemo-resistance pathways. Importantly, our findings establish that F3T3 is an Hsp90 client that shows strong addiction to the Hsp90-Cdc37 complex for cell growth and suggests a novel strategy for targeting F3T3 fusion gene in glioma.

#1737

Association of inherited variants with chromosomal breaks in cancer.

Meng Xiao He,1 Sahar Shahamatdar,2 Amaro Taylor-Weiner,1 Travis Zack,3 Sohini Ramachandran,2 Eliezer Van Allen4. 1 _Harvard University, Boston, MA;_ 2 _Brown University, Providence, RI;_ 3 _University of California, San Francisco, San Francisco, CA;_ 4 _Dana-Farber Cancer Institute, Boston, MA_.

Background: Cancer cells frequently harbor genomic rearrangements and somatic copy number alterations that result from chromosome breaks, which vary in frequency across the genome. The distribution of breakpoint locations depends on features of the local genomic context, such as presence of certain retrotransposons. Moreover, inherited variants have been associated with specific chromosomal alterations in myeloproliferative neoplasms and in blood from cancer-free individuals. Here, we developed and applied computational methodology to broadly identify associations between inherited variants and the location of somatic breakpoints across cancers.

Methods: We analyzed TCGA Affymetrix Genome-Wide Human SNP Array 6.0 data from matched tumor-normal pairs (n = 5,923 samples remaining after quality control and selecting for individuals of predominantly European ancestry). Germline genotypes were called from normal samples using Birdseed v2. Allele-specific copy number analysis using both normal and tumor samples for each individual was performed using ASCAT v2.5.1. Breakpoints were inferred as the intervals between ASCAT-called somatic chromosomal segments.

We assigned individuals binary phenotypes corresponding to whether a somatic breakpoint was detected in non-overlapping 5 megabase bins of the genome. We then performed an association analysis between each phenotype and variants located on the same chromosome as the corresponding bin, adjusting for sex and ancestry via logistic regression with plink2. Analyses were performed separately for each disease type followed by a meta-analysis of association summary statistics with METAL.

Results and Conclusions: We identify several candidate associations between inherited variants and the presence of somatic breakpoints in specific regions of the genome (using a p-value cutoff of 5e-8 for each phenotype). Our results suggest that heritable genetic variation may be associated with risk of development of localized chromosome breaks in somatic cells. Ongoing work is aimed at validating the putative associations (for example using data from the PanCancer Analysis of Whole Genomes study) and identifying potential mechanisms associated with putative risk alleles.

#1738

Characterization of structural variants within MACROD2 in the pathogenesis of colorectal cancer.

Remond J A Fijneman,1 Nienke Mekkes,1 Evert van den Broek,2 Bas Stringer,3 Roel A. Glas,1 Malgorzata A. Komor,1 Christian Rausch,1 Stef van Lieshout,4 Edwin Cuppen,4 Melissa L. Smith,5 Robert P. Sebra,5 William J. Rowell,6 Meredith Ashby,6 Beatriz Carvalho,1 Jaap Heringa,3 Gerrit A. Meijer,1 Sanne Abeln3. 1 _Netherlands Cancer Inst., Amsterdam, Netherlands;_ 2 _University Medical Center Groningen, Groningen, Netherlands;_ 3 _Vrije Universiteit Amsterdam, Amsterdam, Netherlands;_ 4 _Hartwig Medical Foundation, Amsterdam, Netherlands;_ 5 _Icahn Institute of Data Science and Genomics Technology; Icahn School of Medicine at Mount Sinai, New York, NY;_ 6 _Pacific Biosciences, Menlo Park, CA_.

Background: Cancer is caused by somatic DNA alterations, which comprise small nucleotide variants (SNVs), chromosome somatic copy number alterations (SCNAs) and chromosomal breakpoint structural variants (SVs). Previously, we investigated SCNA-associated SVs in colorectal cancer (CRC) and demonstrated that SVs within the MACROD2 gene are highly prevalent. This raises the question whether SVs in MACROD2 may already be present in CRC precursor lesions, i.e. in colorectal adenomas. We have also demonstrated that loss of MACROD2 protein expression is associated with poor response to treatment with 5-fluorouracil-based adjuvant chemotherapy, indicating that MACROD2 function is clinically relevant. The aim of this study is to characterize SVs within MACROD2 in more detail in the pathogenesis of colorectal cancer.

Methods: The frequencies of SCNA-associated SVs in 466 CRCs were compared to those in 118 colorectal adenomas, using array-comparative genomic hybridization. Targeted PacBio long-read sequencing was applied to detect and characterize SVs at nucleotide resolution within MACROD2, in tens of primary CRCs. Illumina whole genome sequencing data of > 450 CRC metastatic lesions, generated by the Hartwig Medical Foundation (HMF; www.hartwigmedicalfoundation.nl), were used for validation purposes.

Results: MACROD2 SCNA-associated SVs were rarely detected among 118 colorectal adenomas (<2%) while being highly prevalent among 466 CRCs (40%). SVs in MACROD2 are currently being characterized at nucleotide resolution by analysis of targeted PacBio long-read sequencing data, the results of which will be presented during the AACR annual meeting. Preliminary analysis of HMF whole genome sequencing data confirms that at least 40% of CRC metastatic lesions are affected by SVs within the MACROD2 gene, most commonly by focal deletions.

Discussion: The current observation that SVs in MACROD2 are nearly absent in adenomas while being highly prevalent in CRCs indicates that MACROD2 is affected at a late stage of colorectal adenoma-to-carcinoma progression. A recent publication by Sakthianandeswaren et al (Cancer Discovery 2018) indicated that loss of MACROD2 promotes chromosomal instability. Taken together, these data support a model in which adenoma-to-carcinoma progression is driven, at least in part, by genomic instability caused by loss of function of the MACROD2 tumor suppressor gene.

#1739

**Diminished** SKP1 **or** CUL1 **expression induces chromosome instability in high-grade serous ovarian cancer precursor cells.**

Chloe C. Lepage, Mark W. Nachtigal, Kirk J. McManus. _University of Manitoba, Winnipeg, Manitoba, Canada_.

High-grade serous ovarian cancer (HGSOC) is the most common ovarian cancer subtype as well as the most lethal. High mortality rates associated with HGSOC are in part due to a lack of reliable early detection methods and effective therapies. Importantly, chromosome instability (CIN; an increased rate of chromosome gains or losses), is causally implicated in cancer development, progression, and resistance to treatment, and is just beginning to be evaluated in an HGSOC context. Overexpression of Cyclin E1 protein induces CIN and genomic amplification contributes to HGSOC pathogenesis in 20% of HGSOC patients. Cyclin E1 protein levels are normally regulated in a cell cycle-dependent manner by the SCF (SKP1-CUL1-FBOX) complex, an E3 ubiquitin ligase that targets substrates for proteolytic degradation. In cases where genomic amplification is absent, we hypothesize that loss of SCF complex function stemming from diminished expression of individual SCF complex components underlies increases in Cyclin E1 levels and induces CIN. The current study focuses on the SCF complex components SKP1 and CUL1 and characterizes their role in early HGSOC pathogenesis within a fallopian tube secretory epithelial cell model (FT; a cell of origin for HGSOC). Using siRNA-based approaches, we show that diminished SKP1 and CUL1 expression results in increased Cyclin E1 protein levels in FT cells. Using quantitative imaging microscopy techniques, we further identify statistically significant changes in CIN-associated phenotypes within the silenced populations, including changes in nuclear areas, increases in micronucleus formation (i.e. small DNA-containing bodies outside of the primary nucleus), and increases in numerical chromosome abnormalities identified in mitotic chromosome spreads. Thus, the current study explores the early origins of HGSOC and identifies SKP1 and CUL1 as two promising new molecular players that may contribute to CIN to drive FT cell transformation, drug resistance and disease recurrence.

#1740

Characterization of CDK9 role in the DNA damage response.

Thales C. Nepomuceno,1 Guilherme Suarez-Kurtz,2 Alvaro N. Monteiro,1 Marcelo A. Carvalho3. 1 _Moffitt Cancer Center, Tampa, FL;_ 2 _INCA, Rio de Janeiro, Brazil;_ 3 _IFRJ, Rio de Janeiro, Brazil_.

Introduction: The DNA damage response (DDR) is a well-coordinated pathway capable of sensing and repairing different types of DNA damage. Double-strand break (DSB) lesions activate the ATM-CHK2 axis, which is responsible for the cell cycle arrest, prompting cells for DNA repair. DSBs are primarily repaired through two distinct pathways: homology-directed recombination (HR) and non-homologous end-joining (NHEJ). BRCA1 and 53BP1 play an important role in DDR by orchestrating the decision between HR and NHEJ, but the precise mechanisms regarding both pathways are not entirely understood. Previously, our group identified the putative interaction of the cyclin-dependent kinase 9 (CDK9) with BRCA1 and BARD1 (BRCA1-associated RING domain 1). CDK9 is a component of the positive transcription elongation complex and has been implicated in genome integrity maintenance associated with the replication stress response. More recently, we characterized CDK9 as a DDR player by modulating BRCA1 recruitment to DNA damaged sites and consequently the HR pathway. We also showed that cells lacking CDK9 failure to form RAD51, but not 53BP1, ionizing radiation-induced foci (IRIF) and are characterized by an increased sensitivity to DSBs

Methods and Results: Here we show that the CDK9-BRCA1 interaction occurs through BRCA1 N-terminal region in a BRCA1-BARD1 heterodimer formation-dependent manner. We also identified a CDK9 mutant (E369A) that abrogates the CDK9-BRCA1 interaction (through BRCA1 C-terminal region). Moreover, we characterized CHK2 as a novel CDK9-interaction partner, through ectopic GST-pulldown and constitutive co-immunoprecipitation assays. Further, cells lacking CDK9 are deficient in promoting G2/M arrest after IR-induced DNA damage. We also identified new CDK9 interactions in a DDR-related context using a TAP-MS approach.

Conclusion: Collectively, our data suggest a role for CDK9 in the DDR through DNA damage repair and cell cycle checkpoint regulation

#1741

Identification of microsatellite instability in hematologic malignant cell lines.

Priyanjali Bhattacharya,1 Naveed Anjum Chikan,2 Arati Sharma,3 Dhimant Desai,3 Charyguly Annageldiyev,3 Pinaki Patel,4 Trupti N. Patel1. 1 _VIT Vellore, Vellore, India;_ 2 _Daskdan Biotech Solutions Ltd., Kashmir, India;_ 3 _Penn State Hershey Cancer Institute, Hershey, PA;_ 4 _Beth Israel Deaconess Medical Centre, Boston, MA_.

Defective DNA repair machinery which is a consequence of chromosomal or molecular alterations is associated with variety of diseases including solid tumors. Cytogenetic analysis is an important tool in diagnosis and treatment of hematologic malignancies. Our aim is to understand the role of microsatellite instability in initiation and progression of blood cancers. Here we hypothesize that leukemogenic cells may carry defective repair mechanisms that contribute to other genomic instabilities. In order to explore the possible role of microsatellite instability (MSI) in leukemia and lymphoma, eight different DNA repair genes involved in mismatch repair (MMR) and direct reversal of damage (DRD) were analyzed in K562 (CML), Molt-4 (T-ALL) and Daudi (Burkitt's lymphoma) cell lines. Fragment analysis was carried out using novel primers considering repeat motifs enriched with (A)n, (CA)n and (AT)n for instability. Almost all the cell lines showed moderate to high level of microsatellite instability and exhibited a shift in selected repeat regions indicating possible loss (66.7%) (MSH2, MSH4, MSH6, MLH1, MLH3, PMS1) or gain (25%) (MSH3, MGMT) of nucleotide repeats as compared to normal DNA. The K562, Philadelphia negative CML cell line appeared to be microsatellite stable (MSS) for genes MSH2 and MLH1. Treatment induced microsatellite instability is known in leukemic patient cells as also seen in Molt-4 cells. Our present and previous observations suggest that MSI is a non-random event in leukemia/lymphoma which needs confirmatory future studies in newly diagnosed untreated patient samples. Considering the fact that both MSI and MSS represents two distinct molecular pathways of carcinogenesis, assessment of MSI, if added as distinct diagnostic criteria, can possibly refine early detection and help in identifying novel targets in management of hematologic malignancies.

#1742

Modulation of genotoxic DNA damage by the ATPase domain of type II topoisomerases.

Amanda M. Johnson,1 Lokha Ranjani A. Boopathy,2 Raveena Gupta,1 Hannah N. Miles,2 Matthew Gilbertson,2 Karin C. Nitiss,2 John L. Nitiss2. 1 _Univ. of Illinois College of Medicine, Rockford, IL;_ 2 _Univ. of Illinois College of Pharmacy, Rockford, IL_.

Type II topoisomerases play key roles in transcription, replication and chromosome segregation. These enzymes modulate DNA structure by generating transient DNA breaks where the protein is covalently bound to DNA. Anti-cancer drugs targeting topoisomerase II (Top2) inhibit the religation of enzyme-induced breaks, and exert their anti-cancer effects through the generation of enzyme mediated DNA damage. In addition to small molecule inhibitors, it has been suggested that aberrant DNA structures or DNA lesions may also lead to Top2/DNA lesions. A mammalian Top2 isoform, Top2β has been suggested to play a key role in hormone-mediated transcription, and in carrying out this function, generates potentially oncogenic DNA damage. We have assessed the generation of potentially oncogenic DNA damage by Top2β in model organisms, and using the purified protein. We found that ectopic expression of Top2β is poorly tolerated in repair deficient yeast strains, while expression of the mammalian Top2α isoform lacks this property. We measured the induction of homologous recombination by Top2β in yeast and found that ectopic expression of the enzyme led to elevated levels of recombination. Recently, a de novo mutation in Top2β was reported in a patient with severe developmental delay resulting from an His63Tyr substitution in Top2β 1. We found that Top2β (His63Tyr) could not complement a defect in yeast Top2, but encoded an enzyme that was catalytically active, and induced elevated levels of recombination. Because of the inferred location of His63 in the ATPase domain of Top2β, and proximity to the ATP binding site of the enzyme, we hypothesized that the mutant protein was partially defective in ATP regulated DNA cleavage. To further explore how ATP regulates Top2β cleavage, we examined the effects of expressing a Top2β variant completely lacking the N-terminal ATPase domain of Top2β. While this mutant did not complement yeast top2 mutants, we found that it s expression induced 10-20 fold higher levels of recombination than cells expressing wild type Top2β. This result indicates that an ATP deficient variant of Top2β induces very high levels of DNA damage. We suggest that regulation of the ATPase activity of Top2β may be critical for maintaining genome stability, and may be responsible for induction of DNA damage during hormone-mediated transcription.

1. C.W. Lam et al., Global developmental delay and intellectual disability associated with a de novo TOP2B mutation. Clinica Chimica Acta 469:63-68, 2017.

#1743

Evaluating the effect of USP22 depletion on chromosome instability in colorectal cancer.

Lucile M. Jeusset, Kirk J. McManus. _University of Manitoba, Winnipeg, Manitoba, Canada_.

Chromosome instability (CIN) is an aberrant phenotype defined as an increase in the rate at which whole chromosomes or large chromosomal fragments are gained or lost, and is observed in virtually all cancer types. Although CIN is associated with aggressive tumors, multi-drug resistance and poor patient prognosis, the genetic defects underlying CIN remain poorly understood. Preliminary data from our laboratory suggest that diminished USP22 (Ubiquitin-Specific Peptidase 22) expression induces CIN. Importantly, USP22 deletions and reduced USP22 expression are frequently observed in multiple cancer types, including colorectal cancer, suggesting diminished USP22 expression/function may be a pathogenic event underlying oncogenesis. Therefore, the present study seeks to evaluate the impact diminished USP22 expression has on CIN by determining whether karyotypically stable colorectal cancer (HCT116) and immortalized fibroblast (hTERT) cell lines can be converted into karyotypically unstable lines following the reduction of USP22 expression. Diminished USP22 expression was achieved by siRNA-based silencing or CRISPR-Cas9-mediated gene knockout (KO) and quantitative image-based single-cell analyses were employed to monitor changes in surrogate markers for CIN, including nuclear areas and chromosome numbers. USP22 silencing induced significant changes in nuclear area as well as increased frequencies of chromosomes gains and losses, which suggest USP22 is a CIN gene. Next, to assess the long-term impact diminished USP22 expression has on CIN, two HCT116 USP22-KO clones were monitored for 10 weeks and revealed dynamic changes in nuclear areas and chromosome numbers over time relative to controls. Collectively, these data identify USP22 as a novel CIN gene and indicate that USP22 deletions in cancer may have long-term impact on CIN and may thereby promote oncogenesis. Further analysis is ongoing to investigate the mechanism through which USP22 depletion induces CIN, and in particular its effect on H2B monoubiquitination. Characterizing the effect diminished USP22 expression has on CIN is the first step towards the development of therapeutic strategies targeting USP22-deficient cancers.

#1744

SAMHD1 expression and impact on clinical outcome in diffuse large B-cell lymphoma: a potential therapeutic target.

Waaqo Daddacha. _Emory, Tucker, GA_.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma. While standard treatment with rituximab, cyclophosphamide, doxorubicin, vincris- tine, and prednisone (R-CHOP) can cure DLBCL, failed patients have poor outcome. Thus, a search for stand-alone or adjuvant therapy to improve patient's outcome is important. Sam and HD domain-containing protein 1 (SAMHD1) is a well-established deoxyribonucleotide triphosphate (dNTP) triphosphohydrolase. Its dNTPase activity has been associated with cellular defence mechanism against infection by depleting cellular dNTP pool bellow level required for viral replication. SAMHD1 mutations have been associated with Aicardi Goutières (AGS) and cancers such as chronic lymphocytic leukemia (CLL), demonstrating its relevance beyond viral infection. In addition, it has been shown that SAMHD1 expression in Recently, our lab has defined a novel role for SAMHD1 in DNA end resection to facilitate DNA double strand break (DSB) repair by homologous recombination (HR). SAMHD1 expression and its diagnostic or therapeutic relevance in DLBCL is not known. Thus, we analyzed SAMHD1 expression in patients with DLBCL and determined its correlation with clinical outcome. In addition, we studied the SAMHD1 expression in germinal center B cell (GCB) and activated B cell (ABC) DLBCL cells lines. Also, we examined the possibility of depleting SAMHD1 using a virus like particles (VLPs) containing viral protein x (Vpx) in DLBCL cell lines. Finally, we determined the effect on cells sensitivity to standard DLBCL therapy, R-CHOP, and other DNA damage inducing agents. Low SAMHD1 expression correlates with improved overall survival. SAMHD1 was downregulated in ABC as compared to GCB DLBCL cell lines and SAMHD1 depletion sensitizes DLBCL cell lines to R-CHOP and other DNA damage inducing agents. These findings suggest that SAMHD1 is a potential therapeutic target to improve the DLBCL patient's clinical outcome.

#1745

Ku70 suppresses alternative end-joining in G1-arrested progenitor B cells.

Richard L. Frock,1 Vipul Kumar,2 Zhouyi Liang,2 Jeff Zurita,2 Zhou Du,2 Sherry G. Lin,2 Cristian Boboila,2 Frederick W. Alt2. 1 _Stanford University, Stanford, CA;_ 2 _Boston Children's Hospital, Boston, MA_.

Classical non-homologous end-joining (C-NHEJ) repairs DNA double-stranded breaks (DSBs) throughout interphase but is thought to predominate in G1-phase when homologous recombination is unavailable. Complexes containing the Ku70/80 ("Ku") and XRCC4/Ligase4 core C-NHEJ factors are required, respectively, for sensing and joining DSBs. While such factors are exclusively required for joining RAG-initiated DSBs during V(D)J recombination in G1-phase lymphocyte progenitors, cycling cells deficient for core C-NHEJ factors join chromosomal DSBs by alternative end-joining (A-EJ) pathways. Restriction of V(D)J recombination to C-NHEJ has been attributed to RAG-mediated exclusion of A-EJ; however, it remains unclear whether A-EJ is similarly excluded from more general DSBs in G1. Here, we report that Ku actively and robustly suppresses A-EJ of both RAG- and Cas9-mediated DSBs in G1-arrested progenitor B cell lines. Thus, while Cas9/gRNA DSBs remain as free broken ends in Ligase4-deficient G1-arrested progenitor B cells, deletion of Ku70 in Ligase4-deficient cells restores DSB re-joining and translocation to levels observed in Ku70-deficient counterparts. Correspondingly, while V(D)J recombination is abrogated in Ligase4-deficient lines, V(D)J-like joining occurs in Ku70-deficient and Ku70/Ligase4-double-deficient lines through a translocation-based A-EJ mechanism. We conclude that in G1, Ligase4-deficient progenitor B cells are functionally end-joining deficient due to a complete Ku-dependent block in A-EJ. Thus, the differential impacts of Ku deficiency versus XRCC4/Ligase4 deficiency on V(D)J recombination, severity of neuronal apoptosis, and embryonic development, including ability of Ku-deficiency to rescue Ligase4-deficient embryonic lethality, may be explained by Ku-mediated inhibition of A-EJ in the G1 cell cycle phase.

#1746

The deubiquitylating enzyme USP15 regulates cancer cell response to PARP inhibitors through participating in homologous recombination repair (HR).

Yihan Peng,1 Qingchao Liao,2 Wei Tan,1 Changmin Peng,1 Huadong Pei1. 1 _George Washington University, Washington, DC;_ 2 _Tianjin University of Science and Technology, Tianjin, China_.

Poly-(ADP-ribose) polymerase (PARP) inhibitors were selectively active in breast and ovarian cancer patients carrying BRCA1/2 mutations who exhibited impairment in homologous recombination(HR) repair. However, PARP inhibitors also show significant clinical benefit in patients without BRCA deficiencies, presumably result from other molecular defects in the BRCA dependent pathway. Therefore, identifying other BRCA-like genomic signatures could expand clinical benefit of PARP inhibitors. Here, we found that the deubiquitylating enzyme USP15 affected cancer cell response to PARP inhibitor through regulating HR. USP15 is recruited to DNA double-strand breaks (DSBs) by MDC1 in an ATM dependent pathway. Subsequently, USP15 deubiquitinates BARD1 BRCT domain, and promotes BRCA1/BARD1 retention at DSBs through facilitating the interaction between BARD1 and heterochromatin reader HP1γ, which in turn promotes HR. USP15 germ line knockout mice exhibited genomic instability in vivo. Furthermore, USP15 loss in breast cancers and pancreatic cancers, or cancer-associated USP15 mutation with decreased USP15-BARD1 interaction, also increased cancer cell response to PARP inhibitor. Thus, our results identify a novel regulator in HR, which is a new potential biomarker for therapeutic treatment using PARP inhibitors in cancers.

#1747

Both somatic and germline BRCA mutations show high degree of bi-allelic loss in breast and ovarian cancers.

Zhongwu Lai,1 Darren Hodgson,1 James Sun,2 Adrienne Johnson,2 Carl Barrett,1 Jonathan Dry1. 1 _AstraZeneca, Waltham, MA;_ 2 _Foundation Medicine, Cambridge, MA_.

PARP inhibitors (PARPi) are promising novel therapies for tumors harboring defects in homologous recombination (HR) DNA repair. Loss of function (LoF) mutations in the tumor suppressors BRCA1 and BRCA2, two critical components in HR pathway, are the most common cause of HR deficiency in tumors. Complete loss of BRCA1/2 function requires bi-allelic loss, and it is hypothesized to be needed for PARPi sensitivity. However, the degrees of bi-allelic loss in breast and ovarian tumors are still lacking consensus, especially when both LoF events are somatic. Recent publications shown a high percentage of germline BRCA2 lacking LOH, including TCGA's breast and ovarian cohorts.

To better understand the loss of wild type BRCA genes, we reanalyzed raw data from TCGA using a novel pipeline to identify both germline and somatic BRCA mutations in breast and ovarian. We then applied mathematical models to determine LOH or bi-allelic loss of those mutations. Our analysis shows a higher degree of bi-allelic loss of BRCA mutations than previously reported, especially germline BRCA2 in breast cancer, which shows nearly 80%. Furthermore, somatic BRCA mutations, which account for about 1/3 of all BRCA mutations, showed a similar degree of bi-allelic loss as germline BRCA mutations. In addition, somatic and germline BRCA were shown to have comparable HRD scores, a measure of tumor genomic instability. Germline BRCA patients are typically diagnosed at younger ages in breast and ovarian cancer, whereas somatic show no difference to non-BRCA patients. In ovarian cancer, patients with somatic or germline BRCA had similar progression-free and overall survival with comparable treatment journeys, suggesting a phenotypic equivalence. Interestingly, BRCA2 tumors showed lower HRD scores than BRCA1 tumors in both breast and ovarian. In breast cancer BRCA1 mutation is significantly enriched in TNBC, but the degree of bi-allelic loss did not show any significant difference between ER+ and TNBC subtypes.

To validate our findings, we examined a much larger cohort of breast (n > 5,000) and ovarian (n > 2,500) cancers sequenced by Foundation Medicine, with germline/somatic and LOH status determined computationally using validated methodology. We found similar prevalence of somatic BRCA mutations and the degree of bi-allelic loss and HRD scores in both germline and somatic BRCA mutations. In addition, potential reactivating reversion mutations were discovered in both somatic and germline mutated BRCA in both breast and ovarian cancers, suggesting they are driver mutations.

In summary, large scale analysis of BRCA mutations in breast and ovarian cancers suggest that about 1/3 of BRCA mutations are somatic, and both germline and somatic BRCA mutations show high degree of bi-allelic loss and similar genomic instability and may benefit from PARPi therapies.

#1748

**RAD51 has an anti-recombinase activity that is countered by BRCA2 -** In vitro **.**

Charles Wang,1 Judit Jimenez-Sainz,2 Ryan B. Jensen,2 Alexander A. Mazin3. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Yale University School of Medicine, New Haven, CT;_ 3 _Drexel University College of Medicine, philadelphia, PA_.

Homologous Recombination (HR) is a high-fidelity process with a range of biologic functions from generation of genetic diversity to repair of DNA double-strand breaks (DSBs). In mammalian cells, BRCA2 facilitates the polymerization of RAD51 onto ssDNA to form a presynaptic nucleoprotein filament. This filament can then strand invade a homologous dsDNA to form the displacement loop (D-loop) structure leading to the eventual DSB repair. This pro-recombinatory feature of RAD51-BRCA2 is well documented. Here, we present biochemical evidence that RAD51 can cause D-loop disassembly. Furthermore, we show that this RAD51 anti-recombinatory activity is countered by BRCA2. These results demonstrate that BRCA2 may have a previously unexpected role: regulation of HR at a post-synaptic stage by modulating RAD51-mediated D-loop dissociation. Our results provide a biochemical mechanistic understanding of homeostasis between RAD51 and BRCA2, which has been suggested to be an important factor of HR based on previously published cellular data.

#1749

Investigating the role of RAD51AP1 in homologous recombination DNA repair.

Elena Pires,1 Neelam Sharma,1 Claudia Wiese,1 Patrick Sung,2 Weixing Zhao2. 1 _Colorado State Univ., Fort Collins, CO;_ 2 _Yale University, New Haven, CT_.

DNA double-strand breaks (DSBs) are considered the most detrimental DNA lesions induced by ionizing radiation (IR). Cells respond to DSBs by activating DNA damage response pathways that include cell cycle arrest and DNA damage repair. Flawless DNA repair through homologous recombination (HR) is essential for correcting DSBs, maintaining genome integrity, and preventing cancer. A pivotal step in HR involves the joining of homologous DNA strands via formation of a presynaptic RAD51 nucleoprotein filament and subsequent displacement-loop. During this step of the HR reaction, RAD51 interacts with RAD51-Associated Protein 1 (RAD51AP1). Our previous work has shown that RAD51AP1 is critical for proper HR and for enhancing the activity of RAD51. Wild-type levels of RAD51AP1 are also essential for protecting human cells from the cytotoxic effects of IR exposure as well as for maintaining DNA replication fork progression and counteracting replication stress. Likely related to its ability to mitigate increased levels of DNA replication stress, RAD51AP1 is found to be up-regulated in many types of cancers, including BRCA1-deficient breast cancers. Significantly, RAD51AP1 is mis-regulated in a variety of tumor types, and Kaplan-Meier plots predict poorer overall survival of breast, ovarian, and lung cancer patients with altered RAD51AP1. Nonetheless, how exactly RAD51AP1 functions during the HR reaction is still incompletely understood. Our main objective is to identify and characterize key attributes of RAD51AP1 in the HR reaction. Here, we have characterized further the DNA-binding properties of recombinant human RAD51AP1. Using the immobilized template assay, we find that RAD51AP1 avidly associates with both naked and chromatinized double-stranded (ds)DNA. Deletional and mutational analyses were used to further define the DNA- and chromatin-binding regions in RAD51AP1 via division of the protein into three fragments: F1 (residues 1-94), F2 (residues 95-187), and F3 (residues 188-335). We show that the N-terminal 94 residues of RAD51AP1, which are able to associate with naked dsDNA avidly, are devoid of binding to a chromatinized dsDNA substrate. In contrast, the C-terminal 148 residues of RAD51AP1 show affinity for both naked and chromatinized dsDNA. Two post-translational modification sites that lie within its C-terminal DNA-binding region were also evaluated and showed decreased affinity to chromatinized dsDNA. Based on these findings and other results, we propose a model in which RAD51AP1 guides homology search and hetero-duplex formation after presynaptic filament formation in the HR reaction. By understanding the biology of this important HR protein, we expect to have direct clinical relevance with knowledge that may lead to the advancement and optimization of targeted cancer therapies.

#1750

USP22 directly interacts with PALB2 and is required for efficient homologous recombination of DNA double-strand breaks.

Isaac K. Nardi, Jeremy Stark, Adrien Larsen, Dan Raz. _City of Hope National Medical Center, Duarte, CA_.

DNA damage by environmental stressors and agents is a huge problem for cells and must be repaired quickly for the cell to maintain its integrity. Cells employ several repair pathways and mechanisms to rectify damaged DNA. Homologous Recombination (HR) repair comprises a series of pathways in a cell that can function to repair DNA double-stranded breaks (DSBs) through template-dependent mechanisms to preserve genomic integrity. Failure to repair DSBs can result in chromosomal loss, carcinogenesis, and apoptosis. Deubiquitylases (DUBs) are a family of proteases that cleave ubiquitin from proteins and have been implicated in several biological processes including tumor development, cell growth, cell cycle regulation, and signal transduction. There is a growing catalog of studies showing the vital role of DUB's in the DNA Damage Response (DDR). But, given that there are over 100 DUBs in the human genome their functions in DDR have still not been fully fleshed out. Along with its role in antibody class switch repair, USP22 is a DUB that has been implicated as a putative oncogene with over-expression observed in various cancer types and is associated with a poor patient prognosis. Furthermore, over-expression of USP22 leads to cisplatin resistance in lung cancer cells strongly suggesting it plays in a vital role in DDR and chemoresistance in recurrent cancers. In this study we show USP22 is necessary for HR, localizes to sites of DNA damage, and is necessary for recruitment of the PALB2-BRCA2-Rad51 complex through stabilizing PALB2 protein levels. Lastly, we show USP22 directly binds to the WD40 domain of PALB2 and this binding stimulates USP22 catalytic activity. Taken together, these findings show a new and novel role of USP22 in the HR pathway.

#1751

The role of SIRT2 in BRCA1 regulation.

Elizabeth Minten,1 Hui Zhang,1 Chunyang Li,1 PamelaSara Head,2 David S. Yu1. 1 _Emory University, Atlanta, GA;_ 2 _NIH, MD_.

The most deleterious type of DNA damage a cell can sustain is the DNA double-stranded break (DSB). One of the major pathways responsible for the repair of DNA DSB breaks is the homologous recombination (HR) pathway, an error-free mechanism of repair that occurs primarily in the S and G2 phases of the cell cycle when a sister chromatid is available to be used as a repair template. Impairment of HR can result in genomic instability, which can increase the risk of tumorigenesis. However, many of the mechanisms by which defects in HR lead to an increased risk for developing cancer are unknown. Mutations or misregulation of the Breast Cancer 1 (BRCA1) protein, a major player in HR and an established tumor suppressor, have been linked to an increased lifetime risk for the development of cancer in both men and women, including colon, breast, ovarian, and pancreatic cancer. Yet, how BRCA1 is regulated in HR is not well understood and thus highlights a major a gap in our understanding of how deficiencies in HR contribute to the development of cancer. Our lab has discovered that the histone deacetylase and putative human tumor suppressor, SIRT2, has a crucial role in the repair of DNA DSBs. We have found that SIRT2 interacts with and deacetylates BRCA1, both in vitro and in cells, and that depletion of SIRT2 decreases BRCA1 protein levels, thus impairing HR. Our results show SIRT2 is a novel regulator of BRCA1 and is critical for the efficient repair of DNA DSBs through HR. These findings will provide invaluable insight into how to exploit the interplay between SIRT2 and BRCA1 as a novel therapeutic approach for the prevention and treatment of cancer.

#1752

BRCA-mediated tumorigenesis is origin and cell-type dependent.

Philip Jonsson, Michael L. Cheng, Chaitanya Bandlamudi, Preethi Srinivasan, Shweta S. Chavan, Noah D. Friedman, Ezra Y. Rosen, Allison L. Richards, Nancy Bouvier, S. Duygu Selcuklu, Craig Bielski, Wassim Abida, Ahmet Zehir, Nikolaus Schultz, Mark T. Donoghue, Jose Baselga, Kenneth Offit, Marc Ladanyi, Eileen M. O'Reilly, Howard I. Scher, Zsofia K. Stadler, Mark E. Robson, David M. Hyman, Michael F. Berger, David B. Solit, Barry S. Taylor. _Memorial Sloan Kettering Cancer Center, New York, NY_.

BRCA1 and BRCA2 mutations predispose to select cancers, yet the interplay between germline and somatic BRCA alterations in driving tumorigenesis and conferring drug sensitivity remain poorly understood. To determine which tumors are dependent on mutant BRCA, we integrated the prospective clinical sequencing of germline blood and matched tumor specimens from 17,152 advanced cancer patients with zygosity analysis, broader somatic molecular features, and treatment outcomes. Tumor lineage dictated BRCA dependence in cancers of both the 2.7% of carriers with germline pathogenic variants and the 1.8% of patients with somatic loss-of-function mutations in BRCA1 and BRCA2 across 38 affected cancer types. The rate of biallelic inactivation of mutant BRCA1/2 varied by mutational origin and tumor lineage. Consequently, BRCA-mediated phenotypes such as homologous recombination deficiency (HRD) were associated with BRCA1/2 mutations in a cell type- and zygosity-dependent manner. Phenotypic penetrance was greatest in tumors of high-risk cancer types and in tumors with biallelic inactivation of mutant BRCA, independent of its germline or somatic origin. Conversely, heterozygous BRCA mutations in other cancer types conferred no HRD phenotype. These lineage-specific differences among hallmarks of BRCA dependence also predicted differential response to PARP-inhibitor therapy. Collectively, only BRCA mutations in tumors of high BRCA penetrance had a strong selective pressure for somatic biallelic inactivation, conferred dose-dependent somatic phenotypic consequences, and PARP inhibitor sensitivity. In contrast, BRCA1/2-mutant patients with cancers not traditionally associated with BRCA susceptibility generally had tumorigenesis independent of mutant BRCA. Overall, mutant BRCA was a founding pathogenic event on which some tumors depended while in others it was likely a dispensable and biologically neutral passenger mutation unrelated to tumorigenesis. This difference was conditioned by lineage, mutational origin, and zygosity, an understanding of which requires integrated germline and somatic molecular characterization in cancer patients with implications for screening, disease pathogenesis, clinical trial design, and therapy.

#1753

Genomic and transcriptomic characterization of congenital trisomy reveal possible role for RB1 and MET.

Maryam A. Bainazar, Sophia E. Maharry, Christopher J. Walker, Luke K. Genutis, Albert de la Chapelle, Ann-Kathrin Eisfeld. _Ohio State University Comprehensive Cancer Center, Columbus, OH_.

Introduction: The vast majority of solid tumors and half of leukemias and lymphomas possess an abnormal number of chromosomes. Individuals with congenital trisomy 21 have an increased risk of developing leukemia and respond differently to treatment when compared to leukemic individuals without congenital aneuploidies. Curiously, the chromosomes that most frequently undergo non-disjunction in malignancy are similar to the chromosomes that occur as trisomies in utero. We aimed to characterize the molecular landscapes of individuals with congenital trisomies to elucidate what changes may lead to an increased risk of cancer development in these individuals.

Methods: We extracted DNA and RNA from fibroblasts of 14 individuals with congenital trisomies, including trisomy 8, 9, 13, 18 and 21 (obtained from Coriell Biorepository). DNA and RNA sequencing were performed with AmpliSeq for Illumina Comprehensive Panel v3. Variants were detected in the DNA using VarScan and annotated with SnpEff, while Kallisto was used for mRNA expression analysis. We applied mRNA context mapping methodology developed by the Computational Cancer Analysis Lab (University of California, San Diego). We used quantitative PCR and Western blotting to validate RB1 and MET expression.

Results: Identification of transcripts highly expressed in multiple trisomies was performed. AXL was the only transcript that was highly expressed in all five trisomy groups, while ACBD5 and CCDC6 were each highly expressed in four. Interestingly, the number of unique highly expressed transcripts differed drastically by trisomy, ranging from n=0 for trisomy 9 to n= 111 for trisomy 13. We then generated a context map of the samples and observed that they cluster together by trisomy, with the exception of two samples. RB1 and MET were up-regulated in eleven samples, and down-regulated in the remaining three samples. We were able to validate these findings at the mRNA and protein level. Finally, we are in the process of identifying rare genetic variants that are unique to or enriched in this population.

Conclusion: Our data indicate that several genes with well-described roles in cancer might also be differentially expressed in individuals with trisomies. RB1 is a tumor suppressor and previous studies have shown that pRB loss leads to centromere dysfunction and chromosomal instability, which could imply dysregulated RB1 plays a role in trisomy acquisition. MET is a proto-oncogene and is implicated as the major driver of oncogenesis in papillary renal carcinomas with chromosomal gain. Further studies that compare congenital trisomy with malignancy-associated trisomy may improve our understanding of non-disjunction and mechanisms of oncogenesis that occur in congenital trisomy patients.

#1754

Monoubiquitinated γ-H2AX - a more specific biomarker of DNA double-strand breaks.

Michal W. Luczak, Anatoly Zhitkovich. _Brown Univ., Providence, RI_.

DNA double-strand brakes (DSBs) are a highly toxic form of DNA damage originating from many endogenous and exogenous sources. Cells employ specialized mechanisms for rapid detection and repair of these lesions. Histone H2AX phosphorylated at Ser139 (known as γ-H2AX) acts as a central platform for recruitment of other components of DSB repair. γ-H2AX assessment is currently the most widely used approach for quantitation of DSBs. However, γ-H2AX can be also induced by certain nongenotoxic stressors and during apoptosis, which can lead to false positive observations, especially in prolonged exposures. γ-H2AX undergoes RNF168-mediated K13/K15 monoubiquitination which is necessary for the recruitment of the downstream DSB repair factors. This ubiquitination event is rarely analyzed despite its functional importance. We examined formation and detection of γ-H2AX and its mono- (ub1) and diubiquitinated (ub2) forms in several human cell lines in response to mechanistically distinct inducers of DSBs and false-positive DSBs stressors. We found that γ-H2AX-ub forms were poorly detected using some common blotting procedures and antibodies. Under optimized conditions, γ-H2AX-ub1 was the predominant form accounting up to 80-90% of total γ-H2AX in primary human cells and its formation was strictly associated with the presence of DSBs. γ-H2AX and γ-H2AX-ub1 showed similar dose dependence and disappearance kinetics whereas H2AX-ub2 form was a more stable and dose-independent product. Unlike γ-H2AX, its ubiquitinated forms were not observed in the absence of DSBs (heat-shock or during apoptotic DNA cleavage). Apoptotic cells showed cleavage of the γ-H2AX-targeting E3 ubiquitin ligase RNF168. Our findings demonstrate that the ub1 form is a major fraction in the overall formation of γ-H2AX and its quantitation offers advantage in specificity for nonapoptotic DSBs.

#1755

Chromatin trapping is a key factor for anticancer cytotoxicity of DNA-binding small molecule drugs.

Elimelech Nesher,1 Alfiya Safina,2 Ieman Aljahdali,2 Scott Portwood,2 Eunice Wang,2 Jianmin Wang,2 Igor Koman,1 Katerina Gurova2. 1 _Ariel University, Ariel, Israel;_ 2 _Roswell Park Comprehensive Cancer Center, Buffalo, NY_.

DNA-targeting small molecules have been widely used for cancer treatment due to their ability to induce DNA damage. The DNA-damaging effect of small molecules depends on chromatin organization, however, the exact mechanism by which small molecules affect the chromatin, as well as the role of chromatin alterations in their biological activity is poorly understood. We previously identified the curaxin CBL0137 which showed a broad anti-cancer activity. CBL0137 is a small molecule which binds DNA but does not cause DNA damage. Rather, the curaxin alters DNA/histone interactions and causes nucleosome disassembly in vitro and in cells, leading to the phenomenon we named "chromatin damage." We have previously shown, that disassembled nucleosomes are bound by the histone chaperone FACT (FAcilitates Chromatin Transcription. We named this massive binding of FACT to different components of disassembled chromatin in curaxin-treated cells "c-trapping". This finding led us to believe that other small DNA-binding molecules may also have the ability to induce c-trapping and chromatin damage. We selected a set of DNA-targeting agents, representing different modes of DNA binding and inhibition of topoisomerases: inhibitors of topoisomerase cleavage activity, inhibitors of topoisomerases re-ligation, compounds that do not bind DNA directly while form a cleavable complex with DNA and topoisomerases I or II, as well as topoisomerases II catalytic inhibitors that do not bind DNA. We investigated this set of compounds examining their cytotoxicity, and their ability to induce c-trapping, which we detected with western blotting, fluorescent imaging and immunofluorescent staining. We examined their effect on nucleosome stability and chromatin destabilization via micrococcal nuclease assays and recombinant mononucleosomes. DNA - damaging activity of tested compounds was also evaluated. We found that only compounds that directly bind DNA are able to induce c-trapping, which in turn correlates with nucleosome destabilization of tested compounds and their cytotoxicity. We showed that this effect was not dependent on DNA damage. Additionally, we showed that FACT can bind DNA at different stages of nucleosome disassembly, including the linker histone removal followed by core histone's eviction, and negative supercoiling accumulation. This work suggests that cytotoxicity of DNA binding molecules is based not on their direct DNA-damage but on the ability of these compounds to induce the chromatin disruption.

#1756

Electronic cigarettes induce DNA damage in the oral epithelium of users.

Vengatesh Ganapathy, Theodore Wagener, Lurdes Queimado. _University of Oklahoma Health Science Center, Oklahoma City, OK_.

Background: The use of electronic cigarettes (e-cigs) has skyrocketed among adolescents and young adults. E-cigs are promoted as safer than combustible cigarettes but their long term effects on human health are still unknown. E-cig aerosols contain significantly lower number of chemicals than tobacco smoke, however they also contain known carcinogens, reactive oxygen species (ROS), and unique chemicals poorly studied. In vitro and animal studies revealed that e-cig aerosols increases DNA damage and reduces DNA repair. There have been no studies assessing e-cig-induced DNA damage in human subjects. This reflects, at least in part, technical limitations that make it quite challenging to measure the in vivo steady-state levels of DNA damage, particularly for relatively low levels of genotoxic exposure. Our lab has previously developed and validated a novel quantitative primer anchored DNA damage detection assay (q-PADDA) to quantify in vivo DNA damage. We have also reported that, in an in vitro setting, q-PADDA has high sensitivity to detect e-cig-induced DNA damage. Here, we take advantage of this assay to start assessing the potential long-term impact of e-cig use on human health.

Aim: To determine whether e-cig users have higher levels of DNA damage on oral epithelial cells than non-users.

Methods: Ethics Committee approval was obtained. Participants were recruited through internet ads, leaflets, flyers and word-of-mouth. After written informed consent, a total of 35 individuals were included in this study: 20 exclusive e-cigarette users and 15 individuals reporting no e-cig or tobacco use. Self-report of tobacco use was biochemically confirmed by salivary cotinine and by measuring exhaled carbon monoxide (CO). Oral mucosa samples were collected by cytobrush and used for DNA extraction and damage quantification. DNA damage was quantified by q-PADDA within p53, the most frequently mutated gene in human cancer. Plasma nicotine levels were analyzed by liquid chromatography-mass spectrometry. Data was analyzed by Student's t test.

Results: The study including participants using high and low voltage e-cig devices. E-cig users (33 ± 8) and non-users (33 ± 7) had similar age distribution. We observed that oral epithelial cells collected from e-cig users have significantly higher levels of DNA damage in both transcribed and non-transcribed DNA strands of the p53 gene when compared to never users.

Conclusion: Our work is the first study to measure DNA damage in e-cig users. Our study shows for the first time that e-cig users have significant levels of DNA damage in their oral mucosa epithelium. These data has major clinical implications and suggest that e-cig users might have an increased cancer risk. Further studies are urgently needed to expand these kind of studies and to characterize the molecular mechanisms underlying e-cig induced DNA damage which may leads to cancer.

#1757

The effect of Zc3h8 expression levels on sensitivity to DNA damage and repair in mouse mammary cells.

Spencer Li, John A. Schmidt, Janice E. Knepper. _Villanova Univ., Villanova, PA_.

Localized in PML nuclear bodies, the fetal liver zinc finger protein (Fliz), or ZC3H8, has been shown to increase cell proliferation, migration, and tumorigenesis in mouse mammary cell lines. While the exact function of ZC3H8 is unclear, PML nuclear bodies are critical centers for genome maintenance. Previous studies have suggested that these nuclear bodies act as deposits of sequestered DNA damage response proteins including SUMO-1, TP53, and BRCA-1. In addition to their localization to PML nuclear bodies, the phosphorylation of ZC3H8 by CK2 leads to dispersion of PML nuclear foci. For these reasons, we speculated that increased expression of Zc3h8, leading to greater protein availability, is involved in the suppression of DNA repair. This in return would increase genome instability and cancer cell aggression. This study uses the single cell comet assay to measure DNA damage repair following treatment with the topoisomerase inhibitor and chemotherapy drug etoposide. Two mouse mammary tumor cells lines, cV1A 01-51 and cV1A 03-31 were transfected to drive an shRNA targeting ZC3H8 (Flizout). In addition, the cV1A 03-31 Flizout line was rescued with a Zc3h8 construct with altered base coding to make transcribed products immune to shRNA targeting (SynFliz). We have also developed a constitutively charged cV1A 03-31 T32E mutant and a non-phosphorylatable T32A mutant. The nontumorigenic Comma1-D cell line was transfected with a vector to increase Zc3h8 expression. Ability to repair after etoposide treatment in these mutants was compared to controls carrying endogenous levels of Zc3h8. Etoposide leads to double strand breaks in the DNA during replication, and all cell lines were sensitive to etoposide and able to undergo repair. For the cV1A 01-51 lines, the control and Flizout mutants had a nonsignificant difference in post-repair comet length (p = 0.06) but a significant difference in tail moment (p = 0.003). Between the control and the mutants, the difference in overall comet length was 4.7%, and the difference in tail moment was 15.8%, with cells expressing higher levels of Zc3h8 being less adept in carrying out repair of double strand DNA breaks. This suggests an inverse relationship between Zc3h8 expression and DNA damage repair. This may indicate that evaluating Zc3h8 expression could be considered for determining the ability of tumor cells to respond to chemotherapy agents. Furthermore, altering its expression may be a novel approach for cancer treatment.

#1758

Splicing component ISY1 interacts with APE1 and regulates base excision repair.

Aruna Jaiswal,1 Elizabeth Williamson,1 Bhavita Patel,2 Gayathri Srinivasan,1 Kimi Kong,1 Carrie Lomelino,2 Satya Narayan,2 Robert Hromas1. 1 _UT Health Science Center San Antonio, San Antonio, TX;_ 2 _University of Florida, Gainesville, FL_.

Cellular genome is continuously challenged by endogenous and exogenous DNA damaging agents. In the genome, base modifications are the most common form of DNA damage. Base excision DNA repair (BER) is the most important pathway for the removal of oxidized or alkylated DNA. If these base modifications continue to persist, the replisome may stall at DNA lesions, causing the replication fork to collapse, leading to genetic instability. While the main components of the BER pathway are well defined, its regulatory mechanism remains poorly understood. We report here that apurinic/apyrimidinic endonuclease 1 (APE1), a multifunctional protein interacts with the pre-mRNA splicing factor ISY1. However, this interaction is independent of DNA or RNA based upon our pull-down experimental results. ISY1 expression is induced by oxidative or DNA alkylation damage, which would then provide an immediate up-regulation of APE1 activity in vivo and enhanced BER of oxidized bases. This was supported from our results of in vitro reconstituted experiment suggesting that ISY1 can stimulate 5'-3' endonuclease 1 activity of APE1 in both the short- and long-patch BER pathways and increase its binding to AP site DNA. The interaction between ISY1 and APE1 also establishes a connection between DNA damage repair and pre-mRNA splicing. Based upon these results, we propose that ISY1 is an important regulator of BER and enhances the ability of APE1 to efficiently recognize abasic sites in DNA. Role of ISY1 becomes important in aging tissues where decreased APE1 activity results in the accumulation of oxidative genomic damages. Therefore, ISY1 may work as an anti-aging factor. Induction of ISY1 in malignancies could constitute a mechanism to avoid apoptotic death after treatment with alkylating agents.

#1759

Studying T-cell force activation and DNA repair dynamics at the single-molecule level with optical tweezers and correlated fluorescence microscopy.

Ali Raja, Leif Anderson, Willem Peutz, Andrea Candelli, Gerrit Sitters. _LUMICKS, Boston, MA_.

DNA repair, the collection of highly regulated mechanisms by which a cell identifies and repairs DNA damage, remains one of the most essential processes of human life. Without DNA repair mechanisms cells lose the ability to transcribe important regions of their genome, resulting in harmful mutations, which could eventually jeopardize cellular wellbeing.

Sources of DNA damage include double-stranded breaks and DNA intra- and interstrand crosslinks, which can ultimately become malignant tumors, leading to cancer. To study DNA repair, single-molecule studies have proven to greatly enhance understanding at the molecular level. However, it is often challenging to obtain adequate sensitivity and resolution, as well as biological conditions mimicking in vivo environments. The C-Trap™ system allows for realtime visualization of the interaction between DNA and DNA repair proteins, under biologically relevant conditions with high spatial and temporal resolution.

The C-Trap™ is the only instrument that integrates optical tweezers, confocal/STED microscopy, and an advanced microfluidics system in a truly correlated manner. It enables live, simultaneous and correlative visualization and manipulation of molecular interactions with sub-picoNewton (pN) force resolution and a kilo-Hz (to mega-Hz) temporal resolution. Previously we have presented applications in: protein (un)folding and conformational changes; DNA-protein interactions and genome modifications; effects of mechanical stress on DNA/RNA structure; motility of cytoskeletal molecular motors; and protein droplet and aggregation dynamics.

By culturing or adhering cells to the surface of the flow chamber, single-molecule cell-based studies can also be performed with the C-Trap. Cell-surface bound mechano-receptors can be activated by forming tethers with an optically trapped particle and then creating a controlled tension on the molecule. Correlated fluorescent imaging can elucidate internal protein cascades and other similar reactions to the applied force.

Here, we present our experiments visualizing and quantifying the mechanisms of DNA repair and the force activation of T-cells using the C-Trap system. These experiments show that the technological advances in hybrid single-molecule methods can be turned into an easy-to-use and stable instrument that opens up new venues in many research areas.

#1760

Plasma vitamin D levels and DNA repair capacity in four molecular subtypes of women with breast cancer.

Carmen Ortiz,1 Jarline Encarnacion,1 Ralphdy Vergne,2 Luis Padilla,2 Jaime Matta1. 1 _Ponce Health Sciences University, Ponce, Puerto Rico;_ 2 _University of Puerto Rico at Ponce, Ponce, Puerto Rico_.

Background: Vitamin D regulates estrogen synthesis among other mechanisms involved in breast cancer (BC) carcinogenesis. An important gap, to our knowledge, is whether blood levels of vitamin D are associated with DNA repair capacity in women with BC. Previously, we reported that a low DNA repair capacity (DRC) is a risk factor for BC and that DRC levels are associated with estrogen receptor (ER) positivity in women with BC. Moreover, we also reported that there is an intrinsic variability in DRC levels among the four principal BC subtypes. Therefore, our aim is to study the link between DRC and plasma vitamin D levels in BC and among BC subtypes.

Methods: Treatment-naïve BC cases and controls were selected from our cohort of Hispanic women (n=1,187). DRC was assessed in lymphocytes through the host-cell reactivation assay. Vitamin D was measured using LC/MS/MS.

Results: Women with BC (n=91) had higher (+6.6 ng/ml) vitamin D than controls (n=22) (p=0.007). There were significant differences in vitamin D levels (p<0.05) between BC cases with high (≥3.8%) (n=17) and low (<3.8%) (n=74) DRC levels. Vitamin D and DRC were negatively correlated in BC cases (r=-0.2296, p=0.03). When women with BC and low DRC levels (n=56) were stratified into: luminal A (n=17), luminal B (n=11), HER2+ (n=10), and triple-negative (n=18), women with HER2+ and TNBC showed the highest levels of vitamin D (p<0.05). Women with ER- had higher levels (+7.6 ng/ml) of vitamin D than women with ER+ tumors (p=0.005). After finding that plasma vitamin D levels from women with BC and controls were significantly different when stratified by DRC levels and BC subtypes, ROC curves were constructed to examine whether these levels could allow us to discriminate between our four study groups. Significant differences (p=0.0126) were found for cases and controls with low DRC; AUC of 0.7178. When considering all women with BC independently of DRC levels, the AUC was 0.7353 (p=0.0300).

Conclusions: This study provides the first evidence of a link between vitamin D and DRC levels in women with BC. We also present data as to how vitamin D varies across four molecular subtypes of BC in Hispanic women.

Impact: Our results fills an important gap on the link between plasma vitamin D and DRC levels in women with BC.

Funding: This study was supported by grants #S06GM008239-20, 9SC1CA182846-04, and 5SC1CA157250-02, 5U54CA163071-04, 5R25GM096955-08.

#1761

Inhibition of PI3K/AKT/mTOR signaling by a dual PI3K/mTOR inhibitor (LY3023414) potentiates the antitumor efficacy of the Chk1 inhibitor prexasertib (LY2606368) in models of human high-grade serous ovarian cancer (HGSOC).

Jack Dempsey, Greg Donoho, Philip Iversen, Jennifer Stephens, Ann McNulty, Ricardo Martinez, Christoph Reinhard, Louis Stancato, Aimee Lin, Wenjuan Wu. _Eli Lilly and Company, Indianapolis, IN_.

HGSOC (characterized by defects in DNA damage repair and high levels of genomic instability and DNA replication stress) is the most lethal gynecological malignancy in the United States. The current treatment options for HGSOC are limited and new approaches are needed. Checkpoint kinase 1 (Chk1), a key protein kinase that regulates the cell cycle, DNA damage and replication stress response, has emerged as an attractive target for anti-cancer therapy. Prexasertib, an ATP-competitive inhibitor of Chk1, is being evaluated in a Phase 2 trial sponsored by NCI (NCT02203513); and the preliminary data showed encouraging results in patients with wild-type BRCA HGSOC. PI3K/AKT/mTOR pathway plays key roles in cancer cell survival, homologous recombination repair and drug resistance. Our previous data showed the expression level of genes related to PI3K/AKT signaling is elevated in prexasertib-resistant TNBC patient-derived xenograft (PDX) tumors. In this study we explored whether the PI3K/AKT/mTOR pathway is involved in prexasertib response of HGSOC and evaluated the combination effect of prexasertib with a PI3K/mTOR inhibitor (LY3023414) on HGSOC in vitro and in vivo. Data from 24 n=1 HGSOC PDX tumors indicated that the tumors with high phospho-AKT were resistant to prexasertib. The PI3K/AKT/mTOR signaling (including p70S6K and S6RP) was activated and correlated with the increased DNA damage (γH2AX) in HGSOC cell lines treated with prexasertib. These data suggest that the PI3K/AKT signaling is associated with prexasertib resistance and the cell survival response to DNA damage induced by prexasertib; and provide the scientific rational for a prexasertib/LY3023414 combination. Indeed the prexasertib/LY3023414 combination induced synergistic or additive inhibition on cell proliferation and colony formation in multiple HGSOC cell lines. This combination enhanced replication stress (phospho-RPA32), DNA damage (γH2AX), S phase arrest and cell death (cleaved PARP and sub-G1) in HGSOC Ovcar-8 cells when compared with single agent activity. Combination efficacy was further tested in HGSOC OV-90 and Cov504 xenograft models. Prexasertib induced tumor inhibition by 58.8% in OV-90 and tumor regression by 37.3% in Cov504, and LY3023414 induced tumor inhibition by 43.37% and 66% in OV-90 and Cov504 tumors, respectively. Prexasertib/LY3023414 combination resulted in tumor regression by 8.9% and 73.7% in OV-90 and Cov504 tumors, respectively, and significantly (p<0.001) enhanced efficacy when compared to singe agent. Taken together, these data support a potential combination strategy of Chk1 inhibitor prexasertib with a PI3K/mTOR inhibitor LY3023414 to treat HGSOC patients. The safety of this combination is being assessed in an ongoing Phase 1b clinical trial (NCT02124148). 

### Kinases, GTPases, and Phosphatases

#1762

The START domain of the DLC1 tumor suppressor binds phosphatidylserine and two proteins, PLCD1 and Caveolin-1.

Beatriz Sanchez-Solana,1 Dunrui Wang,1 Xiaolan Qian,1 Alex Papageorge,1 Parthibane Velayoudame,2 Jairaj Acharya,2 Douglas R. Lowy1. 1 _NIH, Bethesda, MD;_ 2 _NIH, Frederick, MD_.

The tumor suppressor gene DLC1 encodes a multi-domain protein, which includes a Rho-GAP domain (Rho GTPase activating protein domain) that negatively regulates the activity of RhoA, B, and C, and has been hypothesized to be the basis of its tumor suppressor functions. DLC1 also contains a less well characterized START (StAR-related lipid transfer) domain in its C-terminus, whose overall contribution to DLC1 function remains incompletely understood. START domains in other proteins have been shown to bind lipids, but no lipid has yet been identified that binds the DLC1 START domain. In the present study, we attempted to gain further insight into the molecular function and lipid binding properties of the DLC1 START domain.

We previously determined that Caveolin-1, the main structural component and marker of caveolae, interacts with the DLC1 START domain, and this interaction contributes to the full tumor suppressor activity of DLC1. In unpublished studies, we additionally identified Phospholipase C delta 1 (PLCD1), which participates in intracellular Ca2+ mobilization, forming a complex with the DLC1 START domain and with Caveolin-1.

We have now developed evidence that, in addition to binding Caveolin-1 and PLCD1, the DLC1 START domain binds a lipid, phosphatidylserine (PS), and have identified a colon cancer-associated DLC1-START domain mutant (R947C) that results in a protein deficient in forming a complex with all three binding partners and displays reduced tumor suppressor activity but intact RhoGAP activity. The interaction between the DLC1-START domain and PS can occur independently of PLCD1 and/or Caveolin-1, but PS promotes complex formation between the DLC1 START domain and PLCD1 or Caveolin-1, without altering PLCD1 to Caveolin-1 binding. Biological assays with wild type DLC1 show cooperation between DLC1 and PLCD1 or Caveolin-1 to inhibit cell migration, but no cooperation is observed when all three genes are overexpressed, suggesting that Caveolin-1 and PLCD1 occupy the same migration signaling pathway. Thus, we have identified a biologically relevant complex between DLC1 and three macromolecules (PLCD1, PS and Caveolin-1) that contributes to the full tumor suppressor function of DLC1 independently of its RhoGAP activity, and are testing the hypothesis that PS serves as a linker between the DLC1 START domain and PLCD1 or Caveolin-1.

#1763

Tumor mutational burden, mutational signatures and copy number variation in lung cancer driven by the Ras-Raf-MEK-ERK pathway.

Pantelis A. Nicola,1 George Burghel,1 Andrew Wallace,1 Helene Schlecht,1 Eleanor Baker,1 Katie Baker,2 Lynsey Priest,2 Mathew Carter,2 Sharzad Moghadam,2 Jane Rogan,2 Robert G. Bristow,2 William Newman,1 Fiona Blackhall,2 Colin R. Lindsay2. 1 _Manchester Centre for Genomic Medicine, Manchester, United Kingdom;_ 2 _The Christie NHS Foundation Trust, Manchester, United Kingdom_.

Introduction: Lung cancer is the most common cause of cancer-related death. Kirsten rat sarcoma viral oncogene (KRAS) is the most frequently mutated gene in non-small cell lung cancer (NSCLC), occurring in approximately 30% of cases. We characterised the genomic landscape of NSCLCs with an aberrant Ras-Raf-MEK-ERK pathway.

Methods: 121 Greater Manchester patients with resected NSCLC were recruited into the UK 100,000 Genomes Project (Genomics England). Whole genome sequencing (WGS) was performed on tumor specimens and matched blood samples. Data generated was processed by a standard pipeline devised by Genomics England, then mined for mutations in the Ras-Raf-MEK-ERK pathway. NF1 mutation was assessed as a positive control for RAS pathway activation. Tumor mutational burden (TMB), mutational signature profiles and copy number variation (CNV) were also obtained. Clinical characteristics including tumor size, nodal status and stage were documented. Mann-Whitney and Fisher's exact tests were used for statistical comparisons.

Results: Cancers from 42/121 (34.7%) patients (pts) had a RAS variant, of which 40/42 (95.2%) were KRAS alterations. A single NRAS-mutant adenocarcinoma (Q61L) was identified, as was a HRAS variant, not previously observed in squamous lung cancer (Q61L). Codon 12 was the most frequently mutated KRAS site with four mutant alleles (G12C 17/40 pts, G12V 6/40, G12D 4/40, G12A 2/40). Median TMB was not significantly different between KRAS-mutant cases (8.06, range 1.84-55.2) and KRAS-wildtype samples (7.1, range 0.98-45.32) (p=0.3). Smoking-associated signature 4 was the most common mutational process (37/40 pts, median 50%, range 20-70%), appearing in a mutually exclusive fashion from the intrinsic signature 1 (3/40 pts, median 20% range 10-30%). 47/121 pts (38.8%) showed a KRAS CNV with 34/47 (72.3%) being gains. For NF1, 12/121 (9.9%) variants were identified. 6/12 (50%) were missense variants, each with a different codon affected (5/12 splice site variants, 1/12 frameshift). Median TMB was not significantly higher in NF1-mutants (NF1m: median 11.54, range 4.47-28.27; NF1 WT: median 7.1, 0.98-55.2; p=0.065) and mutational signature 4 was again the most common (11/12, median 40%, range 20-60%). 37/121(30.6%) samples showed NF1 CNV, the majority of which were surprisingly gains (25/37 pts, 67.6%). In terms of clinical outcome, neither KRAS-mutant or NF1-mutant tumors were more likely to occur in later stage III disease (KRASm: 9/30 pts, 30%, p=0.817; NF1m: 5/11 cases, 45.5%; p=0.326).

Conclusions: Approximately one half of this NSCLC cohort recruited from Greater Manchester carried a Ras-Raf-MEK-ERK pathway aberration. These KRAS-mutant tumors were often at an early stage and driven by tobacco as their main aetiological process. The mutual exclusivity of signature 4 and 1 suggests there are further complexities to be established in Ras-driven NSCLC.

#1764

Targeting nucleotide exchange to inhibit Gq/11 driver mutations in uveal melanoma.

Michael D. Onken, Carol M. Makepeace, Kevin M. Kaltenbronn, Stanley M. Kanai, Tyson D. Todd, Shiqi Wang, Thomas J. Broekelmann, Prabakar Kumar Rao, John A. Cooper, Kendall J. Blumer. _Washington Univ. School of Medicine, Siteman Cancer Center, St. Louis, MO_.

Constitutively activating mutations in the G-protein alpha subunits GNAQ(Gαq) and GNA11(Gα11) act as oncogenic drivers in over 90% of uveal (eye) melanoma (UM) tumors. We show that constitutively active Gαq and Gα11 can be targeted in UM cells by the cyclic depsipeptide FR900359 (FR). FR inhibits GDP/GTP guanine nucleotide exchange allosterically to trap constitutively active Gαq/11 in inactive GDP-bound Gαβγ heterotrimers. FR inhibits second messenger signaling, arrests proliferation and reinstates melanocytic differentiation in UM cells driven by constitutively active Gαq or Gα11. At higher doses, FR also induces apoptosis. The re-differentiation and anti-proliferative effects of FR are not seen in UM cells that lack mutations in Gαq or Gα11. FR promotes UM cell differentiation by reactivating polycomb repressive complex 2 (PRC2)-mediated gene silencing, and this re-differentiation can be blocked with an EZH2 inhibitor. The effector system regulating PRC2 downstream of constitutively active Gαq/11 in UM is currently under investigation. Preliminary data from human primary tumor samples suggest that targeting constitutively active Gαq/11 with FR could provide an important therapeutic approach for UM.

#1765

**In vivo modeling of** RIT1 **function in development and lung cancer.**

Maria McSharry. _Fred Hutchinson Cancer Research Center, Seattle, WA_.

Ras-like in most tissues 1 (RIT1) encodes a ubiquitously expressed RAS-family small GTPase. Recently, somatic RIT1 mutations were identified in lung adenocarcinoma and myeloid malignancies, and germline RIT1 mutations were found in. RIT1 is also implicated hematopoietic malignancies and in Noonan syndrome, a "Rasopathy" affecting craniofacial development and associated with congenital heart disease. Of a variety of RIT1 mutations discovered, the most recurrent mutation observed was M90I. In this study, murine models were used to determine whether expression of RIT1M90I mutation is sufficient for tumor initiation or developmental phenotypes observed in Noonan patients.

Mice were genetically engineered at the Rosa26 locus to harbor RIT1M90I under the control of a lox-Cre system (LSL-RIT1M90I). The Cre recombinase protein was introduced via intratracheal delivery of adenovirus to the lungs. At one-year post treatment, 7 of the 10 LSL-RIT1M90I mice examined exhibited the expected genetic recombination. Tumors were observed in 3 of these mice, one of which was wild-type for p53. No visible tumors were seen in mice lacking the RIT1M90I gene or in cases where recombination was not observed, regardless of p53 status.

Mouse embryonic fibroblasts (MEFs) were infected by adenovirus to recapitulate the in vivo studies and evaluate transformation. In vitro experiments conducted in RIT1M90I MEFs demonstrated faithful recombination of the transgene upon treatment with Cre. Protein analysis of these same cells reflected higher levels of RIT1 protein in transgenic cells treated with Cre, while in all other conditions, RIT1 was undetectable. Preliminary data from an immortalization assay suggested enhanced proliferation of MEFs expressing the RIT1M90I gene compared to wild-type counterparts.

Cre recombinase was expressed body-wide in tamoxifen (4OHT) inducible mice (UBC-Cre-ERT2) LSL-RIT1M90I animals crossed to the inducible UBC-Cre-ERT2 system exhibited faithful recombination of the transgene upon treatment with 4OHT.

To determine the consequences of constitutive expression of RIT1M90I, RIT1M90I sires were crossed to dams harboring constitutively active Cre recombinase or to a constitutively active Cre SRY-box promoter (Sox2-Cre). Of the 100 pups observed, none contained both RIT1M90I and Cre recombinase (p < 0.0001), indicating embryonic lethality induced by RIT1 activation.

These data demonstrate the powerful effect of RIT1 activation on murine embryonic development and will enable future efforts on the characterization of the mechanism of RIT1-induced Noonan Syndrome and lung cancer.

#1766

High expression of ARHGEF2 is associated with poor prognosis via c-Myc signaling in patients with pancreatic cancer.

Yosuke Nakao, Shigeki Nakagawa, Yo-ichi Yamashita, Rumi Itoyama, Toshihiko Yusa, Naoki Umezaki, Takanobu Yamao, Tatsunori Miyata, Hirohisa Okabe, Katsunori Imai, Hiromitsu Hayashi, Akira Chikamoto, Hideo Baba. _Kumamoto University, Kumamoto, Japan_.

Background: Pancreatic cancer remains a lethal malignancy with a dismal five year survival rate of less than ten percent. ARHGEF2 is one of the Rho guanine exchange factor (RhoGEF). RhoGEF is a microtubule-associated guanine nucleotide exchange factor for the Rho family of small GTPases. We aimed to reveal the association between ARHGEF2 expression and patients' prognosis, or the downstream pathways of ARHGEF in pancreatic cancer.

Methods: For the gene selection, we used the cohort of 102 patients who underwent curative resection for pancreatic cancer from GSE21501 in which the DNA microarray were performed to assess the association with molecular pathways and the prognosis of patients. Pathway analysis was performed by GSEA (gene set enrichment analysis) using HALLMARK geneset.

As a validation cohort, 200 patients underwent curative resection for pancreatic cancer at Kumamoto University from April 2004 to March 2018 were enrolled. The immunohistochemical (IHC) staining was performed to evaluate the expression of ARHGEF2 and c-Myc. IHC was evaluated with the intensity and area of immunoreactivity of ARHGEF2 and c-Myc and scored each factors from 0 to 2. The staining index (ranging from 0 to 4) was calculated by multiplying intensity and area. Low expression was defined from 0 to 1, and high expression was from 2 to 4.

Result: In GSE21501 (n=102), the patients were separated into high- (upper 50%, n = 51) and low- (lower 50%, n = 51) ARHGEF2 groups. High-ARHGEF2 group had significantly poor overall survival than low-ARHGEF group (p = 0.01). Moreover, high ARHGEF2 group was associated with high lymph node metastasis (p=0.03). The GSEA

analysis revealed that the high ARHGEF expression was significantly associated with "HALLMARK-MYC-TARGET-V2" geneset (p=0.04). Also high ARHGEF expression was significantly associated with CDC25A (p=0.006) which is the downstream gene of c-MYC pathway and regulates cell cycle, suggesting that ARHGEF promote cell cycle via MYC signaling.

In immunohistochemistry, the high ARHGEF2 group (n = 122) had shorter recurrence free survival (RFS) (p = 0.0014) and poor overall survival (OS) (p = 0.0036). In addition, in the comparisons of clinicopathological factors between the high ARHGEF2 and the low ARHGEF2 groups, there were significantly larger tumor size and higher lymph node positive rate in the high ARHGEF2 group. In the multivariate analysis for RFS, high CA19-9 value, R1 resection and high ARHGEF2 expression were independent poor prognostic factors. In the multivariate analysis for OS, high CA19-9 value, tumor size (> 40 mm), lymph node metastasis and high ARHGEF2 expression were independently associated with poor prognosis. According to immunohistochemistry, ARHGEF2 and c-Myc have a significant positive correlation.

Conclusion: High expression of ARHGEF2 is associated with poor prognosis via c-Myc signaling in patients with pancreatic cancer.

#1767

Rho-GTPase inhibits claudin-2 expression to promote proximal tubular epithelial cell plasticity and renal cell carcinoma.

Balawant Kumar,1 Rizwan Ahmad,1 Pinelopi Kapitsino,2 Giovanna A. Giannico,3 Roy Zent,3 Raymond Clement Harris,4 Peter Clark,5 Punita Dhawan,1 Amar B. Singh1. 1 _University of Nebraska Medical Center, Omaha, NE;_ 2 _University of Kansas Medical Center, Kansas City, KS;_ 3 _Vanderbilt Medical Center, Nashville, TN;_ 4 _Vanderbilt University School of Medicine, Nashville, TN;_ 5 _Levine Cancer Institute, Omaha, NC_.

The clear-cell renal cell carcinoma (ccRCC) is the most prevalent and malignant type of kidney cancer, for which there are no effective treatment. Moreover, the incidence of ccRCC has been increasing steadily. Disruption of epithelial cell polarity, assisted by tight junction (TJ) deregulation, facilitates rewiring of oncogene and tumor suppressor signaling pathways. In this regard, proximal tubular epithelium (PTE), where ccRCC originates is the sole nephron segment that expresses claudin-2 (hereon Cldn2), a TJ- protein. However, role of Cldn2 in ccRCC remains unknown despite well-recognized role of Cldn2 in regulating other cancers. We here report a novel role for Cldn2 in maintaining epithelial differentiation among PTE cells as its loss induces mesenchymal traits and associates with ccRCC progression. In specific, we found a specific (among claudin proteins) loss of Cldn2 expression in PTE cells subjected to stimuli, like EGF, TGF-ß and hypoxia, known to induce EMT in PTE cells as well as in a murine model of spontaneous renal tumorigenesis. Further analysis of a large renal cancer patient cohort revealed a significant decrease in Cldn2 levels in the ccRCC, and its positive association with cancer metastasis. In vitro, genetic ablation of claudin-2 expression in HK-2 or MDCK-II cells potentiated mesenchymal traits (upregulated Vimentin and Fibronectin; p<0.002) and cell motility (p<0.0002 versus control). In contrast, forced claudin-2 expression in RCC-derived (and claudin-2 low) Caki-2 cells (P<0.002) promoted epithelial differentiation (downregulation of Vimentin and Fibronectin; P<0.0012& P<0.00025) , inhibited cell invasion (P<0.0001) and proliferation (P<0.001). Overall, our findings support a novel and tissue-specific role of claudin-2 in maintaining epithelial differentiation among renal proximal tubular epithelium and tumor suppressor. Further analysis identified a key role of Rho-GTPase in regulating renal claudin-2 expression as ROCK inhibitor (Y-27632) not only reverted EMT-associated loss of Cldn2 but also in Caci-2 cells. An ELISA assay further confirmed an early induction of Rho-GTPase activity in PTE cells subjected to the EMT. Of note, inhibiting Rho-GTPase not only improves renal fibrosis but also inhibits ccRCC. Taken together, we here report a novel role of claudin-2 in maintaining epithelial phenotype in PTE cells with potential clinical significance in acute and chronic renal diseases, and ccRCC progression.

#1768

Biochemical and structural analysis of the Neurofibromin (NF1) protein and a potential role for protein destabilization in Rasopathy diseases.

Mukul Sherekar,1 Sae-Won Han,2 Simon Messing,1 Matthew Drew,1 William Gillette,1 Claire Lorenzo,2 Frank McCormick,2 Dominic Esposito1. 1 _Frederick National Laboratory for Cancer Research, Frederick, MD;_ 2 _University of California San Francisco, San Francisco, CA_.

Neurofibromin is the 320 kilodalton protein product of the NF1 gene which is mutated in the Rasopathy disease Neurofibromatosis Type I. Defects in NF1 lead to abrogation of the GTPase activating activity of the protein, leading to aberrant signaling through the RAS-MAPK pathway. Very little is known about the function of the majority of the NF1 protein—to date only the GAP domain and a region containing a Sec-PH motif have detailed structural information. To better understand the role of this large protein, we have carried out a series of biochemical and biophysical studies which clearly show that full length NF1 protein exists primarily as a dimer. Data from in vivo experiments confirmed that this dimerization also occurs in cells, and presumably has biological implications. A model was generated from negative-stain EM data which suggests that NF1 monomers form both intermolecular contacts to form dimers and intramolecular contacts which stabilize the individual monomers. Deletion analysis permitted us to map these two interacting domains to different parts of the NF1 protein, and crosslinking studies coupled with mass spectrometry helped to identify regions of possible interaction. In addition, introduction of point mutations observed in patients with NF diseases caused a dramatic decrease in the stability of proteins both in vitro and in vivo, potentially arguing for a rationale for loss of NF1 activity by these single mutations

#1769

CSox-based sensors for continuous, homogeneous and quantitative monitoring of protein kinase and phosphatase activity.

Erik M. Schaefer, Susan Cornell-Kennon, Bill Lu. _AssayQuant Technologies, Hopkinton, MA_.

Introduction: Protein kinases are a diverse group of 518 enzymes whose dysregulation lies at the center of many diseases. Currently, 30% of all drug development efforts are focused on protein kinases. Although 41 drugs are approved and >120 in clinical trials, these are predominately ATP-competitive inhibitors. More recently, there has been an expanded focus on kinase inhibitors with different modes of action, where new tools are needed to effectively characterize inhibitor mechanism of action, predict drug potency and to drive decisions earlier in the drug development process. We developed a simple yet powerful method for the generation of peptide sensors that can be used for the continuous, quantitative and homogenous detection of kinase and phosphatase activity with recombinant enzymes and crude lysates to enable target discovery and drug development.

Experimental Procedures: We harnessed chelation-enhanced fluorescence by combining next generation sulfonamido-oxine (Sox) chromophore technology with high-throughput solid-phase peptide synthesis methods to identify optimized sequences based on physiological substrates. Enzyme activity is monitored kinetically using fluorescence intensity (Ex/Em 360/485 nm) or in endpoint mode using Europium and time-resolved fluorescence (Ex/Em 360/620 nm).

Results: We demonstrate the ability to rapidly identify novel optimized substrates, where performance measures included higher reaction rates, lower Km's, higher signal/background, increased sensitivity and specificity. We identified highly generic substrates (for robust detection of 80 Tyrosine kinases) and highly-selective substrates (for quantitative detection of targeted kinases in crude cell or tissue lysates for profiling, potency assessments and SAR). We have developed sensors to monitor activity of high-profile tyrosine kinases, including the EGFR and clinically-relevant mutants, JAK kinases, Tec-kinases, and, serine/threonine kinases, including CDK1-9, MAPK pathway (MAP4Ks, MAP3Ks, MAPKs & MAPKAPKs), PKR/EIF2AKs and PIM1. In addition, CSox-based phosphopeptide substrates are used to monitor protein phosphatases with specificity for tyrosine (PTP1B, SHP1/2) or serine/threonine (PP2A, PP2C, PHLPP).

Conclusions: The generation of robust activity-based sensors, even where peptide assays previously weren't available, opens new areas for effective drug discovery. The Sox-based kinetic assay format is ideal for elucidating drug mechanism of action, potency, and enzyme regulation. The PhosphoSens-Red endpoint format is ideal for HTS, SAR and profiling. Together, these formats can be applied across the entire target discovery and drug development workflow, providing a quantum improvement in performance and productivity needed to address the challenges and opportunities of next generation protein kinase and phosphatase inhibitors.

#1770

RSK2 is required for TRAF6 phosphorylation-mediated colon inflammation.

Ke Yao, Sung-Young Lee, Cong Peng, Do Young Lim, Hiroyuki Yamamoto, Joohyun Ryu, Tae-Gyu Lim, Hanyong Chen, Wei-Ya Ma, Ann M. Bode, Zigang Dong. _Univ. of Minnesota Hormel Inst., Austin, MN_.

Inflammation is a complex biological host reaction to tissue damage, infection and trauma. Extensive study of the inflammatory response has led to the identification of several protein kinases that are essential for signaling and could be potential therapeutic targets. The RSK family of kinases has multiple cellular functions. In our study, we found that RSK2 is a mediator for inflammation signaling and interacts with TRAF6. In vitro kinase assay results indicated that RSK2 strongly phosphorylates TRAF6 at serines 46, 47 and 48. Ectopic over-expression of TRAF6 or knocking down RSK2 expression confirmed that RSK2 is a positive regulator of TRAF6 K63 ubiquitination. TRAF6 is also required for RSK2 ubiquitination. TRAF6 bridges the TNF receptor superfamily and intracellular signaling for the induction of pro-inflammatory cytokines. We developed a colon inflammation model using RSK2 wild type (WT) and knockout (KO) mice. As expected, F4/80 and CD3 infiltration were significantly upregulated in WT mice compared to RSK2 KO mice. Furthermore, inflammation signaling, including Ikkα/β, p38 and JNKs, was dramatically up-regulated in WT mice. Colon tissue immunoprecipitation results further confirmed that TRAF6 K63 ubiquitination was lower in RSK2 KO mice. Overall, these results indicate that phosphorylation of TRAF6 (S46, 47, 48) by RSK2 is required for TRAF6 K63 ubiquitination and inflammation signaling.

#1771

Role of Tsc2-mTORC1 signaling in homeostasis of intestinal epithelium and its relation to inflammation and cancer.

Takenori Kotani, Jajar Setiawan, Yoji Murata, Yasuyuki Saito, Takashi Matozaki. _Kobe Univ. Graduate School of Medicine, Kobe, Japan_.

Intestinal tumorigenesis driven by mutations in adenomatous polyposis coli gene requires the mammalian target of rapamycin complex 1 (mTORC1) activity, whereas the physiological roles of mTORC1 in the homeostasis of intestinal epithelial cells (IECs) remain virtually unknown. We here generated mice, in which tuberous sclerosis complex 2 (Tsc2), a negative regulator of mTORC1, was specifically ablated in IECs (Tsc2 CKO mice). Tsc2 CKO mice manifested the enhanced proliferative activity of IECs in intestinal crypts as well as the promoted migration of these cells along the crypt-villus axis. In vitro study showed that conditional ablation of Tsc2 in IECs promoted development of intestinal organoids, while mTORC1 inhibitor, rapamycin, diminished this phenotype. Tsc2 CKO mice also manifested the increased apoptotic rate of IECs as well as the increased ectopic Paneth cells, which are one of the major differentiated IECs. In addition, such conditional ablation of Tsc2 resulted in a suppressed expression of Wnt target genes in the colonic epithelium. Finally, we found that Tsc2 CKO mice manifested the increased susceptibility for dextran sulfate sodium (DSS)-induced colitis. Although mTORC1 and Wnt signaling pathways are thought to cooperated to promote oncogenesis in the intestinal epithelium, our results thus suggest that the mTORC1 signaling unexpectedly counter-regulates the Wnt signaling with the maintenance of intestinal homeostasis.

#1772

Targeting the stress-activated mitogen-activated protein kinase (MAPK)-activated protein kinase-2 (MK2)/ heat-shock protein 27 (Hsp27) axis suppresses tumor growth of pancreatic ductal adenocarcinoma (PDAC).

Patrick Grierson, Kian Lim, Andrea Wang-Gillam, David DeNardo, Keren Wang, Lin Li, John Herndon, Marianna Ruzinova. _Washington University School of Medicine, Saint Louis, MO_.

PDAC is one of the most lethal cancers and is projected to be the 2nd leading cause of cancer-related death after 2020. Most patients present with advanced disease, with survival of about one year with standard of care chemotherapy. PDAC is characterized by powerful oncogenic events particularly mutations of the KRAS gene that result in constitutive activation of multiple effectors including the MAPK, PI3K and RalGEF pathways to drive intrinsic resistance to therapeutics. Extrinsic to PDAC cells is a desmoplastic tumor microenvironment (TME) rife with cancer-associated fibroblasts (CAF) and immunosuppressive myeloid cells that limits delivery of therapeutics and dampens immune surveillance. Despite decades of effort, effective anti-KRAS inhibitors remain elusive, and targeting of the MAPK pathway has been ineffective, thus identification of novel molecular targets is needed. We performed an unbiased screening phospho kinase array to identify stress-responsive targets up regulated following treatment of PDAC cells with FOLFIRINOX, a standard first-line chemotherapy in PDAC. Our data show upregulation of phospho-Hsp27 as well as its proximal kinase, MK2, following chemotherapy-induced stress. We further show that the combination of chemotherapy with MK2 knockdown or small molecule MK2 inhibition abrogates the adaptive Hsp27 stress response following chemotherapy exposure and leads to increased apoptosis (PARP cleavage) in a panel of PDAC cell lines. Furthermore, treatment with small molecule MEK and ERK inhibitors similarly leads to activation of the MK2/ Hsp27 axis, identifying it as a global stress-responsive pathway in PDAC. Notably, simultaneous inhibition of ERK and MK2 synergistically inhibits 3D colony formation using a panel of PDAC cells. Genetic depletion of MK2 in human PDAC cells profoundly suppresses their tumor forming ability following subcutaneous injection in nude mice. Lastly, tissue microarray of human PDAC shows that elevated baseline phospho-MK2 predicts inferior survival. In conclusion, our data identify the MK2/ Hsp27 axis as a novel stress-responsive pathway in PDAC, and suggest that it may represent a promising target in combination with cytotoxic chemotherapy or targeted MAPK inhibitors in PDAC.

#1773

The tyrosine phosphatase, SHP-2, is involved in regulating PD-L1 expression in non-small cell lung cancer.

Keller Toral. _University of Kentucky, Lexington, KY_.

Immune checkpoint inhibitors (ICIs), specifically those which target the T-cell surface receptor Programmed Cell Death-1 (PD-1) and its ligand, Programmed cell death ligand-1 (PD-L1), have demonstrated substantial clinical benefit in patients with non-small cell lung cancer (NSCLC). Tumoral PD-L1 expression may be important for response to therapy, but the precise mechanism of regulation has yet to be fully elucidated. It is known that PD-L1 expression is elevated in cancers harboring mutations in the RAS family of genes, specifically KRAS. Mutations in the KRAS gene are found in up to 30% of NSCLC tumors, and there are no targeted treatment options available for this subset of patients. The tyrosine phosphatase, SHP-2, has been shown to be a critical regulator of the KRAS signaling cascade. Therefore, genes regulated by the RAS/MAPK signaling cascade, including PD-L1, may be modulated by SHP-2 inhibition. Thus, this study aims to investigate the impact of SHP-2 activity on PD-L1 expression in NSCLC.

The allosteric inhibitor of SHP-2, SHP099, was used to ablate SHP-2 activity in KRAS-mutant NSCLC cell lines, H460, A549, H2122, and EGFR-mutant NSCLC cell line PC9. PD-L1 levels were quantified by western blot and flow cytometry analyses. PD-L1 mRNA was measured by quantitative real time PCR (qRT-PCR) analysis. SHP099 activity was confirmed by SHP-2, siRNA transfection in KRAS active cell lines. Inhibition of SHP-2 led to increased levels of PD-L1 expression in the KRAS-mutant cell lines, but not in the EGFR-mutant cells. Analysis of PD-L1 surface expression by flow cytometry confirmed that surface PD-L1 levels increased following exposure to SHP099. To determine whether SHP-2 controlled expression at the transcriptional level, mRNA quantification by qRT-PCR displayed increased levels of PD-L1 mRNA following SHP099 treatment. Together, these results suggest that SHP-2 regulates PD-L1 expression in KRAS-active NSCLC at the level of PD-L1 gene expression. It is presently unclear whether SHP-2 also has a role in controlling protein turnover. Using SHP-2 mutant constructs that mimic a constitutively active phosphatase, we hope to gain further insight into the roles of both SHP-2 catalytic and scaffolding functionality on PD-L1 expression.

#1774

Targeting aurora A kinase (AAK) in platinum-resistant high grade serous ovarian cancer (HGSOC).

Ram N. Ganapathi, Eric J. Norris, Ashley P. Sutker, Hala Soliman, Mahrukh K. Ganapathi. _Levine Cancer Institute, Carolinas HealthCare System, Charlotte, NC_.

Resistance to platinum-based chemotherapy is a major problem in the clinical management of recurrent HGSOC. While the use of poly ADP-ribose polymerase inhibitors (PARPi) have made an impact in BRCA1/2 mutated and homologous recombinant-deficient HGSOC, resistance and limited response-rates to PARPi persist. Alisertib (AL) an inhibitor of AAK can inhibit entry and progression of cells through mitosis as well as the interaction with N-myc and its degradation (Front. Oncol. doi:10.3389/fonc.2015.00189). In models of HGSOC from patients with recurrent platinum-resistant disease, cisplatin (CP) treatment does not lead to cell cycle traverse perturbations and significant accumulation of cells in G2-M. Since a defect in the G2-M DNA damage checkpoint is a potential mechanism of resistance to cisplatin, we sought to test the hypothesis that inhibition of AAK that is involved in centrosome maturation and entry into mitosis would enhance the anti-tumor activity of cisplatin in platinum-resistant HGSOC. To test this hypothesis, we treated HGSOC cell lines from patients with recurrent platinum-refractory disease with increasing doses of CP (1- 2.5 μM), of AL (0.05 μM) or the combination and evaluated effects on cell proliferation, apoptosis and generation of reactive oxygen species (ROS). The sequential treatment of four different HGSOC cell lines with CP for 3 hours followed by AL for 48 hours and recovery in drug-free medium for 96-120 hours led to a synergistic increase in apoptosis (p<0.01) and a synergistic decrease in cell survival (p<0.001) compared to treatment with either drug alone. The enhanced apoptosis and decreased cell survival with (CP→AL) treatment was accompanied by a significant increase (p<0.05) in ROS. In contrast, sequential treatment with AL followed by CP (AL→CP) led to an antagonistic response. The synergistic effect of (CP→AL) was accompanied by a modest increase in mRNA and protein levels of AAK. However, protein levels of N-myc protein, which interacts with AAK and is degraded, were not altered. Interestingly, (CP→AL) treatment did not elicit the synergistic cytotoxic response or increased ROS in immortalized normal human ovarian surface epithelium or normal human fallopian tube secretory epithelium cells. In platinum-refractory HGSOC cells that are wild-type for BRCA1/2 and resistant to olaparib, treatment with the combination of 0.01 μM AL and 1 μM olaparib for 48 hours resulted in an additive cytotoxic response. Based on the mutation landscape of the HGSOC cell panel evaluated, the synergistic cytotoxicity with the CP→AL treatment was independent of BRCA1/2 or TP53 mutation status. In summary, CP resistance in HGSOC due to a defective G2-M DNA damage response, can be potentially circumvented by sequential treatment with CP followed by AL.

#1775

Targeting RSK4 prevents both chemoresistance and metastasis in lung cancer.

Stelios Chrysostomou,1 Rajat Roy,1 Filippo Prischi,2 Katie Chapman,3 Uwais Mufti,4 Francesco Mauri,1 Guido Bellezza,5 Joel Abrahams,1 Silvia Ottaviani,1 Leandro Castellano,1 Georgios Giamas,6 David Hrouda,7 Mathias Winkler,7 David Klug,1 Sophia Yaliraki,1 Mauricio Barahona,1 Yulan Wang,8 Maruf Ali,1 Michael Seckl,1 Olivier Pardo1. 1 _Imperial College London, London, United Kingdom;_ 2 _University of Essex, Colchester, United Kingdom;_ 3 _Domainex, Essex, United Kingdom;_ 4 _Harrogate and District NHS Foundation Trust, Harrogate, United Kingdom;_ 5 _University of Perugia, Perugia, Italy;_ 6 _University of Sussex, Brighton, United Kingdom;_ 7 _Charing Cross Hospital, Imperial College NHS Trust, London, United Kingdom;_ 8 _Wuhan Institute of Physics and Mathematics, Wuhan, China_.

Lung cancer is the commonest cause of cancer death worldwide with a five-year survival rate of less than five percent for metastatic tumors. Non-small cell lung cancer (NSCLC) accounts for 80% of lung cancer cases of which adenocarcinoma prevails. Patients almost invariably develop metastatic drug-resistant disease and this is responsible for our failure to provide curative therapy. Hence, a better understanding of the mechanisms underlying these biological processes is urgently required to improve clinical outcome.

The 90-kDa ribosomal S6 kinases (RSKs) are downstream effectors of the RAS/MAPK cascade. RSKs are highly conserved serine/threonine protein kinases implicated in diverse cellular processes, including cell survival, proliferation, migration and invasion. Four isoforms exist in humans (RSK1-4) and are uniquely characterized by the presence of two non-identical N- and C-terminal kinase domains. RSK isoforms are 73-80% identical at protein level and this has been thought to suggest overlapping functions.

However, through functional genomic kinome screens, we show that RSK4, contrary to RSK1, promotes both drug resistance and metastasis in lung cancer. This kinase is overexpressed in the majority (57%) of NSCLC biopsies and this correlates with poor overall survival in lung adenocarcinoma patients. Genetic silencing of RSK4 sensitizes lung cancer cells to chemotherapy and prevents their migration and invasiveness in vitro and in vivo. RSK4 downregulation decreases the anti-apoptotic proteins Bcl2 and cIAP1/2 which correlates with increased apoptotic signalling, whilst it also induces mesenchymal-epithelial transition (MET) through inhibition of NFκB activity. A small-molecule inhibitor screen identified several floxacins, including trovafloxacin, as potent allosteric inhibitors of RSK4 activation. Trovafloxacin reproduced all biological and molecular effects of RSK4 silencing in vitro and in vivo, and is predicted to bind a novel allosteric site revealed by our RSK4 N-terminal kinase domain crystal structure and mathematical Markov Transient Analysis.

Taken together, our data demonstrate that RSK4 represents a promising novel therapeutic target in lung cancer.

#1776

An investigation of the effects of the atypical protein kinase C-ζinhibitor ζ-Stat on clear cell carcinoma ovarian cancer proliferation and invasion.

Tracess Smalley, Rekha Patel, Mildred Acevedo-Duncan. _Univ. of South Florida, Tampa, FL_.

Ovarian cancer is the most lethal gynecological cancer diagnosis. There is no efficient screening process for ovarian cancers and the average stage of diagnosis is stage III. The most common ovarian cancer diagnosis is epithelial ovarian carcinomas, which constitute for 85-90% of prognosis; of this percent, clear cell ovarian carcinoma (CCOC) represents 5% of incidence. CCOC presents unique pathological features, has poor prognosis and has a high reoccurrence rate after treatment. An overexpression of atypical protein kinase C isoforms (PKC-ζ and PKC-ι/λ) has been observed in various malignant cells lines and are linked to pathways for cellular proliferation and invasion. In this investigation, ovarian cell lines (TOV21G and ES-2) were treated with the atypical PKC-ζ inhibitor ζ-Stat and assayed to determine the effects on proliferation and cellular invasion. These assays included cell counting, WST-1 assay, Western Blot and endpoint polymerase chain reaction. Mouse xenograft experiments were also performed to determine the effects of ζ-Stat on TOV21G xenograft tumor growth in vivo. Our data showed that ζ-Stat decreased the proliferation and cell viability of clear cell carcinoma ovarian cancer cells. In addition, it knocked down the protein expression of PKC-ζ, as well as the protein and mRNA levels of RhoA (Ras homolog Family Member A). Our results also showed that tumor growth in athymic female mice was decreased when treated with ζ-Stat and that mouse body weight was maintained. This suggests that PKC-ζ is a novel target in the carcinogenesis of CCOC and its inhibition by way of ζ-Stat decreased the rate of proliferation, tumor growth and expression of invasive protein pathways.

#1777

Correlation between different C-KIT exon mutation and clinical outcome to Imatinib Mesylate treatment in patients with gastro-intestinal stromal tumors (GIST).

Abeer A. Bahnnasy, Ola Khorshid, Mona S. Abdellateif, Ghada Y. Zakaria, Nasr M. Allahloubi, Auhood Nassar, Abdel-Rahman N. Zekri. _National Cancer Inst. Cairo Univ., Cairo, Egypt_.

Aim: C-KIT exon mutations were assessed in patients with gastrointestinal stromal tumor (GIST) in relation to patients' characteristics, response to treatment and survival rates.

Methods: C-KIT mutations were assessed in the representative formalin fixed paraffinembedded tissues (FFPETs) of 89 patients with GIST compared to 35 normal control (NC) subjects using the Real Time PCR (Rt-PCR) and immunohistochemistry (IHC).

Results: C-KIT mutations were detected in 61/89 (68.5%) patients, compared to none of the NC subjects (P=<0.001). Most mutations were detected in exon 11 (50.8%), followed by exon 9 (24.5%), exon 13 (19.6%), exon 7 (4.1%), and exon 12 (only 1%). C-KIT protein expression was detected in 69 (77.5%) patients compared to none of the NC subjects (p=<0.001). Significant measure of agreement was detected between C-KIT expression by IHC and and RT-PCR (K=0.774, p=0.0001).

The presence of C-KIT mutations associated significantly with large tumor size (P=0.01), High mitotic rate, lymph nodes metastasis and high tumor risk (p=0.001, for all). Patients with exon 11 mutations 26/31 (83.9%) had a better response to treatment compared to those with exon nine 7 (46.7%) and exon 13 mutation 1/12 (8.3%; P=0.001). Exon 11 mutations associated significantly with better PFS and OS compared to those with exon 9 and 13 mutations (P= 0.001, P= 0.0001; respectively). On univariate analysis, Exon 13 mutations was the most significantly associated with reduced PFS (P=0.0001), whereas advanced disease stage, absence of adequate safety margins and the site of exon mutation associated significantly with reduced OS. (P=0.012, P=0.05 and P=0.005; respectively.

Conclusion: GIST patients with C-KIT exon 11 mutations have a better response to imatinib treatment (predictive value), better OS and lower risk of disease progression than those with exon 9 and 13 mutations. Our specified mutations in exons 9& 13 mutations could be used as an independent prognostic factor for GIST patients.

#1778

5'-nucleotidases are involved in the biology of human lung cancer cell lines.

Muhammad Zawwad Raza,1 Octavia Cadassou,1 Emeline Cros-Perrial,1 Alain Puisieux,1 Charles Dumontet,2 Lars Petter Jordheim1. 1 _Univ Lyon, Université Claude Bernard Lyon1, INSERM U1052, CNRS 5286, Centre Léon Bérard, Centre de Recherche en Cancérologie de Lyon, 69008, Lyon, France;_ 2 _Hospices Civils de Lyon, Lyon, France_.

Cytosolic-5'-nucleotidase II (cN-II) and ecto-5′-nucleotidase (CD73) are enzymes involved in the nucleotide metabolism by dephosphorylating intracellular and extracellular purine nucleotide monophosphates, respectively. Both enzymes have been shown to be involved in cancer by modifying anticancer drug activity, cancer cell biology and immune modulation. To increase the knowledge of cN-II and CD73 roles in cancer cell biology, we developed lung cancer cell models (NCI-H292) with a complete knockout of either or both enzymes using the CRISPR-Cas9 technique. These cell models are used to study cell proliferation and migration, cancer drug sensitivity, cellular behaviour and apoptosis using CFSE staining, IncuCyte relative confluence and wound healing assay, MTT assay, xCELLigence RTCA and caspase-3/7 apoptotic assay. SCID CB17 mice are also used to study the in vivo tumor growth using these cell models. Our results show that there is no significant difference in proliferation between different cell models exposed or not to different concentrations of either adenosine or AMP under normoxic and hypoxic conditions. On the other hand, using xCELLigence RTCA technique, we have observed that CD73-deficient cells have higher cell index as compared to CD73 expressing cells under basal conditions during the first 72 hours of culture. However, adenosine globally decreases the cell index of all NCI-H292 cells, unlike AMP, which influences the cell index of cN-II-deficient cells but not CD73-deficient cells. Under hypoxic condition, cN-II-deficient cells showed higher cell index than the other phenotypes in untreated conditions, whereas cells lacking CD73 showed higher sensitivity to adenosine or AMP. As compared to other phenotypes, CD73 deficient cells are also more sensitive towards vincristine but less sensitive towards mitomycin and fludarabine. These cells are also more prone to adapt to hypoxic conditions and exhibit slower migration rate than their corresponding control cells. However, the effect of adenosine and AMP on migration rate is higher on cN-II-deficient cells. Mice experiments showed no difference in in vivo tumor growth using these cell models. Overall, we show that both CD73 and cN-II are important players in the cell biology of lung cancer cells and in their response to purines. Upcoming experiments should help us understand the molecular mechanisms underlying these observed differences.

#1779

PCDH7 and SET are overexpressed in prostate cancer and promote aberrant MEK and AKT signaling.

Gauri Shishodia, Sweaty Koul, Hari K. Koul. _LSU Health Sciences Ctr. - Shreveport, Shreveport, LA_.

Introduction: Most of prostate cancer (PCa) deaths are a result of distant metastasis. Aberrant activation of AKT and MEK play an important role in PCa progression. PCDH7, a cadherin superfamily transmembrane protein, is involved in cell-cell recognition and adhesion. Recent reports suggest overexpression of PCDH7 promotes metastasis in lung and breast cancer and PCDH7 was shown to interact with SET in lung cancer. Overexpression of SET promoted cell proliferation, survival, drug resistance, invasion and metastasis in different cancers. We evaluated expression and function of PCDH7 and SET in PCa.

Materials and Methods: PCDH7 and SET expression in clinical samples was extracted from publically available data sets (CRPC/NEPC-Trento et al 2016; TCGA, Cell 2015; adenocarcinoma- MSKCC, Cancer Cell 2010 using cBioPortal and UALCAN). PCa cells- LNCaP, C4-2B, DU145, PC3, 22Rv1 and RWPE1 were used in this study. Protein expression was measured by Western blotting in cell lines and immunohistochemistry in FFPE TRAMP and human prostate tissue sections. Gene expression was monitored by quantitative real time PCR. Gene knock down was performed by shRNA using lentiviral expression. Colony formation was assessed by staining with 0.4% crystal violet after 3 weeks of cell seeding. Co-IP studies were performed to check interactions of PCDH7 with SET and PP2A.

Results: Our results show that PCDH7 mRNA is overexpressed in 43% patients in CRPC as compared to 4% of patients in TCGA data set. SET was amplified in 23% of NEPC patients (Trento, 2016), mRNA upregulation in more than 5% adenocarcinoma patients (TCGA, 2015 and MSKCC, 2010). TCGA dataset showed that SET expression is high in tumor samples of adenocarcinoma patients as compared to normal prostate and increase in SET expression was observed with increase in Gleason score (UALCAN). PCDH7 mRNA and protein is overexpressed in CRPC (C4-2B, 22Rv1, DU145 and PC3) cells as compared to castrations sensitive (LNCaP) cells and normal prostate (RWPE1) cells. We also observed increased expression of PCDH7 in prostate tissues of TRAMP mice during PCa progression as well as human PCa as compared to normal tissues. We observed that knocking down PCDH7 and SET decreased colony formation. Co-IP studies revealed that PCDH7 interacts with SET and PP2A. We observed that knocking down PCDH7 reduced ERK and AKT activities, decreased cell migration, reduced cell invasion, and decreased colony formation. Co-IP studies revealed that PCDH7 interacts with SET and PP2A suggesting that PCDH7 could be promoting aberrant ERK and AKT activities upon interaction with SET and PP2A. Collectively, these data suggest PCDH7 and SET are overexpressed in PCa and promotes aberrant activation of ERK/AKT signaling in CRPC.

Conclusion: Our results, for the first time, show overexpression of PCDH7 and SET promotes PCa progression, as such PCDH7 may be an attractive target for therapeutic intervention in subsets of CRPC patients.

#1780

iSCREAM - an unbiased pipeline to screen for activating kinase mutations.

Deepankar Chakroborty,1 Kari J. Kurppa,2 Ilkka Paatero,3 Laura L. Elo,3 Klaus Elenius1. 1 _University of Turku, Turku, Finland;_ 2 _Dana-Farber Cancer Institute, Boston, MA;_ 3 _Turku Center for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland_.

Cancer tissues harbor thousands of mutations, and a given oncogene may be mutated at hundreds of sites. Most of these somatic mutations are expected to be inconsequential passenger mutations that reflect the general instability of the tumors. The discovery of most of the currently known driver mutations has been facilitated by their accumulation in mutation hot-spots within their respective genes. However, a vast majority of mutations in cancer tissues are rare and no information is currently available about their functional significance. Several lines of in vitro and in vivo clinical evidence also indicate that there is a significant number of, as yet unidentified, activating driver mutations that could serve as predictive markers in oncology.

An unbiased iSCREAM (in vitro screen for activating mutations) platform was developed to functionally characterize thousands of variants of a kinase oncogene in a single assay. The functional genetics screen was based on expressing random variants of a cDNA encoding a tyrosine kinase in a cell line in which the activating mutations provide growth-advantage. Targeted next-generation sequencing of the cDNA inserts was used to quantitatively analyze variants that provided growth-advantage.

iSCREAM is able to identify activating mutations from a library of thousands of random mutations of a tyrosine kinase. The pipeline can also be modified to identify mutations conferring resistance to a tyrosine kinase inhibitor.

#1781

Transcriptional activation of CBFβ by CDK11p110 is necessary to promote osteosarcoma cell proliferation.

Yong Feng, Francis Hornicek, Zhenfeng Duan. _UCLA, Los Angeles, CA_.

Aberrant expression and activation of cyclin-dependent protein kinase (CDK) is a hallmark of cancer. CDK11 is a protein kinase in the CDK family and plays a crucial role in cancer cell growth and proliferation. However, the molecular mechanisms of CDK11 in osteosarcoma and CDK11 transcriptional regulated genes are largely unknown. In this study, we performed global transcriptional analysis using gene array technology to investigate the transcriptional role of CDK11 in osteosarcoma. The promoter luciferase assay, chromatin immunoprecipitation assay, and Gel Shift assay were used to identify direct transcriptional target of CDK11. Clinical significance relevance and function of CBFβ were further accessed in osteosarcoma tissue microarray and in osteosarcoma cell lines. We identified a transcriptional role of protein-DNA interaction for CDK11p110, but not CDK11p58, in the regulation of core-binding factor subunit beta (CBFβ) expression in osteosarcoma cells. The CBFβ promoter luciferase assay, chromatin immunoprecipitation assay, and Gel Shift assay confirmed that CBFβ is a direct transcriptional target of CDK11. High expression of CBFβ is associated with poor outcome in osteosarcoma patients. Expression of CBFβ contributes to the proliferation and metastatic behavior of osteosarcoma cells. These data establish CBFβas a mediator of CDK11p110dependent oncogenesis and suggest that targeting the CDK11-CBFβ pathway may be a promising therapeutic strategy for osteosarcoma treatment.

#1782

ANKRD52 inhibited tumor metastasis through dephosphorylation of PAK1 in lung adenocarcinomas.

Ting-Fang Lee,1 Yin-Pu Liu,2 Yen-Fan Lin,2 Chong-Fang Hsu,3 Cheng-Wen Wu4. 1 _National Health Research Institute, Taiwan;_ 2 _National Yang-Ming University, Taiwan;_ 3 _Academia Sinica, Taiwan;_ 4 _National Chiao Tung University, Taiwan_.

PAK1 is an important oncogene involved in the progression of a variety of cancer types including lung cancer. Although PAK1 inhibition is shown to suppress tumor progression, specific PAK1 inhibitors are not available due to the complex structure and insufficient understanding of this protein. In an attempt to find TAZ (WW domain-containing transcription regulator 1) regulated tumor suppressor, a member of ankyrin repeat proteins, ankyrin repeat domain 52 (ANKRD52), is observed to suppress PAK1 phosphorylation. Ankyrin repeat proteins are known to function as a platform for protein-protein interactions, and ANKRD52 was shown to interact with protein phosphatase 6 (PP6). Immunohistochemistry staining in lung cancer tissue revealed prolonged disease-free survival in high ANKRD52 expressing cases. Using lung cancer cell lines, ANKRD52 knockdowns showed significantly increased cell mobility while forced expressed ANKRD52 decreased cell mobility. Using mass spectrometry analysis, p21-activated kinases (PAK1) was identified to interact with ANKRD52. Upregulated PAK1 phosphorylation was detected in the knockdowns of ANKRD52 or PP6. Our model demonstrated a novel anti-metastatic protein, ANKRD52, that regulates cell mobility through interactions with PP6 and dephosphorylation of PAK1.

#1783

A combination therapy of atypical protein kinase inhibitor and phosphatidylinositol-3- kinase inhibitor reduces multidrug resistance in renal cell carcinoma (RCC).

Khandker Mohammad Khalid. _University of South Florida, Tampa, FL_.

Renal Cell Carcinoma (RCC) is the most common type of kidney cancer (85%) of which 75% of the cases involve conventional clear cell RCC (ccRCC). Choice of treatments recommended for clear cell carcinoma and non-clear cell carcinoma are partial nephroctomy (Stage I), radical nephoroctomy (Stage II and III) and chemotherapy (stage IV). Phosphatidylinositol-3- kinase (PI3K) inhibitors are one of the most prospective candidate in the therapy of ccRCC as the PI3CA gene for p110α (a class of the catalytic sub unit of PI3K) is mutated in most of the cases. Hence, PI3K inhibitors like Alpelisib (BYL 719) can be a treatment. But current therapeutic prospects are limited to the fact that RCC is a chemotherapy-resistant cancer type that acts as a barrier for successful treatment with this inhibitor. In addition, of all the other aPKCs, PKC ί/λ and PKC ζ are reported to be frequently overexpressed in most cancer cells and to have different significant roles in tumor progression and carcinogenesis of different type of cancers. Caki-1 metastatic clear cells were treated with PKC ζ inhibitor [8-hydroxynaphthalene-1,3,6-trisulfonic acid] or ζ-stat, Alpelisib and combination of both ζ-stat and Alpelisib at different doses. The results of WST-1 shows that the combination of ζ-stat (7.5 µM) and Alpelisib (10.0 µM) has the most cytotoxicity compared to the single therapy of Alpelisib. Annexin-V/PI assay also suggested that the combination therapy caused greater percentage of apoptosis and cell death compared to the single therapy of Alpelisib. The efflux proteins, (p-glycoprotein (P-gp) and ATP-binding cassette sub-family G member 2 (ABCG2), are responsible for developing cell resistance in Caki-1 cells had reduced expression in the cells when treated with the combination treatment of Alpelisib and ζ-stat. Therefore, combination therapy of Alpelisib and an aPKC inhibitor for metastatic ccRCC can reduce expression of multi drug resistance (MDR) proteins/efflux transporter P-gp and ABCG2. Hence, this combination may be a possible treatment that might help eradicating MDR of ccRCC. Thus, a combination of Alpelisib and an aPKC inhibitor can be a two pronged approach reducing the cell proliferation and resistance.

#1784

High expression of PTK-7 regulates the growth and invasion of triple-negative breast cancer cells.

Lakshmi Reddy Bollu. _UT MD Anderson Cancer Ctr., Houston, TX_.

Background: Triple negative breast cancer (TNBC) is the most aggressive form of breast cancer with poor prognosis. Due to the frequent distant metastasis and lack of successful targeted therapies, the overall survival rate for the patients with TNBC is significantly lower than estrogen receptor positive and HER2 positive breast cancers. To identify potential druggable targets, previously, we performed a gene expression analysis and identified protein kinases that are highly expressed in ER-negative breast cancers. Through these studies, we discovered that the expression of pseudo kinase-7 (PTK-7) is highly elevated in TNBCs and correlated with p53 mutation status and disease progression. We hypothesize that high expression of PTK-7 is required for the growth and invasiveness of TNBC cells.

Experimental design and methods: In this study, we analyzed the expression status of PTK-7 in TNBC and the association of PTK-7 expression with p53 status and disease prognosis using publicly available datasets. Protein levels of PTK-7 were determined through western blotting analysis. The association of p53 and PTK-7 expression was determined using Oncomine analysis. Using specific siRNAs against PTK-7, we determined whether PTK-7 is required for the growth of TNBC cells. The role of PTK-7 in migration and invasion of TNBC cells was determined using Boyden trans-well assays.

Results: Analysis of publicly available datasets reveals that PTK-7 expression is elevated in TNBCs compared with normal breast and ER-positive breast cancers. High PTK-7 expression was found to be associated with p53 mutations. Knockdown of PTK-7 using siRNA significantly reduced the growth of TNBC cells in vitro. In vitro trans-well migration and invasion studies also demonstrated that knockdown of PTK-7 significantly reduced the migration and invasion of TNBC cells. In contrast knockdown of PTK-7 did not affect the growth and invasiveness non-TNBC cells.

Conclusion: Our results suggest that the expression of PTK-7, a critical regulator of growth and invasion of TNBCs. These results suggest that PTK-7 is a promising target for the treatment of women with TNBC. This work was supported by Susan G Komen Promise Grant (PB), SAB Komen grant (PB) and Young Foundation grant (PB).

#1785

Neprilysin: A potential regulator of PI3K/AKT signaling in triple negative breast cancer cells.

Rebecca E. Conway, Katherine Iglesias, Zuhaila Hired, Grace Rutledge, Khadija Kirmani. _Lipscomb University, Nashville, TN_.

Triple-negative breast cancers (TNBC) are among the most aggressive and therapeutically resistant breast cancers and comprise approximately 15-20% of all breast cancer cases. The lack of hormone receptor and HER2 expression makes these cancers inherently resistant to therapies targeting these receptors, yet many TNBC are also resistant to traditional chemotherapies. Thus, identifying molecular targets and predicting sensitivity to these targets is essential for the successful treatment of patients with TNBC. The phosphoinositide 3-kinase (PI3K) pathway is the most commonly mutated pathway in TNBC, suggesting that its inhibition could be a successful therapeutic approach. However, few clinical trials testing PI3K inhibitors in these breast cancers have shown sustained responses. Recent studies classifying TNBC into additional molecular subtypes have identified specific subtypes of TNBC that may be more sensitive to PI3K-targeted therapy, yet early clinical trials reveal heterogeneous patient responses within these subtypes, emphasizing the need to more completely understand the molecular mechanisms driving TNBC to better predict patient response to targeted therapies.

Recent studies in our lab implicate neprilysin, a cell-surface protease expressed on breast epithelial and stromal cells, as a potential regulator of PI3K in TNBC. We previously reported that neprilysin negatively regulates breast cancer invasion and is silenced through promoter methylation in some aggressive breast cancers. The purpose of the current study was to determine whether neprilysin is associated with cancer-related proteins in triple negative breast cancer cells. We initially conducted a bioinformatics screen of 15 TNBC cell lines to compare reverse phase protein array (RPPA) protein expression in cell lines expressing high and low levels of neprilysin mRNA. This analysis revealed 13/26 (50%) significantly differentially expressed proteins that are directly involved in PI3K signaling, suggesting a link between neprilysin and PI3K signaling. To further assess this association, we transfected a neprilysin expression vector in MDA-MB-231 triple negative breast cancer cell lines and measured phosphorylation of all AKT isoforms at T308 and of AKT1 and AKT2 at S473 using Western blots. Our results demonstrate that in MDA-MB-231 cells, neprilysin expression decreases AKT phosphorylation at T308 and decreases AKT1 phosphorylation at S473; no significant differences in AKT2 phosphorylation were observed. Together, our results suggest that neprilysin may be negatively regulating PI3K/AKT signaling, which could have important implications for sensitivity of TNBC with PI3K-pathway mutations and high neprilysin expression to PI3K-targeted therapies.

#1786

Effect of DUSP2 on pancreatic cancer lymphatic dissemination.

Shaw-Jenq Tsai, Chu-An Wang, I-Heng Chang. _National Cheng Kung University, Tainan, Taiwan_.

More than 90% of pancreatic cancers are originated from the exocrine ducts of the pancreas, which is called pancreatic ductal adenocarcinoma (PDAC). PDAC is notorious for the high early metastatic rate and low 5-year survival rate. Vascular endothelial growth factor C (VEGF-C), a member of the VEGF family, was first identified as the master lymphangiogenic factor in embryonic development. Overexpression of VEGF-C in cancers has been tightly linked to lymphangiogenesis and is highly associated with lymphatic invasion and metastasis; however, the underlying mechanism responsible for VEGF-C overexpression remains largely unknown. Constitutive activation of MAPK pathway was characterized as feature of pancreatic cancer. In genetic mouse model of pancreatic cancer, MAPK signaling is required to initiate and maintain the pancreatic intraepithelial neoplasia lesions. In normal cells, MAPKs mediated signaling is crucial to various cell functions and the activation can be balanced by the action of dual specificity phosphatases (DUSPs). Previously, we have demonstrated that hypoxia can suppress DUSP2 by which mediates tumor malignancy and drug resistance. Herein, we further identify that DUSP2 is a master regulator in controlling lymphangiogenesis and early lymphovascular invasion of PDAC. Expression of DUSP2 is silenced in PDAC, which contributes to aberrant production of VEGF-C. VEGF-C expression is controlled by DUSP2 at multiple levels including transcription, maturation, and extracellular vesicle-associated secretion. Our findings provide the proof of concept that blocking DUSP2/Exo-VEGF-C axis may serve as an attractive therapeutic approach to inhibit early dissemination of pancreatic cancer.

#1787

Selective activation of a PI3K catalytic isoform by G protein-coupled receptors in glioblastoma.

Abigail R. Winn,1 Zhi Sheng2. 1 _Virginia Tech Carilion School of Medicine, Roanoke, VA;_ 2 _Virginia Tech Carilion Research Institute, Roanoke, VA_.

Objective: To determine how PI3K catalytic isoforms become dysregulated in glioblastoma.

Background: Recurrence in glioblastoma (GBM) is common and even more resistant to treatment than initial GBM. The Sheng Lab has previously identified that PIK3CB expression levels, but not other PI3K subtypes, are higher in recurrent GBM and are associated with GBM recurrence in newly diagnosed individuals. PI3KCB encodes for PI3K catalytic subunit p110β, and PI3K/AKT pathway activation is a well-known promoter of cell survival. Understanding mechanisms of PIK3CB activation in GBM may identify new therapeutic targets and improve GBM survival. Current knowledge of PI3K isoform activity in GBM is insufficient and contradictory. Given previous studies, it was hypothesized that G-Coupled Protein Receptor (GPCR) promoters preferentially activate PI3K/AKT in PIK3CBhigh GBM cells. Further, that knockdown of PIK3CB, but not other PI3K isoforms, inhibits PI3K/AKT reactivation in PIK3CBhigh cell lines.

Design/Methods: U87MG cells, an established p110βhigh GBM cell line, were starved overnight in serum free media, and then treated with growth factors, promoters or inhibitors of several previously hypothesized pathways. Cell lysates were collected and PI3K/AKT pathway activity was measured with immunoblotting. The ratio of pAKT/AKT/ACTB quantified PI3K/AKT pathway activity.

Results: Overnight serum starvation depleted pAKT, and incubation with 1% FBS restored pAKT signaling in a time dependent manner. Neither EGF nor FGF-2 activated PI3K/AKT signaling. Gefitinib, an EGFR inhibitor, failed to inhibit reactivation when cells were co-treated with 1% FBS. However, GPCR stimulation by Lysophosphatidic acid (LPA) increased pAKT/AKT ratios in a dose dependent manner. More significantly, Gallein, a GPCR inhibitor, suppressed PI3K/AKT signaling when cells were co-treated with 1% FBS. Selective PI3K inhibitors showed little PI3K/AKT reactivation at multiple doses.

Conclusions: These results suggest that GPCR signaling is responsible for PI3K/AKT pathway activation in p110βhigh cells.

#1788

Targeting ornithine decarboxylase down-regulates multiple pathways involved in precancerous lesion formation of esophageal squamous cell cancer.

Yifei Xie,1 Qiong Wu,2 Jimin Zhao,1 Kangdong Liu2. 1 _Zhengzhou University, Zhengzhou City, China;_ 2 _Zhengzhou University & China-US (Henan) Hormel Cancer Institute, Zhengzhou City, China_.

Precancerous lesion of esophageal squamous cell cancer (ESCC) owns the characteristic of bidirectional instability, which can develop to cancer or reverse to normal epithelium by appropriate interference. Therefore, the feature of precancerous lesion provides possibility for ESCC chemoprevention. In the current study, we found that ornithine decarboxylase (ODC) protein level is higher in esophageal precancerous lesions compared with normal esophageal epithelium. Knocking down ODC suppresses cell proliferation and clone formation of ESCC. Moreover, difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, decreases N-nitrosomethylbenzylamine (NMBA)-induced rats' esophageal precancerous lesions. Further molecular study indicated that ODC down-regulates p38, ERK in the MAP kinase pathway and AKT/mTOR/p70S6K pathway, which are involved in the carcinogenesis process. Overall, our results indicate that ODC may be a potential target for ESCC chemoprevention.

#1789

The importance of the phosphatase PRL family sub-cellular localization during the cellular cycle.

Anna K. O'Leary, Jessica Blackburn. _University of Kentucky, Lexington, KY_.

Acute lymphoblastic leukemia (ALL) is one of the most prevalent cancers diagnosed in adolescents. The survival rate for ALL is good during the initial diagnosis but is much less optimistic for patients who have relapsed ALL, due to limited treatment options. Additionally, the high dose cytotoxic chemotherapies given to ALL patients have long term adverse consequences. New and less toxic targeted therapies are needed for the treatment of ALL. We previously performed a large scale transplantation screen in zebrafish to identify genes associated with high relapse potential in ALL, and a top hit from this screen was the protein tyrosine phosphatase 4A3, PTP4A3 or PRL3. PRL3 overexpression in zebrafish enhances ALL progression and penetrance, and small molecule targeting of PRL3 in human cells rapidly induces apoptosis. The mechanisms through which PRL3 drive ALL progression and survival are unknown. PRL3 has a predicted nuclear localization site as well as a predicted prenylation motif, causing its localization to the plasma membrane. My preliminary data suggest that PRL3 shifts its localization between the plasma membrane and nucleus during the cell cycle. I am currently examining the localization of PRL3 and other PRL family members during different phases of the cell cycle and identifying signaling pathways that they may be involved with at these distinct sites to promote ALL progression, using CLIP-tag and SNAP-tag technologies that will allow me to image directly PRL localization in vitro. Biotin ligase approaches (Turbo-ID) will be used to determine the substrates of PRL activity and PRL interacting partners at these distinct locations. My project will provide scientific data that supports our hypothesis that PRL3 can transition between the membrane and nucleus, and plays a role in cell cycle. Identification of substrates of PRL3 that promote ALL progression may provide novel drug targets for this disease.

#1790

**Investigation of oncogenic G-protein coupled receptor signaling pathways in** Kras **dependent pancreatic cancer cell lines.**

Ozhan Ocal,1 Raymond MacDonald,2 Rolf A. Brekken,2 Thomas Wilkie2. 1 _Bilkent University, Ankara, Turkey;_ 2 _UT Southwestern Medical Center, Dallas, TX_.

Pancreatic Ductal Adenocarcinoma (PDA) is the most common form of pancreatic cancer driven primarily by mutations in V-Ki-Ras2 Kirsten Rat Sarcoma Viral Oncogene Homolog (Kras) gene. Its most common mutation, G12D, cause KRAS to remain constitutively active. KrasG12D expressing pancreatic acinar cells undergo transdifferentiation into ductal phenotype and form early tumorigenic lesions called Pancreatic Intraepithelial Neoplasm (PanIN). Coincidentally occurring inactivating mutations of tumor suppressor genes, such as Cyclin-dependent Kinase Inhibitor 2A (Cdkn2a), Tumor Protein p53 (Tp53), and Mothers Against Decapentaplegic Homolog 4 (Smad4) trigger progression of PanINs through stages from 1A to 3, eventually leading to carcinoma in situ and metastasis.

G-protein Coupled Receptors (GPCR) are transmembrane receptors that transfer extracellular signals to intracellular heterotrimeric G-proteins with Gα and Gβγ subunits. Upon activation, dissociated Gα and Gβγ subunits separately induce intracellular second messengers for key cellular responses involved in various aspects of development, metabolism, and cell motility. GPCR signaling is negatively regulated by intracellular Regulator of G-protein Signaling (RGS) proteins that deactivate G-protein by mediating re-association of Gα and Gβγ subunits.

Although GPCRs are the largest non-antibiotic drug targets, their specific role in pancreatic cancer is not much known. GPCR signaling can influence RTK pathways in cross-talk via various second messengers. The interplay between these two major classes of cellular pathways employing both heterotrimeric and monomeric G-proteins has therefore potential to shed new light on pancreatic cancer research.

We have previously identified Rgs16 expression as a marker of pancreatic tumor formation in Rgs16::GFP o/o; Ptf1aCre/+; Lox-STOP-Lox-KrasG12D/+; Cdkn2af/f (Rgs16::GFP-KIC) mice from the onset of earliest PanINs at two weeks of age (Dis. Model Mech. 8, 1201). Using ImageJ software to quantitatively determine eGFP expression in micrographs, we have developed a rapid in vivo assay (RIVA) where we represent pancreatic tumor burden by one month of age and compare outcomes of known and novel therapeutics following two week long drug regimens. We found that just like the recently developed drug, BGB324, targeting an important RTK, called Anexelekto (Axl), the anticoagulant warfarin was more effective in reducing tumor burden than standard chemotherapy alone.

Currently, my research focus is centered on understanding active GPCR pathways that stimulate cell growth, survival, and migration by enhancement of Kras downstream signaling. My aim is comparison of the effects of general G-protein signaling inhibition in Kras mutant and wild-type cell lines, such as AsPC-1 in contrast to BxPC-3, on cell behavior and making connections to active GPCR signaling components.

#1791

RADIL regulates RAS downstream signaling.

Byeong Hyeok Choi, Ziyue Kou, Mark R. Philips, Wei Dai. _NYU School of Medicine, New York, NY_.

RAS proteins regulate a variety of cellular processes including cell survival, proliferation, and differentiation. RAS genes are among the most frequently mutated oncogenes in human malignancies. To date, there are no successful anti-cancer drugs in the clinic that target RAS proteins or their pathways. Therefore, it is imperative to identify and characterize new components that regulate RAS activity or mediates its downstream signaling. Recently, we identified a series of proteins that are associated with HRAS using a combination of immunoprecipitation and mass spectrometry. We first focused on RADIL, a gene product with Ras-Association (RA) and DIL domains. RADIL is known to be a downstream effector of RAP1, inhibiting RhoA signaling in the regulation of cell survival, adhesion, and migration. We demonstrated that RADIL interacted with all three isoforms of RAS, including H, N, and KRAS. Interestingly, constitutively active HRAS (HRASG12V) was strongly associated with RADIL whereas dominant negative HRAS (HRASS17N) displayed a compromised association with RADIL, suggesting that RADIL may be required for RAS activation. We also found that Radil Knockout cells had weakened ERK phosphorylation whereas induced expression of RADIL promoted ERK phosphorylation, suggesting that RADIL is required for the RAS-MEK-ERK signaling cascade in vitro. Taken together, our current studies strongly suggest that RADIL may be important for regulating RAS signaling.

### miRNA Profiling in Cancer

#1792

Exploring new therapeutic options for chemoresistant locally advanced lung cancer.

Roberto Cuttano,1 Elisa Dama,1 Valentina Melocchi,1 Tommaso Colangelo,1 Cristiano Carbonelli,1 Giorgia Maroni,2 Elena Levantini,2 Fabrizio Bianchi1. 1 _Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy;_ 2 _Beth Israel Deaconess Medical Center, MA_.

Background: Non-small-cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide. About one third of NSCLC patients present at diagnosis with locally advanced disease (stage IIIA) which are characterized by lymph node metastases. For this group of patients, neoadjuvant chemotherapy (NACT) followed by surgery is the best therapeutic option. However, clinical response to NACT is heterogeneous including patients with a complete eradication of lymph node metastases (pN0), and patients with persistent disease (pN2). Of note, 'pN0' patients showed a favourable prognosis (~50-60% survival, at 5-year) when compared to 'pN2' patients (~20-30% survival, at 5-year).

Methods: To identify new molecular determinants involved in NACT response, we recently performed whole-microRNA expression profiling of metastatic lung tumor cells, which were collected from mediastinal lymph nodes before NACT either by endobronchial ultrasound transbronchial aspiration (EBUS-TBNA) (n=11) or mediastinoscopy (n=41). In addition, we performed a meta-analysis of a cohort (n=84) of NSCLC (i.e. The Cancer Genome Atlas LUAD and LUSC dataset) with complete follow-up.

Results: We found a set of 20 miRNAs significantly differentially expressed in pN2 vs. pN0 tumors. Hierarchical clustering analysis using this 20-miRNA set revealed a clear separation of pN0 patients from pN2 patients. However, when the 20-miRNA set was used in the primary NSCLCs from TCGA dataset, the chemoresponsive and chemoresistant tumors could not be stratified. This possibly suggests unique properties of this 20-miRNA set and their modulated target genes in metastatic cells, and in the acquisition of a chemoresistant phenotype. Furthermore, by coupling miRNA and mRNA expression profile in metastatic samples, we rewired i) cancer intrinsic gene networks with a role in chemotherapy resistance, and ii) an augmented infiltration of tumor promoting immune cell populations (TILs).

Conclusions: Lung cancer metastases are intrinsically distinct and there exist molecular subtypes with different NACT response, identifiable by miRNA expression profile. Future efforts will be directed to identify the landscape of molecular mechanisms mediated by miRNAs, including the modulation of TILs possibly involved in the acquired chemoresistance.

#1793

Optimal quantification of a circulating miRNA profile predictive for treatment response with droplet digital PCR in patients with multi-organ metastatic colorectal cancer.

Lotte Bakkerus,1 Dennis Poel,1 Elske C. Gootjes,1 Cornelis Verhoef,2 Tineke E. Buffart,1 Hendrik M. Verheul1. 1 _Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam, Netherlands;_ 2 _Erasmus MC Cancer Institute, Rotterdam, Netherlands_.

Introduction: Presence of microRNAs (miRNAs) in blood specimens hold promise for a predictive biomarker for treatment response in clinical practice as they are relatively resistant to degradation. Previously, we identified a six miRNA expression signature from tumor tissue that predicted response to first line systemic treatment in patients with metastatic colorectal cancer (mCRC) when combined with clinicopathological factors. The six miRNA expression signature consists of miR-17-5p, miR-20a-5p, miR-30a-5p, miR-92a-3p, miR-92b-3p and miR-98-5p. The aim of this study is to determine whether circulating levels of these miRNAs will be predictive for treatment outcome as well. Therefore we first studied whether these six miRNAs can be adequately measured in blood samples of patients with mCRC undergoing first line chemotherapy. Droplet digital PCR (ddPCR) technology was used for optimal quantification of miRNAs compared to normal quantitative PCR, because of low abundance of tumor specific miRNAs in blood.

Methods: Serum samples of 120 patients with multi-organ mCRC who participated in a multi-center randomized controlled clinical trial were analyzed. RNA was isolated with miRNeasy Serum/Plasma Advanced Kit of Qiagen. Expression levels of the six miRNA expression signature was determined with ddPCR by using the miRCURY LNA miRNA PCR system combined with EvaGreen. Primer concentration and annealing temperature were first optimized per specific miRNA assay for ddPCR. RNA spike cel-miR-39-3p was used for quality control. Analysis was performed on samples with at least a minimal amount of 10.000 obtained droplets. Absolute quantification per patient is displayed as copies per µl (cop/µl) as defined by Quantasoft software.

Results: In 120 blood samples miRNAs were measured by ddPCR. The most optimal primer concentrations for the different miRNAs was 0,5 µl for cel-miR-39-3p and 1,0 µl for the other 6 miRNAs. For cel-miR-39-3p, miR-17-5p, miR-20a-5p and miR-92a-3p the most efficient annealing temperature was 53 ℃ and for miR-30a-5p, miR-92b-3p and miR-98-5p this was 58 ℃. The mean number of obtained droplets was 15.044 (SD 1922). The highest amount of detected copies were observed in miR-17-5p (644,4 cop/µl), miR-20a-5p (1037,1 cop/µl) and miR-92a-3p (1136,0 cop/µl). Lower expression levels were observed in miR-30a-5p (9,88 cop/µl), miR-92b-3p (1,6 cop/µl) and miR-98-5p (6,9 cop/µl).

Conclusion: We demonstrated that with ddPCR absolute quantification of these six miRNAs in serum samples was efficient and reliable. Further analysis will reveal whether circulating miRNA expression levels can be used as a liquid biomarker for response to first line systemic treatment in patients with multi-organ mCRC.

#1794

A comparative microRNA expression analysis in breast cancer and melanoma tissues.

Yanping Zhang, Guangyong Peng, Eddy C. Hsueh. _St. Louis Univ., St. Louis, MO_.

Recently studies have revealed that a subset of microRNAs (miRNAs) is aberrantly expressed in the development and progression of a variety of cancers including breast cancer and melanoma. Several miRNAs have been implicated in tumorigenesis and progression of human cancer. However, the underlying mechanisms in different types of cancers remain largely unknown. The aim of the study was to compare the expression profiled of miRNA between breast cancer and melanoma patient's tissues. Methods: Expression levels of 7 miRNAs (miR-15b, miR-142-3p, miR-142-5p, miR-142a, miR-199a, miR-221, miR-424, ) were determine in 17 cases of breast cancer and 25 melanoma tissue samples. MiRNA isolation from tissue samples were performed using Trizol reagent ( Sigma, USA). RNA quality and quantity were assessed using a BioMate TM 3 Series Spectrophotometers (Thermo, Madison, WI). Synthesis of cDNA with reverse transcriptase was performed by TaqManTM microRNA Reversed Transcription Kits, For analysis of miRNA expression , real-time q-PCR analyses were using TaqMan MicroRNA Assays. All real-time q-RT-PCR were performed on a 7300 real-Time PCR system (Applied Biosystems, USA). MiR-24 was used as the internal control for normalization. The relative miRNA expression levels were calculated using the 2 DΔCt methods. Values were presented as means ± standard deviation (SD). The comparison of miRNA levels in breast cancer and melanoma tissues were performed using Student's t-test. A p value less than 0.05 was considered statistically significant. The expression of miRNAs in different breast cancer subtypes (infiltrating and Invasive ductal cancer) and melanoma (primary and metastatic) was also performed. Results: Relative miRNA expression levels (miR-15b, miR-142-3p, miR-142-5p, miR-146a, miR-199a, miR-221 and miR-424) were found to be differentially expressed in breast cancer and melanoma tissues. Significantly higher relative miRNA expression levels were detected in melanoma tissues compared with breast cancer tissues in all miRNAs evaluated except for miR-424. Four miRNAs (miR-15b, miR-146a, miR-221, and miR-143-3p) had relative expressions over 1.5 compared with only 1 miRNA (miR-15b) in breast cancer tissue. DΔCt values are plotted following comparison with endogenous levels of miR-24 assessed in each sample. Conclusions: Our study demonstrated that miRNA is not only a potential biomarker, but also a valuable therapeutic target for breast cancer and melanoma.

#1795

MicroRNA sequence analysis reveals signatures of oral cancer lymph node metastasis.

Kelly Yp Liu,1 Denise Brooks,2 Reanne Bowlby,2 Steve Jones,2 Catherine F. Poh1. 1 _University of British Columbia, Vancouver, British Columbia, Canada;_ 2 _Provincial Health Services Authority, Vancouver, British Columbia, Canada_.

Regional lymph node metastasis (LN+) at the time of diagnosis or during post-treatment follow-up has devastating impact on the decimal survival of oral cancer (OC) patients. Early intervention on at-risk patient may improve survival. The purpose of the study was to identify microRNA (miRNA) signatures that contribute to LN+ using miRNA sequencing in OC surgical samples.

The study comprised a Discovery Cohort (n=91) and a Validation Cohort (n=67) of fresh-frozen primary tumors from 158 patients who were surgically treated between 2005 and 2016 with known outcome of LN (LN+, n=76; LN0, n=82). Total RNA was harvested from >70% tumoral areas. MiRNA-seq libraries were constructed from 2µg total RNA per sample followed by adaptor ligation, reverse transcription, PCR, and sequencing on HiSeq 2000 platform. All analyses were performed using R package (v3.4.4) with input matrix of the miRNAs that expressed at a level of at least 10 reads per million (RPM) in at least 10% of samples. A Least Absolute Shrinkage and Selection Operator (LASSO) penalized regression approach was used to identify miRNAs that would best predict nodal-disease free survival (NFS). Random forest classification analysis was used to rank miRNAs for classifying samples as LN0 and LN+.

Among the 158 samples, the LN+ group consisted more poorly differentiated tumors and greater depth of invasion (p=0.0001 and 0.002, respectively). To identify miRNAs that contribute to LN+, we used a penalized regression to generate a signature of NFS. The Discovery Cohort was randomly partitioned into training set (n=72; LN+, n=39, LN0, n=33) to generate a predictive model on NFS. This was then tested in the remaining 19 cases (LN+, n=12, LN0, n=7). The model composed of 2 miRNAs (miR-107 and miR-21-5p) which were significantly overexpressed in LN+. When this was tested in the Validation Cohort (n=67; LN+, 25; LN0, 42), the model was significantly associated with NFS (HR=3.4; 95% CI, 1.5-7.6; 5-year NFS, 24.5%, p=0.001). Additionally, we ranked miRNAs for classifying all samples into LN+ and LN0 by using random forest classification analysis and the two miRNAs from the penalized regression analysis remained to be the top two. The classified LN+ group had significant inferior NFS compared to classified LN0 group (HR=3.2; 95% CI, 2.0-5.1; 5-year NFS, 27.8% vs. 66.7%, p<0.0001). We further investigated diagnostic performance of the two miRNAs using receiver operating curve on all samples which had accuracy of 0.88, sensitivity of 0.82, specificity of 0.80, positive predictive value of 0.82, and negative predictive value of 0.80.

In conclusion, our data demonstrated noticeable dysregulated miRNAs that may serve as predictive biomarkers for LN+.

#1796

p53 regulation of osteoblast differentiation is mediated through specific microRNAs.

Elisha Pendleton, Shivang Shah, Oliver Couture, Teresa Kusper, Mustafa Broachwalla, Lauren Alt, Michael Fay, Nalini Chandar. _Midwestern Univ. - Downers Grove Campus, Downers Grove, IL_.

We have previously shown a role for tumor suppressor gene p53 in the transcriptional activation of several bone-specific genes in osteoblast differentiation. In this study, we used a screen for microRNAs to look for ones that were p53 dependent and differentiation dependent. In a model of invitro osteoblast differentiation, our previous work has established that p53 levels generally increase around day 4 of differentiation and represent the timeline when p53 transcriptionally regulates bone-specific gene expression. In these experiments, we used MC3T3-E1 osteoblasts to stably reduced p53 levels using specific shRNAs. These cells were initially characterized for the reduction in p53 levels and then compared with MC3T3E1 cells stably transfected with a scrambled control (Control MC3T3E1 cells). MicroRNAs were isolated from day 0 and day 4 after treatment with differentiation promoting (DP) media. A microRNA profiling service utilizing a microarray detection system analyzed over 1000 different microRNAs in control and DP treated cells to determine differentiation specific microRNAs expression. This array provided us with information about a number of genes that underwent alterations during differentiation in a p53 dependent manner. We validated several of the changes using realtime PCR and selected two microRNAs to study in detail. These two microRNAs (mIR 34b and 140) were increased two fold during normal differentiation but showed a dramatic reduction in expression when p53 levels were reduced. We ectopically expressed these microRNAs in MC3T3-E1 cells and created stable lines. Cells carrying mIR34b showed a decrease in cell proliferation rates when compared to mIR140 expressing and control cells. P53 levels directly correlated with microRNA 34b expression but not mIR140. MiR 140 ectopic expression affected Bone morphogenetic protein expression (BMP2), an important bone anabolic agent that is also p53 regulated. Putative target binding sites for bone-specific transcription factors Runx2, SP7, Vitamin D receptor (VDR) were found for mIR34b and 140 and were utilized in luciferase reporter assays to confirm microRNA specific interactions. These studies provided evidence and confirmed for us that the p53 regulation of osteoblast differentiation is also mediated through specific microRNAs that directly target important bone-specific genes.

#1797

Serum microRNAs as immune modulators and biomarkers of breast cancer recurrence.

Lauren Chen, Mithil Soni. _University of South Carolina, Irmo, SC_.

MicroRNAs (microRNAs) are short sequences of RNA (about 22 nucleotides) that are involved in the regulation of gene expression. Previous studies have suggested that a number of microRNAs are recognized as new biomarkers for cancers. The aim of this study is to identify specific microRNAs in serum, which may serve as potential diagnostic and prognostic biomarkers and therapeutic targets for breast cancer. Quantitative real-time PCR (qRT-PCR) array analyses of microRNAs in sera from four pairs of recurrent and nonrecurrent breast cancer patients were performed. Those differentially expressed microRNAs were verified in serum samples from 42 breast cancer patients. The prognostic values of the selected microRNAs were statistically analyzed, determined by the correlation between microRNA expression and tumor parameters. High serum levels of miR-134 and miR-483-5p were found to be associated with some aggressive tumor behaviors. Kaplan-Meier analysis of four upregulated microRNAs (miR-134, miR-483-5p, miR-493-3p and miR-139-3p) indicated that serum level of miR-134 can predict tumor recurrence in breast cancer patients after primary treatment. Further functional analysis showed that overexpression of miR-134 has no significant effects on tumor growth but regulates macrophage M1/M2 polarization. Overall, our data suggested that serum microRNAs may promote tumor progression and recurrence via modulating inflammation and immune response and potentially serve as blood biomarkers for prediction of recurrence in breast cancer.

#1798

Dysregulated microRNA expression in pediatric cytogenetically normal acute myeloid leukemia patients.

Vikas Gaur,1 Shilpi Chaudhary,2 Anudishi Tyagi,2 Sameer Bakhshi,2 Pankaj Sharma,1 Sachin Kumar2. 1 _Amity University, Noida, Uttar Pradesh, India, Noida, India;_ 2 _All India Institute of Medical Sciences, New Delhi, India_.

Background

MicroRNAs (miRNAs) have been implicated in various biological processes like proliferation, cell cycle, apoptosis, angiogenesis and differentiation in various human malignancies. However, their role as biomarkers in pediatric cytogenetically normal AML (CN-AML) is still unclear. We aim to identify differentially expressed miRNAs in the bone-marrow of pediatric CN-AML patients.

Methods

For this prospective study, bone marrow (BM) samples from 126 pediatric AML patients and 19 pediatric controls (with solid tumors without BM involvement) were collected from the Dept. of Medical Oncology, AIIMS, New Delhi. Mononuclear cells, isolated using Histopaque-1077 (Sigma-Aldrich), were subjected to cytogenetic analysis and genomic DNA and total RNA isolation. Out of 126 AML patients, 36 CN-AML patients were included in this study. Total RNA from 10 samples (5 CN-AML and 5 controls) was used for global miRNA profiling by small RNA sequencing on Illumina HiSeq2500 platform. After pre-processing of the raw FASTQ data, reads were mapped to mature miRNAs listed in Sanger miRBase v.20. The normalized miRNA read counts were compared between CN-AML and controls using DESeq. For all 36 CN-AML patients, cDNA was prepared using TaqMan advanced miRNA cDNA synthesis kit (Applied Biosystems) followed by qRT-PCR using TaqMan advanced miRNA assay (Applied Biosystems) for pre-selected miRNAs. Further, NPM1 and FLT3 mutation status was determined using RT-PCR. The study was approved by institutes ethics committee. Informed written consent was taken from all the subjects.

Results

Global miRNA profiling revealed differential expression of 168 miRNAs in the BM of pediatric CN-AML patients compared to controls, of which 66 were upregulated and 102 were downregulated (fold change ≥ 2; p ≤ 0.05). Interestingly 40 miRNAs belonging to human 14q32 cluster were significantly downregulated in pediatric CN-AML patients. We shortlisted 16 miRNAs based on their involvement in various signalling pathways relevant to AML and other cancers and validated them in 36 pediatric CN-AML patients with qRT-PCR. Out of 16 miRNAs 11 were found to be dysregulated in patients as compared to controls. Presently, we are correlating the miRNA expression with survival outcomes like event-free survival, disease-free survival, overall survival. Additionally, miRNA expression is also being correlated with clinical parameters (TLC, ANC, platelets, Hb, LDH, MPO) and mutation status (NPM1 & FLT3-ITD). We are also using bioinformatic tools for building a miRNA-gene regulatory network and select target genes for further validation.

Conclusion

Our preliminary findings indicate that several miRNAs are dysregulated in the BM of pediatric CN-AML patients. Since miRNAs are involved in post-transcriptional gene silencing, they might contribute to initiation or progression of CN-AML and might also affect survival outcome.

#1799

MicroRNA hsa-miR-26a expression in hepatocellular carcinoma correlates with patient survival.

Go Nakajima, Kazuhiko Hayashi. _Department of Chemotherapy and Palliative Care, Tokyo, Japan_.

Background: MicroRNAs (miRNAs) are small noncoding RNAs that act as important regulators of gene expression at a post-transcriptional level. Using a next-generation sequencer system, in a previous study, we have identified 49 miRNAs expressed differentially by comparing 26 pair samples of hepatocellular carcinoma (HCC) tumor tissue and nontumor liver tissue. For this validation study, we selected hsa-miR-26a, one of the candidate miRNAs. We measured hsa-miR-26a expression in the validation set HCC samples using real-time quantitative polymerase chain reaction (qPCR) and examined the correlation between miRNA expression and clinical data. Patients and Methods: Overall, 40 patients with HCC who underwent tumor resection from 1991 to 1997 at the Department of Surgery, Institute of Gastroenterology, Tokyo Women's Medical University, Japan were enrolled. Formalin-fixed paraffin-embedded samples were prepared using the standard protocol. The paraffin blocks were cut into 10-μm sections; HCC tumor and adjacent nontumor liver tissues were collected using laser-captured microdissection, and total RNA, including miRNA fractions, were isolated. Following the synthesis of a complementary DNA (cDNA) using an miRNA-specific primer, real-time qPCR was performed using an miRNA-specific primer/probe. Results: The hsa-miR-26a expression tended to be downregulated in HCC tumor tissue compared to nontumor liver tissue (P = 0.0799). Patients in the higher hsa-miR-26a expression group showed a significantly longer overall survival time compared with those in the lower hsa-miR-26a expression group (P = 0.0409). Conclusion: Inhibition of hsa-miR-26a expression might play a role in tumorigenesis, and hsa-miR-26a expression could be a potential prognostic factor for patients with HCC.

#1800

miR-376b and miR-4668 predict therapeutic response to sunitinib in metastatic renal cell carcinoma.

Julia Kovacova,1 Jaroslav Juracek,1 Alexandr Poprach,2 Tomas Buchler,3 Ondrej Fiala,3 Lenka Radova,1 Marek Svoboda,2 Alena Kopkova,1 Marek Vecera,1 Ondrej Slaby2. 1 _Masaryk University, Brno, Czech Republic;_ 2 _Masaryk Memorial Cancer Institute, Brno, Czech Republic;_ 3 _Charles University, Prague, Czech Republic_.

Introduction: Metastatic renal cell carcinoma (mRCC) is routinely treated with sunitinib, a tyrosine kinase inhibitor (TKI) of VEGF signalisation. Although disease eventually progresses in most mRCC patients, length of progression-free survival (PFS) is ranging from few weeks up to more than 24 months. Patients with initial resistance to sunitinib could be redirected to other therapeutical options, as there are several other TKIs available for use in second and third line. However, without a reliable biomarker no result can be predicted. MicroRNAs (miRNAs) belong to class of short non-coding RNAs and could serve as biomarkers of therapy response due to their unique character and presence in tissues and body fluids. Their biomarker potential has been discussed concerning many diseases including mRCC, but current knowledge is very weak, has several discrepancies and is acquired on relatively small cohorts.

Material and method: Candidate microRNAs have been chosen based on global expression profiling using Affymetrix GeneChip 4.0 in 47 samples of FFPE mRCC tissue of patients treated with sunitinib (good response n=25, PFS longer than 17 months; poor response n=22, PFS shorter than 9 months). Validation was performed using qRT-PCR TaqMan assays on an independent cohort of 136 FFPE samples from mRCC patients treated with sunitinib with variable length of PFS. Local ethical committees at all involved centres approved the study protocol. The study was performed according to the World Medical Association Declaration of Helsinki and all patients signed an informed consent.

Results and discussion: Of all tested miRNAs, expression of miR-4668-5p and miR-376b was the most significantly deregulated in non-responding patients with high statistical significance (p>0,005) and combined AUC higher than 0,8. Although other independent validations are necessary, microRNAs presented here seem to be very promising as tools for therapy personalization.

Conclusion: We have successfully validated miR-4668-5p and miR-376b to have predictive potential in estimation of therapeutical response to sunitinib. Further functional analyses could shed more light on their involvement in development of therapy resistance. This work was supported by Ministry of Health of the Czech Republic, grant nr. 15-34678A.

#1801

MicroRNA expression profiling predicts clinical outcome in patients with locally advanced larynx and hypopharynx cancer treated with chemoradiotherapy.

Dennis Poel, Francois Rustenburg, Daoud Sie, Hendrik F. van Essen, Paul P. Eijk, Elisabeth Bloemena, Teresita E. Benites, Bauke Ylstra, Brakenhoff H. Ruud, René C. Leemans, Tineke E. Buffart, Henk M. Verheul, Jens Voortman. _Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam, Netherlands_.

Background

For patients with locally advanced larynx and hypopharynx cancer, organ preservation chemoradiotherapy (CRT) protocols are frequently applied. However, some tumors are resistant to CRT or will recur within a short time frame. For these patients upfront surgical resection may result in an improved cure rate. Currently, there are no molecular markers that can predict CRT response. In this study, tumor microRNA (miRNA) profiling was performed to predict clinical outcome in patients with locally advanced larynx or hypopharynx cancer treated with CRT.

Methods

First, matched fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) tumor biopsies from 12 patients were profiled by Next Generation Sequencing (NGS) to ensure valid miRNA results from FFPE. Next, miRNA profiles of FFPE tumor biopsies from 37 patients with larynx or hypopharynx cancer, treated with primary CRT, were determined. Clinicopathological data, including response to CRT, tumor stage and nodal stage, was collected of all patients. Differential expression analysis was performed to compare patients with poor clinical outcome, i.e residual tumor after treatment or progression free survival (PFS) < 2 years versus patients with good clinical outcome, i.e. complete response and PFS > 2 years. The median follow up of this cohort was 60 months (range 6 - 118). The Cancer Genome Atlas (TCGA) head and neck miRNA expression data of larynx and hypopharynx tumors and normal squamous cell tissues were used to select tumor specific miRNAs. By group-regularized penalized logistic ridge regression analysis, a model to predict clinical outcome was generated using tumor specific miRNAs combined with clinical covariates.

Results

In total, 324 novel candidate and 2,262 mature miRNAs were detected. A comparative analysis of miRNA profiles generated using matched FF and FFPE tumor biopsies showed a high correlation (mean r2=0.75). Using only FFPE samples, 13 miRNAs were significantly differentially expressed between patients with good versus poor clinical outcome (p<0.05, FDR<0.1). Class prediction resulted in a model of three miRNAs combined with AJCC stage, able to predict recurrence with an area under the curve of 0.83. Further validation of the model is ongoing in an additional cohort of patients with larynx or hypopharynx cancer treated with CRT.

Conclusion

NGS miRNA profiling using FFPE tumor biopsies from patients with head and neck cancer is technically feasible, resulting in high quality miRNA expression data comparable to fresh frozen tissue. Based on miRNA expression profiling, candidate miRNAs were identified predictive for clinical outcome following treatment with CRT. A validation study is ongoing.

#1802

Exosomal miRNA as a non-invasive prediction marker of normal tissue toxicity after radiotherapy for prostate cancer.

Pratip Rana,1 Preetam Ghosh,1 Mitchell S. Anscher,2 Ross B. Mikkelsen,3 Vasily A. Yakovlev3. 1 _Virginia Commonwealth University, Richmond, VA;_ 2 _UT, MD Anderson Cancer Center, Houston, TX;_ 3 _VCU Massey Cancer Ctr., Richmond, VA_.

Introduction: A potential limiting feature for radiotherapy (RT) in the treatment of cancer is the toxicity that arises due to damage to normal tissues as a result of the radiation. This risk of toxicity is a particular concern in men with localized prostate cancer. Radiation-induced proctitis (RIP) is a common adverse effect of RT in treatment for prostate cancer. RIP limits radiation dose and treatment volumes, interrupts treatment, and lowers patients' quality of life.

Exosomes are 30-150 nm wide nanovesicles originating from the endosomal network and are found in most body fluids. Exosomes are a fundamental driver of intercellular communication by transferring proteins, lipids and miRNA. Exosomes miRNA signatures may serve as non-invasive prediction biomarkers of late toxicities such as RIP.

Methods: Twelve prostate cancer patients (pts) treated with curative intent with different grades of post-irradiation toxicity were enrolled on an IRB approved study. Pts were evaluated prospectively weekly during RT and at prescribed intervals following completion of RT for the development of toxicity using a standardized instrument of physician reported toxicity (CTCAE v4). Blood samples were collected one day before and one year after RT. The 101Bio kit was used to isolate exosomes from plasma of pts. For the miRNAs expression profiling we used ExiLENT SYBR® Green Maser Mix (Exiqon). The expression of 179 miRNA commonly found in human serum/plasma were analyzed by Serum/plasma PCR Panel (QIAGEN) for each sample.

Results: Exosomes miRNAs were analyzed from the plasma of prostate pts divided into two groups: (i) Low toxicity grade 0-1 (6 pts), and (ii) High toxicity grades 2-4 (6 pts). Out of the 179 miRNAs analyzed, three miRNAs exhibited differential expression at post-RT compared to pre-RT for low toxicity pts (LTP): miR-132-5p (up-regulated, p=0.001), miR-23a-3p (down-regulated, p=0.020) and miR-1-3p (up-regulated, p=0.047). Four miRNAs exhibited differential expression at post-RT compared to pre-RT for high toxicity pts (HTP): miR-132-5p (down-regulated, p=0.003), miR-197-3p (up-regulated, p=0.017), miR-151a-5p (up-regulated, p=0.031), miR-18b-5p (up-regulated, p=0.020). The most interesting results were observed with miR-132-5p. Before RT it showed significantly higher expression in HTP compared to the LTP. However, after RT miR-132-5p expression showed 10.76-fold increase in LTP and 7.87-fold decrease in the HTP. As a result of these dynamic changes, expression of miR-132-5p after RT became significantly higher in LTP compared to the HTP.

Conclusions: Our investigation has revealed miR-132-5p as a potential marker for risk of significant rectal injury following RT for prostate cancer, and changes in the expression of miR-1-3p, miR-18b-5p, miR-23a-3p, miR-151a-5p, and miR-197-3p may reflect the ongoing inflammatory process responsible for radiation induced rectal injury.

#1803

Openarray profiling of the systemic change of microRNAs in rhenium-188 liposome treated human head and neck cancer cells.

Bing-Ze Lin,1 Chun-Yuan Chang,2 Chih-Hsien Chang,3 Yi-Jang Lee4. 1 _National Yang-Ming Univ., Taipei, Taiwan;_ 2 _Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, Taiwan;_ 3 _Isotope Application Division, Institute of Nuclear Energy Research, Taoyuan, Taiwan;_ 4 _Biophotonics and Molecular Imaging Research Center (BMIRC), National Yang-Ming University, Taipei, Taiwan_.

Purpose188Re-liposome has been used for evaluating the theranostic efficacy in various human cancers. However, whether the therapeutic efficacy of 188Re-liposome directly related to microRNAs regulation and specific signaling pathways are largely unknown. In this project, we investigated the 188Re-liposome induced systemic microRNA expressive responses in human head and neck cancer (HNSCC) cells.

Methods

The FaDu-3R cells were implanted into the nude mice at the position of buccal cavity. After two weeks of implantation, the tumors were palpable and the bioluminescent imaging indicated the location of this orthotopic tumor model. The PEGylated 188Re-liposome was intravenously injected single and dual dose respectively. We harvested all of the tumors after 188Re-liposome injection one month ago, and used Taqman Openarray MicroRNA (Thermo, USA) to detect the expression of miRNAs in different groups. The dataset we observed was further analyzed by using Ingenuity Pathway Analysis (IPA) software to establish the molecular signaling pathway of 188Re-liposome radiopharmaceutical.

Results

Quantification of the photon signals showed that the tumor growth of the dual 188Re-liposome treated mice was slower than that of control ones. The survival rate of 188Re-liposome treated animals were also better than control ones. The microRNA Openarray showed that, compared to untreated control, 17 miRNA were significantly changed in expressive levels over 5 fold after 188Re-liposome treatment. Subsequently, we used IPA to determine the role of these miRNA, and found these miRNA with oncogenic and tumor suppressive properties were down-regulated and up-regulated, respectively. Among them, we focused on miR-152-5p that is closed related to cancer occurrence and development, and showed that knockdown of miR-152-5p could decreased the effects of 188Re-liposome on suppressing the growth of HNSCC xenograft tumors.

Conclusion

HNSCC tumor model treated with dual dose of 188Re-liposome exhibited significant response to this radiopharmaceutical. We also found that 17 miRNA could be gsignificantly re-regulated, and miR-152-5p is one example to be involved in mediating the efficacy of 188Re-liposome. Targeting on specific miRNA may enhance the application value of 188Re-liposome.in cancer treatment.

#1804

Role of microRNAs in colorectal adenoma progression.

LaShanale M. Wallace,1 Aneese F. Chaudhry,1 Karen I. Aikhionbare,2 Xuebiao Yao,1 Felix O. Aikhionbare1. 1 _Morehouse School of Medicine, Atlanta, GA;_ 2 _Harrison High School, Kennesaw, GA_.

Colorectal cancer (CRC) survival rates are stage related and majority of patients (75%) with CRC have sporadic of the disease which is in form of adenoma. Most of colorectal adenocarcinomas precursors are adenomatous polyps. Moreover, CRC appears to be an age related disease and studies have shown that mutations in mtDNA appear to accumulate with age because of inadequate repair. Also, oxidative stress has been shown to affect mitochondrial proteins resulting in a decrease of mitochondrial function associated with the precursor lesions of cancer. Recent findings suggest that the mitochondrion has sequences that code for certain miRNA which can regulate different pathways frequently dysregulated in cancer including colorectal cancer (CRC). Isolation of mitochondria RNA from colorectal precancerous polyps, cancer, and normal adjacent tissue samples was performed and assessed for gene expression differences using quantitative reverse transcription PCR. We demonstrate that mitochondria genome expression of miRNAs miR-24, miR 181, miR 210, and miR 21 in colorectal tissue samples varies during disease progression. Expression of microRNAs 24, 181, and 210 progressively increased from the earliest precancerous polyp to cancer. In addition, miR-210 and miR-181 expression increased 3 folds in villous adenomas when compared to tubular adenoma. Meanwhile, miR 21 increased progressively in adenoma tissues but decreased almost 4 folds in cancer tissues when compared to villous adenoma tissue. These results suggest miRNAs within the mitochondria genome may regulate important mitochondrial functional pathways leading to a more favorable environment for transformation or progression of adenomas to cancerous tumors.

#1805

Integrative analysis of microRNAs in blastic plasmacytoid dendritic cell neoplasm.

Maria Rosaria Sapienza,1 Manuela Ferracin,1 Fabio Fuligni,2 Federica Melle,3 Giovanna Motta,3 Maria Antonella Laginestra,4 Maura Rossi,1 Luciano Cascione,5 Alessandro Laganà,6 Claudio Agostinelli,1 Elena Sabattini,4 Alessandro Pileri,1 Carlo Maria Croce,7 Stefano Aldo Pileri3. 1 _University of Bologna, Bologna, Italy;_ 2 _The Hospital for Sick Children, Toronto, Ontario, Canada;_ 3 _Istituto Europeo di Oncologia, Milano, Italy;_ 4 _S.Orsola-Malpighi Hospital, Bologna, Italy;_ 5 _Institute of Oncology Research, Bellinzona, Italy;_ 6 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 7 _Ohio State University, Columbus, OH_.

Background:

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an extremely rare and aggressive hematological disease, deriving from the malignant transformation of plasmacytoid, alpha-interferon producing dendritic cells (pDCs). Recent studies shed new light on BPDCN genomic and trascriptomic alterations, but the microRNA (miRNA) profile is still largely unknown.

We analyzed the miRNA expression profile of BPDCN patients to enhance our understanding of the molecular mechanisms driving this disease and to identify new potential diagnostic and therapeutic biomarkers.

Methods:

We performed the miRNA profiling (NanoString Technologies) of 26 BPDCN patients and 4 normal pDCs samples as controls. To better investigate the functional consequences of miRNAs dysregulation, 9 BPDCN patients, were also analyzed at the transcriptomic level.

We applied a Moderated t-test to find genes and miRNAs differentially expressed between BPDCNs and pDCs. The most deregulated miRNAs and mRNAs were validated by qRT-PCR and immunohistochemistry and analyzed by miRNET, a bioinformatic tool for statistical analysis and functional interpretation of miRNA and mRNA data.

Results:

We found that, according to the supervised clustering analysis of miRNAs, tumor samples display a molecular signature well distinct from their normal counterpart. Indeed, BPDCN patients expressed a set of 175 miRNAs significantly deregulated and potentially involved in essential biological processes and therefore in malignant transformation. Thus, to evaluate the impact of these miRNAs on the BPDCN transcriptome, miRNAs and mRNAs expression profiles were analyzed by miRNET tool.

Thanks to this integrative approach, we uncovered the most relevant miRNA-mRNA networks in BPDCN setting and in particular we identified 9 up-regulated hub miRNAs, targeting multiple genes and synergistically inter-connected: hsa-mir-93-5p, hsa-mir-106b-5p, hsa-mir-19b-3p, hsa-mir-19a-3p, hsa-mir-21-5p, hsa-mir-181a-5p, hsa-mir-25-3p, hsa-mir-155-5p, hsa-mir-17-3p (in order of relevance). These 9 hub miRNAs regulate the expression of genes already described as relevant in BPDCN patients (ex. TCF4, RHOA, EP300) and, according to functional enrichment analysis, that could aberrantly interfere with TLR signaling, protein translation and DNA transcription regulation.

Of interest, most of these hub miRNAs are classified as oncomir (OncoMir Cancer Database) and, if validated in an extended number of cases, promising targets for anti-miRNA based therapy.

In conclusion, we identified a panel of miRNAs that regulate relevant cancer-related pathways and can be also used as new potential biomarkers and therapeutic targets in BPDCN.

#1806

Global analysis and validation of miRNA levels in cerebrospinal fluid of brain tumor patients.

Alena Kopkova,1 Jiri Sana,1 Marek Vecera,1 Tana Machackova,1 Pavel Fadrus,2 Julia Kovacova,1 Ondrej Slaby1. 1 _Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic;_ 2 _Department of Neurosurgery, University Hospital Brno, Masaryk University, Brno, Czech Republic_.

Introduction. Incidence rate of primary brain tumors and brain metastases counts yearly around 40 patients per 100 000 persons in the world and is still growing. Prognosis and therapy differs between brain tumor types and, therefore, accurate and early diagnosis might improve survival and life quality of patients. However, current diagnostic approaches are limited by localization and tissue heterogeneity of brain tumors. Cerebrospinal fluid (CSF) bathes central nervous system (CNS) and thus is supposed to reflect all pathological conditions. From this perspective, CSF looks as ideal source of diagnostic biomarkers of brain tumors. MicroRNAs, short non-coding RNAs involved in the pathogenesis of many cancers including brain tumors, might represent group of new biomarkers. In addition, dysregulated levels of brain tumor specific miRNAs have been already observed also in CSF. Following these facts, analysis of CSF miRNAs in brain tumor patients promises a new diagnostic approach enabling more accurate diagnosis.

Material and methods. Next-generation sequencing was performed for analysis of small RNAs in 89 CSF samples taken from 32 GBM, 14 low-grade glioma (LGG), 11 meningioma, 13 brain metastasis patients and 19 non-tumor donors. CleanTag Small RNA Library Prep Kit (TriLink BioTechnologies) was used for cDNA library preparation. NextSeq 500 instrument together with Next 500/550 High Output v2 Kit - 75 cycles (both Illumina) were used for final sequencing analysis. Subsequently, according to NGS results we measured levels of 13 miRNAs in independent set of CSF samples (35 GBM, 42 meningiomas, 8 metastasis patients and 19 control) using TaqMan Advanced miRNA Assays (ThermoFisher Scientific).

Results. NGS analysis revealed 25, 3, 2 and 14 CSF miRNAs significantly differently expressed in GBM, meningiomas, LGG and metastasis patients (p < 0.001, for LGG p < 0.05), respectively, in comparison with control CSF samples. In addition, 6 miRNAs showed different levels between GBM and LGG (p < 0.05). Subsequent validation of selected CSF miRNAs has confirmed different levels of 7 miRNAs in GBM (miR-10a, miR-196a, miR-196b, miR-30e, miR-30c, miR-7b, miR-7c), 3 in meningioma (miR-140, miR-21, miR-30e) and 2 in brain metastasis (miR-30e and miR-7c) compared to control CSF samples (p < 0.05). Validation of miRNA levels in LGG CSF samples is in progress and will be part of the poster presentation.

Conclusion. We have observed that CSF from patients with various brain tumors (GBM, LGG, meningioma, and brain metastasis) is characterized by specific miRNA signature. Our results suggest potential of CSF miRNAs to be useful biomarkers in brain tumors. This work was supported by Ministry of Health of the Czech Republic grant nr. NV18-03-00398, grant of Czech Grant Agency nr. 17-17636S and by the Ministry of Education, Youth and Sports of the Czech Republic under the project CEITEC 2020 (LQ1601).

#1807

miRNA alterations associated with transition of advanced castration-resistant prostate cancer to neuroendocrine prostate cancer.

Divya Bhagirath, Thao Yang, Laura Tabatabai, Shahana Majid, Rajvir Dahiya, Yuichiro Tanaka, Sharanjot Saini. _Veterans Affair Medical Center, San Francisco and University of California San Francisco, San Francisco, CA_.

Neuroendocrine prostate cancer (NEPC) is an aggressive variant of advanced prostate cancer (PCa) present in ~30% of metastatic castration-resistant tumors, often emerging as a result of AR-targeted therapies such as enzalutamide. NEPC arises via a reversible trans-differentiation process, referred to as neuroendocrine differentiation (NED), wherein cells undergo a lineage switch to an androgen-independent state and exhibit neuroendocrine (NE) features, such as expression of neuronal markers. Clinically, NEPC manifests as the presence of visceral metastatic disease, low serum PSA levels relative to disease burden and limited response to AR signaling inhibitors. The molecular mechanistic basis of NEPC is poorly understood contributing to the lack of effective therapies and robust molecular markers for its diagnosis and prognosis. A few studies have characterized NEPC tumors at a molecular level and identified key genomic events promoting NE states including frequent TMPRSS2-ERG rearrangements, RB1/TP53 loss, amplification/overexpression of Aurora kinase A, N-myc and EZH2. Though these studies have characterized the key genomic lesions associated with NEPC, the role of microRNAs (miRNAs) in NED have not been systematically explored. Considering the important regulatory role of miRNAs, this is a critical drawback contributing to a poor understanding of molecular basis of NEPC. We hypothesize that this switch is associated with significant alterations to the miRNAome, that in turn, drives switching of cellular gene expression patterns towards NE states and in this study, we sought to define the miRNA alterations associated with progression of advanced castration-resistant prostate cancer (CRPC) to androgen-independent neuroendocrine state. We tested our hypothesis by assessing miRNA alterations associated with transition from CRPC with adenocarcinoma features to that with NE features by performing small RNA-NGS in patient samples and in cellular models. Our data suggests that transition to NE states is associated with significant miRNA alterations such as downregulation of miRNAs belonging to miR-379-410 cluster (located on chromosome 14q32) and miR-17/92 cluster (located on chromosome 13q31). Further, our analyses identified that NEPC is associated with upregulation of transcription factors (TFs) SP1, SP4 and POU domain TFs such as POU2F1 (Oct1), POU3F3 (BRN3), POU3F4 (BRN4), in addition to already reported upregulation of POU3F2 (BRN2). We found that miRNAs belonging to miR-17-92 and 379-410 clusters potentially regulate expression of POU domain TFs. To conclude downregulation of these miRNAs upon NED induction, may lead to upregulation of key NE-associated TFs, that in turn, drives the expression of neuronal genes associated with neuroendocrine differentiation states in prostate cancer. 

### Noncoding RNAs 1

#1808

**lncRNA,** PVT-1, **enhances tumorigenesis and cancer stem-like phenotypes in osteosarcoma.**

Susan Tsang,1 Nino Rainusso,1 Lyazat Kurenbekova,1 Motonari Nomura,2 Jason Yustein1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Osaka University, Osaka, Japan_.

Osteosarcoma is the most common pediatric bone cancer and a key genetic characteristic of this particular malignancy is its complex karyotype. Specifically it has been reported that 15-20% of osteosarcoma patients' present with 8q24 amplification. The presence of this specific amplification has been previously associated with a high rate of relapse and poor prognosis for osteosarcoma patients. Within this amplicon resides, a long non-coding RNA gene, PVT-1. Prior studies indicates that PVT-1 has pro-oncogenic properties however the function of PVT-1 in osteosarcoma is not well characterized. To understand PVT-1 copy number Fluorescent In Situ Hybridization was performed on both osteosarcoma cell lines and osteosarcoma patient-derived xenografts. Both models presented a subset of samples with chromosomal balance or amplification of PVT-1.To test PVT-1 pro-oncogenic role in osteosarcoma, several functional assays were performed. Our studies demonstrated that overexpression of PVT-1 in osteosarcoma cell lines promotes multiple tumorigenic behaviors including enhanced proliferation, migration, and invasion. Chemosensitivity assays using osteosarcoma cell lines treated with either doxorubicin or cisplatin correlated lower expression of PVT-1 to increased osteosarcoma sensitivity to cisplatin. However this trend was not seen with treatment of doxorubicin.

PVT-1's ability to mediate metastasis and contribute to chemotherapeutic sensitivity is a shared phenotype of cancer stem cells. Based on this observation, we hypothesize targeting PVT-1 will reduce cancer stem-cell properties. Initial studies were done to test how the increase of PVT-1 regulates cancer stem cell genes. Osteosarcoma lines with increased levels of PVT-1 exhibited higher expression of Nanog, SOX2, c-Myc and Oct4 at both the transcriptomic and proteomic level. Further functional analysis showed increased spheroid formation in osteosarcoma lines with elevated levels of PVT-1. Additional molecular studies were performed in order to gain additional insights into potential mechanism of action for PVT-1 including Reverse Phase Protein Array. Initial analysis suggest a role for PVT-1 in regulating both PI3K-AKT-mTOR and serine synthesis pathways. This suggests two potential oncogenic pathways in which PVT-1 enhances cancer stem cell phenotypes. On-going investigations are addressing potential pathway inhibitors which could be utilized to regulate PVT-1 mediated tumorigenic roles and cancer stem-like properties.

#1809

LncRNA-SOX2-OT contributes to head and neck cancer cell proliferation by regulating DDIT4.

Ru Wang, Yifan Yang, Chen Tan, Ling Feng, Jugao Fang. _Beijing Tongren Hospital, Capital Medical University, Beijing, China_.

Background: Long non-coding RNAs (lncRNAs) have been shown to serve as an oncogenic role in human cancers. However, the role of lncRNAs in head and neck squamous cell carcinoma (HNSCC) is still unclear.

Methods and Results: Our previous microarray analysis showed that lncRNA SOX2-OT was upregulated in HNSCC tumor tissues and was correlated with DDIT4. To determine whether SOX2-OT served as a poor prognosis factor and targeted DDIT4 to facilitate cell proliferation and tumorigenesis in human HNSCC, qRT-PCR tested the relative expression of SOX2-OT and DDIT4 in 60 pairs of LSCC cancer tissues and the corresponding adjacent non-cancer tissues. Cell functional assay was conducted in vitro. We validated the up-regulation of SOX2-OT and DDIT4 in HNSCC tissues. Furthermore, we found that high SOX2-OT expression was significantly associated with advanced tumor differentiation, and worse prognosis. In vitro experiments demonstrated that SOX2-OT functioned as an oncogene by promoting cell proliferation and inhibiting cell apoptosis, and SOX2-OT targeted DDIT4 as its competing endogenous RNA in HNSCC cells.

Conclusions: Our findings for the first time identify the oncogenic role of SOX2-OT in HNSCC, demonstrating that SOX2-OT is a potential prognostic biomarker and promising therapeutic target in HNSCC.

#1810

LncCGU1 and AFP serve as important prognostic marker in non-hepatitis B non-hepatitis C hepatocellular carcinoma.

Kwang-Huei Lin,1 Yang-Hsiang Lin,2 Yu-Chin Liu1. 1 _Chang Gung Univ., Taoyuan, Taiwan;_ 2 _Chang Gung Memorial Hospital, Linkou, Taoyuan, Taiwan_.

Background: Thyroid hormone (T3) and its receptor (TR) are involved in cell metabolism and cancer progression. Hypothyroidism is a significantly elevated risk factor for hepatocellular carcinoma (HCC). Alpha-fetoprotein (AFP) is increased in the majority of patients with HCC and may be useful in the diagnosis and follow-up of cases of HCC. The relationship between T3/TR and AFP levels in HCC is still unclear. Design: We used microarrays to compare expression profiles of lncRNAs in HepG2-TRα1 cells treated with/without T3 and HCC specimens. The profiling results were validated using qRT-PCR. The overall survival and recurrence-free survival were analyzed using Kaplan-Meier method and further confirmed by online available datasets. Results: In the current study, long non-coding RNA CGU1 was downregulated by T3/TR in vitro. Notably, α-fetoprotein (AFP) mRNA levels were downregulated by T3/TR through repression of CGU1. Notably, AFP mRNA is strongly positively correlated with CGU1 and unfavorable prognosis in non-hepatitis B/non-hepatitis C HCC (NBNC-HCC) group patients. Conclusions: our data collectively suggest that T3/TR/ CGU1/AFP act as a potential prognostic marker in NBNC-HCC.

#1811

The long noncoding RNA KIAA0125 is aberrantly expressed in ameloblastomas.

Silvia F. Sousa,1 Marina G. Diniz,1 Josiane A. França,1 Fabricio A. Vilas-Boas,1 Fabricio T. Souza,1 George A. Calin,2 Ricardo S. Gomez,1 Carolina C. Gomes1. 1 _Universidade Federal de Minas Gerais, Belo Horizonte, Brazil;_ 2 _The University of Texas, Houston, TX_.

Long noncoding RNAs (lncRNAs) are transcribed RNA molecules that can interact with DNA or RNA, transcription factors, histones and other chromatin modifying proteins, affecting the expression level of a broad spectrum of genes. Because of their tissue-specific expression characteristics, lncRNAs hold strong promise as novel biomarkers and therapeutic targets for diseases. High-density whole-genome microarray analysis in ameloblastomas, an aggressive odontogenic tumor, and in adenomatoid odontogenic tumor (AOT), an indolent one, showed a high frequency of copy number alteration at chromosome band 14q32.33, which encompasses the lncRNA gene KIAA0125. To understand molecular mechanisms associated with their biological behavior and considering the copy-number gains observed at 14q32.33, we aimed to investigate the expression levels of the lncRNA KIAA0125 in ameloblastomas and in AOTs. The University Ethics Committee approved this study and patients signed informed consent. Thirteen frozen samples were included: five solid/multicystic ameloblastomas, four AOT, and four dental follicles. qPCR reactions were performed in triplicates and the relative changes in gene expression were obtained using the 2^-ΔΔCt formula. The reference gene HPRT1 was used for normalization and the human keratinocyte HaCat cell line was used as calibrator. Differences in the relative changes in gene expression between two groups of samples were assessed and the significance level was set at 0.05. All samples expressed more KIAA0125 than the calibrator. The ameloblastoma group showed higher expression levels of KIAA0125 when compared to dental follicles (p=0.042), while there was no difference between the expression in ameloblastomas and AOT (p>0.05). The expression levels of KIAA0125 in AOT were not different from that of the dental follicle. Our findings suggest that lncRNA KIAA0125 is likely involved in ameloblastoma pathobiology. LncRNAs hold strong promise as therapeutic targets, and experimental validation of this lncRNA functions may lead to tailored therapies targeting KIAA0125 in extensive and recurrent ameloblastomas.

#1812

LncRNAs as prognostic biomarkers in head and neck squamous cell carcinoma (HNSCC).

Ritu Chaudhary, Xuefeng Wang, Biwei Cao, Nicholas T. Gimbrone, Robbert JC Slebos, Christine H. Chung. _Moffitt Cancer Center & Research Institute, Tampa, FL_.

Long noncoding RNAs (lncRNAs) are transcripts that are greater than 200 nucleotides in length and do not encode proteins in contrast to mRNAs. They regulate a variety of cellular processes including cell proliferation and metastasis, and they have a role in cancer development. To identify lncRNAs that may have biological significance in head and neck squamous cell carcinoma (HNSCC), we globally profiled dysregulated lncRNAs in HNSCC. We analyzed RNA-seq data from HNSCC and paired adjacent normal tissue obtained from The Atlas of Noncoding RNAs in Cancer (TANRIC) database. Of 3576 lncRNAs, we identified 927 lncRNAs to be significantly (FDR <0.05) and differentially regulated by 2-fold among the HNSCC samples (upregulated-688, downregulated-239). Next, to evaluate the potential function of the lncRNAs, we performed lncRNA-mRNAs co-expression analysis for the top 10 upregulated lncRNAs and found that three of the lncRNAs (LINC01614, LINC02081, and LINC00460) are associated with cancer-related biological pathways including extracellular matrix organization, cell adhesion, and collagen catabolism. TP53 mutation status and presence of human papillomavirus (HPV) have been frequently associated with the survival in HNSCC. Therefore, we investigated differential expression of lncRNAs in 426 HNSCC based on TP53 mutation and HPV status. We identified 135 lncRNAs (upregulated-105, downregulated-30) to be significantly (FDR <0.05) and differentially regulated by 2-fold among the mutant vs wild-type TP53 samples. Among HPV-negative vs positive samples, we identified 714 differentially regulated lncRNAs (upregulated-203, downregulated-511). Interestingly, we found that LINC01614, LINC02081, and LINC00460 were also upregulated in mutant TP53 and HPV-negative HNSCC compared to wild-type TP53 and HPV-positive HNSCC. Survival analysis of the HNSCC patient data revealed that higher expression of LINC01614, LINC02081, and LINC00460 were significantly associated with poor overall survival. In addition, we were able to validate that higher expression of LINC02081 and LINC00460 were associated with poor overall survival in an independent data of 130 HNSCC generated at Moffitt Cancer Center. In summary, we identified lncRNAs that are differentially expressed in HNSCC compared to the normal tissue and may have a role in cancer development and metastasis pathways. LINC02081 and LINC00460 lncRNAs are associated with poor prognosis. Further evaluation of these lncRNAs is warranted to understand the mechanism and to potentially develop prognostic biomarkers in HNSCC.

#1813

Depletion of PCAT1 in head and neck cancer cells limits tumor growth by regulating c-Myc, AKT1 and MAPK signalling pathways.

Subhayan Sur, Robert Steele, Ratna B. Ray. _Saint Louis University, Saint Louis, MO_.

Head and neck squamous cell carcinoma (HNSCC) represents one of the most common malignancies worldwide with a high mortality rate mainly due to lack of early detection markers, frequent metastasis and aggressive phenotype. Recently, long non-coding RNAs (lncRNAs) have been shown to have important regulatory roles in human cancers. Up-regulation of prostate cancer-associated transcript 1 (PCAT1) in TCGA dataset of HNSCC was noted. In this study, we aimed to evaluate role of the PCAT1 in HNSCC. During validation, we observed up-regulation of PCAT1 in HNSCC patient samples and cell lines. siRNA mediated knock-down of PCAT1 significantly reduced cell proliferation, sphere formation and wound healing in HNSCC cell lines (JHU29 and Cal27). Mechanistic study revealed a significant down-regulation of c-Myc and AKT1 in both RNA and protein levels upon knockdown of PCAT1 in HNSCC cell lines. In-silico analysis suggested a significant positive correlation between PCAT1 and c-Myc or PCAT1 and AKT1 in HNSCC cell lines. Subsequently, we observed modulation of MAPK signalling by activation of p38 and apoptosis signal-regulating kinase 1 (ASK1) upon knockdown of PCAT1, and induction of apoptosis as evident of caspase 9 activation and PARP cleavage. Introduction of PCAT1 into HNSCC xenograft tumors displayed a reduction in tumor volume. Together our data demonstrated an important role of the PCAT1 in HNSCC progression and has potential as a therapeutic candidate.

#1814

Carboxyl-terminal truncated HBx represses lncRNA-LOC to promote cell metastasis.

Yang Hsiang Lin,1 Chau-Ting Yeh,1 Kwang-Huei Lin2. 1 _Liver Research Center, Tao-Yuan, Taiwan;_ 2 _Department of Biochemistry, College of Medicine, Tao-Yuan, Taiwan_.

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancy in the world. The poor prognosis of HCC is partly due to a high incidence rate of extrahepatic metastases. Several studies have shown that the relative risk of HCC among hepatitis B virus (HBV) carrier is higher than non-carrier group. Among the four proteins translated by HBV, X-gene (HBx) has been correlated with oncogenesis of HCC. Meanwhile, it is well known that random HBV genome integration can cause truncation of the HBV genome, especially on the HBx gene locus at the C-terminus (designated as Ct-HBx). The discovery of long non-coding RNA (lncRNA) transcripts has dramatically expanded our understanding of cell biology, especially in cancer. However, the underlying molecular mechanisms of lncRNAs that mediate the metastatic cascade and cancer progression remain largely unclear in liver cancer. In this study, lncRNA-LOC is significantly downregulated in HCC and negatively correlated with overall survival in HCC patients. LncRNA-LOC is negatively regulated by Ct-HBx in vitro and in vivo (HCC specimens). Two novel transcripts of LncRNA-LOC in HepG2 and Huh7 cell lines were identified by 5' and 3' rapid amplification of cDNA ends assays, respectively. Overexpression of lncRNA-LOC induced marked inhibition of cell migration, invasion and cell proliferation in vitro. On the other hand, these phenotypes were reversed upon knockdown of lncRNA-LOC in cells. Mechanically, levels of p-STAT3 (Tyr705 and Ser727), p-c-Jun (Ser63) and Snail were decreased in lncRNA-LOC-overexpressing cells, compared to control cells. Our data collectively highlight that novel regulatory associations among lncRNA-LOC, Ct-HBx, p-STAT3, p-c-Jun and Snail are an important determinant of metastasis in HCC cells and support the potential utility of lncRNA-LOC as a therapeutic strategy for HCC.

#1815

Transcriptomic profiling of VHL-dependent long noncoding RNAs in clear cell renal cell carcinoma.

Joseph N. Samuel, Philip A. Marsden. _St. Michael's Hospital, Toronto, Ontario, Canada_.

INTRODUCTION: Clear cell renal cell carcinoma (ccRCC), the most common subtype of kidney cancer, carries a poor prognosis, with an estimated median overall survival time of only two years. This is often a clinically silent disease and about one third of patients present with metastases. Although inactivation of the tumor suppressor gene von Hippel-Lindau (VHL) is a well-characterized driver event in ccRCC, the exact molecular underpinnings of this disease remain unclear. To address this gap, we performed a transcriptomic analysis, focusing on VHL-dependent long noncoding RNAs (lncRNAs). LncRNAs are known to be involved in cancer progression, but their role in ccRCC oncogenesis has not been extensively studied. Accordingly, the aim of the present study is to characterize lncRNA transcripts that are differentially associated with VHL inactivation status. We anticipate that characterization of the lncRNA expression landscape in ccRCC will enable the identification of biomarkers and novel therapeutic targets for this disease.

METHODS: Transcriptome-wide array-based analyses were performed on total RNA derived from the 786-O (VHL-/-) ccRCC cell line, stably reconstituted with either wild-type VHL (786-O-VHL) or mutant VHL (786-O-C162F). Differential lncRNA analysis was conducted using the Arraystar Human LncRNA V4.0 array. Statistical analyses were performed using Agilent GeneSpring GX v12.1 software, with a false discovery rate (FDR) adjusted p-value < 0.05 used as a threshold for significance of differential lncRNA expression. LncRNAs were cross-referenced to data generated from The Cancer Genome Atlas (TCGA), a publicly available pan-cancer database, to identify those predictive of overall survival. Finally, qRT-PCR was used to validate the most highly differentially dysregulated lncRNAs in another primary ccRCC cell type (RCC4) and in paired kidney tissue.

RESULTS: A total of 360 lncRNA transcripts were differentially expressed four-fold or greater in 786-O-C162F cells relative to 786-O-VHL cells. Of these lncRNAs, 269 were upregulated and 91 were downregulated. Cross-referencing to TCGA, 52 of the upregulated lncRNAs and 23 of the downregulated lncRNAs were predictive of overall survival in ccRCC patients, as evidenced by statistically significant Cox regression and Log-rank p-values (p < 0.05). The top five up- and downregulated lncRNAs were validated with qRT-PCR.

CONCLUSION: To our knowledge, this is one of the only analyses to systematically assess lncRNA expression in ccRCC, and the first to assess lncRNA expression in a VHL-dependent manner. We anticipate that these molecular and clinical findings will provide a framework for utilizing VHL inactivation status as a biomarker to stratify patients with ccRCC and associated molecular targets for further study. Future inter-institutional collaborations are needed to validate these findings in a large cohort of primary ccRCC samples.

#1816

Tumor suppressor LINC02487 inhibits oral cancer cell migration and invasion through directly binding protein USP17.

Lu Feng,1 Feng Qiu,1 Jianjun Zhang,2 Minglei Sun,1 Wantao Chen,2 Weiliu Qiu1. 1 _The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China;_ 2 _Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China_.

Objective: The aim of this study is to explore the functions and mechanisms of long non-coding RNA LINC02487 in oral squamous cell carcinoma(OSCC).

Materials and methods: Relative expression level of LINC02487 in OSCC cell lines and samples were examined by qRT-PCR. Intracellular localization was done using RNA fluorescence in situ hybridization. LINC02487 was cloned into pCMV-puro vector, and transfected into OSCC cell lines HN6 and HN30 by lentivirus. Cell functions as proliferation, apoptosis, cell cycle, migration and invasion were examined subsequently. RNA binding protein was explored by ChIRP-MS. And protein expression was determined by western-blotting assay.

Results: Previous data showed a downregulation of LINC02487 in OSCCs. Here we confirmed the lower expression of LINC02487 in 6 OSCC cell lines comparing with immortalized normal oral epithelial cell lines, and in 50 OSCC samples comparing with paired adjacent normal tissue. LINC02487 was located in cytoplasm, aggregated around nuclei membrane. Overexpression of LINC02487 significantly suppressed cell migration and invasion, increased the ratio of cells in G1 phase, with a minor influence on cell proliferation and apoptosis. Furtherly, LINC02487 was found directly binding to USP17, a deubiquitinating enzyme, and regulating cell migration and invasion through USP17 expression.

Conclusion: Our study confirmed that long non-coding RNA LINC02487 was downregulated in paired OSCC samples and cell lines. It acts as a tumor suppressor through its directly binding protein USP17.

#1817

Differential expression of long non-coding RNA in colon adenocarcinoma RNA-sequence data set.

Stephen J. O'Brien,1 Theodore Kalbfleisch,2 Sudhir Srivastava,2 Shesh Rai,2 Susan Galandiuk1. 1 _Price Institute of Surgical Research, University of Louisville, Louisville, KY;_ 2 _University of Louisville, Louisville, KY_.

Introduction: Colon cancer is the fourth most common cancer in the United States and the third leading cause of cancer-related death. High throughput genomic sequencing has led to a number of significant advances in tumor biology and in identifying novel signaling molecules such as long non-coding RNA (lncRNA). The aim of this study was to identify differentially expressed lncRNA in colon cancer from a large RNA-sequencing (RNA-seq) data set.

Methods: The raw RNA-seq files of 398 patients with colon cancer were downloaded from The Cancer Genome Atlas (TCGA). Sequencing files were aligned using STAR (Spliced Transcripts Alignment to a Reference) to the most recent genome annotation. Using a subset of patients with paired colon cancer and normal colon epithelium RNA-seq data (n=40), an exploratory binomial regression model was used to calculate differential RNA expression. The most differentially expressed lncRNA were identified from the exploratory analysis and verified by comparing the larger colon cancer RNA-seq data set (n=358) with the normal colon epithelium RNA-seq data set (n=40).

Results: 33,514 genes were identified from the comparative analysis, and using differential expression cut off values of > +1.5 or < -1.5 log fold change and a false discovery rate of <0.05; 543 were upregulated and 1822 were downregulated. Within this dysregulated group, 60 lncRNAs were identified. Using the larger data set (n=358 vs n=40), 41/60 lncRNA remained differentially expressed, of which 15 were downregulated and 26 were upregulated. Twenty-four of these lncRNAs have not previously been described in colon cancer, 12 of which are upregulated and 12 of which are downregulated (Table 1).

Conclusions: This analysis of RNA-seq data from TCGA has identified dysregulated lncRNAs which have not previously been described in human colon cancer. These lncRNAs may have significant roles in colon cancer tumor signaling.

Table 1. Differential expression of lncRNA not described in colon cancer | |

|

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

lncRNA | Log Fold Change | P-value | Description in other cancers

KRT16P1 | 4.104 | 1.36E-21 | Increased in lung squamous cell carcinoma

BOK-AS1 | 3.778 | 4.98E-09 | Increased in oral squamous cell carcinoma

CELP | 3.115 | 1.30E-09 | Not available

CLDN10-AS1 | 2.394 | 4.61E-12 | Not available

POU6F2-AS1 | 2.267 | 8.25E-09 | Not available

SLC7A11-AS1 | 2.193 | 1.60E-13 | Decreased in gastric adenocarcinoma

CSAG4 | 2.108 | 3.59E-10 | Not available

LUCAT1 | 2.022 | 4.21E-11 | Increased in Ovarian cancer, renal clear cell carcinoma, head and neck squamous cell carcinoma, Non-small cell cancer, glioma, osteosarcoma, esophageal squamous cell carcinoma

VPS9D1-AS1 | 1.979 | 5.27E-21 | Decreased in gastric adenocarcinoma, Increased in non-small cell lung cancer

LEF1-AS1 | 1.846 | 4.48E-13 | Increased in glioblastoma

CASC8 | 1.648 | 5.64E-06 | Single nucleotide polymorphisms in colorectal adenocarcinoma

LINC00491 | 1.590 | 3.11E-04 | Not available

LINC00648 | -1.502 | 1.02E-15 | Not available

LINC00163 | -1.675 | 1.03E-22 | Not available

SATB2-AS1 | -1.807 | 4.18E-09 | Increased in osteosarcoma

LINC00702 | -2.142 | 4.81E-18 | Not available

TRHDE-AS1 | -2.262 | 3.44E-24 | Not available

LINC00908 | -2.308 | 2.76E-103 | Not available

LINC00483 | -2.401 | 2.87E-39 | Increased in gastric adenocarcinoma

LINC00461 | -2.440 | 1.42E-41 | Increased in glioma, multiple myeloma

FENDRR | -2.687 | 2.12E-58 | Decreased in breast cancer, prostate cancer, osteosarcoma, gastric adenocarcinoma,

ARHGEF26-AS1 | -2.714 | 8.55E-32 | Not available

ADAMTS9-AS2 | -2.904 | 1.78E-74 | Increased in lung cancer salivary adenoid cystic carcinoma, Decreased in glioma, gastric adenocarcinoma

MT1JP | -4.460 | 1.08E-114 | Decreased in retinoblastoma, Decreased in gastric adenocarcinoma

#1818

Up-regulated long non-coding RNA LincIN promotes tumor progression via regulation of NF90/miR-7/HOXB13 in esophageal squamous cell carcinoma.

Zhibo Tan. _Shenzhen Hospital, Southern Medical University, Shenzhen, China_.

Purposes: Long noncoding RNA LincIN is reported to be overexpressed and involved in metastasis of breast cancer. However, the expression and roles of LincIN in esophageal squamous cell carcinoma (ESCC) remain unsolved. The purposes of the study were to determine the expression, functions, and mechanisms of action of LincIN in ESCC.Methods: The expression of LincIN in ESCC tissues, adjacent noncancerous esophageal epithelial tissues, ESCC cell lines, and immortalized normal esophageal epithelial cell line was measured by qRT-PCR. The biological roles of LincIN, microRNA-7 (miR-7), and HOXB13 were assessed by Glo cell viability assay, EdU incorporation assay, transwell migration assay, and transwell invasion assay. The regulatory mechanisms of LincIN on NF90/ miR-7/HOXB13 were investigated using RNA immunoprecipitation, RNA pulldown, luciferase reporter assay, qRT-PCR, and western blot.Results: In this study, we found that LincIN was significantly increased in ESCC tissues and cell lines. Increased expression of LincIN was positively correlated with invasion depth, lymph node metastasis, TNM stages and poor prognosis of ESCC. Functional assays showed that overexpression of LincIN promoted ESCC cell growth, migration, and invasion. Knockdown of LincIN inhibited ESCC cell growth, migration, and invasion. Mechanistic investigation revealed that LincIN physically bound to nuclear factor 90 (NF90), enhanced the binding between NF90 and primary microRNA-7 (pri-miR-7), and further enhanced the inhibitory roles of NF90 on miR-7 biogenesis. Therefore, LincIN down-regulated miR-7 expression in ESCC. miR-7 was decreased in ESCC tissues. The expression of miR-7 was inversely associated with that of LincIN in ESCC tissues. Via down-regulating miR-7, LincIN increased the expression of HOXB13, which is a target of miR-7. Overexpression of miR-7 or depletion of HOXB13 both attenuated the tumor-promoting roles of LincIN in ESCC cell growth, migration, and invasion.Conclusion: Our finding identified LincIN is overexpressed and oncogenic in ESCC via regulation of NF90/miR-7/HOXB13 and implied that LincIN may be a promising prognostic biomarker and therapeutic target for ESCC.

#1819

Non-coding RNAs as key players for understanding oncogenic pathways in ovarian cancer: Studying new therapeutic strategies.

Anaís Sánchez Castillo, Cristina Oliveira Mateos, Marta Soler Riera, Manel Esteller, Sonia Guil. _IDIBELL, Barcelona, Spain_.

The largest part of the human genome is transcribed into non-coding RNAs (ncRNAs) which are functional transcripts displaying relevant roles in many biological processes, and their dysregulation has been shown to be involved in the development of diseases like cancer. The group of long non-coding RNAs (lncRNAs) has been elucidated to be key regulators of gene expression by acting at different levels, as regulators of epigenetic modifications and chromatin remodelling by acting as structural scaffolds, decoys or guides in the nucleus; as well as post-transcriptional regulators of messenger RNA stability and translation in the cytoplasm. Hence, the knowledge about their mechanisms of action may provide a better understanding of cell biology and likewise, lncRNAs may be used as biomarkers or therapeutical tools. The aim of the current project has been to describe the mechanism of action of a lncRNA called RPSAP52 (Ribosomal Protein SA Pseudogene 52) in ovarian cancer, as well as to study the potential use of RPSAP52 as a tool for therapeutical strategies.

Our previous results indicate that RPSAP52, which is an antisense transcript to the well described oncogene HMGA2 (High Mobility Group A2), positively regulates HMGA2 expression through the formation of an R loop structure, and it has been shown that RPSAP52 enhances the oncofetal pathway HMGA2-IGF2BP2-RAS axis through LIN28B-dependent and independent inhibition of let-7 miRNAs in breast cancer and sarcomas. Since the dysregulation of this pathway has been described to be involved in the development of ovarian cancer; which is among the most common types of cancer with poor prognosis, the goal of this project has been to reveal the relevance of RPSAP52 in this system.

The oncogenic potential of RPSAP52 has been confirmed by the analysis of a battery of xenograft samples from ovarian cancer patients, in which significant higher levels of RPSAP52 in those samples versus normal ovary have been observed, and it is positively correlated with higher levels of the sense gene HMGA2. Preliminary in vitro studies have shown that the depletion of RPSAP52 with shRNAs is related with a down-regulation of the oncogenic protein LIN28A in SKOV3 cells. Our findings suggest that RPSAP52 may be involved in the pathogenesis of ovarian cancer, and a further analysis of the link between RPSAP52 and LIN28A needs to be performed, together with the study of other candidate protein partners of RPSAP52 revealed by RNA pull down and RAP-MS techniques. Interestingly, the use of the LNA Gapmers as a strategy for the depletion of RPSAP52 also allows us to use them in vivo to confirm in vitro results as well as to study its potential role as a therapeutic target in ovarian cancer.

#1820

Circular RNAs contribute to neuroblastoma pathogenesis.

Filippos Klironomos, Clara Danssmann, Julian Naderi, Annika Winkler, Uschi Luz, Patrick Hundsdoerfer, Angelika Eggert, Joern Toedling, Falk Hertwig, Johannes H. Schulte, Steffen Fuchs. _Charité - University Medicine, Berlin, Germany_.

Circular RNAs (circRNAs), a noncoding RNA class originating from alternative splicing, are highly abundant in neural tissues and can regulate gene expression by binding to and inhibiting microRNAs and RNA-binding proteins. We explored whether circRNAs influence pathogenesis in neuroblastoma, the most common solid extracranial tumor of childhood. We performed whole-transcriptome sequencing of primary neuroblastoma samples to identify candidate circRNAs, which were validated in a neuroblastoma cell line panel. Overexpression and knockdown models were created to investigate their impact on cell viability, proliferation, apoptosis and differentiation. We identified 4,482 unique circRNA backsplicing junctions involving 2,080 genes in 69 neuroblastoma samples from all risk groups. Candidate circRNA expression did not correlate with host gene expression, indicating independent regulatory mechanisms. Principal component analysis was conducted using tumor circRNA expression. MYCN-amplified tumors clustered tightly together, demonstrating that variations in circRNA expression identify MYCN-amplified neuroblastomas as do gene expression patterns. Comparing our RNA sequencing data with other cancers and healthy fetal brain tissue revealed a circRNA subset specifically upregulated in neuroblastoma that included a circRNA derived from the ARID1A tumor suppressor gene. Cell fractionization and RNA FISH localized circARID1A to the cytoplasm, where it could interact with miRNAs and RNA-binding proteins for functional consequences. Specific circARID1A knockdown reduced cell numbers and viability, and induced neurite outgrowth and differentiation markers. Neither knockdown, nor overexpression of circARID1A influenced ARID1A mRNA and protein levels. Ongoing efforts applying bioinformatics methods to investigate the mechanism of action have identified several binding sites of microRNAs and RNA-binding proteins enriched in the circARID1A sequence. We demonstrate that circRNAs are expressed in neuroblastomas unrelated to host gene expression, and that their expression can be used to cluster MYCN-amplified tumors. As one in a set of upregulated circRNAs, circARID1A acts in a tumor-promoting manner in neuroblastoma cell lines independent of the host gene.

#1821

LINC00346 regulates c-Myc transcriptional activity through CTCF in pancreatic cancer.

Wan-Xin Peng, Yin-Yuan Mo. _Cancer Institute, University of Mississippi Medical Center, Jackson, MS_.

Pancreatic ductal adenocarcinoma (PDAC), a most challenging malignant disease, is estimated to become the second leading cause of cancer-related deaths in the United States between the year 2020 and 2030. Although the aberrant activation of KRAS has been demonstrated as the driving force of PDAC progression, the contribution of other molecules, especially long non-coding RNAs (lncRNAs), to PDAC progression remain largely unknown. In the present study, we interrogated the pancreatic adenocarcinoma dataset of the Cancer Genome Atlas (TCGA) consisting of 186 cases. Among 2,730 lncRNAs analyzed, LINC00346 revealed the highest alterations among these lncRNAs with 11% frequency at the RNA level. In addition, its upregulation was significantly associated with overall survival (OS) and disease-free survival (DFS), respectively. To determine the biological function of LINC00346 in pancreatic cancer, we next performed loss-of-function assay in pancreatic cancer cell lines MIA PaCa-2 and BxPC-3 by CRISPR/Cpf1 technology using dual gRNA approach. The results showed that knockout (KO) of LINC00346 impaired pancreatic cancer cell proliferation, tumorigenesis, migration and invasion ability, while these phenotypes were restored by LINC00346 re-expression in KO cells (i.e., rescue experiment). Mechanistically, LINC00346 functions as a positive regulator of c-Myc. Further characterizations demonstrated that LINC00346 transcriptionally activates c-Myc expression by interaction with CTCF. Together, these results suggest that LINC00346 contributes to PDAC pathogenesis by activating c-Myc, and thus, LINC00346 may serve as a potential target for pancreatic cancer therapy.

#1822

A mitochondrial pfeRNA associates with Far Upstream Element Binding Protein 1 (FUBP1) to promote lung adenocarcinoma tumorigenesis.

Nadege T. Fackche, Yuping Mei, Tomoaki Ito, Matthew Garner, Malcolm Brock. _Johns Hopkins, Baltimore,, MD_.

Background: Protein Functional Effector RNAs (pfeRNAs) are novel small non-coding RNA molecules with great prognostic and diagnostic potential for NSCLC. They feature direct binding to, and regulation of phosphorylated proteins involved in lung cancer tumorigenesis pathways. Using next-generation sncRNA deep sequencing, we have identified a mitochondrial pfeRNA (mtpfeRNA) that could play a key role in tumorigenesis and cancer development of lung adenocarcinoma (ADC).

Methods: Using next-generation sncRNA deep sequencing, we analysed ADC and human normal bronchial epithelial (HBE) cell lines; and biospecimens including matched biopsy-proven ADC tissue, histologically normal adjacent lung tissue, benign lymph node, and (iv) malignant lymph nodes; Identifying a mitochondrial pfeRNA that could play critical roles in lung cancer tumorigenesis. Next, we examined the impact of this mtpfeRNA on lung ADC cell line viability. Liquid chromatography-tandem mass spectrometry analysis and reverse immunoprecipitation assays were used to identify critical interacting protein partners known to drive tumorigenesis. We further correlated our findings to clinical staging of ADC tumor cores by IHC staining.

Results: The expression levels of mtpfeRNA were upregulated in ADC compared to that of squamous cell carcinoma and normal human bronchial epithelial cells. We also noted that blocking the upregulated mtpfeRNA resulted in significant decreases in cell viability in ADC cells. Next, we determined that mtpfeRNA binds to Far Upstream Element Binding Protein 1 (FUBP1). Furthermore, we confirmed that both mtpfeRNA and FUBP1 expression correlated to stages and lymph node metastasis in patients with ADC.

Conclusion: We revealed novel functions of a mtpfeRNA in ADC, providing potential molecules for developing diagnostics and targeted therapy to improve the dismal survival rate of patients with ADC.

#1823

SNORD78 promotes prostate cancer progression and regulates AR expression and signaling.

Alan P. Lombard, Chengfei Liu, Cameron M. Armstrong, Leandro S. D'Abronzo, Wei Lou, Christopher P. Evans, Allen C. Gao. _UC Davis Medical Center, Sacramento, CA_.

While small nucleolar RNAs (snoRNAs) have long been thought of as housekeeping genes, emerging evidence now suggests that snoRNAs are involved in more than previously thought and also in the development of disease. In cancer, it is understood that snoRNA expression and function can be altered, but little is known regarding the role of snoRNAs in these diseases. SNORD78 is a C/D box snoRNA which has been shown to be overexpressed in prostate cancer and associated with disease progression. However, there are no studies functionally characterizing SNORD78 in prostate cancer. In this study we sought to understand the functional contribution of SNORD78 in prostate cancer progression. Oncomine was used to assess SNORD78 expression in clinical prostate cancer samples. LNCaP and C4-2B prostate cancer cell lines were used. We used siRNAs to inhibit SNORD78 expression and cell viability assays to assess cell growth. ELISAs were used to assess PSA secretion and cell death. Western blots and quantitative PCR were used to assess cell death (PARP-cleavage) and AR expression and signaling. We found that SNORD78 expression was up-regulated in prostate tumors and associated with metastatic progression. Cell viability assays demonstrated that inhibition of SNORD78 expression resulted in a drastic reduction in prostate cancer cell growth and lower secretion of PSA into the media. Cell death ELISAs and western blots for cleaved-PARP showed that SNORD78 inhibition induced robust apoptosis in both LNCaP and C4-2B cells. As the androgen receptor (AR) is the primary target for prostate cancer therapy, we tested whether SNORD78 regulated AR expression and signaling. Inhibition of SNORD78 in both LNCaP and C4-2B cells resulted in reduced AR mRNA and protein levels. We also found that SNORD78 knockdown led to decreased levels of PSA and NKX3.1, suggesting that AR downstream signaling was inhibited. Our findings suggest that SNORD78 promotes prostate cancer cell viability and disease progression. We also demonstrate that SNORD78 regulates the AR and may be involved in promoting AR signaling needed for prostate cancer cell survival. Collectively, these data suggest that SNORD78 plays a critical role in prostate cancer progression and may represent a novel therapeutic target.

#1824

Long noncoding RNA NR_027340 promotes head and neck cancer cell proliferation and glycolysis via PFKP.

Yifan Yang, Ru Wang, Ling Feng, Qian Shi, Chen Tan, Jugao Fang. _Beijing Tongren Hospital, Capital Medical University, Beijing, China_.

Introduction: Long noncoding RNAs (lncRNAs) play crucial roles in various cancers. Recent study has indicated that lncRNA NR_027340 was significantly upregulated in head and neck cancer (HNC). However, its multifaceted roles in HNC are unclear. Here we demonstrate the role of lncRNA NR_027340 in HNC cell proliferation and glycolysis.

Methods: qRT-PCR was used to detect lncRNA NR_027340 expression in HNC tissues, and the correlations between lncRNA NR_027340 and clinicopathologic features were then analyzed. RNA pull-down and mass spectrometry analysis were carried out to explore the lncRNA NR_027340-bound proteins. RNA immunoprecipitation (RIP) was used to confirm the association between lncRNA and relevant proteins. lncRNA NR_027340 knockout cell lines were generated using the CRISPR/Cas9. Colony formation, cell cycle analysis and flow cytometry were used to measure the proliferation of HNC cells. The production of lactate was observed to evaluate the glycolysis of HNC cells.

Results: We found lncRNA NR_027340 was significantly up-regulated in HNCs and the elevated lncRNA NR_027340 was correlated with poor clinical prognosis. Mechanistically, we identified PFKP as the protein partner of lncRNA NR_027340 in the cytoplasm. In addition, we found that both lncRNA NR_027340 and PFKP depletion reduced the cell proliferation and glycolysis in vitro. The ingenuity pathway analysis and Western blot assay indicated that lncRNA NR_027340 targeted PFKP to regulate cell proliferation and glycolysis in HNC.

Conclusion: These findings reveal a functional role for lncRNA NR_027340 in altering glycolysis and promoting proliferation in HNC cells, suggesting that lncRNA NR_027340 might serve as a prognostic and diagnostic indicator as well as a potential therapeutic target.

#1825

Epithelial adherens junctions suppress the pro-tumorigenic MIR17HG lncRNA by recruiting RISC.

Mary C. Bridges, Joyce Nair-Menon, Antonis Kourtidis. _The Medical University of South Carolina, Charleston, SC_.

The adherens junctions (AJs) are essential architectural elements of epithelial tissues. Compromised junctional integrity is a common precursor to colon cancer. Recently, we identified a novel mechanism whereby the AJs of non-transformed colon cells recruit the microprocessor and RISC, core elements of the RNAi machinery, as well as miRNAs, to suppress oncogenic mRNAs. Knockdown of the AJ component PLEKHA7, disrupts this RNAi-mediated signaling program, leading to pro-tumorigenic cell transformation. Interestingly, PLEKHA7 RNA-CLIP and subsequent RNA-Seq analysis identified association with numerous long non-coding RNAs (lncRNAs). While a number of lncRNAs have been associated with tumorigenesis, the underlying mechanisms of their regulation during tumor progression are still unclear. As lncRNAs can interact with the RNAi machinery, we hypothesize that the AJs regulate lncRNAs via this localized RNAi mechanism. Examination of PLEKHA7-depleted cells by RNA-seq revealed differential expression of 49 junction-associated lncRNAs. From this set, the top upregulated lncRNA is MIR17HG, an oncogenic polycistronic host transcript of the miR-17-92 cluster. Junctional localization of MIR17HG was confirmed by RNA-FISH. Adherens junctions destabilization by E-cadherin knockdown also led to the upregulation of MIR17HG, demonstrating the role of junctional integrity in MIR17HG suppression. Data from Ago2 knockdown, anti-miR, and miRNA mimic experiments show that PLEKHA7 suppresses the levels of the MIR17HG transcript through the junction-associated RISC and two miRNAs, miR-203a and miR-372. PLEKHA7 depletion also results in increased levels of a subset of MIR17HG hosted miRNAs, including miR-19a and miR-19b, both known cancer promoters. Ectopic expression of PLEKHA7 in the aggressive HCT116 colon cancer cells that lack endogenous PLEKHA7 expression, suppressed anchorage independent growth, as well as MIR17HG levels, which are elevated in these cells. We are currently examining the role of MIR17HG as a functional intermediate of PLEKHA7's loss on colon cell transformation. In summary, our data point towards a novel mechanism of lncRNA regulation that tethers epithelial architecture with cell behavior.

#1826

MALAT1 regulates EGFR translation by KTN1-mediated transcriptomic change in cutaneous squamous cell carcinoma.

Lin Gao,1 Liang Zhou,2 Shanshan Li,2 Zhenhua Ding,2 Junying Qiu,1 Malin Hong,1 Chang Zou1. 1 _Shenzhen People's Hospital, Shenzhen, China;_ 2 _Southern Medical University, Guangzhou, China_.

Introduction MALAT1 is a member of lncRNAs that has been implicated in breast, cervical, colorectal cancers. However, its role in cutaneous squamous cell carcinoma (cSCC) is not clear. In this study, functional studies using in vitro and in vivo confirmed MALAT1 promotes tumor growth, migration and invasiveness of cSCC. MALAT1 regulates EGFR protein expression but not in RNA level. It shows that identified KTN1 as the key mediator for MALAT1 regulation of EGFR.

Methods Total RNAs from cells were extracted using TRIzol for qRT-PCR. Cells were transfected with MALAT1 ASOs or siKTN1_1, siKTN1_2 and cell proliferation were detected using a cell counting kit as described in the manual. Cells were seeded into chambers for transwell assay. Visible cells were photographed and counted after stained for cell migration and invasiveness assays. Total cell or cSCC tissues were prepared and assayed by western blot as previously described. Bound antibodies were visualised using the Luminated Forte Western HRP substrate. For histochemistry staining, 10 μm tissue sections were fixed for 10min with 4% paraformaldehyde, and then incubated with antibodies. The pictures of stained IHC sections were taken under Leica Fluorescence Microscope. Statistical data were presented as mean values ± stand deviation and considered to reflect significant differences.

Results Our results revealed that MALAT1 was markedly higher expressed in A431, HSC-1 and HSC-5 cSCC cells than in HaCaT keratinocytes. MALAT1 exhibited much higher expression in cSCC primary tumors compared to normal tissues. In addition, KTN1 and EGFR protein were higher expression in cSCC tissues. Significant loss of MALAT1 repressed proliferation, migration, and invasiveness but promoted apoptosis of cSCC cells. Loss of MALAT1 led to the significant downregulation of KTN1 through RNA sequencing assay accompanied with lower EGFR protein expression. However, knockdown of KTN1 had effect on the protein expression of EGFR but no effect on RNA levels. The results implied that MALAT1 may regulate EGFR protein expression through inducing KTN1 mRNA transcription.

Conclusions In this study, we revealed a novel mechanism of cSCC treatment, MALAT1 can transcriptionally activates KTN1, positively regulates EGFR protein expression in cSCC. It may provide a new series of drug target for anti-cSCC therapy.

#1827

**LncRNA** TCL6 **/ microRNA155 axis regulates the PI3K/ AKT pathway in clear cell renal carcinoma.**

Priyanka Kulkarni, Pritha Dasgupta, Shahana Majid, Marisa Shiina, Yutaka Hashimoto, Varahram Shahryari, Sharanjot Saini, Soichiro Yamamura, Laura Tabatabai, Guoren Deng, Yuichiro Tanaka, Rajvir Dahiya. _Veterans Affairs Medical Center, San Francisco and University of California San Francisco, San Francisco, San Francisco, CA_.

Background: Kidney cancer is among the 10 most common cancers in both men and women, with clear cell carcinoma (ccRCC) being the most common histologic subtype. It has become increasingly apparent that non-coding RNAs are involved in the regulation of a wide variety of diseases. However, the functional role of non-coding RNAs, such as long noncoding RNA (lncRNAs) and microRNAs (miRNA) in the initiation and progression of kidney tumorigenesis is unclear. Here we show an oncogenic role for miR-155-5p in ccRCC and its novel regulation of RCC through LncRNA TCL6.

Methods: Profiling of TCL6 and miR-155-5p was performed in microdissected renal cancer tissues, matched adjacent normal regions and in human renal cancer cell lines by quantitative real-time PCR. To assess the functional significance of miR-155-5p in RCC, we generated stable miR-155-5p knockdown RCC cells (786-O, Caki-1) and performed functional assays. We also examined the therapeutic potential of miR-155-5p in vivo using a renal cancer xenograft mouse model injected with these stable cells. We performed qPCR and Western analysis to confirm the interaction between miR-155-5p and its targets (TCL6, PIK3R1, FOXO3a, RhoA).

Results: Expression analyses in a cohort of renal cancer clinical specimens showed that LncRNA TCL6 is frequently downregulated and miR-155-5p expression is upregulated in ccRCC. We observed that knockdown of miR-155-5p inhibited cell proliferation, colony formation, migration, invasion and led to G2/M arrest, supporting its oncogenic role in vitro. In ccRCC cells (786-O and Caki-1), inhibition of miR-155-5p significantly upregulated expression, whereas in normal kidney epithelial cell line HK2 over-expression of miR-155-5p decreased levels of its target genes. Over-expression of miR-155-5p in HK2 cells induced cell proliferation, colony formation and enhanced its tumorogenic properties. Furthermore, we found that miR-155-5p may play an important role in the genesis of ccRCC through the PI3K/AKT pathway. In vivo studies demonstrated that the tumor volumes for the mice injected with stable miR-155-5p knockdown cells were significantly less as compared to the mice injected with control parental cells.

Conclusions: Collectively, these data suggest that miR-155-5p plays an oncogenic role in ccRCC. These findings offer new insight into role of lncRNA TCL6/miR-155 axis as a key regulator of the PI3K/AKT pathway in the progression of ccRCC. Thus, inhibition of miR-155 may be an effective therapeutic strategy for treating RCC.

### Signaling Pathways in Cancer Metabolism

#1828

Genetic profiling of breast cancer confirms a pivotal role of EGFR pathway in the development of acquired resistance to tamoxifen.

Abeer A. Bahnnasy, Ibrahim Malash, Nasr Al-Lahlouby, Osman Mansour, Sabry Shaarawy, Mona S. Abdellateif, Hend Yousef, Mai M. Lotfy, Rabab Gaafar, Mohamed. elsesy, Abdel-Rahman N. Zekri. _National Cancer Inst. Cairo Univ., Cairo, Egypt_.

Background: Metastatic breast cancer (MBC) represents a major health problem in Egypt and worldwide. Prognostic and predictive factors for patients with MBC are highly needed, for better management and improved survival. The aim of this study was to assess the prognostic and predictive value(s) of CYP2D6 polymorphisms in MBC patients on Tamoxifen and to determine the genetic profile(s) of Tamoxifen responders and non-responders. Methods: We assessed 157 females with hormone receptor positive, locally recurrent inoperable and/or metastatic breast cancer (MBC) patients from Egypt for 1) CYP2D6 polymorphisms and 2) genetic aberrations in the EGFR pathway (92 genes). All patients received Tamoxifen and according to their response they were divided into Tamoxifen-responders and refractory. RNA was extracted from tumor and normal tissue samples and used to assess the genetic profiles of the patients using the SABioscience array (Qiagen) with four house-keeping genes. Results: 92(58.6%) patients were ER and PR positive, 51 (32.5%) were ER positive and 14 (8.9%) were PR positive. CYP2D6 *3/*4 was significantly prevalent in the refractory group (86.6%).Variants *10/*10 and *10/*3 were more common in the responders (85.5%) compared to refractory (P=0.027). CYP2D6 polymorphism associated significantly with Her-2 amplification (P<0.001), and reduced overall survival rate of patients in both refractory and responder groups (P< 0.001). Fifty-six genes were differentially over-expressed in the refractory group compared to responders, of which only JAK1, COL1A1, GAB1, FN1and MKNK1 showed a significant difference. In the refractory group 34 genes were differentially reduced compared to responders; none of them showed significant difference. Response to Tamoxifen associated significantly with disease site since patients with bone only disease showed better response than those with visceral metastases (P<0.005). Conclusion: A panel of 5 genes in EGFR pathway (MJAK1, COL1A1, GAB1, FN1 and MKNK1) together with CYP2D6 polymorphism scan predict response to Tamoxifen in metastatic BC patients though this has to be verified in an extended study including larger sample.

#1829

HSF1 suppresses AMPK to promote lipogenesis, cholesterol biosynthesis and protein lipidation.

Kuo-Hui Su, Chengkai Dai. _NCI, Frederick, MD_.

AMP-activated protein kinase (AMPK), a key cellular metabolic sensor, suppresses anabolic pathways, including lipogenesis and cholesterol biosynthesis, thereby maintaining energy homeostasis. Heat shock factor 1 (HSF1) is the master regulator of the evolutionarily conserved proteotoxic stress response (PSR), thereby preserving proteostasis in the face of environmental insults. In contrast to its beneficial role in enhancing survival under proteotoxic stress, it has been demonstrated that HSF1 acts as a potent pro-oncogenic factor. Our previous study has shown that metabolic stressors, via AMPK activation, inactivate HSF1 and disrupt proteostasis to suppress tumor growth. Interestingly, our new study now reveals that HSF1, in reciprocal, also suppresses AMPK activation. HSF1 does so independently of its canonical transcriptional action. Furthermore, Hsf1 deficiency promotes the interactions between AMPK and LKB1, a key upstream kinase activating AMPK. Congruent with the suppression of lipogenesis by AMPK, Hsf1 deficiency depletes cellular lipid content and reduces body fat mass in mice. Importantly, these defects can be largely rescued by either pharmacological or genetic inhibition of AMPK. At the molecular level, Hsf1 deficiency leads to inactivation of acetyl-CoA Carboxylase (ACC) and sterol regulatory element-binding transcription factor 1c (SREBP1c), both of which are key players in de novo lipogenesis. In addition to impaired lipogenesis, Hsf1-deficient cells and mice display reduced cholesterol levels. Of particular interest, this defect leads to impaired cholesteroylation of sonic hedgehog (SHH), the best known signaling molecule undergoing this covalent modification. In consequence, SHH secreted by HSF1-deficient cells are less potent in activating its downstream signaling cascade, indicated by reduced DNA binding of GLI1, a transcription factor responding to activation of SHH signaling. Conversely, HSF1 overexpression increases the lipid content, cholesterol levels and the amount of cholesteroylated SHH proteins in xenografted melanomas. Taken together, our results reveal that HSF1, through AMPK suppression, promotes lipogenesis, cholesterol biosynthesis and protein lipidation, thereby supporting malignant growth.

#1830

Role of heat shock factor 1 (HSF1) on metabolic reprogramming of T -cells.

Bhaumik D. Pandya, Nahid M. Mivechi, Dimitrios Moskophidis. _Augusta University, Augusta, GA_.

Heat shock transcription factor 1 (HSF1) is normally activated in response to a variety of environmental stressors. Higher levels of HSF1 expression has been shown in many tumor types and this is associated with poor prognosis. Previous data from our laboratory indicate that genetic inactivation of HSF1 significantly delays ERBB2-induced mammary tumorigenesis and hepatocellular carcinoma induced by chemical carcinogen in mouse models. We have also shown that HSF1 is involved in controlling cellular bioenergetics. CD8-positive T cells plays an important role in anti-tumor immunity response. Our preliminary results show that genetic inactivation of HSF1 in T-cells leads to reduced oxygen consumption rate (OCR) as well as Extracellular Acidification Rate (ECAR). We have also observed that deletion of HSF1 from T cells delayed in-vitro activation of purified CD8-positive T cells upon stimulation with anti-CD3 and anti-CD28. Above characteristics corresponds to reduced cellular glycolysis and T cell proliferation. Further studies will be directed towards exploring the effects of HSF1 deletion in T cell activation induced anti-tumor immunity. This study is supported by the NCI grants; NCICA062130 and NCICA132640.

#1831

Role of autophagy in lymphangiosarcoma initiation and progression.

Fuchun Yang, Shaogang Sun, Jun-Lin Guan. _University of Cincinnati College of Medicine, Cincinnati, OH_.

Lymphangiosarcoma is a rare malignant vascular tumor which is formed from aberrant proliferation of endothelial cells (ECs). We previously established a vascular tumor mouse model by conditional knockout of Tsc1 gene in ECs and revealed that hyper-activated mTORC1 and increased VEGF autocrine are required for lymphangiosarcoma development and progression. However, we also found that there are other unknown factors which contribute to vascular tumor development besides mTORC1 hyper-activation. Autophagy has been shown to play an important role in tumor initiation and progression. However, the role of autophagy in mTORC1-dependent vascular tumor development is unclear. In this study, we studied the potential role and mechanism of autophagy in promoting lymphangiosarcoma development. FIP200 (FAK family interacting protein of 200 kDa) was identified as an essential autophagy gene, which was shown to play a key role in breast cancer development and progression. By crossing Tsc1f/f, Scl-Cre mice with Fip200f/f mice, we generated Tsc1/Fip200 double cKO mice. We found that Fip200 deletion blocked lymphangiosarcoma development induced by mTORC1 hyper-activation in mice. We established an immortalized lymphangiosarcoma cell line named Tsc1ΔEC from Tsc1 cKO mice and examined their growth in xenograft transplant models following CRISPR-Cas9 knockout of Fip200, Atg5 or Atg7 in Tsc1ΔEC cells. Our data showed that KO of each of these autophagy genes impaired vascular tumor growth in nude mice. Mechanistically, we found that Fip200 KO suppressed mTORC1 signaling activation under glucose starvation condition. In addition, we found that both Fip200 KO and lipase inhibitor Orlistat treatment in Tsc1ΔEC inhibited mitochondria respiration and decreased ATP content by seahorse assay. We also found that deletion of Fip200, Atg5, Atg7 or LAL in Tsc1ΔEC blocked lipid droplets (LDs) degradation and decreased glycerol release. Collectively, our data suggest that autophagy is required for lymphangiosarcoma initiation and progression through mTORC1 hyper-activation maintenance under stress conditions and supplying energy consumption by regulating lipid metabolism.

#1832

Ligand-dependent Wnt signaling directs metabolic adaptations to promote pancreatic tumorigenesis.

Kristina Y. Aguilera, Rana Riahi, Edris A. Saadat, Anna R. Lay, Thuc Le, Anthony Cabebe, Timothy R. Donahue, Caius Radu, David W. Dawson. _UCLA, Los Angeles, CA_.

Wnt/β-catenin signaling plays important roles in pancreatic adenocarcinoma (PDA) tumor initiation and progression via broad-ranging effects on proliferation, differentiation, survival, and stemness. Although Wnt has been shown to promote glycolysis in colon cancer, its role in regulating metabolism in PDA has not been explored in any detail. For this study, the AsPC-1 cell line was selected as an exemplar of ligand-dependent autocrine Wnt signaling in PDA. AsPC-1 harbors an inactivating mutation in RNF43 that confers Wnt growth-dependency and response to LGK974, a PORCN inhibitor that blocks Wnt ligand secretion and signaling. Global transcriptomic, proteomic, and metabolomic analysis revealed several metabolites and putative mediators of metabolism that are directly or indirectly linked to autocrine Wnt signaling in AsPC-1. In contrast to colon cancer, pharmacologic inhibition of Wnt by LGK974 in AsPC-1 led to the accumulation of pyruvate and lactate, as well as reduced tricarboxylic acid cycle metabolites and decreased mitochondrial membrane potential as measured by tetramethylrhodamine assay. These metabolic changes were accompanied by cell cycle arrest and downregulation of transcriptional programs involved in cell cycle progression, DNA replication, and nucleotide metabolism. Pyruvate analogs dichloroacetate and oxamate, that respectively target pyruvate dehydrogenase kinase and lactate dehydrogenase, restored mitochondrial membrane potential in the context of LGK974. Importantly, LGK974 results were phenocopied by siRNA-mediated knockdown of WNT7B. Thus, inhibition of mitochondrial oxidative phosphorylation by LGK974 in RNF43-mutant PDA appears mechanistically linked to its inhibition of canonical Wnt signaling, an adaptation and potential metabolic vulnerability that could be leveraged to therapeutic advantage via combinatorial strategies. Altogether, these results suggest tissue context and differing mechanisms of Wnt pathway activation or signal strength may profoundly influence its role in regulating cancer metabolism.

#1833

KLF4 regulates metabolic homeostasis in response to energy stress.

William M. Rosencrans,1 Zachary H. Walsh,2 Andrew C. Blum,1 Engda G. Hagos1. 1 _Colgate, Hamilton, NY;_ 2 _Colorado University School of Medicine, Aurora, CO_.

Krüppel-like factor 4 (KLF4) is a zinc finger-containing transcription factor with diverse regulatory functions in cellular growth, proliferation, differentiation, autophagy and genomic stability. Furthermore, KLF4 exerts a cell-cycle checkpoint effect in part by acting as a transcriptional activator of the cyclin-dependent kinase inhibitor, p21. Our lab has found that KLF4 regulates autophagy, mitophagy, and mitochondrial dynamics. Given the close relationship these processes have with cellular metabolism, we aimed to determine how KLF4 regulates glycolytic and oxidative metabolism. Using various assays, we find that cells lacking KLF4 have both oxidative and glycolytic impairments. Using western blotting, we find that when KLF4 is overexpressed in RKO human colorectal cancer cells, Hexokinase 2 (HK2), a rate determining enzyme in glycolysis, PKM2, a facilitator of lactate production, and MCT4, a lactic acid export protein and marker of glycolysis, are all significantly upregulated. In MEFs, we find that GLUT1, a glucose transport protein, has greater expression in WT than Klf4-null cells. Furthermore, we determined that KLF4 regulates aspects of the basal cell metabolism in a p21-dependent manner. Given that KLF4 has been found to act as an immediate early gene in response to certain stresses, and given the importance of fast metabolic shifts, we also look at how KLF4 influences short term metabolic stress responses. Since metabolic stress tests showed that WT MEFs have an increased glycolytic capacity, we investigate whether the cells have an increased ability to quickly intake glucose in response to particular stresses. Accordingly, we looked at GLUT1 expression and membrane localization with respect to KLF4. Immunostaining in RKO and MEFs suggests that KLF4 plays a role in localizing GLUT1 to the outer membrane, allowing for increased glucose intake. Taken together, our findings provide a rationale for the impaired metabolism of KLF4-null MEFs, where KLF4 plays a role responding to energy stress by modifying the localization of GLUT1, and regulating various metabolic enzymes both directly, and in a p21-dependent manner.

#1835

Nqo1 ablation inhibits activation of the PI3K/Akt and MAPK/ERK pathways and blocks metabolic adaptation in hepatocellular carcinoma.

Manali Dimri,1 Ashley Humphries,1 Archana Laknaur,1 Sawsan Elattar,1 Ashok Sharma,2 Ravindra Kolhe,3 Ande Satyanarayana1. 1 _Georgia Cancer Center, Augusta University, Augusta, GA;_ 2 _Center for Biotechnology and Genomic Medicine, Augusta University, Augusta, GA;_ 3 _Department of Pathology, Augusta University, Augusta, GA_.

According to the World Health Organization, hepatocellular carcinoma (HCC) is the 2nd leading cause of cancer related deaths worldwide. HCC is highly resistant to conventional

chemotherapies; therefore, identification of specific molecular targets for the development of targeted therapies is indispensable. Cancer cells undergo metabolic adaptation to sustain uncontrolled proliferation and depends on the high rate of glycolysis and glutaminolysis. Hence, identifying and targeting proteins that assist cancer cells in metabolic reprogramming is an effective strategy to block their proliferation. We detected a strong upregulation of NAD(P)H Quinone Dehydrogenase 1 (Nqo1), a cytosolic flavoprotein, in vivo in mouse and human liver tumors as well as in liver cancer cell lines in vitro. Knockdown of Nqo1 in liver cancer cells by shRNA caused a significant downregulation of both glycolytic and glutaminolysis genes, impaired cell proliferation, colony formation and in vivo xenograft tumor growth. Nqo1-/- mice with HCC displayed significant reduction in tumor number, size and growth. In Nqo1+/+ mice, histological analysis of liver tumors revealed large tumor cells with hyperchromatic nuclei in a compact growth pattern, whereas Nqo1-/- livers appeared normal. Hyper-activated PI3K/Akt and MAPK/ERK signaling pathways play central role in cancer cell metabolic adaptation since their downstream effectors such as Akt and c-Myc control most of the glycolytic and glutaminolysis genes. Interestingly, at the molecular level, Nqo1 knockdown caused strong upregulation of the tumor suppressor PTEN, and enhanced activation of the serine/threonine phosphatase PP2A, leading to impaired activation of the PI3K/Akt and MAPK/ERK/c-Myc pathways. Our findings indicate that, by targeting PP2A and PTEN, Nqo1 enhances the expression and/or activity of PDK1, Akt, ERK1/2 and c-Myc and promotes cancer cell metabolic reprogramming and hence may function as a key therapeutic target for HCC inhibition.

#1836

Replication stress response dependency is triggered by interferon signaling in pancreatic cancer.

Evan Abt, Amanda Dann, Joseph R. Capri, Soumya Poddar, Chloe M. Cheng, Woosuk Kim, Robert Damoiseaux, Shili Xu, Thuc Le, Johannes Czernin, Timothy R. Donahue, Caius G. Radu. _UCLA, Santa Monica, CA_.

Pancreatic ductal adenocarcinoma (PDAC) tumors are defined by a high rate of KRAS mutation, a dense stromal compartment and an inflammatory microenvironment. The therapeutic vulnerabilities of PDAC have been directly linked to oncogenic KRAS signaling and are influenced by stellate cells, however, the impact of inflammatory mediators on therapy response has not been evaluated. Interferons are pleiotropic cytokines that are well-characterized for their role in regulating tumor cell expression of immune checkpoints and have been linked to both chemotherapy and radiation resistance. Among TCGA solid tumor datasets PDAC ranks highly in interferon signaling biomarker enrichment with a range of observed across specimens. We found that interferon signaling correlates with established PDAC subtypes but is independent of immune cell infiltration. Consistently, we found that interferon signaling is also detectable in a subset of PDAC patient-derived and cell line xenografts. We defined a requirement for PDAC cell cGAS/STING pathway activity in xenograft tumor interferon signaling and implicated PDAC cells as a source of interferon. To identify targetable signaling adaptations elicited by interferon exposure we applied an in vitro integrated analysis of interferon-induced phosphoproteomic alterations using nLC-MS/MS and interferon-induced signaling pathway co-dependencies using a chemical genomics phenotypic screen. We determined that type I interferon exposure increases the activity of replication stress response kinases Ataxia Telangiectasia and Rad3-related protein (ATR) and CHEK1 and that inhibitors of these kinases selectively induce apoptosis in interferon-exposed PDAC cells. We found that both ATR inhibition and interferons trigger a shift in nucleotide metabolism with IFN inducing a shift from a biosynthetic to a predominantly catabolic phenotype limiting the abundance of precursors for DNA replication. This shift is mediated by sterile alpha motif domain- and histidine aspartate domain-containing protein 1 (SAMHD1), however this alteration is not solely responsible for IFN-induced replication stress. These results indicate that PDAC patients harboring tumor interferon signaling biomarker enrichment may be candidates for treatment with inhibitors of replication stress response signaling kinases ATR or CHEK1. Collectively, our work begins to define the crosstalk between cytokine signaling, stress response signal transduction networks and metabolism in PDAC and reveals actionable vulnerabilities masked by cell culture models.

#1837

Tyr phosphorylation activates and inhibits upstream acetyltransferases and deacetylase of 6PGD, respectively, to promote cancer metabolism and tumor growth.

Siyuan Xia, Changliang Shan, Jun Fan, Jing Chen. _Winship Cancer Institute of Emory University, Atlanta, GA_.

Background: We recently reported that 6-phosphogluconate dehydrogenase (6PGD), the third enzyme in the oxidative pentose phosphate pathway (PPP), is commonly activated by lysine acetylation in EGF-stimulated cells but "hijacked" in human cancer cells. Acetylation at K76 and K294 enhances 6PGD activation and is commonly observed in diverse human cancer cells, which is important for coordination of anabolic biosynthesis, redox homeostasis, and glycolysis in cells, providing an overall metabolic advantage to cancer cell proliferation and tumor growth. Moreover, we identified DLAT and ACAT2 as upstream acetyltransferases of K76 and K294, respectively, and HDAC4 as the deacetylase of both sites. However, it remains unclear how oncogenic signals regulate the activity of these acetyltransferases and deacetylase to control 6PGD activity and consequently contribute to tumor cell metabolism and tumor growth.

Methods: Tyrosine phosphorylation sites were identified in Mass Spec-based studies using recombinant purified ACAT2, DLAT and HDAC4 treated with active recombinant oncogenic tyrosine kinases in diverse in vitro kinase assays. Mutational analysis was performed to determine tyrosine phosphorylation sites that are crucial to activate ACAT2 and DLAT or inhibit HDAC4, and/or promote or attenuate 6PGD binding to ACAT2 and DLAT or HDAC4, respectively.

Results: We found that, although tyrosine phosphorylation does not affect 6PGD catalytic activity or binding to its upstream acetyltransferases and deacetylase, diverse growth factor receptors as well as oncogenic tyrosine kinases commonly phosphorylate ACAT2, DLAT and HDAC4, leading to catalytic activation of ACAT2 and DLAT but inhibition of HDAC4. Moreover, tyrosine phosphorylation promotes binding of 6PGD to ACAT2 and DLAT but attenuates HDAC4-6PGD association.

Conclusions: Our findings provide insight into the molecular mechanisms underlying 6PGD regulation, which responds to growth factor stimulation or oncogenic tyrosine kinases through activation and inhibition of upstream 6PGD acetyltransferases and deacetylase, respectively, via tyrosine phosphorylation. Moreover, our studies showcase the beauty of complex signal transduction-based regulation of cellular processes, where hierarchical, distinct posttranslational modifications act in concert to provide precise regulation of a series of sequential events to control catalytic activity of acetyltransferases ACAT2 and DLAT and deacetylase HDAC4 and kinetic formation of protein complex to regulate 6PGD in cancer cells, and subsequently contribute to cancer metabolism and tumor growth.

#1838

Nucleotide metabolism heterogeneity in mutant KRAS pancreatic cancer.

Woosuk Kim, Thuc Le, Joseph Capri, Anthony Cabebe, Wesley Armstrong, Evan Abt, Timothy Donahue, Caius Radu. _UCLA, Los Angeles, CA_.

Mutant KRAS orchestrates major metabolic adaptations critical for pancreatic ductal adenocarcinoma (PDAC) growth and survival including changes in glucose, amino acid, lipid and energy metabolism. However, how nucleotide metabolism in PDAC is impacted by mutant KRAS has received much less attention, even though nucleotides play critical roles in major biological processes including nucleic acid synthesis, phospholipid biosynthesis and protein glycosylation.

Pyrimidine nucleotide metabolism is a highly complex and regulated system with multiple pathways contributing to its plasticity and robustness. Pyrimidine nucleotides can be produced via the de novo pathway (DNP) that utilizes glucose and amino acids, and the nucleoside scavenging pathway (NSP) that utilizes extracellular uridine and cytidine. We hypothesize that nucleotide products of RNA turnover can fuel a third route for pyrimidine biosynthesis, which we term the nucleotide recycling pathway (NRP).

Amongst these three major pyrimidine nucleotide biosynthetic pathways, the DNP has been studied most extensively. The biological and therapeutic significance of the NSP and NRP in PDAC, as well as their regulation and coordination with DNP in the context of mutant KRAS signaling, are poorly understood due to the lack of methodology to profile these convergent pathways. We further hypothesize that since PDAC tumor microenvironment is characterized by insufficient supply of nutrients and oxygen, these tumors have limited DNP capacity and increased reliance on alternate pyrimidine biosynthetic pathways.

Here, we describe a novel mass spectrometric (MS) method to measure the contributions of pyrimidine nucleotide convergent pathways to RNA and DNA synthesis in PDAC models. Applying this method to a panel of PDAC models, including patient-derived primary lines, revealed previously unappreciated pyrimidine biosynthetic heterogeneity, suggesting the existence of distinct pyrimidine nucleotide metabolic subtypes.

Identifying and characterizing pyrimidine nucleotide metabolic subtypes in the context of oncogenic KRAS signaling may enable stratification of PDAC tumors and guide the development of novel therapeutic approaches.

#1839

**Concurrent inhibition of glutaminase (GLS) and metabotropic receptor 1 (GRM1) reduced glutamate bioavailability and led to a decrease in GRM1-expressing melanoma cell proliferation** in vitro **and tumor progression** in vivo **.**

Raj Shah,1 Simar Singh,2 Kevinn Eddy,1 Fabian Filipp,2 Suzie Chen1. 1 _Rutgers University, Piscataway, NJ;_ 2 _University of California Merced, Merced, CA_.

Aberrant glutamatergic signaling has been implicated in multiple metabolic processes and several types of cancer. Our laboratory has previously illustrated the role of metabotropic glutamate receptor 1 (GRM1), a G-protein coupled receptor, in neoplastic transformation of melanocytes in vitro and spontaneous development of metastatic melanoma in vivo. Here, we demonstrate significant overexpression of glutaminase (GLS) in GRM1 expressing melanoma cells, resulting in excess production of GRM1's natural ligand, glutamate, and the establishment of autocrine loops. GLS catalyzes the first step in glutamine metabolism--the conversion of glutamine to glutamate before entering the TCA cycle. Comparison of glutamate levels in circulating blood plasma between melanoma prone GRM1-transgenic and wild type mice showed elevated glutamate levels in melanoma prone GRM1-transgenic mice, suggesting that aberrant GRM1 expression also promotes an increase in circulating glutamate, to ensure constitutive activation of the GRM1 receptor and its downstream signaling effecters in vivo. We also discovered that manipulation of GRM1 expression by genetic means led to robust parallel changes in metabolite expression levels and intracellular glutamate concentrations by mass spectrometry analysis, as well as the endogenous levels of GLS in melanoma cells, suggesting a strong association between GRM1 and GLS. Inclusion of CB-839, a potent, selective, and orally bioavailable inhibitor of GLS, resulted in significant inhibition of GRM1+ melanoma cell proliferation compared to GRM1\- ones. The sensitivity of GRM1+ melanoma cells to modulation of GLS points to the dependency of these cells to glutamate amounts via GLS activity. We also demonstrated that treatment with Riluzole, an inhibitor of glutamatergic signaling by decreasing the available ligand, glutamate, led to significantly reduced melanoma cell proliferation in vitro and tumor progression in vivo. We found that simultaneous targeting of glutamate production (CB-839) and release (Riluzole) in GRM1+ melanoma cells led to additive suppression of cell proliferation in vitro. Most importantly, disruption of GRM1 signaling through combined actions of CB-839 and Riluzole significantly suppressed tumor growth in two independent xenograft mouse models of melanoma, with no obvious symptoms of toxicity detected in these mice. Taken together, these results describe a novel pathway for GRM1-mediated tumorigenesis through rewiring of cellular metabolic networks. We are continuing to investigate the mechanisms and regulation of GLS in our experimental system, with the goal of developing a rational strategy for the treatment of GRM1-expressing cancers.

#1840

Nedd9 **controls glycolysis and autophagy in non-small cell lung cancer (NSCLC).**

Alexander Y. Deneka,1 Anna Nikonova,1 Meghan Kopp,1 Anna Gaponova,2 Anna Kiseleva,1 Harvey Hensley,1 Erica Golemis1. 1 _Fox Chase Cancer Ctr., Philadelphia, PA;_ 2 _Moscow Institute of Physics and Technology, Moscow, Russian Federation_.

Non-small cell lung cancer (NSCLC) has a low survival rate, with metastasis contributing to the vast majority of deaths. Elevated expression of the protein NEDD9 has been reported in a large subset of lung cancers and other malignancies as a promotor of aggressive phenotypes and drug resistance. NEDD9 exerts these pro-metastatic roles by serving as a scaffold for intermediates in the integrin and receptor tyrosine kinase signaling cascades, and other pathways, with NEDD9 knockdown impairing cell migration, motility, and growth. Because scaffolds act by assembling functional complexes in precise stoichiometries, there are cases where both loss and gain result in loss of function. Through analysis of TCGA data, we identified a subset of human NSCLC with low levels of NEDD9 expression, associated with lower overall survival. Therefore, in this study, we for the first time assessed the consequences of a constitutive null genotype for Nedd9 for cancer formation in the 129S/Sv-Krastm3Tyj/Trp53tm1Brn (KP) murine model of NSCLC. Unexpectedly, we found deletion of Nedd9 (in a KPN genotype) significantly accelerated NSCLC tumorigenesis and enhanced tumor invasion. Proteomic analysis of KPN versus KP tumors confirmed these more aggressive phenotypes occurred in spite of depressed activity of the pro-proliferative Nedd9 partners Src and Fak, and surprisingly, suggested depressed expression of activity of enzymes involved in glycolysis in KPN tumors. Functional exploration confirmed a similar protein expression and activity profile, and strongly reduced glycolytic capacity, in multiple human NEDD9-depleted NSCLC cell lines, for the first time demonstrating NEDD9 regulation of glycolysis through control of hexokinase II (HK2) and other enzymes acting upstream of PGK. Compensating for these negative effects of Nedd9 loss during in vivo tumorigenesis, NSCLC tumors arising in KPN mice additionally activated AMPK, causing significantly elevated levels of autophagy, with both aggressive tumor growth and autophagy reduced by the SRC inhibitor dasatinib. Such compensatory activation of autophagy did not occur in cell lines with depleted NEDD9, distinguishing in vitro and in vivo tumor cell signaling response to NEDD9 loss. In sum, these data for the first time identify and define a role for the pro-metastatic protein NEDD9 in control of glycolytic metabolism in NSCLC setting, and establish a mechanism for this control. This data should be useful in guiding selection of patients for specific therapies targeting metabolic pathways.

#1841

Adaptive upregulation of carbamoyl phosphate synthetase-1 (CPS-1) in glucose-deprived metastatic lung adenocarcinoma cells.

Kin Lok Wong, Andrew M. Chan. _Chinese University of Hong Kong, Hong Kong, Hong Kong_.

Advanced lung cancers frequently metastasize to the brain, where nutrient deprivation from tumour microenvironments at either primary, invading or metastatic sites may exert a selective pressure on tumour cells. Our lab has identified a rare brain metastasis cell line of lung cancer origin: A1115, that possessed heightened basal glycolytic activity. Characterization of the metabolic profiles of A1115 and 4 other lung adenocarcinoma cell lines revealed A1115 to have the highest dependence on glycolysis and the lowest utilization of mitochondrial oxidative phosphorylation (OXPHOS) for metabolism. Thus, we hypothesized that A1115 is hypersensitive to glucose starvation. A1115 relied heavily on glycolysis on survival and propagation, while inhibiting mitochondrial OXPHOS had less effect. Glucose deprivation induced the nutrient sensor AMP-activated protein kinase (AMPK) to be elevated in A1115, while the proliferative capacity of A1115 was drastically reduced by an AMPK activator, AICAR, suggesting AMPK to be detrimental to cell viability of A1115 under glucose deprivation. To understand how such metastatic cell line could survive the low glucose environment in blood circulation and during the invasion into the brain parenchyma, we established clonal A1115 sublines that could propagate under low glucose (LG) environment. LG sublines regained hypersensitivity to glucose deprivation upon exposure to normal glucose concentrations suggest an adaptive response. We hypothesised that tumour cells maintain cell viability under glycolytic stress conditions by undergoing metabolic reprogramming, this involves changes in metabolite utilization and transcriptional activity of key metabolic enzymes. A parallel metabolomics (targeted LC/MS) and transcriptomics (RNAseq) analysis was conducted comparing A1115 LG sublines with the parental control. Metabolomics analysis revealed that LG sublines under glucose deprivation harboured a significant metabolite increase in amino acids: glutamate, arginine, aspartic acid and pyrimidine: cytidine, whereas integrated pathway analysis of metabolomics and transcriptomics revealed upregulation in both pyrimidine metabolism and alanine, aspartate and glutamate metabolism. Moreover, increased carbamoyl phosphate synthetase 1 (CPS-1) expression was observed in LG sublines, an initiating enzyme for pyrimidine biosynthesis and urea cycle, silencing CPS-1 decreased cell viability on LG in normal conditioned media, suggesting that the disruption of pathway could impede survival. Overall, our results suggest that lung adenocarcinoma may undergo reprogramming towards a starkly different constituent of transcriptional activation leading to metabolite flux and signalling activation to allow viable adaption to glucose deprivation in the microenvironment.

#1842

**A novel therapeutic approach to overcome resistance to glutaminase inhibition in** NF1 **driven malignant peripheral nerve sheath tumors MPNST.**

Tahir Sheikh, Parag Patwardhan, Chao Lu, Gary K. Schwartz. _Columbia Univ. Medical Ctr, New York, NY_.

Glutamine, one of the most abundant intracellular amino acids, plays an important role in satisfying the biosynthetic needs of proliferating cancer cells by providing carbons to produce tricarboxylic acid (TCA) cycle intermediates, glutathione, fatty acids, and nucleotides. Though recent research has focused on targeting novel pathways in cellular metabolism, targeting of dysregulated metabolic pathways in therapeutic resistance, a highly clinical relevant area in cancer research, has not been specifically addressed. In this study we hypothesized that combinatorial targeting of metabolism and epigenetics may overcome therapeutic resistance to glutaminase (GLS1) inhibitor, CB-839, in NF1 driven MPNST cells. To test this, we cultured NF1-null MPNST cell line (ST8814) sensitive to growth inhibition by CB-839 with the drug in a dose-escalating manner until the emergence of resistant cell populations. Cell viability assays and western blot analysis demonstrated that CB-839-induced inhibition of cell growth and apoptosis (PARP cleavage) were indeed abrogated in resistant ST8814 cells. To rule out the possibility that acquired resistance was due to changes in the uptake, metabolism or target inhibition of CB-839, we also confirmed that the resistant cells were no longer sensitive to growth inhibition and cell death induced by siRNA knockdown of GLS1. We profiled global landscape of histone modifications in ST8814 parental and resistant cells and found that there was a marked decrease in the repressive histone methylation marks H3K9me3 and H3K27me3 and an increase in H3K9ac, a modification associated with active transcription. Accordingly, levels of c-Myc were significantly elevated in resistant cells. RNAi-mediated c-Myc knockdown selectively inhibited growth factor signaling and cell proliferation in CB-839 resistant cells, suggesting that these cells are addicted to the reprogrammed epigenetic and transcriptional state. Expression levels of c-Myc and histone acetylation are connected by the Bromo-domain protein BRD4. Consistently, CB-839 resistant cells demonstrated augmented sensitivity towards treatment with JQ1, a small molecule inhibitor of BRD4. We also carried out receptor tyrosine kinase (RTK) array and immunoblotting analysis which demonstrated strong overexpression and activation of c-Met (HGF-R) in CB-839 resistant cells, compared to the parental cells. Conversely, combination treatment with inhibitors of c-Myc and c-MET resulted in decreased cell viability, and induction of apoptotic markers such as cleaved PARP and cleaved Caspase3 in the resistant ST8814 cells. Taken together, our data strongly indicates that combinatorial targeting of metabolism and epigenetics is a novel potential approach to overcome CB-839 resistance and merits evaluation in the treatment of drug resistant NF1 driven soft tissue sarcoma.

#1843

Computational biology model (CBM) predicts nutrient dependency of cancer patients based on Tumor Genomics: Implication of precision diet in cancer therapy.

Shireen Vali,1 Taher Abbasi,1 Subrat Mohapatra,2 Vishwas Joseph,2 Ashish Kumar Agrawal,2 Anuj Tyagi,2 Neelesh Lunkad,2 Ashokraja Balla2. 1 _Cellworks Group, Inc., San Jose, CA;_ 2 _Cellworks Research India Pvt. Ltd., Bangalore, India_.

Background: Tumor metabolism is the hallmark of cancer cells. Cancer cells utilize different nutrient sources to drive the metabolic pathways to sustain tumor growth. Glucose (Glu) and Glutamine (Gln) are the primary nutrient sources on which cancer cells thrive. Developing precision diet based on patient's molecular characteristics can help treat the cancer with dietary modulations along with traditional approaches.

Methods: Computational Biology Model (CBM) captures the signaling and metabolic pathways to predict cancer phenotypes and biomarkers. Genomic aberrations (Mutations / Copy Number Variations (CNV)) from a patient's tumor are input into the CBM to create the patient disease model. CBM is used for diet prediction based on the molecular characteristics of the patient's disease.

CBM is validated using a data set of 54 cancer cell-lines across indications, by assessing nutrient dependency for Glu and Gln. Simulation based prediction of Glu and Gln dependency is based on the expression of transporters and rate limiting enzymes of cellular glucose (SLC2A1-4, HK2) and glutamine (SLC1A5, GLS) uptake. The enzymes regulating the de-novo synthesis of glucose (PCK1/2, FBP1) and glutamine (GLUL) are negative determinants.

In the CBM, an index is defined to measure Glu and Gln dependency.

Glu Dependency Index = (SLC2A1 + SLC2A2 + SLC2A3 + SLC2A4 + HK2) / (PCK1 + PCK2)

Gln Dependency Index = (SLC1A5 + GLS) / (GLUL)

Threshold values for Glu and Gln dependency was determined based on the simulation correlation with the cell-line data. The validated CBM was then used for diet prediction for patient genomics.

Results: Validation of genomics-based diet prediction by CBM using 54 cancer cell lines had an accuracy, positive predictive value and negative predictive value of 85%, 97% and 44% for Glu dependency and 82%, 94% and 50% for Gln dependency respectively.

Using this validated CBM, we present predictions of patient nutrient source dependency based on their tumor genomics:

Case Study 1: A Glioblastoma multiforme (GBM) patient case with PTEN EGFRVIII and ALK mutation and high copy number of HIF1A and MIR-145. CBM predicted the patient to be Glu dependent and Gln independent.

Case Study 2: A GBM patient case with CTNNB1 mutation and low copy number of PTEN, RB1 and NF1. This patient was predicted to be both Glu and Gln dependent.

Case Study 3: A Triple Negative Breast Cancer (TNBC) patient carrying mutations for MYC, BRD4, EP300 and CREBBP. CBM predicted this patient to be Gln dependent and Glu independent.

The rationales for the nutrient source dependency predictions based on disease pathway characteristics were determined.

Conclusion: Using CBM we could successfully use patient genomic data to predict nutrient dependency of patient's tumor. This analysis enables creating options for precision diet for a patient to be used as an adjuvant alongside traditional approaches.

#1844

xCT promotes tumor formation in airway epithelial cells by activation MYC and WNT pathways.

Xiangming Ji. _Georgia State University, Atlanta, GA_.

INTRODUCTION: Many tumors increase uptake and dependence on nutrients such as glucose, cysteine or glutamine. These basic observations on cancer cell metabolism have opened multiple new diagnostic and therapeutic avenues in cancer. Recent studies demonstrated that smoking can induce the expression of xCT (SLC7A11) in normal epithelial cells and cancer cells, suggesting that overexpression of xCT may support lung tumor progression. These basic observations on cancer cell metabolism have opened multiple new diagnostic and therapeutic avenues in cancer. Here, we investigated the function of SLC7A11 (xCT), a cystine/glutamate anti-porter, in the pathogenesis of lung cancer. Recently, we found xCT to be strongly overexpressed at the cell surface of lung cancers and began to investigate the mechanisms by which xCT may contribute to lung cancer progression.

METHODS: We overexpressed the xCT in mutiple non-tumorgenic cell lines. These cell lines included 16HBE, BEAS2B, HBEC_KTR_P53_C7, and HEK293. We further investigated the role of xCT in cell proliferation, conly formation, and tumor formation in nude mice. Finally, we used RNAseq analysis to indentify the molecular mechanims associated with xCT expression in HEK293 cells.

RESULTS: Our results shows that xCT overexpression transformed the normal airway epithelium cell lines (HBEC3_KTR_P53_C7). The overexpression of xCT in HBEC_KTR_P53C7 induced colony formation in soft agar and promoted cell proliferation. In addition, we found overexpression of xCT reprogrammed multiple metabolic pathways such as glycolysis, glutaminolysis, and pentose phosphate pathway. In addition, we found overexpression of xCT could transform non-tumorigenic cell line HBEC_KTR_P53_C7 to tumor-formation cell line. Our RNAseq data indicated that this transformation is enriched in MYC and WNT pathway.

CONCLUSION: Our results demonstrate that xCT is highly expressed in a subset of NSCLCs. The induction of xCT in transformed aiway epithelial cells causes tumor formation in nude mice. The mechanism of malignant transformation involves the Warburg effect. Further investigation into the mechanisms of transformation may help in answering fundamental questions pertaining to how glutamate/cystine exchange contributes to lung cancer development and progression and also in establishing xCT as a potential novel therapeutic target in lung cancer.

Key words: Non-small cell lung cancer, amino acid transporters, metabolic reprogramming

#1845

BRAF-mediated NAMPT overexpression induces a melanoma cell dedifferentiation program leading to metabolic reprogramming and intrinsic resistant to BRAF inhibitors.

Valentina Audrito,1 Antonella Managò,1 Federica Gaudino,1 Ilaria Manfredonia,1 Vincenzo Gianluca Messana,1 Nicoletta Vitale,2 Lorenzo Brandimarte,1 Enrico Moiso,3 Roberto Piva,2 Nadia Raffaelli,4 Mario Mandalà,5 Silvia Deaglio1. 1 _IIGM Foundation & University of Turin, Turin, Italy; _2 _University of Turin, Turin, Italy;_ 3 _Koch Institute for Integrative Cancer Research, Cambridge, MA;_ 4 _Polytechnic University of Marche, Ancona, Italy;_ 5 _Papa Giovanni XXIII Hospital, Bergamo, Italy_.

INTRODUCTION: In order to support continued proliferation and growth, tumor cells must metabolically adapt to balance their bioenergetic and biosynthetic needs. In metastatic melanoma (MM) oncogenic BRAFV600E signaling is a critical regulator of this process. Consistently, treatment with BRAF/MEK inhibitors (BRAFi/MEKi) leads to a block of glycolysis, promoting energy stress mediated-apoptosis. However, resistance to therapy develops rapidly, with paradoxical activation of the MAPK/ERK signaling pathway and the acquisition of novel metabolic phenotypes.

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

METHODS: NAMPT expression and activity was studied using biochemical, enzymatic, immunohistochemical and ELISA assays. In silico analysis using TCGA database, RNAseq and functional/metabolic analyses were performed. Stable or inducible NAMPT overexpression was established using lentiviral vector. Xenografts of melanoma were set up using immunocompromised mice treated with NAMPT inhibitor (NAMPTi) alone or in combination with BRAFi.

RESULTS: We found that the BRAF oncogenic signature converges transcriptionally on the overexpression of NAMPT, leading to a marked increase in NAD levels, which in turn support a metabolic switch toward glycolysis or OXPHOS, both strategies exploited by BRAF-mutated melanomas to adapt to chronic exposure to BRAFi. NAMPT up-regulation in BRAFi resistant cells was confirmed in serial biopsies from melanoma patients. Furthermore, NAMPT could be dosed in patient plasma where it correlated with disease burden, response to therapy and overall survival.

Treatment of melanoma cells with NAMPTi depleted NAD, inducing mitochondrial stress, cell cycle arrest and apoptosis in vitro, while they were highly effective in controlling the disease in melanoma xenografts. Exogenous overexpression of NAMPT in BRAF-mutated melanoma cell lines leads to a net increase of NAD and ATP, rendering cells intrinsically resistant to BRAFi and supporting a proliferative/invasive phenotype in vitro and in vivo. Mechanistically, NAMPT overexpressing cells switch to a dedifferetiation/epigenetic program leading to a downregulation of microphthalmia-associated transcription factor (MITF).

CONCLUSION: In conclusion, this work links oncogenic BRAF signaling to metabolic reprogramming through NAD biosynthesis and identifies NAMPT as an actionable target for melanoma patients with BRAF mutations.

#1846

Acid sensing G protein-coupled receptor OGR1 is required for acid induced adiposomogenesis in breast cancer cells.

Smitha Pillai, Michael Langsen, Jonathan Nguyen, Jonathan Wojtkowiak, Marilyn Bui, Robert Gatenby, Robert Gillies. _Moffitt Cancer Center, Tampa, FL_.

Malignant tumors exhibit altered metabolism and consume higher levels of glucose compared to surrounding normal tissue, resulting in acidic extracellular microenvironment. Acidity in the microenvironment is a critical stress factor and selection force for the evolution of aggressive tumor types. There are several acid sensing cell surface receptors and ion channels (ASICs) that can sense acidity in the microenvironment; among them proton sensing G protein-coupled receptors (GPCRs) such as OGR1 (GPR68), TDAG8 (GPR65), GPR4 and GPR132 form a major class of acid receptors. Our previous studies demonstrated that acid adaptation is associated with survival mechanisms like chronic activation of autophagy and redistribution of the lysosomal proteins to the plasma membrane. When grown under acidic pH, breast cancer cells accumulate lipids as revealed by staining with Nile Red and perilipin 2, a protein that coats adiposomes or lipid droplets. These are dynamic organelles that store neutral lipids surrounded by a shell of proteins and a phospholipid monolayer. The lipids stored in adiposomes are produced de novo, as acid-induced lipogenic phenotype is maintained, even if cells are grown with de-lipidated serum. Inhibition of fatty acid synthesis was selectively toxic under acidic conditions and attenuated adiposome accumulation. Among the acid sensing receptors, TDAG8 and OGR1 are highly expressed in a panel of breast cancer cell lines compared to non-malignant breast epithelial cells. Highly invasive and acid tolerant MDA-MB-231 cells express significantly higher levels of TDAG8 while MCF7 and T47D cells have high levels of OGR1. We investigated the role of these receptors in transducing the acid signal that results in the accumulation of lipid droplets. CRISPR/Cas9 mediated depletion of the major acid sensors in breast cancer cell lines MCF7 and T47D showed that depletion of OGR1, not TDAG8, specifically inhibited acid induced adiposome accumulation. Additionally, inducing the cells with ogerin, an allosteric activator of OGR1 resulted in adiposomogenesis. Further, OGR1 knock out cells showed inhibition in cell growth under acidic growth conditions compared to OGR1 expressing cells. OGR1 knockout cells were defective in acid induced autophagy. OGR1 is coupled with Gq/11 and, upon ligand (H+) binding, triggers activation of phospholipase C stimulating the formation of the second messenger IP3. Adiposome formation was inhibited in presence of PLC inhibitors, Edelfosine or U73122. These results indicate that OGR1 is the major acid sensing GPCR in these cells that mediate adiposomogenesis. Accumulation of adiposomes is a highly regulated process related to storing autophagic products, and appears to be important in cell survival in acid stress. Taken together, increased dependence on lipid metabolism by cancer cells under acidic conditions reveals novel therapeutic vulnerabilities.

#1847

Cooperativity between glutaminase 1 and histone deacetylase SIRT7 regulates TGF-β mediated lung fibrosis.

Malay Choudhury, Xueqian Yin, Jeong-Han Kang, Mahefatiana Andrianifahanana, Edward B. Leof. _Mayo Clinic, Rochester, Rochester, MN_.

Fibroproliferative diseases are a leading cause of morbidity and mortality featuring localized and systematic tissue/organ fibrosis, which account for nearly 45% of all deaths in the developed world, with an unknown etiology and no effective treatment. Transforming growth factor beta (TGF-β) is a ubiquitous cytokine that contributes to fibroblast activation, myofibroblast differentiation, collagen overproduction and pathological tissue fibrosis. In that mounting evidence indicate that altered glutamine metabolism has critical roles in regulating signaling pathways, maintaining redox balance and macromolecular biosynthesis, we wished to test the general hypothesis that intervention of these metabolic processes could provide novel approaches to developing anti-fibrotic therapies. To that end, in the current work we provide a framework integrating glutamine dysregulation with profibrotic TGF-β signaling. We found that Glutaminase 1 (GLS1), which converts glutamine to glutamate, is induced by TGF-β and is also elevated in (i) fibroblasts isolated from Idiopathic Pulmonary Fibrosis (IPF) patients; and (ii) the murine bleomycin (i.e., TGF-β driven) model of lung fibrosis. While phosphorylation of Smad2 and Smad3 occurs independently of GLS1, TGF-β stimulated expression of profibrotic targets (i.e., Collagen I, PAI-1, CTGF, α-SMA and fibronectin) as well as biological actions (i.e., cell migration and anchorage independent growth) require GLS1 activity. Moreover, knockdown of Smad2 or Smad3 as well as inhibition of MAPK, PI3K, Akt or mTOR abrogates the induction of GLS1 by TGF-β; documenting that TGF-β regulates GLS1 expression via both Smad and non-Smad signaling. As GLS1 represents a critical component mediating the fibroproliferative actions of TGF-β, in order to identify the operative molecular mechanism(s) we focused on (i) a group of histone deacetylases called sirtuins (SIRT) which are known to elicit antifibrotic effects; and (ii) the Forkhead box protein O4 (FOXO4) transcription factor which has been shown to bind the GLS1 promoter. We found that there is not only a decrease in SIRT7 and FOXO4 coincident with the induction of GLS1 by TGF-β, but TGF-β inhibits SIRT7 deacetylation activity which in turn prevents FOXO4 deacetylation and inhibition of GLS1 expression. Last, consistent with SIRT7 and FOXO4 being negative regulators of profibrotic TGF-β signaling, their levels were also decreased in IPF fibroblasts and bleomycin-induced lung fibrosis. These studies point to an exciting, novel and unexplored connection between sirtuins, FOXOs and glutamine metabolism by elucidating the mechanism(s) by which epigenetic and transcriptional processes cooperate to regulate glutamine metabolism and fibrotic development in a TGF-β dependent manner.

#1848

The role of PDK1 in metabolic reprogramming of mesenchymal glioblastoma.

Neha Jain, Maheedhara Guda, Collin Labak, Chase Smith, Simon Park, Andrew Tsung, Kiran Velpula. _University of Illinois College of Medicine, Peoria, IL_.

Despite multimodal therapy and ever-improving surgical technique, glioblastoma (GBM) maintains its poor prognosis due to the chemotherapeutic resistance it builds over time, eventually rendering existing agents ineffective. A well-characterized phenomenon that takes place particularly in mesenchymal GBM tissue is the Warburg Effect, characterized by a shift from oxidative phosphorylation to glycolysis as a primary means to derive energy. Pyruvate Dehydrogenase Kinase 1 (PDK1) is one upregulated molecule in this shift toward anaerobic metabolism that is known for phosphorylating pyruvate dehydrogenase, the "gate keeper" of the TCA cycle. We sought to characterize further the role of PDK1 in terms of its metabolic role and any extra-metabolic functions it may serve. By silencing PDK1 and considering changes in downstream expression, we hope to understand what other factors serve as contributors to metabolic alterations seen in GBM. Elucidating the mechanism of this crucial shift more thoroughly, especially without the prominently cited role of PDK1, can begin to expose new targets for interrupting tumor maintenance and proliferation by affecting its metabolism. In this study we used differential expression of two vectors in which PDK1 was silenced, shPDK1 and SCBT-PDK1. GSEA analysis was done and analyzed to identify classes of genes or proteins that are over-represented and to identify significantly enriched or depleted groups of targets whose expression was vastly changed by silencing of PDK1. Using GSEA heat map data, gene targets whose expression was noted to be consistently changed with silencing of PKD1 targets were identified. Datamining and immunoblot analyses were then conducted to evaluate for expression of these genes in correlating GBM specimens. Ultimately, we further characterize PDK1's global role in GBM and evaluate non-metabolic effects of PDK1 that may still affect survival and proliferation of GBM tumors.

#1849

Cellullar modifications of glucose uptake through the modulation of AMPK & EGFR signaling axes in lung cancer cell lines.

Pedro Barrios-Bernal, Mario Orozco-Morales, Giovanny Soca-Chafre, Oscar Arrieta, Norma Hernández-Pedro. _Instituto Nacional de Cancerología, CDMX, Mexico_.

Background.

The exacerbated consumption of glucose in tumor cells is a widely reported event, this metabolic modification is associated with the activation of anabolic pathways that contribute to uncontrolled proliferation.

Lung cancer tumor cells often present glycolytic metabolism phenotypes with large lactate secretions, due to the fact that they usually develop in hypoxic microenvironments and with low nutrient concentrations.

The inhibition of signaling pathways triggered by growth factors receptors, as well as, the activation of AMP-activated protein kinase (AMPK) pathway has the ability to restore a catabolic phenotype in tumor cells thus controlling proliferation and evasion of the immune system.

It has been observed that treatment with EGFR tyrosine kinase inhibitors (TKIs) reduces the glycolytic phenotype in certain lung cancer cell lines, on the other hand Metformin has shown a reestablishment of oxidative characteristics in several cell lines of solid tumors.

Hypothesis.

The Afatinib-Metformin treatment has the ability to modulate the AMPK and EGFR signaling pathways with a decreasing in the glycolytic phenotype in cell lines with different mutational EGFR status.

Objective.

To determine the effect of combination Afatinib-Metformin on the activation of the EGFR and AMPK pathways and their relationship with glucose consumption in cell lines with different EGFR mutational status.

Methods.

Three different doses of Afatinib and one dose of Metformin were used in lung adenocarcinoma cell lines A549, H1975 and HCC827 (ATCC). Activation of AMPK and EGFR signaling pathways was assessed by Western Blot and Flow Cytometry.

Results.

All cell lines showed decreased expression of EGFR as well as its active form. Interestingly LKB1 inhibition was shown with all treatments in H1975 and HCC827 cell lines that express it basally. The evaluation of downstream protein activation of the EGFR pathway showed inhibition of the S6K effector when treatment with Afatinib was administered. As regards the evaluation of glucose uptake all cell lines showed a reduction of up to 50% compared to the control when the combined treatment was administered.

Conclusions.

The combination treatment Afatinib-Metformin shows regulation of EGFR signaling pathway and inhibition of LKB1 that is one of the activators of AMPK. A down regulation of the incorporation of glucose was shown and this could be associated with a reestablishment of the oxidative character of the metabolic phenotype.

#1850

Role of microRNAs in circulating tumor cells glutamine metabolism pathways.

Ziwen Zhu, Sarah N. Owen, Abhinav Achreja, Sunitha Nagrath, Deepak Nagrath. _University of Michigan, Ann Arbor, MI_.

Circulating tumor cells (CTCs) has an important role in the spread of cancers and metastasis. CTCs are believed to be the most promising model to understand metastatic progression in patients with cancer. Glutamine, an alternative carbon source to glucose, has recently been shown to be important for the cancer cell survival, growth and progression. The role of glutamine metabolism in CTCs is not known. MicroRNAs (miRNAs) are a group of highly conserved noncoding RNAs and approximately 22 nucleotides in length. Here, we postulate that CTCs have unique microRNAs (miRNAs) expression compared to resident tumor cells. And miRNAs control CTC glutamine metabolism for the metastasis. In this study, we used 3 primary CTC cultures to study how miRNA controlling glutamine metabolism. We identified several miRNAs have high expressions in CTCs and these miRNAs are regulating glutamine metabolism in CTCs. Our results demonstrate the metabolic miRNA targets of CTCs on cancer progression. These insights will present a unique opportunity for diagnosing and treating cancer through CTCs.

#1851

Novel roles of DNA-PK in metabolic regulation in prostate cancer.

Emanuela Dylgjeri,1 Jonathan Goodwin,1 Ayesha Shafi,1 Vishal Kothari,2 Giorgia Zadra,3 Erin Seifert,1 Felix Feng,2 Karen Knudsen1. 1 _Thomas Jefferson UNIV., Philadelphia, PA;_ 2 _University of California San Francisco, San Francisco, CA;_ 3 _Dana Farber Cancer Institute, Boston, MA_.

DNA dependent protein kinase catalytic subunit (DNA-PK) is a multifunctional kinase involved in repairing double-strand DNA breaks through non-homologous end joining (NHEJ), with parallel roles as a master transcriptional regulator of gene networks supporting cell migration and invasion. In prostate cancer (PCa), DNA-PK was found to be highly upregulated and hyperactivated. Strikingly, DNA-PK is the most deregulated kinase in metastatic castration resistant prostate cancer (CRPC), and is independently predictive of metastasis and overall survival in patients with high-risk disease. Combined, these findings highlight the importance of understanding DNA-PK functions beyond DNA repair and transcriptional regulation, which promote the acquisition of aggressive tumor phenotypes. Rapid immunoprecipitation of endogenous proteins (RIME) was performed to identify the DNA-PK interactome, which uncovered novel DNA-PK interactors that play key roles in metabolism. To assess the role of DNA-PK in regulation of metabolism, metabolic profiles of CRPC models were investigated upon DNA-PK modulation. Data presented will describe novel roles of DNA-PK in cancer metabolism and subsequent effects of DNA-PK modulation in key metabolic pathways.

#1852

Down-regulation of PRMT6 drives Warburg effect through ERK induced relocalization of PKM2 to promote tumorigenicity and sorafenib resistance in hepatocellular carcinoma.

Tin-Lok Wong, Lok-Hei Chan, Stephanie Ma. _The University of Hong Kong, Pokfulam, Hong Kong_.

Cancer cells have a high dependence of glycolysis for ATP production, especially under hypoxic environment. Metabolic reprogramming in cancer drives an increased glycolytic rate that supports maximal production of nutrients for tumorigenesis. Our group has recently identified the post-translational modification enzyme, protein arginine methyltransferase 6 (PRMT6) to be frequently down-regulated and to exhibit a tumour suppressive effect in maintenance of cancer stemness in hepatocellular carcinoma (HCC) (Chan LH et al. Cell Reports 2018). Our current study finds transcriptome and metabolome profiling of HCC cells with PRMT6 repressed to exhibit enrichment of genes and metabolites involved in glycolysis. In vitro, PRMT6 negatively regulates glycolysis, glucose uptake, lactate production and pyruvate kinase activity in HCC cell lines and patient-derived organoids. Our previous work found PRMT6 to interfere with RAS/RAF binding by directly binding to CRAF and methylating arginine100 residue. Supporting the importance of PRMT6-mediated CRAF methylation, we find overexpression of catalytic inactive PRMT6 to exhibit no effect on glycolysis. MEK/ERK, known downstream effectors of RAS/RAF signalling, has been shown to induce nuclear PKM2 localization. Consistently, we find modulation of PRMT6 to negatively regulate ERK and PKM2 expression and localization. Rescue experiments involving the ERK inhibitor U0126 and shPKM2 further substantiate the importance of ERK/PKM2-mediated glucose metabolic reprogramming in HCC cells with PRMT6 suppressed. Functionally, inhibition of glycolysis by 2-deoxyglucose sensitized PRMT6 knockdown cells to sorafenib treatment in vitro and attenuated tumor growth in vivo. In addition, we also find PRMT6 expression to be regulated via hypoxia by binding of RE1-silencing transcription factor to the promoter region. Rescue experiments by re-expressing PRMT6 under hypoxic conditions further support the involvement of PRMT6 in driving glycolysis in oxygen deprived environments. Our work uncovers a critical function for PRMT6 in driving Warburg effect through regulating expression and localization of PKM2 via post-translational modification of CRAF, thereby promoting HCC initiation and sorafenib resistance.

#1853

Association of IDH genes with prostate cancer progression and their regulation by the androgen receptor.

Kevin Gonthier, Raghavendra TK Poluri, Cindy Weidmann, Etienne Audet-Walsh. _Université Laval, Quebec City, Quebec, Canada_.

A primary function of the prostate is to synthesize and secrete high levels of citrate by way of a unique metabolic profile regulated by the androgen receptor (AR). Prostate cancer (PCa) is an androgen-dependent disease that is characterized by early reprogramming of citrate metabolism. Accordingly, mutations of the isocitrate dehydrogenase genes IDH1 and IDH2, which are key enzymes involved in the regulation of cellular citrate levels, have been demonstrated as important oncogenic events in several cancer types, including in about 3% of all PCa cases. However, if IDH1 and other IDH isoforms are associated with PCa progression as well as the regulatory factors controlling their expression remain mostly unknown. Across the human cancer spectrum, PCa appears to be the cancer type with the highest expression of IDH1, with levels even higher than common cancers associated with IDH1 mutations such as gliomas. Using publicly available datasets and quantitative PCR, we showed that IDH1 is the predominant IDH isoform expressed in PCa cells. In PCa, the androgen receptor was found to regulate several IDH isoforms in both in vitro and in vivo models of PCa, predominantly positively regulating IDH1. Chromatin immunoprecipitation experiments confirmed the recruitment of AR at several regulatory regions of IDH1 and IDH2. IDH1 and other IDH isoforms were shown to be significantly altered during PCa progression, which is consistent with a reprogramming of citrate metabolism in PCa. In addition, modulation of IDH expression significantly altered PCa cell proliferation and metabolism. Overall, our study indicates that IDH gene regulation is associated with PCa progression and that AR plays a significant role in the regulation of IDH genes.

## CANCER CHEMISTRY

### Anticancer Agents from Natural Sources

#1854

Novel pateamine analogs to target the translation initiation factor eIF4A in chronic lymphocytic leukemia.

Rong Chen,1 Mingzhao Zhu,2 Rajan R. Chaudhari,1 Omar Robles,2 Yuling Chen,1 Wesley Skillern,1 Qun Qin,1 William Wierda,1 Shuxing Zhang,1 Kenneth G. Hull,2 Daniel Romo,2 William Plunkett1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Baylor University, Waco, TX_.

The viability of chronic lymphocytic leukemia (CLL) is critically dependent upon staving off death by apoptosis, a hallmark of CLL pathophysiology. The overexpression of the Bcl-2 family proteins likely play a major role in the apoptosis blockade in CLL, and has been an effective target of CLL therapy. The recognition that Mcl-1, a major component of the anti-apoptotic response, is intrinsically short-lived and must be continually resynthesized suggested a novel therapeutic approach. Pateamine A (PatA), a macrolide marine natural product, inhibits cap-dependent translation by binding to the initiation factor eIF4A. We have previously reported the first total synthesis of PatA. Mechanistic studies suggested that binding of eIF4A to PatA caused the stalling of initiation complexes on mRNA, halting the translation initiation process. In this study, we demonstrated that a synthetic derivative of PatA, des-methyl des-amino PatA (DMDAPatA), blocked mRNA translation, reduced Mcl-1 protein and initiated apoptosis in CLL cells. This action was synergistic with the Bcl-2 antagonist ABT-199, by a mechanism to inhibit the two parallel arms of apoptosis control in CLL. However, avid binding to human plasma proteins limited DMDAPatA potency, precluding further development. To address this, we synthesized a new series of 27 PatA analogs with modifications on various regions of PatA, and screened their toxicity against the primary CLL cells. Any modifications on the side chain, or the rigid binding domain of the PatA macrolide ring led to complete loss of activity. Rather, introduction of an amino group at either the C2 or C3 positions of the flexible region of the macrocycle retained the activity and reduced plasma protein binding, likely through a lowered lipophilicity. We identified three new leads with potent inhibition of proteins synthesis and strong CLL cytotoxicity. They also exhibited greater selectivity towards CLL cells over normal lymphocytes comparing to the parental compound PatA. To gain structural insights into the interaction of the PatA analogs with eIF4A, a homology model of the human eIF4A1 was generated using the closed conformation of the eIF4A3 structure (PDB ID: 2HYI) as a template. The predicted PatA binding site is located at the interface of the N-terminal domain and the C-terminal domain, in between the RNA and ATP binding sites. In silico docking analysis of the PatA analogs to eIF4A correlated with their structure-activity relationships and suggested that these compounds may act by stabilizing the closed conformation of eIF4A. Thus, these novel PatA analogs hold promise for application to cancers within the appropriate biological context, such as CLL.

#1855

Re-purposing traditional Chinese anti-asthma formula ASHMI for triple negative breast cancer.

Sanjukta Chakraborty,1 Fei Mo,2 Martin Walsh,3 Changda Liu,3 Mingzhuo Cou,4 Rachana Maniyar,1 Ghada Ben Rahoma,1 Sarnath Singh,1 Tara Jarboe,1 Michelle Carnazza,1 Raj Tiwari,1 Xiu-min Li1. 1 _New York Medical College, Valhalla, NY;_ 2 _Shanghai University of Medicine and Health Sciences, China;_ 3 _Mt. Sinai School of Medicine, NY;_ 4 _Hunan University of Chinese Medicine, China_.

Breast cancer is the most common cancer among women worldwide, contributing to 25.4% of newly diagnosed cancer cases in 2018. Triple negative breast cancer (TNBC) accounts for 10-20% of the breast cancer cases and represents an unmet clinical need because of its higher risk of recurrence and metastasis. Adjuvant therapies have often been employed to prevent secondary recurrence. Traditional Chinese medicine (TCM) formulations have long been used in prevention and cure of many types of diseases because of its anti-inflammatory property. Mainstream treatment modalities for TNBC involve surgery, radiation and chemotherapy (adjuvant or neoadjuvant). Unfortunately, chemotherapy in these patients often lead to resistance, which underscores the importance of alternative therapeutic approaches for this cancer. To this end, we wanted to evaluate anti-carcinogenic property of a TCM formulation ASHMITM developed primarily for asthma treatment. ASHMI™ is an extract of 3 traditional Chinese medicinal herbs-Ganoderma lucidum (Ling-Zhi), Sophora flavescens Ait (Ku-Shen) and Glycyrrhiza uralensis Fischer (Gan-Gao). For this study, triple-negative/basal B mammary carcinoma cell line MDA-MB157 was treated with two different concentrations of ASHMI and cellular proliferation was examined by [3H] thymidine incorporation assay. ASHMITM treated cells showed approximately 74% less [3H] thymidine incorporation indicating dampened proliferation. This observation was validated using western blot analysis with whole cell lysate, showing reduced expression of proliferation markers PCNA and NFκB. Interestingly, an increased expression of tumor suppressor protein cyclin-dependent kinase inhibitor 1 C (p57, Kip2) was observed after ASHMITM treatment. Next, we tested anti-proliferative property of the individual constituents and observed Ganoderma lucidum (GL) extract had a comparable efficacy as the whole formulation at a concentration of 100μg/ml. This observation was further confirmed by western blot and the same expression pattern was observed for PCNA, NFκB and p57. GL was further fractionated into 11 sub-components and each component was tested for the their anti-proliferative activity using similar assays. Fraction 2 with Ganoderenic acid D showed dose-dependent inhibition of cell proliferation and was further tested for its activity at lower concentrations. Ganoderenic acid D had similar efficiency as 100μg/ml of whole GL extract at a concentration as low as 2.5μg/ml suggesting this active compound might be primarily responsible for the anti-proliferative effect of GL. Our studies using an anti-inflammatory formulation of TCM have identified an active anti-carcinogenic compound, Ganoderenic acid D, that could be potentially used as an adjuvant in TNBC therapies.

#1856

Targeting human telomeres by binding of epiberberine to telomeric G-quadruplex.

Clement Lin, Guanhui Wu, Kaibo Wang, Buket Onel, Saburo Sakai, Danzhou Yang. _Purdue University, West Lafayette, IN_.

Human telomeres play critical roles in cancer, aging, and genetic stability. Human telomeric DNA consists of tandem repeats of the sequence d(TTAGGG) and can form G-quadruplexes. G-quadruplexes are non-canonical DNA secondary structures formed in G-rich sequences, built upon the H-bonded G-tetrads and stabilized by monovalent cations such as K+ or Na+. Telomerase is a reverse transcriptase activated in 80-85% of human cancers. Small molecules that stabilize the telomeric G-quadruplex have been demonstrated to inhibit telomerase and disrupt telomere capping and maintenance, resulting in cancer cell apoptosis. Thus, the human telomeric G-quadruplex is considered an attractive target for anticancer drug development. G-quadruplexes formed in human telomeres are structurally polymorphic. The hybrid-2 G-quadruplex is the major form in the wild-type human telomeric DNA in the physiologically relevant K+ solution. Protoberberines are medicinal natural products with anticancer and anti-inflammatory activities. We show for the first time that a small molecule (epiberberine) specifically binds and induces the physiologically relevant hybrid-2 human telomeric G-quadruplex and converts other telomeric G-quadruplexes to the hybrid-2 structure, the first such small molecule reported. We determined the molecular structure of the 1:1 complex of epiberberine and hybrid-2 human telomeric G-quadruplex in K+ solution by NMR, which elucidates the molecular basis for this specific recognition. Epiberberine binding induces extensive rearrangement of the previously disordered 5′ flanking and loop segments to form an unprecedented four-layer binding pocket specific to the hybrid-2 human telomeric G-quadruplex. Epiberberine recruits the flanking (-1) adenine to form a "quasi-triad" intercalated between the external G-tetrad and a T:T:A triad, capped by a T:T base-pair. The crucial hydrogen-bonded pair is observed between epiberberine and the flanking (-1) adenine in the human telomeric sequences. This strong recognition determines the epiberberine's ability to convert other human telomeric G-quadruplex structures to the hybrid-2 structure, regardless of the presence and types of monovalent cation in solution. The deep intercalation of epiberberine in this multi-layer binding pocket explains the significant fluorescence enhancement of epiberberine induced by human telomeric sequences in K+. Our study provides structural insights into rational design of small molecule drugs targeting the hybrid-2 G-quadruplex predominant in the human telomeres in physiologically relevant K+ solution. Furthermore, the human telomeric sequence is polymorphic in nature and various G-quadruplexes can exist in dynamic equilibrium, the discovery of epiberberine provides a potential means to study the specific protein interactions and biological functions of the hybrid-2 telomeric G-quadruplex.

#1857

Proscillaridin A induces apoptosis and suppresses non-small cell lung cancer tumor growth via calcium-induced DR4 up-regulation.

Run-Ze Li, Xing Xing Fan, Xiao- Jun Yao, Elaine L. Leung, Liang Liu. _Macau University of Science and Technology, Macau, Macao_.

Non-small cell lung cancer (NSCLC) is the predominant histological type of lung cancer and is characterized by the highest mortality and incidence rates among these types of malignancies. Cardiac glycosides, a class of natural products, have been identified as a potential type of chemotherapeutic agent. This study aims to investigate the anti-cancer effects and the mechanisms of action of Proscillaridin A (P.A) in NSCLC cells. In vitro sodium-potassium pump (Na+/K+ ATPase) enzyme assays indicated that P.A is a direct Na+/K+ ATPase inhibitor. P.A showed potent cytotoxic effects in NSCLC cells at nanomolar levels. Treatment mechanism studies indicated that P.A elevated Ca2+ levels, activated the AMPK pathway and down-regulated phosphorylation of ACC and mTOR. Subsequently, P.A increased death receptor 4 (DR4) expression and down-regulated NF-κB. Interestingly, P.A selectively suppressed EGFR activation in EGFR mutant cells but not in EGFR wild-type cells. In vivo, P.A significantly suppressed tumor growth in nude mice compared to vehicle-treated mice. Compared with the Afatinib treatment group, P.A displayed less pharmaceutical toxicity, as the body weight of mice treated with P.A did not decrease as much as those treated with Afatinib. Consistent changes in protein levels were obtained from Western blotting analysis of tumors and cell lines. Immunohistochemistry analysis of the tumors from P.A-treated mice showed a significant suppression of EGFR phosphorylation (Tyr 1173) and reduction of the cell proliferation marker Ki67. Taken together, our results suggest that P.A is a promising anti-cancer therapeutic candidate for NSCLC. Acknowledgements This work was supported by the Macau Science Technology Development Fund (project code: 021/2013/A1, 005/2014/AMJ & 010/2016/A1).

#1858

Saffron restricts MACC1-dependent cell proliferation and motility of colorectal cancer cells, and alters the microbiome structure.

Nazli Güllü,1 Dennis Kobelt,1 Hassan Brim,2 Lena Timm,1 Janice Smith,1 Hamid Shoraka,2 Akbar Soleimani,2 Silvia Bisti,3 Marjorie Gondre-Lewis,2 Hassan Ashktorab,2 Ulrike S. Stein1. 1 _Charité Berlin and Max Delbrück Ctr. for Molecular Medicine, Berlin, Germany;_ 2 _Howard University, Washington, DC;_ 3 _Howard University, Berlin, DC_.

Saffron has proven a beneficial effect as a supplement in the treatment of many conditions through its perceived anti-oxidative and anti-inflammatory properties. We previously identified the gene MACC1 and demonstrated its importance as metastasis inducer, prognostic and predictive biomarker for colorectal cancer (CRC). It is confirmed as decisive driver for tumorigenesis and metastasis for a broad range of solid cancers. Therefore, we explored the inhibitory impact of saffron extracts on MACC1-induced cancer cell growth and motility and its effects on the gut microbiome. First, we assessed cell proliferation of MACC1-high and MACC1-low expressing cells using MTT and the IncuCyte real-time cell imaging system. We employed the endogenously MACC1-low expressing colon cancer cell line SW480 and clones with ectopic MACC1 overexpression. In addition, SW620 cells with endogenous high MACC1 expression and CRISPR-Cas9 mediated MACC1 knock-out were used. Cells were treated with increasing amounts of extracts from 4 different saffron species and concentration-dependent proliferation was analyzed by MTT assay. Changes in cell cycle were assessed by FACS. In vitro migration was measured by Boyden chamber assays. Activation of caspases was analyzed by Western blot. In addition, rats treated with saffron had their gut microbiome analyzed through 16S rDNA sequencing of fecal DNA samples. Upon addition of saffron, cells with high MACC1 expression showed a growth delay compared to cells with reduced MACC1 expression. Further, MACC1-dependent migration was reduced when cells were treated with saffron extracts. We did not find activation of caspases, but we showed a cell cycle arrest of MACC1 positive cells treated with saffron extracts. Using real-time measurement of cell proliferation we identified crocin as most active compound, in our system. Furthermore, rats fed with saffron showed major changes at the phylum level of their gut microbiome. A dramatic reduction/depletion of Cyanobacteria and Proteobacteria and a decrease of Bacteroidetes/Firmicutes ratio was found within the saffron treated rats. These reductions were accompanied by enrichment in Spirochaetes, Tenericutes and Candidatus saccharribacteria phyla in these rats. This is the first identification of saffron-based compounds restricting cancer cells proliferation and motility progression via the novel target MACC1. In addition, change of microbiome composition by saffron favors short chain fatty acid synthesis. The use of saffron or some of its extracts might be of therapeutic value to CRC patients.

#1859

Quxie capsule inhibits the colon tumor growth through modulating the gut microbiome and tumor myosin 11 expression.

Dongmei Chen,1 Daoyuan Wei,2 Yufei Yang,3 Peiying Yang2. 1 _Beijing University of Chinese Medicine, Beijing, China;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _Xi-Yuan Hospital, Beijing, China_.

Traditional Chinese Herbal Medicine formula, such as the Quxie Capsule (QXC), has been used for advanced colorectal cancer treatment with favorable clinical outcome at Xiyuan Hospital in Beijing, China. However, the mechanism(s) underlying the anticancer effect of QXC in colon cancer still remain unclear, which hampers its optimal use for the treatment of colon cancer. The purpose of this study is to examine whether, and if yes, how QXC exerts anti-tumor activity via alteration of gut microbiota and related pathways. The antitumor efficacy of QXC was tested in CT-26 syngeneic mouse colon cancer models. Gut microbiome was measured by 16sRNA sequencing in fecal samples. Signaling protein expression profile in tumor tissues was assessed by RPPA and validated by Western blot. QXC gavaged to mice carrying CT26 tumors for 2 weeks significantly reduced the average tumor weight (0.92 ± 0.15g) compared to that of the vehicle control (1.57 ± 0.1689, p < 0.05). In contrast, when mice bearing CT26 tumor were pretreated antibiotics, the tumor weight (0.51±0.11g) in QXC treated mice was not statistically significantly smaller than that of vehicle control (0.76±0.13g, p>0.05). Mice pretreated with vehicle for 12 days then transplanted with fecal from QXC treated mice had smaller tumor compared with vehicle only group (p<0.05). Similarly, the tumor in mice pretreated with QXC and followed by transplantation of fecal from vehicle control mice grew faster than that of QXC treated alone, but not statistically significant (p>0.05). The result of 16sRNA sequencing showed that the ratio of Firmicutes over Bacteriodetes (F/B) was decreased by 2.03 folds in tumor-bearing mice compared to non-tumor-bearing mice (p<0.05). Intriguingly, QXC led to an increase of Firmicutes and reduction of Bacteriodetes and resulted in a 2.0-fold increase in the ratio of F/B compared to that prior to QXC treatment (p<0.05). The OTUs were significantly and markedly decreased (p<0.05) after antibiotics treatment. Both alpha and beta diversity in antibiotic and QXC co-treated mice were similar to that of the vehicle control and antibiotic co-treated group. The result of RPPA showed that protein Myosin 11, which was downregulated in fiber-deprived colon mucosa, was significantly elevated in QXC treated tumor tissues compared to that of control (p<0.05). In contrast, Myosin 11 expression was lower in the tumor tissues of antibiotics and QXC co-treated mice compared to that of antibiotics and vehicle control co-treatment group. QXC elicited upregulation of Myosin 11 protein expression was further validated in QXC treated CT-26 cells in vitro. In conclusion, QXC inhibited the growth of colon tumors, which could be due to its ability to rebalance the intestinal microbiota and upregulate the myosin 11 level in the syngeneic colon cancer model. Note: This study was partially founded by National Natural Science Foundation of China (No. 81373824).

#1860

Muscadine grape extract prevents proliferation of trastuzumab-sensitive and -resistant HER2 positive breast cancer cells and has synergistic effects when combined with trastuzumab.

Jessica Mackert, Patricia E. Gallagher, E. Ann Tallant. _Wake Forest School of Medicine, Winston Salem, NC_.

Human epidermal growth factor receptor 2 (HER2) positive breast cancer, characterized by overexpression of the HER2 protein, is an aggressive type of breast cancer comprising ~20% of diagnosed cases. Although targeted HER2 drugs, such as trastuzumab (TRZ), have reduced mortality by about one third, many patients still experience primary or acquired resistance. Grape seed/skin extracts are popular dietary supplements due to their potent antioxidant and anti-inflammatory properties. However, extracts from muscadine grapes are not extensively studied, and information regarding their therapeutic effects on cancer is limited. The objective of this study was to determine whether a muscadine grape skin and seed extract (MGE) inhibits the proliferation of TRZ-sensitive and -resistant HER2 positive breast cancer cells, and determine if combination treatment with MGE and TRZ has synergistic effects. Treatment with increasing concentrations of MGE for 48h significantly inhibited proliferation of TRZ-sensitive SKBR3 and -resistant HCC1954 human HER2 overexpressing breast cancer cells in a time and dose-dependent manner. HER2 heterodimerization and phosphorylation results in activation of the protein kinase B (AKT) pathway, which regulates breast cancer proliferation and cell survival. Treatment with 20 μg/mL MGE for 24h reduced HER2 protein expression by 52% (p≤0.01) in SKBR3 and 60% (p≤0.01) in HCC1954 cells. AKT activation was also reduced by 63% (p≤0.001) and 65% (p≤0.0001) in SKBR3 and HCC1954 cell lines, respectively. Active AKT stabilizes s-phase kinase-associated protein 2 (SKP2), ultimately leading to increased ubiquitination and degradation of cell-cycle inhibitor cyclin-dependent kinase inhibitor 1B (p27) and its transcription factor forkhead box protein 1 (FOXO1). Treatment with 20 μg/mL MGE for 24h increased total FOXO1 and p27 levels by 4-fold (p≤0.01) and 2.5-fold (p≤0.05), respectively, in SKBR3 cells. In addition, FOXO1 was increased 5-fold (p≤0.0001) and p27 4-fold (p≤0.05) in HCC1954 cells. Furthermore, SKP2 protein expression was reduced by 66% (p≤0.01) in SKBR3 and 73% (p≤0.01) in HCC1954 cells. MGE, in combination with TRZ, was synergistic at five different dose combinations (determined using Chou and Talalay method of synergy), and significantly inhibited proliferation to a greater extent than either agent alone in TRZ-sensitive SKBR3 cells. In summary, treatment with MGE reduced proliferation, inhibited components of the AKT pro-proliferation/survival pathway, and had synergistic effects when combined with TRZ in HER2 positive breast cancer cells. Thus, MGE may serve as an effective therapeutic either administered singly or in combination with targeted therapies for the treatment of HER2 positive breast cancer.

#1861

Aviscumine (ME-503) suppresses the growth of melanoma by potentially targeting c-Myc pathway.

Peiying Yang,1 Tara Conway,1 Patrea Rhea,1 Dongmei Chen,1 Bo Wei,1 Jibin Ding,1 Hans Lentzen,2 Jennifer McQuade1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _2MELEMA Pharma GmbH, Hamburg, Germany_.

Aviscumine, a recombinant mistletoe lectin I, is produced in E. coli and has been evaluated for its antitumor activities in various experimental in-vitro and in-vivo tumor models as well as in clinical trials. A phase II trial demonstrated the safety and efficacy of Aviscumine in pretreated patients with metastatic melanoma (stage IV). However, the mechanism(s) underlying the effect of Aviscumine in melanoma is inconclusive. Here, the antitumor activities and relevant mode of actions of Aviscumine were investigated in human melanoma A375, mouse melanoma Yummer and B16 cells as well as their relevant xenograft animal models. Cells were treated with Aviscumine (0-20 ng/mL) for 48 and 72 hrs, and cell proliferation was measured by MTT assay. Alteration of cell growth regulatory proteins and cell signaling proteins were determined by Reverse Phase Proteomic Array (RPPA) and validated with western blotting. Aviscumine exerted much stronger anti-proliferative activity in both A375 and Yummer cells with IC50 of 0.18 ± 0.03 ng/ml and 0.41 ± 0.06 ng/ml, respectively than that of B16 cells (IC50 15.93 ± 3.48 ng/ml). In A375 cells, Aviscumine significantly increased subG0/G1 population suggesting Aviscumine treatment led to apoptotic and necrotic cell death. Additionally, downregulation of various proteins associated with apoptotic cell death (pCDK1, pRB, MCl-1) was also observed by RPPA. Furthermore, Aviscumine significantly decreased c-Myc protein expression in a concentration-dependent manner measured by both RPPA and western blot. Interestingly, baseline c-Myc protein expression was much higher in the Aviscumine-sensitive A375 and Yummer cells than that in the Aviscumine-resistant B16 cells. Finally, the effects of Aviscumine on tumor growth were tested via subcutaneous injection (30 ng/kg, twice per week for three weeks) in mice bearing A375 or B16 melanoma. At 3 weeks, A375 tumors were markedly smaller in Aviscumune treated mice (383.8 ± 102.2 mg) than in the control-treated group (922.4 ± 296.1 mg). c-Myc protein expression was also significantly reduced (62% vs. control) in Aviscumine treated A375 tumors, as were levels of multiple downstream metabolites (pyruvate, malate, and glutamate) (p< 005). In contrast, similar to the in vitro data, Aviscumine treatment showed no tumor growth inhibitory effects in B16 tumor-bearing mice evidenced by minimum reduction on terminal tumor weight between vehicle control (1455 ± 430 mg) and Aviscumine treated group (1323 ± 270 mg). Together, these preliminary data implicate c-Myc as both a target and predictive biomarker for Aviscumine elicited antitumor activity.

#1862

Natural compound E targets both lung cancer stem and non-cancer stem cells by disrupting HSP70 function.

Seung Yeob Hyun, Huong Thuy Le, HongLan Pei, Simin Chun, Hye-Young Min, Suckchang Hong, Ho-Young Lee. _Seoul National University, Seoul, Republic of Korea_.

Solid malignant tumors are composed of heterogeneous populations of cancer cell, including cancer stem-like cells (CSCs), a specialized cell population characterized by the functional properties of self-renewal and have a potential to differentiate into various cancer subtypes. CSCs contribute to tumor recurrence, metastasis, and drug resistance, affecting efficacy of anticancer therapies. However, treatment strategies specifically targeting CSCs are limited, because current chemotherapeutic drugs mainly kill the bulk of sensitive cancer cells, leaving behind CSCs. These remaining CSCs are the main cause of cancer relapse and drug resistance. Hence, development of novel compounds eradicating CSCs are crucial for effective anticancer therapies. To identify novel CSC-targeting anticancer drugs, we have set-up a reporter system based on promoter activities of CSC markers that allows a standardized identification of CSCs. By using the reporter system, we screened a large natural products chemical library consisting 452 compounds with diverse chemical entity. And we selected 4 compounds. To further assess the inhibitory effect of these 4 compounds, we used sphere-forming assay with NSCLC cell lines. Through these processes, we finally chose Compound E. We found that treatment with Compound E significantly reduced the subpopulation of NSCLC CSC. We confirmed the inhibitory effects of Compound E on NSCLC CSCs by performing sphere formation analysis and flow cytometry-based ALDH detection assay. Treatment with Compound E also suppressed the viability and colony-forming abilities of NSCLC cells (non-CSCs) and their sublines carrying acquired chemoresistance by inducing apoptosis. In line with these in vitro results, orally administered Compound E significantly suppressed the growth of xenograft tumors derived from both NSCLC cell lines and patient-derived tumor tissues. To determine the tumor forming capacity of tumor cells derived from the vehicle or Compound E-treated mice, we carried out a limiting dilution tumor-propagating assay. NOD/SCID mice were transplanted with tumor cells over a range from doses unable to initiate tumor growth to dose that initiated tumor formation. Tumor cells derived from Compound E-treated mice displayed reduced tumor forming capacity compared to vehicle treated mice. Mechanistically, Compound E disrupted Hsp70 function by binding to the ATP-binding pockets of Hsp70. These data collectively unveil the potential of Compound E as a natural Hsp70 inhibitor targeting both CSC and non-CSC populations of NSCLC.

#1863

Identification of anticancer agent from antiallergic treatment regimens of traditional Chinese medicine.

Tara Jarboe, Michelle Carnazza, Sanjukta Chakraborty, Nan Yang, Kamal Srivastava, Jan Geliebter, Raj K. Tiwari, Xiu-Min Li. _New York Medical College, Valhalla, NY_.

Traditional Chinese Medicine (TCM) is a holistic system that has been widely used in the treatment of allergy, and several specific herbal formulations have been used as treatment agents. One such compound, berberine, is a plant alkaloid isolated from Phellodendri chinensis. Berberine has been widely used in both Chinese and Ayurvedic medicine and has antimicrobial and antiprotozoal properties. Additional pharmacologic actions reported include anticancer activity as well as anti-inflammatory effects, specifically suppression of proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha. Berberine has been shown to inhibit IgE production in myeloma cell line U266 without cytotoxicity. In U266 berberine inhibits nuclear translocation of NFκB by dampening phosphorylated IκB expression, subsequently decreasing activation of the ε-germline transcript promoter and suppressing IgE synthesis. Since this pathway plays a crucial role in many aspects of tumorigenesis, we hypothesized that this could have anticancer activity in solid tumors, specifically melanoma and anaplastic thyroid cancer (ATC). Melanoma and thyroid cancer are diagnosed with a high incidence, increasing over the last few decades. Newer therapies being evaluated in melanoma and thyroid cancer include checkpoint inhibitors. These therapeutic modalities, like others, have off target effects and high relapse rates due to acquired resistance. To identify safer alternative therapeutic strategies we characterized berberine for its anticancer properties through an in vitro proliferation assay with XTT on three melanoma cell lines (SKMEL28, SKMEL37, and SKMEL103), four ATC cell lines (T238, SW1736, HTh74, and 8505C), and three papillary thyroid cancer cell lines (TPC1, BCPAP, and K1). These cell lines were treated with 2μM berberine for 24 hrs. Berberine inhibited cellular proliferation in a dose and time dependent manner in all of these tumor types. ATC cell lines showed more sensitivity towards the drug compared to the other morphotypes. This is specifically significant since therapeutic options for ATC are extremely limited. Both ATC and melanoma have a high nuclear NFκB level, potentially contributing towards their aggressive phenotype. Cells were treated with 2μM berberine and expression of cyclinD1, CDK2, Bcl2, TRAF1, TRAF2 and IAP were evaluated by western blot. Berberine showed a differential regulation of these molecules in melanoma and thyroid cancer. This study opens up a new avenue for the adjuvant treatment of these malignancies using naturally occurring compounds.

#1864

Verticillin A causes DNA damage and apoptosis in high grade serous ovarian cancer.

Amrita Salvi, Julia Austin, Daniel Lantvit, Joanna Burdette. _University of Illinois, Chicago, Chicago, IL_.

High grade serous ovarian cancer (HGSOC) is the most lethal gynecological malignancy affecting women worldwide and the fifth most common cause of cancer related deaths among U.S. women. New targeted therapies are needed to prevent HGSOC progression and metastasis related lethality from the disease. The goal of this study was to test the novel natural compound, Verticillin A, for its anticancer properties and mode of action in HGSOC cells. Verticillin A is an epipolythiodioxopiperazine (ETP) alkaloid that is isolated from several terrestrial and marine filamentous fungi and has been shown to be cytotoxic in several cancer cell lines including OVCAR8, OVCAR4 and Kuramochi. Our data indicated that Verticillin A treatment caused cytotoxicity in HGSOC cell lines in a dose-dependent manner.

Furthermore, treatment with Verticillin A in HGSOC cell line OVCAR8 and OVCAR4 enhanced apoptosis, which was demonstrated by PARP cleavage and Annexin V/ Propidium iodide staining. To determine whether Verticillin A caused in vivo tumor growth inhibition, OVCAR8-RFP cells were xenografted in mice to form tumors and the mice were treated with Verticillin A. Encapsulated nanoparticles of Verticillin A decreased tumor growth in vivo and had low cytotoxicity compared to the naked drug. RNA-Seq analysis was performed with OVCAR8 cells treated with Verticillin A and the data found an upregulation of apoptosis signaling pathway and oxidative stress response and downregulation of cancer stemness signaling pathways. A proteomic histone profiling performed in OVCAR8 cells indicated that Verticillin A caused epigenetic modifications with global changes in histone methylation and acetylation marks. Thus, our study identifies Verticillin A as a novel epigenetic modifier in ovarian cancer cells and indicates therapeutic potential for treatment of HGSOC.

#1865

Buserelin trisphenylmethanol derivatives as anti-cancer prodrugs.

Ryan Beni, William Boadi, Renner Antwi. _Tennessee State University, Nashville, TN_.

Buserelin (BUS) is a synthetic nonapeptide agonist analog of LH-RH, first reported by Hoechst in 1976. It is used for the treatment of advanced prostate cancer. Side effects of buserelin include musculoskeletal pain, hearing disorders, increased thirst, worsening hypertension, anxiety, memory and concentration disturbances, fatigue, nervousness, palpitations, leukopenia, thrombocytopenia, altered blood lipids, and glucose intolerance. Therefore, improving of the biological activity of BUS by increasing the cellular uptake and retention is a remedy to this end. In this research, tris(4-methoxyphenyl)methanol (TPM) derivatives were synthesized and linked to BUS through hydrophobic linkers to enhance and optimize the hydrophobicity of this drug with the expectation to improve the cellular uptake. In this regard, several BUS conjugates of TPM derivatives with optimized hydrophobicity were synthesized by the reaction of methoxy benzenes (e.g. anisole, 2-fluoroanisole, 2-trifluoromethylanisole, 2-methylanisole, or 1,2-dimethoxybenzene, and 1,3,5-trioxane, followed by the conjugation with BUS and lauric acid in the presence of HBTU/DIPEA/DIC in moderate yields. Comparative antiproliferative assays between TPM-BUS conjugates and the corresponding non-covalent physical mixtures of the TPM derivatives and BUS were performed against human acute lymphoblastic leukemia (CCRF-CEM), human ovarian adenocarcinoma (SK-OV-3), and mouse pre adipocytes (3T3-L1) cells. TPM-BUS conjugates inhibited the cell proliferation of CCRF-CEM, SK-OV-3 and 3T3-L1 cells by 48-86%, 32-77%, 42-67%, respectively, at a concentration of 1-25 µM after 24-72 h of incubation. These data suggest that TPM-BUS derivatives with optimized hydrophobicity can be used to improve the biological activity of BUS.

#1866

Sea cucumber (Stichopus japonicas) F2 enhanced TRAIL-induced apoptosis via XIAP ubiquitination-ER stress in colorectal cancer cells.

Jung lim Kim, Seong Hye Park, Soyeon Jeong, Bo Ram Kim, Yoo Jin Na, Min Jee Jo, Yoon A Jeong, Hye Kyeong Yun, Dae Yeong Kim, Bu Gyeom Kim, Sang Cheul Oh, Dae-Hee Lee. _KOREA University GURO Hospital, Seoul, Republic of Korea_.

Natural products have shown great promise in sensitizing cells to TNF-related apoptosis-inducing ligand (TRAIL) therapy. Sea cucumber (SC) extracts possess antitumor activity, and hence their potential to sensitize CRC cells to TRAIL therapy was evaluated. This study used Western blotting to evaluate the combination effects of SC and TRAIL in CRC and determined the molecular mechanism underlying these effects. SC fractions and TRAIL alone had no effect on apoptosis; however, combined treatment dramatically induced apoptosis of CRC cells, but not normal colon cells. Combined treatment induced expression of apoptotic proteins (poly (ADP-ribose) polymerase (PARP), caspase 3, and 8), and this effect was markedly inhibited by ubiquitination of X-linked inhibitor of apoptosis protein (XIAP). SC did not affect the mRNA levels, but increased proteasomal degradation and ubiquitination of XIAP protein. Furthermore, SC induced reactive oxygen species (ROS) production, thereby activating c-Jun N-terminal kinase (JNK) and endoplasmic reticulum (ER) stress-related apoptotic pathways in CRC. Taken together, our results demonstrate that the SC fraction may sensitize CRC cells to TRAIL-induced apoptosis through XIAP ubiquitination-ER stress.

#1867

**Liver and urinary bladder cancers: The modifying role of aqueous leaf extract of** Terminalia glaucescens **Planch. ex Benth.**

Jeremiah Olorunjuwon Olugbami,1 Robert Damoiseaux,2 James Kazemier Gimzewski,2 Oyeronke Adunni Odunola1. 1 _University of Ibadan, Ibadan, Nigeria;_ 2 _University of California, Los Angeles, CA_.

Background: The aggressive and asymptomatic nature of liver cancer etiology in blacks is well documented. Furthermore, in Egypt, bladder cancer accounts for as many as 31% of all cancer cases, while in the US, the incidence in whites is higher than in blacks. There has been an increased search for phytochemicals, such as capsaicin (a bioactive component of hot peppers), which are easily available, and possess chemopreventive and chemotherapeutic activities. We therefore investigated the potential scientific relevance of aqueous leaf extract of Terminalia glaucescens (ALET) in cancer management. Terminalia glaucescens has been ethnomedicinally associated with various biological and therapeutic properties.

Methods: Total flavonoid and phenolic contents of ALET were assessed spectrophotometrically using gallic acid and quercetin as standards, in addition to its free-radical-scavenging activity involving 2,2-diphenyl-1-picrylhydrazyl assay with butylated hydroxytoluene and vitamin C as reference compounds. The hydroxyl-radical-scavenging and reducing-power activities were correspondingly assessed. In addition, antiproliferative activities of ALET in comparison with capsaicin on normal and cancer cell lines of human liver (THLE-3 and HepG2) and urinary bladder (HUC-PC and MC-T11) were evaluated using fluorometry. Luminometry was used to determine ATP concentrations, caspase 3/7 activities, glutathione status, and mitochondrial functions.

Results: Quantitative phytochemical assessments indicate the predominance of phenolic compounds (599.61 ± 6.14 µg gallic acid equivalents per mg of ALET) as compared with flavonoids (144.27 ± 3.44 µg quercetin equivalents per mg of ALET). ALET possesses comparable free-radical-scavenging, antioxidant and reducing-power activities in comparison to the standards. Treatment of the four cell lines with ALET for three days results in the following percent total cells: THLE-3 (30.13% at 24 h; 5.65% at 48 h; 5.43% at 72 h), HepG2 (31.09% at 24 h; 4.75% at 48 h; 1.41% at 72 h), HUC-PC (7.33% at 24 h; 2.35% at 48 h; 1.74% at 72 h), and MC-T11 (19.79% at 24 h; 9.20% at 48 h; 0.29% at 72 h). Assessment of the ATP levels after 24 h treatment with ALET resulted in a concentration-dependent depletion with a remarkable effect on HUC-PC and MC-T11 urinary bladder cells. ALET specifically caused a concentration-dependent decrease in caspase 3/7 activities and glutathione levels in HepG2 cells. ALET seems more toxic to the mitochondria at higher concentrations as compared with capsaicin.

Conclusions: ALET could be a natural source of mitocans for the treatment of cancers.

#1868

B-125, a novel berberine derivative, acts as a better retinoid X receptor a agonist with stronger anti-tumor activity in colon cancer.

Beibei Xu, Tian hui Hu, jing Xiong. _Xiamen University, Xiamen, China_.

Our previous study showed that Berberine binds to RXRα to suppress colon cancer growth (Ruan et. al., Oncogene 2017, 36: 6906). To achieve better RXRα binding activity, stronger anti-tumor effect and higher bioavailability, 21 novel berberine derivatives were designed based on the binding information of berberine to RXRα. Among them B-125 performs the best solubility, RXRE luciferase activity and tumor suppressing ability. NMR and fluorescent titration results show that B-125 retain the similar binding ability to RXRα compared with Berberine. CD study suggests that B-125 has stronger allosteric effect on RXRα than berberine. The bioavailability of B-125 is 25 times higher than berberine. In KM12C colon cancer cells, B-125 performs stronger RXRα dependent inhibitory effects than berberine. In vivo results showed that B-125 inhibits the growth of colon cancer xenograft in nude mice, and the effect is stronger than that of berberine. All the above results indicate that B-125 is a better anti-cancer drug than berberine with better RXRα modulation ability, higher bioavailability and stronger tumor suppressing effect. The study promotes B-125 as a potential novel RXRα based anti-cancer drug in the treatment of colon cancer.

#1869

**Mechanistic evaluation of ergosterol peroxide on** in vitro **breast cancer models.**

Camille L. Santiago,1 Fatima Rivas,2 Taotao Ling,2 Ivette J. Suarez-Arroyo,3 Gabriela Ortiz,3 Mercedes Y. Lacourt,3 Anibal Valentin,3 Walter H. Lang,2 Michelle M. Martínez-Montemayor3. 1 _Unviersity of Puerto Rico-Bayamon Campus, Bayamon, PR;_ 2 _St. Jude Children's Research Hospital, Memphis, TN;_ 3 _Univ. Central del Caribe School of Medicine, Bayamon, PR_.

Ganoderma lucidum extract (GLE), an extensively studied mushroom for its anti-cancer and anti-inflammatory properties, has been shown to possess physiologically active compounds such as polysaccharides, triterpenoids and sterols. In our previous studies, we reported that commercially available GLE whole mushroom extract selectively inhibits cancer cell viability, induces apoptosis, reduces invasion, and regulates key signaling molecules in various cell models of breast cancer. Hence, we sought to elucidate the compounds responsible for the observed anti-cancer activity. Among the isolated compounds from GLE, we identified a steroidal compound, Ergosterol Peroxide (EP), which has shown antiproliferative activity against cancer. EP is a member of natural endoperoxides, featuring the peroxy- warhead that can lead to hydroxyperoxy radicals or be activated and react as nucleophile, leading to significant biological cytotoxicity against various cancer models (e.g. ovarian, liver, colon). We hypothesized that EP would promote cell death mechanisms in the aggressive breast cancer phenotype. Thus, in this study, we evaluated EP's anti-cancer potential using immunoblotting, colony formation, cell motility, cell cycle and reactive oxygen species (ROS) production assays against human breast cancer SUM-149, MCF-7, MDA‐MB-231 cellular models. We show that EP displays anti-proliferative effects through cell arrest in G1 phase, and apoptosis induction via caspase 3/7 activation. EP induced ROS and decreased the migratory and invasive phenotype of these cancer cells. Moreover, it inhibited the expression of total AKT1, AKT2, BCL-2, BCL-XL, Cyclin D1 and c-Myc. Our combined results indicate that EP from GLE is a promising molecular scaffold for further exploration as an anti-cancer agent,particularly in aggressive breast cancer models.

#1870

Polyphenols from Korean artemisia annua L (pKAL) showed anti-cancer effects by multiple mechanisms on HCT116 human colorectal cancer cells.

Won Sup Lee,1 Anjugam Paramanantham,1 Eun Joo Jeong,2 Suchismita Raha,2 Hye Jung Kim,2 GonSup Kim,2 Sung Chul Shin2. 1 _Gyeongsang National Univ. Hospital, Jinju, Republic of Korea;_ 2 _Gyeongsang National University School of Medicine, Jinju, Republic of Korea_.

Artemisia annua L., (Gaddongsook in Korean), an annual herb, has been used for a long time in Korean folk medicine for the treatment of infectious or chronic disorder In addition, it possesses anti-inflammatory, and anti-cancer activity. However, the molecular mechanism for anti-cancer properties of Korean Artemisia annua is still unclear. Here, we investigated the anti-cancer effects of we investigated the anti-cancer effects of polyphenols from Korean Artemisia annua (pKAL) on HCT116 human colon cancer cells. pKAL induced cell death associated with the alteration of cell morphology and DNA content. Annexin V and PI staining revealed that late phase apoptosis. pKAL induced ROS generation and the cell death was recovered by NAC. Western blot revealed that pKAL significantly up-regulated the gamma-H2AX, p21, p53, p62 and cleavage of caspases-3 and PARP in HCT116 cells. pKAL induced changes in the expression of p53-dependent targets (p53, p21, Bax, Bak, and Fas). It also induced alteration of the expression of autophagy-related proteins (beclin1, p62, LC3-I, and LC3-II) posttranslational modification of proteins (Lamin A/C, HSP90, GM130) involved in the maintenance of intracellular membrane integrity. Unexpectedly, the effect of pKAL on cell death pKAL was similar in both the P53 wild-type and p53-null HCT116 colon cancer cells, although there was some difference in cell cycle arrest; in P53 wild-type cells, pKAL appeared to induce G1 arrest at lower concentration (50 μg/mL), whereas in p53-null cells, pKAL induced G2/M arrest. The cell death was regulated by JNK and p38 MAPK signaling. The cancer effects of pKAL was similar in p53-null NCI-H1299 cells. We also investigated the protein changes that is related to cell death and cell integrity with anti-body array, which revealed that pKAL induced changes in NF-Kb, PI3K, Ras, TGF-beta signaling too. In conclusion, these findings suggest that pKAL induced anticancer effects by inducing late phase apoptotic cell death at least in part through p38 MAPK and JNK pathways in HCT 1116 human colon cancer cells. It also induced alteration of cell morphology mimic to necrosis in some cells with changes of proteins involved in the maintenance of intracellular membrane integrity. These effects was p53-independent.

#1871

Tannic acid: A natural anticancer agent for non-small cell lung cancer.

Elham Hatami, Prashanth K.B. Nagesh, Pallabita Chowdhury, Advit Bhaskar Shetty, Manish K. Tripathi, Subhash Chauhan, Meena Jaggi, Murali Yallapu. _UTHSC, Memphis, TN_.

Background

Lung cancer is the leading cause of cancer deaths across globe. Statistically, 70-80% of lung cancer cases fall under non-small cells lung cancer (NSCLC) category. Recently, usage of natural compounds (e.g. EGCG, curcumin, resveratrol, apigenin, etc.,) for cancer therapy has gained lots of attention. However, poor dissolution profiles of these natural compounds lead to unfavorable pharmacological doses and administration. For this purpose, we have candidate, a water-soluble natural polyphenol, Tannic acid (TA, C76H52O46), which, a natural polyphenol that is richly found in plants such as green tea. This molecule exhibits anti-oxidant and anti-proliferative activities, which is being used as an alternative therapeutic option in cancer therapy. However, role of TA in NSCLC was not examined. Therefore, the aim of present study is to assess the molecular effects of TA in NSCLC cells.

Methods

For this study, two NSCLC cell lines (A549 and H1299) and one normal lung cell line (BEAS-2B) were used. We investigated the anti-proliferative effects of TA on NSCLC cells using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) tetrazolium (MTT), and colony formation assays. The anti-invasiveness and anti-migratory potential of TA was evaluated through Matrigel and Boyden chamber studies, respectively. Cell cycle and apoptotic effects of TA was evaluated through flow cytometry and western blot studies. The quantification of secreted VEGF was assessed by ELISA. Also, for affirmation results of gene expression profiles we performed q-PCR studies.

Results

Cell proliferation study demonstrated a dose dependent anti-cancer effects of TA on NSCLC cells. However, TA had no significant toxicity effects on human bronchial epithelial cells. Similarly, colony formation results exhibited dose dependence reduction in response to escalated dose of TA exposure. Both the invasion and migration results confirm its anti-metastatic role. Extracellular VEGF quantification showed lower VEGF secretion with TA treatments compared to the control cells. The cell cycle analysis evidently showed TA efficiently induced G1 phase cell arrest in NSCLC cells which was further confirmed through western blotting studies of relevant proteins (Cyclin D1, P53, p21, p18, BAX, BCL-2). We also achieved to show the decreased expression of VEGFR2, p-AKT, and cleaved caspase 7 indicating TA induces apoptosis.

Conclusion

Overall, these results demonstrate illustrate anti-cancer activity of TA by targeting the VEGF related pathways in NSCLC. This approach can lead to an alternative therapeutic strategy for lung cancer, with ideal PK/PD profiles.

#1872

Oleuropein reduces Prdx1 expression, cell proliferation and viability in K562 human leukemia cells.

Natalie Fulco, Sophia Fagan, Shelley A. Phelan. _Fairfield University, Fairfield, CT_.

Oleuropein is one of the two main phenolic compounds derived from olive leaves. Numerous studies have found that the polyphenols contained in olive leaves possess strong antioxidant properties, and increase apoptosis in many types of cancer cells, suggesting its possible efficacy as an anti-cancer treatments. The purpose of our study was to examine the effects of oleuropein extract on cell growth and viability of the K562 human leukemia cell line, and to investigate possible molecular mechanisms of action. Oleuropein of 98% purity was used for our studies. We first demonstrated that K562 cells treated with 200 and 400 μg/ml oleuropein exhibited a significant reduction in cell growth and viability after four days as compared to ethanol-treated controls. Cells treated with 200μg/ml oleuropein showed decreased cell proliferation and viability after two days, and exhibited approximately 90% reduction in cell density and viability after four days of treatment. A concentration of 400 μg/ml oleuropein resulted in 100% toxicity by day four. We also examined the effects of oleuropein on cellular cytotoxicity using an LDH release assay and found a two-fold elevation in LDH release within 24 hours of treatment with 200 μg/mL oleuropein. To begin to explore a mechanisms of action, we used western blotting to examine known antioxidant and signaling pathways for possible regulation by oleuropein in these cells. We examined expression of the Peroxiredoxin (Prdx) family of proteins, a group of thiol-specific antioxidants found to be elevated in many types of cancer. We found that 200 ug/ml oleuropein reduced Prdx1 expression by about 50% after eight and twenty-four hours, while having no significant effect on expression levels of Prdx-2, -3, -4, and -6. We are currently investigating the mechanism of cell death induced by oleuropein, as well as the role of Prdx1 in oleuropein-induced toxicity. Together with previously reported cancer-cell specific effects, this research provides a promising new avenue for the use of natural products as anti-cancer agents.

## TUMOR BIOLOGY

### Epithelial/Mesenchymal Transition (EMT and MET) 2 / Premetastatic Niche

#1873

Water-pipe smoking induces epithelial-mesenchymal transition and enhances cell invasion of human breast cancer cells.

Ala-Eddin Al Moustafa. _MEACR & Oncology Dept./McGill U, Montréal, Quebec, Canada_.

Water-pipe smoking (WPS) has been recently recognized as an emerging risk factor for human health. The exact role of WPS in human breast cancer development and progression has not been investigated yet. We herein explored for the first time the impact of WPS on epithelial-mesenchymal transition (EMT) and cell invasion, which are considered crucial for cancer progression and metastasis in human carcinomas including breast. Our data revealed that WPS can initiate EMT in two human breast cancer cell lines, MCF-7 and BT20. WPS enhanced the cell invasion ability of both cell lines compared with their matched controls. We also found that WPS provoke a down- and up-regulation of two important regulator genes of cancer progression: E-cadherin and focal adhesion kinase (FAK). In addition, our data pointed out that WPS induces the activation of Erk1/Erk2 signaling pathways, which could be behind the stimulation of EMT and deregulation of E-cadherin and FAK genes. Taken together, our results imply the significant effects of WPS on several cellular processes and genes involved in breast cancer progression and metastasis. Further studies are necessary to identify the clinical relevance and exact role of WPS in human carcinogenesis and metastasis. Key words: Water-pipe smoking, breast cancer, EMT, cell invasion, cell adhesion

#1874

A ZEB1-regulated EMT transcriptional program is associated with early nodal metastasis and improved survival for stage N2/N3 breast cancer patients.

C James Block, Guojun Wu. _Wayne State University School of Medicine, Detroit, MI_.

The epithelial-to-mesenchymal transition (EMT) is a transcriptionally-mediated process by which epithelial cells cease expression of cell-cell junction proteins, lose polarity, and begin expressing mesenchymal cytoskeletal and extracellular matrix components. While EMT is critical for normal development and wound healing, this process has been experimentally shown to be pathologically activated in cancer cells, leading to enhanced migratory and invasive capabilities, increased chemoresistance, and distant metastasis in mouse models. Consequently, EMT has been hypothesized to be a driving factor in metastatic progression and tumor recurrence in multiple cancer types, including breast cancer. EMT can be driven by several transcription factors (EMT-TFs) which are independently capable of inducing EMT and can regulate each other. Our group set out to identify EMT-TFs which were commonly activated across EMT models. By performing integrative analysis of multiple datasets, we determined that the EMT-TF ZEB1 demonstrates the most reproducible and consistent association with EMT in experimental models, breast cancer cell lines and in breast cancer patients. We then identified a high-confidence set of genes commonly upregulated in EMT and regulated by ZEB1. We observed that this ZEB1-associated transcriptional program is highly enriched for genes overexpressed in patients with early-stage node metastases compared to patients without lymph node involvement. Paradoxically, we observe that ZEB1 and ZEB1 associated genes exhibit higher expression in adjacent normal tissue samples than in primary tumor samples. Additionally, we discovered a reproducible association between ZEB1 and ZEB1-program gene expression and significantly improved recurrence-free and overall survival for breast cancer patients with distant lymph node involvement. This association was unique to ZEB1, as other EMT-TFs did not predict survival in these patients. In conclusion, our data demonstrate that the ZEB1-driven EMT program may contribute to early nodal metastasis, but that sustained ZEB1 expression may ultimately suppress cancer progression and improve survival. These findings may help clarify the specific contributions of different EMT-TFs to breast cancer progression.

#1875

Polo-like kinase 1 positively correlates with N-cadherin and promotes epithelial-mesenchymal transition in melanoma.

Gagan Chhabra, Mary A. Ndiaye, Shengqin Su, Chandra K. Singh, Nihal Ahmad. _Univ. of Wisconsin, Madison, WI_.

Polo-like kinase 1 (PLK1), a serine/threonine kinase and important cell cycle regulator, is overexpressed in melanoma and has been linked with enhanced cell proliferation. Recent studies have shown involvement of PLK1 in epithelial-mesenchymal transition (EMT) in certain cancer types. However, the role of PLK1 in EMT in melanoma is not well understood. EMT has been shown to promote metastasis, and is associated with an upregulation of the mesenchymal marker N-cadherin (CDH2), downregulation of the epithelial marker E-cadherin (CDH1), and increased cell migration and invasion ability. Earlier, we demonstrated that PLK1 is involved in EMT and its kinase activity is important for EMT-related modulations in A375 melanoma cells (Cancer Res 2018; 78 (13 Suppl): Abstract #2016). In this study, we employed a human tissue microarray (TMA) co-immunostained for PLK1, N-cadherin, E-cadherin and the melanoma biomarker S100 to determine the association between PLK1 and EMT effectors. This TMA, coupled with high-throughput, multispectral Vectra scanning and inForm analysis, allowed us to objectively analyze and quantify protein levels in 57 clinical tissue specimens of nevus (n=15), primary (n=22) and metastatic melanoma (n=20). We observed an increase in PLK1 expression in primary and metastatic melanoma as compared to benign nevi, with a significant increase in metastatic tumors compared to primary tumors (p=0.004). Similarly, N-cadherin expression was markedly higher in metastatic melanoma (fold-change=2.31; p=0.001) and primary melanoma (fold-change=1.8; p=0.03) when compared to benign nevi. Further, we observed a significant decrease in E-cadherin expression in metastatic tumors compared to primary melanoma (fold-change=2.53; p=0.02). Moreover, using single linear regression analyses between expression of two proteins, we found a significant strong positive correlation between PLK1 and mesenchymal marker N-cadherin (correlation co-efficient R=0.75; p<0.01). We also found a weak but negative correlation between PLK1 and epithelial marker E-cadherin (correlation co-efficient R=-0.25). To further confirm the role of PLK1 in EMT, using shRNA-mediated PLK1 knockdown in SK-MEL-2 cells (a human melanoma line that possesses high metastatic potential), we performed cell migration and invasion assays, as well as immunofluorescence staining for expression of N-cadherin and E-cadherin. Interestingly, we found a decrease in expression of mesenchymal marker N-cadherin and an increase in expression of epithelial marker E-cadherin after PLK1 knockdown. These were accompanied by decreased cell migration and invasion, suggesting that inhibition of PLK1 in these human melanoma cells leads to inhibition of EMT. Overall, these results suggest that PLK1 is an important regulator of EMT in melanoma, and therefore can be exploited as a therapeutic target to inhibit melanoma metastasis.

#1876

Mechanism of tyrosine kinase inhibitors resistance and role of epithelial mesenchymal transition in NSCLC.

Sanjana Singh, Nabiha H. Khan, Neelu Puri. _University of Illinois College of Medicine, Rockford, IL_.

EMT is a vital process in the development of metastasis and occurs when epithelial cells lose their polarized structure, by downregulation of adherens junction proteins, E-cadherin and Claudin1, located on the cell membrane. Cells with EMT are elongated spindle-like structures due to upregulation of mesenchymal markers Vimentin and N-cadherin. EMT may be responsible for tyrosine kinase inhibitor (TKIs) resistance to epidermal growth factor receptor (EGFR) in patients with activated EGFR mutations. Our earlier studies indicate that the presence of T790M mutation may induce EMT in erlotinib resistant (ER) cells (H1975). When p120-catenin, a key EMT regulator, is no longer bound to membranous E-cadherin, a complex is formed with Kaiso factor, suppressing its transcription repressor activity and promoting oncogenesis. PRMT1, another key EMT inducer, is also overexpressed in non-small cell lung cancer (NSCLC). Cells undergoing EMT also acquire cancer stem-cell (CSC) like characteristics by expressing CSC markers ABCB1, BMI-1 and Oct-4. Thus, we investigated EMT characteristics in wild type EGFR ER H358 and H2170 cells. The modulation of EMT biomarkers was determined by immunoblotting (WB) and Quantitative real-time PCR (qPCR). Expression of CSC markers was measured using flow cytometry. PRMT1 and p120-catenin were upregulated 1.5 and 6.1-fold in H358ER cells. Hence, knockout of p120-catenin and PRMT1 were performed using CRISPR-Cas9 or an inhibitor and was examined by WB and cell proliferation assay (MTT). Other EMT regulators such as Snail, PRMT-5, Vimentin and Kaiso factor were also upregulated by 2.6 to 10.7-folds and E-cadherin and Claudin-1 were downregulated by 2.6 to 10-fold in H358ER cells. RhoA, a small GTPase downstream of p120-catenin and a negative EMT regulator, was also downregulated by 2.9-fold promoting EMT in these cells. Immunofluorescence (IF) studies showed that there was 95% colocalization of p120-catenin and Kaiso factor in H358ER cells whereas 70% colocalization was seen in H358 parental cells. In ER NSCLC cells with wild type EGFR, flow cytometry studies showed increased expression of CSC markers ABCB1, BMI-1 and Oct-4. Knockdown of PRMT1 by Furamidine increased erlotinib efficacy by 16.5%% in H358ER and 21% in H2170ER cells. Similarly, knockout of p120-catenin by CRISPR-Cas9 increased erlotinib efficacy by 19% in H358ER cells. Also, there was a 54.7% increase in erlotinib's efficiency in p120-catenin knockout H358ER cells treated with Furamidine. Moreover, immunohistochemistry (IHC) studies with p120-catenin and PRMT-1 biomarkers showed that a higher expression of these biomarkers could be related to poor prognosis in NSCLC patients. In conclusion, EMT maybe mediated through biomarkers such as PRMT1, which also upregulates other EMT regulators, and p120-catenin, which represses E-cadherin and Kaiso factor, thus activating EMT in TKI resistant cells.

#1877

The role played by SLUG, an epithelial-mesenchymal transition factor, in invasion and therapeutic resistance of malignant glioma.

Kyung-Hwa Lee,1 Se-Jeong Oh,1 Eun-Jung Ahn,1 Shin Jung,1 Jae-Hyuk Lee,1 Kyung-Keun Kim,2 Hangun Kim,3 Kyung-Sub Moon1. 1 _Chonnam National Univ. Hwasun Hospital, Jeonnam, Republic of Korea;_ 2 _Chonnam National University Medical School, Jeonnam, Republic of Korea;_ 3 _Sunchon National University, Jeonnam, Republic of Korea_.

Purpose: In malignant gliomas, invasive phenotype and cancer stemness promoting resurgence of residual tumor cells, render treatment very difficult. Hence, identification of epithelial-mesenchymal transition (EMT) factors associated with invasion and stemness of glioma cells is critical.

Methods: To address the issue, we investigated several EMT factors in hypermotile U87MG and U251 cells, mouse xenograft model, and human glioma samples. Of several EMT markers, SLUG expression was notably increased at the invasive fronts of gliomas, both in mouse tumor xenografts and human glioma samples. The biological role played by SLUG was investigated using a colony-forming assay after chemotherapy and irradiation, and by employing a neurosphere culture assay. The effect of SLUG on glioma progression was examined in our patient cohort and samples, and compared to large public data from the REMBRANDT and TCGA.

Results: Genetic upregulation of SLUG was associated with increased levels of stemness factors and enhanced resistance to radiation and temozolomide. In our cohort, patients exhibiting lower-level SLUG expression evidenced longer progression-free survival (P=0.042). Also, in the REMBRANDT dataset, a group in which SLUG was downregulated exhibited a significant survival benefit (P<0.001). Although paired gliblastoma multiforme (GBM) samples from our patients did not showed significant increase of SLUG expression, increased mRNA levels of SLUG was found in recurred GBM from TCGA (P=0.052), and in TMZ-treated glioma cells and mouse xenograft.

Conclusion: SLUG may contribute to glioma progression by controlling invasion at infiltrating margins, associated with increased stemness and therapeutic resistance.

#1878

MicroRNA-200c (miRNA-200c) increases adhesive interactions between E-selectin ligands expressed by breast cancer cells and E-selectin under physiological blood flow conditions.

Christian A. Showalter, Monica M. Burdick. _Ohio University, Athens, OH_.

The epithelial-to-mesenchymal transition (EMT), and its reverse process, the mesenchymal-to-epithelial transition (MET), are associated with metastatic tumor progression in breast cancer (BC). E-selectin, a cell adhesion molecule expressed by cytokine-activated vascular endothelium, is also involved in metastasis via binding to counter-receptors (E-selectin ligands) on the surface of BC cells. Consequently, efforts have been made to investigate a link between the EMT/MET and the adhesive interactions between E-selectin and their ligands. Our lab previously demonstrated that BC cells with an epithelial phenotype exhibit more adhesive interactions between E-selectin/ligands under blood flow conditions than cells with a mesenchymal phenotype. Therefore, molecular factors in the blood that activate the EMT or the MET in circulating tumor cells (CTCs) have been of particular interest. These factors may regulate the potential for CTCs to traffic to bone marrow endothelium, a frequent site of BC metastasis that constitutively expresses E-selectin. To advance knowledge in this area, we hypothesized that miRNA-200c, associated with poor prognosis in metastatic BC patients, induces the MET in MDA-MB-231 BC cells and promotes adhesion between E-selectin/ligands. Microscopy images show that MDA-MB-231 cells transfected with miRNA-200c displayed a cell morphology similar to that of epithelial cells, whereas cells transfected with a negative control miRNA (NC miRNA) retained their mesenchymal phenotype. Furthermore, cells transfected with miRNA-200c exhibited greater mRNA expression of E-cadherin and lower expression of Vimentin compared to cells transfected with a NC miRNA by qRT-PCR. The expression of Zeb1/2, both targets for miRNA-200c, were down-regulated in cells transfected with miRNA-200c compared to cells transfected with a NC miRNA. Both the microscopy images and mRNA expression analysis indicate induction of MET in miRNA-200c transfected cells. Shear flow adhesion assays and shear flow detachment assays were performed at bone marrow shear stresses to examine adhesive interactions between E-selectin/ligands. Using these assays, MDA-MB-231 cells transfected with miRNA-200c demonstrated slower rolling velocities, greater firm adhesion, lower detachment, and equivalent tethering activity when compared to cells transfected with a NC miRNA. Moreover, analysis of the mRNA expression of α1,3- and α1,4- fucosyltransferases (FT) revealed increased expression of FT3 and FT6 in cells transfected with miRNA-200c compared to cells transfected with a NC miRNA. Altogether, these results suggest that miRNA-200c increases fluid-shear resistant adhesion and that factors present in the blood may impact the metastatic potential of cancer cells in circulation by regulating cellular phenotypes.

#1879

Novel diphenylamine analogs induce mesenchymal to epithelial transition and enhance the sensitivity of breast cancer cells to conventional chemotherapeutic agents.

Akshita B. Bhatt,1 Thomas D. Wright,1 Katie Anna,1 Mohit Gupta,1 Suravi Chakrabarty,1 Van Hoang,2 Matthew Burow,3 Patrick T. Flaherty,1 Jane E. Cavanaugh1. 1 _Duquesne University, Pittsburgh, PA;_ 2 _Tulane University, LA;_ 3 _Tulane University, New Orleans, LA_.

Epithelial to mesenchymal transition is an important cellular adaptation that helps cancer cells acquire a spindle-like phenotype from a cuboidal phenotype, degrade the extracellular matrix, invade the neighboring tissues, and metastasize to other organs and form secondary tumor. Cellular plasticity is governed by growth factors that act in a paracrine manner to activate downstream oncogenes and regulate the activity of epigenetic factors, which facilitate phenotypic switch from epithelial to mesenchymal, ultimately leading to increased cell migration and invasion. The intracellular phosphorylation cascade that is downstream of growth factor receptors plays an important role in transmitting the signal from the extracellular environment into the nucleus, thereby completing the loop required to elicit a cellular response. Triple negative breast cancer (TNBC), characterized by loss of hormone receptors is a highly aggressive form of cancer and patients show poor prognosis and disease-free survival due to lack of targeted therapy. In contrast, estrogen positive breast cancer can be targeted by estrogen receptor antagonists or CDK4/6 inhibitors, but drug resistance and relapse is often associated with epithelial to mesenchymal transition and poor patient outcome. Hence, targeting the mesenchymal phenotype with small molecule inhibitors is an emerging strategy to attenuate the invasive and aggressive nature of cancer cells. The MEK5-ERK5 pathway is understudied in triple negative breast cancer and there are few research tools available to selectively inhibit this pathway. Diphenylamine derivatives, synthesized as putative MEK5 inhibitors from parent MEK1/2 inhibitor trametinib, are effective in inducing mesenchymal to epithelial transition in MDA-MB-231 triple negative breast cancer cells, as indicated by an increase in E-cadherin expression, which is a marker of epithelial phenotype, and a decrease in spindle index, an important cell shape determinant. Compound 1, a dual MEK1/2 (98%) and MEK5 (59%) inhibitor was further characterized in functional assays; Compound 1 was found to significantly inhibit cell viability, proliferation, migration, spheroid viability, and colony formation in MDA-MB-231 cells. Compound 1 is effective in reversing the mesenchymal phenotype of MDA-MB-231, BT-549, and tamoxifen resistant-MCF-7 breast cancer cells. Signaling crosstalk and drug resistance in cancer limits the applicability of monotherapy. Our current work is focused on treating diverse breast cancer cells with combination of novel compound 1 and paclitaxel, ipatasertib, JQ-1, and LBH589 to induce synthetic lethality at lower concentration, restore drug sensitivity, and enhance selectivity.

#1880

Insulin-like growth factor binding protein-3 (IGFBP-3) differently regulates migration and invasion under normoxic and hypoxic environment in prostate cancer.

Arpit Dheeraj,1 Dhanir Tailor,2 Gagan Deep,3 Rana Singh1. 1 _Jawaharlal Nehru University, New Delhi, India;_ 2 _Stanford University, Palo Alto, CA;_ 3 _Wake Forest Baptist Medical Center, Winston-Salem, NC_.

Prostate cancer (PCa) is the second most frequent cancer persisting in men and fifth leading cause of cancer related deaths in males. It is accounting for 13.5% (1,276,106) of cancer incidence and 6.7% (3,58,989) of total cancer deaths in men in 2018. The high ratio of IGF-1/IGFBP-3 correlates with increased risk of various cancers including prostate cancer. Various studies have shown that IGFBP-3 is down-regulated in PCa cells. In this study, we have evaluated the role of IGFBP-3 on the angiogenesis and epithelial to mesenchymal transition in prostate cancer. Cell growth and clonogenic assays suggested that IGFBP 3 expression inhibits growth and proliferation in DU145 and LNCaP cells. IGFBP-3 overexpression decreased expression of VEGF, an important angiogenic factor. The decreased expression was observed in normoxic and hypoxic conditions as well. IGFBP-3 overexpression down-regulate levels of phospho-Stat3 (Y705), phospho-Akt (S473), phospho-Erk 1/2, mTOR and eIF4G that can result in altered biological functions of cells. We also observed the decreased levels of p65 subunit of NF-κB. IGFBP-3 knockdown increased the growth and proliferation of endothelial cells (HUVECs) under both normoxic as well as hypoxic conditions. IGFBP-3 overexpression inhibited migration of HUVECs and knockdown of IGFBP-3 resulted in an increase in migration under normoxia and hypoxia. Further, we found that IGFBP-3 overexpression inhibited migration under normoxic conditions but when it comes to hypoxic environment, IGFBP-3 overexpression increased the cell migration. IGFBP-3 overexpression subsequently reduced the levels of N-cadherin and FAK, important regulators of migration, under normoxic condition. Knockdown of IGFBP-3 resulted in an increase in levels of E-cadherin, an EMT marker, under hypoxic environment. IGFBP-3 overexpression reduces tumor growth and tumor weight as compared to vector control cells. Overall, our present findings indicate that IGFBP-3 targets different steps of prostate cancer growth and progression and its action depends on the microenvironment.

#1881

Role of E2F activators and mitotic regulators controlled by E2Fs in EMT, invasion, and metastasis of breast cancer.

Shirley Jusino,1 Melanie J. Hidalgo-Vargas,1 Jaya Padmanabhan,2 Srikumar P. Chellapppan,2 Harold I. Saavedra1. 1 _Ponce Health Sciences University, Ponce, PR;_ 2 _H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL_.

The E2 Promoter Binding Factors (E2Fs) are a group of eight transcription factors that are highly deregulated in breast cancer and control several cellular processes, including proliferation and centrosome duplication. Broadly, E2Fs can be divided into transcriptional activators and repressors based on their role in gene transcription. In addition to their traditional cell cycle functions, it is known that deregulated Rb/E2F pathway signals the epithelial-to-mesenchymal transition (EMT); yet the underlining mechanism of how E2Fs drive EMT in breast cancer remains unknown. Published data from our laboratory demonstrates that overexpression of activating E2Fs leads to increased expression of mitotic regulators such as BUBR1, MPS1 (TTK), and SGO1, among others. Furthermore, published data from our laboratory shows that TTK promotes mesenchymal signaling through several mechanisms in breast cancer. Hence, we are investigating to what extent deregulation of E2F activators promotes EMT, invasion, and metastasis through the remaining mitotic regulators. Our hypothesis is that the overexpression of E2F activators contributes to tumorigenesis by promoting EMT through the induction of the transcription of specific mitotic regulators, thus leading to increased invasion and metastasis. To test our hypothesis, E2Fs (1, 2 and 3a) were knockdown individually or in combination of two or three E2F activators simultaneously using specific siRNAs sequences in MDA-MB-231 cells. After 48 hours, western blot analysis was performed to confirm the knockdown and to evaluate the effects of knockdown of these E2Fs on protein levels of mitotic regulators and EMT markers. Real-time PCR was performed to evaluate EMT mRNA expression levels. Additionally, a wound healing assay was conducted to evaluate cell migration, as an indicator of metastasis. All experiments were performed in triplicates and a non-parametric Mann Whitney test was used to evaluate statistical significance. Our results show that knockdown of E2F1 and the combination of all activating E2Fs decreased the expression of mitotic regulators and EMT proteins. Likewise, knockdown of E2Fs decreased the expression of certain EMT genes (but not all) and knockdown of E2F1 decreased cell migration. These results suggest that E2F1 has a prominent role in breast cancer EMT and cell migration. Currently, we are exploring the effects of knockdown of E2F-controlled mitotic regulators in breast cancer EMT and cell migration.

#1882

Molecular context dictates the effects of eribulin on key EMT regulators: Snail and Slug.

Roma Kaul, April L. Risinger, Susan L. Mooberry. _University of Texas Health Science Center at San Antonio, San Antonio, TX_.

Microtubule targeting agents (MTAs) are a mainstay in the treatment of breast cancer and a growing body of evidence demonstrates that they have non-mitotic effects that contribute to their anticancer actions. Even after decades of clinical use, there is much to be learned about the mechanisms of action these drugs, and differences among them, for optimal utility. MTAs rapidly alter microtubule dynamics, often within minutes, leading to significant changes in oncogenic cellular signaling. We evaluated the early effects of eribulin on key oncogenic signaling pathways following a 2 h incubation using clinically relevant concentrations and compared the effects to those initiated by other MTAs. These studies led to the identification of novel mechanisms by which MTAs disrupt oncogenic signaling and contribute to the reversal of epithelial to mesenchymal transition (EMT), including unanticipated differences among drugs. The TGF-β-mediated expression of the Snail transcription factor is a key driver pathway of EMT in breast cancer. The effects of eribulin and other MTAs on TGF-β-induced, Smad-dependent expression of Snail were evaluated. Our results show that eribulin and vinorelbine inhibit the ability of TGF-β to promote the transcriptional induction of Snail by impeding the nuclear transport of Smad2/3 proteins in 4 triple-negative breast cancer cell lines. The effects of microtubule destabilizers contrasted with those of paclitaxel, which had no effect on the expression of Snail. This study begins to explain how microtubule disruption might contribute to the eribulin-mediated EMT reversal. Slug is another member of the Snail family of transcription factors that plays a central role in breast cancer EMT. Although Snail and Slug are often grouped together due to functional similarities, it is now understood that they regulate non-overlapping sets of genes. In contrast to our findings with Snail, eribulin and vinorelbine, but not paclitaxel or ixabepilone, induced Slug over-expression in a subset of breast cancer cell lines. We have identified molecular contexts where eribulin and vinorelbine induced Slug expression, resulting in the identification of a potential biomarker for response to microtubule destabilization. This work highlights the multifaceted nature of MTA-mediated effects on EMT-associated signaling pathways in breast cancer cells. These studies are supported by Eisai Inc.

#1883

Ovarian cancer cells overcome the cytotoxic effect of cisplatin by hyper activating CCR9-mediated signaling in response to cisplatin.

Hina Mir, Neeraj Kapur, Shailesh Singh. _Morehouse School of Medicine, Atlanta, GA_.

Ovarian cancer (OC) has highest mortality rate among the gynecological malignancies. Chemotherapies, in combination with surgery, are standard treatment offered to the patients with advanced OC. Although 80% patients initially respond to platinum-based chemotherapy, clinical responses remain short-lived due to chemoresistance. Most patients experience relapse and 50-60% of them die within 5 years. Therefore, development of novel therapies for OC remains a high priority. Hence, the major focus of this study is to define the biology of CCR9, which is highly expressed in OC in response to platinum drug. Effect of cisplatin on CCR9 expression and impact of CCR9 activation on molecular and cellular changes involved in overcoming the effect of cisplatin was determined by flow cytometric and western blot analyses. Our data show higher expression of CCR9 and CCL25 in OC cells in response to cisplatin treatment. Additionally, our data show higher expression and activation of estrogen receptor-α (ER- α) in OC cell after CCR9 activation. In addition to this, we show CCR9-mediated expression of Smoothened (SMO) known to support stem cell like phenotype by activating Hedgehog signaling and co-expression of CD44 and CCR9 as well as higher CD44 expression in OC cells following CCL25 treatment. CCR9-CCL25 axis promoted epithelial to mesenchymal transition. These observations strongly show an association of CCR9-CCL25 axis in OC pathogenesis, therapeutic resistance, and chemotherapeutic efficacy.

#1884

Development of a novel VIM-RFP reporter line for colorectal cancer EMT study and drug discovery.

Weiguo Shu, Diana Douglas, Sangeeta Kumari, Luis Romero, Luis G. Rodriguez, Chaozhong Zou, Robert Newman. _ATCC, Gaithersburg, MD_.

Epithelial-to-Mesenchymal Transition (EMT) describes a dynamic and reversible process by which cells lose their epithelial characteristics and acquire mesenchymal properties. Accumulating evidence indicates that EMT displays an array of intermediate states, a phenotype referred to as "partial MET". EMT is executed in response to signaling pathway molecules and microRNAs (miRNAs) that induce the expression of specific EMT associated transcription factors (EMT-TFs), including Zeb1/2, Snail1/2, and Twist. There is an ever-growing body of research and clinical evidence indicating that EMT plays an important role in cancer cell dissemination and distal metastasis. Therefore, targeting EMT is considered a novel opportunity in anti-cancer treatment and drug development.

Vimentin, a hallmark of mesenchymal cells, is of increasing interest as a novel anti-cancer therapeutic drug target. In colorectal cancer (CRC) patients, increased expression of the vimentin protein predicts a poor prognosis. Here, we developed a novel CRC HCT-116 VIM-RFP reporter line using CRISPR/Cas9 technology. In this cell line, the RFP reporter was incorporated into the endogenous vimentin gene just before the stop codon at the last exon, enabling real-time monitoring of EMT states in live cells. The VIM-RFP knock-in allele was confirmed at genomic, transcriptional, and translational levels. Bio-functional evaluation data revealed that miRNA-200 inhibitor treatment induced the increased expression of VIM-RFP, and decreased expression of E-cadherin, a hallmark of epithelial cells. The expression of EMT-TFs ZEB-1 and ZEB-2 was also upregulated upon induction. In addition, we showed that induced VIM-RFP cells displayed increased migration capacity. These data suggested that miRNA-200 inhibitor induced VIM-RFP cells have undergone EMT. Azacitidine, a clinically approved demethylating agent, has been extensively evaluated in a number of clinical trials as a treatment for CRC patients. Studies reported that azacitidine can induce MET in a number of cancer cell lines. We showed that azacitidine treatment of VIM-RFP cells can effectively induce RFP expression, suggesting a potential application of this VIM-RFP reporter line as a platform for drug evaluation and compound screening. Taken together, the HCT-116 Vim-RFP EMT reporter line could be a valuable tool for dissecting the molecular mechanisms underlying EMT and for evaluating or screening compounds targeting EMT in CRC.

#1885

CRISPR/Cas9 engineered immortalized breast epithelial MCF10A reporter line for EMT studies and anti-cancer drug discovery.

Weiguo Shu, Sangeeta Kumari, Diana Douglas, Luis G. Rodriguez, Robert Newman. _ATCC, Gaithersburg, MD_.

Metastasis is responsible for most cancer-related deaths. One model for the mechanism of metastasis involves epithelial-to-mesenchymal transition (EMT), a process characterized by the decrease in cell adhesion and increase in cell motility. Cells undergoing EMT often display downregulation of epithelial markers (such as, E-cadherin) and upregulation of mesenchymal markers (such as, Vimentin). Besides metastasis, EMT has also been reported to be associated with other pathological conditions, such as acquired therapeutic drug resistance. Given the roles that EMT may play in these various pathological processes, it is of increasing interest as a target for anti-cancer treatment and drug discovery. In vitro reporter models have proven to be a valuable tool for dissecting the signaling pathways that regulate the EMT process and for screening compounds targeting EMT. In previously developed EMT reporter cell lines, the reporter gene was driven by a truncated EMT marker gene promoter. Therefore, the establishment of a more physiologically relevant reporter cell models is critical for advancing our knowledge of EMT. E-cadherin, a hallmark of epithelial cells, has been implicated in the onset of metastatic dissemination. Utilizing CRISPR/Cas9 knock-in technology, we have generated an E-cadherin (ECAD)-emerald green fluorescent protein (EmGFP) reporter model using immortalized breast epithelial MCF10A cells. In the reporter cells, EmGFP gene was tagged at the C-terminus of E-cadherin, allowing for real-time monitoring of EMT progression in live cells. The targeted knock-in of ECAD-EmGFP allele was verified at the genomic DNA, transcript (mRNA), and protein levels. Bio-functional evaluation of the reporter cell line revealed that treatment of ECAD-EmGFP reporter cells with TGFβ led to EMT induction, as demonstrated by a reduction in E-cadherin-GFP expression and increase in vimentin and fibronectin expressions. Additional functional characterization revealed that the reporter cells possessed an enhanced migration capacity upon EMT induction with TGF-β. In summary, this MCF10A-Ecad-EmGFP reporter cell line can serve as a physiologically relevant in vitro cell model for studying EMT cancer biology and anti-EMT drug discovery.

#1886

Zygote arrest 1 like (ZAR2): An EMT (epithelial-mesenchymal transition) modulator in breast cancer.

Smita Misra, Tanushree Singha, Gautam Chaudhuri. _Meharry Medical College, Nashville, TN_.

Breast cancer is the most common cancer in women and distant site metastasis is the main cause of death in breast cancer patients. EMT is a natural phenomenon in development and wound healing. EMT is also one of the "Hallmarks of cancer". EMT is involved in tumor cell progression, invasion, and metastasis. During the process of EMT, epithelial cancer cells acquire molecular alternations that facilitate the loss of epithelial features and gain of mesenchymal phenotype. Such transformation promotes cancer cell migration and invasion. Decrease in E-cadherin expression and gain of N-cadherin and vimentin expression is indicative of EMT phenotype. Working with our recently characterized C4 zinc finger containing RNA binding protein ZAR2 we observed that levels of ZAR2 are significantly lower in the invasive breast cancer cells compared to non-invasive cells. Knockdown of ZAR2 in the non-invasive breast cancer cells increased the in-vitro invasiveness of these cells whereas forced expression of ZAR2 in the invasive breast cancer cells reduced their invasiveness. To understand the possible mechanism of ZAR2-mediated regulation of invasiveness of the breast cancer cells we studied differential gene expression in the ZAR2 knocked down non-invasive breast cancer cells by RNA-seq analysis. One of the genes that are significantly elevated in the ZAR2 knocked down cells is the invasion determining enzyme ATP6V0A4 along with ERBIN and ABCA12. Here we present our observation regarding ZAR2 regulation of ATP6V0A4 and its effect on metastasis and invasion potential of the breast cancer cells. Supported in part by NIH grant 1U54RR026140, Meharry Bridge and 2U54MD007586-32Revised from Meharry RCMI Program in Women's Health funds to SM and DOD grants BC990678 and BC050641 to GC.

#1887

MDM2 induces a partial/hybrid EMT phenotype switching through TGFβ1-Smad signaling pathway.

Priya Dondapati, Simran Agarwal, Deepthi Gangaram, Appu Rathinavelu. _Nova Southeastern Univ., Fort Lauderdale, FL_.

Epithelial-Mesenchymal Transition (EMT) is a key step in the induction of metastasis, which is critical for the conversion of early-stage prostate tumors into metastatic malignancies. Therefore, identifying the signaling pathways promoting EMT is crucial for the prediction of disease progression and consequently treatment outcomes. The role of MDM2 proto-oncogene was investigated in regulating EMT in LNCaP prostate cancer cells. MDM2 over-expression in LNCaP-MST (MDM2 transfected prostate cancer cells) caused a reduction in epithelial characteristics, by altering the expression levels of proteins that are involved in cell adhesion, cell polarity and cell-ECM interactions. While forcing the loss of epithelial characteristics, the MDM2 oncogene seems to facilitate the gain of mesenchymal properties (such as increased motility, invasive properties, and a spindle-like morphology etc.). These phenotypic changes are generally due to cytoskeletal rearrangements and increased expression of mesenchymal markers, which was clearly identified through our western blotting and fluorescence microscopy imaging analysis in LNCaP-MST cells compared to the LNCaP cells. Nutlin-3, an MDM2 specific inhibitor was able to reverse the expression of mesenchymal markers, which further confirmed the role of MDM2 in phenotype switching. Analysis of cell culture supernatants using ELISA indicated that LNCaP-MST cells secreted two folds higher levels of TGFβ1 compared to LNCaP cells, which appears to cause the activation of downstream Smad signaling pathway leading to EMT. Activation of the TGFβ1-Smad pathway also results in the increased transcription of mesenchymal markers (N-cadherin, vimentin, and fibronectin) while retaining the epithelial characteristics as evidenced by the unaltered levels of E-cadherin. Recent reports have established that, while transitioning between the epithelial and mesenchymal phenotypes, cells can also attain a hybrid, partial, or intermediate EMT phenotypes. Cancer cells in this hybrid phenotype can migrate collectively as clusters (epithelial) or as a single cell (mesenchymal) and exhibit stemness and drug resistance. In addition, these migratory cells with hybrid characteristics can attain the necessary phenotype and growth depending on the microenvironment that is surrounding the metastatic sites. So far, our results have clearly demonstrated that MDM2 could serve as a key regulator to drive the EMT of prostate cancer cells, and this process seems to be aided by the elevation of TGFβ1 signaling. Thus, the pro-metastatic role of MDM2 oncoprotein has been steadily expanding in the last few years, and the newest knowledge acquired through our experiments is the confirmation of its ability to induce hybrid EMT phenotype in prostate cancer cells, (This project was supported by the generous financial support from The Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida)

#1888

Circadian rhythm modulation of breast cancer gene expression depends on stem cell properties induced in culture.

Arpan De,1 Vishal Premdev Sharma,2 Dilshan Harshajith Beligala,1 Angelia Marie Lee,1 Michael Eric Geusz1. 1 _Bowling Green State Univ., Bowling Green, OH;_ 2 _Celsee, Inc, Plymouth, MI_.

It is important to know whether breast cancer cells within tumors have functional circadian clocks. If so, they may benefit from the ability provided by the clock to predict when important daily events occur such as nutrient availability in blood following meals or elevated immune cell surveillance at night. If instead the circadian clock is nonfunctional, then clinical treatments could be directed towards activating the clock because evidence indicates circadian rhythm loss leads to more aggressive tumor growth. Here, we examined the role of circadian clocks in metastasis-related events, focusing on epithelial-mesenchymal transition (EMT), including cancer stem cell (CSC) generation, and mesenchymal-epithelial transition (MET). Because of their differing circadian properties two tumor-forming cell lines were compared, MCF-7 human breast cancer and C6 rat glioma cells. C6 monolayer and tumorsphere cultures express distinct circadian rhythms in gene expression. In contrast, monolayer MCF-7 cell cultures are reported to be arrhythmic and so appear to lack a functional circadian clock. Nevertheless, MCF-7 cells may contain inducible circadian oscillators that fail to generate circadian rhythms under standard culture conditions but may function in tumor microenvironments. Tumorspheres simulate the three-dimensional tumor structure and were used here to test for favorable conditions enabling circadian rhythms in cancer cells. Circadian rhythms in EMT, MET, mitosis, migration, and cell death were evaluated through time-lapse imaging over several days. Bioluminescence imaging and quantitative RT-PCR were used to test for circadian rhythms in expression of the core circadian clock gene Per2 in MCF-7 cells. Following treatment with serum-free culture medium designed for stem cells, EMT and CSC-related protein markers of MCF-7 and C6 cells were confirmed by immunofluorescence: MSI1 and CD133 in MCF-7; OCT4, TWIST1, ZEB1, vimentin in C6. Morphometry showed distinct changes in cell shape following EMT as shown by nearly all MCF-7 cells having an average roundness of 0.815 ±0.09 SD and a C6 roundness of 0.855 ±0.029. This medium increased the number of MCF-7 tumorspheres expressing circadian rhythms by 31.5%, and average amplitude of rhythms increased 11.6-fold. MET was then induced by returning cells to serum-containing medium, which produced cells with a flattened cell morphology indicating epithelial or pseudoepithelial differentiation. A circadian clock in C6 cells appeared to gate MET events after the return to serum (95% confidence limit of period: 20.8-26.9 hrs, p<0.05). The circadian timing properties of MCF-7 tumorspheres suggest that the circadian clock may also be present in tumors and that cell-cell interactions or paracrine factors could sustain otherwise poorly performing circadian clocks in breast cancer.

#1889

Chronic exposure to excess iron promotes EMT and cancer via p53 loss in pancreatic cancer.

Devaraja Rajasekaran,1 Jiro Ogura,2 Mitchell Wachtel,1 Sabarish Ramachandran,1 Ellappan Babu,1 Sathish Sivaprakasam,1 Paul J. Grippo,3 Carolina Torres,3 Thangaraju Muthusamy,4 Jaya P. Gnana-Prakasam,5 Yangzom D. Bhutia1. 1 _Texas Tech University Health Science Center, Lubbock, TX;_ 2 _Tohoku university hospital, Sendai,Miyagi, Japan;_ 3 _University of Illinois at Chicago, Chicago, IL;_ 4 _Augusta University, Augusta, GA;_ 5 _Saint Louis University School of Medicine, Saint Louis, MO_.

Pancreatic cancer especially the pancreatic ductal adenocarcinoma (PDAC) is by far the most lethal of all cancers with a five year survival rate of less than 5%. Gemcitabine is currently used as a first line therapy for locally advanced and metastatic PDAC but with a very low success rate. Hence, there is an urgent need to better understand what actually drives this cancer so as to come up with a better diagnostic and therapeutic strategies. Excess heme and iron are known to be pro-tumorigenic. When present in excess, these molecules become toxic. Fe2+ in free form is a potent oxidant; it catalyzes the Fenton reaction to generate hydroxyl radicals (Fe2+ \+ H2O2 Fe3+ \+ OH \+ OH), a potent reactive oxygen species. Free heme is also toxic as it catalyzes free radical reaction and induces oxidative damage. Accumulation of iron and heme to toxic levels occurs in genetic diseases (hemochromatosis, sickle cell disease), pathological conditions (hemolytic anemia, ischemia reperfusion), infections, and clinical/therapeutic conditions (repeated blood transfusion). Excess intake of iron and heme from dietary sources could also lead to iron/heme overload, particularly in the colon. In this regard, red meat with its ~10-fold higher heme content than white meat is specifically relevant. Among these conditions however, hemochromatosis deserves special mention. Based on the evidence that hemochromatosis, an iron-overload disease, drives hepatocellular carcinoma, we hypothesized that chronic exposure to excess iron, either due to genetic or epigenetic causes, predisposes an individual to cancer. We employed cell culture studies to interrogate the connection between excess iron and cancer in multiple tissues and combined in vitro and in vivo studies to explore the connection in pancreas further. Ferric ammonium citrate was used as an exogenous iron source. Chronic exposure to excess iron induced epithelial-mesenchymal transition (EMT) in normal and cancer cell lines, loss of p53, and suppression of p53 transcriptional activity. In order to further extrapolate our cell culture data, we generated EL-KRASG12D or EL-Kras mouse (pancreatic neoplastic mouse model) expressing Hfe+/+ and Hfe-/- genetic background. p53 target gene expression decreased in EL-Kras/Hfe-/- mouse pancreas compared to EL-Kras/Hfe+/+ mouse pancreas. Interestingly, the incidence of acinar-to-ductal metaplasia and cystic pancreatic neoplasms (CPN) decreased in EL-Kras/Hfe-/- mice, but the CPNs that did develop were larger in these mice than in EL-Kras/Hfe+/+ mice. In conclusion, these in vitro and in vivo studies support a potential role for chronic exposure to excess iron as a promoter of more aggressive disease via p53 loss within pancreatic epithelial cells.

#1890

Calcitonin receptor is required for T-antigen-induced prostate carcinogenesis.

Afaf Aldahish, Arvind Thakkar, Girish V. Shah. _University of Louisiana at Monroe, Monreo, LA_.

Calcitonin & calcitonin receptor axis (CT-CTR) expression is increased in prostate cancer & its levels positively correlate with Gleason grade of prostate cancer. Moreover, activation of CT-CTR autocrine axis promotes several processes with tumor progression such as, tumor growth, invasion, angiogenesis, chemoresistance & metastasis. However, the role of CT-CTR axis in carcinogenesis process has not been investigated. To examine the role of CT-CTR axis in prostate carcinogenesis we examined the impact of CTR deficiency on T-antigen-induced prostate tumorigeneses in transgenic mice. We achieved this by cross-breading CTR-knockout mice with LPB-Tag mice (the transgenic mice that uses long Probasin promotor for prostate-specific expression of Tag T-antigen). The cross-breeding of these two transgenic mice yielded four different groups of mice with the following genotypes 1. Control (which lacked both transgenes), 2. CTRKO (only CTRKO transgene), 3. LPB-Tag (contained LPB-Tag transgene), & 4. CTRKO-LPB-Tag (expressed Both CTRKO & LPB-Tag transgenes). The growth of these mice were monitored until they reached the age of 90 days. The mice were sacrificed, the prostates were collected, fixed & evaluated by histology & immunohistochemistry. The results suggest that the mice of all groups developed normally with the exception of LPB-Tag group which displayed slower growth & the presence of prostatic tumors at the time of necropsy. Although all LPB-Tag mice displayed all prostate tumors, as expected wild type & CTRKO mice did not display change in the size or mass of prostates. Subsequently, H&E histology as well as Ki67 staining confirmed that all LPB-Tag mice displayed well-developed prostatic adenocarcinoma, all CTRKO-LPB-Tag displayed no presence of tumor with the exception of occasional presence of HGPIN. Since our earlier studies have shown that CTR induced epithelial-to-mesenchymal transition (EMT) in prostate cancer cell lines, we also examined the expression of two epithelial markers, ZO-1 & E-cadherin, & two mesenchymal markers, fibronectin & vimentin. The results suggest that prostates of the three groups ( WT, CTRKO, LPB-Tag-CTRKO) displayed higher epithelial markers expression with a remarkably lower expression of mesenchymal markers when compared with those of LPB-Tag group. This was further substantiated by the expression of EMT markers such as snail & N-cadherin. In conclusion, this study shows that the CTR deficiency inhibits prostate carcinogenesis in LPB-Tag mice without reducing T-antigen expression or causing any other apparent harmful side effects. This work was supported by NIH grant CA096534.

#1891

**Oncogenic** MDM2 **-containing exosomes promote pre-metastatic niche establishment.**

Lucia Casadei,1 Federica Calore,1 Ameya Deshmukh,1 Abeba Zewdu,1 Danielle A. Braggio,1 Paolo Fadda,1 Martin Wabitsch,2 Gonzalo Lopez,1 Jennifer Leight,1 Dina Lev,3 Carlo M. Croce,1 Raphael E. Pollock1. 1 _Ohio State University, Columbus, OH;_ 2 _Ulm University Hospital, Germany;_ 3 _The Tel Aviv University, Tel Aviv, Israel_.

Extracellular vesicles (EVs) have an important role in the communication between cells for their ability to transfer proteins and other biologically active molecules. EVs have been also described to have unique functions during pre-metastatic niche establishment and maintenance. MDM2 oncoprotein is the best-characterized cellular antagonist of p53 and its amplification is one of the most known mechanisms for tumor development. MDM2 amplification occurs in more than forty different types of malignancies and in 100% of well-differentiated and dedifferentiated liposarcoma (DDLPS). Retroperitoneal DDLPS is especially concerning given its propensity for multifocal local and distant recurrence, accounting for an overall survival rate of 10% at 10 years. Here, we show the presence of MDM2 in tumor EVs and specifically in both DDLPS cell line-derived EVs and patient-serum EVs. We also show the ability of MDM2-cargo to be transferred to recipient preadipocytes. Furthermore, we also demonstrated that this transfer is able to promote a pre-metastatic niche establishment in the DDLPS tumor microenvironment fat bearing areas, inducing: (I) tumor associated phenotype such as enhanced proliferation and migration, in the recipient preadipocytes, by downregulation of MDM2 downstream pathway; (II) pro-inflammatory phenotype through the release of pro-inflammatory cytokines such as IL-8; (III) release of active metalloproteinases such as MMP2 which is crucial for the invasiveness of many disseminating tumors. In conclusion here we report the presence of MDM2, in the patient circulation; importantly showing that MDM2 triggers the release of active MMP-2, we also propose a possible mechanism for pre-metastatic niche establishment and DDLPS multifocal loco-regional recurrence.

#1892

**Dysfunction of resident memory CD8** + **T cells facilitates breast cancer lung metastasis.**

Erwei Song, Yue Xing, Shicheng Su. _Sun Yat-sen Memorial Hospital, Guangzhou, China_.

Resident memory T cells (TRM) are generally intraepithelially located and manifesting defence potential in diverse existing virus infection models. However, their roles in cancer development, especially metastasis, are poorly understood. Herein, by using a murine model orthotopically inoculated with 4T1 mammary carcinoma cells, we demonstrated the dynamic distribution and functional changes of lung TRM during the formation of lung pre-metastatic niche. Peri-alveolar accumulation of CD8+ T cells, which constitutively express early activation marker CD69 and epithelial-resident marker CD103, is identified. These draining lymph nodes originated and tumor-specific TRM cells, with high expression of IFN-γ and other cytotoxic cytokines, patrol along blood stream and infiltrate in the pre-metastatic lung. IFN-γ coverts alveolar macrophages in the pre-metastatic lung into a immunosuppressive phenotype with high expression of IDO1, which reciprocally compromises the anti-tumor effects of TRM. In conclusion, our study revealed the alteration of lung TRM during the process of metastasis and its interaction with lung pre-metastatic niche, indicating that by finding possible approaches to enhancing TRM function or strengthening local defenses could decrease breast cancer lung-tropic metastasis.

#1893

Consumption of oil derived from frying bacon increases breast cancer metastasis.

Cheng Chen, Joy J. Chen, Liqian Ma, William G. Helferich, Erik R. Nelson. _University of Illinois at Urbana Champaign, Champaign, IL_.

Breast cancer continues to be the most commonly diagnosed cancer among women, with the majority of mortality being associated with the metastatic spread of this disease. In terms of onset, genetic drivers such as mutations in BCR1/2 account for only ~10% of all cases. Thus, environmental factors, including the diet are significant contributors to breast cancer onset and progression. In this regard, elevated circulating cholesterol levels are associated with a poor prognosis, while cholesterol lowering medication (statins) appear protective. We have previously shown that a high cholesterol diet increased both primary tumor growth and metastasis in preclinical models. Interestingly, it was found that the metastatic effects of cholesterol were primarily mediated by its metabolite, 27-hydroxychoelsterol (27HC). In addition to its effects on cancer cells, 27HC also required neutrophils and gamma delta T cells for its pro-metastatic effects. Therefore, we hypothesized that the consumption of foods with high cholesterol, and potentially oxidized cholesterol products, promote breast cancer progression.

Bacon is a common food in the US and is prepared by frying in its own fat. We mimicked the preparation process by using a controlled pan-frying procedure. The used oil was collected and processed into food pellets (5% lipid from bacon frying fat, 5% lipid from soybean oil). A control diet was formulated as 5% fat from rendered pork lard, 5% lipid from soybean oil. We also included a diet with no expected cholesterol, where fat content was matched with soybean oil (10%). In order to determine the relative contribution of cholesterol to observed changes in metastatic colonization and outgrown, we also included groups where mice were treated with ezetimibe, a cholesterol uptake inhibitor. Mice were placed on their respective diets for 4 weeks prior to intravenous engraftment with Met1 cancer cells. There were no significant differences in weight gain observed between the experimental groups. 5 weeks post-engraftment, metastatic burden was assessed by ex vivo imaging. Compared to the no cholesterol control diet, there was a significant increase in metastatic burden in the lard group. Intriguingly, the bacon oil diet increased metastatic burden compared to both the lard and no cholesterol groups, and this increase was attenuated when mice were treated with ezetimibe. Ongoing work is aimed at assessing the circulating cholesterol and oxysterol concentrations, and evaluating changes in the metastatic microenvironment between mice on the different diets. Collectively, this study indicates that while cholesterol consumption increases metastatic progression, its preparation prior to consumption can also have significant impacts. Our work provides further rationale for diets low in cholesterol for breast cancer patients.

This work was supported by the grants from the NCI, AICR and DOD-BCRP to ERN, and Arnold O. Beckman Research Award to WGH. 

### Extracellular Matrix and Mechanotransduction

#1894

Canonical Wnt-associated extracellular matrix in adrenocortical carcinoma.

Morgan K. Penny, Antonio M. Lerario, Chris LaPensee, Thomas J. Giordano, Gary D. Hammer. _University of Michigan, Ann Arbor, MI_.

Adrenocortical carcinoma (ACC) is a rare and often aggressive cancer that affects 1-2 people per million in the United States annually. Genomic alterations activating canonical Wnt signaling occur in approximately 40% of ACCs and are associated with poor prognosis. However, the biological consequences of constitutive canonical Wnt activation in ACC are poorly understood. To better characterize the transcriptional programs that are engaged in ACC, we performed independent component analysis on The Cancer Genome Atlas (TCGA) ACC transcriptome dataset to identify components of coordinately expressed genes. One of the components identified was significantly enriched for Wnt signaling (p = 0.001) and was strongly associated with somatic CTNNB1 (β-catenin) mutations (p = 2.276e-07). Interestingly, this Wnt-enriched component also showed enrichment for extracellular matrix (ECM)-receptor interaction (p = 0.00139), including (but not restricted to) expression of COL11A1 (Collagen alpha-1(XI)), LAMC3 (Laminin, gamma 3), and ITGA2 (Integrin, alpha 2), suggesting that Wnt signaling is regulating cell-ECM interactions and ECM composition in ACC. To follow up on this observation, we performed immunohistochemical staining on tissue microarrays (TMAs) containing 97 ACC samples. We observed a strong correlation of COL11A1 expression and nuclear β-catenin localization (p = 0.0088), suggesting COL11A1 expression may be regulated by canonical Wnt signaling. Furthermore, COL11A1 expression was associated with decreased overall survival (p = 0.0003) and decreased event-free survival (p = 0.0075). To determine whether ECM and ECM-receptor genes are regulated by canonical Wnt activity, the NCI-H295R human ACC cell line, harboring an activating CTNNB1 mutation, was used. Cells were treated with PKF115-584, an inhibitor of canonical Wnt signaling that disrupts β-catenin interaction with TCF/LEF transcription factors. PKF115-584 treatment significantly reduced expression of COL11A1, LAMC3, and ITGA2 in NCI-H295R cells at early timepoints (p≤0.05), consistent with our hypothesis that canonical Wnt signaling regulates expression of ECM components in ACC. At late timepoints following PKF115-584 treatment, NCI-H295R cell viability decreased, indicating that canonical Wnt activity may regulate cell survival in ACC. These results illustrate a novel role for canonical Wnt activity in ACC, and suggest that β-catenin-regulated transcription of ECM and ECM-receptor components may promote cancer cell survival and aggressive disease. Future studies are aimed at characterizing the contribution of extracellular proteins to ACC phenotypes.

#1895

Identification of therapeutic targets in peritumoral microenvironment by single-cell assays using impedance spectroscopy.

Christian Maercker. _Esslingen University of Applied Sciences, Esslingen, Germany_.

Metastasis and tumor growth are very much dependent on the peritumoral microenvironment. Cells in the peripheral cortex send out growth factors and generate an extracellular matrix which is decisive for infiltration of neoplastic cells and, finally, the formation of a functioning tumor tissue. To figure out specific functions of different cell types and their various interactions in in vitro or in vivo assays, which are possibly relevant for the identification of new drug targets, is a challenge, because we deal with a very complex multifactorial network. Therefore, our goal is to develop test systems with a limited complexity, but which still are informative in terms of molecular tumor development. To reach this, we first have to singularize cells from tumor tissue. Within surgical tissue, tumor core tissue has to be discriminated from surrounding tissue by MRI or immunohistochemistry. Afterwards, microdissected tissue samples have to be dissociated by protease digest. In a next step, the mixture of cells has to be separated into the different cell types by immunopanning and/or FACS sorting. RNAseq helps to identify gene expression profiles specific for individual cells within the tumor compared to the tumor microenvironment. For us the most important parameters are the adhesion and migration activities of the various cell types of different origin on different matrices. To establish a test system as a starting point for a more sophisticated experimental setup to characterize tumorigenic tissues, we firstly isolated mesenchymal stem cells (MSC) from fat tissue or bone marrow, before transfer to microwells covered with electrodes to monitor cell adhesion by electrochemical impedance sensing. We found that pre-coating of the microwells with either collagen type I or IV induced different impedance profiles, corresponding to distinct differentiation pathways which was confirmed by histochemical staining as well as protein and gene expression analyses. In another approach, we used impedance sensing to measure prostate or breast cancer cell migration towards the secretome of mesenchymal stem cells. MSC cell culture supernatant was fractionated by size exclusion and ion exchange chromatography. We identified type I and III collagen, fibronectin and Laminin 421 as extracellular matrix proteins to be the main drivers of rapid cancer cell migration requiring as little as two hours for a full migration response. The challenge now is to adjust this experimental approach to individual cell types isolated from different tumor types and also to different matrices simulating the tumor microenvironment.

#1896

Blockade of laminin-411-notch crosstalk as an effective therapy for glioblastoma treatment.

Dmytro Klymyshyn, Anna Galstyan, Rameshwar Patil, Hui Ding, Ekaterina Shatalova, Shawn Wagner, Keith Black, Alexander Ljubimov, Eggehard Holler, Julia Ljubimova, Tao Sun. _Cedars-Sinai Medical Center, Los Angeles, CA_.

Purpose: The extracellular matrix (ECM) in tumor microenvironment including laminins is important for tumor development, and we aimed to develop an effective glioblastoma therapy via blocking the communication between tumor and their microenvironment mediated by laminin-411-Notch axis.

Methods: The glioma vascular protein, laminin-411(α4β1γ1) expression was examined in >200 glioma patient specimens by immunohistochemistry. In glioblastoma bearing mice, laminin-411 expression in tumor cells was disrupted by CRISPR/Ca9 system, and its impact on two glioblastoma cell lines was studied in intracranial xenografts. Laminin-411-Notch crosstalk was further dissected in vitro using cell cultures. Finally, a nanobioconjugate was developed to block laminin-411 expression in vivo. The effects of blocking laminin-411-Notch axis via systemic administration of this nanodrug were investigated in vivo using MRI, immunohistochemistry, western blot and survival analysis.

Results: Laminin-411 (α4β1γ1) expression in a panel of 226 patient brain glioma samples was correlated with higher tumor grade, and expression of cancer stem cell (CSC) markers including Notch pathway, CD133, Nestin, and c-Myc. Laminin-411 overexpression was also correlated with higher recurrence rate and shorter survival of glioma patients. Depletion of laminin-411 α4 and β1 chains with CRISPR/Cas9 in human glioblastoma cells led to slower growth of resultant intracranial tumors compared to wild type tumors as revealed by MRI. Concomitantly, survival of host animals carrying the laminin-411 knockout tumors was significantly increased. Mechanistic studies in vitro and in vivo showed that laminin-411 inhibition suppressed Notch pathway in both normal and malignant human brain cell types. A nanobioconjugate potentially suitable for clinical use and capable of crossing blood-brain barrier was designed to block laminin-411 expression. Nanobioconjugate treatment of mice carrying intracranial glioblastoma significantly increased animal survival and inhibited multiple CSC markers including Notch axis.

Conclusion: The depletion of "malignant" laminin-411 α4 and β1 chains with CRISPR/Cas9 led to slower growth of resultant intracranial tumors compared to wild type tumors, and a systemic nanodrug treatment to inhibit laminin-411-Notch crosstalk provides a new strategy for glioblastoma treatment. This study demonstrates a new strategy of glioblastoma treatment through targeting a critical component of tumor microenvironment, largely independent of heterogeneous genetic mutations in the tumor.

#1897

Exosome mediated intercellular communication in the triple negative breast cancer cell line MB.MDA.231 relies on ITGB3.

Pedro Fuentes Varela,1 Marta Sesé,1 Pedro Jesús Guijarro,1 Marta Emperador,1 Héctor Peinado,2 Stefan Hümmer,1 Santiago Ramon y Cajal1. 1 _Hospital Universitari Vall d'Hebron. UAB, Barcelona, Spain;_ 2 _CNIO, Madrid, Spain_.

Introduction: Tumors are complex systems where cell-cell communication between intra-tumoral and extra-tumoral cells play a crucial role. Extracellular vesicles (EVs), membrane-limited vesicles secreted by normal and malignant cells, have been proposed to act as mediators of intercellular communication in physiological and pathological scenarios. It is now well established that integrins are constantly endocytosed and recycled back to the plasma membrane through multiple routes. Accumulating evidence clearly indicates that endocytosis and recycling of integrins plays a crucial role during cancer progression, invasion, and metastasis. Here we describe a fundamental link between endocytosis mediated integrin trafficking and the integrin-mediated uptake of exosomes.

Objectives: As the integrins are crucial for the role of exosomes in cancer progression and particularly in metastasis we were aiming at delineating the role of ITGB3 in exosome-mediated intercellular communication in the triple negative breast cancer cell line MB.MDA.231.

Methodology: Using stable shRNA of ITGB3 (shITGB3) in the triple-negative breast cancer cell line MDA.MB.231, EVs were isolated by ultracentrifugation and characterized by Western blot analysis to determine the protein composition and by NTA and CryoEM to measure the amount of EVs. An LC-MS/MS-based survey was performed, to determine the differences in the protein composition of EVs after knock-down of ITGB3. Uptake of fluorescently labeled exosomes into recipient cells was measured by flow cytometry. The colony forming capacity induced by conditioned medium was used to assess the functional impact on the role of ITGB3 in intercellular communication.

Results: Analysis of the EVs fraction of shITGB3 cell revealed a striking decrease of classical markers of exosomes as judged by western blot analysis of proteins such as TSG101, CD81, but not others like Flotillin-1. LC-MS/MS analysis of shITGB3-MDA.MB.231 derived-exosomes furthermore revealed clear defects in protein complexes required for exosome formation, namely the ESCRT complex and the Syndecan-syntenin-ALIX complex. Both complexes are required for the formation of endosomal intraluminal vesicles that get released as exosomes. On the other hand, NTA analysis and CryoEM showed the overall amount of EVs was increased. In line with this, the uptake of fluorescently labeled EVs was strongly impaired in shITGB3 cells. By the use of small molecule inhibitors, we can furthermore demonstrate that this defect linked to endocytosis of EVs.

Conclusions: Here we demonstrate that ITGB3 plays a crucial role in controlling the composition of different vesicle populations in the extracellular space, linked to the endocytosis of integrins and interference with this mechanism results in defects in the uptake of EVs and impedes the generation of exosomes. The functional effects are discussed

#1898

TIMP-2 function is regulated by the composition of the cellular microenvironment.

David Peeney. _National Institute of Health, Bethesda, MD_.

The activity of mammalian matrix metalloproteinases (MMPs) is regulated by a family of four endogenous inhibitors known as the tissue inhibitor(s) of metalloproteinases (TIMPs). Since their original discovery and characterization several additional biological roles have been ascribed to TIMP family members, such as regulation of receptor tyrosine kinase-dependent cell growth and migration. TIMP-2 is abundantly expressed in normal tissues with low or absent MMP expression. In addition to this unique expression pattern TIMP-2 demonstrates broad spectrum inhibition of MMPs in vitro. Recent studies in our lab have shown that TIMP-2 is a promising candidate for biological cancer therapy. However, the role of TIMP-2 in cancer progression has been subject to debate with conflicting reports correlating enhanced TIMP-2 expression levels with both good and poor clinical outcomes. TIMP-2 exhibits multiple interactions with components of the extracellular matrix (ECM). Our working hypothesis is that TIMP-2 acts as a homeostatic mediator via modulation of ECM and cellular activities. Analysis of expression levels in tumor samples shows that TIMP-2 displays a strong positive correlation with a number of genes involved in regulation of the ECM. Using sample datasets from The Cancer Genome Atlas, we have identified a multi-gene expression pattern suggesting that the MMP-independent activities of TIMP-2 are sequestered in the tumor microenvironment. Using fluorescent labeled TIMP-2, we have conducted live cell imaging to visualize TIMP-2 localization in vitro. Our data reveals that TIMP-2 is highly dynamic both within the extracellular microenvironment and intracellular compartments. In addition, TIMP-2 displays a diverse pattern of localization within homogeneous cell populations and between cells of epithelial or mesenchymal lineages. Other in vitro observations highlight an inconsistent cellular response following TIMP-2 treatment. We speculated that these variations are a result of irregularities in ECM deposition by target cells. To obviate these inconsistencies, we have 1) generated TIMP-2 knockout cell lines; 2) cultured these cells on purified ECM in the presence or absence of exogenous TIMP-2 and/or mitogenic factors; 3) analyzed cell signaling response under these well-defined culture conditions. Our results show that TIMP-2 can differentially modulate intracellular signaling in a manner that is dependent on specific ECM components and the additional presence or absence of a mitogenic stimulus. Current work focuses on details of extracellular TIMP-2 molecular interactions and cellular uptake/signaling. Through further understanding of the extracellular molecular interactions of TIMP-2, and the factors that regulate its cellular functions, researchers will be able to more effectively target the aberrant extracellular environment that is associated with many human diseases.

#1899

Non-invasive quantification of cross-linked type III collagen products - A measure of extracellular matrix stiffness related to TGF-β signaling in colorectal cancer.

Christina Jensen,1 Sarah Rønnow,2 Jannie M. Sand,2 Lars N. Jørgensen,3 Morten A. Karsdal,2 Nicholas Willumsen2. 1 _University of Copenhagen, Copenhagen, Denmark;_ 2 _Nordic Bioscience, Herlev, Denmark;_ 3 _Bispebjerg Hospital, University of Copenhagen, Copenhagen, Denmark_.

Background: Extracellular matrix (ECM) deposition and stiffening have emerged as an important player in cancer progression. ECM deposition is stimulated by transforming growth factor-beta (TGF-β) that is being widely evaluated as a new therapeutic target in cancer. ECM stiffness is characterized by type III collagen deposition and cross-linking in the tumor microenvironment. Cross-linked type III collagen products reflecting ECM stiffness and TGF-β signaling may therefore have biomarker potential in cancer. By developing an assay to quantify cross-linked type III collagen pro-peptides and applying the "Scar-in-a-jar" (SiaJ) model, we evaluated the impact of TGF-β on this novel biomarker and assessed the biomarker potential in colorectal cancer (CRC).

Methods: A monoclonal antibody was raised against the N-terminal pro-peptide of type III collagen and a sandwich ELISA was developed and technically validated. The cross-linked type III collagen product levels were assessed in supernatants from the SiaJ culture model in which primary human healthy lung fibroblasts were cultured up to 12 days in the presence of ficoll and 1ng/ml TGF-β, with or without addition of 1µM ALK-5/type I TGF-β receptor kinase inhibitor (iTGF-β). The pathological relevance of cross-linked type III collagen products was assessed in serum from patients with CRC (n=34) and healthy controls (n=39). Levels were compared using unpaired, two-tailed Mann-Whitney test.

Results: The newly developed sandwich ELISA was shown to be technically robust and specific towards cross-linked type III collagen pro-peptides. Stimulating lung fibroblasts with TGF-β induced the production of cross-linked type III collagen with levels increasing up to 22-fold compared to without TGF-β (p=0.029), and iTGF-β reduced this induction. Levels of cross-linked type III collagen were significantly elevated in CRC patients compared to healthy controls (7.0 ng/ml vs. 5.6 ng/ml, p=0.0009). Furthermore, the diagnostic accuracy to separate CRC patients from healthy controls was 72% (p=0.0003, AUC: 0.72) with an odds ratio of 8 (95%Cl=2.4-23.2, p=0.0006) for having CRC.

Conclusions: Cross-linked pro-peptides of type III collagen can be quantified in serum from CRC patients as a potential surrogate marker of tissue stiffness and stromal reactivity driving tumor progression. In addition, TGF-β induces increased production of cross-linked type III collagen suggesting that this novel biomarker could potentially interrogate the effect of anti-TGF-β therapy. Future studies are needed to investigate the prognostic and predictive potential.

#1900

A tension-mediated glycocalyx feedback loop promotes glioblastoma.

Shelly Kaushik,1 James Matthew Barnes,1 Russell O. Bainer,1 Jason K. Sa,2 Elliot C. Woods,3 Fuiboon Kai,1 Jonathon N. Lakins,1 Joanna J. Phillips,1 Valerie M. Weaver1. 1 _Univ. of California San Francisco, San Francisco, CA;_ 2 _Institute for Refractory Cancer Research, Seoul, Republic of Korea;_ 3 _Stanford Univ, Palo Alto, CA_.

A considerable challenge in glioblastoma (GBM) therapy is treatment resistance and tumor recurrence, resulting in dismal patient prognosis. GBM aggression and recurrence is often associated with a mesenchymal phenotype, increased expression of glycoproteins and the presence of glioma-initiating "stem-like" cells. Using patient-derived xenograft models, and immune competent syngeneic and transgenic glioma mouse models, we show that aggressive mesenchymal-like GBMs, reminiscent of recurrent GBM, are mechanically stiffer, present with a bulky glycocalyx and demonstrate a stem-like phenotype. We report that the more aggressive mesenchymal GBMs show increased mechanical signaling and contractility, and their tumors are surrounded by a stiffer ECM that maintains and even further enhances integrin mechanosignaling. Since a large proportion of these bulky glycoproteins are also stem markers, upregulation of the glycoproteins and their modulators leads to enhanced GBM stem-ness. This was evident both in our mesenchymal models of GBM as well as multiple patient datasets which compared paired primary and recurrent GBM RNA sequencing and protein data. In addition to stem-ness, we show that the mechanically "enhanced" GBMs also foster a differential immune landscape, infiltrated by type 2 macrophages, believed to be pro-tumorigenic and immune evasive. Our findings suggest that there is a dynamic and reciprocal link between integrin mechanosignaling, the GBM immune landscape and a bulky glycocalyx, which promotes a mesenchymal, stem-like phenotype and likely recurrence in GBM patients. Thus, therapeutic strategies to target GBM tissue tension could prevent recurrence, reduce mortality and improve patient outcome.

#1901

Collagen remodeling in clear cell renal cell carcinoma: Influence of composition on growth and metabolism.

Kyle H. Bond,1 Anna Deck,2 Jennifer Caron,2 Peter Brooks,2 Calvin Vary,2 Sunder Sims-Lucas,3 Leif Oxburgh2. 1 _University of Maine, Westbrook, ME;_ 2 _Maine Medical Center Research Institute, Scarborough, ME;_ 3 _University of Pittsburgh, Pittsburgh, PA_.

The extracellular matrix (ECM) plays an important role in maintaining the structure and function of the kidney. During aggressive fibrotic disease of the kidney, such as chronic kidney disease (CKD), the kidney extracellular matrix is rapidly modified and degraded. Reports have shown that changes in the ECM composition and architecture can have profound influence on kidney function and recovery. The influence of ECM remodeling on disease progression and kidney function in Renal Cell Carcinoma (RCC), is still not well understood. By co-isolating primary cancer cell lines and the extracellular protein fraction from resected ccRCC tumors, we have determined that changes in collagen isoform composition are drastically altered compared to healthy normal kidney via Mass Spectrometry SWATH analysis (NvT COL1 p= 5.2x10-5; COLIII p=0.03; COLVI; COLIV p=0.004). RNA-seq analysis of TCGA data shows that altered collagen composition compared to normal correlates with patient outcomes (5 year survival COL1 HIGH=55%, LOW=72%, p=3.96e-4; COLIII HIGH=60%, LOW=73%, p=6.67e-2; COLIV HIGH=67%, LOW=56%, p=1.06e-2; COLVI HIGH=44%, LOW=71%, p=4.09e-9). Trichrome staining and IHC analysis of COLI, COLIII, and COLIV alongside cryptic-collagen epitopes XL313 and Hu177, suggests that subsets of tumor cell populations degrade surrounding collagen to drastically alter the ECM architecture. H&E scoring of regions of high XL313/Hu177 staining shows increased grade compared to low/no-staining. Isolated primary ccRCC cancer lines were analyzed for differential adherence to collagen isoforms, showing preferential binding preferences. In parallel, isolated normal kidney epithelia and renal proximal tubule epithelial cells (RPTECs) adherence preference was also investigated. Freshly dissociated tumor tissue cultured on a novel ECM cocktail coating in 3D scaffolds show changes in cell viability, growth, and purity compared to standard tissue culture conditions. In this study, we conclude that collagen composition analysis through IHC and Mass Spectrometry can be indicative of patient tumor aggressiveness. Preferential binding to collagen isoforms by tumor cells and the lack of adherence by normal kidney epithelia indicates a novel mechanism for tumor cells to promote local invasion. Additionally, we have generated a novel method by which to isolate human ccRCC cell lines using a collagen cocktail that will be useful for basic research and disease modeling.

#1902

The comparative superiority of a bispecific antibody targeting alpha v and alpha 5 integrins is uniquely characterized by induced degradation of integrins.

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

Background: Bispecific antibody blockade of alpha v and alpha 5 integrins (BsAbαvα5) resulted in superior anti-tumor activity in prostate cancer cells over monospecific αv (MsAbαv) and α5 blockade (MsAbα5), singly or in combination. We sought to define a molecular basis for this observation.

Methods: We examined the comparative impact of monospecific and bispecific antibody strategies on membrane-specific and cellular expression of integrins as well as their transcription, translation and degradation fate in integrin av/a5+ prostate cancer cells (VCAP, C4-2b, DU-145 and PC-3). These dynamic changes were assessed in the context of functional studies including tumor-stromal interactions and downstream signaling.

Results: With MsAbαv or MsAbα5 therapy, alone or in combination, upregulation of target integrin and/or the alternative integrin was observed which suggested both autoregulation as well as a cross-regulation among the target integrins. However, with BsAbαvα5 therapy, either a block in upregulation of target integrin/s or a marked decrease in cellular integrin expression was noted. Unexpectedly, these dynamic changes in cellular integrin expression with the BsAbαvα5 were retained up to 72 hours after antibody exposure with recovery of integrin expression in viable cells noted by 7 days. In cells recovered at 48-72 hours, a marked decrease in clonogenicity, adhesion and migration was observed. Flow cytometry indicated persistent membrane loss of integrin expression at 48-72 hours confirming that the impact of the BsAbαvα5 is not related to altered subcellular integrin trafficking alone. QRT-PCR studies did not identify upregulation of integrin transcription to account for upregulated integrin expression. Blockade of protein translation with cycloheximide failed to abrogate the differential impact of MsAbs or BsAbαvα5 on integrin expression. However, lysosomal inhibition with chloroquine blocked the downregulation of integrin expression with BsAbαvα5 therapy. Variable effects on downstream signaling in the Akt and MAPK pathway observed across cell types contrasted with consistent functional impairment of tumor-stromal interactions.

Conclusions: BsAbαvα5 therapy uniquely induces lysosomal degradation of target integrins compared to MsAbs, alone or in combination. Cellular depletion of integrin expression serves as a pharmacodynamic marker of BsAbαvα5activity. By contrast, upregulated cellular expression secondary to MsAb therapy is likely secondary to a block in both integrin degradation and membrane recycling within the endosomal/lysosomal pathway. These observations provide additional insight into the mechanistic impact of the BsAbαvα5 as well as a pharmacodynamic method for monitoring in vivo activity.

#1903

Tenascin C affects tumor associated macrophage phenotype and tumor invasion in breast cancer.

Esra Guc, Gabriella Sammut DeMarco, Giulia Pieracci, Jeffrey W. Pollard. _MRC Centre for Reproductive Health, Queen's Medical Research Institute, The University of Edinburgh, Edinburgh, United Kingdom_.

Breast cancer metastasis is one of the major causes of cancer deaths among women. Tumor associated macrophages (TAMs) play a role in metastasis by inducing angiogenesis and remodeling the tumor matrix, as well as providing chemotactic, survival and proliferative signals to the tumour cells. Conventional classification of TAMs has been revised due to their complex phenotypic and functional variability dependent on cancer type and location. Therefore the temporal and spatial microenvironment within tumours is crucial to define macrophage subtypes and their effect on the tumor.

In our studies we show that the expression pattern of the extracellular matrix glycoprotein tenascin-C (TNC) correlates with the invasion sites of the tumor and affects the TAM phenotype in breast cancer. TNC is known to promote metastatic seeding and correlates with poor prognosis, however only a few studies have investigated how the tumor and immune cells interact with this matrix. We determined the spatial distribution of TNC using whole slide imaging of mouse breast cancer sections. In a mouse model of breast cancer caused by the mammary restricted expression of the Polyoma Middle T oncoprotein (MMTV-PyMT) we showed that TNC expression is located at the peri-tumor and peri-vascular sites in late stages of disease and is also correlated with angiogenic regions of human breast cancer. We analyzed MMTV-PyMT breast peri-tumor (high tenascin-C and angiogenesis) and intra-tumor (low tenascin-C and angiogenesis) regions separately and found that macrophages at peri-tumor site express more TAM markers associated with pro-tumoural functions such as CD206, CD204, CD11b and CD49d (integrin alpha-4). To better understand how this matrix affects macrophage function, we polarized bone-marrow derived mouse macrophages using the E0771 mammary cancer cell line conditioned medium on collagen, fibronectin and tenascin C coated 2D surfaces. We found that tenascin C significantly increased CD11b, CD49d and CX3CR-1 expression in macrophages. CD49d functions as a co-receptor of CX3CR-1 and its ligand VCAM-1 on E0771 also increases with TNC in macrophage-conditioned medium. It was previously shown that VCAM-1 expression and binding to CD49d promotes breast cancer progression and metastasis. We also show that a 3D matrix with TNC increases transwell migration and spheroid invasion of E0771 cells, suggesting that TNC expression at the invasion site could directly affect breast cancer invasion. Consequently we suggest that TNC not only has prognostic value but could also be crucial to predict invasion sites within breast tumors due its effect on macrophage-tumor interaction and metastasis.

#1904

Versican production is driven by both epithelial and stromal cells in pancreatic cancer.

Hanna R. Rainiero,1 Philip B. Emmerich,1 Chelsie K. Sievers,1 Connor J. Maloney,2 Rosabella T. Pitera,1 Susan N. Payne,1 Mitchell G. Depke,1 Cheri A. Pasch,1 Linda Clipson,1 Jillian K. Johnson,1 Kristina A. Matkowskyj,1 Fotis Asimakopoulos,1 Dustin A. Deming1. 1 _UW-Madison, Madison, WI;_ 2 _UW Madison, Madison, WI_.

Background: Pancreatic ductal adenocarcinoma (PDAC) recently became the third-deadliest cancer due to resistance to chemotherapy and immunotherapies. The tumor microenvironment (TME) in PDAC is thought to contribute to this resistance, with up to 80% of the tumor bulk consisting of stroma. We investigated the role of two major stromal cell types, pancreatic stellate cells (PSCs) and macrophages, in the production of the immunosuppressive proteoglycan versican (VCAN) as a precursor to the identification of novel mechanisms that may enhance therapeutic response.

Methods: B6 KPC mice were utilized as a murine model of PDAC. A human PDAC tissue microarray (TMA) was developed representing normal and neoplastic pancreatic tissue across 131 patients. Immunohistochemistry (IHC) was used to determine levels of VCAN and CD8+ T cells. Bone marrow-derived macrophages (BMDMs) were differentiated from BALB/c mouse femurs and polarized to M1 (antitumor) or M2 (protumor) status. Additionally, PDAC organotypic spheroids were derived from both human and murine tissues whereas PSCs were derived solely from human tissue.

Results: IHC analysis of KPC tumors revealed elevated levels of stromal VCAN compared to normal pancreatic tissue (p<0.001, n=20). VCAN accumulation was increased even in the earliest stages of acinar-to-ductal metaplasia in KPC mice. Areas of intense stromal VCAN staining trended toward reduced CD8+ T cell infiltration (4.9 vs 7.3 cells/high power field (hpf), p=0.3). IHC analysis of human PDAC revealed elevated VCAN accumulation across all stages compared to the normal adjacent tissue (n=231, p<0.001). Areas of high VCAN accumulation demonstrated reduced CD8+ T cells compared to areas of low VCAN (0.6 vs 2.9 cells/hpf, p<0.001). To investigate cell types responsible for enhanced VCAN accumulation in the tumor microenvironment, relative expression (RE) of VCAN was compared in vitro to M0 macrophages. Organotypic cancer spheroids demonstrated increased expression of VCAN from KPC mice (RE=49, n=2) and patient-derived PDAC tissue (RE=14, p=0.01, n=3). M1-polarized BMDMs had increased expression of VCAN (RE=24) compared to M2 BMDMs (RE=8) (p<0.001, n=3). Interestingly, M1 BMDMs cultured in PDAC-conditioned media had reduced RE of M1 markers: TNFα (395 vs 37, p=0.03) and iNOS (24723 vs 4813, p=0.003), and increased M2 markers: Arg-1 (127 versus 1049, p=0.02) and YM-1 (0.5 vs 3, p=0.02). PDAC-conditioned media also reduced VCAN expression of M1 BMDMs (24 vs 9, p=0.02). PSCs derived from human PDAC also demonstrated enhanced RE of VCAN compared to negative controls (RE=68, p=0.017) with no significant change in the presence of PDAC conditioned media (p=0.4).

Conclusions: The accumulation of VCAN is common in PDAC and correlates with CD8+ T cell exclusion. Epithelial and stromal components are responsible for VCAN production. VCAN deserves further investigation as a target for therapeutic interventions for PDAC.

#1905

Chemoradiotherapy promote fibroblasts to a myofibroblastic phenotype in tumor microenvironment in esophageal cancer.

Satoshi Komoto, Kazuhiro Noma, Toshiaki Ohara, Takuya Kato, Yuki Katsura, Hiroaki Sato, Toru Narusaka, Noriyuki Nishiwaki, Hiroshi Tazawa, Yasuhiro Shirakawa, Toshiyoshi Fujiwara. _Okayama Univ. Graduate School of Med., Okayama, Japan_.

Background and aim: Cancer and stromal cells interact each other to promote tumor progression and malignant potential such as proliferation, migration, invasion, and therapeutic resistance of cancer cells. On the other hand, myofibroblast is one of activated subtypes trans-differentiated from normal fibroblast, which is normally triggered by mechanical stress. Cancer-associated fibroblast is thought to be an active form of fibroblast in tumor microenvironment, which mimics transdifferentiation during wound healing. The aim of this study is to clarify whether chemo/radiation-therapy can induce transdifferentiation of fibroblasts to active form, and also the "therapy-induced activated fibroblasts" contribute malignant phenotypes of cancer cells. Method: Human normal fibroblast cell lines (FEF3, NHLF, WI-38) were treated by radiation, cisplatin, 5-FU, docetaxel. And the expression of alpha-smooth muscle actin (αSMA) and fibroblast activation protein alpha (FAP) in treated fibroblasts was evaluated as the marker of activated fibroblasts by immunofluorescence staining (IF), western blotting (WB), and flow cytometry (FCM). To verify a functional impact of treated fibroblasts on cancer cells, we estimated the proliferation, radio-resistance, invasion, and migration abilities of esophageal cancer cell lines (squamous cell carcinoma: TE4, adenocarcinoma: OE33) which were cultured in conditioned medium of FEF3 treated by radiation (CM(FEF3+RT)). Result: IF, FCM, and WB demonstrated up-regulation of FAP and αSMA in CDDP/5-FU/DOC/radiation-treated fibroblasts as a phenotype change to myofibroblasts. In invasion and migration assays, TE4 cells cultured in CM(FEF3+RT) demonstrated more invasive and migrative phenotype than control. Additionally, OE33 cells cultured in CM(FEF3+RT) demonstrated increased proliferation and radio-resistance compared to control. Conclusion: This study shows that chemo/radiation-therapy induces the transdifferentiation of normal fibroblasts to myofibroblastic phenotype, which can promote malignancy in esophageal cancer cell.

#1906

Development of multi-spheroid co-culture 3D tumor assays using real-time live-cell analysis.

Kalpana Patel,1 Minnie Oliver,1 Tim Jackson,2 Nevine Holtz,2 Nicholas Dana,2 Jill Granger,2 Tim Dale,1 Derek Trezise1. 1 _Sartorius, Welwyn Garden City, United Kingdom;_ 2 _Sartorius, Ann Arbor, MI_.

Recapitulation of the tumor microenvironment is widely acknowledged as being key to the development of more predictive 3D in-vitro efficacy models. To facilitate this, we demonstrate simple, robust workflows for creating, monitoring and automatically quantifying features, such as morphology, growth and death of two tumor multi-spheroids co-culture models using real time live cell analysis. 96-well plate-based assays were established using a panel of breast tumor multi spheroids cultured on a base of extracellular matrix (ECM, Matrigel®) in co-culture with either stromal cells or immune cells. SKBr-3 cells, when cultured alone failed to produce spheroids, however inclusion of normal human dermal fibroblasts (NHDFs) had a striking effect on spheroid formation. NHDFs co-cultured with MDA-MB-231 cells reveal a time-dependent morphological effect, transitioning from a stellate appearance at early time-points, before reverting to a more clustered round appearance beyond 6 days. Using a novel brightfield image analysis algorithm, the growth and size (area) of spheroids could be determined over time non-invasively without the use of fluorescent labels. The inhibition of growth caused by a range of standard of care and cytotoxic agents (Lapatinib, ZK164015, camptotecin) aligned with the known expression profile of receptors in MCF-7, MDA-MB-231, SKBr-3 and BT-474 targeted by these agents. When co-cultured with immune cells (PBMCs), multi-spheroid viability could be assessed (over 10 days) by measurement of a fluorescent protein (RFP) loss. In this model, Herceptin induced a concentration-dependent, antibody-dependent cell-mediated cytotoxicity (ADCC) of Her-2 expressing tumor cells. These data highlight the value of live-cell analysis to study the temporal impact of stromal cells on tumor spheroid morphology and pharmacological profiles. In addition, co-culturing with immune cells enables the study of ADCC. Spheroid assays, incorporating ECM and additional cell types have the potential to provide more translational models for the study of the tumor microenvironment.

#1907

Tenascin-C promotes the activation of mammary fibroblasts to calponin-expressing myofibroblasts.

Daisuke Katoh,1 Aya Noro,2 Kyoko Imanaka-Yoshida,1 Tomoko Ogawa,2 Toshimichi Yoshida1. 1 _Mie University Graduate School of Medicine, Tsu city, Japan;_ 2 _Mie University Hospital, Tsu city, Japan_.

[Introduction] Tenascin-C (TN-C), an extracellular matrix molecules, is highly expressed in cancer stroma. Recent studies have demonstrated that cancer associated fibroblasts (CAFs) expressed TN-C in many tumors, including breast cancer. Here, we clarified the effects of TN-C on phenotype changes of the fibroblasts. [Methods] We examined the cell morphology, expression of CAFs markers, and contractile ability in the fibroblasts after TNC treatment in vitro, using human mammary gland fibroblasts (HMFs) and dermal ones (HDFs). We also investigated the correlation between the expression of the CAF markers and TNC deposition in human breast cancer tissues, using immunohistochemical staining. [Results] TN-C (10μg/mL) induced morphological changes of HMFs from spindle-shaped cells to stellate-shaped cells on plastic as well as on glass coverslips. After the treatment of TN-C, the protein expressions of α-SMA and calponin were significantly increased. Immunofluorescence analysis revealed TN-C increased the actin-stress fiber formation and the expression of α-SMA and calponin. Furthermore, TN-C promoted contraction of collagen gel by HMFs. These results indicated TN-C induces transformation of HMFs into high-contractile myofibroblasts. In human breast cancer tissues, α-SMA and calponin-positive fibroblasts were localized in TN-C-positive cancer stroma. Interestingly, calponin-expressing fibroblasts were not observed in the skin metastatic lesions of breast cancer. In addition, the increased expression of α-SMA and calponin after TN-C treatment in HDFs were significantly lower compared to HMFs in vitro. These results suggested the responses of TN-C on fibroblasts differs according to the residential tissues. Next, we examined the receptor to TNC and signaling pathway for TNC-induced change of HMFs. TN-C increased the colocalization of integrin αv and β1 in focal adhesion. Furthermore, phosphorylation of SMAD2 and SMAD3 were significantly increased and SMAD3 translocated from cytoplasm to nucleus after TN-C addition. Inhibition of TGF-β type I receptor (SB-505124) or SMAD3 (SIS3) suppressed the expression of α-SMA and calponin after TN-C treatment. [Conclusion] TNC could contribute cancer stroma formation characteristics of breast cancer tissues, following induction of phenotypical change to myofibroblasts and enhanced stromal contraction via integrin αvβ1/TGF-β/SMAD signaling. Phenotypic differences between HMFs and HDFs after TN-C administration may be present.

#1908

Using hierarchical clustering of differential gene expression patterns to study the contribution of the extracellular matrix in breast cancer.

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

Breast cancer is the most frequently invasive cancer type among women worldwide, accounting for over 2 million new cases annually and 25% of all cancers diagnosed in women. Considering significant advances in the diagnostics and treatment of breast cancer it remains a leading cause of cancer mortality worldwide. The extracellular matrix (ECM) is a meshwork of fibrous proteins and proteoglycans that is essential for providing a physical framework and facilitating biochemical cues to maintain tissue homeostasis. However, the ECM also has a well-established role in tumour development and progression. Increasing evidence suggests that the ECM may impede targeted therapies and novel approaches that target the composition of the ECM may improve drug delivery and efficacy.

Using the xCELLigence real time analysis platform, we monitored the growth of several breast cancer cell lines on the major fibrous proteins of the ECM to elucidate the contribution of individual ECM components on cell behaviour. Coupling this with endpoint assays, we have identified diverse adhesion, proliferation and migration profiles for each cell line on each substrate. RNA was extracted from cells in 2-Dimensional culture on 1) collagen, 2) fibronectin, 3) laminin and 4) stimulated by various growth factors and from cells in 3-Dimensional culture. Using qRT-PCR, we examined the expression of 40 genes that code for proteins known to directly or indirectly regulate the composition of the ECM. We determined that there is significant differential gene expression amongst each cell line in response to individual ECM components. Using a hierarchical clustering analysis of our gene panel, we have identified a strong relationship amongst cell lines maintained on collagen and fibronectin. We used STRING[1] analysis to help us refine a gene signature and are currently examining our gene set in patient tissue samples. Our hypothesis is that the composition of the ECM regulates gene expression and this correlates with advanced cancer stage.

This work has the potential to create a clinically relevant gene signature to be used in the prediction of invasive cancers. Understanding how the ECM influences these proteins may provide information on the design of novel ECM targeted therapies in breast cancer.

1. Szklarczyk, D., et al., The STRING database in 2017: quality-controlled protein-protein association networks, made broadly accessible. Nucleic acids research, 2017. 45(D1):p. D362-D368.

#1909

Stromal cell driven proteolysis of versican is limited by epithelial cells in pancreatic ductal adenocarcinoma.

Philip B. Emmerich, Chelsie Sievers, Hanna Rainiero, Rosabella Pitera, Connor Maloney, Susan Payne, Mitchell Depke, Cheri Pasch, Linda Clipson, Jillian K. Johnson, Kristina Matkowskyj, Fotis Asimakopoulos, Dustin A. Deming. _UW-Madison, Madison, WI_.

Background: Clinical responses to checkpoint inhibitors in gastrointestinal cancers have largely been limited to MSI-high disease. Proteolysis of an immunosuppressive proteoglycan versican (VCAN) by ADAMTS proteases correlates with enhanced CD8+ T cell infiltration in colorectal cancer independent of mismatch repair status. Here we investigate the potential of this biomarker in pancreatic ductal adenocarcinoma (PDAC) as VCAN is abundant VCAN in this disease. Methods: KPC mice were used to generate mouse PDAC tissue and organotypic spheroids. A human PDAC tissue microarray (TMA) was developed representing normal and neoplastic pancreatic tissues across 131 patients. IHC was used to determine levels of VCAN, an immunostimulatory fragment versikine (Vkine), and CD8+ T cells. Bone marrow-derived macrophages (BMDMs) were derived from BALB/c mice. VCAN-specific ADAMTS protease expression profiles were compared between antitumor (M1) and protumor (M2) mouse macrophages, human pancreatic stellate cells (PSCs) and patient-derived PDAC-organotypic spheroids. Results: IHC analysis of normal murine pancreas demonstrated low VCAN and Vkine staining. VCAN is abundant in metaplastic/neoplastic KPC mouse pancreatic tissues. Rare VCAN proteolytic predominant areas (samples with both low VCAN and high Vkine) in KPC tumors had increased tumor-infiltrating CD8+ T cells/ high-powered field compared to weak areas (17 vs 4, p<0.001). Human PDACs had abundant VCAN expression compared to normal pancreatic tissues (p<0.001, n=118). Vkine was absent from normal tissues and present only at low levels in tumor cores (p<0.001, n=117). No human PDAC VCAN proteolytic-predominant samples were identified. M1 BMDMs demonstrated high relative expression (RE) of VCAN-specific ADAMTS proteases relative to unpolarized M0 BMDMs; however, these levels decreased under KPC-conditioned media (ADAMTS4, 101 vs 5, p=0.003; ADAMTS9, 80 vs 1, p=0.02; ADAMTS15, 103 versus 1.6, p<0.001; ADAMTS20, 881 vs 3.2, p<0.001). Similarly, M2 BMDMs had elevated ADAMTS-levels that were reduced under KPC-conditioned media, (ADAMTS4, 42 vs 0.4, p=0.0029; ADAMTS9, 40 vs 1, p=0.002; ADAMTS15, 23 vs 3, p=0.027; ADAMTS20, 310 vs 0.8, p<0.001; n=3). RE levels of ADAMTS proteases in KPC spheroids was consistently lower than reported above in macrophages. RE of ADAMTS proteases in human PSCs were between 18 and 304. No differences in ADAMTS expression were identified under PDAC-conditioned media in the human PSCs (p=0.3). Conclusions: The VCAN proteolysis predominant phenotype correlates with increased CD8+ T cell infiltration in PDAC though is rarely present in the human disease. PDAC epithelial cells influence the stromal cells to reduce VCAN proteolysis through decreased ADAMTS expression by macrophages. Future studies examining measures to enhance VCAN proteolysis could result in a more immune permissive microenvironment.

#1910

Regulation of ovarian tumor microenvironment dynamics by compressive stress.

Marwa Asem, Allison Young Young, Alejandro ClaureDeLaZerda, Matthew Ravosa, Sharon Stack. _University of Notre Dame, South Bend, IN_.

Ovarian cancer (OvCa) is the most fatal gynecologic malignancy and the fifth leading cause of overall cancer death among American women with a low (27%) 5-year survival rate, as 75% of women are diagnosed with disseminated intra-peritoneal (IP) metastasis. OvCa cells detach from the primary tumor and shed into the peritoneal cavity, adhere to the peritoneal mesothelial cell (MC) monolayer, intercalate within this layer, and invade into the submesothelial matrix, where they proliferate and form secondary lesions.

More than one third of OvCa patients develop ascites that correlates with relapse and poor prognosis. The accumulated ascites can reach volumes up to 5 liters, causing a striking elevation in intraperitoneal pressure (IPP) from normally sub-atmospheric ~5 mmHg to as high as 27 mmHg, impacting the tumor microenvironment including host and tumor cells. The pressure created by accumulated ascites alters the force environment in the peritoneal cavity, causing strain and compressive forces on peritoneal structures. Yet, the mechanism through which ascites-induced IPP influences OvCa progression is poorly understood. The aim of this study is to understand how ascites-induced IPP promotes OvCa progression.

To model the compressive force present in vivo due to accumulated tense ascites (~27 mmHg), we evaluated the effects of compressive force on primary human and murine MC monolayers and murine peritoneal explants using the Flexcell Compression System. Strikingly, compressed MC and peritoneal explants exhibited phenotypic alterations and formed abundant Tunneling Nanotubes (TNT), which are cell membrane extensions that modulate transfer of organelles between cells under stress. Furthermore, compression of OvCa cells adherent to murine peritoneal explants show intense formation of TNT between OvCa cells and peritoneal MC. Remarkably, TNT formed under compression displayed membrane distension and modulated the transfer of mitochondria from MC to OvCa cells. Moreover, collected peritoneal explants from OvCa murine model with intense ascites showed TNT formation and changes in the peritoneal sub-mesothelial collagen ultrastructure.

This study reveals a novel response of OvCa cells and the tumor microenvironment to mechanical compression, uncovering a new mechanism by which OvCa metastatic success may be regulated.

#1911

Bi-directional cross-talk of pancreatic cancer and stellate cells, mediated through CXCR4 and hedgehog pathways, promotes chemoresistance via its effect on gemcitabine metabolism, ROS detoxification and cancer stemness.

Mohammad Aslam Khan, Girijesh K. Patel, Sanjeev K. Srivastava, Sumit Arora, Haseeb Zubair, James Elliot Carter, Bin Wang, Seema Singh, Moh'd Khushman, Ajay P. Singh. _University of South Alabama, Mobile, AL_.

Mounting evidence suggests that tumor-stromal interactions play a key role in pancreatic cancer (PC) pathogenesis and chemoresistance. We previously demonstrated that CXCL12, a chemokine secreted by activated pancreatic stellate cells (PSCs), promoted PC chemoresistance upon binding to its receptor CXCR4. Furthermore, activation of CXCL12/CXCR4 pathway in PC cells (PCCs) also increased the expression and secretion of sonic hedgehog (SHH), which is a known inducer of PSCs thus establishing a bi-directional tumor-stromal crosstalk. Here we present data to support that dual targeting of CXCL12/CXCR4 and hedgehog pathways is effective in improving therapeutic outcome in PC by diminishing multiple chemoresistance mechanisms. Monoculture of PCCs (MiaPaCa and Colo357) or coculture of PCCs and PSCs were treated with gemcitabine in the presence and absence of CXCR4 antagonist (AMD3100) and hedgehog inhibitor (GDC-0449). The data demonstrated that PCCs in coculture had better survival against gemcitabine treatment as compared to those grown in monoculture and inhibition of CXCL12/CXCR4 and hedgehog pathways abrogated this co-culture-induced chemoresistance. In further mechanistic studies, we identified co-culture-induced changes in gene expression and confirmed their functional significance in chemoresistance. Our data established important roles of genes associated with gemcitabine metabolism, ROS detoxification and stemness in PC chemoresistance. Inhibition of CXCL12/CXCR4 and hedgehog pathways led to increase in gemcitabine-mediated ROS production, enhanced accumulation of active gemcitabine metabolite (gemcitabine tri-phosphate) and reduced stemness in cocultured PCCs. Finally, we examined the efficacy of our dual targeting approach in vivo in an orthotopic mouse model of pancreatic cancer by treating the mice with gemcitabine alone or in combination with AMD3100 and/or GDC-0449. Our data demonstrated that mice treated with gemcitabine along with AMD3100 and/or GDC-0449 had significantly greater reduction in tumor growth as compared to those treated with gemcitabine only. Studies are ongoing in additional pancreatic tumor models. Together, these findings provide strong evidence in support of a novel combination therapy for pancreatic cancer treatment.

#1912

Inhibition of Exportin-1 function reverses the pro-tumorigenic potential of lung fibrotic microenvironments.

Antonina Bruccoleri, Ornella Rondinone, Francesca Andriani, Federica Facchinetti, Monica Tortoreto, Gabriella Sozzi, Giulia Bertolini, Luca Roz. _Fondazione IRCCS Ist. Nazionale dei Tumori, Milan, Italy_.

The contribution of stromal cells to tumor progression is widely established while less is known about stromal changes that may influence the early phases of tumorigenesis such as incipient growth of (pre)neoplastic lesions or seeding and awakening of metastatic cells. We have previously characterized the pro-tumorigenic properties of lung fibroblasts identifying increased activity of the nuclear transporter Exportin-1 (XPO1) as a key driver in modulating metabolic properties of fibroblasts towards a tumor promoting phenotype through subcellular re-localization of p53 and p62/SQSTM1. Consistently, XPO1 inhibition prevented the ability of activated fibroblasts to stimulate migration of lung cancer cells and to increase the frequency of CD133+ cancer stem cells, thereby also reducing their in vivo pro-tumorigenic activity.

To test the potential of therapeutic strategies based on modification of pro-tumorigenic microenvironments we exploited a mouse model of lung fibrosis based on single intranasal administration of an adenoviral vector expressing activated TGFβ. Fibrosis was efficiently induced in both nude and SCID mice and was associated with the creation of a pro-tumorigenic milieu as tested by tail vein injection of microenvironment-responsive lung cancer cell line LT73 one month after fibrosis induction. Mice treated with AdTGFβ showed increased number of tumor cells and/or CD133+ lung cancer stem cells two months after cancer cell seeding (p<0.05 for AdTGFβ vs. AdLacZ and medium alone). We also evaluated the modulation of immune cell subsets with multi parametric flow-cytometry detecting a consistent increase in neutrophils (Ly6-G+/Ly6-Clow) and a slight increase in monocyte derived macrophages (F4-80high/GR1-/CD11b+) and plasmacytoid dendritic cells (CD11c+/CD11b-) associated with upregulation of fibrosis-related miRNAs (miR-199a-5p) and induction of a pro-tumorigenic milieu. To test the efficacy of microenvironment modulating therapies we used the selective XPO1 inhibitor KPT-330. In vitro pharmacological inhibition of XPO1 was able to modulate expression of miRNAs in fibroblasts (including miR-199a-5p) and reduce fibroblast pro-tumorigenic secretory phenotype (reduction in IL-8, CXCL1, CCL2 and lactate production). For in vivo experiments animals were treated after induction of fibrosis but before injection of cancer cells to avoid confounding effects related to activity on cancer cells. XPO1 inhibition was able to convert the pro-tumorigenic potential of the fibrotic microenvironment and this was associated with a reduction of the levels of miR-199a-5p, decreased expression extracellular matrix-related genes (Sparc, Col1a1, Mmp2, Col6a1, Col6a3) and dampening of the inflammatory milieu.

Overall these findings suggest potential therapeutic efficacy of XPO1 inhibition to revert the pro-tumorigenic activity of fibrotic lung microenvironments.

#1913

Role of glypican 1 in regulating fibroblast activation in the prostate cancer tumor microenvironment.

Sukhneeraj P. Kaur,1 Hee K. Lee,1 Robert D. Arnold,2 Brian S. Cummings1. 1 _University of Georgia, Athens, GA;_ 2 _Auburn University, Auburn, AL_.

Cancer is a heterogeneous collection of neoplastic cells, surrounded by non-malignant, stromal cells, and extracellular matrix (ECM), among other constituents. Cross-talk between tumor and stromal cells forms a dynamic tumor microenvironment (TME) that mediates cell growth, proliferation, and metastasis. The TME can inhibit cancer growth and proliferation during the initial stages of carcinogenesis; however, cancer cells can evade these inhibitory signals, and reshape the TME, making it more amenable to their own growth. This gives rise to cancer-associated stromal cells, including cancer-associated fibroblasts (CAFs). CAFs further modulate the TME by regulating the release of growth signals, cell death, ECM remodeling, angiogenesis, and epithelial-mesenchymal transition. Glypican-1 (GPC1) is a cell surface heparan sulfate proteoglycan (HSPG) that has both pro-mitogenic and pro-metastatic activity. GPC1's role in regulating fibroblasts and their transformation into CAFs has not been investigated. We studied the ability of prostate cancer cells to alter the TME in vitro by exposing human bone fibroblasts (HS-5 cells) to tumor conditioned media (TCM) isolated from a human neuroendocrine (DU-145) and bone (PC-3) metastatic prostate cancer cell lines and assessed changes in cell morphology and cytotoxicity (using MTT). Exposure of HS-5 cells to TCM from DU-145 and PC-3 cells altered cellular morphology to a more mesenchymal phenotype similar to that seen in active myofibroblasts. TCM also decreased MTT staining in a time-dependent manner. We also determined the effect of GPC1 knockdown (KD) in HS-5 cells on cell morphology, migration and cytotoxicity. Interestingly, we saw phenotypic switching of GPC1 KD HS-5 cells to a myofibroblast like morphology akin to that when treated with TCM. There was no change in cytotoxicity in GPC1 KD HS-5 cells as compared to control cells. However, GPC1 KD enhanced the migration of HS-5 cells in wound healing assay. Migration was also increased in GPC1 KD HS-5 cells upon treatment with TCM. GPC1 KD also increased HS-5 cell contractility significantly. Collectively, these data support the hypothesis that GPC1 plays an inhibitory role in the TME of prostate cancer by regulating the activation of fibroblasts.

#1914

Acute myeloid leukemia cell and stem-progenitor cell behavior studied in mimetic bone marrow microenvironment.

Giorgia Simonetti,1 Marco Malferrari,2 Maila Becconi,2 Samantha Bruno,3 Giovanni Martinelli,1 Stefania Rapino2. 1 _Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) Srl - IRCCS, Meldola (FC), Italy;_ 2 _Department of Chemistry G. Ciamician, University of Bologna, Bologna, Italy;_ 3 _Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Institute of Hematology "L. e A. Seràgnoli", Italy_.

The bone marrow (BM) microenvironment plays a pivotal role in the survival and chemoresistance of leukemia stem cells (LSC) in acute myeloid leukemia (AML). However, conventional culture approaches do not reflect physiological conditions of crucial anatomical districts and signals of incubation-induced dysregulation may confound leukemic transcriptome results. In vivo mimetic cultures of the LSC districts will significantly aid in the disentanglement of LSC mechanisms and in the assessment of the therapy. We engineered Petri dishes able to mimic the "hypoxic" conditions found in the stem cell niches and in BM district. Our approach is based on a patented method which controls the physiological concentrations of oxygen, glucose and pH in the culture environment. Cell proliferation and hypoxia-induced apoptosis in the engineered device were investigated for OCI-AML3 and KG1 AML cell lines. We observed a slight but significant reduction in cell proliferation compared with control cells for OCI AML-3, which is in line with a more quiescent cell condition. No significant differences were obtained by culturing KG-1 cells, which are more resistant to low oxygen concentrations. We have analyzed the effects of cell culturing in the hypoxic dishes on the expression of MYC and p21. Immunofluorescence analysis of HIF-1α levels and HIF-1α nuclear localization as a function of oxygen concentration were determined. Time-lapse experiments of AML cell lines and primary BM cells were performed in the in vivo mimetic device to elucidate the response of CD34+ AML cells to different oxygen concentrations. We tested an innovative cell culturing device able to mimic the dynamical conditions of oxygen content, which are found in vivo. Our results shed light on AML cells and progenitor-stem cells behavior in response to oxygen conditions in the BM niche and allow to disentangle the LSCs complex biology. This "in vivo mimetic" approach will generate crucial information on the molecular mechanisms preserving LSC in AML, thus providing novel potential therapeutic targets to eradicate leukemia initiating cells in their own microenvironment, contrasting therapy resistance.

Supported by: Associazione Italiana per la Ricerca sul Cancro, MFAG id. 19044.

#1915

In vivo **prostate tumor tissue stiffness differs by tumor region and can be recapitulated in bioengineered prostate tumor tissues.**

Nicole L. Habbit,1 Benjamin Anbiah,1 Luke S. Anderson,1 Joshita Suresh,1 Iman Hassani,1 Matthew Eggert,1 Shanese L. Jasper,1 Balabhaskar Prabhakarpandian,2 Robert D. Arnold,1 Elizabeth A. Lipke1. 1 _Auburn University, Auburn University, AL;_ 2 _CFD Research Corporation, Huntsville, AL_.

Throughout the tumorigenic process, locational heterogeneities in tumor tissue microarchitecture develop as a result of aberrant angiogenesis and a subsequently induced oxygen and nutrient gradient within the three-dimensional (3D) mass. This phenomenon often results in differential tissue stiffness between the necrotic, quiescent, and proliferative tumor regions. In vitro, strong correlations have been found to exist between cell culture platform stiffness and acquired chemoresistance and varied drug response. Therefore, to accurately recapitulate the tumor microenvironment, biomimetic models must provide a mechanically similar scaffold. This study reports novel quantification of the in vivo prostate tumor stiffness and the ensuing development of tunable 3D bioengineered tumor tissue (BioTT) to successfully recapitulate in vivo mechanical cues in vitro.

In vivo samples were generated by subcutaneously injecting Matrigel-suspended metastatic prostate cancer cells (PC-3) into the flank of athymic NCr nude mice. Resultant tumors (300 – 1,500 mm3) were excised from the murine host and geometrically dissected to provide samples from the tumor core, midpoint, and periphery. The Young's modulus was quantified via parallel plate compression under physiological conditions. The 3D BioTT model is comprised of poly(ethylene glycol)-fibrinogen (PF) with varying amounts of excess poly(ethylene glycol) diacrylate (PEGDA) to modulate the mechanical properties of the scaffold. PC-3 cancer cells and BJ-5ta human fibroblasts were encapsulated within the covalently crosslinkable biomaterial and co-cultured for 29 days in vitro. Cell viability was assessed by LIVE/DEAD staining and cellular morphology was visualized with Hoechst 33342, Phalloidin, and the anti-fibroblast immunomarker, TE-7. Temporal variations in cell populations were quantified by flow cytometry and mechanical stiffness characterization was again performed by parallel plate compression.

In vivo prostate cancer tumors presented a wide range of tissue stiffness heterogeneity (200 – 5,750 Pa), characterized by an increasing modulus with respect to locational progression from the core to the periphery (n = 48 per tumor region). The BioTT model successfully recapitulated the full tumor stiffness range through biomaterial composition modulation; the addition of excess PEGDA significantly stiffened the PF scaffold (p ≤ 0.05, n = 3). PC-3 and BJ-5ta cells survived the encapsulation process and remained viable throughout long-term co-culture. Visualization of the 3D cellular microenvironment revealed both cancer and stromal cells maintained characteristic morphology. In future studies, the BioTT will be extended to a microfluidic chip platform, thus augmenting the physiological relevancy of the model by incorporating dynamic shear conditions and the ability to monitor cancer cell metastasis.

#1916

Actomyosin-YAP/TAZ signaling regulates the viability and chemoresistance of circulating tumor cells in hemodynamic shear stress.

Youhua Tan, Ying Xin, Keming Li. _The Hong Kong Polytechnic University, Hung Hom, Hong Kong, Hong Kong_.

Background: Tumor cells metastasize to distant organs mainly through hematogenous dissemination, where they experience considerable levels of shear stress. However, the influence of hemodynamic shear stress on the biophysical properties of circulating tumor cells (CTCs) remains elusive. The mechanisms underlying the survival of a subpopulation of CTCs in fluid shear flow and chemoresistance are unclear.

Methods: A circulatory microfluidic system was developed to mimic blood circulation in vitro. The survival of suspended breast tumor cells in fluid shear flow and tumor cells after chemotherapy was tested by MTS assay. The expressions of the related genes and proteins were measured by qPCR and western blotting. The biophysical properties of surviving tumor cells were examined by various methods.

Results: The majority of suspended breast CTCs could be eliminated by fluid shear stress and the survival rate depended on the magnitudes of shear stress and circulation duration. As the driving force for metastasis, cancer stem cells held survival advantages in shear flow. Higher fluid shear stress facilitated more nuclear translocation of YAP/TAZ in suspended tumor cells and induced lower cell survival. Inhibiting the entry of YAP/TAZ into the nucleus enhanced cell survival in shear flow. Further, suppressing actomyosin attenuated YAP/TAZ activity, which promoted the survival of suspended tumor cells in shear flow. In contrast, activating actomyosin enhanced the nuclear translocation of YAP/TAZ, which suppressed the survival of CTCs and cancer stem cells. Mechanistically, the nuclear YAP/TAZ interacted with p73, which then initiated the transcription of pro-apoptosis genes (including BAX) and promoted cell apoptosis.

Compared to untreated cells, tumor cells surviving shear stress exhibited unique biophysical properties: the adhesion of surviving tumor cells was significantly retarded; these cells exhibited elongated morphology, enhanced spreading, and up-regulated genes related to epithelial-mesenchymal transition; surviving tumor cells showed reduced F-actin expression and stiffness. Soft surviving tumor cells held survival advantages in shear flow and higher resistance to chemotherapy. Inhibiting actomyosin activity and YAP/TAZ in untreated cells enhanced chemoresistance, while activating actomyosin and YAP/TAZ in surviving tumor cells suppressed this ability, which may be associated with the corresponding changes in the expressions of the genes related to multidrug resistance.

Conclusion: This study unveils the regulatory roles of actomyosin-YAP/TAZ signaling in the survival and drug resistance of CTCs in hemodynamic shear flow, which suggest the importance of fluid shear stress and actomyosin-YAP/TAZ activity in tumor metastasis.

### Models and Drug Discovery

#1917

High-throughput evaluation of treatment response in patient-derived glioma stem cell models.

Ze-yan Zhang,1 Yingwen Ding,1 Ravesanker Ezhilarasan,1 Jie Yang,1 Lihong Long,2 Lawrence Bronk,2 Qianghu Wang,2 Erik P. Sulman1. 1 _Laura and Isaac Pelmutter Cancer Center at NYU Langone Health, New York, NY;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Although significant progresses in molecular oncology are being made using conventional cell lines, most therapies still fail in phase III clinical trials. Patient-derived models are being used more frequently as they are more faithfully representing the genomic features of primary tumors. However, one by one test of each model from large biobanks is extremely economy and time consuming. In this study, each of a panel of patient-derived glioblastoma stem cell (GSC) models was uniquely tagged by a lentiviral Cas9D10A and paired-gRNA targetable unique reporter (CAPTURE) barcoding system. Barcoded GSCs were then pooled evenly and following by radiation treatment (RT) in vitro. Amplicon sequencing was employed to count the barcodes distribution, which represent the relative cell number. The results showed that this approach faithfully identified the RT resistant GSCs from a mixing pool when comparing to the results from canonical clonogenic assay. In addition, a fluorescence marker will be switched by delivery of corresponding barcodes targeting CRISPR so that we can re-isolate interested cell models from the treated pool for investigating the treatment sensitivity and resistance mechanism. This study will provide a robust approach for therapeutic discovery take advantage of patient-derived models from large biobanks.

#1918

**InnoPanel** TM **-a well characterized panel of 1000 cancer cell lines for anticancer drug development.**

Feng Hao, Changpeng Liu, Hao Peng, Xiaolin Zhou, Yunhui Fu, Qiang Hou, Jinying Ning. _KYinno Biotechnology Co., Ltd, Beijing, China_.

In vitro cell line assays are broadly used in drug screening to assess drug efficacy, probe drug mechanism, discover and validate predictive biomarkers. High-throughput screen can be performed on cell line collections such as the well-known NCI-60 panel that is consisted of 60 human cancer cell lines developed by the National Cancer Institute (NCI).1Cell lines in such panels are usually well annotated for basic information such as cancer type and subtype, histopathology, and marker gene expression; many experimental parameters are also measured such as doubling time and inoculation density. The utility of cell panels is greatly enhanced with more cell lines that are thoroughly characterized for their genomics by next generation sequencing2-3. To this end, we have developed InnoPanelTM—a panel of 1000 cancer cell lines, including 800 commonly used cancer cell lines, 100 primary cell lines, and 100 engineered cell lines. These cell lines cover all major cancers and majority are genomically characterized with gene expression, mutation and copy number data. Flow cytometry data were also available for engineered cell lines to validate and characterize designed modifications, for example, Siglec-15 overexpression in the CHO-K1 human Siglec-15 cell line. All data are openly available to researchers at our online database InnopediaTM.To make InnoPanelTM-based screening more valuable and accessible to biologists, we developed a bioinformatics pipeline to automate analysis that combines genomics and efficacy data. The utility of such analysis is demonstrated by case studies. InnoPanelTMcan also be customized for a subset of the 1000 cell lines. Subpanel for selected cancer types or cell lines bearing certain features can be constructed. In summary, we developed a well characterized 1000-cell line panel as a valuable resource for researchers working on anticancer drug development.

References1. Shoemaker RH. The NCI60 human tumour cell line anticancer drug screen. Nat Rev Cancer 2006; 6: 813-23.2. Barretina J, Caponigro G , Stransky N , et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature, 2012, 483(7391):603.3. Consortium CCLE. Pharmacogenomic agreement between two cancer cell line data sets. Nature, 2015, 528(7580):84-7.

#1919

Molecularly annotation of mouse avatar models derived from patients with colorectal cancer liver metastasis.

Jingyuan Wang,1 Wei Liu,2 Jing Gao,1 Baocai Xing,2 Lin Shen1. 1 _Key laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Department of Gastrointestinal Oncology, Peking University Cancer Hospital and Institute, Beijing, China;_ 2 _Key laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Department of Hepato-Pancreato-Biliary Surgery I, Peking University Cancer Hospital and Institute, Beijing, China_.

Background: Liver is the most common metastatic site in advanced colorectal cancer. Most patients with colorectal cancer liver metastasis (CRLM) do not benefit from current treatment. Patient-derived xenograft (PDX) with defined molecular signatures are attractive models for preclinical studies.

Methods: Successfully established PDXs were evaluated to elucidate their fidelity of patients' biologic characteristics (pathologic, genetic and protein properties, together with chemosensitivity). The genomic variations of PDXs were analyzed by next-generation sequencing to explore the underlying molecular mechanism of metastasis and potential therapeutic targets.

Results: CRLM (N=73) showed a significantly higher successful PDX establishment rate than primary specimens (N=26; 76.7% vs. 57.7%). CRLM PDXs recapitulated the pathologic, genetic and protein properties of parental tumors, as well as chemosensitivity. Frequent altered genes in PDXs showed high consistency compared to patients' genomic alterations and were enriched in MAPK, ErbB, cell cycle, focal adhesion pathways for CRLM PDXs, whereas primary tumor-derived PDXs only exhibited genomic variations involving ErbB and cell cycle. The genetic alterations showed high concordance between paired PDXs from primary and metastatic tissues, except for recurrent gene mutations (ARID1A, CDK8, ETV1, STAT5B and WNK3) and common copy number gains in chromosomes 20q (e.g., AURKA/TPX2). Several potential drug targets such as KRAS, HER2, and FGFR2 were validated using corresponding inhibitors. Additionally, PDX models could also be used in screening efficient regimens for patients with no druggable alterations.

Conclusion: This study has successfully established and validated a large panel of molecularly annotated platforms from patients with CRLM for preclinical studies.

#1920

PC9 AZD9291-resistant (PC9-OSIR) cell line, a useful cell model for novel drug discovery.

Zhaoshuai Bai, Guoqian Wang, Xiaolin Zhou, Zhongquan Mu, Feng Hao, Jinying Ning. _Kyinno Biotechnology co., Ltd, Beijing, China_.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and the leading cause of lung cancer-related mortality. Identification of EGFR as a driver gene of NSCLC has led to the development of EGFR inhibitors, such as Gefitinib, Erlotinib, Afatinib and Osimertinib (AZD9291). AZD9291, a third-generation EGFR inhibitor, has been approved as the first-line treatment for patients with EGFR T790M mutation-positive NSCLC. Resistance to AZD9291 was observed clinically and C797S mutation was reported to be a major mechanism for resistance to AZD9291.Our group has generated PC9 AZD9291-resistant (PC9-OSIR) cell line using a dose-escalation method after treatment of the cells with mutagenic agent. Specifically, PC9 cells was treated by AZD99291 with increasing dose for 2 weeks, and remaining live cells was retrieved and designated as PC9-OSIR. Cell survival assay showed that IC50 of PC9-OSIR was over 2μM, 100-fold more resistant than the parental PC9 cells. We explored the resistance mechanisms using whole exome sequencing (WES) and RNA-Seq, but did not find EGFR C797S mutation—the most common resistance mechanism to AZD9291—in PC9-OSIR, suggesting a likely novel resistance mechanism, possibly the activation of a bypass signaling pathway, which will be elucidated by further studies.The PC9-OSIR cell line can be a useful cell line model, in addition to cell lines with C797S mutation, in the development of future EGFR inhibitors. It can also be used to explore new mechanisms of resistance to AZD9291 resistance.

#1921

Establishment of organoids derived from biliary tract carcinomas and its application for drug screening.

Yoshimasa Saito, Toshihide Muramatsu, Nao Yoshikawa, Ryoei Uchida, Ryo Furukawa, Aya Kitahara, Tomoko Yamaguchi, Masaki Kimura, Hidetsugu Saito. _Keio University Faculty of Pharmacy, Tokyo, Japan_.

Background

Biliary tract carcinomas (BTCs) are epithelial malignancies arising in the region between the intrahepatic bile ducts and the ampulla of Vater. Patients with inoperable BTCs generally receive chemotherapy regimens including gemcitabine. However, the effect of these drugs is limited, and the 5-year survival rates of patients are very low. Currently, however, there are no defined models that can recapitulate the features of BTCs. The newly developed 3D culture system known as "organoid culture" allows long-term expansion of stem cells into cyst-like structures (organoids) with properties resembling those of the original tissues. To develop in vitro preclinical models of BTCs, we established patient-derived BTC organoids and applied them for drug screening.

Methods

We established organoids using surgically resected cancer tissues obtained from patients with BTC. Driver gene mutations and gene expression profiles were analyzed in these BTC organoids. To investigate the possibility of drug repositioning, we screened a compound library consisting of 339 drugs employed clinically for their ability to suppress BTC organoids.

Results

We successfully established organoid lines derived from intrahepatic cholangiocarcinoma, gallbladder cancer and neuroendocrine carcinoma of the ampulla of Vater. These patient-derived BTC organoids were cultured stably for over one year and closely recapitulated the histopathological features of the primary tumors. Gene expression profiling of BTC organoids revealed enrichment of cancer-associated genes such as HOXB7, SOX2 and miR-17-92 cluster, and suppression of MMP1 and CD24. From a group of 339 medicines already in clinical use, we successfully screened 22 compounds that were able to significantly suppress BTC organoids. As expected, these compounds were mainly anticancer agents, including antimicrotubule agents, tyrosine kinase inhibitors, antimetabolite agents, mTOR inhibitors and proteasome inhibitors. Interestingly, four compounds that are not classified as anticancer agents showed significant suppression of the growth of BTC organoids.

Conclusions

These drugs that are not classified as anticancer agents but suppress BTC organoids could be potentially applied for the prevention and treatment of BTC patients with minimal side effects. Patient-derived organoids may be a powerful preclinical model for clarification of molecular pathogenesis and discovery of biomarkers and therapeutic drugs for refractory cancers.

#1922

MYB mimic peptides targeting human and murine MYB-NFIB positive tumor cells.

Yue Jiang,1 Lauren Forbes,2 Jianping Li,1 Shelby Freeberg,1 Chunxia Cao,1 Maria Zajac-Kaye,1 Jonathan Licht,1 Alex Kentsis,2 Frederic Kaye1. 1 _University of Florida, Gainesville, FL;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Adenoid Cystic Cancer (ACC) is the most aggressive salivary gland tumor with no effective systemic therapy. MYB/MYBL1 fusion event underlies the etiology of ACC with few recurrent variants focused on NOTCH and chromatin remodeling pathways and low overall mutational tumor burden. No chemotherapy or targeted drugs have been approved for ACC. Therefore, there is an urgent need to develop novel therapeutic strategies. To study MYB biology and test new therapeutic agents we have developed and characterized the phenotype of a new murine conditional MYB-NFIB transgene model and have also generated a mammary tumor cell line expressing the human MYB fusion transgene with ACC-like features. We have also generated new human MYB-NFIB positive ACC tumor cell lines and xenografts from patient samples and have tested these tumor cells with newly developed MYB mimic peptides. We analyzed the phenotype of mice carrying conditional MMTV-CRE MYB-NFIB transgene alone or after crossing to heterozygosity with p16/ARF null alleles. We observed shortened survival in two different mouse transgene strains due to development of B-cell leukemia manifested by elevated WBC, low platelets, and marked splenomegaly. We also noted sporadic solid tumors including the development of a murine mammary tumor which generated a MYB-NFIB positive tumor cell line for testing new therapies. In addition, we have generated patient derived xenografts (PDX) and the successful generation of a human ACC tumor cell line from fresh ACC patient biopsies. Using shRNA reagents we observed reduced tumor cell growth and viability assayed by MTS assay following Notch1 and MYB knockdown. We also tested four candidate MYB inhibitors, celastrol, all-trans retinoic acid, and the peptidomimetic inhibitors, MYBMIM and CRYBMIM, against murine and human ACC tumor samples with immortalized normal cells as controls and will discuss these results.

#1923

KRAS-mutant (mt) colorectal cancer (CRC) organoid models generated from patient-derived xenografts (PDX) show response to combination of trametinib (Tm), neratinib (N), and trastuzumab (Tz).

Rekha Pal,1 Ashok Srinivasan,1 Peter C. Lucas,1 Carmen J. Allegra,2 Angela M. Davies,3 Alshad S. Lalani,4 Samuel A. Jacobs,1 Katherine L. Pogue-Geile1. 1 _NSABP Foundation, Inc., Pittsburgh, PA;_ 2 _NSABP Foundation, Inc., and The University of Florida Health, Pittsburgh, PA;_ 3 _Champions Oncology, MD;_ 4 _Puma Biotechnology, Inc., CA_.

Background: KRAS-mt CRC has constitutively activated RAF-MEK-ERK pathways and resistance to anti-EGFR therapies. In pre-clinical models, we found that cells with inflammatory subtype tumors were sensitive to the MEK-inhibitor, MEK162, plus N, regardless of KRAS mutations. However, both KRAS-mt and -wt stem-like CRC cells were resistant to this combination. In C-07 and C-08 clinical studies, patients (pts) with stem-like subtype tumors were resistant to chemotherapy and had a very poor prognosis. We hypothesized that dual-HER2 targeting may provide more robust ERBB inhibition than N alone. We therefore tested Tz combined with Tm+N using PDX organoids (PDXOs) derived from KRAS-mt CRC tumors. PDOs have emerged as powerful preclinical models to predict clinical outcomes. The goal of this study was to identify more efficacious drug combinations for KRAS-mt tumors and stem-like subtype tumors using PDXOs.

Methods: PDXO's were generated using recently described methods from Hans Clevers' laboratory. Three KRAS-mt PDX tissues from Champions Oncology were used to generate four PDXOs for drug testing. The CTG-0406 PDX model was used to develop PDXOs from two different mice.

Results: All three KRAS-mt PDXOs: CTG-0406 (inflammatory), CTG-1170 (inflammatory), and CTG-0079 (stem-like), were resistant to Tm, N, and Tz, as single agents. N+Tz was effective in inhibiting cell viability in all four models; however, more significant viability inhibition occurred when Tm was added to N+Tz in three of the PDXOs including the stem-like model. In all PDXOs, Tm+N+Tz inhibited 67-76% viability.

Conclusion: We demonstrate that KRAS-mt PDXOs were inhibited to a greater extent with Tm+N+Tz compared to any of these drugs alone. PDXOs provide a rapid and cost-effective preclinical platform for screening of unique drug combinations for sensitivity or resistance.

Table 1. % PDXO cell viability | |  | |

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

Drugs | CTG-0406 (1) (KRAS mt) Inflammatory | CTG-0406 (2) (KRAS mt) Inflammatory | CTG-1170 (KRAS mt) Inflammatory | CTG-0079 (KRAS mt) Stem-Like

Tm (10nM) | 96% | 116% | 95% | 94%

N (125nM) | 109% | 97% | 96% | 92%

Tz (20 µg/ml) | 92% | 119% | 97% | 93%

Tm (10nM) + N (125nM) | 64% | 59% | 31% | 56%

Tm (10nM) + Tz (20 µg/ml) | 74% | 75% | 41% | 70%

N (125nM) + Tz (20 µg/ml) | 34% | 70% | 23% | 43%

Tm (10nM) + N (125nM)+ Tz (20 µg/ml) | 26% | 33% | 24% | 31%

Support: NSABP Foundation, Inc.

#1924

Rapid screening of anti-OX40 antibodies for cancer immunotherapy.

Tian Gan,1 Yanan Li,2 Jian Ni,3 Benny(Yi) Yang4. 1 _Biocytoge, Beijing, China;_ 2 _Biocytogen, Wakefield, MA;_ 3 _Eucure, Beijing, China;_ 4 _Biocytogen, Beijing, China_.

OX40, also known as CD134 or TNFRSF4, is a member of the TNFR superfamily. OX40 is a co-stimulatory receptor, and it interacts with its ligand to provide positive signal for T cell activation. OX40 is not expressed on naïve T cells, however its expression goes up after 24 to 72 hours following activation. Disruption of the OX40 pathway led to defective T cell responses, and overexpression of OX40 caused massive immune activation. Due to its importance in keeping immune homeostasis, strategies modulating the OX40 pathway hold great promise in efforts against cancer and autoimmune diseases.

Based on the promising nature of this target, we focused on developing novel OX40 agonists that would effectively stimulate anti-tumor activity. Dozens of high-affinity candidate antibodies were generated using classic hybridoma technology. In order to directly screen for antibodies for anti-tumor effects in animals, we ranked these antibodies in vivo using the drug screening platform based on humanized mouse models. Specifically, we used B-hOX40 mice and implanted syngeneic tumors subcutaneously, followed by treating mice with purified candidate antibodies. We quickly identified several lead antibodies that effectively inhibited tumor growth using this approach, without prior knowledge of their in vitro activities. Their ability to stimulate T cells was confirmed by an engineered reporter line of Jurkat T cells in vitro. Furthermore, we showed that one clone works in concert with Keytruda in a double humanized model of h-OX40 and h-PD-1. We are currently investigating the mechanisms of action underlying these potentially valuable antibodies and carrying out more combination evaluation studies using other available humanized immune-checkpoint mouse models.

#1925

**Establishment and characterization of 3D cancer organoids as clinically relevant** ex vivo **drug screening tools for cancer translational research and drug discovery.**

Xiaoxi Xu,1 Satya Pathi,2 Limei Shang,1 Yan Liu,1 Peng Han,1 Likun Zhang,1 Binchen Mao,3 Davy Ouyang,3 Henry Li,2 Wenqing Yang3. 1 _Crown Bioscience Inc, Beijing, China;_ 2 _Crown Bioscience Inc, San Diego, CA;_ 3 _Crown Bioscience Inc, Taicang, Suzhu, China_.

Patient derived cancer organoids (PDO), a form of comprehensive 3D spheroid cultures that harbor cancer multicellular components and mimic cancer lesion structures and tumor heterogeneity, have been proven to be innovative preclinical model system for use in both academic research and industrial cancer drug development. It has been demonstrated by a large amount of independent literature reports that 3D PDOs can better predict drug response than 2D cell line settings due to their clinical relevant tumor micro-environment. We have the world's largest patient derived xenograft (PDX) tumor model collection (~2500 models), which were annotated and validated genomically and phenotypically. However, there appears to be limitations when using PDX models to evaluate compounds in large scale fashion with narrow time windows and limited budget allocation. To address these limitations and to fully leverage preclinical values of these PDX model assets, we have established and characterized a series of PDX-derived cancer organoid (PDXO) models so that they could be used as scalable and high throughput compatible drug screening platforms.

As first phase of development, we started with low passage PDX primary tumors from 5 major cancers types, including colorectal, lung, breast cancer, gastric and pancreatic carcinoma. The PDX tumor tissues were mechanically and enzymatically dissociated and the digested tumor fragments were cultured and passaged in Matrigel containing culture medium supplemented with stem cell niche factors. Technical protocols for 3D cultures including culture propagation procedures and medium optimization have been established. Characterization and QC

of these PDXO models were done by gross morphology, histopathology, and genomic profiling. Histopathological analysis showed cellular/structural similarities (ductal, mucous or carcinoid) between PDXO models and original PDX tumor tissues, suggesting that tissue specific structural features were maintained in the 3D organoids. Moreover, ~10 PDXO models were tested for ex vivo cytotoxicity with SOC drugs. The preliminary data indicated that majority of (73%, 24/33) the PDXO ex vivo drug sensitivity IC50 datasets correlated with the in vivo PDX drug sensitivity TGI data. The negative and positive predictive values for PDXO are 94% and 53%, respectively. Genomic characterization including RNAseq and WES, is in progress. In summary, we successfully established PDXO models that were stably passaged from PDX primary tumors and resembled original PDX tumors both morphologically and histopathologically. In addition, the PDXOs apparently had encouraging predictive values for SOC drug response. Further in-depth model validation/characterization is warranted.

#1926

Ba/F3 NTRK cell panel, a powerful cell model for NTRK inhibitor discovery and development.

Feng Hao, Zhaoshuai Bai, Guoqian Wang, Feng He, Changpeng Liu, Jinying Ning. _KYinno Biotechnology Co., Ltd, Beijing, China_.

NTRK (Neurotrophic tyrosine kinase receptor)family, also named as TRK (Tropomyosin receptor kinase),is composedof three transmembrane protein receptors, TrkA, TrkB and TrkC,which play important rolesin neuronal differentiation and survival. Over-activation of NTRK1, NTRK2 and NTRK3 due to chromosomal rearrangement and mutations, was found in congenital fibrosarcoma, secretory breast carcinoma cancer and other types ofcancer. The identification of NTRK fusions as diver cancer genes has led to rapid development of anticancer therapeutic agents, including LOXO101, LOXO195 and TPX0005.Ba/F3 line is a popular system for studying both kinases and their inhibitors, because some protein kinases can render the Ba/F3 cells to be depended on the activation of the kinases instead of IL-3 supplement, while their inhibitors can antagonize the kinase-dependent growth effects. Our group used Ba/F3 cells togenerate a celllinepanel with 14 different NTRK fusion genes or mutations for NTRK inhibitor screening and development. Inaddition to use the cell lines for in vitro assays, thetransformed Ba/F3 linescan grow well in immune-deficient miceas in vivo models. LOXO101—a NTRK inhibitor—was used to test tumor growth inhibition, the results showedthat threewild-type fusion cell lineswere sensitive to LOXO101, but not other tumor models containing NTRK gatekeeper mutations. All the tumor models were sensitive to TPX0005, another NTRK inhibitor. Ourdata indicatethatthe celllinepanel of Ba/F3is apowerful cell model for new NTRK inhibitor discovery and development.

#1927

High-throughput cell line panel drug screening in organoids and 3D systems.

Tim M. Scales, Anett Rada-Kovacs, Helen N. Pemberton, Ceri M. Wiggins, Nicola J. McCarthy, Annette S. Little, David A. Sorrell. _Horizon Discovery Ltd, Cambridge, United Kingdom_.

Cell panel screening of cancer lines grown in standard 2D conditions is a well-established component of the drug discovery toolbox. A sufficiently large cell line panel, reflecting the molecular heterogeneity within a cancer type/sub-type, is required to detect correlations with robust statistical power that aid biomarker identification and patient stratification. However, the heterogenous exposure of tumor cells to nutrients, oxygen and other physical or chemical stresses found in three-dimensional solid tumors also has a role to play in patient response and this is not well mimicked in 2D monolayer cell cultures.

To meet the need for high-throughput cell-panel screening in more complex culture systems, we have developed 3D cell-based screens to more closely model the complex physiological environment found in tumors. We have characterised our OncoSignature panel of 300 cell lines for growth as spheroids to produce a 200 strong panel of spheroid forming lines. Using our high-throughput platform to compare drug activity in 2D and 3D cultures, we have identified clear differentials for some compounds in single agent and drug combination screens. For example, MEK and EGFR inhibitors have significantly higher activity in 3D cultures than 2D cultures, with a greater than 10-fold shift in EC50 values. These results were validated in 3D soft agar assays, with similar compound potencies being observed in the HTS spheroid assay and the lower throughput soft-agar assay.

We have further extended our 3D screening into organoid cultures. Organoid cultures, with increased complexity in terms of structure and cell heterogeneity compared to spheroids, have historically been challenging to use in high-throughput screening. However, our initial results show encouragingly robust data, with compound activity that is congruent with the genetic characteristics of the organoids.

Finally, we used our integrated screening platform to evaluate the power of CRISPR approaches to examine genetic dependencies in KRAS mutant cells in 2D, 3D spheroid and organoid cell cultures. Pooled, next-generation sequencing-linked CRISPRko allows an alternative and complementary genomics approach to arrayed cell panel screening, providing a powerful and robust target identification and validation strategy.

Overall, Horizon's 3D screening platforms will enable large scale interrogation of both drug and gene interactions in more complex cell culture systems.

#1928

Radiation response in preclinical orthotopic models of brain cancer.

Erin E. Trachet, Sumithra Urs, Alden Wong, Scott Wise, Maryland Rosenfeld Franklin. _MI BioResearch, Ann Arbor, MI_.

Glioblastoma (GBM) is a fast-growing, aggressive type of central nervous system tumor that forms on the supportive tissue of the brain. Primary treatment options for patients with GBMs have not changed much in many years, and still include surgery, radiation and/or chemotherapy treatment with temozolomide (TMZ). GBM is an incurable disease where there is a desperate need for continued development of novel therapeutic options. A number of preclinical models exist to support the early work needed to develop these new drugs. Here we describe three different preclinical models of brain cancer, U87MG-Luc, GL261-Luc and BT142. Each model was evaluated following orthotopic implantation into the brain. As radiation therapy remains a steadfast approach to GBM, we evaluated each model for its response to radiation and tracked disease progression and response to treatment with either bioluminescence imaging (BLI) or magnetic resonance imaging (MRI) along with traditional survival (morbidity/mortality) analysis.

U87MG-Luc is a human glioblastoma cell line that was developed from an astrocytoma. We found that orthotopic implant of U87MG-luc results in reliable growth with a tumor doubling time of 4 days and a median survival time of 45 days. We tested this model with radiation therapy (2.5Gy for 5 days on, 2 days off, for 2 cycles) and found a 70% partial response rate as measured by BLI. We also tested this model with TMZ therapy (33.3mg/kg; orally every day for 5 days) and found a significant delay in disease progression, resulting in a 93% increase in life span.

GL261-Luc is a murine glioblastoma model that grows quite aggressively in the mouse brain. We find a tumor volume doubling time of 2 days and a median survival time of 14 days. As this model grows in a mouse strain with an intact immune system it allowed us to evaluate the tumor infiltrating immune cell populations. While we found a low number of CD45+ cells infiltrating into GL261-Luc tumors, immune population changes over time will be described. In this model we tested increasing doses of radiation and found that a single dose of 15Gy was curative whereas the tumors were more moderately responsive to a single dose of 7.5Gy (30% tumor regressions). We then combined 7.5Gy with the immune checkpoint inhibitor anti-PD-1 and saw an improved outcome with a 100% response rate.

BT142 is a human anaplastic oligoastroytoma that is relatively unique due to its isocitrate dehydrogenase (IDH1/2) mutational status which results in an accumulation of the oncometabolite 2-hydroxygluarate (2-HG) in the tumor. This tumor line grows in the NOD SCID mouse strain and we found a tumor doubling time of 5 days, as measured by MRI. The BT142 model has proven to be sensitive to radiation following a single dose of 10Gy but less responsive when lower dosage levels were administered on a fractionated schedule. Thus, a variety of human and mouse GBM models exist to enable further drug discovery and development efforts for this intractable disease.

#1929

Establishment, molecular and pharmacological profiling of novel prostate cancer-derived xenografts from patients with advanced, treatment-refractory, disease to identify resistance mechanisms and new therapeutic options.

Olivier Deas,1 Ludovic Bigot,2 Emilie Dassé,1 Fabrice André,2 Jean Charles Soria,2 Benjamin Besse,2 Stefano Cairo,1 Marie Tavernier,1 Katell Mevel,1 Enora Le Ven,1 Luc Friboulet,2 Jean Gabriel Judde,1 Yohan Loriot2. 1 _XenTech, Evry, France;_ 2 _Institut Gustave Roussy, Villejuif, France_.

Prostate Cancer (PCa) is the most frequently diagnosed cancer in men and remains one of the leading causes of cancer death worldwide. It is a highly heterogenous and complex disease, presenting serious challenges to preclinical drug development and biomedical research. The normal development, growth and function of the prostate and most of its malignancies are under androgen regulation. However, after initiation of androgen deprivation therapy (ADT), most patients progress to intractable castration-resistant PCa (CRPC) within 2 to 3 years. Therefore, understanding the mechanisms of resistance that cause PCa to progress to a castration-resistance stage is the key to developing future beneficial therapies. Patient Derived Xenografts (PDX) mouse models show greater fidelity to the disease than any other in vivo models in terms of histopathologic, genomic and metastatic features, facilitating the translation of preclinical results into the clinical setting. However, PCa PDXs have been challenging to develop and a major limitation in PCa research is the lack of relevant preclinical models. The development of PCa PDX models is a mandatory requirement to explore new treatments targeting castration-resistant tumor cells and to improve anti-androgen therapies. To overcome the limitations of the number of relevant available models, in the framework of the MATCH-R clinical trial, which enrolls patients with oncogene-driven cancer who had previously responded to targeted therapy and subsequently experienced disease progression, XenTech and Gustave Roussy Institute are collaborating to develop a panel of extensively characterized PCa PDX models. Using biopsy material from metastatic lesions implanted into the sub-renal capsule of NSG mice, we report the successful establishment of 8 advanced PCa PDX models. All PDX models are characterized at the molecular level by whole exome sequencing and RNA-sequencing. Their response to physical castration and to the androgen receptor inhibitor enzalutamide is compared to the response observed in patients. This collection of PCa PDX models will be a useful preclinical tool to identify pivotal mechanisms underlying acquired resistance to current targeted therapies and develop novel treatment strategies.

#1930

High specific characterization of patient-derived tumor xenograft models for accelerating drug development in muscle-invasive bladder cancers.

Hervé Lang,1 Claire Beraud,2 Myriam Lassalle,3 Véronique Lindner,1 Michel Soulié,4 Xavier Gamé,4 Pascal Rischmann,4 Yves Allory,5 François Radvanyi,5 Isabelle Bernard-Pierrot,5 Philippe Lluel,6 Thierry Massfelder7. 1 _Strasbourg Universitary Hospital, Strasbourg, France;_ 2 _Urolead, Toulouse, France;_ 3 _Urosphere, Toulouse, France;_ 4 _Hôpital Rangueil, Toulouse, France;_ 5 _Institut Curie, Paris, France;_ 6 _Urosphere/Urolead, Toulouse, France;_ 7 _Inserm UMR 1260, Strasbourg, France_.

Muscle-invasive bladder cancers (MIBCs) constitute a heterogeneous group of tumors with a poor outcome. Recently, MIBC molecular subtyping efforts from an international consortium led to the identification of six subtypes to improve prediction of clinical outcomes and treatment responses. These subtypes can be schematically divided into luminal (differentiated) and non-luminal subtypes. FGFR3 alterations (mutations and translocations) are among the most frequent genetic events in bladder carcinoma and are found mainly in one subtype, the luminal papillary that respond poorly to chemo- and immuno-therapy. Here we describe the development and characterization of patient-derived primary MIBC xenografts (PDX) belonging to these different subtypes. Bladder primary tumors and normal corresponding tissues were directly obtained from patients at surgery. Tumor fragments were subcutaneously xenografted into immune-compromised mice. After the first growth in mice, they were serially passaged. PDXs tumors at multiple passages and patients' primary tumors from which they are derived were processed for analyses including growth characteristics, histopathology (H&E, CK5/6, FOXA1 and GATA3), gene expression (Affymetrix U133 plus 2.0 microarray), genetic stability (STR profiling). Specifically, hotspot oncogenic mutations including FGFR3, PIK3CA, HRAS, KRAS, NRAS, and PPARG were also explored. Additionally, pharmacological responses to standards of care and targeted therapies were characterized. Since 10 years, we have collected 152 MIBC tumors at all stages and grades. Up to now, 32 PDX models have been successfully established (> P3 in mice), i.e. 21.1 % success rate. This take rate seems not to be correlated to any classical tumor characteristics. Importantly, transcriptomic analysis allowed us to identify PDX models belonging to the different molecular subtypes including the basal-like and the luminal papillary subtypes (which include several PDX with FGFR3 mutations). All histological, genetic and molecular features validated the stability of the PDX models compared to the parental tumors. Histological analyses were correlated with the molecular classification. These models reproduced the response to cisplatin-based therapies observed in the clinic and FGFR3-mutated PDX models were shown to be highly responder to FGFR3 inhibitors. We have developed highly relevant preclinical models for MIBCs corresponding to the main subtypes which have been described. They represent essential tools for developing adapted and efficient therapies against this deadly disease.

#1931

**Investigating the PK/PD/efficacy relationship of PI3K inhibitors** in vitro **, enabled by a microfluidic addition and removal device.**

Tomasz M. Kostrzewski, Dharaminder Singh, Paul Golby, Alysha Bray, David Hughes. _CN Bio Innovations, Welwyn Garden City, United Kingdom_.

Characterizing the relationship between pharmacokinetics (PK), pharmacodynamics (PD) and efficacy is critical in the discovery and development of new drugs, schedules and combinations. The PK/PD/efficacy relationship has historically been characterized in xenograft models, owing to an absence of viable alternatives. The study of this relationship in vitro, has to date been problematic as the generation of time varying concentrations in multi-well plates has not been possible.

We have explored an in vitro methodology utilizing a device (Microformulator, Viibre, Vanderbilt University) capable of precision addition and removal of medium from the wells of a microtiter cell culture plate. Through stepwise addition and removal of medium the device was able to recapitulate PK-like, time varying concentration profiles of one, or more drugs in individual wells. We used this approach to explore the effects of PI3K pathway inhibitors, on p-AKT levels, and viability in a number of cancer lines. The PI3K/mTOR pathway is a central oncogenic pathway deregulated in cancer and p-AKT is a marker of PI3K pathway activity.

BYL719 and PI-103, are PI3K inhibitors with varying PK profiles and varying target coverage against PI3K isoforms. The compounds were tested on a pair of cell lines, T47D (breast, sensitive) and Colo205 (gut, insensitive). Both compounds were potent in a bolus dose 72hr assay with BYL719 having an IC50 of 0.63 µM for T47D and 15.8 µM for Colo205. Whilst PI-103 gave IC50 of 0.72 µM for T47D and 4.16 µM for Colo205. Both compounds also reduced, p-AKT in a dose-dependent manner after 2 hours. Using the microfluidic device, we generated PK-like dosing profiles for the two compounds, (BYL719 - t1/2 6 hours) (PI-103 - t1/2 3 hours) which mimicked their in vivo clearance profiles over a 24-hour period. p-AKT levels were initially reduced by both compounds but recovered at 24 hours as the concentration of the compound in the well declined. This replicates findings in xenograph models. The shorter t1/2 of PI-103, resulted in a more rapid recovery of p-AKT than observed for BYL719. Efficacy tests were conducted in which a 24 hour PK-like profile was applied for 3 consecutive days, mimicking daily dosing. For both compounds in T47D cells, the inhibition of cell growth was reduced as compared to a bolus addition at the Cmax concentration. These data demonstrate how PI-103 initially appeared to be an efficacious molecule but its rapid in vivo metabolism meant it could not be progressed in clinical studies.

In conclusion, this study demonstrates the microfluidic addition and removal device can be used to recapitulate PK-like profiles in vitro and allow exploration of the PK/PD/efficacy relationship. The availability of an in vitro method will enable these important parameters to be determined at an earlier stage of the drug discovery process.

#1932

Cationic amphiphilic drugs as a potential new treatment option for patients with high risk non-muscle invasive bladder cancer.

Geertje van der Horst,1 Arjanneke F. van de Merbel,1 Eline Ruigrok,1 Maaike H. van der Mark,1 Emily Ploeg,1 Marja Jäätelä,2 Janneke van Uhm,1 Rob C. Pelger,1 Chris H. Bangma,3 Joost Boormans,3 Gabri van der Pluijm,1 Ellen C. Zwarthoff3. 1 _Leiden University Medical Center, Leiden, Netherlands;_ 2 _Danish Cancer Society Research Center, Copenhagen, Denmark;_ 3 _Erasmus MC Cancer Institute, Rotterdam, Netherlands_.

Urothelial carcinoma of the bladder (UCB) is the fifth most common cancer in the western world. UCB present either as non-muscle-invasive BC (NMIBC) or as muscle-invasive carcinomas (MIBC). NMIBC is characterized by a high risk of recurrence and even progression to MIBC based on clinicopathological risk factors. Despite radical surgery and extensive chemotherapy, the outcome of MIBC patients is poor. High-risk NMIBC is treated with adjuvant intravesical instillations with Bacillus Calmette-Guérin (BCG). However, a substantial number of patients do not tolerate BCG or do not respond. Novel therapy for patients who do not tolerate BCG or in who are BCG unresponsive is a clear unmet clinical need. Drug repositioning of existing FDA-approved drugs could be a promising opportunity. Recently, cationic amphiphilic drugs (CADs), including commonly used antidepressants, antihistamines and antipsychotics were found to preferentially induce lysosomal cell death in transformed cells. In this study, we evaluated the potential anti-tumor efficacy and toxicity of these drugs in in vitro assays, in vivo preclinical models, and in and ex-vivo cultured patient-derived bladder cancer tissue slices. Treatment with CADs resulted in a significant dose-dependent decrease in proliferation and viability of multiple human UCB cell lines (a.o. RT4, TCC-SUP, and UM-UC-3) in vitro. Consistent with previous data regarding the ability of CADs to induce lysosomal membrane permeabilization in other solid cancer cells, we observed lysosomal puncta formation, which is a hallmark of lysosomal leakage. Strikingly, instillation of CADs in preclinical in vivo models of orthotopically growing human UM-UC-3 UCB resulted in a significant reduction in orthotopic as well as metastatic tumor burden. In line with these data, treatment of 39 patient-derived ex vivo cultured UCB slices with penfluridol resulted in significant anti-tumor responses ranging from complete regression to a significant reduction of the tumor.In vivo experiments revealed that normal mouse urothelium is not affected upon bladder instillation of CADs (weekly instillation of penfluridol for 5 weeks) in non-tumor bearing nude mice. In addition, penfluridol treatment of ex vivo cultured normal human urothelium only marginally affected the tissue. In conclusion, we demonstrated that CADs can target UCB cells and might be a promising novel approach for treatment of UCB.

### Novel Imaging Techniques

#1933

**Tissue print V** mem **imaging: Visualizing bioelectric signatures in cancer.**

Dany S. Adams,1 Brian H. Tracey,1 Larry Takiff,2 James Kearns,3 Stephen P. Naber,3 Sandra M. Gaston4. 1 _Tufts University, Medford, MA;_ 2 _Akita Innovations, Billerica, MA;_ 3 _Tufts Medical Center, Boston, MA;_ 4 _New England Baptist Hospital, Tufts Med Ctr and Tufts Univ School of Medicine, Boston, MA_.

Work in models of embryonic development shows that membrane voltage, or resting potential (Vmem), regulates cell behaviors that are disrupted in cancer, including proliferation, migration, apoptosis, homeostasis, and cell-cell signaling. Normal cells and tissues maintain stereotypical patterns of Vmem and these patterns are recognizable in Vmem Imaging as bioelectric signatures. In embryonic tissues, cells that are relatively undifferentiated are usually less negative (depolarized) than fully differentiated cells, and different cell lineages have characteristic bioelectric signatures. Recently, the development of innovative imaging technologies using Voltage Sensitive Dyes (VSDs) allows the visualization of Vmem in living cells and tissues. In culture, Vmem Imaging using VSDs can readily differentiate between sister breast cancer cell lines of low and high metastatic potential (EpH4 and EpH4.2, respectively). Although the cultures are identical in bright field images, Vmem Imaging shows a dramatically more heterogeneous bioelectric signature for EpH4.2 than for EpH4 cells, consistent with reduced gap junctional communication. One of the challenges of Vmem Imaging has been the limited thickness of tissue that can be analyzed using the VSDs (about 50 microns). To overcome this limitation, we have combined innovative Tissue Print technologies with Vmem Imaging in order to visualize patterns of electric potential in living cancer cells obtained from human surgical specimens. Tissue Prints are touch preps that transfer a thin layer of viable cells to a nitrocellulose membrane as an oriented imprint; biomarkers identified on these tissues prints can be mapped back to the source tissue to establish pathology and gene expression correlations. Tissue Print Vmem Imaging of radical prostatectomy specimens shows differentiation of bioelectric signatures between cancer and adjacent benign tissue and between cancer of different grades (Gleason pattern 3 and pattern 4). Such differences in bioelectric signatures represent unexplored cancer phenotypes that are made accessible by Tissue Print Vmem Imaging technologies.

#1934

Development of spectrally distinct silica coated gold nanorods for detection of cancer using MSOT.

Karl N. Thomas,1 Abhilash Samykutty,2 Molly McNally,2 Lacey R. McNally3. 1 _Williams College, Williamstown, MA;_ 2 _Wake Forest Univ. Comp. Cancer Ctr., Winston-Salem, NC;_ 3 _Wake Forest Univ. Comp. Cancer Ctr., Winston Salem, NC_.

Traditional cancer imaging devices are limited in their ability to screen for multiple contrast agents simultaneously in real time. Multispectral Optoacoustic Tomography (MSOT) is an emerging imaging modality capable of real-time imaging of numerous contrast agents with enhanced spatial resolution of 75µm at depths of 5 cm. The use of exogenous contrast agents in MSOT remains largely unexplored, so we developed two species of spectrally distinct gold nanorod as contrast agents for use in MSOT. Our goal was to evaluate the potential of MSOT to spectrally differentiate two exogenous contrast agents simultaneously. Two gold nanorod species were created using hydrogen peroxide (GNR-H2O2) or ascorbic (GNR-ASC) acid as reducing agents to modify the length of each species to create nanorods with individual light absorbance spectra in the IR range (680-900 nm). These gold nanorods were highly stabilized via encapsulation with mesoporous silica along with a subsequent chitosan capping. Human epidermal growth factor receptor 2 positive (HER2+) cells were specifically targeted by conjugating these mesoporous silica-coated chitosan capped gold nanorods (CMGs) to Trastuzumab resulting in TRA-CMG particles. Both TRA-CMG-ASC and TRA-CMG-H2O2 resulted in optoacoustic spectrally distinct signals when imaged in tissue phantoms both individually as well as mixed within the same well after multispectral processing using linear regression. Treatment of HER2+ breast cancer cell lines, DY36T2Q and SKBR3, with TRA-CMG-H2O2 resulted in 2.5x and 3.1x enhanced signal, respectively, as compared to HER2- MDA-MD468 cells. Treatment of DY36T2Q and SKBR3 cells with TRA-CMG-ASC demonstrated 3.7x and 6.9x, respectively, compared to MDA-MD468. In all three cell lines treated with a combination of TRA-CMG-H2O2's and TRA-CMG-ASC's clear and distinct signals were observed for each particle, demonstrating that each TRA-CMG possessed and maintained a detectibly distinct optoacoustic spectrum, in the IR range, allowing them to be detectable as separate contrast agents in MSOT while proximate to other targeted contrast agents. Both particles have demonstrated that they can be simultaneously administered and targeted at HER2+ cell while also maintaining distinct photoacoustic signals in MSOT upon consolidation. Each particle species, targeted to the same cells, were capable of being monitored individually in the presence of the other gold nanorod contrast agent.

#1935

Tracking angiogenesis induced microvascular changes in a lymphoma model via a new high throughput non-invasive dual modality imaging platform.

Juan D. Rojas,1 Rajalekha Rajamahendiran,1 Tomasz J. Czernuszewicz,1 Brian Velasco,2 Jonathan Perdomo,1 Max Harlacher,1 Graeme O'Connell,1 James Butler,1 Blossom Damania,3 Paul A. Dayton,2 Ryan C. Gessner1. 1 _SonoVol, Inc., Durham, NC;_ 2 _The University of North Carolina and North Carolina State University, Chapel Hill, NC;_ 3 _The University of North Carolina School of Medicine, Chapel Hill, NC_.

This study presents a novel dual-modality imaging system for assessing cancer progression in rodents. The system incorporates bioluminescence imaging (BLI), used to assess tumor growth, and contrast-enhanced ultrasound (CEUS), used to assess anatomical information and map microvasculature. The combination of the two modalities has previously been shown to reduce inter-user variability of BLI quantification, and in this work, we demonstrate that a dual BLI/US system can provide a more holistic assessment of disease.

NSG (NOD/scid/gamma) female mice were implanted with luc-tagged lymphoma cells (BCBL-1, RRID: CVCL_0165, 1x105 cells, intraperitoneal (IP) injection, N = 8 mice) and imaged using the US and BLI hybrid modality system (SonoVol, Inc.), and a BLI-alone system (Perkin Elmer, Inc.) for comparison to a widely available commercial BLI system. BLI sensitivity was evaluated using a weakly luminescent tritium phantom to find the shortest exposure required to detect signal. In vivo studies consisted of an IP injection of D-luciferin (250 µL at 15 mg/mL) and serial captures of images with exposure times of 60 s every 3 min. Acoustic Angiography (AA), a high-resolution CEUS technique, was used to acquire 3D volumes in the abdomen surrounding the tumor site to assess angiogenesis-induced vascular remodeling associated with tumor growth.

In vitro BLI sensitivity experiments showed that the dual-modality system required an exposure of 3 sec to detect signal (p < 0.05) and the BLI-alone system required an exposure of 1 sec (p< 0.05). For in vivo studies, the change in luminescence occurring between week 2 and 3 post-cell implantation was calculated (a surrogate measurement for tumor growth), and the difference in signal was 17.15 ± 10.1 photons/sec and 16.04 ± 7.6 for the dual-modality and BLI-alone systems, respectively. Images of the vascular remodeling arising during the first two weeks of tumor growth were captured with AA and demonstrated an increase in perfusion in the vicinity of BLI signal by a factor of 1.4 ± 0.38, with vascular remodeling being evident even at the periphery of BLI signal.

This work demonstrates that non-invasive measurements of in vivo microvascular remodeling can be precisely mapped to changes in tumor growth with a hybrid modality system. The system has comparable sensitivity to a BLI-alone system and provides similar assessments of longitudinal tumor growth. Adding quantitative metrics for vascular remodeling to the widely used luminescent imaging could provide a more comprehensive assessment for tumor functional status than either modality could individually. This should prove valuable when using antiangiogenic therapies because changes in vasculature will precede cell death, and the ability to monitor both the cells and their blood supply might help to elucidate underlying biological processes.

#1936

Label-free optical imaging and characterization of cancer-associated urinary extracellular vesicles: Implications for biomarker discovery.

Ronit Barkalifa, Sixian You, Haohua Tu, Alison Masyr, Rebecca Kamerer, Marina Marjanovic, Jaena Park, Kimberly Selting, Stephen Boppart. _University of Illinois at Urbana-Champaign, Urbana-Champaign, IL_.

The discovery of extracellular vesicles (EVs) in all bodily fluids is rapidly increasing the fundamental knowledge of disease mechanisms and the ways in which cells communicate systemically to facilitate tumor progression and metastasis. The type, content, and magnitude of tumor-associated EVs have been linked to tumor invasiveness both in-vitro and in-vivo. In this study we utilized a novel label-free multimodal multiphoton optical imaging method to detect and characterize isolated urinary EVs and classify their optical signatures. Blinded analysis conducted on urinary EVs isolated from 4 healthy dogs and 19 dogs diagnosed with multiple types of cancer showed different optical signatures; cancer-associated urinary EVs were found to have significantly higher NADPH concentrations in comparison to those isolated from healthy dogs. These results suggest a potential label-free optical methodology to detect and characterize EVs by their optical signatures, which can be utilized as possible diagnostic and prognostic biomarkers for cancer.

#1937

pH-responsive tumor-targeted mesoporous silica nanoparticle for the identification of pancreatic cancer using optoacoustic tomography.

William MacCuaig,1 Abhilash Samykutty,1 Molly McNally,1 Kylie Nairon,1 Surya Banks,1 William Grizzle,2 Lacey R. McNally3. 1 _Wake Forest Univ. Comp. Cancer Ctr., Winston-Salem, NC;_ 2 _University of Alabama Birmingham, Birmingham, AL;_ 3 _Wake Forest Univ. Comp. Cancer Ctr., Winston Salem, NC_.

Purpose: Due to inadequate early detection and inability to operate at advanced stages, pancreatic ductal adenocarcinoma (PDAC) has remained one of the most difficult types of cancer to treat. A small range of non-specific symptoms coupled with quick metastasis rate result in a poor 5-year survival rate; 14% for those diagnosed within stage IA, and as low as 1% for those diagnosed during stage IV. Nanoparticles have recently emerged as a potential delivery agent for diagnostic and therapeutic agents, and although clinical success has not been ample due to targeting accuracy issues. This work shows a nanoparticle that has been functionalized with a pancreatic cancer-specific targeting ligand and exhibits specific particle release in pancreatic malignant environment (pH 6.6) as compared to non-malignant environments (pH 7.4). Methods: Wormhole-pored mesoporous silica nanoparticles were formed at 80°C using Tetrapropyl orthosilicate (TPOS) and a scaffold of hexadecyltrimethyl-ammonium bromide (CTAB). Acetic acid and ethanol were used in dialysis procedures to remove the CTAB scaffold and create worm-like pores. Chitosan was added to coat the silica particles and serve as a gatekeeper. IR-780 was added before the solution was acidified to load the dye into the worm-hole particles created. Shortly after, the solution pH was raised back to physiological levels (pH=7.4) to trap the dye within the particle. The particle surfaces were functionalized to attach a targeting ligand pH-low insertion peptide (V7) to conjugate the dye-loaded nanoparticles. Pancreatic adenocarcinoma cells (S2VP10 line) were plated in pH-7.4,6.8, and 6.6 PBS solutions with the loaded particles to assess uptake via near-infrared fluorescence and multispectral optoacoustic imaging. Results: Zeta potential and dynamic light scattering were used to ensure the 63nm size nanoparticle and proper coating. Near-infrared fluorescence imaging showed ~10X increased signal at pH 6.6 as compared to pH=7.4. MSOT imaging ~5X increased signal in the malignant microenvironment environment that is acidic as compared to the non-malignant environment at pH 7.4. Conclusion: The functionalized wormhole mesoporous silica nanoparticles coated with chitosan demonstrated pH-sensitivity in terms of cellular uptake via NIR fluorescence and MSOT imaging.

#1938

Implantable nanosensor for non-invasive ovarian cancer biomarker detection with wearable interface.

Daniel A. Heller,1 Ryan M. Williams,1 Douglas A. Levine2. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _NYU Langone Health, New York, NY_.

Patients with high-grade serous ovarian carcinoma (HGSC) exhibit poor 5-year survival rates, which may be significantly improved by early-stage detection. The US FDA-approved biomarkers for HGSC—CA-125 and HE4—do not generally appear at detectable levels in the serum until advanced stages of the disease. An implantable device placed proximal to disease sites, such as in or near the fallopian tube, ovary, uterine cavity, or peritoneal cavity, may constitute a feasible strategy to improve detection of HGSC. Furthermore, a sensor implant under the skin may allow real-time measurements after diagnosis, to monitor treatment for example, via interrogation using a wearable device. We engineered a prototype optical sensor composed of an antibody-functionalized carbon nanotube complex, which responds quantitatively to HE4 via modulation of the nanotube optical bandgap. The complexes measured HE4 with nanomolar sensitivity to differentiate disease from benign patient biofluids. The sensors were implanted into four models of ovarian cancer, within a semipermeable membrane, enabling the optical detection of HE4 within the live animals. We thus present the first in vivo optical nanosensor capable of noninvasive cancer biomarker detection in orthotopic models of disease. This technology may potentially be implanted locally in the uterine cavity, for point-of-care measurement, or under the skin, to then interface with a wearable device.

#1939

Non-invasive prediction of glioma tumor stemness using multimodal MRI.

Esmaeil Davoodi-Bojd, Tathiane Malta, Brent Griffith, James Snyder, Tobias Walbert, Houtan Noushmehr, Hamid Soltanian-Zadeh. _Henry Ford Health System, Detroit, MI_.

Introduction: Stemness indices and sub-classifications of glioma have been proposed based on the epigenomic and transcriptomic makeup of these tumors, to inform prognosis and guide therapeutic management and drug discovery for this devastating disease. They reveal close association with tumor aggressiveness, which reflects clinical outcome. Stemness indices reveal tumor biology, which may affect sensitivity of individual tumors to therapy, and ultimately help to develop effective targeted therapies against this deadly disease. Due to the difficulty and cost of brain biopsy and molecular profiling, we proposed a noninvasive approach to estimate glioma stemness indices solely by numerical MRI features.

Methods: We used four MRI modalities (pre- and post-contrast T1-weighted, T2-weighted, and T2-FLAIR images) of 73 glioma patients from the cancer genome atlas (TCGA) respiratory. For each patient, the tumor was segmented into four sub-volumes: necrosis, edema, contrast enhancing tumor (CE), and non-enhancing tumor (NE) using BraTumIA software. From each of these four sub-volumes, 25 features (1 volume, 4 histogram, and 20 texture features) were extracted from the four MRI modalities, generating a total of 400 noninvasive imaging features. A linear regression model was used to model each stemness index using the imaging features. Irrelevant/uncorrelated imaging features were discarded through a hierarchical feature selection-regression algorithm, which was developed to find the best subset of features predicting a particular stemness index linearly. The mean squared error (MSE) and Akaike information criterion (AIC) were used to optimize the regression models and to find an optimal number of features, respectively.

Results: The resulting regression models were more accurate for the DNA-based stemness indices than the RNA-based stemness indices. This is not surprising, since it was reported that mDNAsi correlated with glioma outcome more than mRNAsi. Interestingly, ENHsi which defined stemness indices based on genomic enhancer stem signatures (targets of Sox and Oct transcription factor binding) had the strongest correlation with the MRI features. This suggests that MRI can detect glioma subtypes with strong stem like features, which were shown to coincide with more aggressive subtypes of brain cancer. Moreover, features extracted from post-contrast MRI contributed to the resulting models more than the other MRI modalities. This confirms the crucial role of contrast-enhanced MRI in the diagnosis and treatment of brain glioma tumors.

Conclusions: Stemness indices can be estimated using MRI features and used for glioma diagnosis, treatment planning, and prognosis instead of repeated brain biopsies and molecular profiling, which are invasive and costly.

#1940

Comparison of anti-hepatocellular carcinoma efficacy between regorafenib and sorafenib using multimodality molecular imaging: A preclinical study.

Yang Du, Jie Tian. _Chinese Academy of Sciences, Beijing, China_.

Purpose: Sorafenib has been used as a targeted therapy for HCC for more than a decade, and regorafenib has recently been approved for HCC treatment and shown to prolong survival for advanced HCC patients after treatment failure with sorafenib. However, there has been no systematic study to directly compare the therapeutic effects of regorafenib and sorafenib against HCC. In this study, we systematically compared the therapeutic effects of sorafenib and regorafenib against HCC in vitro and in vivo using multimodality molecular imaging.

Experimental Design: we systemically investigated and compared the anti-tumorigenic and anti-angiogenic efficacy of regorafenib and sorafenib in vitro and in vivo using multi-modality molecular imaging as bioluminescence imaging (BLI), bioluminescence tomography (BLT), fluorescence molecular imaging (FMI), and computer tomography angiography (CTA) etc. Moreover, the safety and side effects of two drugs were simultaneously systemically evaluated.

Results: The results showed that compared with sorafenib treatment, regorafenib exerted stronger antitumor and antiangiogenic effects and significantly increased the survival rate of HCC model mice. Although both drugs caused side effects, their severity was usually higher with sorafenib, except that regorafenib caused hypertension, which was not observed with sorafenib.

Conclusions: The study suggests that multimodality molecular imaging can help sensitively evaluate the drug treatment efficacy against HCC before tumor anatomical changes occur and also helps understand the underlying antitumor mechanism of a drug at the molecular and cellular levels. Our data may provide an experimental basis for clinical application of HCC targeted treatment with regorafenib and sorafenib.

#1941

Fluorescence lifetime imaging of treatment response in neuroendocrine tumor organoids.

Amani A. Gillette, Christopher P. Babiarz, Cheri A. Pasch, Dustin A. Deming, Melissa C. Skala. _University of Wisconsin - Madison, Madison, WI_.

Gastroenteropancreatic neuroendocrine tumors (GEP-NET) are now the second most common form of gastrointestinal cancer, and account for roughly 60% of all neuroendocrine tumors. Unfortunately, no animal models or cell lines exist that reflect the in vivo characteristics of human GEP-NET's for use as predictive models. Therefore, pre-clinical work is often not representative of the clinical outcome, so clinical trials begin in patients without rigorous pre-clinical validation of the treatment regimen. There is an established need to predict the most effective treatments prior to clinical trials for GEP-NET patients, to increase treatment efficacy and reduce toxicities. Here we show that a three-dimensional organoid model of primary patient tumors is representative of the original patient tumors. Furthermore, this study aims to use the organoid model to test a novel pre-clinical combination of ABT263, an experimental cancer drug that has been shown to enhance the effects of other chemotherapeutic agents, and the standard drug everolimus, on GEP-NET organoids. Previous studies have shown that ABT263, which targets Bcl-2, Bcl-XL and Bcl-w, induces apoptosis when used in combination with everolimus, making this a promising drug combination for GEP-NET patients. Treatment response to ABT263, everolimus, and the combination, was tested in three-dimensional organoids derived from six primary GEP-NET patients, using two-photon fluorescence lifetime imaging microscopy (FLIM). FLIM captures the fluorescence lifetime of metabolic cofactors NAD(P)H and FAD. Further, the fluorescence intensity of NAD(P)H divided by that of FAD (the redox ratio) was measured in all samples. Altogether, two-photon imaging of NAD(P)H and FAD fluorescence intensities and lifetimes (optical metabolic imaging, or OMI) in primary patient GEP-NET organoids provides a novel tool to measure metabolic changes with drug treatment. OMI measured metabolic differences induced by treatment in primary patient GEP-NETs when compared to control, at 72 hours post-treatment. All patients except one showed a decrease in redox ratio, which corresponds with a response, to the combination treatment. As metabolic imaging is performed to collect treatment response from more patients, this OMI data will be integrated into a multivariate analysis of variance to predict treatment response. Independent measures of organoid response, including changes in organoid diameter and immunohistochemistry, have been used to validate OMI predictions. We have shown that ABT263 plus everolimus is a promising combination treatment for GEP-NET's. Overall, this work establishes the feasibility of personalized drug screens for GEP-NET's using OMI in primary tumor organoids. Additionally, this platform can be used to identify new treatment regimens for improved GEP-NET patient outcomes, such as the ABT263 plus everolimus combination tested here.

#1942

Volume and vascular evolution assessed with ultrasound in tumor growth: First steps for a future tool of predictive responses of treatment.

Jerome Griffon, Zixin Yang, Delphine Le Guillou-Buffello, Alexandre Dizeux, Michele Lamuraglia, Lori Bridal. _Laboratoire d'Imagerie Biomedicale (UPMC, CNRS, INSERM), Paris, France_.

Background: Mathematical models have been developed to simulate tumor growth, as a goal to predict therapy response. We implemented three models and we investigated their agreement with tumor size assessed by ultrasound. We also investigated the link between microvascular flow parameters and future tumor size evolution.

Methods: Three ordinary differential equation tumor growth models were implemented in Matlab. The exponential-linear model (EL) assumes growth rate tends to linear after initial exponential growth as nutrient support becomes limited. The Gompertz model (G) describes demographic growth with a fixed carrying capacity because growth cannot exceed the capacity of the environment. The dynamic carrying capacity model (DCC) assumes this carrying capacity can change with time, for example, due to angiogenesis or necrosis formation. Experimental data were acquired in 11 control and 10 antiangiogenic (AA) treated mice with ectopic murine colorectal carcinoma (CT26) on Days 7, 9, 13, 15 and 21 after implantation (therapy started at day 7). Ellipsoidal volume (V) was estimated from B-mode measurements of the major transverse and longitudinal axes and used to estimate Relative Growth Rate (RGR): (t{n}) = [V(t_{n}-V(t_{n-1})]/[(t_{n}-t_{n-1}).V(t_{n-1})]. Contrast-Enhanced Ultrasound (CEUS) data were acquired along the longitudinal axis with Sequoia 512, 7-14 MHz probe. Regions with no contrast-enhancement were identified. Average contrast intensity vs. time curves in the perfused zone were fit to a lognormal model to estimate Area Under the Curve (AUC), Peak Enhancement (PE), Time to Peak (TTP), Mean Transit Time (MTT), Wash In Rate (WIR) and Wash Out Rate (WOR). The Akaike Information Criterion (AIC) was used to evaluate the relative quality of the models to describe the tumor size evolution. AIC penalizes models with more parameters to deal with the trade-off between goodness of fit and the model's simplicity.

Results: Initial implementation of the models using a fixed (1 mm3) and free initial tumor size were compared. In this data set where earliest measured tumor size (day 4) varied from 15 to 39 mm3, the additional free parameter resulted in improved mean AIC (EL: 37 vs. 40 ; G: 37 vs. 41 ; DCC: 43 vs. 45 - free and fixed initial volume respectively). AIC was lowest on average for the G model in control mice. The TTP, WIR and WOR at t_n were linked with future RGR between t_n and t_{n+1} (RS = -0.51, 0.44 and -0.46 respectively for C (TTP and WIR) or AA (WOR) group, p<0.01, non-parametric Spearman correlation coefficient).

Conclusions: The microvascular function includes additional information related to tumor growth. The integration of size and vascularization parameters into the mathematical models is the next step for predictive tumor growth. Such a model based on predictive quantitative ultrasound images has potential application in treatment follow-up.

#1943

Highly specific tumor-targeting near-infrared persistent luminescence nanomaterials (ZGO and LGGO) for lung cancer bioimaging.

Ming-Hsien Chan,1 Ru-Shi Liu,2 Michael Hsiao1. 1 _Acadamia Sinica, Taipei, Taiwan;_ 2 _National Taiwan University, Taipei, Taiwan_.

Lung cancer cells grow in-situ usually results in metastasis and is frequently recurrent after therapy. Moreover, treatment of lung cancer using surgery or chemotherapy is difficult to determine that all tumor tissue has been removed. ZnGa2O4/La3GaGe5O16 (ZGO and LGGO) persistent luminescence nanomaterials (PLNs) which are based on the biological characteristics of metastatic or remaining lung cancer cells, to promote the clinical application of these nanomaterials for long-term specific tracking. A silica shell-assisted synthetic route for mono-disperse, near infrared-PLNs, will be developed and applied in the, in vitro cell model and in vivo mouse model. The Cr3+-doped PLNs can hold the emitted light longer than several hours. These NIR-PLNs will absorb the energy of UV or X-ray light first and retain fluorescence emission. Moreover, we will identify the specific aptamer, MAGE-A3, and modify the PLN's design accordingly, to increase the ability of cancer targeting. These targeted molecules can track the metastatic cell with specific binding. The advancement of nanoscale techniques in lung cancer is an urgent need. Hence, this research will deliver a credible diagnostic strategy and a novel method for therapeutic administration. To evaluate the luminescent effect in vitro and in vivo of NIR-PLNs, repeated in vivo NIR persistent luminescence images of a mouse after intravenous injection (I.V.) and intratracheal injection (I.T.) of NIR-PLNs. After the NIR-PLNs is irradiated with UV light for 120 seconds in vitro, the particles are entering in the mouse by I.V. and I.T. It is seen that NIR persistent luminescence signal could be repeatedly observed over the mouse's lung tumor tissue for more than 6 hours. Interestingly, no significant NIR luminescence signal can be observed in the other parts of the mice. The major task is, providing a potential strategy for diagnostic in lung cancer. The current diagnostic approach does not easily distinguish the metastatic and remaining tumor cells. Moreover, we anticipate that our designed nanoplatform or accessed procedure

could be applied to other types of materials or cancers, leading NIR-PLNs nanomaterials to the exploration of gene-scale research.

#1944

Detection of metabolic change in glioblastoma after radiotherapy using hyperpolarized 13C-MRI: Glycolytic metabolism in cancer stem cell-like cell-derived tumor model.

Tatsuya Kawai, Murali C. Krishna, Tamalee Kramp, Jeffery R. Brender, Philip Tofilon, Kevin A. Camphausen. _National Cancer Institute/NIH, North Bethesda, MD_.

OBJECTIVES: Since the cancer stem-like cells (CSCs) play an important role in the resistance against radiation therapy due to its different metabolic profile, investigation of the specific metabolic characteristics of CSCs is valuable. Dynamic nuclear polarization (DNP) technique using 13C-labeled substrates enables magnetic resonance imaging (MRI) to monitor specific enzymatic reactions in tumors. In this study, DNP-MRI along with hyperpolarized [1-13C] pyruvate was conducted to evaluate the metabolic change in glycolytic profiles after irradiation between a glioblastoma cell line and a CSC-derived glioma using orthotopic xenograft mouse model. A microarray analysis was also performed to compare to the imaging study.

METHODS: DNP-MRI A 3T MRI scanner along with a custom-made head coil was used. To obtain hyperpolarized [1-13C] pyruvate, 30 μL of [1-13C] pyruvic acid containing 15 mM of OX063 (trityl radical compound) and 2.5 mM of the gadolinium chelate were polarized at 3.35 T and 1.4 K in the Hypersense DNP polarizer. The hyperpolarized sample dissolved in a buffered medium was injected via the tail vein followed by an acquisition of 13C-two-dimensional MR-spectroscopy to measure the pyruvate-to-lactate ratios (Lac/Pyr) in the tumor region and the contralateral normal brain separately.

Cell lines A human glioblastoma cell line, U251 and a glioma CSC line, NSC11 were cultured for stereotactic intracranial injection.

Animal model Orthotopic brain tumor model was developed in athymic nude mice by intracranial implantation. When the tumor volume reached 50 +/- 10 mm3 on T2-weighted MRI, a sequence of DNP-MRI scans was performed 24 hours before and 6, 16 and 24 after 6-Gy whole-brain irradiation (N = 4 each with a non-irradiated control group). Independently, a microarray analysis using the tumor tissue samples excised 6 and 24 hours after irradiation was performed on Affymetrix Human Genome U133 Plus 2.0 array.

RESULTS: DNP-MRI showed a decrease in Lac/Pyr 6 and 16 hours after irradiation compared to the control group in U251 whereas no significant change was observed in the NSC11 tumor. The microarray analysis showed a significant down-regulation of lactate dehydrogenase A (LDH-A) expression in U251 6 and 24 hours after irradiation, consistent with the finding of DNP-MRI. In the microarray analysis, SIRT2 and KLF4 expression also changed in the U251 tumor, suggested to regulate the LDH-A activity/expression.

CONCLUSION: DNP-MRI demonstrated the difference in radiation response in glycolytic metabolism between the glioblastoma cell line and the CSC-derived glioma. This technique might be a feasible diagnostic method evaluating metabolic profiles of the brain tumors in the clinical settings of pretreatment and post-treatment.

#1945

Conjugated biosynthesized magnetic nanoparticles for specific targeting and imaging of triple negative breast cancer.

John D. Obayemi,1 Jingjie Hu,2 Ali Salifu,1 Vanessa Uzonwanne,1 Winston Soboyejo1. 1 _Worcester Polytechnic Institute (WPI), Worcester, MA;_ 2 _Mayo Clinic, Mayo Clinic, AZ_.

Some of the major problems associated with current diagnoses of triple negative breast cancer (TNBC) are due to poor spatial resolution and false positive diagnosis leading to delayed prognosis and metastases. The use of magnetite nanoparticles (MNPs) as an MRI contrast agent have generated tremendously interest within the last decade due to their potential clinical application in cancer diagnosis and treatment. In this study, we present the use of biosynthesized MNPs that were conjugated with luteinizing hormone releasing hormone (LHRH), a molecular recognition unit whose receptors are overexpressed on triple negative breast cancer (TNBC). In vivo study of LHRH-conjugated MNPs for the specific targeting of TNBC and as MRI contrast enhancement agent were explored. These biocompatible LHRH-conjugated BMNPs were found to specifically attached to subcutaneous xenograft tumor two hours after their tail vein intravenous injection to the six-weeks-old tumor bearing female nude mice. Significant LHRH-conjugated nanoparticles uptake were revealed through T2 enriched MRI contrast of the mice xenograft tumor. The attachment of the nanoparticles is shown to be due to the presence of LHRH binding sites/receptors that are overexpressed on the surfaces TNBC that are explored via Immunofluorescence (IF) staining and Immunohistochemistry (IHC). The implications of this work were discussed for the use of LHRH-conjugated biosynthesized nanoparticles as an enhanced MRI contrast agent for the specific targeting of early stage TNBC.

#1946

Quantifying trastuzumab-induced alterations of intratumoral heterogeneity using MRI-derived tumor subregions in the preclinical setting.

Anum Syed,1 Jennifer Whisenant,2 Anna Sorace,1 Thomas Yankeelov1. 1 _University of Texas at Austin, Austin, TX;_ 2 _Vanderbilt University, Nashville, TN_.

Introduction: Heterogeneity in the tumor microenvironment affects therapeutic delivery, providing a major challenge for cancer treatment. We investigate the ability of multi-parametric, voxel-based characterizations of tumor heterogeneity from quantitative magnetic resonance imaging (MRI) to spatially resolve physiological tumor subregions of response to anti-HER2 targeted therapy in a murine model of HER2+ breast cancer.

Methods: BT474 cells were implanted in nude athymic mice (n=20) and tumors grown to ~225 mm3. Mice were randomly assigned to saline control or trastuzumab groups, with treatments given on days 0 and 3. Dynamic contrast enhanced (DCE) MRI and diffusion weighted (DW) MRI data were collected on days 0 (pre-treatment), 1 and 4. Tumors, excised on day 4, were processed for histology with anti-CD31 staining. Apparent diffusion coefficients (ADC, a measure of cell density) were extracted from DW-MRI data. DCE-MRI data was modeled to extract the extravascular, extracellular volume fraction, ve, and the volume transfer coefficients, Ktrans and kep, which correspond to the rate of wash-in and wash-out of contrast agent, respectively. Hierarchical clustering of tumor voxel data (ADC, ve, Ktrans, and kep) was used to identify physiological tumor subregions. The contribution of each subregion to tumor volume was quantified as percent tumor volume for each mouse and time point. Image analysis of histology data was used to generate vessel area and nuclei maps for whole-slice histology data. CD31 stained slices were subsequently divided into physiological subregions in terms of vessel and nuclei density, and the contribution of each subregion to the whole-slice area was quantified as percent tumor area.

Results: Hierarchical clustering of the MRI data yielded four clusters: low vascularity - low cellularity (LV-LC), low vascularity - high cellularity (LV-HC), high vascularity - low cellularity (HV-LC), high vascularity - high cellularity (HV-HC). At day 0, no significant differences in cluster percent tumor volume were observed between control and treated tumors (p>0.05). At day 4, a significant decrease in LV-HC percent tumor volume was observed in treated tumors compared to control (mean 13.7% vs. 35.0%, p=0.04). Histology subregion analysis corroborated in vivo imaging findings, with treated tumors having significantly lower LV-HC percent tumor area compared to control (mean 26.8% vs 53.6%, p<0.01).

Conclusion: High-dimensional analysis of quantitative MRI parameter maps can be utilized to identify physiological subregions of tumor response, and can be biologically validated using histology data. The results suggest trastuzumab therapy decreases the hypoxic (LV-HC) tumor volume. Quantifying tumor microenvironment alterations in response to therapy can potentially be used to predict response for patients with HER2+ breast cancer.

#1947

Modelling of lipid-based nanoparticle pharmacokinetics and tumor accumulation using preclinical models of ovarian cancer.

Michael S. Valic, Wenlei Jiang, Lili Ding, Juan Chen, Stéphanie Lheureux, Amit M. Oza, Gang Zheng. _Univ. Health Network Princess Margaret Hospital, Toronto, Ontario, Canada_.

Background: The development of lipid-based nanoparticles for the delivery of drugs, imaging agents, ect. to solid tumors continues to yield promising candidates for clinical testing. However, concerns have been raised over the poor translation of preclinical datasets with nanoparticle-enabled therapies into clinical outcomes. In an effort to support the utility of preclinical tumors models for predicting nanoparticle accumulation, we have created a multi-compartmental pharmacokinetic model for lipid-based nanoparticles using experimental data in two prevalent xenograft models of ovarian cancer.

Methods: Two ovarian cancer xenograft models, SK-OV-3 and OV-90, were orthotopically established in a single ovary of athymic nude mice. Once the tumors had reached a volume of 0.5-0.7 mm2, the mice were intra-venously administered equivalent doses of radio-labelled nanoparticles, either porphyrin-lipid-containing PEGylated nanovesicles (Porphysomes) and PEGylated liposomes. At six timepoints post administration (6, 12, 18, 24, 36, and 48 hours) a full necropsy was preformed, and the organs measured for radio-activity. The concentrations of nanoparticles in tumors and major organs were calculated and plotted versus time. A multi-compartmental pharmacokinetic model was constructed using the time-dependent concentration of nanoparticles for either xenograft model.

Results: The pathology of the orthotopic SK-OV-3 and OV-90 models were remarkably different in their vascular-permeability and cellular density, and these differences contributed to diverging nanoparticle biokinetics. Porphysome accumulation (%I.D./g), exposure (A.U.C.), and delivery efficiency (%) were significantly greater in the SK-OV-3 tumors compared with control organs (e.g., healthy ovary, muscle, etc.). Liposomes, however, did not exhibit this trend in SK-OV-3 tumors. In the OV-90 tumors, both nanoparticles exhibited increased but statistically insignificant accumulation and exposure compared with control organs. Only Porphysomes demonstrated improved delivery efficiency in the OV-90 tumors. Modelling the pharmacokinetics of the nanoparticles in a multi-compartmental model revealed distinctive kinetic parameters specific to each nanoparticle type and tumor pathology. In particular, differences were observed in the clearance rates of Porphysomes and liposomes, providing a possible explanation for their dissimilar tumor accumulations and exposures.

Conclusions: Differences in the accumulation of lipid-based nanoparticles in preclinical models of ovarian cancer appear most sensitive to vascular permeability and cellular density of the xenograft. Adjusting for xenograft-dependent kinetic parameters, the Porphysome demonstrated improved but statistically insignificant tumor exposure and delivery efficiency compared with liposomes.

#1948

**A near-infrared, highly specific NQO1-activated turn-on fluorescent probe for the detection and imaging of cancer cells** in vivo **.**

Surendra Reddy Punganuru, Viswanath Arutla, Kalkunte S. Srivenugopal. _Texas Tech Univ. Health Sciences Ctr., Amarillo, TX_.

Biomedical imaging plays an important role in all phases of cancer management including screening, guidance for biopsy excisions, malignancy staging, prognosis, therapy planning, follow-up and therapeutic responses. Unfortunately, the present detection threshold for solid tumors is approximately 109 cells (1 g = 1 cm3) growing as a single mass. The Near-infrared Fluorescence (NIRF) molecular imaging of cancer is known to be superior in sensitivity and resolution compared to other imaging modalities because of insignificant absorption by macromolecules, deeper tissue penetration and less auto-fluorescence. NAD(P)H Quinone oxidoreductase 1 (NQO1) is expressed in many human solid tumors at levels >200-fold than in normal tissues and its elevated activity has been closely associated with tumor progression, aggressiveness, resistance to chemotherapy, and poor prognosis. Therefore, NQO1 has been recognized as a potential biomarker of malignant tumors, accurate detection of which is highly desirable to improve diagnosis, efficacy and predict drug responsiveness. Fast, selective, and noninvasive reporting of intracellular cancer-associated events and species will lead to a better understanding of tumorigenesis at the molecular level and development of precision medicine. Therefore, we synthesized an NQO1 activatable NIR fluorescent probe (NQ-DCI) by conjugating dicyanoisophorone (DCI) fluorophore with the NQO1 substrate quinone propionic acid (QPA). The probe remained non-fluorescent until activation by NQO1, whose expression is largely limited to malignant tissues. With a large Stokes shift (186 nm) and a prominent near-infrared emission (646 nm) in response to NQO1, NQ-DCI was capable of monitoring NQO1 activity in vitro and in vivo with a high specificity and selectivity. We successfully employed the NQ-DCI to differentiate cancer cells from normal cells based on NQO1 activity using fluorescence microscopy and flow cytometry. Chemical and genetic approaches involving the use of ES936, a specific inhibitor of NQO1 and siRNA and gene transfection procedures unambiguously demonstrated NQO1 to be the sole target activating the NQ-DCI in cell cultures. NQ-DCI was successfully used to detect and image the endogenous NQO1 in three live tumor-bearing mouse models (A549 lung cancer, Lewis lung carcinoma, and MDMAMB 231 xenografts) with a high signal-to-low noise ratiometric NIR fluorescence response. When the NQO1-proficient A549 tumors and NQO1-deficient MDA-MB-231 tumors were developed in the same animal, only the A549 malignancies activated the NQ-DCI probe with a strong signal within 10 minutes. No other host tissues revealed fluorescence signals at this time. Because of its high sensitivity, rapid activation, tumor selectivity and nontoxic properties, we suggest that the NQ-DCI is a promising probe with clinical applications (supported by CPRIT RP170207).

#1949

Mouse model of metastatic colorectal cancer by orthotopic transplantation of patient derived organoids.

Takuya Okamoto,1 Katsuyuki Yaginuma,1 Satoshi Nagayama,2 Ryoji Yao1. 1 _The Cancer Institute of JFCR, Tokyo, Japan;_ 2 _Cancer Institute Hospital of JFCR, Tokyo, Japan_.

Cancer metastasis proceeds through multiple steps and metastatic colonization is formed by disseminated tumor cells with proliferative capacity in distant tissues. In the analysis of metastasis, macroscopically completed lesions are often used for subjects, which is attributed to the difficulty to capture the early process occurring in living body and the analysis using human specimen is particularly limited. To capture the metastatic process of colorectal cancer (CRC) and to identify cell population involved in it, we produced the mouse model for CRC by orthotopic transplantation of patient derived organoids (PDOs). Unlike ectopic transplantation models, this model precisely recapitulates the metastasis process in human patients. To evaluate the metastasis potentials, the patient matched organoids from the primary, the synchronous liver metastasis and the post-chemotherapy liver recurrence lesion were established. No significant difference in macroscopic morphology or pathogenic mutations were noted among the organoids. The organoids were marked by fluorescent protein and luciferase fusion gene to be visualized the process over time by in vivo imaging system and injected under rectal mucosa on prolapse. Each graft reflected histopathological feature of the surgical specimen. At the results of observation, the lung metastases were frequently occurred in recurrence organoids mouse models, but little occurred in primary or metastasis ones. The lung metastases were considered to be in the early stage of metastasis, because they spread diffusely in each lung lobes, and could not be confirmed macroscopically. They were forming two state; one appeared to be the embolism in blood vessels around the bronchus and others were the micro-metastases in lung tissue. The orthotropic transplantation of patient-matched PDOs provides unique opportunities to explore the metastasis process of CRC.

#1950

Differential expression of MUC16 mucin (CA125) depending on the architecture of the tumors developed in an orthotopic mouse model of high-grade serous ovarian cancer.

Alicia A. Goyeneche, Michael A. Lisio, Zu-hua Gao, Carlos M. Telleria. _Faculty of Medicine, McGill University, Montreal, Quebec, Canada_.

The histopathology of high-grade serous ovarian cancer (HGSOC) in orthotopic mouse models has received limited attention. Using immunosuppressed mice, we generated intraperitoneal HGSOC disease that includes formation of ascites and/or pleural effusions, together with a myriad of intra-abdominal growths. We studied 3 HGSOC cell lines evolved from the same patient along disease progression—a platinum-sensitive, PEO1; a platinum-resistant after further relapse, PEO4; and another at the end of life, PEO6. We found that the animals were prone to develop the disease sooner when implanted with cells obtained from the most advanced disease. Using these models, we characterized the histopathology of the masses preferably developed in the stomach-spleen-pancreas region enclosing the omentum. Other sites of tumor formation we found where the lower pelvic cavity, the peritoneal wall, the liver base, the diaphragm, the lungs, the uterus, and the ovaries. The architecture of the tumors was more often represented by slit-like fenestrations, yet certain zones displayed papillary, glandular, or solid structures sometimes including infiltrating lymphocytes. When we studied the expression of antigen CA125, we found that its pattern of staining was highly heterogeneous. In PEO1/4/6 cells maintained in culture CA125 was expressed in all cell types, yet with a major abundance aligned with disease evolution. In vivo, within the tumors, the expression of CA125 was mostly limited to the apical region of cells facing slit-like spaces generated in between solid sheets of tumor cells surrounded by fibrovascular stroma. When tumor cells were located deep within homogeneous solid masses devoid of slit-like fenestrations, they usually did not express CA125. In contrast, when cells were found gathered as multicellular structures free-floating in open spaces surrounding target organs—e.g. within the bursa around the ovaries—CA125 was highly expressed. This is coincident with the high expression of CA125 observed in vitro in cells spontaneously forming multicellular aggregates in suspension. It is likely that HGSOC cells express CA125 to face or invade into open spaces. In summary, this work reveals tissue heterogeneity and polarity in the expression of CA125 in solid tumors of HGSOC developed in immunosuppressed mice. This selected pattern of expression may be related to the oncogenic functions of CA125 (a.k.a. MUC16, a glycoprotein), such as protecting cells from immunological attacks, or promoting metastases by binding to mesothelin produced by mesothelial cells that line the peritoneal cavity.

#1951

Increased glioma collagen is associated with greater blood flow and peri-tumoral fluid flux, but less infiltration.

Tavarekere N. Nagaraja,1 Rasha Elmghirbi,2 Stephen L. Brown,1 Ian Y. Lee,1 Glauber Cabral,1 Swayamprava Panda,1 Robert A. Knight,1 James R. Ewing1. 1 _Henry Ford Hospital, Detroit, MI;_ 2 _Oakland University, Rochester, MI_.

Introduction: Composition of the glioma microenvironment (TME) determines tumor growth rate, invasion, metastasis and resistance to treatment. Compared to normal brain, tumor extracellular matrix (ECM) has a higher concentration of structural proteins including collagen, laminin, tenascin, and vinculin. We compared collagen expression in two rat models of glioma, human U251 grown in immune-compromised rats and 9L in syngeneic Fischer-344 rats, representing primary and recurrent glioma features, respectively.

Experimental procedures: The U251 and 9L cells were implanted in the right brain hemisphere of female RNU rats (n=10) and Fischer-344 rats (n=7), respectively. Rats were imaged 2 to 3 weeks after implantation by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Tumor size, blood flow, apparent diffusion coefficient (ADC), blood-to-tumor forward volumetric transfer constant (Ktrans), peri-tumoral contrast flux (Flux), hydraulic conductivity (K), and extracellular volume fraction (VD) in the tumor (VD-tumor), tumor rim (VD-rim) and its periphery (VD-peri) along with tumor interstitial fluid pressure (TIFP) were measured. Immediately after imaging, brains were processed for paraffin embedding and histopathology. Brain sections (6 µm) containing the tumor were stained for collagen using Picrosirius red and adjacent brain sections with hematoxylin and eosin (H&E). MRI data were compared by t-tests and significance inferred at p≤0.05. Digital images of Picrosirius red staining were analyzed using ImageJ for collagen expression expressed as % fraction of total tumor area. It was compared to the MRI biomarkers, and to the patterns of tumor cell dispersion into normal brain observed on H&E stained images.

Results: Tumor diameters averaged 4 mm for both models at the time of imaging. The U251 tumors showed greater Flux (p=0.03), higher blood flow (p=0.02), smaller VD_tumor (p=0.01) and VD_rim (p=0.02) values than the 9L tumors. TIFP also tended to be higher in the U251 model (p=0.06). Collagen expression in U251 tumors was significantly higher than in 9L tumors (p=0.002). On H&E stained sections, the U251 tumors showed a well delineated tumor margin with very few cells invading the host tissue. In contrast, the 9L tumors showed cancer cells infiltrating singly and in clusters, along white matter tracts as well as in perivascular spaces.

Conclusions: Increased collagen content was associated with elevated blood flow, Flux and TIFP, but less tumor cell infiltration in the U251 glioma. Observations of low collagen content in conjunction with increased tumor cell invasion in the 9L model suggest that treatment with collagenase, while increasing drug penetration may also make the tumors more infiltrative. A closer examination of TME to determine the composition of ECM proteins that regulate glioma aggressiveness and modulate its response to treatments is warranted.

#1952

Tumor microenvironment targeted Rosella nanoparticle for the detection of triple negative breast cancer by multispectral optoacoustic tomography.

Abhilash Samykutty,1 Molly McNally,1 Alexandra Thomas,1 William Grizzle,2 Lacey R. McNally3. 1 _Wake Forest Univ. Comp. Cancer Ctr., Winston-Salem, NC;_ 2 _University of Alabama Birmingham, Birmingham, AL;_ 3 _Wake Forest Univ. Comp. Cancer Ctr., Winston Salem, NC_.

Purpose: Recent advances in the nanotechnology and molecular imaging provides an excellent opportunity for development of delivery vehicles and imaging probes to improve real-time assessment and early detection of breast cancer progression.The absence of estrogen receptor (ER), progesterone receptor (PR), or HER-2 genes, represents a major clinical challenge for triple negative breast cancer (TNBC). Because of the absence of reliable markers, there is an unmet clinical need for developing efficient methods to identify TNBC. We have developed an acidic pH targeted Rosella nanoparticles that can actively release IR780 dye into orthotopically implanted TNBC tumors to improve tumor detection using multispectral optoacoustic imaging technology (MSOT).

Methods: The Rosella nanoparticles are a mesoporous silica base with wormhole pore architecture containing a chitosan gatekeeper and V3 pHLIP targeting peptide. The particles were synthesized using the sol-gel method and characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The Rosella particles were loaded with propidium Iodide (PI) or IR780 infrared imaging dye to evaluate pH-sensitive cargo release. Female athymic mice were implanted with MDA-MB-468 breast cancer cells by the mammary fat pad injection. Once the tumor reaches 3mm in size, athymic mice were intravenously injected with ROSELLA nanoparticles carrying IR780 dye and were imaged with MSOT.

Results: We have synthesized the Rosella nanoparticle with wormhole pores that is 27 nm diameter and can encapsulate the IR780 imaging probes for the detection of the TNBC. The Rosella particle contains a chitosan gatekeeper which can degrade upon contact with acidic pH tumor to prevent off-target release and is further sensitized to pH using V3 peptide. The Rosella nanoparticles can detect acidic tumor microenvironment and can penetrate inside the tumor cells. As a model of TNBC, we have injected MDA-MB-468 cells into the mammary fat pad of the female athymic mice to develop breast tumors. Once the tumor reached 3mm in size, we have intravenously delivered ROSELLA nanoparticles to the athymic mice with TNBC tumors. Our results were demonstrated that the intravenous injection of the ROSELLA particles could detect the orthotopically implanted TNBC tumors (p<0.0001,n=5).

Conclusion: most aggressive subtypes of breast cancer, TNBC has a poor response for majority of the FDA-approved breast cancer drugs. To overcome these limitations, the distinct nanoformulations with potent imaging technology will enable to develop tumor-penetrating nanoparticles to more effectively deliver chemotherapeutics or imaging agents with least off-target effects. Future translation of these technologies has a high clinical impact concerning our current treatment options for TNBC patients.

#1953

**HER2 functionalized nanoparticles are safe and specific for** in vivo **HER2+ breast tumor cell detection.**

Marie Zhang,1 Jose Vargas,1 Antimone Dewing,1 Farideh Bischoff,1 Kelsey Mathieu,2 John Hazle2. 1 _Imagion Biosystems, Inc., San Diego, CA;_ 2 _MD Anderson, Houston, TX_.

Superparamagnetic Relaxometry (SPMR) is a highly sensitive in vivo detection technology that can localize and quantify superparamagnetic iron oxide (Fe3O4) nanoparticles when bound to tumor cells. Nanoparticles that reach and bind to the target cells are measureable by superconducting quantum interference device (SQUID) magnetometers (MRX instrument developed in house), while unbound nanoparticles such as those freely circulating in the bloodstream are not detected and bone and normal tissue do not produce any magnetic signal. In this study we demonstrate that anti-HER2 antibody conjugated nanoparticles exhibit great specificity and selectivity towards HER2+ tumor cells, and are safe (non-toxic) in preclinical models.

Anti-HER2 antibody conjugated nanoparticles consist of a 25nm Fe3O4 core covered by a polymer shell with PEG and antibody conjugated on the surface. The overall size of particles is 70-80nm with PDI <0.1 as measured by DLS. Each particle has between 3-5 antibody molecules covalently attached to the surface with PEGylation to reduce opsonization.

Our results have shown that our antibody conjugated nanoparticles can distinguish high, medium and low HER2+ expression cell lines, such as BT474 (3+), ZR75 (2+), MCF7(0/1+) in an in vitro cell based assay. Furthermore, positive signal can be competed out by pre-incubation with free HER2 antibody. In additional, negative cell lines, such as MCF10 produce undetectable SPMR signal, demonstrating good sensitivity, specificity and selectivity. Incubation with PBMC indicates that the nanoparticles do not produce an SPMR signal, suggesting minimum non-specific interaction with lymphocytes. Extensive in vivo studies using xenograph tumor mouse models have shown the nanoparticles can selectively distinguish between MCF7 vs BT474 dual implanted tumors. In addition, as a control, we used PEG only nanoparticles and showed they do not generate measurable signal in tumor. Results are corroborated using other orthogonal detection methods, such as IHC and ICP-MS.

The safety profile of the nanoparticles has been demonstrated by analyzing nanoparticle accumulation in organs when injected at 20mg/kg dose via intravenous, intraperitoneal and subcutaneous delivery routes. All major organs except liver do not generate measurable MRX signal at multiple time points post injection, indicating no significant accumulation of nanoparticles in major organs. A full GLP toxicity study using rats is being conducted to demonstrate the safety of the anti-HER2 antibody conjugated nanoparticle prior to first-in-human use.

Together, these results suggest that our HER2 nanoparticles are safe; can provide targeted and specific delivery to cancerous tissue in vivo and generate measurable signal on our MRX detection instrument. These studies lay out ground work for our future human clinical study for breast cancer detection.

#1954

Tracking of orally-administered particles within the gastrointestinal tract of murine models using multispectral optoacoustic tomography.

Neal Bhutiani,1 Abhilash Samykutty,2 Kelly McMasters,1 Nejat Egilmez,1 Lacey R. McNally3. 1 _University of Louisville, Louisville, KY;_ 2 _Wake Forest Univ. Comp. Cancer Ctr., Winston-Salem, NC;_ 3 _Wake Forest Univ. Comp. Cancer Ctr., Winston Salem, NC_.

While particle carriers have potential to revolutionize disease treatment, using these carriers requires knowledge of spatial and temporal biodistribution. The goal of this study was to track orally administered particle uptake and trafficking through the murine gastrointestinal (GI) tract using multispectral optoacoustic tomography (MSOT).

Polylactic acid (PLA) particles encapsulating AlexaFluor 680 (AF680) dye conjugated to bovine serum albumin (BSA) were orally gavaged into mice. Particle uptake and trafficking were observed using MSOT imaging with subsequent confirmation of particle uptake via fluorescent microscopy. Mice treated with PLA-AF680-BSA particles exhibited MSOT signal within the small bowel wall at 1 and 6 h, colon wall at 6, 12, and 24 h, and mesenteric lymph node 24 and 48h. Particle localization identified using MSOT correlated with fluorescence microscopy. Despite the potential of GI tract motion artifacts, MSOT allowed for teal-time tracking of particles within the GI tract in a non-invasive and real-time manner.

Future use of MSOT in conjunction with particles containing both protein-conjugated fluorophores as well as therapeutic agents could allow for non-invasive, real time tracking of particle uptake and drug delivery.

#1955

A preclinical ultrasound platform for widefield 3D imaging of rodent tumors.

Tomasz Czernuszewicz,1 Virginie Papadopoulou,2 Juan D. Rojas,1 Rajalekha Rajamahendiran,1 Jonathan Perdomo,1 James Butler,1 Max Harlacher,1 Graeme O'Connell,1 Dzenan Zukic,3 Paul A. Dayton,2 Stephen Aylward,3 Ryan C. Gessner1. 1 _SonoVol, Inc., Durham, NC;_ 2 _The University of North Carolina and North Carolina State University, Chapel Hill, NC;_ 3 _Kitware, Inc., Carrboro, NC_.

Background: Preclinical ultrasound (US) and contrast-enhanced ultrasound (CEUS) imaging have long been used in oncology to noninvasively measure tumor volume and vascularity. While the value of preclinical US has been repeatedly demonstrated, these modalities are not without several key limitations that make them unattractive to cancer researchers, including: high user-variability, low throughput, and limited imaging field-of-view (FOV). Herein, we present a novel robotic preclinical US/CEUS system that addresses these limitations and demonstrates its use in evaluating tumors in 3D in a rodent model.

Methods: The imaging system was designed to allow seamless whole-body 3D imaging, which requires rodents to be imaged without physical contact between the US transducer and the animal. To achieve this, a custom dual-element transducer was mounted on a robotic carriage, submerged in a hydrocarbon fluid, and the reservoir sealed with an acoustically transmissive top platform. Eight NOD/scid/gamma (NSG) female mice were injected subcutaneously in the flank with 8×109 786-O human clear-cell renal cell carcinoma (ccRCC) cells. Weekly imaging commenced after tumors reached a size of 150 mm3 and continued until tumors reached a maximum size of 1 cm3 (∼4-5 weeks). An additional six nude athymic female mice were injected subcutaneously in the flank with 7 × 105 SVR angiosarcoma cells to perform an inter-operator variability study. Imaging consisted of 3D B-mode (conventional ultrasound) of the whole abdomen (< 1 min), as well as contrast-enhanced acoustic angiography (< 10 min) to measure blood vessel density (BVD). Tumors were manually segmented in 3D (< 2 min) and inter-operator and inter-reader reliability was assessed with Krippendorff's alpha.

Results: Wide-field US images reconstructed from 3D volumetric data showed superior FOV over conventional US. Several anatomical landmarks could be identified within each image surrounding the tumor, including the liver, small intestines, bladder, and inguinal lymph nodes. Tumor boundaries were clearly delineated in both B-mode and BVD images, with BVD images showing heterogeneous microvessel density at later timepoints suggesting tumor necrosis. Excellent agreement was measured for both inter-reader and inter-operator experiments, with alpha coefficients of 0.914 (95% CI: 0.824-0.948) and 0.959 (0.911-0.981), respectively.

Conclusion: We have demonstrated a novel preclinical US imaging system that can accurately and consistently evaluate tumors in rodent models. The system leverages cost-effective robotic technology, and a new scanning paradigm that allows for easy and reproducible data acquisition to enable wide-field, 3D, multi-parametric ultrasound imaging.

#1956

ICG pHLIP: A novel agent for fluorescence-guided surgery.

Troy Crawford,1 Anna Moshnikova,1 Sean Roles,1 Lukas M. Carter,2 Jason S. Lewis,2 Donald M. Engelman,3 Oleg A. Andreev,4 Yana K. Reshetnyak1. 1 _University of Rhode Island, Kingston, RI;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 3 _Yale, New Haven, CT;_ 4 _University of Rhode Island, Saunderstown, RI_.

Introduction: Fluorescence imaging has applications in medicine for many image-guided procedures such as mapping and visualization of lymph nodes, targeting and marking cancerous lesions and assessment of tumor margins. Several fluorescent molecules are approved for clinical use including the ICG (indocyanine green) near infrared (NIR) fluorescent dye. We conjugated ICG to a pH Low Insertion Peptide (pHLIP®), which senses and targets the acidity at the surfaces of cancer cells, and evaluated ICG pHLIP® as a targeted imaging agent for tumor visualization. Our construct is progressing toward clinical trials.

Experimental Procedures: ICG pHLIP® was manufactured for proof-of-concept tumor-targeting imaging studies in mice, and toxicity evaluation in mice, dogs and rats. Tumor targeting was assessed in various human and murine tumor models. Biodistribution was evaluated at different time points from 5 min to 48 hours after a single intravenous administration of 0.5 mg/kg of ICG pHLIP®. The distribution of the agent within tumors was evaluated in tumor sections.

Results: The fluorescence of ICG is enhanced about 15 times when pHLIP® tethers it to a cell membrane, significantly improving the contrast index. No toxicity is seen at 25-30x of the proposed human dose, evaluated in mice and dogs. ICG pHLIP® demonstrates excellent tumor targeting, including targeting of 1-3 mm sized tumors in breast. About 10-15% ID/g was observed within tumors at 4 hours p.i., and the signal persists for up to 24 hours with a slight decline at 48 hours. The background signal in major organs decayed after agent administration. The highest uptake, of about 50% ID/g, was observed in liver at 4 hrs p.i. and decayed after that. An excellent correlation was established between ICG pHLIP® NIRF imaging and H&E histopathology at the indicated tumor location.

Conclusions: ICG pHLIP® is a promising new imaging agent for fluorescence-guided surgical applications. It is on the path to clinical translation for accurate resection of breast tumors. Since pHLIP® targeting of tumors is based on the general principle of cancer cell surface acidity, ICG pHLIP® is an agent that may be used in a range of fluorescence-guided surgical interventions.

#1957

**Novel substrates for NanoLuc luciferase with improved brightness and signal duration for bioluminescence imaging** in vivo **.**

Joel R. Walker,1 Yunhee Park,2 Michael Lin,2 Thomas A. Kirkland,1 Mary P. Hall,3 Lance P. Encell,3 Younghee Oh,2 Lan Liu2. 1 _Promega Biosciences, San Luis Obispo, CA;_ 2 _Stanford University, Stanford, CA;_ 3 _Promega, Madison, WI_.

NanoLuc® luciferase, and its substrate furimazine, are becoming a preferred bioluminescent system in cellular and biochemical assays due to its brightness and glow-type kinetics. The construct Antares, made by fusing NanoLuc to the cyan-excitable orange-red fluorescent protein CyOFP, combines the high catalytic rate of NanoLuc with the high quantum yield and red-shifted emission of CyOFP (584 nm), which is well suited for in vivo imaging. Transgenic mice were created to constituently express Antares and these mice were used to screen coelenterazine analogues for a substrate that showed appropriate distribution and brightness in vivo. Our new Antares (NanoLuc) substrates are administered i.p., have improved solubility over furimazine, exhibit long (>30 min) signal kinetics, and are brighter than D-luciferin/Luc2 expressed on the same promoters.

#1958

Early treatment response detected in a murine clear cell renal cell carcinoma model in response to combination therapy with antiangiogenic and notch inhibition therapy using a non-invasive imaging tool.

Juan D. Rojas,1 Virginie Papadopoulou,2 Tomasz Czernuszewicz,1 Rajalekha Rajamahendiran,1 Anna Chytil,3 Yun-Chen Chiang,4 Diana Chong,5 Victoria L. Bautch,4 Wendy K. Rathmell,3 Stephen Aylward,6 Ryan Gessner,1 Paul Dayton2. 1 _SonoVol, Inc., Durham, NC;_ 2 _The University of North Carolina and North Carolina State University, Chapel Hill, NC;_ 3 _Vanderbilt University Medical Center, Nashville, TN;_ 4 _The University of North Carolina Lineberger Comprehensive Cancer Center, Chapel Hill, NC;_ 5 _The University of North Carolina, Chapel Hill, NC;_ 6 _Kitware, Inc., Carrboro, NC_.

Background: Functional and molecular changes often precede gross anatomical changes in cancer, so early assessment of a tumor's functional and molecular response to therapy can help reduce a patient's exposure to the side effects of ineffective chemotherapeutics or other treatment strategies. Clear-cell renal cell carcinoma (ccRCC) is an aggressive and hyper-vascular form of renal cancer that is often treated with anti-angiogenic and Notch Inhibition therapies, which target the vasculature feeding the disease. The purpose of this work is to show that ultrasound microvascular imaging can provide indications of response to antiangiogenic and Notch Inhibition therapies prior to measurable changes in tumor size.

Methods: Mice bearing 786-O ccRCC xenograft tumors were treated with SU (Sunitnib malate, Selleckchem, TX), an antiangiogenic drug, and a combination of SU and the Notch inhibitor GSI (Gamma secretase inhibitor, PF-03084014, Pfizer, New York, NY) therapies (n=8). A 3D ultrasound system (SonoVol Inc., Research Triangle Park, NC), in addition to microbubble ultrasound contrast agents, was used to obtain a measurement of microvascular density over time and assess the response of the tumors to the therapies. CD31 immunohistochemistry was performed to serve as a gold standard for comparison against imaging results. Statistical tests included: Spearman correlation to compare imaging and histology; Kruskal-Wallis analysis with Tukey multiple comparison post-test to determine if the vessel density or tumor volume were significantly different between the treatment groups; and receiver operating characteristic (ROC) curve analysis to determine sensitivity/specificity for separating treated/untreated groups.

Results: Data indicated that ultrasound-derived microvascular density can detect response to antiangiogenic and Notch inhibition therapies a week prior to changes in tumor volume. Furthermore, the imaging measurements of vasculature are strongly correlated with physiological characteristics of the tumors as measured by histology (p=0.75). Moreover, data demonstrated that ultrasound measurements of vascular density can determine response to therapy and classify between-treatment groups 1 week after the start of treatment with a high sensitivity and specificity of 94% and 86%, respectively.

Conclusion: This work shows vascular density measurements that are strongly correlated with histology can be obtained using ultrasound, and that imaging-derived vessel density metrics may be a better tool for assessing the response of ccRCC to antiangiogenic and Notch inhibition therapies than anatomical size measurements.

#1959

Translational imaging findings in a pediatric patient-derived orthotopic xenograft brain tumor model.

Tuulia Huhtala,1 Julia Schueler,2 Jussi Rytkönen,1 Daniela Lötsch,3 Dorothee Lenhard,3 Artem Shatillo,1 Kimmo Lehtimäki,1 Pekka Poutiainen,4 Diana Miszczuk,1 Johannes Gojol,3 Walter Berger3. 1 _Charles River Discovery, Kuopio, Finland;_ 2 _Charles River Discovery, Freiburg, Germany;_ 3 _Medical University of Vienna, Vienna, Austria;_ 4 _Kuopio University Hospital, Kuopio, Finland_.

Malignant brain tumors are the most common cause of solid cancer death in children. Innovative therapies are vital to improve treatment outcomes, but must be developed to enable trafficking across the blood brain barrier (BBB). For this advent, animal models provide important information prior to clinical studies. Among the different in vivo models orthotopic patient-derived xenograft (PDX) models represent the diversity seen in patient tumors and hence replicate response rates in the clinical trials better as compared to other more simplistic models.

Especially in the brain tumor field, imaging has a central role in clinical diagnosis and as a prognostic factor to monitor therapy response. It enables longitudinal patient monitoring in a fully translational manner. Magnetic resonance imaging (MRI) and positron emission tomography (PET) are widely used for clinical diagnosis and disease follow up. Choosing the most suitable imaging application depends of the target of interest or mechanism of action. MRI offers unprecedented soft tissue contrast, high spatial resolution and non-invasive nature renders MRI in rodents a perfect tool for preclinical work in oncological applications. In case of orthotopic brain tumor models, MRI offers the state-of-the-art quantitative volumetric tumor size analysis over disease progression. PET is an excellent tool to study tumor proliferation, metabolism, metastasis as well biodistribution of novel antibodies.

The purpose of this work was to analyze volumetric, metabolic and functional changes in orthotopic PDX brain tumor model using MRI, MRS and PET imaging. During the course of the experiment, volume, perfusion within the tumor as well proliferation and metastasis were monitored. Perfusion measurement indicates angiogenesis in tumor, one hallmark of most malignant gliomas. Also, alterations in glucose and amino acid metabolism between tumor and healthy tissue has been previously identified. By direct comparison of the imaging data derived from the preclinical mouse model with similar data-sets from the donor patient the translational value of the model as well as the read-out system will be achieved.

As a conclusion, translational in vivo imaging techniques were applied to study orthotopic tumor progression. These readouts provide a powerful and translational research tool together with oncological disease animal models allowing comprehensive evaluation of disease progression and treatment interventions for in vivo studies.

#1960

Diffusion MR imaging detects differential progression of pathology in two mutant KRas mouse models of pancreatic neoplasia.

Palamadai N. Venkatasubramanian,1 Matthew Smith,1 Emman Mascrinas,2 Andrew Diaz,2 Karla Castellanos,2 Brian DeCant,2 Ron McKinney,2 Paul J. Grippo,2 Alice M. Wyrwicz1. 1 _NorthShore University HealthSystem, Evanston, IL;_ 2 _University of Illinois at Chicago, Chicago, IL_.

A significant clinical need exists for understanding the early changes in pancreatic cancer (PC) progression and employing imaging of cellular and tissue phenotypes to detect neoplasia. We have investigated the development of early pathology in EL-Kras and p48-Cre/LSL-Kras (EK and KC, respectively) mice using diffusion MR imaging. These models express mutant Kras via different mechanisms yet recapitulate pancreatic precursor lesions seen in human PC. Our ex vivo investigation shows that diffusion MR imaging based only on endogenous contrast not only detects pre-neoplastic pathology in EK and KC mice, but also differentiates the progression of pathology in these mouse models. Fixed pancreata from EK and KC mice at 4M, 8M and 12M (n=4/group) were examined by diffusion imaging on a 14.1T MR microimager. Mean diffusivity was calculated for acinar lobules and lesions. Pancreata evaluated by MRI were processed for histology (H&E and trichrome) to determine percent area of tissue containing normal, acinar-ductal metaplasia (ADM), fibrosis and neoplasia. Diffusivity was lower in the acinar compartment of EK mice at 4M (0.83x10-3mm2/s) relative to KC mice (1.21x10-3mm2/s) and normal controls (1.17x10-3mm2/s), suggesting that Kras-related pathology targets the acinar cells of EK mice at a younger age than KC mice. Acinar diffusivity, however, did not change further in EK mice at 8 and 12 months, indicating little or no progression of pathology. In contrast, acinar diffusivity progressively decreased with age in KC mice (8M: 0.68x10-3mm2/s; 12M: 0.59x10-3mm2/s) indicating a later onset, but more advancing pathology. Changes in tissue diffusivity arise from a complex combination of cytological changes induced by pathology. Lower diffusivity in EK mice at 4M probably arises from higher levels of ADM (31.7%) compared to 4M KC mice (ADM 11.7%). Reduced diffusivity in KC mice at 8 and 12M most likely is related to elevated fibrosis in those mice (60.0-62.5%) which is relatively low in EK mice (13.3-18.3%). While diffusion MR images revealed the presence of fluid-filled cysts in both models, only KC mice showed lesions with much lower diffusivity (0.55-0.39x10-3mm2/s). Lower diffusivity indicates high cellularity in these neoplastic lesions which are likely mPanINs. Histology revealed that mPanINs which occupied 11.7-18.8% of the tissue in the KC mouse pancreas, are mostly absent in EK mice. Based on our imaging and histology results, MR-measured diffusivity might be a useful marker to assess the onset and progression of PanIN and cystic disease in PC.

#1961

PET imaging with zirconium-89 labeled 3,2-HOPO mesothelin antibody-chelator conjugate reflects biodistribution of mesothelin targeted thorium-227 conjugate.

Linda N. Broer,1 Baard Indrevoll,2 Christine Ellingsen,2 Alexander Kristian,2 Roger M. Bjerke,2 Liv-Ingrid Oedegaardstuen,2 Olav B. Ryan,2 Urs B. Hagemann,3 Alan S. Cuthbertson,2 Patricia E. Cole,3 Elisabeth G. de Vries,1 Marjolijn N. Lub-de Hooge4. 1 _Departments of Medical Oncology, University Medical Centre Groningen, University of Groningen, Groningen, Netherlands;_ 2 _Thorium Conjugate Research, Bayer AS, Oslo, Norway;_ 3 _Bayer AG, Pharmaceuticals Division, Berlin, Germany;_ 4 _Clinical Pharmacy and Pharmacology, University Medical Centre Groningen, University of Groningen, Groningen, Netherlands_.

INTRODUCTION Cell surface glycoprotein mesothelin (MSLN) is highly expressed in many human cancers. MSLN targeted thorium-227 conjugate (MSLN-TTC, BAY 2287411), comprising the MSLN targeted antibody anetumab, covalently attached to 3,2-HOPO chelator, enabling specific complexation and delivery of the alpha particle emitter thorium-227 (227Th) to tumor cells, is currently in a phase 1 clinical trial (NCT03507452). To study whether positron emission tomography (PET) imaging can guide 227Th radio-immunotherapy, the antibody-chelator conjugate was radiolabeled with PET isotope zirconium-89 (89Zr) to compare MSLN-TTC and 89Zr-MSLN biodistribution.

METHODS The antibody-chelator conjugate was radiolabeled with 89Zr. NMRI-Foxn1nu female mice were subcutaneously (sc) engrafted with low MSLN expressing BxPc-3 human pancreatic cancer cell line (4,200 receptors per cell) at the right flank. Mice received a total antibody dose of 0.75 mg/kg or 1.5 mg/kg of 89Zr-MSLN and underwent microPET imaging 24, 72 and 168 h post injection (pi). PET scan data are presented as mean standardized uptake values (SUVmean), calculated from the mean activity in the region of interest divided by the injected dose (corrected for decay) per gram body weight. Thereafter mice were terminated and ex vivo biodistribution was performed. Additionally, mice were engrafted with medium MSLN expressing human ovarian cancer cell line OVCAR-3 (38,000 receptors per cell). These animals received 0.75 mg/kg 89Zr-MSLN or MSLN-TTC, followed by ex vivo biodistribution 0.5, 2, 6, 24, 72 and 168 h pi. In tumors and organs high energy gamma rays of 89Zr (909 keV) and 227Th (236 keV) were measured in a gammacounter to determine % injected dose per gram (%ID/g) of 89Zr-MSLN or MSLN-TTC, respectively.

RESULTS PET images showed 89Zr-MSLN BxPc-3 tumor accumulation at 168 h pi with a SUVmean of 1.6, a tumor to blood ratio of 1.2 and limited uptake in other organs at 0.75 mg/kg dose (n = 6). Ex vivo biodistribution revealed tumor uptake of 7.6 % ID/g, liver uptake of 4.9 % ID/g and limited uptake in other organs. No major differences were observed between the 0.75 mg/kg and 1.5 mg/kg tracer dose. In the OVCAR-3 model ex vivo biodistribution of 89Zr-MSLN resembled biodistribution of MSLN-TTC at different time points with increasing tumor uptake and decreasing blood levels over time and limited uptake in other organs. 168 h pi tumor uptake was 75 % ID/g for 89Zr-MSLN and 64 % ID/g for MSLN-TTC (n = 5, no statistical difference).

CONCLUSION 89Zr-MSLN biodistribution reflects PET imaging in BxPc-3 xenografts and 89Zr-MSLN biodistribution reflects MSLN-TTC biodistribution in OVCAR-3 xenografts. Therefore 89Zr-MSLN PET imaging might aid in clinical guidance for MSLN-TTC therapy.

#1962

Visualizing the efficacy of PI3K inhibitor combination treatment on tumor metabolism.

Manushka Vaidya, Olmo Sonzogni, Cody Callahan, Jocelin Pinto, Gerburg M. Wulf, Aaron Grant. _Beth Israel Deaconess Medical Center, Boston, MA_.

Purpose: Many types of cancer exhibit an upregulation in glycolysis. Cancer therapies that target tumor metabolism include PI3K inhibitor combinations, which are currently in clinical trials. Real-time metabolic imaging can be possible using hyperpolarization, which is a signal enhancement technique for MRI that increases the signal by a factor of 10,000 or more. The purpose of this work was to determine whether the anti-metabolic effect of such therapies on preclinical breast cancer models could be visualized non-invasively by MRI of hyperpolarized 13C-pyruvate to lactate conversion.

Methods: Experiments were carried out on a 9.4 T animal MRI scanner (Biospec 94/20, Bruker, Billerica MA), and all animal studies were done with IACUC approval. Two models of triple negative BRCA1 mutant breast cancer, either resistant (T1R1, n=4) or sensitive (T2S, n=4) to PARP inhibition, were implanted in the inguinal fat pad of mice. Animals with tumor sizes of approximately 10x10x10 mm3 were then imaged prior to and after four consecutive days of combination treatment with a PI3K inhibitor and PARP inhibitor. In order to visualize the interconversion of pyruvate to lactate in vivo, a bolus of hyperpolarized 13C pyruvate (Hypersense, Oxford Instruments, Oxfordshire UK) with concentration=100 mM and volume=200uL was administered to the mouse via tail vein. 13C MR images were acquired using echo-planar spectroscopic imaging (Flip angle=4 degrees; matrix size=16x16; resolution = 2.5mm; slice thickness=5mm; spectral points=512; spectral width=4kHz; repetitions=32) with a 13C radiofrequency transmit/receive surface coil (Bruker, MA) and overlaid on proton anatomical images obtained using a proton volume coil (78 mm diameter, Bruker, MA). Data were reconstructed (Mathematica, Wolfram) to obtain pixel by pixel time integrated pyruvate and lactate spectra for an axial slice. The area under the curve for both metabolites was calculated for all pixels and summed within the tumor to obtain a lactate to pyruvate ratio for each case. Statistical analysis was carried out using a paired t-test.

Results: For the animals with T2S tumors, the lactate to pyruvate ratio reduced from 1.65 ± 0.44 to 0.84 ± 0.32 (mean ± std. dev.) after treatment with significance p = 0.0378. For animals with T1R1 tumors, the ratio before and after treatment was 0.91 ± 0.44 and 0.93 ± 0.20 (p = 0.9553).

Conclusions: Noninvasive visualization of pyruvate to lactate interconversion can be achieved using 13C MRI, allowing for imaging the efficacy of treatments on cancer metabolism. 

### Secreted Changes in the Tumor Microenvironment: Exosomes and Chemokines

#1963

Feasibility and efficacy of using self-delivering RNAi against TGFB1 to reduce TME immunosuppression.

Winnie F. Tam, Dingxue Yan, Melissa Maxwell, James Cardia, Gerrit Dispersyn. _Phio Pharmaceuticals, Marlborough, MA_.

One of the leading causes of non-responding tumors with existing immunotherapies is an immunosuppressive tumor micro-environment (TME). The suppressive nature of the TME is, in part, mediated by immunoregulatory cells (e.g. MDSC cells) and cytokines/chemokines. TGFB1 has recently been shown to be a key suppressive factor responsible for facilitating "cold" or immune-excluded tumors. In these tumors, T cell activity is restricted and by reducing TGFB1 levels we aim to support anti-tumor T cell activity. We evaluated if downregulation of TGFB1 with a self-delivering RNAi (sd-rxRNA) in MDSCs (in vitro) or TME (in vivo) promotes T cell activity and increase anti-tumor activity.

sd-rxRNA is a chemically modified RNAi which incorporates drug like properties allowing for self-delivery of RNAi compounds to immune cells (including MDSC, DC, T and NK cells) in the absence of electroporation or formulation in a delivery vehicle. Tumor induced MDSCs were incubated with sd-rxRNA targeting TGFB1, its downstream target COX2 or in combination for 2 days. The cells were washed and added to a co-culture with OT-1 T cells and OVA expressing B16 melanoma. The anti-tumor activity of T-cells was monitored using a Real-Time Cell Activity assay (RTCA). TGFB1 and COX2 downregulated MDSCs had reduced immune-suppressive activity as shown by the enhanced B16-OVA tumor cell killing by CD8 OT-1 T cells in the RTCA assay while the combination of TGFB1 and COX2 MDSCs did not further improve the anti-tumor activity.

To evaluate the effect of anti-TGFB1 sd-rxRNA in vivo, we first studied the tumor distribution and TGFB1 downregulation by intratumoral injection of sd-rxRNA to immunocompetent Balbc mice bearing 4T1-RL-GFP breast cancer in a fat pad model. DY547 labeled sd-rxRNA targeting PPIB or unlabeled sd-rxRNA targeting TGFB1 was injected into tumors. Two days later, tumors were resected, and fluorescence signal was analyzed by microscopy and TGFB1 downregulation was evaluated by RT-PCR. Efficient delivery of the DY547 labeled sd-rxRNA was observed in the vast majority of cells as shown by fluorescence microscopy. In addition, fluorescence signal could be detected in tumors up to 28 days after intratumoral injection, suggesting that sd-rxRNA was stable in tumors. Downregulation of TGFB1 in resected tumors, and an increased anti-tumor activity, was seen after three injections of sd-rxRNA. A follow up study was initiated investigating the efficacy of TGFB1 sd-rxRNA in combination with local tumor radiation on anti-tumor and metastatic activities in a 4T1 orthotopic breast cancer model. Preliminary data shows promising results.

Our results suggest that sd-rxRNA compounds targeting TGFB1 can enhance the anti-tumor activity of T cells. Local injection of sd-rxRNA into tumors can effectively target TGFB1 in the TME resulting in less immunosuppression and increased anti-tumoral activity.

#1964

C-C chemokine receptor type 1 is a prognostic indicator in ovarian cancer.

Anita M. Chanana, Venkatesh Krishnan, Supreeti Tallapragada, Oliver Dorigo. _Stanford University School of Medicine, Stanford, CA_.

Targeting CC-chemokine receptor type 1 (CCR1) in pre-clinical cancer models has shown reduction in tumor burden. Similar agents have also been safely used in clinical trials for autoimmune conditions. Despite promising results in cancer models and adequate safety profiles, the role of targeting CCR1 in ovarian cancer is poorly understood, warranting further evaluation. We have identified a macrophage-driven chemokine receptor axis, CCL23/CCR1 that may play a critical role in ovarian cancer metastasis. We hypothesize that CCR1 expressed by ovarian cancer cells and the differential expression of this chemokine receptor may have a prognostic role in survival and response to chemotherapy. We performed immunofluorescence and immunohistochemistry to profile the expression of CCR1 in a cell array containing 53 ovarian cancer cell lines and in three tissue microarrays containing greater than 1000 cores from 192 patients diagnosed with serous, endometrioid, or clear cell ovarian carcinoma. We measured the percent of CCR1 3,3′-Diaminobenzidine (DAB) staining using HALO image analysis software's area quantification algorithm (Indica Labs, Corrales, NM) and correlated with survival. We analyzed three tissue microarrays containing varied histologic subtypes of ovarian cancer and found that CCR1 was highly expressed, defined as greater than 30% DAB staining, in 53% of serous (N=93), 68% of endometrioid (N=66), and 45% of clear cell carcinoma (N=22) averaged tissue cores. In comparing different ovarian cancer histologic subtypes, averaged tissue cores from patients with endometrioid carcinoma have significantly higher expression of CCR1, compared to serous carcinoma and clear cell carcinoma, 10.5% (p≤0.02) and 12.5% (p≤0.002), respectively. In all histotypes, we observed a significant lower median progression-free survival (p≤0.05) in patients with high CCR1 expression. Interestingly, in evaluating the level of CCR1 expression and response to platinum chemotherapy, we found that patients with platinum resistance (N=36) (defined as recurrence after 12 months of completing chemotherapy) had significantly higher expression of CCR1 compared to patients that were platinum sensitive (N=92) (p≤0.04). In addition, we analyzed a cell array and found differential expression of CCR1 across phenotypically different lines. For example, the cell lines OVCA420, OVSAHO, and OVCAR3 showed higher expression of CCR1 compared to TOV-21G, MOVCAR18, and OVCAR5. Given the potential prognostic significance of CCR1 in ovarian cancer, our future studies are focused on determining the functional role of CCR1 inhibition using both in vitro migration and in vivo colonization assays. Outcomes from pre-clinical studies will inform us on the clinical translatability for targeting CCR1 in patients with epithelial ovarian cancer.

#1965

Inhibiting the CCR2/MCP-1 chemokine pathway blocks MDSC recruitment and promotes anti-tumor immunity.

Payal Mittal,1 Liqing Wang,1 Tatiana Akimova,1 Craig A. Leach,2 Jose C. Clemente,2 Mathew Sender,2 Yao Chen,2 Brandon Turenen,2 Wayne W. Hancock1. 1 _CHOP/U Penn, Philadelphia, PA;_ 2 _GSK, Philadelphia, PA_.

Host anti-tumor immunity, including the actions of cytotoxic T cells, plays a key role in curtailing the growth of "hot" tumors but can be hindered by multiple mechanisms. One such mechanism involves the local recruitment and expansion of myeloid derived suppressor cells (MDSCs) within the tumor microenvironment. We investigated the role of the CCR2/MCP-1 axis in MDSC-associated tumor progression using the TC1 lung tumor cell line implanted into syngeneic C57BL/6 mice. Phenotypic profiling of TC1 tumors revealed maximal expression of CCR2 by tumor resident MDSCs (p<0.01 vs. neutrophils, tumor-associated macrophages/TAM or T cells), and MCP-1 by transplanted tumor cells (p<0.01 vs. all types of leukocytes), respectively. Additionally, utilization of CCR2 knockout (CCR2KO) mice showed the dependence of progression of TC1 tumor on CCR2 signaling (p<0.01). Tumors in CCR2KO mice had fewer CCR2lowMDSCs (p<0.01), CD4 T cells (p<0.01), and Foxp3+ Treg cells (p<0.05). Conversely, CD8 T cell infiltration was significantly augmented in tumors from CCR2KO mice (p<0.05), and these CD8 T cells were capable of making higher levels of IFN-γ (p<0.01) and granzyme-B (p<0.05) upon restimulation than CD8 T cells from WT tumor-bearing mice. These effects were tumor-specific and not the result of genetic ablation of CCR2 on T cells, since the adoptive transfer of WT or CCR2KO cells into B2D6/F1 mice showed that CCR2KO cells had comparable activation, proliferation, inflammatory cytokine production, and Treg-mediated suppressive function as WT cells (all p>0.05). We next used a thioglycolate-induced peritonitis model of inflammation to validate the role of CCR2 and MCP-1 in trafficking of CCR2+ cells to the site of inflammation. We showed the ability of a CCR2 antagonist to inhibit trafficking of CCR2+ cells to the site of inflammation, in a dose-dependent manner, with a maximal effect at a dose of 10 mg/kg (p<0.05). We then tested the compound at this inhibitory concentration for its ability to impair CCR2+ cell recruitment to MCP-1 expressing TC1 tumors. Use of the CCR2 antagonist blocked growth TC1 tumors (p<0.001) and markedly suppressed intratumoral MDSC numbers (p<0.01), while boosting the numbers of cytotoxic CD8 T cells (p<0.005) and intratumoral expression of IFN-γ, TNF-α, granzyme-B and perforin (all p<0.01). In summary, fully complementary genetic and pharmacologic data indicate that CCR2 targeting may be an important new component of immuno-oncology based therapies.

#1966

Obesity and prostate cancer progression: Role of the chemokine CXCL12.

Songyeon Ahn,1 Achinto Saha,1 Mikhail G. Kolonin,2 John DiGiovanni1. 1 _University of Texas at Austin, Austin, TX;_ 2 _The University of Texas Health Sciences Center at Houston, TX_.

Obesity is a significant risk factor for several cancers in both men and women. A number of studies have shown that obesity is associated with increased prostate cancer (PCa) progression and higher mortality. However, the mechanism(s) for obesity-driven PCa remain unclear. In earlier studies, we showed that obesity enhances PCa progression in the HiMyc mouse model by changing the surrounding tumor microenvironment, especially the white adipose tissue (WAT). WAT consists of many cells including inflammatory cells, mature adipocytes and adipose stromal cells (ASCs). Here, we further investigated the role of ASCs and ASC derived CXCL12 in obesity-driven PCa progression. In HMVP2 cells, a PCa cell line derived from HiMyc mice expressing high levels of CXCR4, we observed activation of MAPKs (JNK and ERK), AKT, STAT3 and NFkB following treatment with CXCL12. These CXCL12-induced signaling pathways were inhibited by treating with AMD3100, a CXCR4 antagonist, and also by CXCR4 knock-down. To confirm this in vivo, we compared PCa tumor growth in lean and obese mice using the HMVP2 allograft model. Our data demonstrates that obesity promotes EMT in tumor cells and increases invasion of surrounding WAT into the tumor stoma. Treatment of mice with AMD3100 significantly inhibited the growth of HMVP2 tumors and suppressed EMT in both the obese and control diet groups. Furthermore, D-CAN, a hunter-killer peptide developed for targeting ASCs, also suppressed obesity-induced EMT and tumor growth in HMVP2 tumor allografts. Lastly, AMD3100 in combination with D-CAN, showed a significant reduction in PCa growth in obese mice compared to either agent used alone. These results provide evidence that ASCs activate the CXCL12/CXCR4 signaling as well as other pathways playing an important role in obesity-driven PCa progression. Our data suggests this pathway as a novel therapeutic target for inhibiting obesity-driven PCa growth and progression. Research supported by NCI Grant CA196259.

#1967

Nab-paclitaxel chemotherapy of breast tumors upregulates chemokines that increase chemotaxis of myeloid-lymphatic progenitors.

Lisa D. Volk-Draper, Sophia Ran. _Southern Illinois University School of Medicine, Springfield, IL_.

Introduction: Lymph node metastasis is the main prognostic indicator for poor survival of breast cancer patients. Metastasis is promoted by tumor-induced lymphangiogenesis mediated, in part, through recruitment of myeloid-derived lymphatic endothelial cell progenitors (M-LECP). We previously showed that differentiation of M-LECP is induced by activated Toll-like receptor-4 (TLR4). The goal of this study was to identify TLR4-induced chemokines produced by tumors alone or in response to nab-paclitaxel (nab-PXL) chemotherapy that enhance bone marrow (BM) differentiation and tumor recruitment of M-LECP.

Methods: Profile of BM composition for lymphatic-like, myeloid and progenitor cells was assessed by flow cytometry in normal and tumor-bearing mice treated either with saline or nab-PXL. We used two TLR4 overexpressing tumor models: MDA-MB-231 and HCC1806-TLR4+. RT-qPCR was used to determine differences in expression of 130 cytokines in cultured tumor cells and tumors from mice treated either with saline or nab-PXL. Targets with >2-fold upregulation were analyzed for protein expression by ELISA. ELISA-validated cytokines were tested for their ability to induce migration of M-LECP.

Results: Both MDA-MB-231 or HCC1806-TLR4+ tumors significantly increased the levels of differentiated M-LECP in the BM, and that was further enhanced by treatment with taxane chemotherapy. Expression of progenitor markers CD117, CD33, and Sca-1 was increased up to 7-fold in both tested tumor models, and further upregulated by nab-PXL treatment by additional 3 to 9-fold. Out of 23 cytokines measured in tumor lysates by ELISA, 40% were upregulated by nab-PXL. Migration assay showed that several of these cytokines increased chemotaxis of M-LECP with CXCL1 being the strongest. Neutralization of CXCL1 in the conditioned medium of nab-PXL-treated MDA-MB-231 cells inhibited migration of lymphatic progenitors by 75%.

Conclusion: Taxane chemotherapy acting presumably as a ligand for TLR4 significantly expanded M-LECP population in the BM. Our analysis suggests that these lymphatic precursors can mobilize to tumors by chemokines such as CXCL1 that are highly upregulated in TLR4-positive tumor cells, particularly when activated by paclitaxel. These data suggest that CXCL1 plays an important role in recruiting myeloid-lymphatic progenitors to breast tumors, which, in turn, promotes lymphatic vessel formation and metastasis. Blocking this cytokine might have clinical utility for prevention of tumor mobilization of M-LECP, and by extension, breast cancer metastasis to lymph nodes.

#1968

Tumor necrosis factor-α upregulation of CCR7 induces prostate cancer cell migration in lymphatic metastasis.

Tomoyuki Makino,1 Kouji Izumi,1 Aerken Maolake,2 Ariunbold Natsagdorj,1 Hiroaki Iwamoto,1 Suguru Kadomoto,1 Renato Naito,1 Kaoru Hiratsuka,1 Yoshifumi Kadono,1 Atsushi Mizokami1. 1 _Kanazawa University Graduate School of Medical Science, Kanazawa, Japan;_ 2 _Roswell Park Comprehensive Cancer Center, Buffalo, NY_.

Objectives: Prostate cancer (PCa) with lymph node (LN) metastasis has a poor patient prognosis. Broader understanding of the causes of LN metastasis and disease dissemination will assist in developing novel treatments. C-C chemokine receptor 7 (CCR7) and its ligand CCL21 are abundantly expressed in LN metastasis and during PCa progression. Tumor necrosis factor-alpha (TNF-α) is present at low levels within the tumor microenvironment. TNF-α and C-C chemokines interact in tumorigenesis. The study aim was to determine whether TNF-α might promote PCa dissemination from metastatic LN by activating the CCL21/CCR7 axis.

Methods: TNF-α and CCR7 mRNA and protein expression were assayed in PC-3, DU145, and LNCaP, and human PCa cells by reverse transcription-polymerase chain reaction and western blotting. Promotion of transmembrane migration by TNF-α was assayed in CCL21-pretreated PCa cells. Phosphorylation of several mitogen-activated protein kinases expressed downstream of TNF-α was assayed.

Results: Low-dose TNF-α induced CCR7 expression in PCa cells and promoted migration of CCL21-pretreated PCa cells. TNF-α-mediated CCR7 upregulation via the ERK signaling pathway. p38 was a downstream target of CCL21/CCR7 signaling and a key factor in the promotion of PCa cell migration.

Conclusions: This is the first demonstration of the influence of the TNF-α-CCL21/CCR7 axis on PCa cell migration in the microenvironment of LN metastasis. Because it is activated in PCa LN metastasis, the CCL21/CCR7 axis might be a novel therapeutic target and biomarker of PCa.

#1969

Role of the CXCL17-CXCR8 (GPR35) axis in cutaneous squamous cell carcinoma.

Alok R. Khandelwal, Md Maksud Alam, Tara Moore-Medlin, Hillary A. Savage, Cherie-Ann O. Nathan. _LSU Health-SHV, Shreveport, LA_.

Cutaneous squamous cell carcinoma (cSCC) is a keratinocyte-derived invasive and metastatic tumor of the skin and is the second-most commonly diagnosed form of skin cancer (16%) striking 200,000 Americans annually. Increased exposure to Ultraviolet-B (UVB) from solar radiation as a result of the depletion of the ozone layer and the widespread use of tanning beds are the primary reasons for the increased incidence of cSCC. Novel mechanism-based therapeutics are needed to prevent UVB-induced cSCC. Chemokines are a group of small chemotactic cytokines that can exert extensive and complex effects on cancer via interaction with their receptors. Chemokine (C-X-C motif) ligand 17 (CXCL17) is the latest member of the chemokine family, and recent studies have identified both a causative and suppressive role of CXCL17 and its cognate receptor GPR35 in tumorigenesis. The purpose of this study was to determine the role of CXCL17 and GPR35 in the pathogenesis of cSCC. CXCL17 and GPR35 were significantly overexpressed in cSCC cells (SCC12A and SCC118) compared to HaCaT cells. Stimulation with CXCL17 significantly induced cSCC cell proliferation, migration, and motility suggesting an essential role in tumor growth, invasion, and metastasis. Tumor-specific overexpression of CXCL17 was detected in human cSCC and UVB-induced mouse cSCCs that further bolsters our in vitro findings. Recent studies have also identified the role of CXCL17 in tumor cell infiltration and immune invasion leading to tumor progression. In our preliminary studies, increased expression of CXCL17 in cSCC correlated with an augmented number of CD3+, CD4+ and CD8+ T-cells infiltrates. Our data suggest that CXCL17 could potentially exert differential effects based on cell types in the tumor microenvironment. Our studies underscore the importance of CXCL17/GPR35 signaling in cSCC and provide a novel target for the treatment of non-melanoma skin cancer.

#1970

Melatonin inhibits osteolytic bone metastasis through interrupting cancer cells-derived osteolytic factor and reduce osteoclast differentiation.

Jyun-Lin Lai,1 An-Chen Chang,1 Po-Chun Chen,2 Chih-Hsin Tang1. 1 _China Medical University, Taichung, Taiwan;_ 2 _Shin-Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan_.

Osteolytic bone metastasis is seen in lung, breast, and prostate cancer and is associated with severe complications such as bone pain, fractures and hypercalcemia. In the bone microenvironment, osteoclasts play an important role in bone remodeling and bone resorption. Metastatic cancer cells may increase osteoclast differentiation and activity by releasing inflammatory cytokines including receptor activator of nuclear factor kappa-Β ligand (RANKL), tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6) and IL-8, leading to bone resorption and osteolytic lesions. The endogenous hormone melatonin is known to modulate bone resorption. Increasing evidence suggests that melatonin inhibits cancer cell proliferation, angiogenesis and metastasis. Up until now, the inhibitory role of melatonin in osteolytic bone metastasis has remained unclear. In In vitro study, we observed that melatonin inhibits the differentiation of precursor osteoclasts and promotes apoptosis in mature osteoclasts. We injected mice tibia with luciferase-labeled osteolytic cancer cell lines (lung cancer: A549, prostate cancer: PC-3) to establish bone metastasis. After 4 weeks of intraperitoneal injection with melatonin, IVIS fluorescence signaling indicated that melatonin inhibited tumor growth. X-ray imaging of the bone revealed an improvement in bone resorption after melatonin treatment. In In vitro melatonin also reduced RANKL protein and mRNA expression in the A549 and PC-3 cell lines. Our data indicate that melatonin improves bone mass by inhibiting the expression of cancer-derived osteolytic factors and by promoting apoptosis in mature osteoclasts. Our future investigations will seek to determine the signaling pathway involved in melatonin-regulated RANKL expression in cancer cells. In summary, our findings provide insight into the benefits of melatonin treatment in osteolytic bone metastasis.

#1971

A multi-variate transcriptome analysis of CXCR5-CXCL13 signaling in ovarian cancer.

Kaylin Carey, Tiara Griffen, Corey Young, Courtney Dill, James Lillard. _Morehouse School of Medicine, Atlanta, GA_.

Ovarian Cancer (OvCa) is a disease that affects postmenopausal women, with high-grade serous ovarian carcinoma being the most common and lethal histopathological type. High-grade serous carcinoma develops from malignant cells that originate from tubal epithelium. Many patients are asymptomatic in the early stages of OvCa, thus going undetected, leading to higher patient mortality rates. Currently, 51% of women are not diagnosed until Stage III and 29% are diagnosed during Stage IV. One of the major limitations in current research is the lack of understanding of key drivers of clinical phenotypes and determination diagnostic biomarkers to provide targeted therapeutic approaches. Immunotherapy has emerged as a major treatment modality for cancer. Chemokines and their receptors play a significant role in immune cell chemotaxis that drives inflammation and immunity. Our laboratory was the first to show that CXCL13-CXCR5 signaling mediates prostate, breast and lung cancer cell growth, migration, invasion, and survival. Furthermore, we provided evidence that CXCL13 and CXCR5 are highly elevated in OvCa cell lines and clinical samples. These previous findings provided the rationale to support the hypothesis CXCL13-CXCR5 signaling promotes OvCa progression, metastasis and survival. In this study we further analyzed transcriptome data to determine possible molecular mechanisms and CXCL13-CXCR5 signaling pathways controlling OvCa progression. Weighted Gene Network Co-expression (WGCNA) analysis was used to identify gene co-expression networks correlated with aggressive OvCa clinical phenotypes. Using Ingenuity Pathway Analysis (IPA), we identified several CXCL13-CXCR5 associated hub genes that reveal gene-gene interactions, upstream regulators and biological pathways that drive OvCa progression. Of interest, WGCNA-designated Blue module contained CXCR5 along with various co-expressed genes, including RB1, PIK3 C3, GNAI1 that are known to facilitate the progression of OvCa as well as LTB and TNFRSF11B, which have been shown to mediate tertiary lymphoid structure formation. Furthermore, the Blue module genes were significantly (p = 0.04) associated with age at diagnosis. The Salmon module, closely related to the Blue module and, significantly (p = 0.01) correlated with relapse. Importantly, the Salmon module co-Expressed genes, included AURKB and CDCA8 and cell cycle checkpoint regulatory genes, which have shown to be prominent in OvCa recurrence. Taken together, our study suggest an important role of CXCL13-CXCR5 signaling and reveals an important set of co-expressed epigengenes involved in OvCa relapse.

#1972

CXCR5-CXCL13 signaling contributes to CLL relapse and patient survival.

Tiara Griffen, Corey Young, Courtney D. Dill, Kaylin Carey, Olayinka Adebayo, Sha'kayla Nunez, James Lillard. _Morehouse School of Medicine, Atlanta, GA_.

Chronic Lymphocytic Leukemia (CLL) is characterized by the appearance of mature-appearing B cells in the blood, bone marrow, and secondary lymphoid organs. There is an average of 19,000 new cases, in the United States annually, but the molecular pathogenesis of CLL is incompletely understood. Chemokines and their receptors have been shown to contribute to the progression and metastasis of several types of cancers. Chemokine receptor 5 (CXCR5) and chemokine ligand 13 (CXCL13) are produced by cells in the microenvironment of secondary lymphoid organs such as lymphatic endothelial cells, which promote disease progression. There is a gap in understanding the molecular mechanisms and functions resulting from CXCL13-CXCR5 interactions during CLL. This gap is important to fill, because these molecules could be additional therapeutic targets to treat this disease. Together this provided the rationale to test the hypothesis CXCR5-CXCL13 signaling contributes to the growth, metastasis, and survival of CLL. The role of CXCR5, CXCL13, and associated genes was determined by conducting Weighted Gene Network Co-expression (WGCNA) and upstream regulator, canonical pathway, and gene interaction analyses of 297 International Cancer Genome Consortium (ICGC) CLL patients. Our previous studies show CXCR5-CXCL13 signaling activates CTNNB1, JUN, and VAV2 transcriptional factors. These factors promote the expression of GNA, GRK, PIK3, RAC and RHO family genes, which were significantly associated with the WGCNA-designated Yellow signaling module. Interestingly, the WGCNA-designated Grey module contained independently expressed genes, e.g., CXCL13, LTB, IGHV, ZAP70, which were significantly associated (p = 0.00003) and correlated (R2 = 0.29) with relapse. Genes in this module significantly correlated with CLL relapse and survival. Taken together, our data demonstrate the clinical significance of the CXCL13-CXCR5 pathway in CLL progression.

#1973

RIP1 and its signaling network regulate radiation-induced modification of cancer microenvironment.

Jeong Hyun Cho,1 Jung Lim Kim,2 Hong-Duck Um,1 Sang-Gu Hwang,1 EunAh Lee,3 Sang-Churl Oh,2 Jong Kuk Park1. 1 _Korea Inst. of Radiological & Medical Sci., Seoul, Republic of Korea; _2 _Korea University Guro Hospital, Seoul, Republic of Korea;_ 3 _Kyung Hee University, Seoul, Republic of Korea_.

Radiotherapy is the principal treatment method for inducing cell death in various cancer types such as lung cancer; however, this therapeutics is frequently associated with occurrence of resistance such as recurrences or metastasis due to recent studies that radiotherapy might promote cancer progression and metastasis. Recurrences or metastasis is major cause of treatment failure of cancer, but its intra-/inter-cellular molecular mechanisms was not identified clearly yet due to the fact that genetic characteristics of cancer are extremely diverse according to individuals. Therefore, the objective of this study is to investige the modifications of intracellular signaling/genetic profiling in ionizing radiation (IR)-irradiated microenvironment of non-small-cell lung cancer (NSCLC), and to provide new therapeutic target for treatment of metastatic cancer. Previously, we have studied that IR treatment increased migration and invasion via induction of epithelial-mesenchymal transition (EMT) in A549 cells. With the same system, we found the increase of expression level of receptor interacting protein 1 (RIP1) in IR-treated A549, and this increased expression of RIP1 is associated with IR-induced EMT induction in vitro. NCI-H460 and A549 overexpressing RIP1 was established, and these transfectants also showed increased of migration and invasion via EMT induction. Signaling analyses indicated activation of EGFR-Src-STAT3 pathway is required for the EMT induction, and RIP1 exerts effect on this pathway resulting in activation of EGFR- RIP1-Src-STAT3 pathway. The down-regulation of RIP1 expression by treatment of siRNA, pharmaceutical inhibitor for RIP1 kinase, or dominant negative mutant of the kinase suppressed not only the activation of EGFR-RIP1-Src-STAT3, but also the EMT induction; this suggests that RIP1 might be one of key molecules in IR-induced migration/invasion. Additionally, DNA profiling analyses of RIP1-overexpressed NCI-H460 and A549 indicated that IL-1β is the most prominent over-expressed gene in common. IR treatment also stimulated IL-1β expression and its upstream NF-κB activation via increase of RIP1 followed by induction of EMT and cancer invasion/migration. Supernatants of stable cells including secreted IL-1β treatment promoted invasion/migration of NCI-H460 and A549 cells via EMT induced by activation of EGFR- RIP1-Src-STAT3 pathway. Taken together, our present results imply that RIP1 signals through EGFR- RIP1-Src-STAT3 axis and also through RIP1-NF-κB-IL-1β pathway. This also suggest that these two pathways are connected with common molecule, RIP1, which is novel signaling pathway and activations of these two pathways contribute to new autocrine signaling involving IL-1β secretion; therefore, RIP1 has a role as a novel biomarker as well as a molecular target for suppressing lung cancer metastasis.

#1974

Autocrine TIMP-1-mediated chemoresistance via induction of IL-6 in NSCLC.

Wei Xiao, Amyn M. Rojiani, Sampa Ghoshal Gupta, Mumtaz V. Rojiani. _Augusta University- Medical College of Georgia, Augusta, GA_.

Our previous studies have confirmed the well-defined antiapoptotic functions of TIMP-1. We have shown that overexpressing TIMP-1 in NSCLC cells increases cell survival with activation of the ERK pathway via phosphorylation of BAD at serine 112. Knocking down TIMP-1 in NSCLC cells increased the levels of miR-125a-5p with concomitant upregulation in apoptosis. These studies beg the question- does modulation of TIMP-1 affect chemotherapy in NSCLC? To address this question we examined two cell lines, A549 and H460, and their specific shRNA-mediated TIMP-1 gene knockdown clones. We found that TIMP-1 mediated chemoresistance against two front line chemotherapeutic agents (Gemcitabine and Cisplatin) as determined by increased apoptosis in the KD clones. In order to determine any contribution of cytokine activity towards this antiapoptotic function of TIMP-1, we carried out a cytometric bead array assay. The results of this screening assay which included potential inflammatory cytokines and growth factors, indicated that IL-6 plays a role in mediating the chemoresistance in TIMP-1 expressing NSCLCs. We then determined endogenous IL-6 levels in our array of NSCLC cell lines and found a direct correlation of IL-6 levels with TIMP-1 levels. It is important to note that IL-6 expression is precisely regulated with TIMP-1 production at both protein and mRNA levels. The effect of TIMP-1 on IL-6 levels is more pronounced under hypoxic versus normoxic conditions. Importantly, IL-6 expression is upregulated by addition of exogenous human recombinant TIMP-1 in a time- and dose- dependent manner. Blocking of TIMP-1 signaling with specific neutralizing antibodies delayed the IL-6 upregulation. Moreover, addition of rhTIMP-1 or rhIL-6 rescued apoptosis induced by chemotherapeutic agents albeit to a limited extent. Although early in the investigation, our studies suggest that interventions impacting the TIMP-1/IL-6 axis in NSCLCs may provide an efficient target to boost effects of clinical chemotherapy.

#1975

Multivariate exome analysis identifies CXCR5-CXCL13 signaling as a key driver in early breast cancer development and prognosis.

Courtney D. Dill, Tiara Griffen, Corey Young, Kaylin Carey, James W. Lillard. _Morehouse School of Medicine, Atlanta, GA_.

Breast cancer (BrCa) is the most frequently diagnosed cancer and the second leading cause of cancer-related death among women worldwide despite new developments in prevention and treatment. More recently, rapid increases in BrCa incidence have been reported in countries of the Asia-Pacific region. Age of diagnosis in Asian BrCa patients is much younger than age of diagnosis in western countries, such as the United States, and Asian early BrCa patients tend to be pre-menopausal, whereas only 15-30% of western BrCas are pre-menopausal. The mechanisms responsible for early BrCa remain unknown. Furthermore, it remains controversial whether early breast cancer has unique tumor biology. The goal of this study was to characterize the molecular phenotype of early BrCa in the context of CXCL13, CXCR5 and associated gene expression. We hypothesized that CXCR5-CXCL13 signaling contributes to the aggressive phenotype of early BrCa. A bioinformatic approach was used to aid in characterizing this condition. Our patient cohort data (50 females of Korean descent, age ≤ 35 years) was obtained from The International Cancer Genome Consortium (ICGC). DESeq analysis was used to identify genes differentially expressed among normal tissue and primary tumor groups. Weighted Gene Network Co-expression (WGCNA) analysis was carried-out to identify gene networks associated with factors influencing BrCa prognosis. Finally, canonical pathway, upstream regulator, and gene interaction analysis was conducted using Ingenuity Pathway Analysis. Our data show CXCL13, CXCR5, and associated genes (LTA, LTB, IL10, IL27RA, TNFRSF13B, TNFRSF17, and TNFRSF9), which have been previously shown to drive tertiary lymphoid structure (TLS) formation, were significantly associated with the molecular phenotype of early BrCa in our patient cohort. Taken together, our findings suggest TLSs contribute to the tumor immune microenvironment and contribute to early BrCa progression.

#1976

β-arrestin-1 function in CAFs is necessary for enhancement of self-renewal of NSCLC stem-like cells.

Mohan Kumar Durai Raj, Srikumar P. Chellappan. _Moffitt Cancer Center, Tampa, FL_.

Background: Cigarette smoking is a major risk factor in the genesis of non-small cell lung cancer (NSCLC). Nicotine, the addictive component of tobacco smoke, promotes growth and metastasis of NSCLCs. The scaffolding protein β-arrestin-1 (ARRB1), mediates the proliferative effects of nicotine through nicotinic acetylcholine receptors (nAChR) signaling. Nicotine induces nuclear translocation of ARRB1 and increases E2F-driven proliferative, EMT and survival genes to promote NSCLC growth. Further, nicotine promotes NSCLC stemness by inducing SCF (Stem cell factor) in a β-arrestin-1-dependent manner. While all these studies describe β-arrestin-1 role in cell-autonomous functions, its role in NSCLC microenvironment is largely unknown. Cancer associated fibroblasts (CAFs) and human mesenchymal stem cells (hMSCs) promote the self-renewal and proliferation of cancer stem cells in vitro and in vivo. In the present study, we address if the function of β-arrestin-1 in CAFs/hMSCs is necessary to enhance the self-renewal of stem like cells from NSCLCs.

Methods: GFP stable expressing NSCLC cell line A549 (A549-GFP) was generated. The side population (SP) cells from A549-GFP was used in 3D co-culture with cytotracker red labeled lung CAFs or hMSCs in stem cell selective medium and the sphere growth was monitored for 10 days. Cytokine arrays were used to test the changes that occur in CAFs/hMSCs upon β-arrestin-1 depletion. RNAseq analysis was carried out for differential expression of RNA in β-arrestin-1 depleted lung CAFs.

Results: Our results show that CAFs/hMSCs can promote the self-renewal of SP cells from NSCLCs, while β-arrestin-1 depleted CAFs/hMSCs are incompetent to promote SP cells self-renewal. Cytokine array results showed reduced levels of multiple cytokines including CXCL1/GROα, CXCL5, ICAM-1, VCAM-1 and CCL5 in β-arrestin-1 depleted lung CAFs/hMSCs. Further, RNAseq analysis of β-arrestin-1 depleted lung CAFs revealed differential expression of 577 genes. Interestingly, the data showed significantly altered expression of 23 genes involved in cytokine-cytokine receptor interaction.

Conclusions: β-arrestin-1 in CAFs/hMSCs appears to enhance the stemness of SP cells from NSCLCs. β-arrestin-1 depletion in CAFs/hMSCs significantly altered the expression of multiple cytokines/growth factors. In good correlation, RNAseq results displayed a significant difference in the expression of genes encoding cytokines/cytokine receptors, suggesting the potential role of CAFs/hMSCs secreted cytokines in mediating β-arrestin-1-mediated self-renewal. Experiments are under way to identify cytokines that act downstream of β-arrestin-1 to enhance NSCLC stemness. These studies will shed new light on the mechanisms by which CAFs/hMSCs promote self-renewal and tumor growth, enabling the identification of pathways downstream of β-arrestin-1 that can potentially be targeted for NSCLC therapy.

#1977

**Chemokines expression in an** in vitro **model of melanoma brain metastasis.**

Renata Santos, Maria Clara Sampaio, Amanda Albuquerque, Michelly Pereira, Maira Pitta, Moacyr Rêgo. _Universidade Federal de Pernambuco, Recife, Brazil_.

Melanoma brain metastasis is still a great challenge. This work aimed to characterize the secretion profile and understand the possible role of chemokines in a xenogeneic in vitro model of melanoma brain metastasis. Brain primary mixed cultures from cerebral cortex were prepared from the neonates Wistar rats and maintained at 37°C, 5% CO2until 80% of confluency and maturity. Melanoma lineages used were UACC62 and SKMEL-28. Co-cultures were performed to study the direct interactions between melanoma and cortical cells. The indirect effects in the cerebral microenvironment were evaluated through conditioned medium (CM) from brain cells on melanoma lineages. It was used an early CM collected at 24h (CM24) and a late CM collected after 48h (CM48), whose both were incubated during more 24h (24CM24 and 24CM48) or 48h (48CM24 and 48CM48) on melanoma cultures. The chemokines IP10, CCL5 and IL8 were studied through Cytometric Bead Array (CBA),MIG and CCL2 also, however their values were undetectable. After the characterization of the model, IP10 was silenced to evaluate its influence in the model. CBA demonstrated that in SKMEL-28 the late soluble factors during 48h (48CM48) were capable of rase IP10 levels (p=0,0286). On the other hand, direct and indirect contact with brain cells reduced IL8 levels, compared to SKMEL28 alone with respectively p = 0.02 and p=0,0286, where both chemokines were not detected. On UACC62, IP10 levels were higher only in the co-culture (p=0,02), suggesting the need of contact between the different cells. Also, IL8 was decreased in all conditions compared to UACC62. After IP10 silencing, SKMEL-28 presented higher levels of IL8 in 48CM48. On UACC62, the IP10 silencing resulted in elevation of IL8 levels when the cells were in co-culture. RANTES did not present alterations in its levels in any of the conditions after IP10 knock-down. The IP10 silencing was also capable of impair the transwell invasion of SKMEL-28 in the presence of CM24 and CM48. Together, these results confirmed that IP10 alters IL8 levels, but no RANTES in a mimetic microenvironment of melanoma brain metastasis. Furthermore, IP10 is capable of influencing SKMEL-28 migration in this xenogeneic model. More studies to better understand the role of IP10 and IL8 in the establishment of melanoma brain metastasis in this model are in progress.

#1978

Sarcoma invasion associated with heparan sulfate degradation.

Donghong Ju,1 Leonard Lipovich,1 Mary A. Kosir2. 1 _Wayne State University, Detroit, MI;_ 2 _Barbara Ann Karmanos Cancer Inst., Detroit, MI_.

INTRODUCTION: Sarcomas are locally aggressive, angiogenic and spread hematogenously. The purpose of this study was to quantify the effect of heparan sulfate degrading activity on invasion of K-ras-transformed sarcoma cells.

METHODS: Heparan degrading enzyme assays were performed in triplicate on lysate from Balb/c 3T3 (fibroblasts) and K234 cells (K-ras-transformed 3T3) cells (sarcoma) using an enzyme assay kit (cat# MK412;Takara). Invasion assays were performed using Corning Matrigel Invasion Chamber (cat#354480; ThermoFisher ). 7.5 x 104 cells were placed in serum-free DMEM in the upper chamber (insert), and DMEM with 10% FBS as attractant in the lower chamber. After incubation for 24 h at 37 °C, the insert was removed and cells on the undersurface of the insert were fixed with 5% glutaraldehyde for 10 min, then stained with 1% crystal violet in 2% ethanol for 20 min. After the insert was dry, the cells on the filter were counted by microscope over multiple fields. For experiments with inhibitors, 10 ug/ml λ-carageenan (cat# 22049; Sigma), anti-NAP-2 antibody (cat# sc-19224; Santa Cruz Biotech), or anti-HPA1 antibody were used.

RESULTS: Heparan sulfate-degrading activity was 0.0625 U/ug for K234 cell lysate compared to 0.0125 U/ug for Balb/c3T3 cells. λ-carageenan inhibited the activity completely confirming glycanase inhibition. Anti-NAP2 and anti-HPA1 antibody inhibited heparan sulfate degrading activity in 3T3 lysate. Invasion was two-fold greater by K234 cells than for 3T3 cells. When anti-NAP2 antibody was added to the invasion assay, both K234 and 3T3 cell invasion was inhibited by 55% and 54%, respectively. Less than 1% inhibition of invasion occurred with anti-HPA1 antibody.

CONCLUSION: The invasion of heparan sulfate-containing Matrigel by K234 cells is partly influenced by heparan sulfate-degrading activity. The inhibition of the degrading activity by an anti-chemokine antibody decreased invasion, and invokes other processes for the malignant sarcoma phenotype.

#1979

Interaction of atorvastatin and CX3CR1-fractalkine in androgen-dependent prostate cancer cells: Effect on PI3K pathway.

Belal Al-Husein. _JUST, Irbid, Jordan_.

Prostate cancer (PC) management lacks any targeted therapies. Statins have shown anticancer effects against PC in both in vitro and in vivo studies. CX3CR1 is a receptor expressed by many cancer cells, including PC. In this research, we studies the interaction between atorvastatin and CX3CR1/fractalkine on androgen-dependent PC cell line 22Rv1 vs. normal prostatic cells, RWPE1. Experimental settings were based on testing effect of atorvastatin in presence of fractalkine or by removing its effect using fractalkine neutralizing antibodies. MTT proliferation assay and adhesion assay, together with testing the expression of CX3CR1, p-AKT and p-GSK-3 were used. Results showed that fractalkine partially blunts the effect atorvastatin on proliferation and adhesion of PC cells. Similar effects were not noticed on normal prostatic cells. Western blotting showed a dose-dependent reduction in the expression of CX3CR1, p-akt and p-GSK-3 in presence of fractalkine, that was further enhanced when fractalkine was neutralized using specific antibodies. Results suggest the deprivation of fractalkine as a synergistic mechanism to enhance effects of atorvastatin and probably other anticancer agents.

#1980

The critical role of interleukin-8 chemokine axis in the development of benign prostatic hyperplasia (BPH).

Diandra K. Smith, Natasha Venugopal, Martha K. Terris, Bal L. Lokeshwar. _Augusta University, Augusta, GA_.

Benign prostatic hyperplasia (BPH), a non-malignant proliferative disease of the prostate, has a global incidence of over 210 million men aged 50 years or older. BPH is essentially a non-cancerous (non-malignant tumor) with significant morbidity. The severity varies from mild urinary retention to severe lower urinary tract symptoms (LUTS) accounting for greater than 50% of hospital visits for difficulty with urination, retention, and pain, and close to 40% of the prostate surgeries. BPH strongly associates with prostate volume that begins to increase in men aged 40 or higher. We investigated the role of chemokine and chemokine receptors in three immortalized cell lines and primary cultures derived from fresh BPH tissue following transurethral resection of the prostate. We analyzed the levels of chemokines and chemokine receptors secreted in the culture medium and in the mRNA from cells by qPCR, western blotting and Cytokine arrays. The epithelial identity of primary cultures was established by analyzing cytokeratin expression. Two natural products isolated from Allspice were identified that inhibit the growth of BPH cultures. A cell line identified as the BPH progenitor, a BPH intermediary cell line and a cell line with frank BPH features secreted a large excess of chemokine, predominantly CXCL-VIII or Interleukin-8 (IL-8). Unusually high amount of IL-8 was secreted by BPH progenitor cells and intermediate cells as compared to a stable normal prostate cell line (RWPE1) derived from the peripheral zone of the prostate, which did not secrete any IL-8 or express its receptor. The investigation found that expression of CXC receptors CXCR1, CXCR2 and an IL-8 inducible CXCR (CXCR7) was absent in progenitor cells and in intermediate cells, but was highly expressed in the cell line derived from BPH tissue and more significantly, in primary cultures of BPH tissue. Further, primary cultures and progenitor cells expressed high levels of immune stimulatory factors indicating PMN and monocyte infiltration leading to potential chronic inflammation of the transitional zone of the prostate. The natural products from Allspice inhibited all members of the IL-8 axis and proliferation, suggesting their potential preventative potential for BPH. These results demonstrate how a novel chemokine axis is likely to enhance pathogenesis of BPH and suggest several avenues to control the abnormal growth of prostate in aging male.

#1981

M2-macrophage derived exosomes mediates tumor progression via GATA3-IL-4-IL-13 axis in ovarian cancer.

Pinar Kanlikilicer, Amr El-Arabey, Merve Denizli, Recep Bayraktar, Bulent Ozpolat, Gabriel Lopez-Berestein. _Md Anderson Cancer Center, Houston, TX_.

Tumor microenvironment is the key factor for tumor progression. Stromal cells which consist of, tumor associated macrophages(TAMs), tumor associated fibroblast as well as cancer stem cells has been implicated in the acquisition of oncogenic phenotype via cell-cell communication. Exosomes are the major players in tumor microenvironment that contributes to tumorigenesis. Exosomes carry genetic information from host cells to another cell to reprogram the recipient cell. Exosomes from TAMS has emerged as important mediators of tumor growth. Here, we report that GATA3 is released abundantly from TAM cells via exosomes and promote oncogenesis. GATA3 acts as an oncogenic protein and silencing GATA3 by siRNA led to decrease in tumor cell viability. Our data showed that silencing GATA3 increased the release of IL-4 and IL-13 from EOC cells, which leads to M2 cell polarization from M0 cells. Our results suggest that GATA3 released from macrophage exosomes contributes to tumor growth by affecting intracellular processes as well as tumor microenvironment.

#1982

Sustained CXCR2 signaling promotes secretory phenotype in cancer-associated fibroblasts of pancreatic cancer via activation of NFkB.

Mohammad Awaji, Michelle Varney, Abhilasha Purohit, Lingyun Wu, Sugandha Saxena, Sushil Kumar, Surinder K. Batra, Rakesh K. Singh. _University of Nebraska Medical Center, Omaha, NE_.

Pancreatic cancer (PC) is the fourth leading cause of cancer-related deaths in the US and remains one of the most challenging malignancies. Desmoplasia and tumor-supporting inflammation are hallmarks of PC. In addition to the autonomous aggressiveness feature of PC malignant cells, host tumor microenvironment contribute greatly to tumor progression and spread. Cancer-associated fibroblasts (CAFs), represent the major component of the tumor microenvironment in PC, are implicated in facilitating therapy resistance and metastasis. Recent reports emphasize the concurrence of multiple subtypes of CAFs that carry out different functions. CXCR2 is a chemokine receptor that is known for its role during inflammation by recruiting innate immune cells and the induction of angiogenesis. In PC, oncogenic Kras upregulates CXCR2 and its ligands (CXCL1-3 and 5-8), which are linked to inducing tumor proliferation and immunosuppression as well as induction of malignant cells stemness contributing to therapy resistance. Deletion of CXCR2 in a syngeneic mouse model (Cxcr2-/-) of PC increased the abundance of fibrosis revealing a potential undescribed role of CXCR2 in regulating CAFs. We hypothesize that CXCR2 regulates CAFs function in PC and contribute to CAFs heterogeneity. Co-culture of PC tumor cells in the conditioned media of CAFs and co-culturing CAFs in conditioned media of tumor cells revealed an oncogenic Kras-dependent interaction. CAFs enhanced the growth of PC cells with oncogenic Kras; whereas, conditioned media of the PC cells with oncogenic Kras has inhibited CAFs growth. Paracrine factors derived from tumor cells decreased the expression of the fibrotic CAFs-associated markers including αSMA and collagen I, and increased the expression of immunosuppressive cytokines and tumor-promoting chemokines including IL-4, IL-10, IL-13 and CXCL7. Utilizing recombinant CXCR2 chemokines and CXCR2 inhibitors, we confirmed the involvement of CXCR2 in this secretory phenotype. CXCL1 and CXCL8 exhibited a similar effect on CAFs by downregulating αSMA and upregulating IL-4, IL-13 and CXCL7. CXCR2 inhibitors reverted some of the observed effects. Examining downstream signaling pathways revealed that this effect is mediated through activation of NFκB. We further confirmed these findings in-vivo. Immunohistochemistry revealed higher αSMA expression in the tumors implanted in Cxcr2-/- compared to wildtype mice. Altogether, we demonstrate that sustained signaling through CXCR2 activates NFκB and induces a secretory phenotype of CAFs in PC that upregulates pro-tumor immunosuppression growth factors.

#1983

Cancer exosomes immunosuppressive activity in the absence of contaminating soluble factors is multifactorial.

Shin La Shu,1 Junko Matsuzaki,1 Cheryl Allen,1 Yunchen Yang,2 Edward Hurley,2 Kunle Odunsi,1 Yun Wu,2 Marc S. Ernstoff1. 1 _Roswell Park Comprehensive Cancer Center, Buffalo, NY;_ 2 _State University of New York at Buffalo, Buffalo, NY_.

Cancer derived exosomes, along with soluble factors such as growth factors, cytokines and chemokines have been identified as integral immunosuppressive pathways employed by cancer cells. Using melanoma cell lines as a model for generating human melanoma exosomes (HMEX), we studied the soluble factors associated with exosome purification methods. A Luminex 13-plex T cell activation kit was used to detect the presence of soluble factors in exosome isolates. We have demonstrated that combining Ultrafiltration and Size Exclusion Chromatography (REIUS method) can yield up to 58-fold more exosomes than ultracentrifugation (UC) with greatly reduced concentrations of contaminating soluble factors (IL-8: 4.6 pg/ml by REIUS versus 489 pg/ml by UC | IL-10: 1,052 pg/ml by REIUS versus 34,500 pg/ml by UC). A novel exosome-based Compact Surface Plasmon Resonance Biosensor demonstrated that REIUS-isolated exosomes abundantly express PD-L1 on their surface compared to UC-purified exosomes. These HMEX significantly inhibited a cloned NY-ESO tumor antigen-specific patient derived T cell function when co-cultured with a NY-ESO positive melanoma cell line for 4 h (IFN-γ positive T cell population: 78.5 ± 0.03 % versus 49.05 ± 0.07 %). Blocking with anti-PDL1 or anti-IL-10 failed to restore T cell function (IFN-γ production), while both anti-PD-L1 and anti-IL-10 antibodies partially reverse this suppression, thereby identifying IL-10 and PD-L1 as a novel collaborating inhibitory pathway. Taken together, we report that different exosome isolation methods impact the nature of the exosomes and contaminating soluble factors and should be chosen with caution. In addition, our data suggests a novel role for tumor-derived exosomes in the IL-10 and PD1-PDL1 axis crosstalk that impacts functional activity of T cells.

#1984

Anisotropy of collagen I matrix influences therapeutic response and secretome of Luminal A cells.

Ana M. Reyes,1 Jorge Almodovar,2 Elaine Alarid,3 Maribella Domenech1. 1 _University of Puerto Rico-Mayaguez, Mayaguez, PR;_ 2 _University of Arkansas, AR;_ 3 _University of Winsonsin-Madison, Madison, WI_.

High degree of collagen I fiber orientation has been associated with guided tumor cell invasion, but little is known about its impact in therapeutic response. Anisotropic substrates have been shown to influence the phenotype of stem cells which suggests that it can influence tumor cell behavior beyond cell migration. We hypothesized that the anisotropy of collagen I matrix impacts therapeutic response by modulating the secretome of breast cancer cells and this effect is mediated by integrin signaling. To test our hypothesis, the cell secretome, response to hormone ablation therapy and integrin inhibitors were evaluated in breast tumor cells cultured in collagen type 1 substrates of defined orientation. The orientation of electrospun collagen I fibrils was regulated by adjusting the syringe flow rate and collector velocity to obtain random (<70%) or aligned (>80%) oriented fibrils. Average porosity (45± 3.4 %), matrix stiffness (0.8 ±0.24 MPa) and fiber diameter (1.6 ± 0.86μm) were kept constant across collagen substrates. Estrogen-dependent Luminal A cells (MCF-7 and T-47D) cells were cultured on collagen substrates in a hormone-deprived environment for 3-6 days. Polycaprolactone (PCL) fibrous substrates of aligned and random orientation were used as negative fibrous controls for integrin signaling. Evaluation of cell growth showed that a fibrillar structure in combination with a high degree of fibril orientation significantly increased cell proliferation (>75%) as compared to gelatin-coated tissue culture plastic (TCP). Collagen fibers significantly supported hormone-independent tumor growth at levels equivalent to or above those observed in the estrogen-treated control. Increased proliferation levels were supported on collagen fibrous substrates treated with Tamoxifen and Fulvestrant, and this effect was enhanced in aligned collagen I. Inhibition of α2β1 integrin resulted in enhanced proliferation rates in random fibers and suppression of cell growth in aligned fibers. PCL aligned substrates where not sufficient to support hormone-independent cell growth. MCF-7 cells on collagen substrates significantly stimulated the growth (>7 fold) of adjacent cells on TCP in a dose-response manner as follows: aligned > random > gelatin. Expression levels of 1000 factors were quantified in conditioned medium to identify differences in the secretome signatures associated with cell-matrix signaling. MCF-7 cells cultured on collagen fibrous substrates displayed secretome signatures that were different across all substrates. Fourteen factors were identified to be upregulated in fibrous substrates by more than ten folds including apoptotic regulators RalA and BCL-2. Our results show for the first time the influence of collagen I fibril orientation in supporting hormone-independent growth and as a novel regulator therapeutic response in luminal A cells.

#1985

Reassessment of exosome composition.

Dennis K. Jeppesen, Jeffrey L. Franklin, James N. Higginbotham, Qin Zhang, Robert J. Coffey. _Vanderbilt University Medical Center, Nashville, TN_.

The heterogeneity of small extracellular vesicles and the presence of non-vesicular extracellular matter is a major obstacle to the study of exosomes. Here we employ two complementary methods, high-resolution density gradient fractionation and direct immunoaffinity capture, to demonstrate separation of small extracellular vesicles from non-vesicular material, and exosomes from other types of small extracellular vesicles. Cells secrete Argonaute 1-4 independently of exosomes. Extracellular RNA and RNA-binding proteins are differentially expressed between exosome and non-vesicle compartments. Exosomes do not possess a cytoskeleton and exclude glycolytic enzymes. Annexin A1 is a novel and specific membrane-associated protein marker of microvesicles shed directly from the cell plasma membrane, distinct from exosomes in vitro and in vivo. Small extracellular vesicles are not vehicles of active DNA release. Instead we propose a new model for active secretion of extracellular DNA through an autophagy- and multivesicular endosome-dependent but exosome-independent mechanism. A reassessment of exosome composition is necessary.

#1986

Breast cancer-secreted exosomes stimulate beige/brown differentiation and reprogram metabolism in stromal adipocytes to promote tumor progression.

Juanjuan Li,1 Qi Wu,1 Zhiyu Li,1 Si Sun,1 Shan Zhu,1 Lijun Wang,1 Juan Wu,1 Yimin Zhang,1 Jingping Yuan,1 Shengrong Sun,1 Changhua Wang2. 1 _Renmin Hospital of Wuhan University, Wuhan, China;_ 2 _Wuhan University School of Basic Medical Sciences, Wuhan, China_.

Background: Emerging evidence supports the pivotal roles of adipocytes in breast cancer progression. Beige/brown adipose tissue that activates thermogenesis is suggested to contribute to the hypermetabolic state. However, the mediators and mechanisms remain unclear.

Methods: Survival probabilities for recurrence-free survival (RFS) of 106 breast cancer patients were estimated using the Kaplan-Meier method based on the immunohistochemistry results of several biomarkers. Biochemical studies were performed to characterize the novel interrelation between breast cancer cells and adipocytes.

Results: In the survival analysis, high MCT4 expression in stromal adipose tissue and overexpression of CD36 or FATP1 in malignant tissue predict poor prognosis for breast cancer patients. We show that tumor-surrounding adipocytes exhibit an altered phenotype in terms of upregulated beige/brown characteristics and increased catabolism. This increase in catabolism is associated with an activated state characterized by the release of metabolites, including free fatty acids, pyruvate, lactate and ketone bodies. Likewise, tumor cells cocultivated with mature adipocytes exhibit metabolic adaptation and an aggressive phenotype in vitro and in vivo. Mechanistically, we show that tumour cells induce beige/brown differentiation and remodel metabolism in resident adipocytes by secreting exosomes that carry high levels of miRNA-144 and miRNA-126. Cancer cell-secreted miRNA-144 promotes beige/brown adipocyte characteristics by downregulating the MAP3K8/ERK1/2/PPARγ axis. We also found that exosomal miRNA-126 remodels metabolism by disrupting IRS/Glut-4 signalling, activating the AMPK/autophagy pathway and stabilizing HIF1α expression in imminent adipocytes. In vivo inhibition of miRNA-144 or miRNA-126 decreases adipocyte-induced tumour growth.

Conclusions: These results demonstrate that cancer-derived exosomal miRNA-144 and miRNA-126 can reprogram the systemic energy metabolism to facilitate tumor progression by inducing beige/brown differentiation and enhancing catabolism in recipient adipocytes.

#1987

Exosome from CAF-ESCC aggravates tumor progression by its containing molecular miR-3656.

Xiaojia Chen,1 Yuan Jin,1 Bihui Zhang,1 Xiaocen LI,2 Baoqing Tian,1 Qiang Wang,1 An Hong1. 1 _Institute of Biomedicine, Jinan University, Guangzhou, China;_ 2 _Department of Biochemistry and Molecular Medicine, University of California Davis, Sacramento, CA_.

Background: Exosome plays an important role in cell communication and cell environment. But the mechanism of how exosome derived from cancer-associated fibroblast (CAF) influent esophageal squamous cell carcinoma (ESCC) progression is not clear. In this work, compared with normal fibroblast-derived exosomes (NDEs), we found that CAF derived exosomes (CDEs) can enhance the growth, migration, and invasion of ESCC cells obviously. Therefore, we would like to clarify the role of CDEs in ESCC progression.

Methods and Results: Using microRNA array to analyze the different miRNAs in the CDEs and NDEs, we got a series of up-regulation miRNAs and two down-regulation miRNAs in CDEs. Among them, miR-3656 shows a significantly highest level. Then, we transfected miR-3656 mimics to the ESCC cells and found the mimics increased the growth, migration, and invasion of ESCC cells in cell proliferation assay. cell scratch tests and chamber invasion experiments. We established recombinant ESCC cells with miR-3656 high expression by the lentiviral system. The cells were injected into mice to form the xenograft tumor. The results showed the miR-3656 can enhance the tumor formation. After that, aided by TargetScan software, we found ACAP2, one of miR3656 targets, was significantly down-regulating in ESCC cells at the high level of miR-3656. When ACAP2 expression was knocked-down by siRNA, the proliferation and migration of ESCC cells was increased, which indicated ACAP2 a potential anti-tumor factor. Furthermore, we detected the different proteins between ESCC cells with high expression miR-3656 and normal ESCC cells through LC_MS; the results were analyzed by Integrate Pathway Analysis (IPA) system and many differential proteins were found to be involved of PI3K/AKT and Wnt pathways.

Conclusions: In this study, we found the CDEs promote the progression of their sounding ESCC cells in vitro and in vivo. The main reason is that CDEs carry some tumor-promoting miRNAs that influent the target ESCC cells. Especially, miR-3656 expresses the highest level of miRNAs in CDEs. It not only decreases the expression of ACAP2 but also impacts the expression of proteins involved PI3K/AKT and Wnt pathways in ESCC cells. The results indicate the CDEs playing a very important role in ESCC progression.

#1988

Metastatic v non-metastatic cancer exosomes in brain metastasis.

Megan Sayyad, Madhavi Puchalapalli, Chris Canal, Theresa Swift-Scanlan, Andrew Ottens, Jennifer E. Koblinski. _Virginia Commonwealth University, Richmond, VA_.

Brain metastasis affects 10-30% of breast cancer patients, but it is unclear how breast cancer cells can colonize the unique and mostly cellular brain environment. Exosomes, endosomal-derived extracellular vesicles, have been reported to support tumorigenesis, mediate tumor-stromal interactions, and drive pre-metastatic niche formation, ultimately creating sites conducive to cancer development. Still, their role in brain metastasis remains to be fully understood. Glial cells, namely astrocytes, are important contributors to brain metastasis and secrete exosomes that support metastatic outgrowth in the brain. Thus, our study aims to investigate if breast cancer-derived exosomes can transform astrocytes towards a pro-metastatic phenotype. To do this, we isolated exosomes from 4T1 metastatic and 67NR non-metastatic mouse mammary carcinoma cell conditioned media using ultracentrifugation. Exosomes were characterized using Western immunoblot, dynamic light scatter analysis, and transmission electron microscopy. Using confocal microscopy, we found that mouse astrocytes can internalize 4T1 exosomes in vitro. Preliminary studies using qPCR analysis revealed that mouse astrocytes treated with 4T1 exosomes compared to those treated with 67NR exosomes exhibit differential gene expression, suggesting that there is a unique response profile associated with metastatic versus non-metastatic breast cancer-derived exosomes. To further assess this, we are investigating changes in the astrocyte secretome in response to 4T1 and 67NR exosome treatment using protein mass spectrometry. Taking a global approach, we have also been assessing differences between 4T1 and 67NR exosomal miRNA, and protein that could promote a shift in astrocytes towards a pro-metastatic phenotype. For these studies, we have implemented the Nanostring nCounter Analysis System and protein mass spectrometry, respectively. Proteomics assessment revealed distinct differences between the 4T1 metastatic and 67NR non-metastatic exosomes, whereby we identified 259 proteins that were enriched in 4T1 exosomes and 71 proteins that were decreased in 4T1 exosomes compared to 67NR's. Proteins enriched in 4T1 exosomes were found to be associated with RNA binding and translation machinery, as well as abnormal cell adhesion, and BRCA1 interactions. Proteins identified to be decreased in 4T1 exosomes were found to play a role in IL-6 and IL-12 signaling events, the estrogen signaling pathway, and syndecan-2 signaling. These findings indicate that 4T1 and 67NR exosomes differ in protein cargo and these differences may be important in facilitating brain metastasis. Altogether, these studies will provide a more comprehensive assessment of the differences between metastatic versus non-metastatic cancer exosomes, and ultimately, provide novel insight into understanding breast cancer metastasis to the brain.

#1989

Breast cancer-derived exosomes modulate the endocytic pathway in brain endothelial cells.

Golnaz Morad,1 Christopher V. Carman,2 Marsha A. Moses3. 1 _Harvard University, Boston Children's Hospital, Boston, MA;_ 2 _Harvard T.H. Chan School of Public Health, Boston, MA;_ 3 _Boston Children's Hospital, Harvard Medical School, Boston, MA_.

Breast cancer brain metastasis is associated with high mortality and has shown a rising trend in incidence during the recent years. Given the rapid progression of brain metastasis, elucidation of the early events that lead to brain metastasis will pave the way to identifying potential diagnostic and therapeutic targets for early intervention. To explore the early mechanisms of breast cancer brain metastasis, we focused on the role of breast cancer-derived exosomes in this process. While extensive studies have explored the role of tumor-derived exosomes in tumor progression, the current understanding of the contribution of tumor-derived exosomes to brain metastasis remains limited. Using a mouse model of brain metastasis, we showed that pre-treatment of mice with exosomes derived from the brain-seeking MDA-MB-231 breast cancer cell line (Br-Ex) increases brain metastasis growth. This facilitation of brain metastasis was not observed with exosomes derived from the parental MDA-MB-231 cells (P-Ex). It is widely acknowledged that during early stages of brain metastasis, tumor cells grow along the brain vasculature, the blood brain barrier (BBB). To understand the mechanisms of the exosome-derived facilitation of brain metastasis, we studied the interactions between exosomes and the BBB. Using state-of-the-art models of the BBB and high-resolution microscopy, we have identified, for the first time, the mechanisms by which Br-Ex modulate the endocytic pathway in brain endothelial cells to decrease exosome degradation. Interestingly, our mechanistic studies showed that these effects are induced through the transfer of exosomal miRNAs enriched in Br-EX. Moreover, we have shown that Br-Ex can exclusively change the expression of integrins in brain endothelial cells in a way that it could alter the microenvironment around the BBB in favor of metastatic tumor cells. These findings indicate that exosomes derived from a brain-seeking subpopulation of breast cancer cells can exclusively modify the physiological regulation of the BBB at multiple levels to accelerate metastatic growth in the brain microenvironment. Taken together, these studies increase our understanding of the early events that facilitate brain metastasis and provide multiple potential targets for diagnostic and therapeutic applications in brain metastasis. (This work was supported by the Breast Cancer Research Foundation and NIH R01CA185530.)

#1990

Cjc-1134-pc, a long-acting glp-1r agonist, effects in modulating pro-inflammatory cytokines in the tumor environment.

Xiping Liu, Sean Hong, Shijuan Wu, Zhiwen Yao, Qiang Xie. _ConjuChem, Irvine, CA_.

The incidence rates of The incidence rates of diabetes and cancer are growing globally. It is reported that type 2 diabetes (T2D) is associated with increased risk of cancers, which suggests a synergy. There is a strong association among inflammation and cancer on diabetes, which is reflected by the high cytokine prevalence in the microenvironment. Previously our studies showed that IL-2 and IFN-γ were downregulated but IL-6 and TNFα were upregulated in diabetic mice. The levels of IL-2, IFN-γ, IL-6 and TNFα returned to baseline levels similar to that of non-diabetic mice after the treatment of CJC-1134-PC. In order to explore the benefits of CJC-1134-PC beside its glucose lowering effects, the level of pro-inflammatory cytokines has been measured and compared between vehicle and CJC-1134-PC treated BALB/c mice bearing CT26 colon tumors. All mice bearing tumors showed high IL-6 and TNF-r compared with the normal mice. Interestingly, after CJC-1134-PC treatment, IL-6 and TNFα levels decreased significantly (P < 0.05), which makes the cytokine profile close to the reported levels in normal mice. Overall, the data suggests that CJC-1134-PC plays a role in modulating inflammation, which could benefit cancer treatment in addition to achieving glycemic control.

#1991

Exploring the potential of exosomes for cancer diagnosis and management: Comparative analysis of isolation methods for optimum yield purity and downstream applications.

Girijesh K. Patel. _University of South Alabama Mitchell Cancer Institute, Mobile, AL_.

Exosomes have received significant attention due to their involvement in several diseases, including cancer, and are being explored as a tool for disease diagnosis and management. As a result, several kits based on different principles are now available in the market for exosome isolation. In this study, we have compared the relative efficacy of four commercial kits from Invitrogen, 101Bio, Wako and iZON along with conventional ultracentrifugation-based method for exosome yield, purity and quality for downstream applications. Cancer cell culture supernatant was used as an abundant and uniform source of exosomes for all methods and exosome quantity, size distribution, zeta potential, marker expression, RNA (qRT-PCR) and protein (mass spectrometry) quality was determined. The Invitrogen method showed the highest yield but the preparation showed broader size distribution likely due to co-precipitation of microvesicles and had the least magnitude of zeta potential (dispersion stability). Exosome preparations from other methods showed accepted size range (<150 nm) with a variable zeta potential (-12mV to -29mV) with good purity. The gel filtration method; however, showed a broader size distribution in the lower size range, which could be due contamination of aggregates of proteins and vitamins molecules. Quality of RNA isolated from all preparations was comparable; however, proteins isolated from Invitrogen method-based exosomal preparation showed contamination of polyethylene glycol (PEG) in mass spectrometry analyses. Similarly, other chemical impurities coming from the precipitant in Invitrogen kit could also be the reason for toxicity of resultant exosomal preparation in biological growth measurement assay. Together, these findings from comparative analyses should serve as a guide for choice and further optimization of exosome isolation methods for their desired downstream applications in cancer biology, therapy and/or biomarker discovery.

#1992

Mesenchymal stem cell-derived interleukin-28 can drive the selection of apoptosis resistant bone metastatic prostate cancer.

Jeremy J. Mcguire,1 Leah Cook,2 Jeremy Frieling,1 Ayaz Muhammad,1 Harshani Lawrence,1 Nicholas Lawrence,1 Conor Lynch1. 1 _Moffitt Cancer Center, Tampa, FL;_ 2 _University of Nebraska Medical Center, Omaha, NE_.

Bone metastatic prostate cancer is painful and incurable. We believe that understanding the interactions of tumor cells and the surrounding stromal cells may provide new therapeutic options. Bone metastatic prostate cancer promotes mesenchymal stem cell (MSC) recruitment and differentiation into osteoblasts, leading to the clinically often-observed osteogenic phenotype. However, the reciprocal roles of bone- marrow derived MSCs on prostate cancer cells are less explored. Here, we discovered that MSCs significantly suppress prostate cancer growth in vitro and in vivo. Mechanistically, we show that MSC-derived interleukin-28 (IL-28) promotes prostate cancer cell apoptosis via the IL-28 receptor alpha (IL-28Rα). However, chronic exposure to MSCs yields prostate cancer cell populations that are resistant to IL-28 induced apoptosis and therapeutics such as docetaxel. In vivo, we observed that MSC selected prostate cancer cells rather than being suppressed, grow at a significantly enhanced rate in bone. The increase in apoptosis resistance is accompanied by a shift in downstream signaling of the IL-28Rα from STAT1 to STAT3. Reduction of STAT3 levels or pharmacological inhibition (STI-201) significantly reduces the growth of MSC selected prostate cancer cells in vitro and in vivo. Collectively, these data shed light on how MSCs in the bone marrow microenvironment initially protect against prostate cancer cell establishment but in doing so, help select for aggressive, apoptosis resistant bone metastatic prostate cancer cells that are more aggressive. Given that the majority of prostate to bone metastases are positive for STAT3 expression and activity, our data provide rationale for the therapeutic targeting of STAT3 in bone metastatic prostate cancer.

### Therapeutic Metastasis Prevention

#1993

Gluthathione peroxidase 2 downregulation in the PyMT mammary tumor model leads to an aggressive phenotype due to stimulation of angiogenesis and tumor stemness via HIF1 alpha signaling.

Zuen Ren,1 Huizhi Liang,1 Outhiriaradjou Benard,1 Kimita Suyama,1 Joseph M. Albanese,1 Larry Norton,2 Wei Zheng,1 Rachel B. Hazan1. 1 _Albert Einstein College of Medicine, Bronx, NY;_ 2 _Memorial Sloan-Kettering Cancer Center, New York, NY_.

Breast tumors are known to undergo high levels of mitochondrial oxidative phosphorylation leading to accumulation of reactive oxygen species (ROS), which in turn potentiate cancer stem cells and chemoresistance. This was shown to be due to HIF1α stabilization by ROS; however, the signaling events downstream of HIF1α regulating the cancer stem cell (CSC) phenotype remain unknown. Kaplan Meier survival curves upon the gene profiling database of breast cancer patients showed that reduced levels of the ROS scavenging enzyme, glutathione peroxidase 2 (GPx2) in breast cancers was associated with shorter patient survival duration. Consistent with this idea, we found that GPx2 was downregulated in metastatic mammary tumor cells, suggesting GPx2 loss enhances metastasis. Indeed, GPx2 knockdown in poorly metastatic cells increased ROS production, shortened tumor latency, and stimulated dramatic mammary tumor growth and metastasis. These effects were associated with marked increases in angiogenesis, CSC-like properties, and chemoresistance. Here, we show that GPx2 downregulation in mammary epithelial cells increases ROS production and HIF1α stabilization which were accompanied by VEGF upregulation and enhanced angiogenesis. On the other hand, GPx2 loss caused dramatic mammosphere formation and resistance to doxorubicin, implying effects on stemness leading to chemoresistance. These data suggest that GPx2 loss is a critical event that drives mammary tumorigenesis and metastasis via HIF1α signaling leading to stimulation of angiogenesis and stemness properties. Understanding the ramifications of GPx2 loss in breast cancer will allow us to identify key regulators of stemness and chemoresistance, with the goal of identifying novel drug targets in metastasis.

#1994

Dual inhibition of JAK and Src in pancreatic cancer: A novel and synergistic combination to target the tumour and its microenvironment.

Ashleigh Parkin, Angela Steinmann, Danielle Froio, Nicolas Vogel, Jennifer Man, Kendelle Murphy, Erin Mosmann, Julia Yin, Jaswinder Samra, Anthony Gill, Paul Timpson, Marina Pajic. _The Garvan Institute of Medical Research, Australia, Darlinghurst, Australia_.

Introduction: Pancreatic cancer (PC) has a 5-year survival of only 6%, and persists as the 4th most common cause of cancer-related death in Western societies. A more tailored treatment approach may be beneficial as the current standard-of-care therapies offer only a modest increase in overall patient survival. Recent large-scale genomic studies have revealed that the Src/JAK/STAT3 signalling pathway is deregulated in up to 35% of PC, and is yet to be systematically examined in this disease. Consequently, we hypothesized that targeting pancreatic tumours with activated JAK/STAT3 signalling with selective JAK1/JAK2 or JAK3 inhibitors and an Src inhibitor represents a promising novel therapeutic strategy for this disease.

Materials and methods: We utilized well-annotated patient-derived cell-line models (ICGC), along with cell-lines generated from the aggressive KPC mouse model. Using these pre-clinical models we assessed the in vitro efficacy of therapeutic strategies involving Src/JAK/STAT3 inhibition, using cell proliferation assays, 2D-drug synergy screens, 3D organotypic invasion assays, and 3D organoid cultures. Extracellular matrix integrity post-treatment was assessed using second-harmonic generation (SHG) imaging and picrosirius staining. Multiplex cytokine arrays, and single-cell transcriptomics were used to delineate mechanisms and assess pathway modulation following treatment. To examine in vivo efficacy, we utilized a syngeneic KPC mouse model as well as patient-derived xenograft models.

Results: We show that selected JAK and Src-inhibitors inhibit cell proliferation in candidate PDCLs and KPC lines, characterized by activated Src/JAK/STAT3 signalling, with combination therapy being synergistic in the majority of these cell-lines. Cell invasion was significantly inhibited in organotypic matrices, and there was decreased collagen contractility, and reduced fibrillar collagen coverage. We show significant changes in cytokine production, and have also identified several pathways that are modulated following treatment, via the use of single-cell transcriptomics. We also demonstrate the in vivo efficacy of these therapies, and show their ability to reduce regulatory T-cells, MDSCs and tumour-associated macrophages.

Conclusion: Our findings demonstrate the potential for tailored therapeutic strategies involving Src/JAK/STAT3 inhibition in PC, and suggest that therapeutic efficacy may be the result of targeting both tumour cells and the tumour microenvironment, as well as by overcoming tumour-induced immunosuppression.

#1995

**Low-dose eribulin suppresses lung metastasis of osteosarcoma** in vitro **and** in vivo **.**

Kenta Watanabe,1 Yoshihiro Yui,2 Satoru Sasagawa,2 Kayo Suzuki,1 Masahiko Kanamori,1 Taketoshi Yasuda,1 Tomoatsu Kimura1. 1 _University of Toyama, Toyama, Japan;_ 2 _Research Institute Nozaki Tokushukai Hospital, Osaka, Japan_.

Osteosarcoma is the most common malignant bone tumor among children and adolescents. The prognosis of osteosarcoma remains poor in these thirty years because the effective therapeutic option for lung metastasis has not been established. Therefore, the development of effective metastatic therapy is required. Eribulin, a new microtubule targeting agent, has been indicated for recurrent breast cancer and malignant soft tissue sarcoma. Clinical trials of eribulin showed that it elongated overall survival period rather than disease-free survival period. These results motivated us to investigate the effect of eribulin on metastasis suppression of osteosarcoma. Here, we examined the inhibitory effect of eribulin on lung metastasis and its mechanism using murine osteosarcoma metastasis model. In vivo experiments: Eribulin was intravenously injected by two administration methods, (1) high dose administration on standard schedule; 1 mg/kg q7d × 2, (2) frequent low dose administration; 0.3 mg/kg q4d × 4. Both methods sufficiently reduced circulating tumor cells in blood and inhibited lung metastasis. Method (1) also inhibited primary tumor growth, but induced severe body weight loss. Method (2) had little effect on primary tumor growth and showed little body weight loss. In vitro experiments: The IC50 concentration of eribulin for metastatic osteosarcoma cell line, LM8, was about 20 nM. Pharmacokinetics studies have shown that eribulin intravenously administered at 1 mg/kg presents with a brief high-concentration phase which surges to ≥100 nM followed by a long low-concentration phase which stabilizes at ~10 nM for one week. These two phases sandwiched the IC50 concentration of eribulin. Thus, we separately investigated that the effect of high and low eribulin concentrations on LM8. High eribulin concentrations induced cell cycle arrest and apoptosis in LM8, whereas low eribulin concentrations changed cell morphology and decreased cell migration through reducing directionality and focal adhesion turnover. Reduced peripheral localization of adenomatous polyposis coli (APC) protein by eribulin might cause these effects. In a three-dimensional collagen culture system, low eribulin concentrations inhibited tumor cell infiltration and colony formation. The anti-metastatic effects at low concentrations backed-up the results of our in vivo experiments by frequent low dose administration method. Eribulin is a potential therapeutic option for lung metastasis of osteosarcoma. Frequent low dose administration method with fewer side effects enables the combination with other chemotherapeutic agents and broadens the application of eribulin as an anti-metastatic drug for osteosarcoma.

#1996

Epigenetic reactivation of BAI1/ADGRB1 suppresses tumor invasion by preventing TGFβ1-induced mesenchymal switch in glioblastoma.

Satoru Osuka,1 Liquan Yang,1 Dan Zhu,1 Hideharu Hashimoto,2 Narra S. Devi,1 Erwin G. Van Meir1. 1 _School of Medicine and Winship Cancer Institute, Emory University, Atlanta, GA;_ 2 _Thomas Jefferson University, Philadelphia, PA_.

Glioblastoma (GBM) is the most common and lethal type of malignant brain tumor in adults. GBM cells are highly invasive and diffusely infiltrate throughout the brain, which strongly restricts multimodal therapies. Acquiring a better knowledge of molecular defects underlying GBM invasion is essential for the development of effective therapies.

Brain-specific Angiogenesis Inhibitor 1 (BAI1/ADGRB1) is a transmembrane receptor of the adhesion GPCR family widely expressed in normal brain, but its expression is lost in the majority of glioblastoma through epigenetic silencing and restoration of its expression can inhibit glioma growth (Zhu D. et al, Cancer Res, 2012). Recently, we reported that BAI1 protects p53 from Mdm2-mediated degradation and regulate tumor growth in medulloblastoma (Zhu D. et al, Cancer Cell, 2018). However, it is unclear whether BAI1 loss is important for tumor invasion and the mesenchymal phenotype in GBM.

Microarray analysis of the GBM TCGA dataset revealed that low BAI1 mRNA expression correlates with poor outcome and with expression of many key mesenchymal genes, including Fibronectin1, SLUG, and TWIST1. Restoration of BAI1 expression in human GBM cells suppresses mesenchymal gene expression in culture, and dramatically decreases brain tumor invasion in mice xenografts. Mechanistically, we found that the N-terminal thrombospondin type 1 repeat (TSR#1) of BAI1 inhibits the maturation process of TGFβ1, a key growth factor involved in EMT. BAI1 is silenced epigenetically in GBM cells by methylated CpG-binding protein MBD2, and its expression can be reactivated by KCC-07, a blood-brain barrier permeable MBD2 inhibitor. We found that restoration of BAI1 expression by KCC-07 treatment dramatically reduced tumor cell invasion in orthotopic xenografts model of GBM cells.

These experiments demonstrate that epigenetic silencing of BAI1 is important for activation of the GBM invasive phenotype through TGFβ1 pathway activation. Epigenetic targeting of this process by KCC-07 can reduce GBM invasion and improve patient survival.

#1997

The Rac inhibitors HV-107 and HV-118 as potential therapeutics for metastatic breast cancer.

Grace E. Velez,1 Cornelis Vlaar,2 Eliud Hernandez,2 Anibal Valentin,1 Linette Castillo-Pichardo,1 Suranganie Dharmawardhane2. 1 _UCC, Bayamon, Puerto Rico;_ 2 _University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico_.

Breast cancer is the first cause of death in women globally. Metastatic breast cancer is stimated to affect more than a quarter of million of women in the US. Due to the lack of effective treatment, metastasis remains the main cause of breast cancer mortality. Therefore, it is imperative to develop novel effective strategies against metastasis. A promising target for anti-metastatic therapy is the Rho GTPase Rac because it plays a key role in metastatic cancer progression by regulating cellular processes such as adhesion, migration, proliferation and survival. Our group developed Ehop-016, a small molecule that inhibits Rac in metastatic breast cancer cells with an IC50=1µM and significantly reduces tumor growth, angiogenesis, and metastasis in a mouse model of metastatic breast cancer. However, its relative bioavailability is moderate and should be improved. Therefore, to find a compound with increased potency and bioavailability, we tested several Ehop-016 derivatives. Using Rac pulldown assays we show that HV-107 and HV-118 inhibit Rac activation by 60% in MDA-MB-231 and MDA-MB-435 metastatic breast cancer cells at 100 and 250nM, respectively. MTT assays show HV-107 (at ≥500nM) and HV-118 (at ≥50nM) significantly inhibit metastatic breast cancer cell viability, while showing minimal toxicity towards non-cancerous cells. Cell cycle analysis by flow cytometry demonstrates a G2-M arrest and a prominent sub-G1 population, indicative of cell death, in metastatic breast cancer cells treated with HV-107 (1000 nM) and HV-118 (100 nM). To evaluate apoptosis as a potential cell death mechanism, we measured caspase 3 activity. Our results show HV-107 and HV-118 significantly induce caspase 3 activity by approximately 1.6-fold at 1000 and 100nM, respectively in metastatic breast cancer cells. Therefore, these Rac inhibitors affect cell viability by inhibiting cell cycle progression and inducing apoptosis. Finally, we tested HV-118 (at 1mg/kg BW) in a mouse model of metastatic breast cancer and found a 30% reduction in tumor growth and a 90% inhibition in metastasis. Taken together, our results indicate HV-107 and HV-118 have potential as anti-breast cancer metastasis therapeutics. This study was supported by awards from the Susan Komen for the Cure, NIH/NIMHHD U54MD008149, and the Puerto Rico Science and Technology Trust to SD; NIH/NCRR R25GM061838 to UPR MSC; NIH/NIMHHD RCMI 8G12MD007583RCMI, Title V PPOHA 031M10505 and Title V Cooperative P031S130068 from U.S. Department of Education to UCC; and and PRINBRE (NIH/NIGMS P20GM103475-13) Sub-Award to LCP.

#1998

Selinexor effectively inhibits tumor growth in a triple negative breast cancer brain metastasis mouse model.

Hua Chang, Marsha Crochiere, Sophie Debler, Trinayan Kashyap, Thaddeus J. Unger, Erkan Baloglu, William Senapedis, Sharon Shacham, Yosef Landesman. _Karyopharm Therapeutics, Inc., Newton, MA_.

Introduction: Breast cancer brain metastasis (BCBM) is a devastating disease and remains largely incurable. Patients with triple negative breast cancer (TNBC) develop brain metastasis at a significantly higher incidence (46%) than patients with other breast cancer subtypes. Median survival after brain metastases in TNBC can be as short as 5 months. Selinexor is a first-in-class oral SINE (Selective Inhibitor of Nuclear Export) drug that inhibits XPO1 (exportin-1/ CRM1) activity and exhibits remarkable anti-cancer activity in advanced clinical trials. Moreover, selinexor can penetrate the blood-brain barrier and shows promising anti-tumor activity in animal models of glioblastoma and central nervous system lymphoma. Previous studies have shown that most TNBC cell lines tested in vitro and in vivo are sensitive to selinexor-induced growth inhibition and cell death. To explore the possibility of using selinexor as an effective treatment for TNBC brain metastasis, a mouse model was established and tested.

Methods: TNBC cell line HCC1806 (ATCC# CRL-2335) was transformed with RediFect Red-Fluc-Puromycin and selected using puromycin to generate HCC1806-Fluc cells that expressed both puromycin resistance gene and a luciferase gene. Twenty-four nu/nu mice were inoculated intracranially with 1 x 104 HCC1806-Fluc cells. Four days post implant, mice were allocated to three groups of eight mice and treated with either vehicle, selinexor 10 mg/kg, or selinexor 15 mg/kg through oral gavage, three times per week (Mon, Wed, Fri). Body weight and condition of animals were recorded daily, and tumors were imaged twice per week (Tue and Thu). Brain tissues were collected at the end of study and analyzed by immunohistochemistry (IHC).

Results: HCC1806-Fluc cells injected intracranially successfully implanted in the brains of host mice. All mice survived the duration of the study (day 16 after treatment initiation) and no significant difference in body weight was observed between different treatment groups. Mice treated with selinexor had a significant reduction in tumor area relative to vehicle controls. Tumor growth inhibition (TGI) was 103% for selinexor at 10 mg/kg and 97% for selinexor at 15 mg/kg compared to vehicle controls. IHC showed reduced tumor cell proliferation, decreased expression of Myc and Survivin, and increased nuclear expression of p21 and p27 in tumor samples from the brain of mice treated with selinexor as compared to that of controls. Decreased expression of HDAC1 and HDAC5 was also observed in brain tumor samples from selinexor-treated mice.

Conclusions: Oral intake of selinexor effectively inhibited TNBC cell growth in a mouse model of brain metastasis. The anti-cancer effect is likely achieved through multiple signaling pathways, including cell cycle regulation, down-regulation of oncogenes and HDACs.

#1999

**As** 4 **O** 6 **suppresses triple-negative breast cancer via inhibiting EGFR/mTOR mediated EMT** in vitro **and** in vivo **.**

Illju Bae,1 Zenglin Lian,2 Ziyao Liu,3 Zhaoyan Wu1. 1 _CHEMAS Co., Ltd., Seoul, Republic of Korea;_ 2 _Institute of Biological Chinese Medicine, Beijing Yichuang Institute of Biotechnology Industry, China, Seoul, Republic of Korea;_ 3 _School of Chinese Medicine, Beijing University of Chinese Medicine, Beijing, China_.

Objective:The present study aimed to verify the anti-metastasis effects and its mechanisms of As4O6 on triple-negative breast cancer(TNBC)in vitro and in vivo.

Methods:MTT assay was used to evaluate cell proliferation. Wound healing assay and the invasion assay were carried out to investigate cell migration and invasion. The xenograft zebrafish model and orthotopic xenograft nude mouse model derived from MDA-MB-231-Green fluorescent protein (GFP) cells was established to analysis the anti-metastasis effect of As4O6in vivo. Western blot and immunohistochemistry were performed to analysis the metastasis-related proteins expression.

Result: MTT assay showed cell proliferation of TNBC cell MDA-MB-231 was inhibited in a dose-dependent manner by As4O6 treatment. Also, As4O6 suppressed cell migration and invasion by a Wound healing assay and the invasion assay, resulted in the reduction of MMP-2 and MMP-9 expression, especially for the inhibition of EGFR and mTOR phosphorylation. Meanwhile, As4O6 suppressed cancer metastasis in xenograft zebrafish model. The number of metastatic lesion in mouse lung was significantly reduced by the treatment of As4O6 in the orthotopic xenograft nude mouse model. The expression of p-mTOR, p-EGFR and EMT-related proteins such as Vimentin and E-cadherin were significantly reversed in As4O6 treated mouse lung tissues than that of non-treatment group. Furthermore, As4O6 treatment suppressed the expression of angiogenesis related proteins VEGF-a, VEGFR, CD34 and MMP2/9 in mouse lung tissues.

Conclusion:Thus, the data suggests that As4O6suppresses triple-negative breast cancer metastasis via inhibiting EGFR/mTOR mediated EMT in vitro and in vivo. As4O6 might be served as a potential useful agent for TNBC metastasis in the future.

#2000

Cancer metastasis driven by the novel MEK1 substrate MACC1 is restricted by clinically applicable MEK1 inhibitors.

Dennis Kobelt,1 Daniel Perez-Hernandez,2 Claudia Fleuter,1 Mathias Dahlmann,1 Fabian Zincke,1 Janice Smith,1 Rebekka Migotti,2 Susen Burock,3 Wolfgang Walther,1 Gunnar Dittmar,2 Ulrike S. Stein1. 1 _Charité Berlin and Max Delbrück Ctr. for Molecular Medicine, Berlin, Germany;_ 2 _Max Delbrück Ctr. for Molecular Medicine, Berlin, Germany;_ 3 _Charité Comprehensive Cancer Center Berlin, Berlin, Germany_.

Cancer metastasis is responsible for >90% of cancer deaths and remains a major treatment challenge. In search of new drivers of metastasis we identified the novel, previously undescribed gene Metastasis-Associated in Colon Cancer 1 (MACC1) in human colorectal cancer (CRC). MACC1 induces fundamental processes like proliferation, migration, invasiveness and metastasis in xenografted and transgenic mice. MACC1 has been established by us and many other groups as key player, prognostic and predictive biomarker for tumor progression and metastasis in >20 solid cancer types. Proof-of-concept for MACC1 as a therapeutic target to restrict cancer progression and metastasis was provided by transcriptional and post-transcriptional downregulation of MACC1 for several solid cancers, including CRC. Specific inhibitors targeting MACC1 post-translational protein modifications to restrict tumor growth and metastasis are not identified so far. Here we report the identification of MACC1 as a newly identified substrate of the kinase MEK1 (MAP2K1). MEK1 directly phosphorylates MACC1 leading to accelerated and increased ERK1 activation. Mutating three potential hierarchical MACC1 tyrosine phosphorylation sites abrogates MACC1 dependent target gene expression like MET, cell proliferation and motility in cell culture and importantly, metastasis in mouse xenograft models. Targeting MEK1 by RNAi or by clinically applicable MEK1 inhibitors like AZD6244 and GSK1120212 reduces MACC1 tyrosine phosphorylation and restricts MACC1-induced metastasis formation. Our findings demonstrate that MAP kinase signaling is not linearly leading only to ERK activation, but branches at the level of MEK1. In summary, MACC1 tyrosine phosphorylation is decisive for tumor growth and metastasis. The fundamental finding of MACC1 being a newly identified MEK1 substrate opens new therapeutic options with potential for clinical translation. Targeting MACC1 tyrosine phosphorylation using MEK1 inhibitors thereby intervening in MACC1-induced metastasis aims at the ultimate goal of personalized therapies for inhibition of cancer progression and metastasis, resulting in improved patient survival. Since MACC1 is confirmed as decisive driver for tumor growth and metastasis in a variety of solid cancers, the findings made here for CRC might be translated to further solid tumor types. The usefulness of MACC1 as therapeutic target towards MEK1 inhibitor treatment requires confirmation in clinical trials.

#2001

Inhibiting voltage-gated sodium channel activity in medullary thyroid cancer using small molecule compounds.

Paras Ahuja, Rachael Guenter, Jaden Cowan, Yazen Shihab, Jason Whitt, Herbert Chen, Sadanandan Velu, Renata Jaskula-Sztul. _University of Alabama at Birmingham, Birmingham, AL_.

Medullary thyroid cancer (MTC) is a type of neuroendocrine cancer (NE) with increasing incidence worldwide. Compared with well-differentiated thyroid cancers, distant metastases are more commonly observed in MTC patients, ranging from 7-23%. The inability to treat metastatic MTC contributes to a decreased patient survival rate. Currently, at least nine mammalian voltage-gated sodium channels (VGSCs) named Nav1.1-Nav1.9 have been identified as potential safe drug targets for treating metastasis in various cancers. Herein, we investigate the effects of targeting VGSCs in MTC using small molecule inhibitors. Two MTC cell lines, TT and MZ, were treated with four compounds and the changes in mRNA and protein expression of VGSC isoform Nav1.7 were assessed through RT-PCR and western blotting. Treatment by one of the compounds exhibited a 28-fold decrease in mRNA expression of VGSC isoform 1.7. Furthermore, a cell proliferation assay ,proliferation assay (MTT) a MTT, assay to study cell proliferation after treatment showed decreasing cell viability resulted from the treatment. and Additionally, morphological changes characteristic of apoptosis in fluorescently labeled MTC cell lines after 48 hours of treatment with VGSC inhibitory compounds were observed. A Boyden chamber assay was also performed on MTC cell lines to examine changes in migration and invasion after administering the potential inhibitory compounds. This study has generated preliminary evidence that voltage-gated sodium channels (VGSCs) may indeed be potential targets for treating metastatic MTC. The results may be translated to future studies focused on controlling metastasis through specified drug treatment in MTC, leading to the limitation of cancer progression by means of targeting VGSCs.

#2002

Computational drug repositioning identifies bortezomib as a novel metastatic inhibitor of lung cancer.

Ok-Seon Kwon,1 Haeseung Lee,2 Wankyu Kim,2 Hyuk-Jin Cha1. 1 _Seoul National University, Seoul, Republic of Korea;_ 2 _Ewha Womans University, Seoul, Republic of Korea_.

Although many molecular targets for cancer therapy have been discovered, they often show poor druggability, which is a major obstacle to develop targeted drugs. As an alternative route to drug discovery, we adopted an in silicodrug repositioning (in silicoDR) approach based on large-scale gene expression signatures, with the goal of identifying inhibitors of lung cancer metastasis. Our analysis of clinicogenomic data identified GALNT14, an enzyme involved in O-linked N-acetyl galactosamine glycosylation, as a putative driver of lung cancer metastasis leading to poor survival. To overcome the poor druggability of GALNT14, we leveraged Connectivity Map approach, an in silicoscreening for drugs that are likely to revert the metastatic expression patterns. It leads to identification of bortezomib (BTZ) as a potent metastatic inhibitor, bypassing direct inhibition of poorly druggable target, GALNT14. The anti-metastatic effect of BTZ was verified in vitroand in vivo. Notably, both BTZ treatment and GALNT14knockdown attenuated TGFβ-mediated gene expression and suppressed TGFβ-dependent metastatic genes, suggesting that BTZ acts by modulating TGFβsignaling. Taken together, these results demonstrate that our in silicoDR approach is a viable strategy to identify a candidate drug for undruggable targets, and to uncover its underlying mechanisms.

#2003

Evaluation of 6-mercaptopurine in a cell culture model of adaptable triple-negative breast cancer with metastatic potential.

Balraj Singh, Vanessa N. Sarli, Anthony Lucci. _UT MD Anderson Cancer Center, Houston, TX_.

Even after an early diagnosis, surgery, radiation, and adjuvant chemotherapy, some triple-negative breast cancers (TNBC) relapse within a year. Some patients, e.g., those with persistent minimal residual disease (MRD) at the end of currently offered therapies after surgery, have a high likelihood of disease relapse. The biggest challenge is to identify the therapies that are safe and effective in halting the progression of poor-prognosis MRD (which efficiently switches between quiescence and proliferation) to metastasis/recurrence. Progenitor-like cancer cells that can survive in reversible quiescence when faced with various challenges in the body, including immune attacks and currently offered therapies, are often behind disease progression to metastasis. A lack of glutamine in culture medium, which eliminates >99.9% of proliferating SUM149 TNBC cells, selects such adaptable, panresistant cells, similar to a selection of highly resistant cancer cells in poorly perfused regions of cancer. Our data support the hypothesis that a lack of glutamine forces the selection of an epigenetic state that does not require a high level of TET2, thus selecting an "undifferentiated" therapy-resistant phenotype as seen in TET2-mutant cancers. Investigations of these adaptable cells (functional studies and molecular analyses) have revealed a variety of ways (genetic mutations, modifications of epigenome, transcriptome, proteome, etc.) that could generate a tremendous cellular heterogeneity and confer survival advantage under various bottlenecks in the body. Our data suggesting that highly adaptable cells are generated through reprograming of the epigenome and transcriptome (indicated by gene expression changes in ZEB1, TET2, ESRP1, ESRP2, FTO, ADARB1, ADARB2, etc.) led us to evaluate low-dose 6-mercaptopurine (6-MP) as a potential therapy in our model of adaptable cancer cells. We chose 6-MP based on its ability to induce and maintain remission in inflammatory bowel disease and childhood acute lymphoblastic leukemia, thus having a potential to be repurposed for treating the TNBC that is at high risk of relapse. We found that long treatment with low-dose 6-MP inhibited these adaptable cells to a greater extent than it inhibited parental cells. Specifically, 6-MP inhibited progenitor-like cancer cells from proliferating thus keeping them arrested in reversible quiescence. Our results suggest that safe and effective drugs like 6-MP could halt progression of poor-prognosis MRD toward recurrence/metastasis. Supported by a State of Texas Grant for Rare and Aggressive Cancers.

#2004

**Identification of a novel small molecule inhibitor of the Six1/Eya2 complex and** in vivo **metastasis.**

Hengbo Zhou,1 Melanie Blevins,1 Lingdi Zhang,1 Deguang Kong,1 Michael Oliphant,1 Daniel L. Gustafson,2 Samarjit Patnaik,3 Juan Marugan,3 Rui Zhao,1 Heide L. Ford1. 1 _University of Colorado, Anschutz Medical Campus, Aurora, CO;_ 2 _Colorado State University, Fort Collins, CO;_ 3 _National Institutes of Health, Bethesda, MD_.

Metastasis is the #1 cause of death for cancer patients. It is now well accepted that misregulation of developmental signaling pathways in tumor cells can contribute to the metastatic process. The SIX1/EYA transcriptional complex acts as a master regulatory complex that is involved in the development of numerous organs. This complex has been shown to promote metastasis through activation of TGF-β signaling and consequent induction of epithelial-mesenchymal transition (EMT) in breast cancer. More recently, Six1 has been shown to enhance metastasis in a non-cell autonomous manner, through activation of GLI-signaling in neighboring tumors cells, resulting in increased aggressiveness of cells not directly expressing the transcription factor. The fact that Six1 can regulate metastasis both cell and non-cell autonomously suggests that inhibiting its function would be an effective anti-metastatic approach. Herein, we describe the use of an alpha screen, and the subsequent discovery of a class of small molecule compounds, that disrupt the Six1/Eya2 interaction. We show that our lead compound from the class not only disrupts the SIX1/EYA2 interaction, but also decreases nuclear EYA2 levels, inhibits TGF-β signaling, and reverses EMT. Moreover, our lead compound confers cytostatic effects on tumor cells in a SIX1-dependent manner. To determine whether this novel Six1/Eya2 inhibitor reverses Six1-mediated transcription, we performed RNA-sequencing in the presence and absence of the compound, and found a substantial overlap between targets induced by SIX1 and targets inhibited by the Six1/Eya2 inhibitor. Targets altered by our novel inhibitor are highly enriched in critical developmental process mediated by SIX1. In addition, the Six1/Eya2 inhibitor partially reverses major pathways altered by SIX1 in cancer, including p53 and Hedgehog signaling, as well as pathways associated with cancer stem cells, invasiveness, and EMT. Most critically, our novel Six1/Eya2 inhibitor is well tolerated when delivered IP or IV to mice, and preliminary in vivo experiments demonstrate that the compound can dramatically suppress breast cancer associated metastasis, without significantly altering primary tumor growth. In summary, our study identified a novel class of compounds that target the SIX1/EYA2 complex, and demonstrate for the first time that pharmacological inhibition of this complex is sufficient to suppress breast cancer metastasis.

#2005

Optimized range of osmotic pressure and viscosity for a lymphatic drug delivery system.

Tetsuya Kodama,1 Shiro Mori2. 1 _Tohoku University, Sendai, Japan;_ 2 _Tohoku University Hospital, Sendai, Japan_.

The lymphatic drug delivery system (LDDS) is a method that allows the injection of drugs into sentinel lymph nodes (SLNs) to facilitate their delivery to downstream lymph nodes (LNs) via the lymphatic network to treat or prevent cancer cell development in both LNs. A high concentration of drug is effectively maintained in both LNs. Here we show that therapeutic effects in metastatic LNs depend on the osmotic pressure and viscosity of the administered drug in a metastatic mouse (MXH10/Mo-lpr/lpr) model. We found using an in vivo bioluminescence imaging system that indocyanine (ICG) solutions were retained in the proper axillary lymph node (PALN) at an osmotic pressure > 1,700 kPa and 6 mPa·s viscosity, when ICG solution was injected into the SiLN to deliver it to the (PALN). Edematous swelling was detected near the SiLN at > 3,500 kPa osmotic pressure and 400 mPa·s viscosity. Furthermore, we established that the therapeutic effects of drugs on metastatic PALN were observed in the osmotic pressure range 1000 - 2400 kPa and viscosity range 2 - 30 mPa·s, after tumor cells were injected into the SiLN to induce metastasis in the PALN. Cisplatin solution was injected into the accessory axillary lymph node to deliver cisplatin to the metastatic PALN. Our results demonstrate the optimized values of osmotic pressure and viscosity for LDDS. We anticipate our results to be a starting point for more sophisticated drug administration by a LDDS.

#2006

Targeting AXL kinase with TP-0903 successfully reverses the mesenchymal phenotype and extends survival in preclinical models of advanced ovarian cancer.

Nozomi Tomimatsu,1 Ken Fujimura,1 Yuta Matsumura,2 Hiroki Umehara,1 Lars Mouritsen,2 Steven L. Warner,2 David J. Bearss2. 1 _Sumitomo Dainippon Pharma Co., Ltd., Osaka, Japan;_ 2 _Tolero Pharmaceuticals, Inc., Lehi, UT_.

Epithelial-to-mesenchymal transition (EMT) is a critical step in the progression of ovarian cancer (OVCA), which drives local invasion, metastasis and drug resistance leading to poor survival. AXL, a receptor tyrosine kinase, functions as a molecular driver of EMT in multiple cancers, including OVCA. The high mortality of this tumor is explained by the fact that the majority (75%) of patients present at an advanced stage, with widely metastatic disease within the peritoneal cavity often associated with ascites. The extent of ascites is a prognostic factor for survival, impacts quality of life, and correlates with the EMT phenotype. Approaches to block or reverse EMT are needed to improve the prognosis of OVCA patients, particularly those with refractory or early relapse (<6 months) disease following debulking surgery and conventional adjuvant treatments. TP-0903 is a potent, small molecule AXL inhibitor that has activity in reversing EMT in multiple models of disease and embryonic development. The objective of this study was to explore the anti-tumor activity in preclinical mesenchymal OVCA-derived models, which may provide rationale to evaluate TP-0903 in treatment-resistant or refractory OVCA patients. TP-0903 showed potent effects on cell viability in multiple OVCA-derived cell lines. The IC50 values in cell viability studies ranged from 33 nM (OVTOKO cells) to 840 nM (OVMANA cells). Kuramochi cells represent the most common ovarian cancer subtype, high-grade serous ovarian cancer and was particularly platinum-resistant (>5 μM), yet sensitive to TP-0903 (210 nM). Baseline phospho-AXL (Y702) was exceptionally high in Kuramochi cells and TP-0903 inhibited this phosphorylation at concentrations as low as 100 nM. TP-0903 reversed EMT in OVCA cells as evident by the down-regulation of mesenchymal markers including Snail and Slug. Consistent with the reversal of the EMT, TP-0903 inhibited the migration of ES-2 OVCA cells in a scratch assay. TP-0903 suppressed ascites development in preclinical models of OVCA and inhibited metastatic intraperitoneal tumor dissemination. Treatment suppressed ascites development (ascites volume, vehicle: 3.34 mL, TP-0903: 0.28 mL; abdominal circumference, vehicle: 7.96 cm (day 15), TP-0903: 6.82 cm (day 15), p<0.05) in an orthotopic ES-2 model and extended overall survival compared to vehicle or cisplatin alone. Phospho-AXL modulation and changes in other mechanism-validating biomarkers were observed in the ES-2 model. Similar preclinical activities, including decreased ascites volume, were observed in A2780cis and OVCAR-3 xenograft models. These results support further clinical evaluation of TP-0903 in either single-agent or combination regimens treating recurrent or refractory OVCA. TP-0903 is investigated in a Phase I trial that includes an expansion cohort of this patient population (NCT02729298).

#2007

Validation of a preclinical model for bone metastatic triple negative breast cancer.

Justyna Zdrojewska,1 Mari I. Suominen,1 Katja M. Fagerlund,1 Jussi M. Halleen,1 Jenni Bernoulli,1 Johann Zimmermann2. 1 _Pharmatest Services, Turku, Finland;_ 2 _Polyphor Ltd, Allschwil, Switzerland_.

Metastatic breast cancer (MBC) is the most advanced stage of breast cancer, diagnosed as stage IV. Typically, MBC metastases occur in the bones, liver, brain, and lungs. Bone metastases cause significant morbidity and mortality in late-stage breast cancer patients. At present, there is no efficient curative or preventive treatment for bone metastases. Therefore, establishment and validation of well characterized in vivo models of breast cancer bone metastasis is of utmost importance. The aim of this study was to validate an intracardiac model of bone MBC utilizing GFP tagged triple negative (estrogen receptor; ER, progesterone receptor; PR and human epidermal growth factor receptor 2; HER2 negative) human breast cancer cells, which could be used to study efficacy of new potential treatments. Female athymic nude mice (Hsd: Athymic Nude-Foxn1nu) were used in this study. Bone seeking MDA-MB-231(SA)-GFP human breast cancer cells were inoculated into the left cardiac ventricle of 4-6 weeks old female mice. Allocation to treatment groups (n=15) was performed by stratification procedure based on animal weight prior to the beginning of the study. To validate the model, the current standard-of-care (SOC) treatment (Paclitaxel) was administered between study days 13-17 at the therapeutic dose. Serum bone biomarker TRACP5b was measured before cancer cell inoculation and at sacrifice. Planar X-ray of hind limbs and ex vivo fluorescence imaging of the whole body were performed at sacrifice. The observed tumor take rate was 100%. 33% of mice in the vehicle group were cachectic, but mice treated with SOC exhibited only mild and temporary weight loss. Tumors were observed mainly in skeletal sites but also in soft tissues. The SOC treatment decreased both total and skeletal tumor burden. Based on X-ray images, tumor induced osteolytic bone lesions were smaller in the SOC treated group compared to vehicle though no differences were observed in total bone lesion area. Serum TRACP5b was increased in the vehicle group as a result of osteolytic lesions, and decreased with the SOC treatment. In conclusion, an intracardiac model of MBC was successfully validated with the SOC treatment paclitaxel that efficiently inhibited disease progression. Therefore, this intracardiac model provides a promising tool for testing new treatments against bone and soft tissue MBC in vivo.

#2008

CENP-E kinesin facilitates chemokine ligand CCL18 translocation to promote migration in triple negative breast cancer.

McKay M. Mullen. _Morehouse School of Medicine, Atlanta, GA_.

Our research is focused on the molecular mechanisms responsible for enhanced cellular proliferation and migration as it relates to triple negative breast cancer (TNBC). Advances in visualization techniques and technologies work to provide an in-depth view of various cellular mechanisms, allowing us to capture molecular level protein-protein interactions previously unknown. There are more than 40 kinesin motor proteins found within the spindle assembly checkpoint, classified into 14 families, which each play essential roles in microtubule-dependent intracellular trafficking. This assembly is a critical component to the cell cycle control complex that acts during mitosis to prevent chromosome missegregation and aneuploidy. Centromere associated protein E (CENP-E) is a kinesin motor protein that shows aberrant overexpression in TNBC which facilitates exponential cell growth and eventual metastasis that occurs in TNBC. Growing awareness through scientific discoveries sheds light on the fact that it is a rare occurrence for biological functions to be attributed to a single mutated macromolecule. Most physiological processes arise from a complex orchestration of cellular events occurring between multiple molecules acting in concert. The nature of protein-protein transactions that drive gene activation appear to be dynamic in TNBC cells thus enabling uncontrolled proliferation. We believe that a pleiotropic network of small molecules may be working cooperatively with CENP-E to facilitate the exponential cell growth and eventual metastasis that occurs in TNBC cells. Currently, we are carrying out spectral imaging and biophysical force measurements to delineate CCL18-elicited cell mechanics and breast cancer metastasis. To address this, we have incorporated syntelin, a small inhibitory molecule targeted to CENP-E in an effort to analyze the resultant effect it has on this network of proteins we believe may be involved in the CENP-E signaling cascade. Taken together, these results indicate that syntelin may comprise a novel targeted chemotherapeutic intervention for TNBC.

#2009

Leronlimab, a humanized monoclonal antibody to CCR5, blocks breast cancer cellular invasion and enhances cell death induced by DNA damaging chemotherapies.

Xuanmao Jiao, Min Wang, Richard G. Pestell. _Baruch S. Blumberg Institute, Doylestown, PA_.

Purpose of the study. To assess binding and functional interaction of the humanized monoclonal antibody to CCR5 (Leronlimab) with human breast cancer cell lines. The G protein coupled receptor CCR5, is normally expressed on a subset of T cells and serves as a co-receptor for HIV infection. During malignant transformation CCR5 expression is known to increase in a number of cancers (breast cancer (BCa), prostate cancer, colon cancer, melanoma). CCR5 targeted cancer clinical trials using small molecular inhibitors opened to accrual in late 2018. CCR5 is expressed in >50% of human BCa, primarily in triple negative BCa. Its expression in human BCa correlates with poor outcome and CCR5+ BCa epithelial cells have characteristics of cancer stem cells, forming mammospheres and initiating tumors with >60-fold greater efficiency in mice. Reintroduction of CCR5 expression into CCR5 negative BCa cells promotes tumor metastases and induces DNA repair gene expression and activity. The CCR5 inhibitor Leronlimab has been used for treatment of >550 patients HIV, including meeting its primary endpoints in a phase III study, without significant adverse events reported.

Results. Leronlimab bound to CCR5 expressed in human breast cancer cell lines with 98% efficiency. Leronlimab abrogated CCL5 induced Ca+2 flux and blocked 3-d matrigel invasion of MDA-MB-231 cells. Leronlimab also augmented cell killing by DNA damage inducing agents Doxorubicin.

Conclusions. Leronlimab binds CCR5 in BCa cells, blocking breast cancer cellular invasion and augmenting cell killing by DNA damage inducing chemotherapies. As CCR5 augments DNA repair and is expressed selectively on cancerous but not normal breast epithelial cells, Leronlimab may enhance the tumor specific activities of DDR-based treatments, allowing a reduction in dose of standard chemotherapy and radiation.

#2010

SKI-G-801, an AXL kinase inhibitor, blocks metastasis and induces anti-tumor immune responses in various syngeneic cancer models.

Chun-Feng Xin,1 Sung Eun Kim,2 Kyoung-Ho Pyo,2 Ha Ni Jo,2 Jae Seok Cho,2 Jae Hwan Kim,2 Wongeun Lee,3 Hee Kyu Lee,4 Jung-Ho Kim,4 Ho-Juhn Song,5 Jong Sung Koh,5 Byoung Chul Cho6. 1 _Brain Korea 21 PLUS project for Medical Science, Yonsei University College of Medicine, Seoul, Republic of Korea;_ 2 _Yonsei University College of Medicine, Seoul, Republic of Korea;_ 3 _JEUK Institute for Cancer Research, Gumi, Republic of Korea;_ 4 _Oscotec, Seongnam, Republic of Korea;_ 5 _Genosco, Cambridge, MA;_ 6 _Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Republic of Korea_.

Introduction: Aberrant AXL expression plays a critical role in cancer cell migration, metastasis and drug resistance. Researchers have revealed that AXL signaling is also over expressed on cells associated with tumor microenvironment. These findings highlight AXL as an attractive drug candidate for targeting tumor evasion and metastasis. Here we present SKI-G-801, a small molecule inhibitor that targets phosphorylation of AXL (IC50 = 20 nM) and its downstream signals.

Methods: Inhibitory effects of SKI-G-801 on cancer viability (MTT and colony formation assay), invasion and migration (trans-well invasion assay) were examined in AXL high-expressing lung cancer cells in vitro. LLC2 lung and 4T1 breast cancer bearing mouse models were established. in addition, C57BL/6 mice were injected intravenously with B16F10 melanoma cells to establish lung metastasis model. Mice were administrated with 30 mg/kg of SKI-G-801 orally before (metastasis model) or after (syngeneic model) tumor injection. To elucidate the involvement of AXL inhibitor on tumor microenvironment, the population of T cells and myeloid cells was analyzed by flow cytometry from the LLC2 tumor.

Results: Treatment of SKI-G-801 showed strong inhibition of cancer migration and invasion, when its direct killing effect on cancer cells was modest. These results were reproduced in vivo test that pretreatment of SKI-G-801 significantly reduced metastatic burden in B16F10 model (p < 0.05). LLC2 and 4T1 tumors were decreased in SKI-G-801 treatment group (p < 0.05), but not in anthemic nude mice. CD3+CD8+ T cell population and memory Tc cells were increased in SKI- G-801 treatment group (p < 0.05). Especially, granzyme B+ Tc and gp70+ tumor specific Tc were increased (p < 0.05). SKI-G-801 increase the helper T cell population; CD3+CD4+ (p < 0.05), and CD44+ memory Th cells (p < 0.01).

Conclusion: SKI-G-801 demonstrates great potential in anti-cancer activity though immune responses. The anti-cancer effects lead to a reversal of the metastatic phenotype in animal model. Our results suggest that SKI-G-801 is a promising drug for prevention against metastatic cancer.

#2011

Potassium usnate inhibits invasion and metastasis in colorectal cancer: Implication of clinical application of a water-soluble form of usnic acid in anticancer therapy.

Yi Yang,1 Woo Kyun Bae,2 Kyung Hwa Lee,2 Kyung-Sub Moon,2 So-Yeon Park,1 Rui Zhou,1 Isa Taş,1 Chathurika Gamage,1 Sueun Lee,2 Chaeyong Jung,2 Kyung Keun Kim,2 Hyung-Ho Ha,1 Hangun Kim1. 1 _Sunchon National Univ., Sunchon, Republic of Korea;_ 2 _Chonnam National Univ. Medical School, Gwangju, Republic of Korea_.

Usnic acid (UA), a lichen secondary substance, has considerable anticancer activity in vitro, whereas its effect in vivo is limited. Here, potassium usnate (KU) was prepared by the salinization of UA to enhance its water solubility. KU showed increased bioavailability compared with UA in the tumor, liver, and plasma of a CT26 syngeneic mouse tumor xenograft model after oral administration, as determined by LC-MS/MS analysis. KU exhibited potent anticancer effects on colorectal cancer cells and inhibited liver metastasis in an orthotopic murine colorectal cancer model. KU treatment downregulated the epithelial-mesenchymal markers Twist, Snail, and Slug and the metastasis-related genes CAPN1, CDC42, CFL1, IGF1, WASF1, and WASL in cells and tumor tissues. The present results suggest the potential application of the water-soluble form of UA, KU, in anticancer therapy.

#2012

**Interrogating the** in vivo **anti-metastatic action of tinzaparin and simvastatin in an orthotopic surgical resection mouse model of triple negative breast cancer.**

Ian S. Miller,1 Liam P. Shiels,1 Grace Buckley,1 Kate Connor,1 Annette T. Byrne,1 Fionnuala Ní Ainle2. 1 _Royal College of Surgeons in Ireland, Dublin, Ireland;_ 2 _University College Dublin, Dublin, Ireland_.

Background: One of the key events in the progression of cancer metastasis is the migration of tumor cells through the vascular endothelium. Tumors induce abnormal endothelial permeability to promote cancer cell invasion. Recently, inhibition of tumor‐induced vascular permeability has been shown to attenuate metastasis in vivo (Tichet M et al. Nat Commun. 2015;6:6993). Low molecular weight heparins (LMWHs) inhibit metastasis in vivo (Klerk et al. J Clin Oncol. 2005;23(10):2130-5) but may cause bleeding complications when used at effective doses. Consequently, LMWHs are unlikely to be used for prevention of metastatic progression unless bleeding side-effects can be diminished. To address this issue, we have recently demonstrated that a LMWH/statin combination (tinzaparin and simvastatin) attenuates abnormal vascular permeability and reduces epithelial cell transmigration at concentrations unlikely to cause bleeding side-effects in vivo. (Kevane et al. Res Pract Thromb Haemost. 2017;1(1)). Here, we aim to interrogate the anti-metastatic effect of the LMWH/statin combination (tinzaparin and simvastatin) in an orthotopic surgical resection model of triple negative breast cancer implementing a LMWH/stain dose that does not increase bleeding risk.

Methods: NOD/SCID mice were surgically implanted with 5x105 MDA-MB-231-LUC2 triple negative breast cancer cells in the right inguinal mammary fat pad. Tumors were allowed to grow until they reached 250mm3. Subsequently, primary tumors were surgically resected. 5 days post-resection, mice were randomized into 4 groups (group 1: 0.5% methylcellulose (vehicle), group 2: 150IU/kg TNZ, group 3: 3mg/kg SVS and group 4: TNZ (150IU/kg) and SVS (3mg/kg)) and treated daily for 4 weeks. Post-resection metastatic progression was monitored by weekly bioluminescence imaging (BLI) [IVIS Spectrum]. Animals that displayed metastatic progression or significant regrowth of primary tumour were euthanized. At time of death, apical lymph nodes and lungs were removed and fixed in 10% formalin for quantification of metastatic burden

Results: Prior to study commencement it was determined that a combination of 150IU/kg TNZ and 3mg/kg SVS did not significantly increase bleeding time in a cohort of tumour naïve animals. Following primary tumor resection, BLI assessment of metastatic progression revealed that 150IU/kg TNZ, 3mg/kg SVS and combination therapy of 150IU/kg TNZ and 3mg/kg SVS significantly (P= 0.0373, P=0.0086 and P=0.0409 respectively N=11) increased the time to metastasis compared to the vehicle treated animals.

Conclusion: For the first time we demonstrate that tinzaparin and simvastatin may be combined to significantly delay time to metastasis while diminishing the associated bleeding risk of LMWHs in a clinically relevant mouse model of aggressive triple negative breast cancer.

#2013

A carbonic anhydrase inhibitor methazolamide potentiates efficacy of gemcitabine by modulating anti-proliferative activity and cancer stem cell markers in pancreatic carcinoma.

Kalpana Joshi, Jeevan D. Ghosalkar, Vinay R. Sonawane, Siddhika R. Raut, Geena Malhotra. _Cipla Ltd, Mumbai, India_.

Pancreatic cancer is the fourth cause for cancer related deaths globally. By 2020, incidence of new cases is expected to be 420,000 with equal number of associated mortalities. Overall 5-year survival has marginally improved over past despite of our growing understanding of the disease. Surgery remains standard of care for resectable tumors followed by adjuvant chemotherapy which resulted in median survival of 26 months. Newer therapies targeting stroma and immunotherapies hold promise for the future. Noticeably, a high unmet medical need warrant novel therapy to extend survival over a year in metastatic settings.

Drug repositioning holds promise over the conventional drug discovery, as it reduces development time and cost. Using this approach, we report promising anticancer activity of Methazolamide (MZM) in pancreatic cancer. MZM, a synthetic derivative of sulphonamide and a carbonic anhydrase IX (CA) inhibitor is used for the treatment of ocular conditions for lowering intraocular pressure. Overexpression of CA is implicated in acidification of tumor microenvironment leading to hypoxia which in turn promotes growth and metastasis of the tumor. Therefore, modulation of extracellular tumor pH via inhibition of CA represents a promising course to anticancer therapy.

Here, we report anti-proliferative activity of MZM in 12 pancreatic cell lines including patient derived (PDX) in 2D models and anchorage independent growth inhibition in 3D models. Combination treatment with GEM yielded significant growth inhibition than drug alone in all tested cell lines. Additive effects were observed for the combination in PDX-1986, PDX-546, Capan-2, Mia-Pa-Ca and PANC-1 cells. Further, in vivo efficacy of MZM in combination with GEM was performed using PDX-546 in nude mice. Combination group showed significant tumor growth inhibition (72%, p<0.05) as against drug alone, MZM at 60 mg/kg, p.o. (50%, p<0.05) and GEM at 50 mg/kg i.v. (61%, p<0.05). There was no significant body weight loss. For PK/PD studies, MZM levels were measured in tumors and PD effect was evaluated using various markers in tumor samples. Expression of target genes was measured by qRT-PCR. Significant reduction in expression of stem cell markers viz. RAC1, OCT4, CD34, CD14, Nanog and proliferation marker (Ki67) was observed in combination group as compared to drug alone. In addition, anti-angiogenic potential viz. would healing, tube formation and expression of important anti-angiogenic markers like HIF1α, VEGF were profoundly modulated in MZM and combination group. Thus, our study indicates that the combination of MZM, a CA inhibitor with GEM is a promising novel approach for the treatment of pancreatic cancer especially in metastatic settings.

#2014

A novel small molecule inhibitor to suppress Notch1 activation in CRC.

Chendil Damodaran,1 Ashish Tyagi,1 Balaji Chandrasekaran,1 Venkatesh Kolluru,1 Ibrahim Jojua,1 Srinivasa Ramisetti,2 Arun K. Sharma,2 Murali K. Ankem1. 1 _University of Louisville, Louisville, KY;_ 2 _Pennsylvania State University, Hershey, PA_.

Introduction: Earlier we reported that aberrant overexpression of Notch1 in colon cancer patients is responsible for aggressiveness and progression of the disease. Current, therapeutic interventions of notch1 inhibitors that binds catalytic unit of γ-secretase and disrupts the cleavage activity cause gastrointestinal toxicity in clinical trial. Therefore, our study aims to develop a small molecule inhibitor, which inhibits Notch1 signaling but binds outside of γ-secretase's catalytic unit thereby substantially reducing associated toxicity attributes. Through structure-activity relationship (SAR) studies based on naturally occurring Withaferin A (WA) structure, we have recently identified a WA-difuroate analog ASR458 as one such small molecule.

Methods: To analyze the effect of ASR490 on HCT116 and HCT116/Notch1 cells, we performed phenotypic, western blot and in vivo analysis.

Results: Notch1 overexpressing CRC cells (HCT/Notch1) showed aggressive cell growth (42%) as compared to HCT116 cells, however, ASR490 treatment inhibited cell growth in both HCT116 (IC50: 600 nM) as well as HCT/Notch1 cells (IC50: 850 nM). ASR458 significantly inhibited Notch1 expression and its downstream events in both the transfected cell lines. As a result, we observed shutdown of pro-survival machinery and induction of pro-apoptotic events (Bax, caspases and PARP) resulting in apoptotic cell death in ASR456 treated HCT116 and Notch1/HCT116 cells. Since Notch1 is responsible for epithelial mesenchyme transition (EMT); we analyzed EMT drivers and readout invasion/migration assays. ASR458 treatment reversed the mesenchymal characteristics by restoring the function of E-cadherin, which resulted in the suppression of invasion and migration in readout assays of HCT116 transfectants. Our ongoing Xenotransplanted mice experiments may suggest whether ASR458 inhibits the growth of HCT116 and HCT116/Notch1 tumors.

Conclusion: Restrained gastrointestinal toxicity of Notch1 targeting therapeutics is achievable with novel small molecules displaying a differential binding affinity to γ-secretase. ASR458 inhibits cell growth in Notch1 overexpressing cells and abrogates Notch1 induced EMT. Ongoing molecular docking and in vivo studies may confirm ASR458 binding sites in γ-secretase and validate our in vitro findings respectively.

#2015

Downregulation of MZB1 by aminoguanidine, APOE mimetic and nitroxyl compounds associated with reduction in invasive breast tumor volume and metastasis.

Yuk Sing Robert Cheng, Debashree Basudhar, Rdinour Lisa, Veena Somasundaram, David Wink. _National Cancer Inst., Frederick, MD_.

Triple negative breast cancer (TNBC) is notoriously known for poor prognosis due to a lack of actionable molecular target(s). Remarkably, the majority of TNBC patients suffer the cancer relapse even they receive treatment given for early stage breast cancers. Up-to-date, lots of therapeutic agents have been developed and actionable molecular targets have been proposed. Nevertheless, the survival rate for metastatic invasive breast cancer is low because of poor prognosis. In this study, we determined whether different classes of chemo-agents (or compounds) interact with different cellular and molecular pathways they might share some common pathway(s) if their therapeutic outcome is similar. We first tested the effect of aminoguanidine (AG), APOE mimetics (COG), and nitroxyl (HNO) compounds on carcinogenesis and metastasis using an orthotopic breast cancer model. In vivo imaging analysis indicated that these compounds significantly reduced the tumor loading in the xenografts (MDA-MB-231-GFP). Moreover, compounds AG and COG are able to reduce brain metastasis significantly. RNA-Seq analysis identified only one gene which is commonly altered in all treatment groups. This gene encodes marginal zone B and B1 cell specific protein (MZB1). MZB1 was downregulated after treatment and its expression was inversely correlated with the efficacy of brain metastasis. In addition, pathway comparison analysis of RNA-Seq data revealed there are three top canonical pathways including (1) Role of NFAT in Regulation of the Immune Response, (2) PKC theta Signaling in T Lymphocytes, and (3) iCOS-iCOSL Signaling in T Helper Cells, downregulated in all treatment groups. In conclusion, our findings demonstrated three different classes of compounds can reduce not only tumor loading but metastasis. MZB1, identified from our transcriptome analysis, may serve as an actionable molecular target for TNBC. All-in-all, it provides new insights into the development of new therapeutic targets and biomarkers for TNBC.

#2016

HDAC inhibition significantly reduces primary and lung metastatic osteosarcoma progression.

Niveditha Nerlakanti, Jeremy McGuire, Diana Yu, Damon R. Reed, Conor C. Lynch. _H. Lee Moffitt Cancer Research Center and Institute, Tampa, FL_.

Osteosarcoma (OS) is the most common bone malignancy in adolescents. At the time of initial diagnosis, approximately 15% of the patients present with metastatic disease in lungs. Prognosis for this cohort is grim with 5-year overall survival rates at 30%. Frustratingly, these statistics have changed little over the past three decades, in part due to the lack of progress in identifying effective therapies and difficulty in coordinating robust clinical trials in this relatively rare malignancy. To address this, we previously screened 54 FDA approved drugs against 5 different OS cell lines and identified the histone deacetylase inhibitor (HDAC), panobinostat (Pano) as having potent effects in vitro. Here, we explore whether Panobinostat is effective in preventing multiple stages of OS progression in vivo using the K7M2 and SAOS2LM7 pre-clinical models of the disease. Pano prevents the growth of primary OS and lung metastasis. Mice were intratibially inoculated with K7M2-Luc cells and then treated with vehicle (n=11) or panobinostat (n=10; 10 mg/kg for all in vivo studies). Tumor growth was measured by bioluminescence (RLU). The clinical endpoint was 1x106RLU (IVIS-200). Primary OS growth was significantly slower in the panobinostat treated group (p<0.05 by day 17) with the median time to clinical endpoint being 2.5-fold longer than the controls (control median: 21 days vs. Pano median: 53 days, p<0.05). Micro-CT analysis demonstrated panobinostat limited OS induced bone disease. Panobinostat also impacted spontaneous lung metastasis (Time to detection; control, 10 days vs. Pano, 21 days). Histological analyses showed significantly lower metastatic burden and growth rates (pHistone-H3/cleaved caspase-3) in the panobinostat group. Pano pretreatment prevents OS lung seeding. Mice were pretreated with vehicle (n=9) or panobinostat (n=5) for 5 days prior to tail vein inoculation of 1x106K7M2-Luc cells. Bioluminescence demonstrated that panobinostat significantly (p<0.05) delayed tumor growth compared to control animals. This translated into higher overall survival rates (Control median: 54 days vs. Pano median: 93 days, p<0.05). Despite halting panobinostat treatment at day 38, 44% (4/9) of the mice remained alive at day 100. Pano halts the growth of established lung metastases. Mice were tail-vein inoculated with 1x106K7M2-Luc cells and randomized after three days into control (n=12) and panobinostat (n=15) treated groups. Bioluminescence readings demonstrated that panobinostat significantly reduced the growth of established OS lung metastases (p<0.05). These data were confirmed using the SAOS2LM7 model. Mechanistically, we are now exploring the contribution of individual HDACs to the progression of OS. Taken together we demonstrate that panobinostat is effective for the treatment of OS and our findings provide rationale for the initiation of broad spectrum HDAC inhibitor focused clinical trials.

#2017

Blocking ErbB-c-Met-FAK signaling axis suppresses brain metastasis of breast cancer.

Shailendra K. Gautam,1 Ranjana Kanchan,1 Pranita Atri,1 Jawed Siddiqui,1 Sanchita Rauth,1 Sidharth Mahapatra,1 Maneesh Jain,1 Ponnusamy P. Moorthy,1 Ravi Salgia,2 Vimla Band,1 Surinder K. Batra,1 Mohd W. Nasser1. 1 _University of Nebraska Medical Center, Omaha, NE;_ 2 _City of Hope, Duarte, CA_.

Background: Brain metastasis is associated with the leading cause of cancer related death and worse quality of life in the breast cancer (BC) patients. Among different molecular subtypes, ErbB2-positive patients show highest incidences of brain metastases. Inability of trastuzumab to cross blood-brain barrier and altered phenotype of metastasizing cells lead to poor therapeutic outcome in ErbB2-positive BC brain metastatic patients. In this regard, new approaches are warranted to overcome challenges associated with brain metastasis.

Experimental design:To understand the molecular expression profile in ErbB2+BC brain metastasis, we compared RNA-Seq expression data of ErbB2+ BC brain-seeking cell line (SKBR3-BrM3) to its parental cell line. Highly expressed targets, epidermal growth factor receptor (EGFR), tyrosine kinase receptor c-Met, and focal adhesion kinase (FAK) were selected for therapeutic intervention. In vitroanalysis of combination therapies was performed using parental and their brain seeking derivative BC cell lines. Neratinib in combination with c-Met inhibitor (Cabozantinib) or FAK inhibitor (PF-573,228) was tested in Hu-ErbB2-postive transgenic (Tg)-mouse derived organoids and in orthotopic nude mouse models.

Results: RNA-Seq data revealed that ErbB2 expression is substantially decreased in ErbB2+ BC brain seeking SKBR3-BrM3 cell line as compared to its parental counterpart SKBR3 cell line. In contrast, EGFR, c-Met and their downstream target, FAK are significantly upregulated in SKBR3-BrM3 cell line, suggesting their role in brain metastasis. Altered expression of these selected targets was further validated by western blotting and immunofluorescence assays. When tested in combination with FDA approved EGFR inhibitor neratinib, both c-Met or FAK inhibitor synergistically inhibited the proliferation and metastasis of metastatic cell lines as observed in colony formation assay and invasion assays, respectively. In addition, the combinational therapy showed efficacy in various preclinical models of breast cancer brain metastasis.

Conclusions:These studies conclude that EGFR, c-Met, and FAK are potential targets in ErbB2-positive brain metastasis. These studies also demonstrate that FDA approved drugs such as Neratinib or c-Met inhibitor and preclinical FAK-inhibitor in different combinations could be used as a novel preventive strategy to attenuate breast cancer brain metastasis.

#2018

Cannabinoid receptor 2 agonist JWH-015 inhibits growth and metastasis of triple negative breast cancers through regulation of autophagy mechanism.

Nabanita Chatterjee, Subhadip Das, Dinesh K. Ahirwar, Sanjay Mishra, Ramesh Ganju. _The Ohio State University, Columbus, OH_.

Metastasis to distant organs is the major cause of mortality associated with breast cancer. Triple-negative breast cancer (TNBC) subtype has been shown to be associated with worst patient survival due to high metastasis rate and lack of targeted therapies. Therefore, there is an utmost need to develop novel therapeutic strategies against TNBC growth and metastasis. Cannabinoids are known to possess anti-cancer activity. However, the associated psychotropic activity limits the therapeutic use of Cannabinoids in breast cancer patients. Here, we used synthetic cannabinoid JWH-015 which activates cannabinoid receptor 2 (CB2) and is devoid of psychotropic activity. In vitro molecular analysis revealed that JWH-015 induces TNBC cell death by activating autophagy associated apoptosis. In addition, we have further shown using in vivo tumor models, that JWH-015 potentially inhibits TNBC growth and metastasis through activation of autophagy mechanisms. Further analysis showed that JWH-015 treated tumors recruited the reduced number of macrophages compared to vehicle control treated tumors. In addition, there were more anti-tumor M1-type macrophages present in JWH-015 treated tumors. Conversely, vehicle control treated tumors possessed a higher number of pro-tumor M2-type macrophages. These observations have established the anti-metastatic potential of JWH-015 in different TNBC pre-clinical mouse models. The mechanistic studies have elaborated the role of JWH-015-induced autophagy in promoting tumor cell death to inhibit TNBC growth and metastasis.

#2019

Enhancing the effect of immunotherapy by inhibiting tumor promoting effect of HDAC6.

Debarati Banik, Melissa Beaty, Erical Palmer, Maria Del Mar Gracia Hernandez, Satish K. Noonepalle, Prathima Vembu, Alan P. Kozikowski, Alejandro Villagra. _George Washington Univ., Washington DC, DC_.

Histone deacetylases (HDAC) are recognized for diverse functions beyond remodeling of chromatin landscape. It may range from regulating the cellular-health to immune-diseases like cancer, positioning the HDAC inhibitors (HDACi) at a crucial junction of immunotherapy. The toxicity among pan-HDACi has led to the development of selective inhibitors, which helped to understand the roles of specific HDACs in immune responses. For example, HDAC6 is reported to promote the pro-tumorigenic STAT3 pathway. By using specific HDAC6i, the downstream immune-modulatory pathways of STAT3, such as PDL1, could be targeted. HDAC6 has been associated with numerous structural functions, including cellular motility, shape, antigen processing, and intracellular transport through the acetylation of tubulin and cortactin, among others. This function suggests that HDAC6 could also a regulator of cellular processes involved in metastasis. In this respect, we found that the selective HDAC6i NextA was able to reduce the primary tumor growth and the appearance of spontaneously metastatic nodules in the murine Triple Negative Breast Cancer (TNBC) tumor model 4T1. Additionally, the in vitro use of NextA in multiple murine and human breast cancer cell lines was found to reduce invasion and modulate multiple EMT-specific genes without exhibiting excessive cytotoxicity. PDL1 expression was also reduced, as described previously in melanoma models. Given that 4T1 is a TIL-infiltrated tumor, we tested the efficacy of αPD-1 immune checkpoint inhibitors (ICI) and found a lower dose of αPD-1 to be more effective than the higher doses to reduce primary and secondary tumor growth. However, the expression of IFNγ & PD-L1 were enhanced with the monotherapy. In an in vitro setting, we were able to nullify the upregulation of PD-L1 by αPD-1 with either NextA or IFNγ neutralization. To apply this insight in vivo, we tested the combination of NextA and αPD-1 to find a significant reduction in tumor growth, both in primary & secondary nodules. Analysis of the effector molecules revealed a reduction in intra-tumoral PD-L1 and IDO1 along with a moderate level of IFNγ, as well as a reduction in several key EMT signature genes, including cMYC, MMP9, vimentin, and twist. We also identified E-cadherin, a negative regulator of the metastatic process, as a novel target of NextA that was found to be upregulated in vivo. This was corroborated by the in vitro observations in several murine and human breast cancer cell lines, irrespective of hormonal receptor status. In conclusion, we propose that combining HDAC6i along with ICI such as αPD-1 may offer a novel avenue to enhance the efficacy of immunotherapy, as well as alter the tumor-intricate physiology, without incurring unnecessary toxicity.

### Tumor-Stroma Interactions

#2020

Adipocytes up-regulates CD36 expression, fatty acid uptake and oxidation enhancing breast cancer cell progression.

Junjeong Choi, Jones Gyamfi. _Yonsei University College of Pharmacy, Incheon, Republic of Korea_.

The role of the tumor microenvironment in tumor progression has recently gained enormous attention, with studies reporting heterogeneous interactions between tumor cells and tumor stroma cells driving tumor progression. The breast cancer microenvironment is unique since the breast tissue within which the tumor originates comprises predominantly of adipocytes. This makes adipocytes a unique component in the breast cancer microenvironment. Adipocytes secrete various growth factors and cytokines that influence tumor progression by induction of epithelial-mesenchymal transition (EMT) which enhances cancer cell migration, invasion and metastasis. This study presents an alternative approach by which adipocytes may enhance cancer progression via the transfer of free-fatty acids from tumor-associated adipocytes to breast cancer cells. The uptake of free fatty acid subsequently serves as a secondary source of energy to drive breast cancer progression. The study aimed to provide an in-depth understanding of the molecular mechanism by which free fatty acid are imported and metabolized in cancer cells in an adipocyte-rich microenvironment. Human adipocytes were co-cultured with cancer cells and the fatty acid (FA) translocase (CD36) activity was inhibited by the chemical inhibitor sulfosuccinimidyl oleate (SSO). Silencing RNA and expression plasmids were used to evaluate CD36 activity in cancer cells. Expression levels of key genes and proteins involved in lipid transfer and metabolism were evaluated by qtPCR and western blot. Inhibition of CD36 reduces free fatty acid uptake and induces intracellular lipogenesis with upregulation of fatty acid synthase (FASN). Breast cancer cells co-cultured with human adipocytes express high levels of the CD36 facilitating free fatty acid uptake from adipocytes. Co-cultured cells had increased levels of fatty acid oxidation (FAO) and oxygen consumption, with increased migration and invasion characteristics. We also observed a significant increase in the expression of CPT1A required for mitochondrial fatty acid import and ATGL required for triglyceride metabolism. Our findings indicate an enhanced dependence on FAO in co-cultured cancer cell. Indicating a potential bioenergetic adaptation of breast cancer cells to the adipocyte-rich microenvironment. Inhibition of CD36 and CPT1 significantly decreased breast cancer cell migration and invasion. These results suggest that cancer-associated adipocytes induce free fatty acid uptake via upregulation of CD36 and reprogram breast cancer cell metabolism. Thus, targeting CD36 may have a potential targeted therapy.

#2021

Role of MMP1-PAR-1 crosstalk in the pro-tumorigenic senescent fibroblasts in large cell carcinoma of the lung.

Marta Gabasa,1 Rafael Ikemori,1 Evette Radisky,2 Noemí Reguart,3 Derek Radisky,2 Jordi Alcaraz1. 1 _Univ. of Barcelona, Barcelona, Spain;_ 2 _Mayo Clinic, Jacksonville, FL;_ 3 _Hospital Clínic de Barcelona, Barcelona, Spain_.

Tumor associated fibroblasts (TAFs) are key effector cells of cancer progression. Intriguingly, senescent TAFs have been reported in a growing list of agressive cancer subtypes including large cell carcinoma (LCC) of the lung. We previously reported that LCC cells induce fibroblast senescence in indirect co-cultures with transwells, revealing that paracrine signaling must be involved. A follow-up study identified MMP1 as an important regulatory protein in fibroblast senescence, since knocking-down MMP1 in LCC cells was sufficient to abrogate the induction of fibroblast senescence in co-cultures as well as the growth and invasion enhancement elicited by the conditioned medium of fibroblasts in LCC cells. Here we examined the potential role of the protease activated protein 1 (PAR-1) in fibroblast senescence by MMP1, since PAR-1 can be activated by MMP1 and has been reported in the stroma of lung cancer patients. We found that PAR-1 expression was down-regulated in fibroblasts that become senescent upon co-culture with LCC cells. In addition, forcing a reduced PAR-1 expression in fibroblasts by siRNA increased their expression of markers of the senescence associated secretory phenotype (SASP) during co-culture with LCC cells, whereas the percentage of senescence-associated beta-galactosidase positive fibroblasts remained largely unaffected. These results suggest that fibroblast senescence elicited by MMP1 secreted by LCC cells is mediated by processes other than PAR-1 activation and/or overexpression in fibroblasts.

#2022

Chronic UV damage of the stroma improves melanoma survival.

Eduardo Nagore,1 Katharina Roeck,2 Timothy Budden,2 Stephen P. Smith,3 Sarah Craig,2 Jean Krutmann,4 Martin Lotz,5 Simon Furney,6 Amaya Viros2. 1 _Department of Dermatology, Instituto Valenciano Oncologico, Valencia, Spain;_ 2 _Cancer Research UK Manchester Institute, Manchester, United Kingdom;_ 3 _Department of Pathology, University of Cambridge, United Kingdom;_ 4 _IUF – Leibniz-Institut für umweltmedizinische Forschung, Dusseldorf, Germany;_ 5 _The University of Manchester, Manchester, United Kingdom;_ 6 _Royal College of Surgeons, Dublin, Ireland_.

Introduction Skin that is exposed to chronic UV presents a distinct pattern of structural degradation compared to age-matched, sun-protected skin. The morphological change underpinning chronic UV cutaneous damage is collagen degradation of the dermis. Molecularly, UV-aged skin accumulates primarily UV-driven mutations, in contrast to UV-protected skin where mutations are rare. The enrichment of mutations in non-neoplastic, aged cells is a defining feature of skin ageing. Melanoma increases with age, and most patients who die are older than 55. Age is the most important independent marker of adverse outcome together with tumor thickness. Old patients commonly present melanoma over chronic UV-damaged skin with a high number of mutations, and some studies suggest these may be linked to better outcome. Here we explore the relationship between tumor mutation burden, stromal DNA damage and outcome of elderly primary melanoma patients.

Results We examined the types of mutations contributing to the somatic mutation load in melanoma. We inferred the signature linked to accumulated UV DNA damage (signature 7 from COSMIC), as well as the intrinsic, proliferation and age-related signature (signature 1). Logistic regression models of signature 1 and 7 demonstrate both correlate with age, and elderly patients are at higher risk of death in this cohort. These data are in contrast to studies suggesting melanomas arising at sites of chronic sun exposure (CSE) have an improved survival, and studies showing higher mutation rates in melanoma are linked to better outcome. To test this, we generated isogenic melanoma cell lines with high versus low UV-mutation burden, and found CSE melanoma cells were more proliferative, invasive and migratory. We next investigated if a higher rate of UV damage to dermal fibroblasts affects outcome by generating isogenic CSE and noCSE dermal fibroblast lines and found melanoma cells exposed to noCSE fibroblasts were more invasive than melanoma cells exposed to CSE fibroblasts. We validated this in human dermal fibroblasts. Dermal fibroblasts from protected sites presented few mutations and promoted melanoma invasion, whereas fibroblasts from chronic UV skin had more mutations and did not drive melanoma invasion. Furthermore, CSE fibroblasts with many mutations present a distinct expression and metabolic profile linked to the sunburn response genes that blocks melanoma invasion. We validated our findings in 490 specimens of elderly primary cutaneous melanoma patients. Multivariate analysis shows the presence of degraded collagen, a surrogate marker of UV damage, is an independent marker of better outcome in the elderly population.

Discussion Our study shows UV-driven damage in aged dermal fibroblasts protects from primary melanoma invasion and humans with extensive dermal UV damage present an improved outcome. Our finding that excessive UV can improve outcome of patients with melanoma is a paradigm shift.

#2023

Cellular senescence, represented by expression of caveolin-1, in cancer-associated fibroblasts promotes tumor Invasion in pancreatic cancer cellular.

Takanobu Yamao, Yo-ichi Yamashita, Kensuke Yamamura, Naoki Umezaki, Rumi Itoyama, Yosuke Nakao, Toshihiko Yusa, Tatsunori Miyata, Shigeki Nakagawa, Hirohisa Okabe, Katsunori Imai, Hiromitsu Hayashi, Akira Chikamoto, Takatoshi Ishiko, Hideo Baba. _Kumamoto University, Kumamoto, Japan_.

Background: The role of cellular senescence of cancer associated fibroblasts (CAFs) in the development of cancer is controversial. In this study, we investigated how senescence of CAFs, represented by Caveolin-1 (CAV1) expression, affects tumor progression in pancreatic cancers (PC).

Methods: We used CAV1 expression to monitor cellular senescence, because CAV1 has been reported to play a major role in cellular senescence. A total 157 consecutive patients with PC underwent curative resections between January 2004 and December 2017 were enrolled. Patients were divided into two groups according to CAV1 expression in CAFs (high and low CAV1, measured by immunohistochemistry). We investigated the relationship between the CAV1 expression in CAFs and the patients' clinicopathological characteristics, including survival. We also established ten CAFs cell lines using PC clinical samples and and evaluated the relationship between CAV1

and senescence marker expression, such as p21 and p53. We chose high CAV1 CAFs to knock down CAV1 expression using siRNA. Finally, we cultured a PC cell line (MIAPaCa-2) with CAFs-conditioned medium (CM) to evaluate the effect of cellular senescence in CAFs on proliferation and invasion of PC cell lines.

Results: The high CAV1 group counts of 49 patients (31%), and the low CAV1 group counts of 110 patients (69%). As for patients' clinicopatholoical factors, the serum levels of CA19-9 (p=0.024) and pathological T factor stage (T0,1/T2,3,4) (p=0.014), defined by Japan Pancreas Society as the "General Rules for the Study of Pancreatic Cancer" were significantly higher in the high CAV1 group. The high CAV1 group had significantly worse outcomes in overall (log-rank p=0.0062) and disease-free survivals (log-rank p=0.0018). Multivariate analysis revealed that high CAV1 group was the independent prognostic factors for OS (p=0.043) and DFS (p=0.018). In coculture assays using CAFs-CM

and MIAPaCa-2 cells, we found that knockdown of CAV1 in CAFs negatively affected the invasion of the PC cells compared to that with si control (p<0.0001).

Conclusion: The present results suggest that, in PC, CAV1 expression in CAFs is associated with patients' poor prognosis and the downregulation of CAV1 in CAFs reduces the invasive ability of PC cells. Therefore, CAV1 of CAFs might be a new target for the treatment of PC.

#2024

Obesity induces breast cancer cell invasion through stromal secretion of IGF-1.

Lauren Hillers, Joseph D'Amato, Tamara Chamberlin, Gretchen Paderta, Lisa Arendt. _University of Wisconsin-Madison, Madison, WI_.

Obese women diagnosed with breast cancer have an increased risk for metastasis, and the underlying mechanisms are not well established. Within the mammary gland, adipose stromal cells (ASCs) are a group of heterogeneous cells with the capacity to differentiate along multiple mesenchymal lineages. ASCs are recruited and activated within tumors, but it is unknown how obesity may influence ASCs in both normal and cancerous tissue. To study the effects of obesity on ASCs, we isolated the stromal vascular fraction from the mammary glands of mice fed either a control diet (CD) or high fat diet (HFD) to induce obesity and plated the cells to select for adherent growth. We observed that obesity increased ASCs proliferation, decreased differentiation potential, and upregulated expression of α-smooth muscle actin, a marker of activated fibroblasts, compared to ASCs from lean mice. To determine how ASCs from obese mice impacted tumor growth, we mixed ASCs isolated from CD- or HFD-fed mice with mammary tumor cells, Met-1 cells, and transplanted them into the mammary glands of lean FVB female mice. Tumors cells mixed with ASCs from obese mice grew significantly larger tumors and had increased invasion into surrounding adipose tissue than tumor cells mixed with control ASCs. To study the effects of factors secreted by ASCs on tumor cell behavior, we collected conditioned media (CM) from lean and obese ASCs and treated Met-1 cells. Compared with lean ASCs, obese ASCs expressed higher levels of insulin-like growth factor-1 (IGF-1), which has been implicated in tumor cell invasion. Met-1 cells treated with recombinant IGF-1 were more invasive than vehicle-treated cells. In addition, Met-1 cells treated with obese ASCs CM supplemented with an IGF-1 neutralizing antibody had significantly reduced invasion, compared to controls. Weight loss induced in obese mice significantly decreased expression of IGF-1 from ASCs and reduced the ability of the ASCs to induce an invasive phenotype. Together, these results suggest that obesity enhances local invasion of breast cancer cells through increased expression of IGF-1 by mammary ASCs, and weight loss may reverse this tumor-promoting phenotype.

#2025

Nicotine promotes lung metastasis in triple negative breast cancer through paracrine signaling in the tumor microenvironment.

Abhishek Tyagi,1 Sambad Sharma,1 Kerui Wu,1 Shih-Ying Wu,1 Fei Xing,1 Yin Liu,1 Dan Zhao,1 Ravindra Pramod Deshpande,1 Yin-Yuan MO,2 Kounosuke Watabe1. 1 _Wake Forest Baptist Medical Center, Winston-Salem, NC;_ 2 _University of Mississippi Medical Center, Jackson, MS_.

Triple-negative breast cancer (TNBC) is responsible for a majority of the mortalities through aggressive metastatic spread, which accounts for a poor 5-year survival. Despite progress in the development of drugs that efficiently target cancer cells, treatments for TNBC are often ineffective. Increasing evidence from epidemiologic studies indicate that cigarette smoking is a major risk factor of TNBC with increased metastatic burden and recurrence. Nicotine, the major addictive component of cigarettes, is not carcinogenic but can promotes cancer progression. However, how nicotine promotes breast cancer lung metastasis is not well understood. To address this question, we orthotopically implanted 4T1 cells (TNBC) into Balb/c mice followed by i.p. injection of nicotine and measured tumor growth and metastatic progression. We found that nicotine increased the growth of primary tumor by 10-fold while it promoted the lung metastasis burden by 100-fold in vivo. Similar results were obtained in experimental metastasis. Immunohistochemical analysis of the primary and lung metastasis tissues showed predominant infiltration of neutrophils in lung metastasis lesions. This observation was further validated by depleting neutrophils using anti-Ly6G antibody, which showed significant decrease in lung metastasis burden in vivo. Intriguingly, infiltrated neutrophils in the lung metastasis tissues selectively overexpressed N2-polarization phenotype. Furthermore, in vitro treatment of primary neutrophils with nicotine induced them into N2 phenotype in a Stat3-dependent manner. To explore further the functional contribution of neutrophils to metastasis, we cultured MDA-MB-231 and MCF10CA (TNBC) cells in primary neutrophils conditioned medium (CM) prior treated with or without nicotine. We found that both MDA-MB-231 and MCF10CA cells displayed epithelial phenotype with loss of mesenchymal markers and gain of epithelial markers in nicotine-induced primary neutrophils CM. These results strongly suggest that nicotine-induced polarization of N2-neutrophils plays a critical role in regulating cancer cell plasticity. We then examined secretory factor(s) from nicotine-treated primary neutrophils using a customized qPCR array system. We found that secretion of LCN2 was significantly augmented in nicotine treated primary neutrophils conditioned medium. In addition, clinical data of stage-II breast cancer patients also showed high LCN2 serum level in smokers than non-smokers. Collectively, our results suggest that nicotine promotes N2 polarization of neutrophils thereby supporting MET transition in a paracrine manner via secreting LCN2 that facilitate metastatic colonization of TNBC cells.

#2026

Loss of fibroblasts-dependent androgen receptor activation in prostate cancer cells develops castration-resistant prostate cancer.

Kenichiro Ishii,1 Izumi Matsuoka,1 Takeshi Sasaki,1 Manabu Kato,1 Kohei Nishikawa,1 Hideki Kanda,1 Yoshifumi Hirokawa,1 Kazuhiro Iguchi,2 Kiminobu Arima,1 Masatoshi Watanabe,1 Yoshiki Sugimura1. 1 _Mie Univ. Graduate School of Medicine, Tsu, Japan;_ 2 _Gifu Pharmaceutical University, Gifu, Japan_.

Well-differentiated prostate cancer (PCa) cells are generally androgen/androgen receptor (AR)-dependent, i.e. AR signaling regulates cell cycle and differentiation. Loss of AR signaling after androgen deprivation therapy (ADT) triggers AR-independent outgrowth, generating poorly differentiated uncontrollable PCa cells. To prevent development and progression of castration-resistant prostate cancer (CRPC), we hypothesize that preservation of AR signaling after ADT is an important target. Tumor stroma surrounding PCa cells is enriched in fibroblasts secreting AR-activating factors, e.g. EGF, IGF1, and IL-6. Thus, we investigated the role of fibroblasts in AR activation of human prostate cancer LNCaP sublines differing in androgen sensitivity under androgen deprivation. Androgen-low-sensitive E9 and F10 cells were obtained from the parental androgen-sensitive LNCaP cell population through use of a limiting dilution method in regular culture condition. In contrast, androgen-insensitive AIDL cells were established from LNCaP cells by continuous passaging under hormone-depleted condition. Original fibroblasts pcPrFs (-M5, -M6, -M7) were isolated from needle biopsy samples of patients with PCa. The PCa cells alone or PCa cells plus pcPrF-M5 were grafted beneath the renal capsule of male athymic nude mice, and then mice were castrated on 14 days post transplantation. Tumor volume of E9 cells + pcPrF-M5 became diminished post castration but that of F10 and AIDL cells + pcPrF-M5 was gradually increased even post castration. In E9 cells + pcPrF-M5 tumors, serum PSA became detected on day 21 post castration. Interestingly, serum PSA was gradually increased even in F10 cells inoculated alone. In AIDL tumors with or without pcPrF-M5, serum PSA was not detected because of mutated AR in AIDL cells. Cell growth of E9 and AIDL cells was directly increased by treating with EGF, whereas that in F10 cells was not affected. Phosphorylation of AKT and ERK1/2 in E9 and AIDL cells was also significantly increased by treating with EGF, whereas that in F10 cells was not affected. In the condition of co-cultures with fibroblasts, PSA production was directly increased in E9 cells but not in F10 and AIDL cells. Protein expression of AR splice variant 7 (AR-V7) was only detected in F10 cells. The reduced AR-dependency of PCa cells is an important clinical development because of its association with the cell' progression to CRPC. Here our results showed that fibroblasts secreting AR-activating factors preserved AR signaling in E9 cells after ADT, indicating that this type of PCa cells can be controlled by ADT. In contrast, we suggest that loss of fibroblasts-dependent AR activation in F10 and AIDL cells may be responsible for development and progression of CRPC.

#2027

Understanding the role and regulation of newly synthesized Annexin A2 in the progression of breast cancer.

Amira F. Mahdi,1 Beatrice Malacrida,1 Joanne Nolan,1 Kieran McGourty,1 Aoife J. Lowery,2 Patrick A. Kiely1. 1 _University of Limerick, Limerick, Ireland;_ 2 _National University of Ireland, Galway, Galway, Ireland_.

In Ireland, almost three thousand women are diagnosed with invasive breast cancer every year. This makes it the most common form of malignant tumour found in Irish females and accounts for 17% of all female cancer deaths. Although treatment for breast cancer when diagnosed early is highly effective, for those who develop late stage and metastatic breast cancer, survival rates decline rapidly. As a result, a large effort is being made to understand the mechanisms involved in the progression of breast cancer and identifying the changes that take place during the in-situ to metastatic transition.

Using 2-D and 3-D in vitro models of cancer metastasis, we conducted a mass spectrometry screen to identify proteins that are newly synthesised by breast cancer cells as they migrate towards a growth factor gradient. From this screen, we identified Annexin A2 as a newly synthesised protein. Annexin A2 is a calcium-ion regulated, phospholipid-binding protein, which is involved in cell trafficking, cell-to- cell contact, cell motility and regulation of the cytoskeletal structure. Annexin A2 has been reported to be overexpressed in a range of cancers including breast cancer and has also been reported to contribute to cancer-associated processes such as proliferation, migration and chemoresistance.

While investigating the role of Annexin A2 in the progression of breast cancer, our findings suggest that through modulation of the cell culture environment, subjecting cells to stress conditions and altering growth factor supplementation, that the intracellular expression of Annexin A2 remains relatively constant. However, in breast cancer patients we see dysregulation of Annexin A2 expression at a gene level, which correlates with disease progression. We are currently investigating how alterations in the composition of the extracellular environment, particularly the extracellular matrix, regulates Annexin A2 expression and cellular location.

We believe that delineating the function of newly synthesised proteins such as Annexin A2 has the potential to direct novel anti-cancer therapies aimed specifically at halting the progression of breast cancer. Ultimately, these insights may lead to improvements in survival rates for those affected with this disease.

#2028

CCL18 signaling from breast tumor-associated macrophages fosters the activation of fibroblasts into a chemoresistance-inducing phenotype.

Erwei Song, Jianing Chen, Shicheng Su. _Sun Yat-sen Memorial Hospital, Guangzhou, China_.

Carcinoma-associated fibroblasts (CAFs) are abundant and heterogeneous stromal cells in tumor microenvironment critically involved in cancer progression. Our recent study identified a specific CAFs subset by two cell-surface molecules, CD10 and GPR77. We proved that CD10+GPR77+CAFs could promote tumor formation and chemoresistance by providing a survival niche for cancer stem cells (CSCs). However, their origin and underlying activating initiation mechanism are unclear. Tumor-associated macrophages (TAMs) are the most abundant immune-related stromal cells and the important source of inflammatory cytokine. Here, we demonstrated that CCL18 secreted by TAMs could induces the activation of normal breast fibroblasts into a CD10+GPR77+CAFs phenotype, which are not only resistant to chemotherapy themselves, but also can induce chemoresistance in cancer cells by abundantly producing IL-8 and IL-6 and enriching the population of cancer stem cells. In conclusion, our studies have identified an inflammatory signaling network in the interaction of different kind of stroma cells in tumor microenvironment and highlighted its potential values as predictive markers and therapeutic targets to abrogate tumor formation and improve chemotherapeutic efficacy.

#2029

Identification of cancer-associated fibroblasts that suppress pancreatic cancer progression.

Atsushi Enomoto,1 Yasuyuki Mizutani,1 Hiroki Kobayashi,2 Tadashi Iida,1 Susan L. Woods,2 Daniel L. Worthley,2 Masahide Takahashi1. 1 _Nagoya University Graduate School of Medicine, Nagoya, Japan;_ 2 _University of Adelaide and South Australian Health and Medical Research Institute, Adelaide, Australia_.

Cancer-associated fibroblasts (CAFs) constitute a major component of the cancer stroma. They can promote cancer progression through a variety of mechanisms including the production of growth factors, chemo/cytokines, and extracellular matrix (ECM). Recent observations from genetically engineered mouse models and clinical studies have suggested that there may exist at least two subpopulations of CAFs, i.e., cancer-promoting CAFs (pCAFs) and cancer-restraining CAFs (rCAFs). Although various pCAF markers have been identified, the identity of rCAFs is unknown due to a lack of specific rCAF marker(s). Here, we show that Meflin, a glycosylphosphatidylinositol (GPI)-anchored protein that maintains the undifferentiated state of mesenchymal stromal/stem cells (MSCs) (Maeda et al., Sci Rep, 6:22288, 2016), is a marker of pancreatic stellate cells (PSCs). We find that Meflin-positive CAFs represent rCAFs in pancreatic ductal adenocarcinoma (PDAC). Infiltration of Meflin-positive CAFs correlated with favourable prognosis in patients with PDAC, consistent with our observation that Meflin deficiency led to tumour progression with poorly differentiated histology in a PDAC mouse model. Notably, Meflin-positive cells gave rise to α-smooth muscle actin (SMA)-positive CAFs during cancer progression. Both genetic ablation of Meflin-positive CAFs and delivery of a Meflin-expressing lentivirus into the stroma suppressed xenografted tumour differentiation and growth, respectively. Mechanistically, Meflin interacts with lysyl oxidase (Lox) to inhibit collagen crosslinking activity, and Meflin deficiency led to straightened and wide stromal collagen fibers as demonstrated by a second harmonic generation microscopy. These data demonstrate the presence of rCAFs in the cancer stroma and the significance of their differentiation to pCAFs in cancer progression, which may be exploited for the development of therapeutic strategies to specifically target pCAFs or reprogram them into rCAFs.

#2030

Inhibiting the GAS6/AXL axis suppresses tumor progression by blocking the interaction between cancer-associated fibroblasts and cancer cells in gastric carcinoma.

Dagyoeng Lee,1 Jongsu Woo,1 Cheong A Bae,1 In-Hye Ham,1 Hye Jung Oh,1 Sang-Yong Son,1 Sang-Uk Han,1 Tae-Min Kim,2 Jung Hwan Yoon,2 Won Sang Park,2 Hoon Hur1. 1 _Ajou University School of Medicine, Suwon, Republic of Korea;_ 2 _College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea_.

Cancer-associated fibroblasts (CAF) are a major component of tumor stroma and their effect on the progression of gastric carcinoma (GC) has recently been demonstrated. However, agents targeting the interaction between CAF and GC cells have not been applied in a clinical setting. The aim of this study was to investigate the effect of an AXL inhibitor on CAF-induced GC progression. We examined growth arrest specific factor 6 (GAS6) expression and phosphorylation of AXL in various cell lines, including GC cells and non-cancerous cells, by qPCR, western blot, and immunocytochemistry. We investigated the role of CAF-derived GAS6 on the migration and proliferation of GC cells using migration and cell survival assays. The effect of the AXL inhibitor, Bemcentinib, on the CAF-induced aggressive phenotype of GC cells was also investigated. Finally, we performed immunohistochemistry to measure the phosphorylation of AXL in a tissue microarray composed of 175 GC tissues, and evaluated its correlation with the prognosis of GC patients. qPCR and western blot analysis showed that GAS6 expression was higher in CAF relative to other cells. We found that CAF increased the phosphorylation of AXL (P-AXL), differentiation into a mesenchymal-like phenotype, and cell survival in GC cell lines. When the expression of AXL was inhibited in GC cells, the effect of CAF was reduced. In addition, Bemcentinib, a small molecule inhibitor of AXL, suppressed the effects of CAF on GC cell lines. In GC tissues, P-AXL positively correlated with the expression of vimentin (P < 0.001) and high levels of P-AXL were significantly associated with poor overall survival (P = 0.022).These findings suggest that CAF is a major source of GAS6 in the GC microenvironment and actively promotes an aggressive phenotype in GC cells through AXL activation. We conclude that an AXL inhibitor may be a novel agent for GC treatment.

#2031

Pancreatic cancer associated fibroblast-released pyruvate blocks the activity of mitochondrially targeted therapies.

Samuel A. Kerk, Lin Lin, Amy Myers, Yatrik M. Shah, Costas A. Lyssiotis. _University of Michigan, Ann Arbor, MI_.

Tumor cells repurpose normal metabolic pathways to meet the energetic and biosynthetic demands imposed by aberrant proliferation [1]. The malate-aspartate shuttle (MAS) is required in normal cells to transfer electrons between the cytosol and mitochondria to maintain redox homeostasis and mitochondrial respiration. PDA has been shown to instead repurpose the MAS for protection from reactive oxygen species (ROS) in a Kras-driven mechanism [2].

We have found that inhibition of glutamate oxaloacetate transaminase 2 (GOT2), an enzyme in the MAS, impaired colony formation across a panel of PDA cell lines in vitro but paradoxically had no effect on tumor formation in vivo. Previous studies have shown that certain metabolites, such as pyruvate, are able to support aspartate synthesis and proliferation when mitochondrial respiration is impaired [3,4]. Additionally, Kras has been shown to shape crosstalk between cancer cells and normal cells in the tumor stroma [5]. Therefore, we hypothesized that cancer-associated fibroblasts (CAFs), which are present in vivo but not in vitro, release pyruvate to promote PDA proliferation when mitochondrial respiration is impaired.

To test this hypothesis, we cultured GOT2 KD PDA cells in pancreatic stellate cell (PSC) conditioned media (CM), the most prominent CAFs present in pancreatic tumors [6]. PSC CM rescued colony formation, while PDA or tumor-educated macrophage (TEM) CM did not. PSC CM maintained this activity when subjected to boiling or molecular weight cut-off filters. We also found that exogenous pyruvate rescued colony formation in GOT2 KD cells. Strikingly, we found that PSCs release nearly 1 mM of pyruvate. In future studies, we will inhibit pyruvate uptake in PDA GOT2 KD cells to determine if this blunts the tumor promoting effects of PSC CM. Additionally, we plan to test whether PSCs are able to provide resistance in PDA against inhibitors of mitochondrial respiration.

[1] Pavlova et al, Cell Metabolism 2016; [2] Sousa et al, Nature 2016; [3] Sullivan et al, Cell 2015; [4] Birsoy et al, Cell 2015; [5] Tape et al, Cell 2016; [6] Omary et al, JCI 2007

#2032

Investigating the contribution of mTORC1-dependent signaling in the microenvironment to cancer onset in Li-Fraumeni syndrome .

Camilla Giovino, Nish Patel, Sangeetha Paramathas, Gaetano Zafarana, Ran Kafri, David Malkin. _The Hospital for Sick Children, Toronto, Ontario, Canada_.

We are investigating the role of stromal mTORC1 signaling in the development of malignancy in individuals with Li-Fraumeni Syndrome (LFS). LFS is a hereditary cancer predisposition syndrome that is associated with germline mutations in TP53. Individuals with LFS are faced with a significant lifetime risk of developing a wide spectrum of early-onset malignancies, thus necessitating the identification of pharmacologic agents to prevent tumour onset.

Chemical inhibitors of the mTORC1 pathway have been shown to promote lifespan extension and delay or even prevent tumour onset in LFS mouse models. These findings are noteworthy, since they suggest that mTORC1 signaling plays a central role in favoring tumorigenesis in LFS.

mTORC1 signaling has been implicated in promoting the pro-tumorigenic senescence-associated secretory phenotype (SASP). The SASP constitutes the release of soluble factors, such as cytokines, growth factors, and proteases from senescent cells into the microenvironment, thus promoting oncogenesis in a paracrine manner. Mutant p53 has also been shown to promote the secretion of a variety of pro-tumorigenic factors from various cell types.

We believe that mTORC1 signaling in LFS stromal cells favors the production and secretion of pro-tumorigenic factors, which may induce changes in the surrounding tissue to promote development of a precancerous niche, thus favoring tumorigenesis. Our objectives are to investigate the contribution of stromal cells and stromal secreted factors in favoring tumorigenesis in LFS, and to investigate the role of mTORC1 signaling in favoring these altered stromal phenotypes.

To determine whether patient-derived LFS fibroblasts with TP53 mutations secrete more pro-tumorigenic cytokines than non-LFS fibroblasts, we performed protein arrays with conditioned media (CM) from fibroblast cultures. We saw that LFS fibroblasts secrete markedly higher levels of pro-tumorigenic, SASP-related cytokines.

Next, we treated spheroids formed from cancer cells of a common LFS subtype (osteosarcoma) with CM from LFS and non-LFS fibroblast cultures. LFS fibroblast CM had a robust pro-proliferative effect on spheroids, while non-LFS fibroblast CM inhibited spheroid growth.

To further address these aims in vivo, we co-injected LFS fibroblasts with cancer cells of a common LFS subtype [rhabdomyosarcoma (RMS)] into Nude mice. We found that tumours formed from the co-injection of RMS cells with LFS fibroblasts grew to be larger than tumours formed from co-injection with non-LFS fibroblasts.

Thus far, we have observed that LFS stromal constituents favor cancer cell proliferation in vitro and in vivo. We are currently investigating the role that mTORC1 signaling plays

in these processes by repeating these studies following chemical and genetic perturbation of mTORC1 signaling in the LFS fibroblasts.

#2033

Bidirectional paracrine IL-33-TGF-beta signaling underlies a tumor-promoting stem cell niche.

Naoki Oshimori,1 Sachiko Oshimori,2 Ajit Elhance,2 Justin Leitenberger,2 Sushil Kumar2. 1 _Knight Cancer Inst. Oregon Health & Science Univ., Portland, OR; _2 _Oregon Health & Science University, Portland, OR_.

Malignant tumor progression and aggressive tumor recurrence after treatment are driven by subsets of cancer stem cells (CSCs), but targeting CSCs remains a major challenge due to their plasticity. CSCs in squamous cell carcinoma reside at the tumor-stromal interface, a paramount tumor microenvironment (TME) for interactive crosstalk between cancer cells and various other cells via soluble mediators, and tumor-infiltrating immune cells are pivotal regulators of tumor progression. However, the molecular link between tumor-associated immune-inflammatory responses and the regulation of CSCs are largely unknown. To define a framework of the CSC niche, we have been focusing on crosstalk between TGF-beta-activated CSCs and their neighboring immune cells. By transcriptomic analysis, we identify interleukin-33 (IL-33) as the most upregulated cytokine in TGF-beta-activated CSCs. We find that IL-33 is secreted in response to the activation of antioxidant responses, and IL-33 mobilizes a subset of tumor-promoting macrophages that express the IL-33 receptor in the adjacent stroma. Tumor-specific IL33 knockdown in vivo dampens both invasive carcinogenesis and paracrine TGF-beta signaling in tumor cells, indicating that IL-33 has a crucial role in the formation of TGF-beta-rich tumor-promoting TME by affecting tumor-infiltrating bone marrow-derived monocytes. Together, these findings on a bidirectional paracrine signaling axis provide novel insights into mechanisms of the formation a CSC privilege niche, which may hold great potential to drastically improve the therapeutic efficacy of current and future cancer treatments.

#2034

Activation of cancer-associated fibroblasts via the transfer of miR-125b containing extracellular vesicles from tumor cells.

Luyen Tien Vu,1 Boya Peng,1 Daniel Xin Zhang,1 Victor Ma,2 Andrew Grimson,3 William C. Cho,2 Judy Lieberman,4 Minh TN Le1. 1 _City University of Hong Kong, Kowloon, Hong Kong;_ 2 _Queen Elizabeth Hospital, Hong Kong;_ 3 _Cornell University, NY;_ 4 _Harvard University, MA_.

Tumor cells secrete elevated amounts of extracellular vesicles (EVs) as a mean of communication between them and other cells in the tumor microenvironment. Here, we established a series of breast cancer cell lines releasing fluorescent EVs to identify the common cell types that receive tumor EVs within the tumor microenvironment. Among the different cell types, cancer-associated fibroblasts consistently take up the most EVs from cancer cells. Using next generation sequencing, we find that miR-125b is among the top enriched miRNAs in EVs released by mouse isogenic triple-negative breast cancer 4T1 and 4TO7 cell lines. Co-injection of 4TO7 cells with 4T1 EVs results in an upregulation of fibroblast activation in vivo while blockage of miR-125b in 4T1 EVs rescues the effect indicating that EV-delivery of miR-125b is crucial for the fibroblast activation in mouse breast cancer models. Moreover, EVs from human breast cancer cells also contain abundant miR-125b and fibroblasts within the xenografted tumor that take up these EVs gain higher levels of miR-125b and upregulation of cancer-associated fibroblast markers. Both mouse and human fibroblasts that are transfected with miR-125b mimics show an activated phenotype similar to the knockdown of established miR-125b targets. In summary, normal fibroblasts within the tumor microenvironment actively take up the miR-125b-containing EVs from breast cancer cells and develop into cancer-associated fibroblasts.

#2035

ELF3 is required for IL-1alpha activation of mesenchymal stem cells in the perivascular niche to immune reactive fibroblasts.

Linda L. Tran, David R. Rowley. _Baylor College of Medicine, Houston, TX_.

Tissue resident mesenchymal stem cells (MSC) have been identified in many adult tissues and are a potential origin for key components of the reactive stroma adjacent to cancer foci. Attempts to target cancer associated fibroblasts as a form of cancer treatment have had mixed results. This may be due in part to our lack of understanding in the heterogeneity of stromal populations and mechanisms that regulate their co-evolution in tumors. Previous work in our lab indicates that tissue-resident MSCs may occupy a perivascular stem cell niche that becomes activated in the presence of IL-1α. We developed a Cspg4 lineage tracing mouse to address the cell ontogeny of reactive stroma. We have shown that Cspg4+ cells occupy the perivascular niche in multiple tissues and that the cells isolated from the microvasculature are more responsive to IL-1α than prostate fibroblasts. Employing a syngeneic tumor model, we've shown Cspg4+ cells can be recruited from the perivascular niche and expand into the tumor.

To elucidate IL-1α regulated mechanisms, co-cultures using normal human prostate, CSPG4+ MSCs (HPS-19I) and epithelial cells (PNT1A) engineered to overexpress IL-1α were performed. A genome-wide gene expression microarray was utilized to determine IL-1α regulated gene expression in the stromal compartment. Among the ~2400 genes upregulated, gene ontology analysis presented enrichment for the immune and inflammatory responses. E74-Like Factor 3 (ELF3) was identified as a candidate transcription factor that may mediate the MSC response to inflammatory signals and subsequent differentiation. Transient knockdown of ELF3 using small interfering RNA in HPS-19Is showed a greatly reduced response to IL-1α and inhibited cellular proliferation. Of interest, exposure to IL-1α induced MSC differentiation to a pro-inflammatory immune fibroblast cell type that was restricted from further differentiation to prototypical myofibroblasts by TGFβ. While responsive to TGFβ, these cells continue to express inflammatory genes, including IL-1β, IL6, IL8, and CSF2, although they were refractory to induced smooth muscle α-actin and collagen expression, hallmarks of myofibroblast differentiation. Moreover, co-activation by TGFβ and IL-1α was associated with activation of the Hippo pathway and inhibition of YAP resulted in restricted myofibroblast differentiation of HPS-19Is. Together these data suggest that activation and differentiation of tissue-resident MSCs by reactive stroma regulators IL-1α and TGFβ results from complex interplay between ELF3 and Hippo pathway components.

MSCs represent one source of the stromal cells that compose reactive stroma in tumors. By more fully understanding the ontogeny and specific regulatory pathways of MSC expansion and differentiation to stromal cell types in the tumor microenvironment, we can develop better therapeutics that target the stromal compartment.

#2036

Osteoblasts use multiple mechanisms of cellular communication to regulate breast cancer cell proliferation in bone.

Alexus D. Kolb, Alison B. Shupp, Dimpi Mukhopdhyay, Karen M. Bussard. _Thomas Jefferson Univ. Kimmel Cancer Ctr., Philadelphia, PA_.

Breast cancer (BC) preferentially metastasizes to bone. While the mechanism for preferred metastasis is unknown, bone likely provides a hospitable environment that attracts BC cells (BCCs) and allows them to colonize and grow. Bone metastases perturb the balance of normal bone remodeling. Emerging evidence suggests that OBs may be altered and respond differently to cancer cells during metastatic BC progression. According to one study, OBs suppress cancer cell growth in early metastasis. Our work suggests OBs reduce BCC growth in bone prior to macrometastatic colonization. Thus, our data suggest OBs have tumor-inhibitory properties. We have new evidence to suggest that communication between OBs and BCCs 'educates' OBs to produce factors that suppress BCC proliferation in bone. We have in-vitro and in-vivo mouse-model evidence that 'educated' osteoblasts (EOs) have a unique secretory protein profile compared to 'uneducated' OBs. We also identified EOs as being present in the bone tissue samples of human patients with bone metastatic BC via multi-plex immunofluorescence. When we treated BCCs with EO conditioned media (CM), BCC proliferation was reduced in both triple negative and ER+ metastatic BCCs, while CM from 'uneducated' OBs did not affect BCC proliferation. This effect was mediated through alterations in EO production of decorin and NOV. We identified EO CM as a rich source of exosomes (exo) and confirmed the presence of an exo population via iodixanol density gradient and western blotting for specific exo protein markers. We found that EO-derived exo, but not 'uneducated' OB-derived exo, decreased proliferation of ER+ and triple negative BCC. Also, exposure to EO-derived exo 1) decreased the number of Ki67 positive metastatic BCC, and 2) decreased the number of metastatic BCC in the S phase of cell cycle as measured by EdU incorporation. Moreover, we labeled EO exo with RFP-conjugated CD63 to visually confirm exo transfer from EO cells to BCCs using confocal microscopy. And, co-culture with EOs increased triple negative and ER+ metastatic BC expression of p21 compared to co-cultures with 'uneducated' OBs. Our data suggest that EOs use multiple mechanisms of cellular communication to regulate BCC proliferation in bone. Impact: Our data suggest OBs produce factors that suppress metastatic BCC growth. Much less attention has been given to OB interactions with tumor cells at sites of bone metastasis due to observations that OB populations are reduced at sites of advanced osteolysis. However, we propose that OBs may be valuable endogenous targets to aid in suppression of metastatic BC growth in the niche in concert with therapeutic drugs to kill the cancer cells. Our data suggest there is a population of OBs that demonstrate a functional role in suppressing metastatic BCC growth; a property capable of exploitation. For these reasons, OBs and EOs are strong candidates for therapeutic targeting.

#2037

Macrophage Inflammatory Protein-3 beta (MIP-3β) from cancer-associated fibroblasts promote the proliferation of melanoma cells.

Heishiro Fujikawa, Masakazu Yashiro, Takaharu Hatano, Shusaku Maeda, Hisashi Motomura. _Osaka City University, Graduate School of Medicine, Osaka-shi, Japan_.

Introduction: Malignant tumors are composed of cancer cells and various types of stromal cells, including fibroblasts, vascular endothelial cells, and immune cells. It has been reported that fibroblasts in the tumor stroma termed as cancer-associated fibroblasts (CAFs) may affect cancer progression. However, the currently available evidence regarding the function of CAFs remains controversial, with some studies reporting stimulatory activity and some studies reporting inhibitory activity in malignant melanoma cells. Previously, we reported that CAFs possibly exert a stimulatory effect on the proliferation of melanoma cells. In this study, we identified a factor from CAFs that are involved in melanoma progression.

Materials and Methods: We used three malignant melanoma cell lines, namely MMAc, COLO679, and A375. Three CAFs and one type of non-affected fibroblasts (NAFs) were established from sebaceous carcinoma/skin malignant melanoma and normal skin, respectively. Serum-free conditioned medium from CAFs (CAF-CM) was prepared as follows: CAFs were seeded into 100-mm plastic dishes with 10 ml of Dulbecco's modification of Eagle medium (DMEM) containing 5% fetal calf serum. To obtain CAF-CM, CAFs were washed using phosphate-buffered saline and subsequently incubated for 3 days with serum-free DMEM, and the conditioned medium was collected as CAF-CM. Additionally, we obtained NAF-CM from NAFs using the same procedure. These fibroblasts were cultured and used before the 15th passage. The cytokines included in each conditioned medium were determined using cytokine assay. Subsequently, we selected some cytokines, which were released more abundantly in CAFs than in NAFs, as potential factors affecting the proliferation of melanoma cells. The effects of these cytokines on the proliferation of melanoma cells were determined using MTT assay and cytometry assay using a Coulter Counter.

Results: CAF-CM contained significantly high concentration of MIP-3β, in compared with that in NAF-CM. In addition, MIP-3β significantly increases in the proliferation of melanoma cells, A375 and COLO679, at 48 h and 72 h culture.

Conclusion: CAFs from skin cancer might promote the proliferation of malignant melanoma cells via MIP-3β signaling.

#2038

NG1/NGL1 engagement supports PDAC development via CAF to PDAC nutrition and CAF-regulated immunosuppression.

Ralph Francescone,1 Débora Barbosa Vendramini-Costa,1 Janusz Franco-Barraza,1 Jessica Wagner,1 Alex Muir,2 Linara Gabitova,1 Tatiana Pazina,1 Tiffany Luong,1 Neelima Shah,1 Dustin Rollins,1 Ruchi Malik,1 Sapna Gupta,1 Roshan Thapa,1 Diana Restifo,1 Allison Lau,2 Yan Zhou,1 Kathy Q. Cai,1 Harvey Harvey Hensley,1 Emmanuelle Nicolas,1 Warren D. Kruger,1 Karthik Devarajan,1 Siddharth Balachandran,1 Wafik S. El-Deiry,1 Matthew Vander Heiden,2 Kerry Campbell,1 Igor Astsaturov,1 Edna Cukierman1. 1 _Fox Chase Cancer Ctr., Philadelphia, PA;_ 2 _MIT/Koch Institute, Philadelphia, PA_.

Pancreatic ductal adenocarcinoma (PDAC) is predicted to soon become the second killer cancer in the USA. One of the hallmarks of PDAC is desmoplasia, which consists of an expansion in activated stellate cells, known as cancer associated fibroblasts (CAFs), and remodeling of their extracellular matrix. Desmoplasia is a major culprit for the unique PDAC microenvironment, which both collapses blood vessels restricting nutrients and fosters immunosuppression. Crosstalk between CAFs, PDAC, and immune cells, within this microenvironment, is not fully understood. To study the functions of CAFs on PDAC development, we first performed a microarray comparing patient matched normal pancreatic stellate cells and CAFs cultured using our stromal 3D system. We identified proteins upregulated in CAFs that have not been previously implicated in desmoplasia. To understand the functional role of these proteins, CRISPR/Cas9 mediated deletion of target proteins was undertaken and cells were subjected to a number of co-culture assays, with PDAC cells and NK cells. Assays consisted of material transfer, cell engagement, proliferation, survival, NK cell activation and PDAC killing. Syngeneic/orthotopic in vivo models, RNAseq analyzes and quantitative multi-plex microscopy, on pathological samples, were also used. We uncovered the ectopic expressions of NetrinG1 (NG1), a neural pre-synaptic protein, in CAFs, and of NG1's post-synaptic binding partner, NGL1, in PDAC cells. Using our 3D system, we observed that heterotypic CAF-to-PDAC interactions, via NG1/NGL1 engagement, are critical for providing survival advantages to nutrient deprived PDAC cells and for CAFs to protect PDAC from NK cell-driven elimination. Mechanistically, we uncovered that PDAC starvation is overcome by NG1 expressing CAFs that provide nutrition via material transfer. Further, knockout of NG1, in CAFs, significantly reduced the production of immunosuppressive cytokines, such as IL-6, IL-8, and TGF-β. Moreover, loss of NG1 from CAFs resulted in anti-tumor NK cell activation. Unexpectedly, we observed a link between glutamate metabolism in CAFs and the inactivation of anti-tumor NK cells, and this was dependent on CAFs' NG1 expression. These results were confirmed in an orthotopic PDAC mouse model in which ablation of NGL1, in syngeneic mouse PDAC cells, significantly halted tumor growth. RNAseq comparing CON or NG1+ CAFs vs. knockout NG1- CAFs rendered changes in neural, metabolic and fibrous signature pathways. Translationally, we saw clear expression of stromal NG1 only in PDAC affected pancreata. Overall, this study identifies two novel targets for PDAC; a cancer that lacks effective therapies. Inhibition of the NG1/NGL1 axis, constitutes a potential future therapeutic approach, as it alters CAF mediated metabolism and reverts immunosuppression, stunting PDAC growth in vitro and in vivo.

#2039

Direct interactions between multiple myeloma cells and osteocytes in the hypoxic myeloma microenvironment increase vegf-a production by osteocytes.

Patrick L. Mulcrone, Daniela N. Petrusca, Keith W. Condon, Jesus Delgado-Calle, Garson D. Roodman. _Indiana University School of Medicine, Indianapolis, IN_.

About 80% of multiple myeloma (MM) patients develop bone disease (MMBD), characterized by severe bone-destructive lesions, decreased bone formation, and increased marrow angiogenesis. Osteocytes, the most abundant cells in bone, regulate bone homeostasis and play a major role in MMBD. We previously found that osteocytes directly interact with MM cells, which induces bidirectional Notch signaling that fuels tumor growth, increases osteoclast resorption, and suppresses osteoblast activity. In addition, the MMBD tumor microenvironment is very hypoxic and facilitates angiogenesis to increase MM growth. Although osteocytes produce pro-angiogenic factors during mechanical load stress and in pro-inflammatory environments, it is unclear if osteocyte:MM interactions also contribute to enhanced bone marrow vascularization associated with MMBD. We hypothesize that hypoxia and direct interactions of osteocytes with MM cells increase pro-angiogenic signaling in osteocytes, which in turn may contribute to the enhanced vascularization in MMBD. We found that MLOA5 murine osteocyte-like cells cultured in 1% O2 (hypoxic) had increased expression of pro-angiogenic genes by qPCR array. Validation studies showed that Vascular endothelial growth factor-a (Vegf-a), a key protein for blood vessel formation, was increased at the mRNA and protein levels in hypoxic MLOA5s compared to normoxic cells. MLOA5s were then co-cultured with different MM cells for 24hrs in either normoxia or hypoxia, and murine Vegf-a levels were analyzed in the conditioned media (CM) from these co-cultures by ELISA. Co-culture of MM:MLOA5 in hypoxia induced greater than two-fold production of Vegf-a when compared to normoxic mono-culture and a 42-55% increase compared to normoxic co-cultures. In vivo, we detected an increased number of Vegf-positive osteocytes in 5TGM1 MM tumor-bearing mice compared to controls 8 weeks after inoculation. To test if elevated secretion of Vegf-a promoted blood vessel formation, CM from normoxic or hypoxic MLOA5s were collected and used to treat HUVECs grown on growth factor-reduced matrigel. Hypoxic CM increased HUVEC network branching by 56% at 8hrs when compared to normoxic CM. This increase was abrogated when a neutralizing antibody targeting murine Vegf-a was added to the hypoxic CM. Together, these results demonstrate that hypoxia and MM cells increase osteocyte production of Vegf-a, which supports angiogenesis in vitro. Thus, angiogenic signals from osteocytes may contribute to MM progression and resistance to therapy, and may be potential targets for the treatment of MM.

#2040

Acute myeloid leukemia cells induce new bone formation by expanding osteoprogenitor niche in the bone marrow.

Bin Yuan, Stanley Ly, Ivo Veletic, Kiersten Maldonado, Xin Zhou, Benoit deCrombrugghe, Michael Andreeff, Venkata Lokesh Battula. _UT MD Anderson Cancer Ctr., Houston, TX_.

Genetic alterations in osteoprogenitor cells have been shown to induce myeloid leukemia in mouse models. We reported recently that acute myeloid leukemia (AML) cells induce osteogenic differentiation in mesenchymal stromal cells (MSC) in the bone marrow (BM) to facilitate faster AML engraftment in mice (Battula et al., JCI Insight, 2017). However, specifics of this osteogenic niche generated by AML are not known. Here, we hypothesize that AML cells induce new bone formation by expanding osteoprogenitor-rich niche in the BM. To investigate the effect of AML cells on osteoprogenitor cells and mature osteoblasts, we generated transgenic mice by crossing Osx-CreERt2 mice with Ocn-GFP; ROSA-tdTomato mice. The resulting triple transgenic mice had the genotype of Osx-CreERt2;Ocn-GFP;ROSA-tdTomato. In these mice the tdTomato (red) positive cells represented cells that originated from Osterix-expressing (Osx+) cells, whereas a GFP+ (green) cell represented an osteocalcin-expressing (Ocn+) mature osteoblast. We implanted murine AML cells with MLL-ENL fusion proteins into these mice. Three weeks after implantation of AML cells, the femurs and tibia of these mice were subjected to histological evaluation using whole-tissue fluorescence microscopy. Interestigly, in the BM of mice implanted with AML cells, we found a 3-4 fold increase in Osx+ cells compared to control animals. However, no significant difference in the number of GFP+ cells on the endosteum and trabecular bone surface was observed, suggesting that AML cells expand osteoprogenitor cells in the BM. Next, to investigate AML-induced alterations in bone, we implanted AML patient-derived xenograft (PDX) cells with the FLT3-ITD translocation into non-obese diabetic scid interleukin-2Rγnull (NSG) mice. When the percentage of AML blasts in peripheral blood increased over 95%, we performed micro-computed tomography (µCT) to analyze changes in the bone architecture. Of note, we observed a massive increase in cortical bone thickness and new medullary bone formation in the diaphysis area. Quantitative analysis revealed that the bone volume and bone mineral content in AML mouse femurs were about twofold higher than those in healthy mouse femurs. To validate these findings, we stained femurs from mice with and without AML with Masson-Goldner's trichrome reagents. Notably, staining of the femurs from mice with AML revealed massive web-like bone formation within the medullary cavity, which is usually not seen in normal BM. Also, bone that was not fully formed, but in the process of maturation, stained light blue. In conclusion, our data suggest that AML cells expand osteoprogenitor cells resulting in thickening of the cortical bone and new bone formation in mouse models. In-depth analysis of bone remodeling in AML patients could result in new insights into the pathobiology of the disease and provide novel therapeutic avenues for AML.

#2041

Lung metastases from breast cancer recruit CD31+/CD45-/CD44+ large platelets and regulate their chemotactic function.

Wei Zheng. _Albert Einstein College of Medicine, Bronx, NY_.

Anti-angiogenic therapy that aims at cutting off the blood supple has shown effects in some primary cancers but is ineffective for metastatic breast cancer. More than being passive conduits, blood vessels can positively regulate neighboring cells by forming perivascular niches and through angiocrine signaling. To test if vascular endothelial cells (ECs) associated with lung metastases express a distinct gene expression program that promotes growth either directly or via regulation of macrophages/monocytes, we isolated ECs by FACS using CD31+/CD45- for RNA sequencing and found that CD44 is upregulated in these cells from lung metastases. Unexpectedly, we found that the increased CD31+CD45-/CD44+ cells were not authentic resident ECs, as they were rarely incorporated into the endothelium. Rather, they were large platelets that originated from the CD45+ hematopoietic lineage in the bone marrow. These anuclear CD44+ CD41+ large platelets were recruited to the lung and upregulated a variety of myeloid cell-regulating chemokines upon metastatic challenge. These data suggest that analysis of ECs based on flow cytometry should be viewed critically and should be coupled with robust validation. Importantly, these results also indicate that the large platelets recruited to the lung may promote metastatic growth via indirect mechanisms.

#2042

Modeling estrogen receptor-positive breast cancer bone metastasis to query osteolytic effects of tumor ER signaling.

Julia N. Cheng, Jennifer B. Frye, Susan A. Whitman, Andrew G. Kunihiro, Madison M. Egan, Julia A. Brickey, Lily A. Alvord, Janet L. Funk. _University of Arizona, Tucson, AZ_.

Estrogen receptor α-positive (ER+) breast cancers (BrCAs) have the greatest predilection for forming clinically evident bone metastases (BMETs). To query ERα's role in osteolytic BrCA BMET progression, tumoral vs bone microenvironment effects of 17β-estradiol (E2) were determined in a human xenograft murine model of ER+ BrCA BMET. Female athymic mice aged 4 weeks (young) or 15 weeks (mature) were inoculated with 1x105 human ER+ MCF-7 BrCA cells via the left cardiac ventricle 2 days post-placement of 60-day release E2 pellets (0.05, 0.10, 0.18, 0.36, & 0.72 mg E2). Osteolytic BMET formation was assessed radiographically and E2 effects on bone were determined by DXA and microCT at various time points. The BMET size and proliferation were assessed by immunohistochemical (IHC) analyses at the end of the experiment (42 days). The effect of E2 on tumoral secretion of the osteolytic factor, parathyroid hormone related protein (PTHrP), was determined using a commercially available radioimmunoassay. The incidence and size of osteolytic BMET, which were not evident in the absence of E2 supplementation, were E2 dose-dependent in young mice. In contrast, E2 effects on the bone microenvironment were not dose-dependent, and resulted in identical increases in bone mineral density (BMD) and bone volume (BV/TV). In skeletally immature (young) mice vs skeletally mature mice treated with an identical E2 dose (0.72 mg), which caused significantly different effects on bone turnover, progression of E2-dependent BMET was identical. These results suggest that E2 effects on the tumor, rather than bone, were driving E2-dependency of BMET progression. IHC analysis demonstrated that neither the size of human cytokeratin-positive tumors nor the proportion of Ki67-positive proliferating tumor cells in bone were E2 dose-dependent, suggesting that proliferative effects of E2 could not explain E2-dose dependent differences in osteolytic BMET formation. PTHrP, an osteolytic factor expressed in most clinical BrCA BMET, was E2-inducible ex vivo and secreted in higher levels by tumor cells isolated from BMETs. These results suggest that during ER+ BrCA BMET progression, tumoral effects of E2 not only support ER+ BMET proliferation, but may also have bone-specific effects due to the induction of PTHrP, which may explain the marked osteolytic capacity of the ER+ tumor cells in this model despite a microenvironment where bone volume is markedly increased.

#2043

Fundamental differences in tumor stromal biology causes racial disparities in cancer susceptibility and survival.

Paris Jafari,1 Xiaoying Xu,2 Beatrice Tassone,2 Luigi Mazzeo,2 Gian-Paolo Dotto1. 1 _Harvard Medical School, Charlestown, MA;_ 2 _University of Lausanne, Lausanne, Switzerland_.

Many cancer types show substantial racial disparity in the incidence and survival rate. African (Black) population shows higher incidence of different types of malignancy and are more vulnerable to aggressive forms of cancer with higher rate of death. The cellular and molecular mechanisms for such race-based disparity in the incidence and survival from cancers are poorly understood. Squamous Cell Carcinomas (SCCs) are epithelial tumors that occur in many internal organs as well as skin and share a common genetic basis. Black Africans have a higher incidence and lower survival for all SCC types. Cancer-associated fibroblasts (CAFs) are the main components of tumor stroma and have a major role in tumor progression and metastasis. Using skin as a benchmark to study SCC development and progression, we tested the hypothesis that the differences in stromal fibroblast biology from White Caucasian versus Back African individuals are important determinants of their different susceptibility to cancer development. We used an integrated approach based on combining gene expression analysis of primary Human Dermal Fibroblasts (HDFs) from a large number of White versus Black individuals with functional assessment of key regulatory gene networks involved in CAF activation. We observed that 225 genes are differentially expressed (DEGs) between Black and White HDFs. GO analysis showed that genes involved in positive regulation of cell proliferation and especially keratinocyte proliferation and migration are significantly enriched in Black DEGs (with higher expression in Black). Also, several specific growth factors were highly enriched in Black DEGs. GSEA analysis revealed a CAF signature enriched in the gene expression pattern of Black HDFs at native state. Cell proliferation analysis showed that not only Black HDFs have higher proliferation rate as compared to White HDFs, but also their paracrine effect on SCC cell and human primary keratinocytes (HKC) enhances the proliferation of these cells in a significant manner. In a set of functional assays, serum-free culture media conditioned by Black or White HDFs (SF-CM) was used to assess the paracrine effect of these cells on the stemness potential of HKC through sphere formation assay. Incubation with SF-CM from Black HDFs significantly increased the ratio of large spheres formed by HKCs in a 3D Matrigel matrix. Further, we observed that co-cultures of Black HDFs with SCC cells, enhanced the cluster formation and expansion rate of these cells. Our results suggest that the fundamentals of tumor biology and the cross talk between stromal fibroblasts and the tumor cells in two populations might be different with more tumor promoting effect from cancer stroma in Black population, where fibroblasts are more susceptible to CAF conversion. This could be due to a higher activity of growth factor-dependent signaling pathway in these cells.

#2044

CD44 facilitates metastasis by promoting co-clustering of breast cancer cells and cancer associated fibroblasts.

Benjamin Troness,1 Angela Spartz,1 Utsav Sharma,2 Philip Miller,2 Kelsie Medina Saenz,2 Marc Lippman,2 James McCarthy,1 Dorraya El-Ashry1. 1 _University of Minnesota Masonic Cancer Center, Minneapolis, MN;_ 2 _Georgetown University Lombardi Cancer Center, Washington, DC_.

Background: Transport of tumor cells via the circulation (CTCs) is a key step in metastasis. CTC clusters and in particular, cancer stem cell-like cell (cCSC) clusters exhibit enhanced metastatic capacity over individual CTCs or cCSCs. Cancer associated fibroblasts (CAFs) are critical players in the breast tumor microenvironment (TME) and are also found in metastases. In clinical and pre-clinical studies, we have identified circulating CAFs (cCAFs) and cCAF/CTC co-clusters as potential biomarkers for breast cancer progression and metastasis. Our data suggest that the ability of breast cancer cells (BCCs) to cluster with CAFs depends on their intrinsic metastatic capacity, and specifically, their CSC-like phenotype, characterized by expression of the CD44 cell surface marker: both in vivo and in in vitro cluster assays, only metastatic BCCs could form co-clusters while non-metastatic BCCs could not. Further, both in vivo and in vitro, co-clustering potential was marked by a stem cell-like phenotype, including increased CD44 expression. We hypothesize that cCAF/cCSC co-clusters are the functional units of metastasis. The goal of this current study is to identify mechanisms that influence co-clustering between CTCs and cCAFs and specifically to investigate the role of CD44 in co-clustering.

Methods: Co- and mono-clusters of CAFs and BCCs were established in vitro by plating equal numbers of CAFs/BCCs into ultra-low attachment plates. After 24 hrs, clusters were collected and injected into female NSG mice via tail vein. Metastases was monitored by IVIS and confirmed by IHC. Mice were sacrificed at specific time points and blood collected by cardiac puncture. In vitro-generated clusters and blood were filtered using the faCTCheker microfluidic filtration instrument (Circulogix) to capture circulating cells with an 8-micron pore filter. Filters were stained by immunofluorescence for tumor cells, CAFs, and CD44. Hyaluronin (HA) was visualized with biotinylated HA binding protein. anti-CD44 siRNA (Dharmacon smart pool) was used to knockdown CD44 in BCCs or in CAFs

Results: Injection of CAF/BCC co-clusters resulted in accelerated metastases compared to mono-cluster injection. In both co-clusters captured from mouse blood and established in vitro, HA, a CD44 ligand secreted by CAFs as well as produced by cancer cells, was observed. Elimination of HA in CAFs and BCCs reduced co-cluster formation. Knockdown of CD44 expression in BCCs via siRNA abrogated BCC co-clustering ability.

Conclusion: In vitro and in vivo models of induced and conditional knockout of CD44 are defining the role of cancer cell-autonomous or CAF-autonomous CD44 in cCAF/CTC co-clustering and BC metastasis. These studies implicate CD44 as a driver of co-clustering between BCCs and CAFs. Inactivation of CD44 may abrogate co-clustering as well as impair the CSC phenotype, thus impacting critical pathways in BCC metastasis.

#2045

**A factor ( >** MR **30 kDa) from cancer-associated fibroblasts may stimulate the progression of schirrous gastric cancer without FGFR2 overexpression.**

Syuhei Kushiyama, Masakazu Yashiro, Tomohisa Okuno, Kenji Kuroda, Ryota Tanaka, Shingo Togano, Sadaaki Nishimura, Takahiro Toyokawa, Hiroaki Tanaka, Kazuya Muguruma, Kosei Hirakawa, Masaichi Ohira. _Osaka City University Graduate School of Medicine, Osaka, Japan_.

Purpose: Scirrhous gastric cancer (SGC), diffusely infiltrating carcinoma, proliferate rapidly with fibrosis, when the cancer cells invade into the submucosa of the stomach. It has been reported that the interactions between the cancer cells and stromal cells, such as cancer-associated fibroblasts (CAFs), surrounding them advance the proliferation and invasion of some types of solid carcinomas. We previously clarified that fibroblast growth factor-7 (FGF7) produced by CAFs promotes the proliferation and tumor progression of SGC. However, the FGF receptor type 2 (FGFR2), which is a specific receptor of FGF7, is just expressed on only 20% of SGC, suggesting that about 80% of growth-stimulating signaling by the interaction between the SGC cells and CAFs might be different from FGF7/FGFR2 signaling.

Materials and Methods: In this study, we aim to clarify the interaction between SGC cells and CAFs except FGF7/FGFR2 signaling in the tumor progression of SGC. We used 4 SGC cell lines, OCUM-1, OCUM-8, OCUM-9, and OCUM-12, which do not overexpress FGFR2. Five CAF cell lines, CAF82, CAF88, CAF90, CAF91, and CAF92, were established by a primary culture from individual gastric carcinoma specimens. Serum-free conditioned medium (SF-CM) from CAFs was added to each SGC cell lines and evaluate them. The interaction of SGC and SF-CM was analyzed by CCK assay and the wound healing assay. The humoral factors in SF-CM which stimulate the proliferation of SGC is divided according to the size of the molecular weight and evaluated by the CCK assay.

Results: CCK assay revealed that CAF82 SF-CM promoted the proliferation of OCUM-9 and OCUM-12 by each 27 % and 20 %. CAF88 SF-CM promoted the proliferation of OCUM-9 by 17 %. CAF90 SF-CM promoted the proliferation of OCUM-1 and OCUM-9 by each 33 % and 21 %. CAF91 SF-CM promoted the proliferation of OCUM-1 and OCUM-9 by each 29 % and 25 %. CAF92 SF-CM promoted the proliferation of OCUM-1 and OCUM-9 by each 37% and 5 %. According to the wound healing assay, the invasion activity of OCUM-12 was promoted by SF-CM from CAF82, CAF90, CAF91, and CAF92 by each 56%, 37%, 34%, and 52%. CAF82 SF-CM which contain humoral factors that molecular weight is more than 30kDa promote the proliferation of OCUM-12 by 16%.

Conclusion: The factors over 30kDa from CAF might stimulate the progression of SGC cells without FGFR2 expression.

#2046

Egr-1 promotes bone marrow fibrosis in primary myelofibrosis.

Rekha M. Rao,1 Amar Kumar,1 Pratikkumar Vekaria,2 Abdulraheem Yacoub,1 Barry Skikne,1 Joseph McGuirk1. 1 _Univ. of Kansas Medical Ctr., Kansas City, KS;_ 2 _Redmond Regional Medical Center, Rome, GA_.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) characterized by JAK-STAT-activated proliferation of leukemia cells and progressive bone marrow fibrosis. However little is known about the bone marrow stromal cell (BMSC) intrinsic factors that work in concert with leukemia stem cells (LSCs) to promote fibrosis of the bone marrow in PMF. In this study we developed an in vitro model to simulate bone marrow fibrosis in PMF by exposing cultured BMSC, HS-5 cells to PMF-patient plasma. We observed that exposure of BMSCs to PMF plasma but not normal serum increased total cellular and mitochondrial reactive oxygen species (ROS), as well as Early growth response 1 (Egr-1) and its target gene expression in BMSCs. Egr-1 is a transcription factor that upregulates fibrosis-associated genes, and is acutely induced by numerous cytokines (e.g. TGF-β, PDGFα) that are elevated in PMF. Utilizing this model, we performed RNA sequencing of HS-5 cells exposed to PMF plasma. We observed that PMF plasma exposure alters pathways responsible for multiple DNA damage responses, G1/s cell cycle checkpoint regulation and protein homeostasis. Since PMF progression is associated with enhanced oxidative stress which induces both DNA damage and Egr-1 expression, we determined the effect of anti-oxidants on the induction Egr-1 and fibrosis associated genes in BMSCs following treatment with PMF plasma. We report that treatment with N-acetyl-cysteine (a ROS scavenger) and mitotempo (a mitochondrial ROS scavenger) abrogated the up-regulation of PMF plasma-induced fibrosis-associated genes in BMSCs. Additionally, knockdown of Egr-1 in BMSCs resulted in the down-regulation of collagen 1A1 and connective tissue growth factor (CTGF) following exposure to PMF plasma. These findings suggest that PMF plasma induces ROS and promotes the upregulation of pro-fibrotic genes in an Egr-1-dependent manner in PMF. Our studies suggest that inhibition of ROS and Egr-1-induced gene transcription could be used as a strategy to alleviate bone marrow fibrosis; a disease complication in PMF for which there are no approved therapies.

#2047

Identification of endometrial cancer specific immuno-therapeutic targets by comprehensive In Silico analysis.

Yuji Ikeda. _Nihon University, Tokyo, Japan_.

Background; Immune-therapeutic targets specific to endometrial cancer has been unclarified. In term of HLA allele type, about 60% and 40% of Japanese has HLA-A*24 and -A*02,respectively. Development of new immune-therapeutics taking account of these types is required. We herein aimed to identify immune-therapeutic targets specific to endometrial cancer and commonly available for Japanese patients using in Silico analysis, which can search comprehensively peptides binding to HLA molecule in an allele-specific manner. Methods; mRNA sequencing data obtained from 582 samples of endometrial cancer and 53 control samples from 31 normal organs were extracted from cancer genome atlas (TCGA) database and Genotype-Tissue Expression project, respectively. After filtering of protein coding genes, 8 to 12 peptides with high binding affinity to HLA-A*24 and -A*02 alleles were identified by an artificial neural network soft trained for peptide-MHC class I binding. Results; Based on mRNA sequencing data, we identified 71 genes that are expressed in endometrial cancer but not in control organs. Of them, top ten genes with high mRNA levels in the cancer are selected. Analysis of binding affinity revealed that 21 and 146 peptides derived-from ten genes of interest showed high binding affinity to HLA-A*24 and -A*02, respectively. Especially, one target gene showed the highest expression levels in endometrial cancer, and possessed 15 and 58 candidate peptides in HLA-A*24 and -A*02, respectively. Conclusion; By comprehensive analysis using mRNA data, we identified reliable candidates target of immune-therapeutics for HLA-A*02 and -A*24 patient with endometrial cancer.

#2048

Stat3 downstream transthyretin stimulates tumor growth through regulation of tumor, immune and endothelial cells.

Xinchun Ding. _IUPUI, indianapolis, IN_.

Lung cancer is a very aggressive malignant form of cancer, and is one of the biggest public health challenges facing the United States and many other countries. Although incidence rates have been stabilized, an estimated 154,050 Americans are expected to die from lung & bronchus cancer in 2018, accounting for approximately 25.3 percent of all cancer deaths. Lung cancer is a difficult disease to detect in its early stages, with greater than 50% of patients diagnosed with lung cancer presenting with metastatic disease. Early detection of lung cancer is an important opportunity for decreasing mortality while it is still treatable and curable. The overall 5-year survival rate is ~15 percent. Thus, it is essential to better understand the mechanisms that initiate lung carcinogenesis and find easy-use biomarkers for more accurate lung cancer detection. Due to heterogeneity of lung cancers, a panel of biomarkers should be used for more accurate lung cancer detection and classification. Signal transducer and activator of transcription 3 (Stat3) is well known for its lung cancer-promoting activity. To assess the consequences of STAT3 persistent activation in the lung, a doxycycline-controlled CCSP-rtTA/(tetO)7-Stat3C bitransgenic mouse model was generated that over-expresses STAT3C (a constitutively active form of STAT3) in alveolar type II (AT II) epithelial cells. In sequential steps, Stat3C over-expression up-regulated pro-inflammatory molecules, increased inflammatory cell infiltration and caused adenocarcinomas in the lung. Using this lung tumor model, thirteen secretory proteins that are Stat3 downstream gene products were identified as a panel of biomarkers for lung cancer detection in human sera. This panel of biomarkers potentially differentiates different types of lung cancer for classification. Among them, the transthyretin (TTR) concentration was highly increased in human serum of lung cancer patients. TTR concentration was also induced in the serum, bronchoalveolar lavage fluid, alveolar type II epithelial cells and alveolar myeloid cells of the CCSP-rtTA/(tetO)7-Stat3C lung tumor mouse model. Recombinant TTR stimulated lung tumor cell proliferation and growth, which were mediated by activation of mitogenic and oncogenic molecules. TTR possesses cytokine functions to stimulate myeloid cell differentiation, which are known to play roles in tumor environment. TTR demonstrated a great influence on a wide spectrum of endothelial cell functions to control tumor and immune cell migration and infiltration. TTR-treated endothelial cells suppressed T cell proliferation. Taken together, Stat3 downstream inducible secretory protein biomarkers potentially can be used as clinical targets for lung cancer personalized treatment if their expression levels are increased in a given lung cancer patient in the blood.

## EXPERIMENTAL AND MOLECULAR THERAPEUTICS

### Apoptosis, Necrosis, and Cancer Cell Survival

#2049

LKB1 regulates BH3-mimetics vulnerability of KRAS mutant non-small cell lung cancer by alternating mitochondrial apoptotic protein interactions.

Chendi Li, Audris Oh, Varuna Nangia, Aaron N. Hata. _Massachusetts General Hospital, Charlestown, MA_.

Over 30% of lung adenocarcinomas harbor KRAS mutation. Despite progress in targeted therapies for specific genetic subsets of lung cancer (e.g. EGFR or ALK), there are no clinically effective targeted therapies for KRAS non-small cell lung cancer (NSCLC). Interpatient heterogeneity of KRAS mutant lung cancers, with large variability in co-occurring mutations, may contribute to its refractory phenotype. Specifically, KRAS mutant lung cancers with concurrent loss of the tumor suppressor LKB1 (KRAS-LKB1) have increased metastatic frequency and resistance to chemotherapy in pre-clinical models, and these patients have poor responses to immune checkpoint inhibitor. Thus, there is a critical need to develop novel therapeutic approaches for this subset of NSCLC. Intrinsic resistance to apoptosis limits the efficacy of therapies targeting KRAS signaling. Previous studies reported BCL-2/BCL-XL \+ MEK inhibition can increase apoptotic responses of some KRAS mutant cancers. Recently, potent and selective inhibitors of MCL-1 have been developed, creating additional possibilities for targeting apoptotic machinery for cancers that dependent upon MCL-1 for survival.

We recently reported that novel MCL-1 inhibitor AMG-176 combined with MEK inhibitor trametinib can induce tumor regression in subsets of KRAS mutant NSCLC pre-clinical tumor models. In addition to commercially available cell lines, we established 20 patient-derived cell line/xenograft mouse model and showed that KRAS-LKB1 cell lines are particularly sensitive to MEK + MCL1 inhibition with high synergy score. Moreover, restoring LKB1 expression in LKB1-/- cell lines hampers the synergy and blocks mitochondrial depolarization and apoptosis. BH3-profiling reveals high dependency on MCL-1 in KRAS-LKB1 cell lines. We reported that trametinib increase intercellular Bim (pro-apoptotic protein) and subsequent loading of Bim onto pro-survival proteins BCL-XL and/or MCL1. MCL1 inhibitor releases Bim from MCL1 and initiates the apoptotic cascade. Interestingly, trametinib induces preferential sequestration of Bim by MCL-1 in KRAS-LKB1 models. Restoration of LKB1 in LKB1-/- cell lines reduces the trametinib-induced Bim:MCL-1 protein-protein interactions. Combined inhibition of MEK + MCL1 caused dramatic tumor regression in LKB1-deficient xenograft mouse models compared to LKB1-restored models.

Our results reveal fundamental insights into a novel role for LKB1 in the regulation of mitochondrial apoptosis and will lay a solid pre-clinical foundation for the clinical investigation of the MEK + MCL-1 inhibitor combination. Our data may also support using LKB1 as a genetic biomarker for guiding the selection of BH3 mimetics in targeting KRAS mutant NSCLC.

#2050

Cell penetrating peptide, ST101, disrupts ATF5 regulation of anti-apoptotic Bcl-2 family proteins, resulting in induction of cancer cell death in vitro and tumor growth inhibition/regression in vivo.

Jim A. Rotolo, Rick Ramirez, Mark Koester, Siok Leong, Lila Ghamsari, Gene Merutka, Barry J. Kappel. _Sapience Therapeutics, Inc., Harrison, NY_.

Anti-apoptotic B cell lymphoma 2 (Bcl-2) family proteins are frequently overexpressed across a variety of tumors, resulting in tumor cell survival and resistance to therapy. Inhibition of the expression or activity of these survival factors is an attractive approach for cancer therapy. Activating transcription factor 5 (ATF5) regulates gene transcription of anti-apoptotic Bcl-2 family proteins in neural progenitor cells and a wide range of human cancer cells. Here, we describe ST101, a rationally designed, synthetic, D-amino acid, cell penetrating peptide therapeutic designed to disrupt the protein-protein interactions driving ATF5-regulated gene transcription. Exposure of HL60 promyelocytic leukemia cells and MCF7 breast adenocarcinoma cells to low micromolar concentration of ST101 resulted in a decrease in MCL-1, BCL-2 and BIRC5 (Survivin) mRNA expression at 4 and 24 hrs post exposure. Further, exposure to ST101 resulted in a dose-dependent loss of viability across a panel of human cancer cells, including MCF7, HL60, U251 glioblastoma, A375 melanoma, DU145 prostate cancer, and A549 lung adenocarcinoma, characterized by an increase in annexin V and PI staining by flow cytometry peaking 48 hrs post exposure, resulting in a median half maximal effective concentration (EC50) value of 4.0 micromolar. In contrast, normal human peripheral blood mononuclear cells and bone marrow mononuclear cells were resistant to ST101-mediated cell death, with >80 micromolar EC50 values. In mouse xenograft experiments, 25mg/kg ST101 administered three times per week for three weeks resulted in significant tumor regression in MCF7 and U251 subcutaneous tumors as well as tumor growth delay in HL60 subcutaneous tumors. Tumor growth remained significantly inhibited weeks after the last treatment in the MCF7 and U251 models. In summary, ST101 selectively kills cancer cell lines by decreasing BCL-2 family gene expression, resulting in significant reductions in tumor growth in mouse models. Taken together, these data validate ST101 as a potent peptide therapeutic candidate for a variety of solid tumor and hematologic malignancies.

#2051

BI 905711, a novel CDH17-targeting TRAILR2 agonist, effectively triggers tumor cell apoptosis and tumor regressions selectively in CDH17-positive colorectal cancer models.

Juan Manuel Garcia-Martinez,1 Andreas Wernitznig,1 Joerg Rinnenthal,1 Maria Antonietta Impagnatiello,1 Frank Hilberg,1 Craig Giragossian,2 Norbert Kraut,1 Mark Pearson,1 Klaus-Peter Kuenkele1. 1 _Boehringer Ingelheim RCV GmbH & Co KG, Wien, Austria; _2 _Boehringer Ingelheim Pharmaceuticals, Inc., CT_.

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has the capability to induce apoptosis in a wide variety of tumor cells and has emerged as an important therapeutic concept for cancer treatment. To date however, TRAILR2 agonistic antibodies, designed to trigger tumor cell apoptosis, have only had limited clinical success due either to lack of efficacy or liver toxicity. BI 905711, a tetravalent bispecific antibody targeting both TRAILR2 and CDH17, was designed to overcome the disadvantages of current TRAILR2 antibodies. CDH17-dependent clustering of TRAILR2 permits BI 905711 to selectively induce apoptosis in CDH17-expressing tumor cells. CDH17 was selected as the anchor target due to its restricted expression, in particular a lack of expression in normal liver tissue, thereby avoiding the clinical hepatotoxicity associated with TRAILR2 agonism. Here, we report the preclinical activity of BI 905711 using colorectal cancer (CRC)-derived cell lines and patient-derived xenograft (PDX) models. We demonstrated that BI 905711 effectively triggered apoptosis in a broad range of CDH17-positive CRC tumor cells in vitro. Furthermore, BI 905711 potently initiated the apoptosis cascade as evidenced by a strong post-treatment increase of both caspase-8 and caspase-3/7. Importantly, induction of extrinsic apoptosis signaling by BI 905711 was strictly CDH17-dependent, as further demonstrated using a pair of CDH17-positive and -negative clones of the CRC cell line GP2d. When comparing the CDH17-negative clone with the parental cell line, the absence of CDH17 translated into a more than 1000-fold drop in BI 905711-dependent efficacy. Moreover, BI 905711 demonstrated single-agent tumor regressions in the GP2d colorectal cancer xenograft model. The antitumor efficacy of BI 905711 was further investigated in vivo in different CRC PDX models, showing effective tumor growth inhibition in a q14d dosing schedule. In summary, we demonstrated that BI 905711 potently triggers the extrinsic apoptosis pathway specifically in CDH17-positive tumor cells, which translates into significant tumor growth inhibition in CRC patient-derived xenograft tumors. BI 905711 is a first-in-class molecule that shows superiority to existing TRAILR2 agonists, represents a targeted strategy for the treatment of CRC and additional CDH17-positive cancer types. Together with its potential for a favorable safety profile, these data support our plan for a phase I trial of BI 905711 in these patient populations.

#2052

A novel antibody blocks anti-apoptotic activity of MUC1 in pancreatic cancer cell lines.

Mukulika Bose, Pinku Mukherjee. _University of North Carolina at Charlotte, Charlotte, NC_.

Introduction: The third leading cause of cancer-related deaths in the United States is pancreatic cancer, >95% of which is Pancreatic Ductal Adenocarcinoma or PDAC. MUC1 is a transmembrane glycoprotein expressed on normal glandular epithelial cells. In PDAC, MUC1 is overexpressed and aberrantly glycosylated and is designated tumor associated MUC1 (tMUC1). Over 80% of human PDAC expresses tMUC1. In a NCI initiated study, out of 75 tumor antigens, MUC1 was ranked the second most targetable antigen. Furthermore, the 72 amino acid cytoplasmic tail of MUC1 (MUC1 CT) is reported to aid in oncogenic signaling leading to tumor progression and metastasis by blocking apoptosis. A novel monoclonal antibody, TAB004, has been developed specifically against tMUC1. TAB004 detects tMUC1 with a high rate of specificity and sensitivity and spares recognition of normal epithelial MUC1. HYPOTHESIS: Treatment with TAB004 curbs PDAC cell survival by blocking MUC1 CT associated oncogenic signaling and renders the cells more susceptible to standard chemotherapy drugs.

Methods: Several human PDAC cell lines based on their tMUC1 expression were grown in media with heat-inactivated serum to ensure that it is devoid of complement proteins. Cells were treated with various concentrations of TAB004 antibody, control IgG, 5-FU, paclitaxel, or gemcitabine and the IC50 was determined using the MTT or SRB cell survival assays. Once IC50 was determined, cells were treated with combinations of TAB004 and the chemotherapy drugs. To determine apoptosis, treated cells were stained with Annexin V-FITC and PI, and analyzed by flow cytometer. tMUC1 expression and localization was determined by staining cells with TAB004.Cy5.5 and imaged by confocal microscopy. Data analysis was performed using GraphPad Prism 7.0 and a p value of <0.05 was considered significant.

Results: By itself, TAB004 treatment had minimal effect on most of the cell lines tested except for Capan2. The IC50 of TAB004 was ~400ugs/ml for Capan2. However, when combined with Gemcitabine, PTX, or 5-FU, TAB004 significantly increased anti-tumor efficacy of the drugs, especially 5-FU in Capan2, and 5-FU and gemcitabine, and PTX in BxPC3.MUC1. Annexin V-FITC and PI staining confirmed the MTT data in Capan2 cells. Fluorescent images showed that TAB004 internalizes in MUC1-positive PDAC cell lines but not in MUC1-negative cells.

Conclusions: TAB004 has a dose-dependent, complement-independent growth-inhibitory effect on Capan2 cells (IC50 is ~400ug/ml). TAB 004 internalizes and activates apoptosis in Capan2 cells. The combination of TAB004 with chemotherapy drugs increases the efficacy of drug-induced killing of PDAC cells. Future studies will determine the mechanism of TAB004 induced apoptosis and sensitization of PDAC cells to drug-induced killing.

#2053

Targeting GRK2 increases chemotherapy-induced apoptosis in medulloblastoma.

Anup S. Pathania,1 Xiuhai Ren,2 Min Mahdi,2 Gregory M. Shackleford,2 Anat Erdreich-Epstein3. 1 _Children's Hospital Los Angeles/University of Southern California, Los Angeles, CA;_ 2 _Children's Hospital Los Angeles, Los Angeles, CA;_ 3 _Children's Hospital Los Angeles/Norris Comprehensive Cancer Center/University of Southern California, Los Angeles, CA_.

Background: G protein receptor kinase-2 (GRK2; ADRBK1) belongs to the family of G protein-coupled receptor (GPCR) kinases (GRKs). GRK2 phosphorylates GPCR and non-GPCR targets including cell surface receptors, cytoskeletal and mitochondrial proteins and transcription factors. It also functions in embryonic development, heart function, metabolism, and some cancer models. Here we hypothesized that targeting GRK2 will sensitizes cancer cells to therapy. We tested this in medulloblastoma, the most common malignant brain tumor in children.

Methods: siRNA transfections, shRNA viral infections, SDS-PAGE, western blotting, flow cytometry. Cells: UW228 and DAOY medulloblastoma.

Results: We found that GRK2 inhibition promotes anti-tumor effects of chemotherapeutic drugs cisplatin and etoposide in UW228 and DAOY medulloblastoma cell lines. GRK2 silencing increased apoptosis induced by cisplatin and etoposide and GRK2 overexpression decreased apoptosis induced by these drugs in UW228 and DAOY. Time dependent studies revealed that GRK2 overexpression delayed cisplatin- and etoposide-induced apoptosis measured by the induction of Annexin V positive population and by PARP-1 cleavage. GRK2 mutants, GRK2 K220R (kinase deficient mutant) and GRK2 S670A (cannot be phosphorylated) were similar to wildtype GRK2 in delaying the cisplatin and etoposide induced apoptosis. GRK2 knockdown antagonized serum induced AKT and ERK phosphorylation in both lines whereas GRK2 overexpression promoted phosphorylation, suggesting that GRK2 is important in medulloblastoma survival pathways.

Conclusions: These results demonstrate that targeting GRK2 enhances cisplatin- and etoposide-induced apoptosis in medulloblastoma cell lines and suggests that GRK2 inhibitors may enhance responsiveness of medulloblastoma to chemotherapy.

#2054

A novel Bcl-2/Bcl-XL inhibitor APG-1252-12A as a potential therapeutic strategy for gastric carcinoma.

Xianglei Yan,1 Hanjie Yi,1 Qiuyun Luo,1 Luping Yuan,1 Suna Zhou,1 Wentao Pan,2 Lin Zhang,1 Miaozhen Qiu,1 Dajun Yang1. 1 _Sun Yat-sen University Cancer Center, Guangzhou, China;_ 2 _Ascentage Pharma Inc., Suzhou, China_.

Background: Gastric cancer (GC) is the fourth most common type of malignant tumor and is the second leading cause of cancer-related mortality. Surgery and chemotherapy play important role in the treatment of GC, however the overall survival of gastric cancer patients is not promising. APG-1252-12A specifically binding to Bcl-2 and Bcl-xl has shown efficacy in some solid tumor. We intend to investigate the anticancer effect and combination enhancement of Fluorouracil (5-FU) in gastric cancer.

Materials and methods: Human gastric cancer cell lines (AGS, NCI-N87, SGC7901, BGC823, MKN45 and NUGC3) were treated with different concentrations and time of APG-1252-12A. CCK-8, survival assays, flow cytometry and western blot were used to assess cell survival, cell cycle progression and apoptosis. In vivo, xenograft mouse models (NCI-N87) were established to analyze the anticancer effect and combination enhancement of 5-FU.

Results: In vitro, APG-1252-12A inhibited proliferation of gastric cancer cell lines, without cell-cycle arrest. Meanwhile, APG-1252-12A increased protein expression of apoptosis signal pathway. The combination of APG-1252-12A and 5-FU resulted in the accumulation of apoptosis protein such as PARP and cleaved caspase3, which indicating an increasing of chemosensitivity. In xenograft mouse models, single therapy with APG-1252-12A obviously suppressed tumor growth, especially in 100mg/kg group. Combination group delayed tumor growth more significantly compared with APG-1252-12A and 5-FU single therapy. By analyzing proliferation marker Ki67 in xenograft tumor specimens, we found that the number of proliferating cells significantly decreased in tumors receiving combination treatment of APG-1252-12A and 5-FU compared with tumors receiving single treatment. The results strongly confirmed the antitumor capacity of APG-1252-12A combined with 5-FU.

Conclusions: The results strongly suggest that Bcl-2 and Bcl-xl inhibitor APG-1252-12A may serve not only as a single therapy, but also as an effective adjunct to current chemotherapeutic regimens for treating gastric cancer through apoptosis signal pathway.

#2055

Differential expression of TP73 isoforms modulates cell survival and therapeutic response in diffuse large B-cell lymphoma.

Hesham M. Hassan,1 Michelle L. Varney,2 Sugandha Saxena,2 Rakesh K. Singh,2 Bhavana J. Dave2. 1 _Assuit University, Assuit, Egypt;_ 2 _Univ. of Nebraska Medical Ctr., Omaha, NE_.

Rearrangements of the chromosome locus 1p36 with ensuing deletion or disruption of TP73, one of the most distally located putative tumor suppressor genes, is frequent in non-Hodgkin lymphoma (NHL) and confers inferior prognosis. TP73 shares homology to TP53 and is capable of transactivating p53 target genes. Earlier reports in normal B-cells and lymphoma animal models suggest an important role of p73 in B-cell lymphomas, however, the biological significance of p73 isoforms in Diffuse large B-cell lymphoma (DLBCL) is not clear. Diffuse large B-cell lymphoma is the most common form of NHL, and although long-term remission is achieved with current therapies, relapse occurs in almost one-third of the patients. Previously, we have demonstrated that the differential expression of p73 isoforms (TAp73 and ∆Np73) correlate with proliferation and apoptosis in DLBCL. In the present study, we examined whether modulating the expression of these p73 isoforms in DLBCL cells regulates cell survival, proliferation, and response to the chemotherapeutic agent, doxorubicin. We performed in vitro studies in DLBCL cells and modulated the expression of the two opposing p73 isoforms (TAp73 and ∆Np73) using expression vectors and siRNA in DHL16 (GCB subtype) and OCI-Ly3 (ABC subtype) cells. Under normal conditions TAp73-transfected cells showed comparable growth to control vector-transfected cells; however, the TAp73-transfected cells were more susceptible to serum deprivation as well as doxorubicin. In contrast, ∆Np73-transfected cells had accelerated growth when compared with control vector-transfected cells under normal conditions and were more resistant to serum deprivation and doxorubicin treatment. Subsequently, we analyzed the p73 direct transcriptional pro-apoptotic targets in cells transfected with TAp73 and ∆Np73 and vector control. Both the common p53 and p73 targets (PUMA and NOXA) as well as the exclusively p73 targets GRAMD4 and BIM, were up-regulated in the TAp73-transfected cells compared to control cells. A reduction of PUMA, BIM, and GRAMD4 was observed in the ∆Np73-transfected cells as compared to control cells. Furthermore, we observed that p73 knockdown using p73 siRNA altered the growth and the response to doxorubicin in both DLBCL cell lines. Together, these data demonstrate the significant biological role of p73 isoforms in DLBCL pathogenesis and therapeutic response. Our findings illustrate that modulation of TP73 isoforms induces apoptosis and expression of pro-apoptotic targets, thus establishing p73 as a potential therapeutic target in DLBCL.

#2056

Combined FLT3 and Pim kinase inhibitor treatment downregulates c-Myc early in apoptosis induction in acute myeloid leukemia with FLT3-ITD.

Jonelle K. Lee,1 Mario Scarpa,1 Shivani Kapoor,1 Maria R. Baer2. 1 _University of Maryland School of Medicine, Baltimore, MD;_ 2 _University of Maryland Greenebaum Comprehensive Cancer Center, Baltimore, MD_.

Background: fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is present in acute myeloid leukemia (AML) in 30 % of patients, with poor treatment outcomes due to rapid relapse. FLT3 inhibitors have limited activity and rapid onset of resistance. The oncogenic serine/threonine kinase proviral integration site for Moloney murine leukemia virus 1 (Pim-1) is upregulated downstream of FLT3-ITD and phosphorylates and stabilizes FLT3 in a positive feedback loop in cells with FLT3-ITD. Dual inhibition of FLT3 and Pim kinases is therefore a promising treatment strategy for AML with FLT3-ITD. The proto-oncogene c-Myc, upregulated in many cancer types, drives proliferation and causes resistance to apoptosis. We previously showed that concurrent FLT3 and Pim inhibition in cells with FLT3-ITD increases apoptosis through downregulation of the anti-apoptotic protein Mcl-1. Here we demonstrate downregulation of c-Myc upstream of Mcl-1.

Methods: Ba/F3-ITD and MV4-11 cells, with FLT3-ITD, were cultured with the FLT3 inhibitors gilteritinib (15 nM) or quizartinib (1 nM) and the pan-Pim inhibitor AZD1208 (1 μM) alone and in combination, and with DMSO control. c-Myc and Mcl-1 protein expression was measured by immunoblotting and c-Myc mRNA by qPCR using SybrGreen. Cells were cultured with cycloheximide (100 μg/mL) with and without the proteasome inhibitor MG-132 (20 μM). Ba/F3-ITD cells were stably transfected with a lentiviral c-Myc plasmid or a retroviral Mcl-1 plasmid and corresponding empty vectors. Apoptosis was detected by Annexin V and propidium iodine staining, measured by flow cytometry.

Results: Combined FLT3 and Pim inhibitor treatment decreased c-Myc protein expression in Ba/F3-ITD and MV4-11 cells within 30 minutes, preceding Mcl-1 downregulation. c-Myc mRNA levels did not change, but c-Myc protein half-life decreased with combination treatment, and this decrease was abrogated by proteasome inhibitionMcl-1 overexpression did not abrogate c-Myc downregulation by combination treatment, and c-Myc overexpression decreased apoptosis induction by combination treatment from 65% to 45%.

Conclusion: Combined FLT3 and Pim kinase inhibitor treatment enhances apoptosis in FLT3-ITD cells through c-Myc downregulation, which precedes Mcl-1 downregulation. c-Myc downregulation is post-translational, via increased proteasomal degradation.

#2057

Small molecule CB001 induces extrinsic apoptosis via downregulation of c-FLIP expression in mutant p53-expressing cancer cells.

Shengliang Zhang,1 Lanlan Zhou,1 Marie D. Ralff,2 Wafik S. El-Deiry1. 1 _Fox Chase Cancer Center, Philadelphia, PA;_ 2 _Lewis Katz School of Medicine, Temple University, Philadelphia, PA_.

Cellular FADD-like IL-1β-converting enzyme-inhibitory protein (c-FLIP) is a master anti-apoptotic regulator in cancer cells. c-FLIP inhibits caspase 8 activation and suppresses cell apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL) in malignant cells. Targeting c-FLIP is an attractive strategy for cancer therapy. We previously identified a small-molecule CB001 as a p53-restoring compound and found that CB001 induces cell death with little or no genotoxicity in human colorectal cancer cells. We now show that CB001 downregulates c-FLIP expression in cancer cells at the post-translational level via protein degradation. CB001 treatment induces an increase in caspase 8 activity which correlates with the decrease in c-FLIP at the protein level in mutant p53-expressing cancer cells. Knockdown of caspase 8 rescues cancer cells from CB001-induced apoptosis in mutant p53-expressing cancer cells, suggesting that downregulation of c-FLIP is one of the major mechanisms by which CB001 induces extrinsic cell apoptosis. Furthermore, our studies reveal a synergy between CB001 and TRAIL to induce apoptosis in TRAIL-resistant cancer cells, but not in normal cells at the tested doses. These results taken together suggest that CB001 is a promising lead for drug development with a strong capability to suppress tumor growth through targeting c-FLIP in cancer cells.

#2058

BCL-2 selective inhibitor APG-2575 synergizes with BTK inhibitor in preclinical xenograft models of follicular lymphoma and diffuse large B-cell lymphoma.

Douglas D. Fang, Guoqin Zhai, Shoulai Gu, Ran Tao, Qiuqiong Tang, Ping Min, Qixin Wang, Dongmei Yang, Jiaxing Gu, Yinfeng Li, Dingxiong Chen, Jiajun Li, Guangfeng Wang, Dajun Yang, Yifan Zhai. _Ascentage Pharma (Suzhou) Co., Ltd, China_.

Follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) are among the most prevalent B-lymphocyte neoplasms with Bruton's tyrosine kinase (BTK) often found abnormally activated in these patients. BTK inhibitor ibrutinib has showed proven efficacy in both indications, but its clinical application is limited to gradually acquired resistance. Recent studies reported that ibrutinib-resistant cells exhibited higher BCL-2 expression and therefore increase their sensitivity to BCL-2 inhibitors. In this study, using a BCL-2 selective inhibitor APG-2575, we asked whether its combination with ibrutinib enhanced antitumor activity in FL and DLBCL in preclinical studies. Indeed, the combination treatment with APG-2575 and ibrutinib synergistically enhanced antitumor activity in DOHH2 cell-derived FL xenograft models. Similarly, enhanced activity was achieved in DLBCL xenograft models derived from OCI-LY1 cells. Interestingly, in OCI-LY1 xenografts, the combination treatment achieved a 100 % response rate, which include partially (PR) and complete tumor regression (CR), with ~80 % of CR and ~20 % of PR recorded. Durable response was continuously observed till the end of the study after dose suspension. Pharmacokinetic studies revealed that there were no significant changes in drug concentrations between single agents and the combination treatment, suggesting there was no drug-drug interaction between these two agents. On-target pharmacodynamic (PD) modulations of BTK and apoptosis pathways were observed in corresponding tumor tissues co-treated with ibrutinib and APG-2575. An increase in the levels of cleaved PARP-1 protein was observed under the combination treatment suggesting enhanced induction of apoptosis occurred, which may be the biological event contributing to the synergistic effect of the combination treatment. In summary, our study suggests that APG-2575 may be applied to the combination therapy with ibrutinib in the treatment of FL and DLBCL.

#2059

Long-term survival of mice with relapsed ALL treated by oncolytic measles virus is terminated by expansion of persistently infected virus-resistant blasts.

Annika V. Goß,1 Carmen Dorneburg,1 Medhanie Mulaw,2 Chun Xu,3 Christine Landthaler,1 Irmela Jeremias,4 Jiwu Wei,3 Klaus-Michael Debatin,1 Christian Beltinger1. 1 _University Medical Center Ulm, Ulm, Germany;_ 2 _Ulm University, Ulm, Germany;_ 3 _Medical School Nanjing University, Nanjing, China;_ 4 _Ludwig-Maximilians-Universität München, München, Germany_.

Background: The prognosis of patients with relapsed and refractory ALL is poor. Oncolytic virotherapy is a promising treatment modality combining cancer cell lysis with immunomodulation.

Aims: To determine whether clinical-grade attenuated measles virus (MV) controls relapsed and refractory ALL in vitro and in vivo, to assess resistance mechanisms and to outline targets for immunotherapy in resistant cells.

Methods: The clinical-grade MV-NIS strain was used. ALL cell lines insensitive to chemotherapy and ALL xenografts derived from patients that had died from their disease were investigated. In vitro, cells were treated with MV-NIS and viability was determined. NSG mice were transplanted with a relapsed ALL PDX. Treatment was started when the leukemic load was high, i.e. when 20-30% blasts were present in the peripheral blood, and when the disease had heavily infiltrated spleen, liver, bone marrow and CNS. One dose of i.v. MV-NIS was given. Peripheral blast counts and survival of mice were determined. At time of death the presence of MV in leukemic infiltrates was determined by immunohistochemistry and qRT-PCR, MV was isolated from blasts and serum, and infectivity of isolated MV was tested on Vero indicator cells. mRNA from the blasts, including the genome of the MV they were infected with (MV is a mRNA virus), was subjected to mRNA sequencing. Gene set enrichment and mutation analyses were performed.

Results: In vitro, MV-NIS effectively killed ALL cell lines insensitive to chemotherapy and cells from relapsed patient ALL. In NSG mice with a high and disseminated load of relapsed patient ALL cells, one injection of i.v. MV-NIS sufficed to rapidly decrease leukemic load leading to long-term survival of the mice. Survival was terminated by late disease exacerbation due to expansion of virus-resistant leukemic cells persistently infected with infectious MV. Genome-wide mRNA sequencing revealed strong enrichment of antiviral pathways in the resistant cells, without mutations in the viral genome that could explain the resistance of the blasts.

Conclusions: MV-NIS is preclinically effective against relapsed and refractory ALL in vitro and, initially, in vivo. Late expansion of virus-resistant cancer cells persistently infected with MV is a phenomenon hitherto not described. The viral and antiviral proteins strongly expressed in these cells, which include cell surface proteins, can serve as targets for subsequent immunotherapy.

#2060

Combination of CD20 targeted engineered toxin body, MT-3724, with chemotherapy or IMiDs for the treatment of non Hodgkin's lymphoma.

Jack P. Higgins, Aimee Iberg, Caleigh Howard, Erin Willert. _Molecular Templates Inc., Austin, TX_.

MT-3724 is a novel recombinant fusion protein consisting of a CD20 binding variable fragment (scFv) fused to Shiga-like toxin-I A1 (SLTA) ribosome inactivating protein. Engineered Toxin Bodies (ETBs) are unique as they can induce internalization after binding to non- or poorly-internalizing receptors with subsequent enzymatic and permanent ribosome destruction through SLTA, representing a novel mechanism of direct cell-kill in oncology. MT-3724 has demonstrated single agent anti-tumor activity in heavily pre-treated relapsed/refractory (R/R) Non-Hodgkin's lymphoma NHL patients in a Phase I clinical study. Specifically, in R/R diffuse large B cell lymphoma (DLBCL) patients who had low pre-existing serum levels of rituximab, an objective response rate (ORR) of 30% (3 of 10) and disease control rate of 70% (7 of 10) has been observed thus far (NCT02361346).

Given the unique ribosomal inhibition mechanism of action for MT-3724, we hypothesized that combination of MT-3724 with agents possessing a differentiated mechanism of action could result in additive or synergistic cellular cytotoxicity in CD20 positive NHL cell lines. Preclinical studies were conducted to assess MT-3724 in combination with cytotoxic chemotherapeutic agents (doxorubicin, gemcitabine, bendamustine, and vincristine) or an immunomodulatory (IMiD) agent (lenalidomide) on selected NHL cell lines. The combination of MT-3724 with all agents demonstrated additive or synergistic cytotoxicity of NHL cell lines.

The single agent clinical activity of MT-3724 in heavily pre-treated R/R NHL patients along with this preclinical combinatorial effect of MT-3724 with chemotherapy and IMiDs used in the treatment of R/R NHL have lead the opening of a Phase 2a study of MT-3724 in combination with Gemcitabine and oxaliplatin (GEMOX, NCT03488251) and another planned Phase 2a study of MT-3724 in combination with lenalidomide (NCT03645395).

MT-3724 has demonstrated good tolerability and early signs of clinical activity as monotherapy in patients with R/R NHL along with additive and/or synergistic effects in combination with chemotherapy and IMiDs preclinically. Additional clinical studies to evaluate MT-3724 as single agent and in combination with GEMOX or lenalidomide are underway.

#2061

A novel small molecule inhibitor of MDM2-p53 (APG-115) has antitumor activity in gastric adenocarcinoma.

Lin Zhang,1 Luo Qiuyun,1 Yan Xianglei,1 Xueping Wan,1 Luping Yuan,1 Yuxing Zhang,1 Suna Zhou,1 Wentao Pan,2 Mengxian Pan,2 Miaozhen Qiu,1 Shijuan Mai,1 Dajun Yang1. 1 _Sun Yat-Sen University Cancer Center, Guangzhou, China;_ 2 _Suzhou Ascentage Pharma Inc, Suzhou, China_.

Background: The amplification of Murine Double Minute 2 (MDM2) and mutation of P53 are significant processes in the incidence of gastric cancer. APG-115, a small molecular inhibitor of MDM2-P53, is a novel potential anticancer agent. We aim to investigate the anticancer effect of APG-115 in gastric cancer.

Methods: We selected 87 pairs of gastric cancer tissues (primary tumors and metastatic tissues). Using Immunohistochemistry (IHC) and Fluorescence in situ hybridization (FISH), we examined the expression and amplification of MDM2 and P53. Using MTS, we examined the anti-proliferation activity of APG-115 in five gastric cancer cell lines (AGS, MKN-45, BGC-823, NCI-N87, NUGC-3) by CCK-8 assay. We used western blot analysis and siRNA interference for mechanism exploration to examine the anti-cancer activity of APG-115 in p53 wild type gastric cancer cell lines. Gastric cancer cell xenograft nude mice models were used for in vivo efficacy evaluation.

Results: We examined 87 pairs of gastric cancer tissues in IHC. The results suggested that the primary tumors and metastatic tissues with high expression of MDM2 and P53 have shortened survival. The proliferation inhibition of APG-115 on p53 wild-type gastric cancer cells of AGS and MKN45 was concentration and time dependent. APG-115 induced apoptosis in gastric cancer cells. APG-115 can induced cell-cycle arrest in G1/G0 phase in the p53 wild-type of gastric cancer cells. APG-115 exhibited potent antitumor activity against established human gastric cancer xenografts.

Conclusions: APG-115 can inhibitor tumor growth in p53 wild-type gastric cancer cells by inducing apoptosis and cell cycle arrest both in vitro and vivo. APG-115 can serve as an effective and precise therapeutic strategy for gastric cancer.

#2062

Evaluating the effect of tumor necrosis on glycolytic flux and cell survival in glioblastoma.

Evan Noch, Isaiah Yim, Lewis Cantley. _Weill Cornell Medicine, New York, NY_.

Glioblastoma continues to rank as one of the most lethal primary human tumors. The degree of necrosis in glioblastoma remains one of the most powerful predictors of poor prognosis, but the relationship between necrosis and poor prognosis is not known. It is unclear whether endogenous tumor necrosis is merely an indicator of an aggressive phenotype or whether tumor necrosis itself propagates the aggressive phenotype. Determining the role of necrosis in mediating the malignant potential of glioblastoma therefore represents an important aspect of therapy. We describe a model of patient-derived glioblastoma spheroids expressing a genetically encoded fluorescent NADH/NAD+ biosensor. This biosensor reports glycolytic flux and can be imaged with fluorescent microscopy. We imaged spheroids using multiphoton microscopy and differentiated cells by high versus low NADH/NAD+ levels. FACS analysis of dissociated spheroids followed by RT-PCR analysis demonstrated increased expression of genes known to be upregulated in peri-necrotic regions, including procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 (PLOD2), activating transcription factor 5 (ATF5), and protein O-fucosyltransferase 2 (POFUT2). These cells also had increased expression of hypoxia-responsive genes, including vascular endothelial growth factor (VEGF) and jumonji domain-containing protein 6 (JMJD6). We created an intracranial glioblastoma patient-derived xenograft model, with cells expressing this NADH/NAD+ biosensor. When imaging these tumors in vivo, we were able to demonstrate that cells with high NADH/NAD+ levels are more likely to survive within the peri-necrotic environment. These findings indicate that increased glycolytic flux facilitates survival from expanding regions of necrosis. Future experiments will focus on biopsy of cellular populations with high versus low NADH/NAD+ levels within the peri-necrotic niche. We will then perform RNAseq and metabolomics analysis to determine the genetic and metabolic adaptations that facilitate survival within this microenvironment. This model to study cell survival within the peri-necrotic region will improve awareness of necrosis-mediated growth proliferation and may lead to treatments targeted to tumor necrosis that could improve patient prognosis.

#2063

GO-Chl induce necroptosis death in A549 lung cancer cells through p62/SQSTM1 mediated DNA damage response.

Braham D. Arya, Surinder P. Singh. _CSIR- National Physical Laboratory, New Delhi, India_.

Autophagy is a highly regulated intracellular catabolic pathway for maintenance of cell homeostasis through the process of degrading proteins and organelles. Inhibition or modulation of autophagy may leads to accumulation of DNA damage and genomic instability. Recently, it has been reported that DNA damage or repair response by autophagy machinery can switch the cell signalling cascade from apoptosis to necroptosis. The synergistic effect of autophagy machinery with DNA damage pathways may provide a therapeutic window for next generation nanomedicine. Graphene oxide (GO) alters the autophagy response through toll like receptor signaling, lysosomal dysfunction, mitochondrial destabilization and NF- κB pathways. On the other hand, Chloroquine (Chl) an FDA approved drug inhibits the autophagy and has also shown anticancer potential. In the present study, we endeavored the conjugation of Chl onto GO nanosheets and evaluated the antiproliferative efficacy of GO-Chl nanoconjugate on human lung cancer A549 and normal lung BEAS-2B cell lines. Morphological properties of GO and GO-Chl have been analyzed through TEM, FESEM and AFM and formation of nearly monolayer highly exfoliated GO nanosheets has been observed. Further, structural, functional and optical properties of GO, Chl and GO-Chl have been investigated using Raman, FTIR and UV-Vis spectroscopy respectively. MTT assay has been performed for in- vitro cytotoxicity evaluation of GO, Chl and GO-Chl exposure on A549 and BEAS-2B cell lines and demonstrated that GO-Chl treatment exhibits significant cell death in A549 lung cancer cells. DCFDA assay reveals that GO-Chl exposure enhance generation of ROS. Flow cytometry based Propidium Iodide (PI) assay reveals the plasma membrane disruption leading to alteration in the cell cycle. Further, flow cytometry based annexin V/PI assay for cell cycle analysis indicate towards the halts of the cell cycle at G1 phase and possible DNA damage response. The integrity of A549 cells DNA was accesed using Comet assay. The results reveals a significant increase in Olive tail moment with increasing concentration of GO-Chl. Further, the autophagy response in A549 cells due to GO-Chl treatment is investigated through fluorescence microscopic analysis (MDC staining and GFP-LC3 plasmid), TEM observations and immunoblot analysis. Enhanced level of LC-3 I/II and Atg-5 markers signifies the autophagosomes formation and elevated expression of p62/SQSTM1 indicates the inhibition of autophagy at later stage. Recently, it was reported that enhanced level of p62/SQSTM1 may inhibit the DNA repair mechanism leading to decreased growth in-vitro and in-vivo. On the basis of results obtained, it is envisioned that GO-Chl nanoconjugate could be used as an effective cancer therapeutic agent, by targeting the DDR response through modualtion of autophagy.

### Cellular Responses to Anticancer Agents 2 / Overcoming Resistance

#2064

CHRM1-mediated acetylcholine signaling protects prostate cancer cells from the chemotherapy agent docetaxel.

Tyler Bland, Karen Vo, Baron Bechtold, Boyang Wu. _WSU, Spokane, WA_.

Background: Chemotherapy-resistant prostate cancer (PCa) remains a leading cause of cancer-related deaths in advanced PCa patients. The current study investigates the role of acetylcholine (ACh) signaling in the development and maintenance of chemotherapy resistance in PCa.

Methods: Human PCa cell cultures were used to harvest total RNA and protein for qPCR and Western blot analyses, respectively. The chemotherapy drug docetaxel's (DTX) ability to induce apoptosis was measured with crystal violet survival assays and Western blot analysis of pro-apoptotic proteins.

Results: Treatment of the androgen-independent human PCa cell lines 22Rv1 and DU145 with a low concentration of DTX at 1 nM increases the expression of the key ACh production and secretion enzymes choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), and the high-affinity choline transporter 1 (CHT1), which is accompanied by a 4-fold increase in ACh secretion. These genes also show increased expression in DTX-resistant 22Rv1 cells. The muscarinic receptor system further shows alterations in gene expression with low-concentration DTX treatment and acquired DTX resistance. Specifically, across all 5 muscarinic cholinergic receptors (CHRM1-5), the expression of CHRM1 is increased in both circumstances. Treatment of both DTX-sensitive and -resistant cell lines with a combination of DTX and the CHRM1 antagonist dicyclomine decreases cell survival, lowers DTX IC50 values (DU145: 62%; 22Rv1: 48%; DTX-resistant 22Rv1: 23%), and enhances the expression of the pro-apoptotic proteins cleaved caspase-3 and cleaved PARP1 compared to treatment with DTX alone. This is paralleled by the observations that treatment with the ACh mimetic carbachol (CCh) protects cells from DTX treatment. Mechanistically, ACh signaling protects PCa cells from DTX through activation of the extracellular signal-regulated kinase / cAMP response element binding protein 1 (ERK/CREB) pathway. Treatment of 22Rv1 cells with CCh increases levels of the active phosphorylated forms of both ERK1/2 and CREB within 30 min. This increase is inhibited by the pan muscarinic receptor antagonist atropine and is not affected by the pan nicotinic receptor antagonist tubocurarine. Treatment of 22Rv1 cells with a combination of DTX and either the ERK inhibitor UO-126 or the CREB inhibitor 666-15 decreases cell survival in a synergistic manner.

Conclusion: DTX induces an enhancement of ACh signaling in PCa cells, which may contribute to the development and maintenance of DTX resistance through CHRM1 and the downstream ERK/CREB pathway. Antagonism of CHRM1 and its downstream effectors may be useful in preventing and reverting chemotherapy resistance in PCa.

#2065

Biomarker-targeted novel combination for renal cell carcinoma.

Jiaojiao Wang,1 Andre R. Jordan,2 Sarrah S. Lahorewala,1 Vinata B. Lokeshwar1. 1 _Augusta University, Augusta, GA;_ 2 _University of Miami, Miami, FL_.

INTRODUCTION: The survival of patients with metastatic renal cell carcinoma (mRCC) is < 10% at 5-years, despite treatment. Sorafenib (SF) is a second-line oral treatment which improves overall survival by 12-18%. However, mechanism for SF failure is not known. Hyaluronic acid (HA) promotes cell proliferation, invasion and motility through its receptors CD44 and RHAMM. Hymecromone (HC) is a non−toxic dietary supplement that inhibits HA synthesis and down regulates CD44 and RHAMM mRNA levels. In this study we examined the mechanism of SF resistance and the efficacy of HC+SF combination as a targeted therapy for advanced RCC.

METHODS: HAS3 levels were measured in kidney specimens (normal=45; tumor= 86) by qPCR. HAS3 promoter was cloned and effect of SF and HC on HAS3 promoter activity was determined. Response of Vector-only (EV) or HAS3-overexpressing (HAS3) or HAS3-knockdown (HAS3-KO) transfectants of 786-O and Caki-1 cells, primary RCC spheroids and endothelial cells to SF+HC combination was examined in proliferation, apoptosis, motility and invasion assays. Immunoblotting was performed for target analysis. Treatment efficacy was evaluated in the Caki−1 subcutaneous (s.c.) and orthotopic xenograft models.

RESULTS: HAS3 levels were 5-10-fold elevated in mRCC patients' tumors as compared to non-mRCC patients and normal kidney tissues, respectively. HAS3 levels were an independent predictor of metastasis (P=0.043; sensitivity: 85.7%). Combination of SF (5 µM) with HC (20, 40 µg/mL) downregulated (>80%) HAS3 expression, HAS3 promoter activity and HA synthesis in RCC cells. HC+SF combination inhibited RCC cell and primary tumor spheroid proliferation > 90%, motility/invasion (>80%) and induced apoptosis (5-fold). While HAS3 transfectants were resistant to SF+HC treatment, HAS3-KO transfectants were sensitive to SF. HA addition also attenuated the effect of HC+SF observed in EV cells. Endothelial cells co-cultured with HAS3 transfectants were resistant to SF+HC treatment. SF+HC up-regulated apoptosis effectors, but downregulated CD44, RHAMM, and phospho-Met levels in EV but not in HAS3 transfectants. HC (100 or 200 mg/kg) and SF (30 mg/kg) oral combination abrogated Caki-1 tumor growth (>80%), without serum/tissue toxicity. HC+SF treatment decreased HAS3/HA levels, Ki67 index and microvessel density. HAS-overexpressing tumors were resistant to treatment.

CONCLUSION: This is the first study to demonstrate that HAS3 overexpression in mRCC contributes to SF failure and that HAS3/HA downregulation is a mechanism to re-sensitize cells to SF. HC+SF is an orally bioavailable and non-toxic combination that may be an effective therapeutic strategy for mRCC.

#2066

Targeting NUPR1: Response to a common compensatory pathway in triple negative breast cancer.

Jeffrey P. Solzak, Milan Radovich. _Indiana Univ. School of Medicine, Indianapolis, IN_.

Introduction: Triple negative breast cancer (TNBC) is a devastating disease responsible for a higher rate of morbidity and mortality compared to all other breast cancer subtypes. With few effective treatments, many have moved towards combination therapies. Using a genomically driven methodology, we show that Nuclear Protein 1 (NUPR1) is upregulated and activated in response to several targeted therapies and to standard chemotherapy. In this work, we show that targeting NUPR1 in conjunction with the chemotherapeutic paclitaxel, results in a synergistic combination.

Methods: To evaluate a cohort of individuals with TNBC, 58 tumors were analyzed pre- versus post-chemotherapy using Ingenuity Pathway Analysis (IPA, Qiagen) after whole transcriptome sequencing. Six TNBC cell lines were utilized for in vitro work to reflect the heterogeneity of disease presentation. Four targeted therapies (Dasatinib, Erlotinib, Everolimus, and Trametinib) were chosen based on prior data suggesting activation of SRC, EGFR, mTOR, and MEK1/2 in TNBC respectively. Paclitaxel was also used as a standard chemotherapy. IC50s were calculated using standard log dose escalations and Prism 7 (GraphPad). Next, whole transcriptome sequencing and pathway analysis using IPA was performed on treated cells at their designated IC50s for each drug and compared to vehicle controls. Trifluoperazine (TFP, a small molecule inhibitor of NUPR1) and paclitaxel combination experiments were completed on TNBC cell lines and CI values were determined using the Chou-Talalay method. To observe the effects of NUPR1 inhibition, a NUPR1 siRNA was utilized in combination with paclitaxel at increasing doses with mock transfected cells for control.

Results: We observed NUPR1 activation and RNA overexpression in all six cell lines treated with four targeted therapies. Combining the RNA sequencing analysis of these cell lines and focusing on common responses, NUPR1 was in the top three responses across all treatments. Our analysis of TNBC patient tumors (N=58) before and after chemotherapy resulted in observing an activation of NUPR1 (p=5.32E-10). In TFP and paclitaxel combination experiments, we observed CI values across cell lines ranging from .45-.90 denoting synergy. Experiments performed that knocked down NUPR1 with a siRNA displayed similar significant synergy with paclitaxel.

Conclusion: Heterogeneous cell lines treated with targeted therapies as well as patients treated with chemotherapy demonstrate upregulation of the somatic pathway governing NUPR1. Targeting NUPR1's ability to communicate using TFP in combination with paclitaxel increased the potency of paclitaxel at nanomolar doses. While further in vivo experiments testing this combination are an obligate next step, the combination of TFP and paclitaxel has the potential to be a viable treatment for TNBC that may be effective in a wider population.

#2067

**Trifluridine/tipiracil can overcome the resistance of gastric 5-fluorouracil-refractory cancer cells with various cancer driver-genes alterations** in vitro **and** in vivo **.**

Kazuaki Matsuoka, Takashi Kobunai, Teiji Takechi. _Taiho Pharmaceutical Co., Ltd., Japan_.

Background: Trifluridine (FTD) is a key component of the antitumor drug combination trifluridine/tipiracil (FTD/TPI, also known as TAS-102), which is approved for the treatment of patients with metastatic colorectal cancer refractory to standard chemotherapies. Recently, a global phase III Trial (TAGS trial) revealed that FTD/TPI also prolongs overall survival in patients with metastatic gastric cancer. In this study, we explored the efficacy of FTD/TPI in 5-fluorouracil (5-FU)-resistant gastric cancer cell lines in vitro and in vivo, revealing their unique genomic signatures by a multi-omics profiling approach.

Method: 5-FU-resistant cell lines were established by continuously exposing the parent cell lines (MKN45, MKN74, and KATOIII) to escalating concentrations of 5-FU over a 1-year period. The sensitivities of the cell lines to FTD were evaluated by a cell growth inhibitory assay using crystal violet staining. Total RNA was purified using a silica membrane spin column-based centrifugation procedure. The extracted RNA was used to establish a cDNA library for transcriptome sequencing using a HiSeq 2500 platform. The resulting fastq files were used for mapping, gene mutation, and expression analysis. We especially focused on "pyrimidine metabolism" in Kyoto Encyclopedia of Genes and Genomes analysis.

Results: The KATOIII/5FU, MKN74/5FU, and MKN45/5FU were 2.4-fold, 4.7-fold, and 14.3-fold more resistant to 5-FU than their parent cell lines, respectively. MKN74/5FU and KATOIII/5FU did not show any cross-resistance to FTD, while TS-overexpressing MKN45/5FU showed partial cross-resistance to FTD in vitro. FTD/TPI exhibited significant antitumor activity in both MKN45/5FU and MKN74/5FU cells in vivo. As compared with corresponding parental cell lines, the transcriptome analysis revealed that ENTPD3 gene expression level changed by more than 2-fold in only MKN45/5FU, while ENTPD8 and ENPP3 gene expression changed by less than 0.5-fold in both MKN74/5FU and KATOIII/5FU. In addition, there are genes (UMPS, POLR2B, POLR2D, POLR2H, POLR2L, POLR2J2, POLR3G, PRIM1, POLE, and UPP1) that had non-synonymous mutation within their coding sequences. Interestingly, mutation status of POLR2L and PRIM1 in MKN45/5FU, of UMPS, POLR2D, and POLR2H in MKN74/5FU, of POLR2B, POLR2J2, and POLR3G in KATOIII/5FU differed from those of parental cell lines.

Conclusions: FTD/TPI was effective against 5-FU-resistant gastric cancer cells harboring various genetic alterations in vitro and in vivo. This might be a reason why FTD/TPI is expected to show significant benefits against heterogeneous metastatic gastric cancer in a clinical setting.

#2068

A highly selective dual Haspin-kinase and PIM-inhibitor overcomes RAF/MEK-inhibitor resistance in melanoma.

Johannes C. Melms, Sreeram Vallabhaneni, Caitlin E. Mills, Peter K. Sorger, Kai Wucherpfennig, Benjamin Izar. _Dana-Farber Cancer Institute, Cambridge, MA_.

Intrinsic or acquired resistance to targeted therapies, such as RAF/MEK inhibitors in melanoma, represents a significant clinical challenge. We performed a small molecule screen in isogenic sensitive (S) and RAF/MEK-inhibitor-resistant (RMR) melanoma cell lines and identified a nominal PIM kinase inhibitor (Comp-1) with high activity against A375-S and A375-RMR, with an EC50of ~100 nM. While the phosphorylation of some PIM targets, including 4EBP1 and MYC, were significantly inhibited in a dose-dependent fashion, phosphorylation of BAD was not reduced as one would expect. To determine the on-target activity of this compound, we generated A375 cell lines overexpressing WT PIM isoforms 1-3 or their kinase-dead (KD) forms, followed by treatment with PIMi-1. We found only modest shifts in EC50and hypothesized that the beneficial effects of this compounds were due to inhibition of an alternative target. A screen of Comp-1, along with structurally related compounds, against 468 human kinases revealed exquisite affinity for Haspin kinase (HK). We determined the IC50of Comp-1 against HK to be 14.9 nM. The only described role of HK is phosphorylation of Histone H3 at Thr 3 (H3T3) and thereby regulation of mitotic progression. Quantitative single-cell immunofluorescence (IF) analysis of Comp-1 treated A375 confirmed complete abrogation of p-H3T3, indicating on-target activity in vitro. To understand the temporal effects of HK inhibition with Comp-1, we performed live-cell imaging and single-cell analysis of A375 transduced with a mitotic phase-specific reporter. Cells experienced mitotic stress or mitotic slippage resulting in DNA-damage and an increase gamma-H2AX foci per nuclei. In line with these observation, we observed increased phosphorylation p53 and CHECK2. Furthermore, a portion of cells developed micronuclei in a dose-dependent fashion providing a substrate for the cytosolic DNA-sensing machinery, including the cGAS-STING pathway, and rationale for combining Comp-1 with immunotherapies. Ongoing experiments in tumor-bearing BALB/c mice indicate safety and potentially enhanced efficacy of Comp-1 when combined with either anti-CTLA-4 or anti-PD-1 therapy. Ex vivotreatment of patient-derived tumor-infiltrating lymphocytes (TILs) with Comp-1 revealed no toxic effects. A pan-cancer RNA-seq analysis revealed significant upregulation of HK in 34 of 37 tumor types supporting its role as a potential therapeutic target. Overall, our work identified a highly selective Haspin Kinase inhibitor with activity against RAF/MEK inhibitor resistant melanoma, insights into the underlying mechanisms, and potential in vivo benefit when combined with immune checkpoint inhibitors.

#2069

Both target and warhead sensitivity are critical components of activity for c-Met targeting ADC TR1801-ADC.

Julia A. Coronella,1 Marco Gymnopoulos,1 Oscar Betancourt,1 Vincent Blot,2 Diana Ly,1 Jose Villicana,1 Neki Patel,3 Balakumar Vijayakrishnan,3 Roland Newman1. 1 _Tanabe Research Laboratories, Inc., San Diego, CA;_ 2 _Abbvie, San Francisco, CA;_ 3 _MedImmune, San Diego, CA_.

Antibody drug conjugates (ADCs) have demonstrated robust clinical activity, leading to strong growth in ADC development. However, it is an oft-noted phenomenon that the clinical activity of ADCs correlates poorly with quantity of the ADC target protein. Understanding the mechanistic factors involved may lead to more effective clinical strategies, and ultimately, improved patient outcomes. In the current study, we sought to explore factors that might contribute to sensitivity or resistance to TR1801-ADC, a c-Met targeted ADC that utilizes a pyrrolobenzodiazepine (PBD) payload. TR1801-ADC was potently cytotoxic in a panel of c-Met expressing cell lines in vitro, but no correlation between IC50 and c-Met expression was observed, beyond a requirement for a baseline number of c-Met receptors per cell as measured by qFACS. Beyond this threshold level, quantity of cell surface c-Met did not correlate significantly with in vitro IC50. Instead, TR1801-ADC activity in specific cell lines correlated with sensitivity to the PBD warhead when assayed as a free small molecule in cytotoxicity assays. Corresponding data were observed for a series of 20 c-Met expressing colorectal and gastric patient-derived xenograft (PDX) models. PDX in vivo tumor growth inhibition (%TGI) by TR1801-ADC was associated with ex vivo sensitivity to the PBD warhead, but not the level of c-Met as measured by IHC. TR1801-ADC is thus a targeted cytotoxic where sensitivity to the toxin is an important mechanistic component. However, individual cytotoxic agents such as commonly used chemotherapy drugs have well-documented differential activity across specific indications and tumors. To explore this, the PBD warhead was assayed as a free small molecule in 320 human cancer cell lines in vitro. Potent cytotoxic activity was observed broadly across cell lines and indications of origin, with a 4-log range in IC50. Of 50 comparator control drugs, the pattern of PBD cell line specific activity most closely resembled platinum compounds, although unlike the platinums, the PBD was equally active in KRAS mutant and wildtype lines. Sensitivity to the PBD in vitro was associated with alterations in DNA repair pathway genes, as might be expected for a DNA cross-linking agent, while elevated ABCB1 expression was associated with decreased sensitivity. Strikingly, in vivo PDX efficacy studies with TR1801-ADC were completely consistent with these findings. Gastric and CRC PDX models containing KRAS mutations and wt models were equally sensitive to TR1801-ADC in vivo. Further, models with meaningful mutations within the DNA mismatch repair (MMR) pathway were significantly more sensitive to TR1801-ADC in vivo, while resistance was associated with ABCB1 overexpression. In summary, we conclude that the profile of sensitivity to the ADC warhead is a critical component of ADC activity. These and other data will be discussed.

#2070

Exploration of markers of synergistic lethality of PARP and PI3K-Akt-mTOR inhibitors in women's cancers.

Marilyne Labrie,1 Zhenlin Ju,2 Jennifer K. Litton,2 Tae-Beom Kim,2 Sanghoon Lee,2 Ken Chen,2 Pamela T. Soliman,2 Michael Frumovitz,2 Larissa A. Meyer,2 Stacy Moulder,2 Amir A. Jazaeri,2 Karen H. Lu,2 Anil K. Sood,2 Robert L. Coleman,2 Gordon B. Mills,1 Shannon N. Westin2. 1 _OHSU, Portland, OR;_ 2 _The University of Texas, MD Anderson Cancer Center, TX_.

Introduction: The objective was to identify molecular mechanisms and protein networks involved in synthetic lethality as well as mechanisms of adaptive changes to the combination of olaparib (O) combined with inhibitors of the PI3K-AKT axis.

Methods: 110 patients with recurrent ovarian, endometrial, and triple negative breast cancer were treated with a combination of O and vistusertib (V) or capivasertib (C) (mTORC1/2 and AKT inhibitors, respectively). We have previously reported durable clinical efficacy of these combinations, regardless of BRCA status. Tumor samples were collected pre-treatment and 30 days post-treatment and reverse-phase protein array (RPPA) was performed. Baseline expression was assessed and change in expression was calculated. Bioinformatics and statistical methods were developed to assess activity of signaling pathways. Pathway scores were determined based on the expression of sets of proteins known to be involved in a specific pathway, including PI3K, RAS/RAF, and DNA damage repair. Association between protein expression and treatment efficacy was determined.

Results: We analyzed a total of 55 paired samples from patients treated with O/V (n=25) and O/C (n=30). These included 20 endometrial (37% PR, 31.5% SD, 31.5% PD), 21 ovarian (10% PR, 55% SD, 35% PD) and 14 triple negative breast (15% PR, 23% SD, 62% PD) tumors. Response to O/V was associated with low pre-treatment mTOR pathway activity and led to altered immune activity based on decreased CD4 and PD1 expression. Resistance to O/V was associated with high pre-treatment cell proliferation (high CCNB1, CDK1 and PLK1 protein expression). In contrast, response to O/C was associated with adaptive responses indicated by decreased mTOR activity and induction of the DNA damage checkpoint (high phospho-CHK1, -WEE1 and -CDC2). Resistance was associated with high pre-treatment RTK activity levels (high phospho-HER3, -EGFR, -IGFR, -MET) and AKT-independent activation of mTOR in the on-treatment samples.

Conclusion: Analysis of pre- and on-treatment samples from responders and non-responders to PARP and PI3K pathway combination therapy identified biomarkers of patients likely to benefit from therapy and helps to understand molecular mechanisms involved in response and resistance to these drug combinations.

#2071

Protein aggregate formation predicts clinical responses to EGFR tyrosine kinase inhibitors.

Pim French, Ya Gao, Maurice de Wit, Darlene Mercieca, Iris de Heer, Bart Valkenburg, Martin van Royen, Joachim Aerts, Peter Sillevis Smitt. _Erasmus MC, Rotterdam, Netherlands_.

Although many tumours depend on EGFR signalling for growth, benefit from EGFR tyrosine kinase inhibitors (TKIs) is restricted to patients harboring specific activating mutations and even then restricted to specific TKIs only. We here performed functional analysis on EGFR to understand why only specific mutation-TKI combinations respond. We find that the addition of TKIs (n=5 inhibitors tested) to mutated-EGFR expressing cells resulted in a rapid and massive formation of protein aggregates, but only with EGFR-mutations where clinical responses have been documented (L858R, ΔE746-A750) and only with TKIs with proven clinical benefit (n=4). No aggregates are formed with inhibitors that are clinically inactive (lapatinib) or with clinically unresponsive mutations (EGFRvIII, EGFRwt). Constructs harboring secondary resistance mutations only formed aggregates in the presence of osimertinib. Western blot, quantitative imaging analysis, revers phase phosphoprotein arrays (RPPA) and RT-qPCR was performed on five different lung cancer cell lines to demonstrate that all TKIs effectively inhibit EGFR phosphorylation and downstream pathway activation. Aggregate formation therefore occurs in addition to EGFR dephosphorylation. We next tested whether our assay is able to predict clinical responses to EGFR TKIs. For this, we first generated eleven different mutation constructs and show that our in-vitro assay directly correlates with cell survival in eleven different EGFR-mutated cell-lines, with striking concordance of IC50 values. We then generated more mutation constructs (total n=31) to show that our in-vitro assay predicts clinical benefit to EGFR TKIs in pulmonary adenocarcinoma patients (median survival 7.0 vs 13 months, HR 0.21, P=0.0004), including prediction of mutations where the response to inhibitors has thus-far not been documented (n=7). Our in-vitro assay therefore provides a clinically important asset to predict whether a tumor harboring an unknown mutation will respond to EGFR-TKIs, and if so, which TKI is most effective. We propose a model whereby only clinically effective inhibitors induce a conformational change, which occurs only in the context of specific activating mutations and in addition to EGFR dephosphorylation. Since this model only depends on the mutation present and the inhibitor used, response to EGFR TKIs is largely independent of the genetic background of the tumor. All patients with sensitive EGFR mutations should, regardless of the type of tumor, be considered for treatment with EGFR-TKIs.

#2072

Selinexor in combination with chemotherapy or idelalisib elicits a synergistic cytotoxic effect in primary CLL cells.

Maria Todaro,1 Valentina Griggio,1 Chiara Salvetti,2 Chiara Riganti,2 Yosef Landesman,3 Mario Boccadoro,1 Candida Vitale,1 Marta Coscia1. 1 _Division of Hematology, University of Turin, Turin, Italy;_ 2 _University of Turin, Turin, Italy;_ 3 _Karyopharm Therapeutics Inc, Boston, MA_.

Despite the therapeutic efficacy of new targeted drugs in chronic lymphocytic leukemia (CLL), treatment of high-risk patients remains an unmet clinical need. Tumor suppressor proteins (TSPs) and growth regulatory proteins (e.g. p53, p21, FoxO3a - the effector of Akt signal transduction, and IκB - the endogenous inhibitor of NF-κB) bind the nuclear export protein XPO1 and are carried through the nuclear pore complex into the cell cytoplasm. XPO1 overexpression plays a pathogenic role in CLL by mislocalizing TSPs and other cargoes in the cytoplasm. Selinexor is an oral inhibitor of XPO1, which is active as single agent in different hematologic malignancies. The purpose of this study was to evaluate the in vitro cytotoxic effects of selinexor, used in combination with chemotherapy drugs or with the PI3K inhibitor, idelalisib, against primary CLL cells. Specifically, this study aimed at identifying combination regimens that might overcome single agent resistance. Purified CLL cells from 29 patients were exposed to selinexor (Sel) in combination with fludarabine (F-ara-A), bendamustine (Ben) or idelalisib (Ide). Drug concentrations and exposure times were selected based on data obtained in an initial cohort of 5 patients. In selected experiments, the M2-10B4 murine stromal cell (SC) line was used in co-culture with CLL primary cells. Cell viability was analysed by Annexin-V/propidium Iodide immunostaining and flow cytometry. Combination index (CI) was determined using Calcusyn software. NF-kB and Akt activity was tested through an ELISA assay. After 72-hour culture, a significant decrease in CLL cell viability was observed when samples were treated with drug combinations (i.e. Sel 0.1 µM + F-ara-A 1 µM/F-ara-A 10 µM/Ben 30 µM/Ben 50 µM/Ide 0.1 µM/Ide 1 µM/ Ide 10 µM; Sel 1 µM + F-ara-A 1 µM/Ben 30 µM/Ben 50 µM) compared to the single compounds. The majority of combinations exerted synergistic cytotoxic effects. Interestingly, the addition of selinexor was effective in restoring the fludarabine sensitivity of CLL cells showing in vitro resistance to fludarabine. Sel was also capable to overcome the SC-mediated drug resistance, as shown by the significantly increased and synergistic cytotoxicity exerted by drug combinations, compared to single agents, toward leukemic cells co-cultured with SC. From the molecular standpoint, preliminary data showed that Sel significantly potentiated the inhibitory effect exerted by single-agent idelalisib on NF-kB and Akt activity. Our data demonstrate that the combination of Sel with chemotherapy or idelalisib has synergistic cytotoxic effects, also counteracting intrinsic or SC-mediated drug resistance. In vivo experiments in the Eμ-TCL-1 mouse model are currently ongoing to further corroborate the efficacy of selected drug combinations.

#2073

Profiling and comparison of breast cancer standard of care agents using the OncoPanel™ cell-based profiling service.

Alastair J. King, Lee R. Cavedine, Alyssa M. Croff, Kristin C. Dempsey, Brogan A. Epkins, Steven M. Garner, Tracy Lu, Victoria V. McBain, Joseph W. Murphy, Vanessa L. Norman, Natiya E. Robinson, Kayla A. Stehle, Charles R. Wageman, Jesse J. Parry. _Eurofins Panlabs, Inc., St. Charles, MO_.

Oncology drugs with clinical utility for the treatment of breast cancer span a wide range of different mechanisms and cancer type profiles. Anti-cancer therapies have come a long way from the early use of broad spectrum, cytotoxic agents, with mechanisms that often result in harsh side-effects. The advent of more focused, targeted therapies has afforded the potential for not only fewer side effects, but also the ability to provide more efficacious combination therapies with a greater potential for clinical efficacy. Recent advances have seen the approval of agents targeting CDK4/6, Her2, and PARP over the last ten years. Profiling of candidate molecules for eventual use in a therapeutic setting is an essential component of the drug discovery and development process. Understanding not only the biological potency of a candidate drug, but also the breadth of indications for which it may provide clinical benefit, is paramount in the advancement of molecules for clinical testing. Furthermore, understanding something of both the molecular mechanism and the possibility for combination synergy is an essential part of being able to visualize the full clinical potential of a drug. In the current era of personalized medicine, and understanding of the effect of genomic composition of a particular patient's tumor on clinical outcome, the ability to evaluate a vast array of genomic signatures is important in being able to successfully direct therapeutic application. Eurofins' OncoPanel cell-based profiling service provides a convenient way for drug hunters to evaluate not only the potential indication spectrum of a candidate molecule by high-content imaging analysis, but also to identify potentially predictive genomic biomarkers for both sensitivity and resistance to these agents. We have evaluated a range of different approved breast cancer therapeutic agents, using the OncoPanel platform, for their effect on the growth of human tumor cell lines in vitro. These agents span a range of different mechanisms and therapeutic modalities, from broad spectrum cytotoxics to more targeted small molecule and antibody drugs. In this poster, we compare and contrast the different utilities of these drugs, both from the perspective of which tumor types and sub-types are sensitive to these agents, but also with regard to which genomic biomarkers are associated with sensitivity toward them. In doing so, we show how these biomarkers can be useful in not only identifying potentially responsive patient subsets, but also potential combination studies that could be used to improve therapeutic outcome.

#2074

Bioactive marine drugs target acquired oncogenic burden in resilient pancreatic cancer.

Sheeja Aravindan,1 Somasundaram T. Somasundaram,2 Mohan Natarajan,3 Terence S. Herman,4 Natarajan Aravindan4. 1 _Stephenson Cancer Institute, Oklahoma City, OK;_ 2 _Annamalai University, Chidambaram, India;_ 3 _University of Texas Health Sciences Center, San Antonio, TX;_ 4 _University of Oklahoma Health Sciences Center, Oklahoma City, OK_.

The upsurge of marine-derived therapeutics for cancer treatment is evident, with many drugs in clinical use and in clinical trials. Seaweeds harbor large amounts of polyphenols and their anti-cancer benefit is linear to their anti-oxidant activity. Our studies identified three superlative anti-cancer seaweed polyphenol drug candidates (SW-PD). We investigated the acquisition of oncogenic burden in radiotherapy-resilient pancreatic cancer (PC) that could drive tumor relapse, and elucidated the efficacy of SW-PD candidates as adjuvants in genetically diverse in vitro systems (Panc-1, Panc-3.27, BxPC-3, MiaPaCa-2) and a mouse model of radiation-residual disease. For residual PC studies, PC xenografts developed in nude mice were selectively exposed to clinically relevant fractionated irradiation, (2 Gy/day for 5 days per week for a total of 3 weeks), to a total dose of 30 Gy. QPCR profiling of custom archived 88 oncogenes in therapy-resilient PC cells identified a 'shared' activation of 40 oncogenes. SW-PD pre-treatment inflicted a significant mitigation of acquired (shared) oncogenic burden, in addition to drug- and cell-line-specific repression signatures. Tissue-microarray with IHC of radiation-residual tumors in mice signified acquired cellular localization of key oncoproteins (Fosb, cFos, Fra2, cMYC, MYB, NF2) and other critical architects (SELE, CD73, Cytokeratins, Col3, Decorin, and Sulf2). Conversely, SW-PD treatment inhibited the acquisition of these critical drivers of tumor genesis, dissemination, and evolution. Heightened death of resilient PC cells with SW-PD treatment validated the translation aspects. The results defined the acquisition of oncogenic burden in resilient PC and demonstrated that the marine polyphenols effectively target the acquired oncogenic burden and could serve as adjuvant(s) for PC treatment.

#2075

MAPK pathway expression in platinum-treated cancers.

Rebecca A. Georgiadis,1 Amy Stark,1 Amanda B. Hummon2. 1 _University of Notre Dame, Notre Dame, IN;_ 2 _The Ohio State University, Columbus, OH_.

The Mitogen-Activated Protein Kinase (MAPK) Pathway is a critical cell signaling pathway in normal cell function. It is involved in cell proliferation, cell survival, and differentiation. It is a common pathway that is mutated in many cancers, and drug targets have already been developed and tested in clinical trials for many components of this pathway. Further understanding of how gene expression in this pathway is altered by treatment of chemotherapeutic agents can potentially impact resistance to chemotherapeutic agents. The half-maximal inhibitory concentration (IC50) was found for A549 cultured lung adenocarcinoma cells treated with cisplatin and for DLD1 cultured colorectal adenocarcinoma cells and HCT116 colorectal carcinoma cells treated with cisplatin and oxaliplatin. The IC50 concentration of cisplatin-treated A549 cells was 5.429 µM, of DLD1 cells was 5.017 µM, and of HCT116 cells was 4.42 µM. The IC50 concentration of oxaliplatin-treated DLD1 cells was 37.53 µM, as compared to 10.36 µM for HCT116 cells. Therefore, the HCT116 cell type is more sensitive to both chemotherapies. qPCR was then conducted on platinum-treated A549 cultured cells, DLD1 cultured cells, and HCT116 cultured cells to evaluate expression of 6 MAPK pathway genes: RAF1, MAP2K1, MAPK1, STAT1, CREB1, and LAMTOR3. A different pattern of induced gene expression was observed across all three cell types. The 24 hour time point of cisplatin-treatment of A549 cells demonstrated a consistent upregulation of expression in all genes, which is starkly contrasted with a significant downregulation at the 48 hour time point. In HCT116 cells, cisplatin treatment led to the opposite trend, as there was downregulation at the 18 hour time point and significant upregulation at the 48 hour time point. DLD1 had inconsistent results. This demonstrates that level of expression of genes in the pathway is cell-type dependent and time-dependent. Naringenin is a flavonoid that has been implicated in MAPK signalling. We tested this compound as an inhibitor to determine the effect on platinum induced gene expression in lung and colorectal cells. These results have implications for treatment of multiple cancers, especially lung and colorectal cancer.

#2076

Targeting vulnerable aberrant cancer-associated pathways by rationalized triple combination regimens to effectively control urothelial carcinoma cells in vitro and in vivo.

Hwa-Chain R. Wang, Pawat Pattarawat, Jinquan Wang, Robert Donnell. _Univ. of Tennessee, College of Veterinary Medicine, Knoxville, TN_.

Urinary bladder cancer (UBC) is the fifth-most common cancer in the United States, and urothelial carcinomas (UCs) account for more than 90% of UBCs. Although current therapies are effective in the short-term treatment of UCs, the high tendency (>50%) for them to recur and progress to life threatening, advanced muscle-invasive UCs requires life-long surveillance with extended medical care. Thus, it still requires extensive therapeutic development to achieve an effective control of UC recurrence and improve the response rate and overall survival for UC patients. To develop effective and safe regimens to control the development and recurrence of advanced UCs, we investigated the efficacy of rationalized combination regimens in controlling UC cells in vitro and UC cell-derived xenografts (CDXs) in vivo. Our in vitro studies identified a triple combination of gemcitabine, romidepsin, and cisplatin, which are all FDA-approved anticancer agents, effective in synergistically inducing death and reducing drug resistance of various UC cell lines. Our studies also revealed that UC-associated aberrant pathways, including mitochondrial reactive oxygen species (ROS) and the ERK-Nox pathway, which were already induced in UC cells, were enhanced by the triple combination regimen to play essential roles for inducing apoptosis and reducing resistance. We also detected that the unfolded protein response played a role in modulating cell death induced by the regimen. Our in vivo studies used a tolerable protocol for administering combination regimens into animals. Our studies verified the efficacy of the triple combination gemcitabine plus romidepsin and cisplatin regimen, versus double combination regimens, in controlling UC CDXs in immune-deficient mice. In conclusion, our studies indicated that a rationalized triple combination of gemcitabine plus romidepsin and cisplatin regimen was more effective than double combination regimens in modulating UC-associated vulnerable aberrant pathways and controlling UC tumor development and recurrence in animals with low tolerable/ reversible adverse effects. Thus, the triple combination gemcitabine plus romidepsin and cisplatin regimen should be promptly considered as an advanced treatment over the conventional double combination gemcitabine plus cisplatin regimen in clinical trials in order to control advanced UCs.

#2077

A novel assay for PARP-DNA trapping provides insights into the mechanism of action (MoA) of clinical PARP inhibitors (PARPi).

Giuditta Illuzzi, Mark J O'Connor, Elisabetta Leo. _AstraZeneca, Cambridge, United Kingdom_.

Treatments with PARPi in cancers with impaired DNA repair mechanisms (i.e. with Homologous Recombination Repair Deficiency, HRD) causes unsupportable genomic instability resulting in tumor cell death. PARPi act via a dual mechanism: 1) they block PARylation activity that normally occurs in response to DNA damage; 2) they trap PARP onto DNA lesions creating potentially cytotoxic PARP-DNA complexes. The longer PARP is inhibited and trapped onto the DNA, the greater the cytotoxic effect of PARPi in preclinical models. However, differences in preclinical PARP trapping potency have not translated into increased clinical efficacy with standard clinical doses used. Therefore, the ability to assess PARP-chromatin trapping in cancer models is critical for understanding the MoA of existing clinical PARPi. To date, this has been achieved by low throughput assays in non-HRD models using high PARPi concentrations and the addition of exogenous DNA damage.

Here, we describe the development of a novel assay, a high throughput in situ cell extraction platform, where PARP-chromatin trapping is monitored by immunofluorescence and can be multiplexed with the analysis of other relevant biomarkers. Our novel assay has several advantages compared to conventional methods: the overall experimental process is simpler and less time consuming with results being quantitative and less error prone. Most importantly, the higher throughput allows a thorough evaluation of PARP1-chromatin trapping kinetics and their effects with dose-response in a time-dependent manner for clinical PARPi following both continuous treatment or after a wash out of drug.

Using this new assay, we tested the kinetics in isogenic cell line pairs (BRCA2-/- and WT) and followed in parallel the appearance of biomarkers of DNA damage (e.g. γ-H2AX). The data have revealed important elements of differentiation between the MoA of clinical PARPi including the important insight that the strongest PARP trapper (talazoparib) has clearly reduced cytotoxic specificity for HRD cells, which likely explains the significantly reduced dose used in the clinic compared to other PARPi. These along with other data presented highlight how this next generation PARP trapping assays can provide important insights into PARPi MoA.

#2078

Mitochondrial DNA copy number correlates with trifluridine sensitivity of human tumor cell lines.

Takashi Kobunai, Kazuaki Matsuoka, Mamoru Nukatsuka, Hiroshi Tsukihara, Teiji Takechi. _Taiho Pharmaceutical Co., Ltd., Tokyo, Japan_.

Background: Trifluridine (FTD) is a key component of the novel oral antitumor drug trifluridine/tipiracil, which was approved for the treatment of patients with metastatic colorectal cancer refractory to standard chemotherapies. FTD, an antineoplastic thymidine analogue, is incorporated into the genomic DNA of tumor cells. However no detailed analysis of the relation between mitochondrial DNA (mtDNA) in human tumors and FTD's cytotoxic effects has been reported yet. In this study, we analyzed mtDNA copy number of 23 human cancer cell lines and its relation with FTD sensitivity.

Method: Twenty-three human cancer cell lines, representing the nine main tumor types, were tested for sensitivity to FTD by WST-8 assay. The cell lines included lung (NCI-H226, NCI-H23), colon (COLO 205, HCT 116, HCT-15, SW620), pancreas (PANC-1), breast (Hs 578T, MCF7, MDA-MB-231, T-47D), melanoma (Malme-3M, SK-MEL-28, SK-MEL-5), prostate (DU 145, PC-3), renal (786-O, A-498), leukemia (CCRF-CEM, HL-60, MOLT-4), and ovary (OVCAR-3, SK-OV-3). Genomic DNA was purified using a silica membrane spin column-based centrifugation procedure. The copy number of mtDNA was quantitated by array-based digital PCR and real-time PCR. Furthermore, the mtDNA heterogeneity of each cell line was assessed by deep-sequencing as follows: a DNA panel targeting 222 amplicons covering the entire mitochondrial genome was used for NGS library preparation; NGS libraries were subjected to cluster generation in a flow cell and paired-end sequencing for 300 cycles; and finally, the resulting fastq files were uploaded to mtDNA-server for mapping and heteroplasmy evaluation.

Results: The IC50s of FTD ranged from 0.6 uM to 52 uM (Median: 5.6 uM). While cell lines were arbitrarily classified as FTD-high- and FTD-low sensitive cell lines according to their respective IC50 values at median, mtDNA copy numbers of FTD-high sensitive cell lines were significantly lower than those of FTD-low sensitive cell lines (p = 0.01). Median heteroplasmy level and counts were 0.057 and 137, respectively. Interestingly, significant negative correlation was found between mtDNA copy number and heteroplasmy level. (Spearman's Rho = -0.46, p= 0.02). While 6 out of 23 cell lines were microsatellite instable, MSI status also significantly correlated with mtDNA copy number, FTD-sensitivity, and heteroplasmy level (p< 0.05).

Conclusions: Integrated analysis of mtDNA copy number and mutational profiling may be useful to predict FTD sensitivity of cancer cells.

#2079

Enhanced potency of the polymeric fluoropyrimidine CF10 to KRAS-mutant colorectal cancer cells.

William H. Gmeiner,1 Mandira Manandhar,1 Amanda M. Hinds,1 Julija Holmes,1 Pierre Vidi,1 Toshiyuki Tsunoda,2 Senji Shirasawa2. 1 _Wake Forest Univ. School of Medicine, Winston-Salem, NC;_ 2 _Faculty of Medicine Fukuoka University, Fukuoka, Japan_.

Background: Mutant KRAS is a key stratification factor in determining colorectal cancer (CRC) treatment. Nearly 50% of CRC tumors have KRAS mutations and these patients are unlikely to respond to anti-EGFR therapy. 5-fluorouracil (5-FU)-based chemotherapy is the preferred treatment for CRC patients with KRAS mutations; however, response rates remain very low for patients with advanced disease. Further, a sub-set of patients treated with 5-FU-based regimens suffers from serious side effects resulting from RNA-directed effects that do not contribute to the anti-cancer activity. We are developing polymeric fluoropyrimidines, such as CF10, to treat CRC more effectively and with reduced side effects. CF10 is >300-fold more potent than 5-FU across the NCI 60 cell line screen with particularly strong potency to KRAS-mutant CRC cells. We investigate the role of KRAS mutation on CF10 and 5-FU response as part of an overall effort to determine if CF10-based regimens can more effectively treat CRC patients for whom 5-FU-based regimens are currently the preferred treatment option.

Methods: HKe3-wtKRAS and HKe3-mtKRAS were generated from HCT-116 human CRC cells (ATCC) by transfection with the pMSCVpuro vector encoding HA-tagged mutant or wild-type KRAS. Cytotoxicity was determined using an Alamar blue assay. Thymidylate synthase (TS) inhibition was evaluated by detecting ternary complex formation by Western blot and using a TS activity assay. Topoisomerase 1 cleavage complex (Top1cc) was evaluated using a RADAR assay and by immunofluorescence. DNA double strand breaks were detected by IF for γH2AX.

Results: CF10 is highly potent to CRC cells regardless of KRAS mutation status. In isogenic cell lines that differ only in KRAS, CF10 is more potent to KRAS-mutant relative to wild type cells. The improved potency of CF10 relative to 5-FU and increased sensitivity of KRAS-mutant cells is enhanced for cells grown in 3D culture. CF10 cytotoxicity is mediated by TS inhibition and Top1cc formation that induces DNA DSBs and activates apoptosis. In vivo, CF10 displays significantly improved anti-tumor activity relative to 5-FU in HCT-116 orthotopic tumors and causes minimal systemic toxicity.

Conclusions. KRAS mutation confers increased sensitivity to the DNA-directed fluoropyrimidine CF10. The overall mechanism of CF10 cytotoxicity to CRC cells is similar in both KRAS-WT and KRAS-MT CRC cells and involves TS inhibition, and misincorporation of FdU into DNA followed by generation of Top1-mediated DNA DSBs. Our studies indicate FP polymers are likely to be effective in CRC regardless of KRAS-mutation. The increased sensitivity of KRAS-mutant CRC to CF10 together with the very low systemic toxicity indicates that CRC patients currently being treated with 5-FU-based therapy could more effectively be treated with CF10-based regimens.

#2080

Novel molecularly targeted combination for renal cell carcinoma.

Andre R. Jordan,1 Jiaojiao Wang,2 Luis E. Lopez,2 Daley S. Morera,2 Vinata B. Lokeshwar2. 1 _UNIV. Of Miami. Sch Of Medicine, Miami, FL;_ 2 _Augusta University, Augusta, GA_.

INTRODUCTION: Despite many available treatments, >90% of metastatic renal cell carcinoma (mRCC) patients die within 5 years. Due to its modest efficacy, Sorafenib (SF) is a second line treatment for mRCC. Molecular basis for SF failure is unknown. Hymecromone (HC) is a non−toxic orally bioavailable agent used in Europe for improving liver health. We examined the molecular basis of SF failure in clinical specimens and provided mechanistic evidence for why SF+HC could be a potent therapeutic combination against mRCC.

METHODS: UGT1A9 (A9) levels and SF glucuronidation were measured in kidney specimens (45 normal; 86 tumor) by qPCR and a glucuronidation assay using tumor microsomes. Response to SF+HC combination in vector-only (EV), A9-overexpressing (A9) and A9-knockdown (A9-KO) 786-0 and Caki-1 transfectants, primary RCC spheroids, and endothelial cells was examined by proliferation, apoptosis, motility and invasion assays. Target analysis was performed by immunoblot. Treatment efficacy was evaluated in Caki−1 s.c. and orthotopic xenograft models, with bioluminescence imaging.

RESULTS: A9 has been shown to glucuronidate and inactivate SF. A9 levels were 8-10-fold elevated in mRCC patients' tumors compared to non-mRCC patients and normal kidney tissues. A9 levels were an independent predictor of metastasis (P=0.022; efficacy: 73%; sensitivity: 93.8%). Tumor microsomes from mRCC patients' glucuronidated SF 5-fold higher than from non-mRCC patients. In RCC cells, HC treatment downregulated A9 levels, A9 promoter activity and inhibited SF-glucuronidation. SF (5 µM) and HC (20-40 µg/ml) combination inhibited RCC cell and primary tumor spheroid proliferation (>90%), motility/invasion (>80%), and induced apoptosis (5-fold). While A9 transfectants were resistant to SF+HC treatment, A9-KO cells were sensitive to SF alone. Endothelial cells co-cultured with A9 transfectants were resistant to SF+HC treatment. SF+HC upregulated apoptosis effectors, but downregulated CD44, RHAMM, and phospho-Met levels in EV but not A9 transfectants. HC (100 or 200 mg/kg) and SF (30 mg/kg) oral combination abrogated Caki−1 tumor growth (>80%) without serum/tissue toxicity. HC+SF treatment decreased A9 levels, Ki67 index and microvessel density. A9-overexpressing tumors were resistant to treatment.

CONCLUSION: This is the first study to demonstrate that A9 overexpression in mRCC is likely a mechanism of SF failure and A9 downregulation can re-sensitize cells. HC+SF is an orally bioavailable and non-toxic combination that may be an effective therapeutic strategy for mRCC.

#2081

NUC 3373 induces endoplasmic reticulum stress in colorectal cancer cells.

Fiona G. McKissock, Asmithaa Prabhakaran, Peter Mullen, David J. Harrison. _University of St Andrews, St Andrews, United Kingdom_.

Background

Although 5-fluorouracil-based (5-FU) chemotherapies remain the cornerstone of combination therapies for colorectal cancer (CRC), their clinical utility is limited by key cancer resistance mechanisms associated with breakdown, transport, and activation. This includes the intracellular conversion of 5-FU into its active metabolite, fluorodeoxyuridine-monophosphate (FUDR-MP), before core anti-cancer activity can be exerted via inhibition of the enzyme thymidylate synthase (TS). NUC-3373, a phosphoramidate transformation of FUDR-MP, is designed to bypass the key resistance mechanisms associated with 5-FU, resulting in a potentially more effective and safer treatment option. In a Phase I dose-finding study, plasma obtained from patients with advanced cancers revealed that NUC-3373 has a longer half-life than 5-FU (9.7 hours vs 8-14 minutes). Herein, we aimed to investigate the potential role of endoplasmic reticulum (ER) stress pathways in the cancer cell response to NUC-3373.

Methods IC50 values for NUC-3373 were established in nine CRC cell lines by sulforhodamine B assay, after which one sensitive (HCT116) and one resistant (SW480) cell line were selected for further characterization. Control experiments were conducted with thapsigargin and tunicamycin. Cells were examined by transmission electron microscopy and, using stereological principles, changes in cell ultrastructure were quantified. Expression of the ER chaperone protein binding immunoglobulin protein (BiP) was detected by Western blot analysis.

Results Ultrastructural analysis revealed that NUC-3373 had differential effects on ER stress that were dependent on cell line sensitivity to the drug. Following treatment, NUC-3373-sensitive HCT116 cells responded by dilation of their ER, while the SW480 resistant cells responded by lengthening of ER but without dilation. These changes were reflected with the two classical ER stress inducers, thapsigargin and tunicamycin. NUC-3373 activated the unfolded protein response (UPR), as evidenced by upregulation of BiP. Both the magnitude and the duration of the BiP response were higher in SW480 cells.

Conclusions This study supports a novel mechanism of action for NUC-3373-induced anti-cancer activity through modulation of cell stress. NUC-3373 induces UPR and ER stress in colorectal cancer cells. Results of the ultrastructural analysis suggest that the sensitivity of cells to NUC-3373 may be determined by the differential adaptation of ER to stress conditions.

#2082

NUC 3373 induces a cytoplasmic translocation of thymidylate synthase in colorectal carcinoma cell lines.

Fiona G. McKissock, In Hwa Um, Peter Mullen, David J. Harrison. _University of St Andrews, St Andrews, United Kingdom_.

BACKGROUND: Although 5-fluorouracil-based (5-FU) chemotherapies remain the cornerstone of combination therapies for colorectal cancer (CRC), their clinical utility is limited by key cancer resistance mechanisms associated with breakdown, transport, and activation. This includes the intracellular conversion of 5-FU into its active metabolite, fluorodeoxyuridine-monophosphate (FUDR-MP), before core anti-cancer activity can be exerted via inhibition of the enzyme thymidylate synthase (TS). Although widely studied, there are mixed reports as to whether high or low basal tumor TS levels are prognostic of response to 5-FU therapy. NUC-3373, a phosphoramidate transformation of FUDR-MP, is designed to bypass the key resistance mechanisms associated with 5-FU, resulting in a potentially more effective and safer treatment option. This study aimed to determine the prognostic value of cancer cell TS protein expression in predicting therapeutic response to NUC-3373 and to further explore the effects of NUC-3373 on TS biology.

METHODS: IC50 values for NUC-3373 were established in nine CRC cell lines by sulforhodamine B assay, after which one sensitive (HCT116) and one resistant (SW480) cell line were selected for further characterization. In these two cell lines, endogenous, induced, and ternary complex TS expression was measured by Western blot analysis and the cellular localization of TS was confirmed by immunocytochemistry and assessed by stereological methods.

RESULTS: TS protein expression was measured in nine CRC cell lines treated with NUC-3373 and no correlation was found between TS protein levels and the IC50 for NUC-3373. This indicates that the sensitivity of these cell lines to NUC-3373 is not dependent on basal TS protein expression levels. When the two cell lines selected for their sensitivity/resistance to NUC-3373 were treated with NUC-3373, TS expression was induced after 12 hours and remained elevated at 48 hours. In addition, treatment with NUC-3373 was associated with a translocation of TS from the nucleus to the cell cytoplasm, which was more pronounced in the NUC-3373 sensitive cells. Translocated TS did not localize to vesicles or the Golgi apparatus; the cytoplasmic localization remains to be determined.

CONCLUSIONS: These results suggest that basal TS levels are not prognostic of response to NUC-3373 in CRC cells, instead NUC-3373 induces TS expression and causes nuclear to cytoplasmic translocation. This novel finding is indicative of an alternative mechanism of action for FUDR-MP in promoting anti-cancer activity that is independent of the DNA damage pathway.

#2083

Human normal and tumor colon organoids: a 3D high content screening platform for anticancer drug development.

Mariusz Madej, Jara Garcia Mateos, Kuan Yan, Lidia Daszkiewicz, Bram Herpers, Leo S. Price. _OcellO BV, Leiden, Netherlands_.

Background: Recent advances in establishing organoids from human specimens have enabled further development of advanced 3D cell culture models such as normal and tumor colon organoids derived from the same patient. These organoid cultures increase the scope for predicting treatment responses in patients, enabling the testing of cancer (immune)therapies, discriminating different drug responses and flagging off-target effects. Here we present a high throughput 3D human intestinal organoid culture platform combined with high content image-based analysis. This platform allows visualization and quantification of various treatment effects on healthy and diseased gut epithelium.

Methods: Intestinal organoids, obtained from HUB Organoid Technology, are cultured in natural extracellular matrix scaffolds and show gene expression patterns, differentiation and functional characteristics that closely resemble the in vivo biology. To investigate the effect of standard-of-care (SoC) treatments like 5-FU, Oxaliplatin and Irinotecan and/or (in combination with) novel targeting antibodies or other small molecules on the function, formation and integrity of the gut epithelium, we set up the following intestinal organoid test systems: 1. Colorectal cancer organoids with a broad heterogeneity of mutations for sensitive and high throughput phenotypic screening that enable accurate compound profiling and 2. Human normal vs. tumor organoid pair assays for off-target effect studies.

Results: High content 3D image analysis of the organoid models enables sensitive detection of treatment-induced and compound-specific morphological changes such as (inhibition of) growth, development, lumen formation, epithelial integrity and cell death. This enables distinction between mechanisms of action and determining compound synergy. The image-based measurements can be complemented with detection of secreted factors (e.g. cytokines, chemokines) or expressed genes in response to various therapeutic compounds.

Conclusion: OcellO's advanced 3D image analysis of in vitro cultured organoids represents a rapid and biologically relevant approach to test various candidate compounds (e.g. antibodies, antibody-drug conjugates and small molecules) that can treat intestinal disorders such as Inflammatory Bowel Disease and colon cancer. This organoid technology also allows identifying potential adverse effects that can occur during in-vivo studies. Therefore, our intestinal organoid screening platform represents a significant advance on conventional in vitro models and helps bridge the translational gap between in vivo studies.

#2084

Cell panel profiling of 162 small molecule therapeutics identifies response biomarkers for PARP, BET-family and proteasome inhibitors.

Joost C. Uitdehaag, Jeffrey J. Kooijman, Jeroen A.D.M. de Roos, Martine B.W. Prinsen, Jelle Dylus, Nicole Willemsen-Seegers, Jos de Man, Suzanne J.C. van Gerwen, Rogier C. Buijsman, Guido J.R. Zaman. _Netherland Translational Research Ctr. BV, Oss, Netherlands_.

The collective effort of cancer research has resulted in a large diversity of small molecule therapeutics that are tested clinically as precision medicine in specific patient populations. The applications of these therapeutics could be further extended and refined by the identification of new genomic biomarkers that are predictive for response. One of the best ways to do this, is by in vitro profiling in cancer cell line panels.

Oncolines is a panel of 102 genetically characterized cell lines from diverse tumour origins, on which proliferation assays are run in parallel. Earlier analysis has shown that the Oncolines workflow generates highly reproducible data, as required for biomarker discovery [1]. In this cell panel, we profiled 162 different cancer therapeutics, including many standard of care chemotherapeutic agents, approved and pre-clinical kinase inhibitors, epigenetic modulators and compounds acting by other mechanisms.

Assays were based on ATP-lite read-out, with a nine-point duplicate dilution series of the compounds. Drug response was quantified by manual curve fitting. Response was associated with the genomic status of the cell lines as retrieved from the COSMIC and Cancer Cell Line Encyclopedia (CCLE) databases. Mutations in patient hotspot locations and copy number changes in well-characterized cancer genes were used as input for ANOVA tests. Basal gene expression levels of 383 clinically actionable genes and sets of perturbation-response genes were applied to pathway analysis.

Because various alternative metrics were proposed recently to quantify cell line response, we first investigated which metric resulted in the most sensitive identification of biomarkers. Responses were quantified as IC50, GI50, growth rate inhibition (GR50), DSS score, AUC and maximum effect. Known responder populations were analysed such as BRAF(V600E) mutant cell lines for BRAF inhibitors and EGFR amplified lines for EGFR inhibitors, to identify the optimal response metric for biomarker identification.

The full spectrum of cell line responses was analyzed with a variety of clustering and principal component methods to define groups of therapeutic agents based on common biological mechanisms. We further analyzed novel clusters of the Poly (ADP-ribose) polymerase (PARP) inhibitors olaparib, niraparib, rucaparib, and talazoparib, the proteasome inhibitors MG-132, bortezomib and carfilzomib, the ubiquitin activating enzyme inhibitor MLN-7243 (TAK-243) and the BET-family inhibitors JQ1 and I-BET-762 and identified several response biomarkers based on mutation, gene expression and pathway analysis.

References [1] Uitdehaag et al. (2016). Cell Panel Profiling Reveals Conserved Therapeutic Clusters and Differentiates the Mechanism of Action of Different PI3K/mTOR, Aurora Kinase and EZH2 Inhibitors.Mol. Cancer Ther. 15, 3097-3109.

#2085

Treatment of human melanoma cells with dasatinib requires inactivation of both mTOR and MAPK pathways to achieve high cell eradication efficacy.

Jiri Vachtenheim, Jiri Réda, Kateřina Vlčková, Lubica Ondrušová. _Institute of Medical Biochemistry and Laboratory Diagnostics, First Faculty of Medicine, Charles University, Prague, Czech Republic_.

Advanced stages of melanoma are resilient to therapy and new strategies for the treatment are required. About 60% of melanomas harbor the mutated BRAF oncogene, which activate the MAPK pathway. This BRAF driver mutation can be targeted by a small molecule drug vemurafenib. However, most of patients develop resistance during treatment and even drug addiction is acquired. The nonreceptor SRC tyrosine kinase is highly expressed and deregulated in melanoma and can be efficiently inhibited by the inhibitor dasatinib, an anticancer drug which is already used for the treatment of some tumor types. SRC does not have its own canonical pathway but is capable of activating and augmenting different signaling routes in cancer cells. We found that melanoma tumor cell lines analysed were generally resistant to dasatinib irrespective of BRAF or NRAS mutational status. The exception was the cell line Hbl which was very sensitive even to low dasatinib doses and Hbl cells were completely eradicated by 10 nM of dasatinib in several days. The sensitivity of Hbl to dasatinib was dependent on the drug ability to inhibit completely both the MAPK and AKT/mTOR signaling pathways. Dasatinib was capable of inhibiting SRC activation resulting in the repression of AKT activation in Hbl cells. AKT phosphorylation at Ser473 was fully abolished and Thr308 was severely attenuated through inhibiting SRC by dasatinib in Hbl cells. These two phosphorylations are required for AKT activity. MAPK pathway (ERK1/2 phosphorylation) was also inhibited in Hbl cells. Dasatinib also inhibited the phosphorylation of SRC at Tyr416 (a hallmark of SRC activity) in Hbl cells and, surprisingly, also in other dasatinib-resistant melanoma cells. Importantly, although SRC was dephosphorylated at Tyr416, dasatinib was unable to inactivate AKT and MAPK pathway (ERK1/2) in all other melanoma cells. Molecular changes closely correlated with proliferation assays. Our data indicate that the combination therapy of melanoma with dasatinib would bring benefit to patients only by blocking both MAPK and AKT signaling pathways in melanoma cells. Altogether, more molecular knowledge of the effects of the powerful anticancer drug dasatinib is required in order to be used for the treatment of melanoma. This work was supported by the Institutional research project PROGRESQ25 from Charles University Prague.

#2086

Targeting the MNK effector hnRNPA1 enhances the efficacy of BET inhibitors in cancer cells.

Thao Pham, Sophie Stempel, Mario Shields, Christina Spaulding, Krishan Kumar, David Bentrem, Hidayatullah Munshi. _Northwestern University, Chicago, IL_.

Bromodomain and extraterminal domain (BET) proteins are epigenetic readers that regulate gene expression and are involved in cancer pathogenesis. While a number of BET inhibitors are currently in preclinical development or in early phase clinical trials, BET inhibitors show limited single-agent activity. Thus, there is increasing interest in identifying combination regimens to enhance the efficacy of BET inhibitors. Since MNK kinases can enhance the efficacy of BET inhibitors in vitro and in vivo, in this report we evaluated whether targeting effectors downstream of MNK kinases can enhance the efficacy of BET inhibitors. We show that there is co-expression of the BET protein BRD4 and the MNK effector hnRNPA1 in human tumors. We show that hnRNPA1 knockdown can potentiate the effects of BET inhibitors, with enhanced apoptosis and suppression of proliferation and sphere-forming ability by cancer cells. We also show that the plant flavonoid Quercetin, which reduces hnRNPA1 protein levels in cancer cells, can enhance the efficacy of BET inhibitors. Co-treatment with BET inhibitors and Quercetin results in enhanced apoptosis, further suppression of proliferation, and decreased sphere-forming ability by cancer cells. Significantly, Quercetin synergizes with BET inhibitors at suppressing tumor growth in vivo. Together, these results demonstrate that targeting the MNK effector hnRNPA1 can enhance the efficacy of BET inhibitors, and we identify combination therapy with BET inhibitors and Quercetin for the treatment of cancer patients.

#2087

Mechanistic studies reveal novel molecular insights on anticancer properties of F10 compound in human colon cancer cells.

Chinnadurai Mani,1 William Gmeiner,2 Komaraiah Palle1. 1 _Texas Tech University Health Sciences Center, Lubbock, TX;_ 2 _Wake Forest School of Medicine, Winston-Salem, NC_.

Fluoropyrimidine (FP) drugs, mainly 5-Fluorouracil (5-FU), constitute the backbone of combination chemotherapy regimens (e.g. FOLFOX and FOLFIRI) for treating colorectal cancer (CRC) and provide a survival benefit for patients with stage II, III, and IV colorectal cancer. However, 5 year survival rate for CRC patients with metastatic disease is less than 10%. This dreadful situation suggests urgent need for novel and more potent FPs to improve the outcomes of these patients. We have developed novel fluoropyrimidine drug polymers (e.g. F10) that show promising antitumor activity in multiple pre-clinical models even while displaying reduced systemic toxicities. F10 is taken up by malignant cells via active transport compared to 5-Fluorouracil, which is taken up by diffusion in malignant and non-malignant cells. Additionally, F10 compound is primarily incorporated into DNA, and produced thymidylate synthase and topoisomerase toxic metabolites at greater levels, whereas 5-Fluorouracil is primarily incorporated into RNA, and only has about 5-10% production of active form of thymidylate synthase toxic metabolites. MMR is involved in repairing the DNA mismatches and FP drugs induced DNA damage. Interestingly, our studies show that F10 acts by different mechanisms compared to 5-FU. Particularly, F10 treated CRC cells exhibit replication stress as evidenced by paused and slow progression of replication forks and R-loops formation at the active transcriptional regions, suggesting replication and transcription dependent generation of DNA lesions in these cells. F10 treatment induced several fold increased DNA double strand breaks (DSB) as measured by COMET assays and about 1000 folds more cytotoxicity to CRC cells compared to 5-FU. Similarly, F10 treatment activated distinctive DNA damage responses in MMR proficient CRC cells compared MMR deficient cells. Additionally, our studies suggest F10 as a promising therapeutic agent for CRC therapy either alone or in combination with CHK1 and PARP inhibitors.

#2088

Comprehensive CRISPR screening unravel the different mechanisms of IMiDs resistance.

Wenrong Zhou,1 Tianyu Song,2 Min Long,2 Jiye Liu,3 Qunsheng Ji,1 Yong Cang1. 1 _WuXi AppTec, Shanghai, China;_ 2 _Zhejiang University, Hangzhou, China;_ 3 _Jerome Lipper Multiple Myeloma Center, Harward Medical School, Boston, MA_.

Immunomodulatory drugs (IMiDs) including lenalidomide and pomalidomide bind cereblon (CRBN) and activate the CRL4CRBN ubiquitin ligase to trigger proteasomal degradation of the essential transcription factors IKZF1 and IKZF3 and multiple myeloma (MM) cytotoxicity. To have a comprehensive understanding of mechanism underlying sensitivity of MM cells to IMiDs, we performed genome-wide CRISPR-Cas9 screening in three MM cell lines: MM.1S, NCI-H929 and OPM2, which showed resistance to IMiDs at different extent. In the published screening in MM.1S cell, we found that CSN9 signalosome complex, a deactivator of Cullin-RING ubiquitin ligase, regulates MM sensitivity to IMiDs by modulating CRBN expression via SCFFbxo7 ubiquitin ligase. With the newly outcome from H929 and OPM2 cells, we identified 5 top ranking genes with more than 2 sgRNAs ranking in the top 5% sgRNAs in all the three cell lines. CRBN was the top ranking gene with all CRBN-targeting sgRNAs ranking in the top 1% enriched sgRNAs. UbE2G1, reported as an E2 of CRL4-CRBN E3 ligase recently, was another top ranking gene in all the three cell lines. DEPDC5, a component of the GATOR1 complex which inhibits mTORC1 pathway, and SLC35G2, classified as a member of nucleotide sugars transporters (NSTs), were also identified in the top list of all the three cell lines, indicating that mTOR pathway as well as Glycosylation may play an important role in the MM cytotoxicity induced by IMiDs. We also identified 76 top genes shared in two of the three cell lines with more than 2 sgRNAs. Taken together, these results provide a research framework leading to distinguish the cancer patients who become resistant to IMiDs therapy.

#2089

Off-target toxicity is a common mechanism-of-action of clinical cancer drugs.

Jason M. Sheltzer. _Cold Spring Harbor Laboratory Cancer Ctr., Cold Spring Harbor, NY_.

97% of drug-indication pairs that are tested in clinical trials in oncology never advance to receive FDA approval. While lack of efficacy and dose-limiting toxicities are the most common causes of trial failure, the reason(s) why so many new drugs encounter these problems is not well-understood. Using CRISPR/Cas9 mutagenesis, we investigated a set of cancer drugs and drug targets in various stages of clinical testing. We show that - contrary to previous reports obtained predominantly with RNAi and small-molecule inhibitors - the proteins ostensibly targeted by these drugs are non-essential for cancer cell proliferation. Moreover, the efficacy of each drug is unaffected by the loss of its putative target, indicating that these compounds kill cells via an off-target effect. By applying a genetic target-discovery strategy, we validate the cyclin-dependent kinase CDK11B as a novel cancer dependency and as the in cellulo target of one mis-characterized clinical kinase inhibitor. We suggest that stringent CRISPR-mediated validation of cancer drugs and drug targets in the preclinical setting may decrease the number of therapies tested in human patients that fail to provide any clinical benefit.

#2090

A biomarker development for NSCLC patients based on pattern identification of traditional Korean medicine: A pilot study.

Ji Hye Kim,1 Myeong-Sun Kim,1 Sooyeon Kang,1 Yu-Jeong Choi,1 Seo Yeon Lee,1 Sukjoong Oh,2 Seong-Gyu Ko1. 1 _Department of Preventive Medicine, College of Korean Medicine, Kyung Hee University, Seoul, Republic of Korea;_ 2 _Department of Internal Medicine, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea_.

Background: In the study, we studied a biomarker development for lung cancer based on the pattern identification for the personalized Korean medicine.

Methods: We recruited a total of 30 subjects including 4 Xu, 11 Stasis, and 15 not assigned group. Using the tissue sample of the subjects, we performed a whole exome sequencing to examine the genetic differences among three groups. Subjects were composed of normal and cancer tissues to identify cancer specific polymorphisms. A total of seven in silico analyses (GERP++, SIFT, LRT, Mutation Assessor, Mutation Taster, PhyloP and PolyPhen2 HDIV) were performed to predict functional role of significant SNPs.

Results: The exome sequencing identified among 23,490 SNPs germline variants, 12 variants showed significant frequency differences between Xu and Stasis groups (P < 0.0005). As similar, 18 and 10 variants were identified in analysis for Xu vs. Gentleness group and Stasis vs. Gentleness group, respectively (P < 0.001). Our exome sequencing also found 8,792 lung cancer specific variants and among the groups identified 6, 34, and 12 variants which showed significant allele frequency differences in the comparison groups; Xu vs. Stasis, Xu vs. Gentleness group, and Stasis vs. Gentleness group. As a result of PCA analysis, in germline data set, Xu group was divided from other groups. Analysis using somatic variants also showed similar result. And in gene ontology analysis using pattern identification variants, we found genes like as FUT3, MYCBPAP, and ST5 were related to tumorigenicity, tumor metastasis, carbohydrate biosynthetic were showed most significant association (P = 0.004) in comparison between Xu and Stasis. Other significant SNPs for two groups (Xu vs. Gentleness group and Stasis vs. Gentleness group) were responsible for eye morphogenesis and olfactory receptor activity (P < 0.01). Classification of somatic pattern identification variants showed close relationship in multicellular organism reproduction, anion-anion antiporter activity, and GTPase regulator activity (P < 0.02).

Conclusions: Taken together, our study suggests that this is first attempt to explore the specific variants discovery in whole exome regions for development of lung cancer biomarker based on pattern identification of Korean Traditional Medicine. Our study identified 40 variants in 29 genes in association with germline difference of pattern identification groups and 52 variants in 47 genes in somatic cancer tissues.

#2091

Lysosomal targeting of doxorubicin induces different membrane permeabilization and cytotoxicity in two breast cancer cell lines.

Mohammed Alvi, Rachel Nicoletto, Bayan A. Eshmawi, Clyde M. Ofner. _Univ. of the Sciences in Philadelphia, Philadelphia, PA_.

Background: Lysosomal targeting has been investigated for the treatment of cancer but details of the resulting cytotoxicity remain unclear. The induced lysosomal membrane permeabilization (LMP) has been shown to induce apoptosis and cytotoxicity. The purpose of this study is to elucidate details of doxorubicin (DOX) induced LMP in breast cancer cells and identify correlations with cytotoxicity.

Methods: A gelatin – doxorubicin conjugate (GDOX) to target lysosomes was synthesized by EDC chemistry in formamide and purified by precipitation and desalting. GDox and free Dox uptake studies at 10 µM or its equivalent were conducted in MCF7 and MDA-MB-231 (triple negative breast cancer-TNBC) cells in growth media. Intracellular localization was followed by fluorescent markers. LMP was shown by cytosolic release of 10 kD Dextran-Alexa Fluor 488 previously loaded into lysosomes. Intra-lysosomal release of Dox from GDOX was examined by rupture of lysosomes and nuclear accumulation of released Dox followed by fluorescent microscopy and ImageJ. Released Dox accumulation in the nucleus without lysosome rupture was analyzed by UHPLC. Viability, growth inhibition profiles, and IC50 values were determined by the MTT procedure and GraphPad Prism 7.

Results: GDox localized substantially to the lysosomes in both MCF7 and TNBC cells while free DOX localized to the nucleus, as expected. GDOX induced LMP by 24 hrs in 100% of the TNBC cells but only 20% of the MCF7 cells even up to 48 hrs. Intra-lysosomal DOX release in the MCF7 cells was notable only by 24 hrs and was estimated by fluorescent intensity (300,000 integrated density units per cell) at one-half the amount of free DOX accumulation in the nucleus. Nuclear accumulation of released DOX escaping intact lysosomes in MCF7 cells by 24 hrs was one-half that at 48 hrs which reached 0.13 µg Dox per 106 cells by 48 hrs representing only one-tenth that achieved by free DOX. MCF7 cell viability in GDOX was reduced to 62±3% of controls by 24 hr but reached 19±4% by 48 hrs compared to 11±1% for free Dox. The IC50 values of GDOX after 48 hr were 1.3±0.7 µM and 4.5±2 µM in TNBC and MCF7 cells, respectively.

Conclusions: Taken together these results indicate that LMP is not induced by pooling of released DOX in MCF7 lysosomes nor does LMP contribute to cytotoxicity in these cells. The lysosomal accumulation of GDOX in MDA cells, in contrast to MCF7 cells, leads to extensive LMP which may contribute to the greater cytotoxicity in these cells compared to the MCF7 cells. The small amount of released DOX accumulating in the MCF7 nuclei may induce a nuclear pathway of cytotoxicity in these cells.

Acknowledgements: Financial support is appreciated from the Agnes Varis Trust for Women's Leadership and Health, King Abdul-Aziz University, and NIH R15 CA135421.

#2092

Metabolic approach to EGFR-targeted therapy in non-small cell lung cancer.

Yoonseok Kam, Pamela Swain, Natalia Romero, Brian P. Dranka. _Agilent Technologies, Lexington, MA_.

Mutation of the epidermal growth factor receptor (EGFR) is a major genetic driver of non-small cell lung cancer (NSCLC). While first-line tyrosine-kinase inhibitors (TKIs) have improved patient survival, many patients eventually develop resistance to these drugs. Combination therapies are now being researched to overcome this resistance. Pathways that may synergize with EGFR signaling are of interest since a liability created with one compound may then be exploited by a second. It is well known that glycolysis is controlled by EGFR signaling in cancer cells. However, the interplay of cell metabolism and EGFR inhibitors is not fully understood. Using PC9 cells which have known constitutive EGFR activation, we examined the relative poise of ATP production from mitochondria and glycolysis. In response to any of four different TKIs: afatinib, CO-1686, dacomitinib and erlotinib, a rapid decrease in glycolytic activity was induced. However, the total ATP production rates were not changed significantly as a result of increased mitochondrial ATP production. We next examined the mitochondrial fuel sources which support this increase by selectively blocking individual fuel pathways. In the presence of either a mitochondrial pyruvate carrier or glutaminase inhibitor, total ATP production rate decreased, and cell viability followed. Taken together, these data suggest that a combinational application of EGFR-targeted therapy and mitochondrial-targeted therapy may provide metabolic stress to cancer cells. Further study is required to understand the interplay of the tumor microenvironment and metabolic niches that may be found therein.

#2093

Drug induced autophagy and transcriptional gene expression signatures in breast cancer cell lines.

Francesca Mascia, Kostantinos Karagiannis, Wells W. Wu, Rong-Fong Shen, Ashutosh Rao. _FDA, Silver Spring, MD_.

Cancer cells can bypass the desired cell death response to chemotherapy through cytoprotective autophagy. In support of this hypothesis, drug resistant clones display higher basal autophagy than the correspondent drug sensitive cell lines. Autophagy therefore represents a potential target to reduce drug resistance and to enhance drug efficacy. We induced autophagy in several breast cancer cell lines with differing ER/PR/Her2 status by exposing them to tamoxifen (MCF-7/ER positive cells), trastuzumab (SKBR3/Her2 positive cells), bortezomib and rapamycin (MDA-MB-231 triple negative cells) in the presence and absence of an autophagy inhibitor such as hydroxychloroquine. While the ongoing autophagy flux process was confirmed by the amount of LC3II at the lipidated protein level, total RNA was extracted at the same time to perform RNA sequencing analysis. We performed differential expression analysis between all the drug treatments and the relative basal level of each cell type obtaining a cell and drug-specific gene list. We then compared all the gene lists to find a common, cell and drug-independent signature. Bioinformatic analysis was performed using the software IPA (Ingenuity Pathway Analysis) and setting a threshold of 0.01 for the p value. This analysis suggested a set of canonical pathways that are significantly and commonly affected in all the samples tested. The top canonical pathways disrupted in all the gene sets were the one identified as tRNA charging, CHK proteins in cell cycle check point control, hypoxia signaling and unfolded protein response. We plan to further identify the smallest gene list that is associated with the disruption of all these pathways across all the cells. This signature may help to uncover a consistent biomarker of autophagy that can be used during preclinical and clinical drug development aiming to reduce or eliminate drug resistance and improve cancer treatment outcomes.

### Drug Resistance 3

#2094

Prostaglandin E receptor 3 mediates resistance to tumor treating fields in glioblastoma cells.

Dongjiang Chen,1 Son Le,1 Nagheme Thomas,1 Changwang Deng,1 Dan Jin,1 Mathew Sebstian,1 Jie Ren,2 David Tran1. 1 _Division of Neuro-Oncology, Preston A. Wells, Jr. Center for Brain Tumor Therapy, Lilian S. Wells Department of Neurological Surgery, University of Florida, Gainesville, FL;_ 2 _Shanghai Jiaotong University School of Medicine, Shanghai, China_.

Glioblastoma (GBM) is the most common and deadliest malignant brain cancer in adults despite surgery and aggressive chemoradiotherapy. Tumor Treating Fields (TTFields) have been approved in combination with adjuvant temozolomide chemotherapy for newly diagnosed GBM. The addition of TTFields resulted in a significant improvement in overall survival. TTFields are low-intensity alternating electric fields that are thought to disturb mitotic macromolecules' assembly, leading to disrupted chromosomal segregation and cell death. However, treatment resistance develops in many TTFields responders. The mechanism of TTFields resistance remains largely unexplored. Understanding how GBM cells circumvent the biophysical forces of TTFields and their downstream effects will provide new opportunities to improve therapeutic efficacy of this novel anti-cancer treatment. To accomplish these objectives, we have developed several human GBM cell lines that demonstrated relative resistance to the cytotoxic effects of TTFields compared to the parental cells. Importantly TTFields-induced chromosomal instability such as the formation of cytoplasmic micronuclei was preserved in resistant cells compared to their sensitive counterparts, indicating resistance to TTFields is mediated through a non-bioiphysical mechanism. Indeed, TTFields-induced inflammatory response was severely suppressed in resistant cells, supporting the hypothesis that that resistance to TTFields is conferred by a selective loss of the deleterious effects induced by the biophysical insults. Importantly, this acquired TTFields resistance phenotype was associated with a transition to a stem-like state as determined by a standard neurosphere assay. Using a systems approach aided by a suite of innovative computational platforms, we methodically dissected this stemness program in resistant cells to identify master regulators of the resistance mechanism. Interestingly, 3 networks were found disrupted, including nervous system developmental regulation, inflammatory response and cell-cell adhesion, all of which play critical roles in GBM stem-like cells, thus confirming our initial hypothesis. Utilizing a unique master regulator ranking system, we successfully identified Prostaglandin E Receptor 3 (PTGER3) as a key master regulator at the apex of these pathways and responsible for the resistant phenotype. PTGER3 is rapidly upregulated in GBM cells when exposed to TTFields and appears to channel treated cells away from the beneficial inflammatory pathways that TTFields also activates in parallel. Ongoing experiments are aimed at understanding the molecular mechanism of PTGER3-dependent TTFields resistance. Our goal is to develop targeted therapies to overcome resistance to TTFields.

#2095

**AsiDNA** TM **, a targeted therapy with no acquired resistance.**

Wael Jdey,1 Maria Kozlac,2 Sergey Alekseev,2 Danielle Johnson,3 françoise Bono,1 Srividya bhaskara,3 frederic thomas,4 Marie Dutreix2. 1 _Onxeo, Paris, France;_ 2 _Institut Curie, Orsay, France;_ 3 _University of Utah School of Medicine and Huntsman Cancer Institute, Salt Lake City, UT;_ 4 _Université de Montpellier, Orsay, France_.

The Achilles heel of conventional and targeted anticancer treatments is intrinsic or acquired resistance following Darwinian selection, i.e. treatment toxicity place the surviving cells under intense evolutionary selective pressure to develop resistance. Here, we demonstrate that AsiDNA, the first antitumor drug with an agonist activity, can instead drive the evolution of malignant cells toward a different trajectory. Specifically, long term exposure of cancer cells to the strong alarm signal, generated by the AsiDNATM DNA repair inhibitors, did not promote resistance emergence. It induces a stable new state characterized by the down regulation of the targeted pathways that persists for months after treatments. This property is due to the original mechanism of action of AsiDNATM, which acts by over-activating a "false" signaling of DNA damage through DNA-PK and PARP enzymes, and is not observed with other DNA repair inhibitors such as the PARP inhibitors olaparib and talazoparib. Transcriptomic analysis of AsiDNATM treated independent populations derived from lung and breast cancers revealed the common evolution of independent AsiDNA-treated samples, resulting in a general down-regulation of enzymes involved in DNA repair and oncogenesis, and a specific down regulation of DNA-PK targets. Similar down regulation of DNA-PK targets was observed in xenografted tumor in animal models after three cycles of AsiDNA treatment. Chromatin analysis revealed a major change in AsiDNATM treated populations with an increased nucleosome stability that could be responsible for the epigenetic modification observed. Long term treatment with AsiDNATM induces a new "alarm down" state in the tumor cells that increase its efficacy. These results suggest that agonist drugs such as AsiDNATM could promote a state-dependent tumor cell evolution by lowering their ability to respond to damage signal as predicted by the ecological and evolutionary "smoke detector principle".

#2096

Resistance to APTO-253 caused by internal deletion and alternate promoter usage of the MYC gene in malignant B cells.

Andrea Local,1 Cheng-Yu Tsai,2 Hongying Zhang,1 Susan Sheng,1 Khalid Benbatoul,1 Stephen B. Howell,2 William G. Rice1. 1 _Aptose Biosciences, Inc., San Diego, CA;_ 2 _Moores Cancer Center, University of California San Diego, San Diego, CA_.

APTO-253 is a clinical stage small molecule that acts in part by down-regulation of MYC at mRNA and protein levels. MYC onco-protein dysregulation, which leads to increased proliferation and survival, is implicated in the etiology of numerous cancer subtypes. In the current study, we explored sensitivity of B-cell cancer cells to APTO-253 and sought to understand how changes in MYC can yield resistance to APTO-253. APTO-253 showed potency against B-cell cancer cell lines (IC50 30-600 nM) and bone marrow samples from patients. Sensitivity of cell lines correlated with MYC over-expression, amplification or translocation. APTO-253 resistant Raji cells (Raji253R) are characterized by upregulation of the ABCG2 transporter. Treatment with ABCG2 inhibitors only partially reversed the resistant phenotype suggesting additional contributing mechanisms. RNA-seq analysis revealed that while levels of MYC mRNA were unchanged in Raji253R, a truncation of exon 2 was present. This internal deletion occurred at the DNA level, not by alternative splicing, and resulted in a truncated MYC protein (45 kDa) lacking the conserved core sequence of MYC box III (MBIII). At early stages in the evolution of resistance residual full length MYC mRNA and protein was detectable; however, this expression was eventually lost in Raji253R cells as selection continued. Deletion of MBIII has been implicated in MYC protein stability, in vitro and in vivo transformation, gene repression, and MYC dependent apoptosis. Cycloheximide treatment of Raji and Raji253R cells demonstrated a modest increase in stability of truncated over full length MYC protein. In addition, Raji253R cells exhibited hypersensitivity to etoposide. The internal truncation is flanked by sites of microhomology suggesting a method for deletion. We hypothesize that DNA damage in response to APTO-253 treatment led to error prone repair by microhomology end joining and resulted in a MYC internal deletion. Interestingly, transcription from the canonical P1/P2 promoter, as seen in WT Raji cells, was systematically lost and transcription initiation shifted to a downstream promoter, P3. APTO-253 inhibits MYC expression via binding and stabilizing the G-quadruplex (G4) sequence in the P1/P2 promoter therefore switching of transcriptional initiation to P3 should relieve APTO-253 mediated repression. Indeed, APTO-253 treatment of Raji253R cells did not repress MYC expression, even at concentrations 50-fold higher than required for MYC repression in Raji WT cells. Presence of the active intra-cellular form of APTO-253, Fe(253)3, was confirmed in Raji253R cells. In summary, MYC driven Raji cells become resistant to APTO-253 by acquisition of a more stable MYC protein and by utilization of an alternate promoter not inhibited by G4 binding and stabilization, thereby confirming the essential role of MYC repression in the mechanism of action of APTO-253.

#2097

LIN9 regulation of NEK2 underlies taxol resistance in triple-negative breast cancer.

Melyssa S. Shively, Ruth A. Keri. _Case Western Reserve University, Cleveland, OH_.

Currently, there are no targeted strategies to combat triple negative breast cancer, resulting in poor patient survival. This disease initially responds well to cytotoxic chemotherapies such as paclitaxel, yet resistance and metastatic recurrence are common. Taxanes cause defects in centrosome function and chromosome segregation leading to cell death. While greatly effective, they are also associated with high toxicity. Thus discovering new therapeutic targets that provide a selective vulnerability for TNBC should yield novel approaches for improving patient outcomes. We discovered that LIN9, a transcriptional regulator of mitosis, is overexpressed in 66% of TNBC and is associated with poor survival. We now report that both LIN9 mRNA and protein expression are upregulated in paclitaxel-resistant versus sensitive cells and directly correlates with paclitaxel IC50 values across eight breast cancer cell lines. In MDA-MB-231 and MDA-MB-468 cell lines, LIN9 silencing results in multi- and micronucleation, and supernumerary centrosomes. Moreover, LIN9 silencing increases sensitivity to paclitaxel in TNBC cells with intrinsic (BT549) or acquired (MDA-MB-231 and -468) resistance. We previously reported that Bromodomain and ExtraTerminal protein inhibitors (BETi) treatment reduces LIN9 expression, and thus determined if BETi could reverse paclitaxel resistance. Treatment with the BETi, JQ1, in conjunction with paclitaxel caused a greater induction of apoptosis, abnormal centrosomes, multi- and micronucleation compared to either drug alone. To identify the mechanism(s) by which suppression of LIN9 reverses paclitaxel resistance, we compared the transcriptomes of MDA-MB-231 and HCC70 TNBC cells transiently transfected with non-targeting or LIN9-targeted siRNAs. The resulting gene list was then filtered to include only genes whose expression correlates with LIN9 in breast cancer, are bound to LIN9 in a published ChIP-Seq dataset, and are associated with breast cancer survival. Using this approach, we identified NIMA-related Kinase 2 (NEK2), a serine/threonine kinase required for centrosome separation during mitosis, as a potential mediator of LIN9-associated paclitaxel resistance. NEK2 is overexpressed in 47% of basal breast cancers and is associated with poor patient outcomes. Additionally, NEK2 is upregulated in paclitaxel-resistant cells and LIN9 silencing decreases expression of NEK2. Importantly, silencing NEK2 expression also restores sensitivity to paclitaxel in resistant cells. Together, these data indicate that increased LIN9 expression in TNBC promotes paclitaxel resistance by upregulating NEK2 and that loss of LIN9 or NEK2 contributes to centrosome dysfunction, potentiating paclitaxel sensitivity. They also indicate that suppressing LIN9 expression using agents such as BET inhibitors may be a viable therapeutic approach for improving paclitaxel efficacy in TNBC patients.

#2098

IFIT1 and IFIT3 modulate the drug response in human oral squamous cell carcinoma through interaction and activation of Hsp90.

Vijaya Kumar Pidugu,1 Meei-Maan wu,2 Hima Bindu Pidugu,3 Te-Chang Lee3. 1 _Taiwan International Graduate Program in Molecular Medicine, National Yang-Ming University, and Academia Sinica, Taipei, Taiwan;_ 2 _Department of Public Health, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan;_ 3 _Institute of BIomedical sciences, Academia Sinica, Taipei, Taiwan_.

Interferon-induced protein with tetratricopeptide repeats (IFITs) family, well-known interferon-stimulated genes, have multiple tetratricopeptide repeats with helix-turn-helix structural motifs that mediate a variety of protein-protein interactions. Four IFIT genes have been identified in humans: IFIT1, IFIT2, IFIT3, and IFIT5. While the importance of IFIT1 and IFIT3 in the prognosis of cancer has been reported, the molecular basis of IFIT1 and IFIT3 in cancer progression remains unexplored. We have previously shown that high IFIT1 and IFIT3 expression associated with advanced T-stage, lymph node metastasis, perineural invasion, lymphovascular invasion, extranodal extension, and poor overall survival rate in oral squamous cell carcinoma (OSCC) patients. Also, we have demonstrated that high IFIT1 or IFIT3 expression promoted OSCC invasion and metastasis by EGFR signaling via enhancing EGFR endocytic recycling. Intriguingly, our recent studies have revealed the involvement of IFIT1 and IFIT3 in the resistance to chemotherapeutic agents in OSCC cells. The failure of chemotherapy or targeted therapy was often associated with the intrinsic or acquired drug resistance in cancer cells. In the present study, we intended to explore the biological role of IFIT1 and IFIT3 on drug resistance in OSCC cells. Our results showed that ectopic expression of IFIT1 or IFIT3 proteins in OSCC cells significantly increased the resistance to various therapeutic drugs such as cisplatin, carboplatin, 5-FU, and doxorubicin. Whereas, silencing of IFIT1 and IFIT3 by shRNA enhanced the sensitivity to these drugs. We further demonstrated that enhanced expression of IFIT1 and IFIT3 significantly increased the levels of C-terminal phosphorylation of Hsp90 and activation of its client proteins, such as EGFR, AKT, p38, and SAPK/JNK. Using mass spectrophotometric, immunoprecipitation and immunofluorescence analyses, we identified the interactions of IFIT1 and IFIT3 with Hsp90. Suppression of Hsp90 by a specific inhibitor resulted in decreased activation of its downstream targets in IFIT1- or IFIT3-overexpressing cells. These results suggested a novel mechanism of IFIT1 and IFIT3 in activating Hsp90 and several downstream signaling regulators which are crucial for OSCC tumor progression and drug resistance. Thus, IFIT1 and IFIT3 may act as co-chaperones and serve as potentially important prognostic biomarkers for OSCC progression.

#2099

N-Myc-mediated epigenetic reprogramming drives lineage plasticity in advanced prostate cancer.

Adeline Berger,1 Nicholas Brady,1 Rohan Bareja,1 Brian Robinson,1 Vincenza Conteduca,1 Michael Augello,1 Loredana Puca,1 Adnan Ahmed,1 Xiaodong Lu,2 Inah Hwang,1 Alyssa Bagadion,1 Andrea Sboner,1 Olivier Elemento,1 Jihye Paik,1 Jindan Yu,2 Christopher Barbieri,1 Noah Dephoure,1 Himisha Beltran,1 David S. Rickman1. 1 _Weill Cornell Medical College, New York, NY;_ 2 _Northwestern University, Chicago, IL_.

Background:

Despite recent advances in the development of highly effective androgen receptor (AR)-directed therapies for the treatment of prostate cancer, nearly 37% of patients develop resistance. A further third of these patients progress to develop aggressive neuroendocrine prostate cancer (NEPC) for which no effective therapies exist. Lineage plasticity, a process by which differentiated cells lose their identity and acquire alternative lineage programs, has been proposed as a mechanism of resistance to targeted therapies in epithelial tumors such as prostate cancer. Although epigenetic deregulation has been implicated as a driver of the epithelial to neuronal switch observed in NEPC, the molecular programs are poorly understood. Previously, we observed that the majority of NEPC and 20% of castration-resistant prostate cancer (CRPC) aberrantly overexpress the transcription factor MYCN (N-Myc). Despite this frequent occurrence, the role of N-Myc in driving lineage plasticity and the epigenetic mechanisms which regulate disease progression remain to be elucidated.

Methods:

We analyzed overall survival and whole transcriptome data from a cohort of over 200 prostate cancer patients, including the largest-to-date population of NEPC patients. We also analyzed epigenetic modifications along with the N-Myc transcriptome, cistrome and chromatin-bound interactome by performing ChIP-seq, RNA-seq and RIME in a combination of mouse models, human prostate cancer cell lines, and NEPC patient-derived organoids.

Results:

Expression of MYCN in CRPC and NEPC patients correlates with reduced overall survival. NEPC tumors are significantly enriched for stem cell genes associated with normal neuroendocrine cell precursors, embryonic stem cells and neural lineage-defining genes from activated neural stem cells. The integration of next-generation sequencing data revealed that the N-Myc cistrome is androgen-dependent and drives a transcriptional program leading to epithelial plasticity and the acquisition of clinically relevant neuronal lineage markers. Our data also reveal that N-Myc interacting proteins as well as AR co-factors and pioneering factors (e.g. FOXA1 and HOXB13) explain the observed dynamic binding of N-Myc. Interestingly, histone marks and EZH2 activity specifically associated with lineage-defining genes were reprogrammed by N-Myc. Finally, we demonstrated that N-Myc-induced gene expression and epigenetic changes accurately classify our patient cohort.

Conclusions:

We describe a novel role for N-Myc in prostate cancer, characterized by changes in the N-Myc cistrome and cooperation with AR co-factors, as well as reprogramming of the epigenome in an androgen context-dependent manner. This reprogramming is associated with induction of a lineage plastic state and a switch towards a neural identity that favors the development of AR independence and NEPC.

#2100

Resistance of gastric carcinoma cells towards c-met inhibition is mediated by compensatory HER3 upregulation involving SATB1 and PKC.

Robert Jenke,1 Thomas Büch,1 Miriam Rein,1 Stefanie Müller,1 Florian Lordick,2 Achim M. Aigner1. 1 _Leipzig University, Leipzig, Germany;_ 2 _University Hospital Leipzig, Leipzig, Germany_.

Introduction: In patients with gastric cancer expressing high levels of c-met, inhibitors of the receptor tyrosine kinase (RTK) c-met are explored as targeted therapeutics. Unfortunately, clinical trials have failed to improve survival so far. This might be due to compensatory stimulation of other oncogenic RTKs. Here, we highlight the role of HER3 upregulation and its specific stimulation as resistance mechanism. On the mechanistic side, we demonstrate the genome regulator special AT-rich sequence-binding protein-1 (SATB1) as a critical mediator of this HER3 upregulation, with further manipulation being executed by contrary roles of Protein kinase C (PKC) and Phosphatidylinositol 3-kinase (PI3K).

Methods: We used gastric cancer cell lines with a c-met amplification and evaluated the impact of pharmacological or siRNA-mediated c-met inhibition on cellular proliferation and survival. Moreover, we analyzed the expression of HER family receptors after inhibitor treatment. Furthermore, we used transient knockdown of SATB1 as well as inhibitors of PKC and PI3K to gain insight into signaling mechanisms.

Results: The use of c-met kinase inhibitors or RNAi-induced downregulation of c-met led to a complete disruption of cellular proliferation. Without addition of c-met inhibitors, the investigated gastric cancer cells expressed HER1, HER2, and HER3 receptors, but no HER3 ligands. After pretreatment with c-met inhibitors, HER3 expression was markedly upregulated. Treatment of gastric cancer cells with the HER3 ligand beta-heregulin, usually secreted by cancer associated fibroblasts, partially reversed the anti-proliferative effects of c-met inhibitors. The induction of HER3 was reduced after pretreating cells with SATB1 siRNA. SATB1 is a known regulator of HER receptor expression in other tumor entities. The regulatory functions of SATB1 can be further modulated by phosphorylation via PKC or PI3K. Therefore, we investigated the effect of PKC and PI3K inhibition on HER3 induction. Of note, PKC inhibition markedly reduced HER3 upregulation, whereas PI3K inhibitors even increased HER3 levels.

Conclusions: PKC and SATB1 are involved in the compensatory upregulation of HER3 upon c-met inhibition. Since this represents a critical resistance factor, interference with PKC and SATB1 signaling is a potential avenue to prevent resistance towards c-met inhibitors in gastric cancer.

#2101

Targeting the root of cancer persister cells using an expressed barcode library.

Yaara Oren,1 Pratiksha Thakore,2 Mike S. Cuoco,2 Heidie Frisco Cabanos,3 Aaron Hata,3 Joan S. Brugge,4 Aviv Regev2. 1 _The Broad and Harvard Medical School, Boston, MA;_ 2 _The Broad, Cambridge, MA;_ 3 _Massachusetts General Hospital, Boston, MA;_ 4 _Harvard Medical School, Boston, MA_.

Despite a favorable initial response, many cancer patients will experience recurrence of disease within months or years after diagnosis. Recurrence largely arises as a result of the growth of residual cancer cells that remain after treatment. The ability of a subset of cells to survive is attributed frequently to genetic heterogeneity, however recently it was shown that in multiple cancer types relapse can arise due to the presence of persister cells. Persisters are a subpopulation of transiently drug-tolerant cells that are able to survive therapy through reversible, non-mutational mechanisms. Tumor dormancy, stochastic cell state shifts and stem cell-like populations are amongst the mechanisms hypothesized to underlie persister phenotype. However, given the lack of high-throughput methods to concurrently track cell state and lineage, it is not currently feasible to distinguish the relative contribution of each of these factors. To address this need, we generated the Watermelon library. The Watermelon library is a high-complexity expressed barcode library that enables simultaneous tracing of lineage as well as the transcriptional and proliferative state of each cell in the population during drug treatment. We have applied the watermelon system to study the mechanisms underlying the ability of a small population of cells to regain proliferative capacity under constant treatment with EGFR tyrosine kinase inhibitors. We combine time-lapse imaging with single-cell RNA sequencing to show that early drug-cyclers do not acquire a facilitating resistance mutation but rather transition to a new cell state. We find that this non-genetic drug-proliferative state is not restricted to a certain clonal lineage and can be reached by distinct transcriptional paths. We anticipate that this unique library, which can be applied to other systems, would facilitate a better understanding of the cellular and molecular pathways that affect non-inherited drug resistance.

#2102

Assessing glioma-protein inhibition in sunitinib-resistance using a 3-dimentional tissue-engineered renal cell carcinoma patient derived model.

Ninadh M. D'Costa, Zheng Tan, Claudia Chavez-Munoz, Alan So. _Univ. of British Columbia, Vancouver, British Columbia, Canada_.

Objective: To identify the role of glioma-proteins (Gli) in sunitinib-resistant metastatic renal cell carcinoma and the effect of Gli-inhibition using a 3D tissue-engineered patient-derived model.

Background: Renal cell carcinoma (RCC) is the 6th most common malignancy with 2.3% annual increase in Canada. Metastatic RCC (mRCC) patients are treated with anti-angiogenic tyrosine kinase inhibitors (TKIs, sunitinib) as the first-line of treatment because mRCC tumor is heavily depend on VEGF pathway. Therefore, identifying the factor(s) responsible for sunitinib-resistance development and disease advancement in mRCC is imperative. Cancer reflect aspects of developmental patterning and the Hedgehog family proteins are known to play a central role. Among other proteins in the family, the inappropriate activation and/or maintenance of Sonic hedgehog (Shh)-Gli pathway results in various tumors. However, the potential of Shh-Gli pathway inhibitors in sunitinib-resistant mRCC have never been tested. To screen for effective Gli-inhibitors, we will use a 3D tissue-engineered patient-derived tumor model utilizing decellularized kidneys as matrices. Organ decellularization is the process of complete cell removal from the organ and maximum preservation of the extracellular matrix (ECM) microarchitecture, leaving some reported growth factors in which tumor cells from patients can be reseeded, regenerating the organ. This 3D-model is an economic and reliable platform to screen the Gli-inhibitors, and identify tumour-specific optimal therapies.

Results: We established sunitinib-conditioned cell-line (Caki-1DC) from the parental cell-line (Caki-1WT) and a mouse model of acquired sunitinib-resistance. Proteomics and microarray results from these two models showed transcriptional and translational changes between the sensitive and resistant phenotypes, particularly in the Hedgehog signalling. In Caki-1DC, an upregulation of Gli-2 was observed, which could be abrogated with Shh pathway inhibitor, cyclopamine. Furthermore, in our decellularization and reseeding techniques of kidney, we have assessed cell viability for up to a month exhibiting adequate cell viability. This 3D platform will be used to assess the effectiveness of Gli-inhibitors.

Conclusion: Our data from in vitro and in vivo models established Shh-Gli pathway activation in sunitinib-resistant mRCC. Testing the effectiveness of Gli-inhibitors will help tailor drugs for the patients to improve progression free survival. The 3D patient-derived mRCC platform will enable us to generate many in vitro avatars to accurately recreate the tumor of a patient, and simultaneously screen for suitable drugs and dose for the patient. The identification of the Shh-Gli driver pathway and the effective therapy will translate into improving patient's quality of life and survival.

#2103

NETs promote tumor resistance to anthracyclines.

Cindy Lin,1 Sarah E. Herlihy,2 Marina Li,1 Hui Deng,1 Luca Bernabei,3 Dmitry I. Gabrilovich,1 Dan T. Vogl,3 Yulia Nefedova1. 1 _The Wistar Institute, Philadelphia, PA;_ 2 _Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania, Philadelphia, PA;_ 3 _Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA_.

Multiple Myeloma (MM) is an incurable blood cancer characterized by the clonal proliferation of plasma cells that accumulate in the bone marrow (BM). Resistance to chemotherapy represents one of the main challenges in the treatment of this disease. We have previously shown that neutrophils, one of the major cellular populations in the BM, protect MM cells from chemotherapies including anthracyclines; however, the mechanisms of this effect remain unknown. Thus, we set out to investigate the mechanisms by which neutrophils reduce sensitivity of tumor cells to anthracyclines. Neutrophil extracellular traps (NETs) are structures composed of chromatin and protein formed by neutrophils that are a part of an innate immune mechanism of binding and killing pathogens. Recently, NETs have been shown to form in cancer patients as well as tumor-bearing animals and have been implicated in solid tumor progression; however, their role in hematological malignancies remain poorly understood. Utilizing an in vitro culture system, we demonstrated that MM cells were able to induce NET formation by BM neutrophils. Interestingly, neutrophils isolated from the BM of patients with MM have a significantly higher ability to produce NETs in response to tumor cells as compared to BM neutrophils from donors. NET formation was accompanied by an increase in the level of extracellular DNA. Using imaging flow cytometry and confocal microscopy we demonstrated that extracellular DNA is internalized by tumor cells and localizes to the cytoplasm. This internalized DNA was able to bind to doxorubicin and other anthracyclines thereby reducing their cytotoxic effects. Significantly increased cell-free DNA levels were detected in BM aspirates from MM patients as well as in tumor-bearing mice. Targeting the extracellular DNA in vivo using the clinically available DNase, Pulmozyme, restored the sensitivity of tumor cells to doxorubicin resulting in prolonged survival of tumor-bearing animals. Here, we have identified a novel mechanism of resistance to anthracyclines that is mediated by neutrophils in the tumor microenvironment and provided evidence to support the premise that targeting this mechanism would be of significant benefit in the treatment of MM.

#2104

Treatment persistence in the 3-dimensional tumor spatial niche is mediated by a distinct cellular state with suppressed Myc activity and resembling embryonic diapause.

Eugen Dhimolea,1 Ricardo De Matos Simoes,1 Xiang Weng,1 Pallavi Awate,1 Joseline Raja,1 Shruti Sharma,1 Huihui Tang,1 Juliette Bouyssou,1 Dhvanir Kansara,1 Michal Sheffer,1 Jennifer Roth,2 Aedin Culhane,1 Constantine Mitsiades1. 1 _Dana Farber Cancer Inst, Boston, MA;_ 2 _Broad Institute, Boston, MA_.

Complete responses to anti-cancer therapies are frequently prevented by residual tumor foci that persist despite continuation of treatment administration and consist the "seed" for the eventual relapse. Eradicating the drug-persister malignant foci could lead to cures, but their therapeutic vulnerabilities have so far remained elusive, mainly because bona fide models of this cancer cell state, amenable to pharmacogenomic interrogation, are lacking. We show that prolonged treatment with cytotoxic chemotherapeutics eradicated cancer cells in conventional 2-dimensional (2D) cultures but led to the emergence of persister organoid fractions (typically the organoid cores) in 3D cultures of cell lines and patient-derived cells. In contrast to previously-described 2D culture models of drug persistence, reflecting pre-existing resistant clones or putative stochastic epigenetic states of rare cells, the proneness to drug persistence was manifested in most or all 3D organoids and was lost when the cancer cells were transferred back in 2D culture; indicative of distinct putative molecular mediators of drug-refractoriness in the 3D niche. Importantly, the drug-persistent tumor organoid fractions phenotypically and molecularly simulated the emergence of drug-refractory residual tumors in in vivo preclinical and clinical settings. The persister 3D cancer organoids acquired a transient dormant state with downregulated Myc activity, low biosynthetic rates, and a molecular profile strikingly similar to embryonic diapause, a dormant but reversible state of suspended epiblast development triggered by unfavorable conditions and driven by suppression of Myc. Congruously, the genetic ablation of MYC or the pharmacological inhibition of Myc transcriptional co-activators, BET bromodomains, abrogated the cytotoxic effect of chemotherapeutics in cancer cells. The chemoprotective effect of BET bromodomain inhibitors was cell cycle-independent and induced in cancer cells below-baseline redox stress levels which were maintained during drug exposure. These observations call for careful consideration of therapeutic approaches combining chemotherapy with BET bromodomain inhibitors in solid tumors. While the transcriptional activity of Myc is necessary for cancer cells to grow, our data reveal an unexpected role of Myc as regulator of cancer cell entry into a transient drug-persistent state that prevents the complete tumor eradication, co-opting developmental survival mechanisms that enable facultative survival and reproductive strategies under environmental stress. The diapause-like 3D organoid cancer models provide ex vivo tractability for studying the otherwise elusive, dormant, drug-persister residual tumors, with potential implications in personalized medicine and drug discovery.

#2105

Single cell RNA sequencing reveals multiple adaptive resistance mechanisms to regorafenib in colon cancer.

Anuja Sathe, Billy T. Lau, Sue Grimes, Stephanie Greer, Hanlee Ji. _Stanford University, Stanford, CA_.

Relapses in cancer therapy occur due to the presence of sub-populations that exhibit intrinsic or acquired resistance to treatment. Acquired resistance can arise via non-mutational adaptive mechanisms in response to a perturbation. Identification of these heterogenous mechanisms mediated via stochastic changes in cellular signaling is challenging using traditional bulk methods that average signals at a cellular population level. We harnessed the power of single-cell RNA sequencing (scRNA-seq) to identify the adaptive resistance mechanisms to regorafenib, a clinically approved multi-tyrosine kinase inhibitor, in a colon cancer model.

HCT116 cells were treated with an IC50 dose of regorafenib or vehicle control for 1 hour, 3 days, 7 days or 12 days and subjected to scRNA-seq using the 10X Genomics platform. Cells were sequenced at an average depth of 50000 reads/cell using Illumina sequencing. We controlled for batch effects by incorporating independent biological replicates and multiplexing using cell "hashing" with barcoded antibodies (BD Biosciences). Graph-based clustering, differential expression analysis and cell cycle phase assignment was performed using the Seurat algorithm. Proliferation was assessed using PI staining for cell cycle status, CellTiter Blue viability assay and clonogenic assays.

We analyzed a total of 19,879 single cells following a time-course of regorafenib or vehicle treatment. Regorafenib treatment at one hour resulted in no significant transcriptional changes. At all further time points, we could confirm the down-regulation of MAPK signaling, reflecting the mechanism of action of regorafenib. At 72 hours, we detected sub-populations with varying activation levels of ER stress response and increased lipid metabolism, together with upregulation of transcription factors SOX4 and ID3. These features continued to be detected till 7 and 12 days after treatment. By 12 days, we observed upregulation of keratins 7, 8, 10, 18, 19 together with markers of G2M/S phase progression. Survival of clonal cell populations at 12 days was confirmed by crystal violet staining. Small molecule-based inhibition of ER stress (azoramide) and lipid synthesis (fatostain) was synergistic with regorafenib at day 3 and successfully inhibited the emergence of these resistant clones by day 12. Our results reveal multiple signaling events that lead to a resistance phenotype characterized by increased proliferation and keratin upregulation that can be counteracted by combination therapies. For future studies we will continue to dissect the molecular networks underlying these events by gene silencing and protein over-expression studies.

#2106

Role of organic cation transporter-1 (OCT-1) in drug resistance condition of ph+ chronic myeloid leukemia (CML).

Saumya Patel,1 Krupa Shah,2 Rakesh Rawal1. 1 _Gujarat University, Ahmedabad, India;_ 2 _The Gujarat Cancer and Research Institute, Ahmedabad, India_.

A solute carrier (SLC) superfamily member, a major influx protein Organic Cation Transporter-1 (OCT-1), reported to be involved in influx of the Imatinib and other drugs in target cells. Despite the excellent responses to Imatinib and other tyrosine kinase inhibitors (TKIs) in CML, around a quarter of patients demonstrate primary resistance or suboptimal response within one year of therapy. Hence, present study mainly emphases the possible translocation mechanism of Imatinib, major TKIs and other chemotherapeutic agents that is mediated by OCT-1 transporter and OCT-1 gene expression level in progressive phases of CML. Homology based comparative modeling of OCT-1 protein, and its in-depth systemic interaction binding pattern analysis with Imatinib and other TKIs were conducted using computational modeling and molecular dynamics studies, respectively. Quantitative expression analysis of OCT-1 has also been evaluated in patients receiving Imatinib alone, hydroxyurea alone or both drug in different CML phases such as chronic, accelerated and blastic crisis phases for both in bone marrow and PBMNC samples (n=120). The interaction profile of Imatinib with OCT-1 revealed major conformational between ligand and protein interaction sites. However, generated protein-ligand complexes proceeded for molecular dynamics studies (100ns) that have confirmed the selectivity of Imatinib and other drugs towards the OCT-1 and possible translocation path of ligands in the OCT-1 cavities. In quantitative gene expression analysis, downregulation of OCT-1 expression as evident by decreased fold change values in patients with blastic crisis compared to chronic phase responsible for lower uptake of Imatinib into the cells. Hence, cells can not achieve optimum Imatinib inhibitory concentration which may be responsible for disease progression and ultimately leads to drug resistance. Thus, proposed combined computational and experimental approaches suggests OCT-1 mediated activation of bcr-abl independent mechanism, which may be potential confounder of developing Imatinib resistance in CML patients.

#2107

Integrative molecular and functional genomic analysis of chemotherapy resistant bladder cancer cell lines identifies novel mediators of therapeutic response.

Robert T. Jones,1 Tahlita C. Zuiverloon,2 Hedvig Vekony,1 Andrew Goodspeed,1 Teemu D. Laajala,3 Molishree Joshi,1 Colin Sempeck,1 Megan Tu,1 James C. Costello,1 Dan Theodorescu4. 1 _University of Colorado, Aurora, CO;_ 2 _Erasmus MC, Rotterdam, Netherlands;_ 3 _University of Turku, Turku, Finland;_ 4 _Samuel Oschin Comprehensive Cancer Institute, Los Angeles, CA_.

Gemcitabine and cisplatin (GC) combination neoadjuvant chemotherapy (NAC) followed by radical cystectomy (RC) is the standard of care for treating muscle-invasive bladder cancer (MIBC). Approximately 25% of patients experience complete pathologic response (pT0), while the remaining ~75% have residual disease and poorer prognosis. Therefore, defining mechanisms that enable bladder cancer cells to survive NAC will allow us to I) identify predictive biomarkers of response to NAC and improve MIBC patient stratification for NAC, and II) define novel genetic dependencies in GC-resistant bladder cancers for future drug development. We have performed RNA sequencing on a panel of six genetically diverse urothelial cancer cell lines, each with matched derivatives possessing acquired resistance to single agent gemcitabine, cisplatin, and combination GC. Initial analyses of RNA sequencing data has revealed hundreds of differentially expressed genes and associated pathways as potential drivers of resistance to these agents. Examples of significantly dysregulated pathways seen in gene set enrichment analyses implicate a range of pathways known to be associated with many diverse cellular processes such as: extracellular matrix organization, invasion and migration, interferon signaling, lipid and cholesterol metabolism and cellular differentiation status, among others. Importantly, these candidates include both shared and context-specific resistance phenotypes, which vary based on the genetic and molecular alterations found in the parental cell line. To complement these transcriptomic profiling experiments and to better understand how these changes in gene expression relate to mutation status and pathway activation status on the protein level, we have performed whole-exome sequencing, and are currently conducting phospho-proteomic profiling with reverse-phase protein microarray to enable more integrative analyses. Together the complement of transcriptomic, genomic and proteomic analyses will facilitate a more comprehensive assessment of which molecular patterns are drivers of resistance in our resistant cell lines. Finally, we have taken a more systematic approach to define functional genetic dependencies acquired in these NAC-resistant cells. To accomplish this we have performed whole-genome CRISPR knockout screening on the combined GC-resistant cells to identify vulnerabilities associated with GC-resistance. Taken together, our data provide a deep look into the context-specific and shared molecular mechanisms driving resistance to GC chemotherapy and provide a rational basis for the future development of molecularly guided therapies for NAC resistant bladder cancers.

#2108

Chromatin state as a mechanism of anthracycline response in breast cancer.

Jose A. Seoane, Jacob G. Kirkland, Jennifer L. Caswell-Jin, Gerald R. Crabtree, Christina Curtis. _Stanford University, Stanford, CA_.

The aim of this study is to determine the effect of chromatin regulators on anthracycline response in breast cancer. Anthracyclines are a highly effective component of curative breast cancer chemotherapy, but can be associated with substantial side effects. Because anthracyclines work in part via inhibition of topoisomerase II (TOP2) on accessible DNA, we hypothesized that expression levels of chromatin regulatory proteins that mediate DNA accessibility might predict anthracycline response. We used RNA-sequencing data from the TCGA breast cancer cohort to infer a breast cancer chromatin regulatory network using the ARACNE algorithm. A panel of 45 breast cancer cell lines with accompanying gene expression and growth inhibitory (GI50) data was used to build a genome-wide signature of anthracycline response. Based on the GI50, cell lines were classified as sensitive or resistant. The VIPER algorithm was then used to identify chromatin regulatory genes (CRGs) from the ARACNE network whose targets were significantly enriched in the anthracycline resistance signature. We further compiled a metacohort of 1006 clinically annotated early-stage breast cancer patients with gene expression data, allowing us to identify CRGs whose expression levels were associated with outcome after adjuvant treatment with anthracyclines. 24 CRGs associated (P<0.1) with anthracycline response in vitro and 54 CRGs associated (P<0.05) with anthracycline response in the patient cohort, 9 of which overlapped with the in vitro CRGs. In the patient cohort, CRG expression associated strongly with anthracycline response in all major subgroups of breast cancer (ER+/HER2-, HER2+, and triple-negative). In general, CRGs that promoted open chromatin (BAF and COMPASS) were, at high levels of expression, associated with anthracycline sensitivity, while CRGs associated with closed chromatin (PRC2) were, at high levels of expression, associated with anthracycline resistance. We functionally validated KDM4B as a predictor of anthracycline, but not taxane, sensitivity in a breast cancer cell line model through shRNA knockdown. We present a set of CRGs that control the chromatin accessibility in breast cancer both in vitro and in vivo; we infer that those tumors with more open chromatin as determined by the expression of these CRGs are intrinsically more sensitive to anthracyclines, while those with more closed chormatin are more resistant. Future work into the predictive power of these CRGs may allow us to target anthracyclines to only those patients who will benefit.

#2109

**A novel role for** ESRP1 **in regulating proliferation in therapy-resistant ER-positive breast cancer.**

Yesim Gökmen-Polar, Yuan Gu, Xiaoping Gu, Sunil S. Badve. _Indiana Univ. School of Medicine, Indianapolis, IN_.

Epithelial Splicing Regulatory Protein 1 (ESRP1) is a key splicing factor that plays an important role in cancer. We have previously demonstrated that ESRP1 is associated with poor prognosis and tamoxifen resistance in human ER-positive (ER+) breast tumors. Knockdown of ESRP1 decreased the tumor growth in ER+ breast cancer models resistant to endocrine therapy. In this study, we explored the upstream/downstream regulatory mechanisms of ESRP1 in tamoxifen-resistant breast cancer using ESRP1 knockdown model. Probe based-Human Transcriptome Array 2.0 (HTA; Applied Biosystems/Thermo Fisher) was performed using RNAs from control and ESRP1 knockdown cells [tamoxifen-resistant LCC2 cells (2-control) versus LCC2 cells with ESRP1 knockdown (2C3)] in ER+ breast cancer. Transcriptome profiling of ESRP1 in 2C3 knockdown model revealed that CEBP gamma, a transcription factor belonging to the CCAAT/enhancer-binding protein (C/EBP) family, was significantly decreased in ESRP1 knockdown cells (p value=2.33E-06). The expression of other related proteins C/EBP family including C/EBP alpha, C/EBP beta, and C/EBP delta that form homodimers and heterodimers with each other was also assessed. In knockdown cells, C/EBP alpha was increased (p value=0.0022), while two different C/EBP beta transcripts were decreased (p values=0.0010 and 0.03, respectively). C/EBP delta expression was not significantly altered. Using a competitive promoter-binding transcription-factor assay, we measured the transcription activity of C/EBP gamma in the absence and presence of ESRP1 promoter DNA fragment. We identified strong binding of C/EBP gamma to ESRP1 promoter region indicating that it is upstream regulator of ESRP1. CEBP transcription factor family members are important regulators of proliferation via cyclin-dependent kinases (CDKs). We further demonstrated that CDK4/CDK6 inhibitor abemaciclib decreases the expression of ESRP1 and C/EBPG levels significantly (p values= 0.0009 and 0.0019, respectively). In conclusion, for the first time, we identify that C/EBP gamma upregulates ESRP1 expression, which further regulates proliferation through control of expression of CDKs.

#2110

WEE1 inhibition could reverse trastuzumab resistance by downregulation of PD-L1 in HER2-positive cancers.

Meihua Jin, Ah-Rong Nam, Ji-Eun Park, Ju-Hee Bang, Kyoung-Seok Oh, Do-Youn Oh, Yung-Jue Bang. _Seoul National Univ. College of Medicine, Seoul, Republic of Korea_.

Background: Trastuzumab in combination with chemotherapy is a standard of care for patients with HER2-positive breast and gastric cancer. Resistance mechanism to trastuzumab, anti-HER2 therapy, includes multiple pathways. Cancer cells could evade immune surveillance through high expression of programmed cell death ligand 1 (PD-L1) on cancer or immune cells. Among resistance mechanisms to trastuzumab, the role of PD-L1 modulation has not yet been discovered in HER2-positive cancers. WEE1 is involved in cell cycle progression and DNA damage response (DDR), and, interestingly, beyond DDR, it could modulate immunes.

We aimed to evaluate the role of PD-L1 in trastuzumab resistance in HER2-positive cancers with/without immune cells and to explore WEE1 inhibitor as a trastuzumab resistance overcoming strategy by modulation of PD-L1.

Methods: Four trastuzumab-resistant (HR) cells (SNU216HR, N87HR, SNU2670HR, SNU2773HR) were established from 2 HER2-amplified gastric cancer cells (SNU216, NCI-N87) and 2 HER2-amplified biliary tract cancer cells (SNU2670, SNU2773). For WEE1 inhibition, AZD1775 was used. MTT assay, colony formation assay, cell cycle analysis by FACS Calibur flow cytometer, and western blot were done.

Results: All four HR cells showed PD-L1 upregulation compared with parental cells. When PD-L1 was knocked-down by transfection with si-PD-L1, anti-growth/proliferation effects were observed in MTT assay and colony forming assay. PD-L1 itself may promote cancer cell growth without interacting with immune cells.

AZD1775 downregulated PD-L1 expression and induced cell cycle arrest at sub-G1 and G2/M phases in all 4 HR cells. The synergistic anti-proliferative effects were found in all 4 HR cells with co-treatment of trastuzumab and AZD1775.

Conclusion: PD-L1 upregulation may contribute to trastuzumab resistance in HER2-positive cancer cells. Targeting WEE1-PD-L1 pathway might be a candidate strategy to overcome trastuzumab resistance in HER2-positive cancers.

#2111

Increased heme influx dictates tumorigenic functions of non-small cell lung cancer cells.

Sagar Shashikant Sohoni, Poorva Ghosh, Li Zhang. _UT Dallas, Richardson, TX_.

Heme, iron protoporphyrin IX, is a central metabolic and signaling molecule that regulates diverse molecular and cellular processes relating to oxygen utilization and metabolism. Heme serves as a cofactor or a prosthetic group for complexes II, III, and IV in electron transport chain. Hence, it has a direct role in oxidative phosphorylation and energy generation in cells. Most human cells can synthesize and uptake heme from circulation. A number of epidemiological studies have shown that high heme intake is associated with an increased risk of cancer, including lung cancer. Our lab has previously shown that an array of non-small cell lung cancer cells (NSCLCs) exhibit high heme synthesis and uptake. This increased heme flux correlates with an enhanced oxygen consumption and ATP production. NSCLCs also exhibited significantly elevated levels of hemoproteins and mitochondrial biogenesis proteins compared to normal lung cell lines. Expression levels of these proteins correlate with increased oxygen consumption and the total heme content of NSCLCs suggesting a possible role of heme in energy metabolism and mitochondrial biogenesis.

In order to assess the involvement of heme in tumorigenicity of the NSCLC cell lines, we overexpressed heme synthesis enzyme, ALAS1 or heme transporters, HCP1 or HRG1 using lentivirus system. NSCLC cell lines A549 and H1299 were infected with a lentivirus carrying ALAS1 or HCP1 or HRG1 plasmid. Stable clones were obtained with antibiotic selection and checked for overexpression with western-blots. Cells overexpressing ALAS1, HCP1 and HRG1 showed significantly higher rates of heme influx as well as intensified oxygen consumption and ATP formation compared to eGFP controls. Tumorigenic properties such as invasion, migration and colony formation were also found to be significantly increased in these cell lines compared to their eGFP counterparts. Subcutaneous xenografts generated with overexpression cell lines also showed significantly high tumor growth rates and mass compared to eGFP controls. Our data from both in vitro and in vivo experiments show that heme directly improves the tumorigenic properties of NSCLC cells by substantially improving the respiration rates and energy metabolism.

#2112

Structural characterization of transmembrane protein TMEM205.

Xiaoyun Bai, Di Xia. _National Institute of Health, Bethesda, MD_.

Cisplatin resistance is notorious in leading to therapeutic failure of many solid malignancies. Various resistance mechanisms have been proposed while many more are being added, based on the growing number of resistance-inducing genes identified. TMEM205 is an integral membrane protein with unknown function, but its intracellular level was found markedly elevated in cisplatin resistance cells, the mechanism of which is unknown. Here for the first time we characterized the molecular mechanism of TMEM205-mediated cisplatin resistance through structural and functional studies of TMEM205. The crystal structure of the full-length TMEM205 was obtained at 2.5 Å resolution and shows a 4-helix bundle with a highly hydrophilic tunnel. Strong anomalous signals were observed indicating platinum binding sites. Our structural and functional study of TMEM205 provides novel insight into the complexity of cisplatin resistance.

#2113

KRAS **amplification mediates resistance to osimertinib in acquired afatinib-resistant NSCLC harboring exon 19 deletion/T790M in EGFR.**

Toshimitsu Yamaoka,1 Ohba Motoi,1 Yasunari Kishino,2 Sojiro Kusumoto,2 Junji Tsurutani,1 Tohru Ohmori2. 1 _Showa University, TOKYO, Japan;_ 2 _Showa University School of Medicine, TOKYO, Japan_.

Background: The second mutation of T790M in epidermal growth factor receptor (EGFR) exon 20 mediates resistance to first- and second-generation EGFR tyrosine kinase inhibitors (TKIs; gefitinib, erlotinib, and afatinib). The critical T790M mutation in EGFR has facilitated the development of third-generation mutation-selective EGFR TKIs (rociletinib and osimertinib). We previously reported heterogeneous afatinib resistance in lung adenocarcinoma PC-9 cells, harboring 15bp deletions in EGFR exon 19. Among the afatinib-resistant PC-9 cells, we noted the emergence of T790M in EGFR exon 20 and responses to third-generation EGFR-TKIs. Here, we used afatinib-resistant lung adenocarcinoma PC-9 cells, called AfaR, harboring the exon 19 deletion/T790M in EGFR.

Purpose: To identify novel resistance mechanisms in post-afatinib treatment, two osimertinib-resistant cell lines, OsiR1 and OsiR2, were established using increasing concentrations of osimertinib by stepwise dose escalation up to 1 μmol/L over 10 to 12 months.

Results: A linear and reversible KRAS amplification and overexpression with increased osimertinib concentrations in OsiR1 and OsiR2 cells, respectively, was detected. OsiR1 cells maintained osimertinib resistance with KRAS amplification after osimertinib withdrawal for 2 months. After osimertinib withdrawal for 2 months, OsiR2 cells exhibited KRAS attenuation and osimertinib sensitivity was entirely recovered in vitro and in vivo. Phospho-EGFR (Y1068) and growth factor receptor-bound protein 2 (GRB2)/son of sevenless homolog 1 (SOS1) complex was found to mediate osimertinib resistance in OsiR1 cells with sustained KRAS activation. The association of phosphorylated EGFR with the GRB2/SOS1 complex leads to KRAS activation. After 2 months of osimertinib withdrawal, the GRB2/SOS1 complex was dissociated, and the EGFR signal but not the GRB2/SOS1 signal was activated. Concomitant inhibition of mitogen-activated protein kinase kinase and EGFR could overcome osimertinib-resistance in OsiR1 cells.

Conclusion: We characterized novel acquired resistance mechanisms for third-generation EGFR-TKI, osimertinib, providing insights into the development of novel treatment strategies.

#2114

Co-delivery nanoparticle to overcome therapeutic resistance in human head and neck cancer promoted by insufficient Src-targeted treatment.

Liwei Lang,1 Chloe Shay,2 Xuli Wang,3 Yong Teng4. 1 _Department of Oral Biology and Diagnostic Sciences, Augusta Univ., Augusta, GA;_ 2 _Emory Children's Center, Emory University, Augusta, GA;_ 3 _Department of Radiology and Imaging Sciences, School of Medicine, University of Utah, Salt Lake City, UT;_ 4 _Georgia Cancer Center, Augusta Univ., Augusta, GA_.

Head and neck cancer (HNC) exhibiting resistance to molecular-targeted therapeutics poses a challenge to their effective clinical management and alternative treatment strategies are actively sought to improve results. Src is commonly hyperactivated in HNC cells and has been implicated as an oncogenic driver in the development and progression of HNC. Unfortunately, the clinical benefit is dampened significantly in HNC patients because only a small fraction of patients respond positively to Src-targeted treatment, and many develop drug resistance. Here, we demonstrate that the addition of AKT blockade in Src-targeted treatment may prevent activation of alternative signaling pathways to bypass the Src inhibition in HNC cells. The combination of Src and AKT inhibitors significantly suppressed HNC growth more efficiently than either drug alone. However, the properties and pharmacokinetics of two inhibitors required a novel approach to optimal delivery into the same tumor cells. To address this, we developed novel tumor-targeting nanoparticles to selectively co-deliver Src and AKT inhibitors to the tumor site in an orthotopic mouse model of HNC. This nanotherapeutic modality significantly improved the efficacy of tumor repression, which was mainly attributed to the highly specific tumor-targeted drug delivery system and synergistic anticancer effect by co-inactivation of AKT and Src. The results of this study will enable us to take a major step toward addressing the urgent need for single drug resistance and failure, and strongly impact on the development of clinically acceptable therapeutics to combat HNC.

#2115

FOS contributes to 5-FU resistance and reversal effects of natural flavonoid GL-V9 in colon cancer.

Li Zhao,1 Li Zhaohe,1 Ding Youxiang2. 1 _China Pharmaceutical University, Nanjing, China;_ 2 _China pharmaceutical university, Nanjing, China_.

5-FU is widely used in the treatment of colon cancers. Although 5-FU can improve response rates and survival, response rates for 5-FU-based chemotherapy as a first-line treatment for advanced colorectal cancer are only 10-15%. Meanwhile, cancer drug resistance often occurs, which is an important reason for poor treatment effect of 5-FU. Therefore, it is significant to uncover the mechanisms underlying 5-FU resistance and develop effective but nontoxic agent candidates for reversing cancer drug resistance. Here, we found that proto-oncogene FOS was activated in 5-FU-resistant colon cancer cells compared to parental cells. Additionally, FOS overexpression using lentivirus plasmid obviously triggered 5-FU resistance in colon cancer cells. Moreover, by analyzing two GEO datasets, we also found that FOS expression was associated with poor overall survival of colon cancer. Taken together, FOS confers 5-FU resistance to colon cancer and it is potential to be exploited as a therapeutical target. Lastly, we studied the reversal effects of natural flavonoid GL-V9 on the FOS activation and 5-FU resistance in order to develop candidates for FOS inhibitor which would be helpful for 5-FU-resistant colon cancer. Consequently, nontoxic doses of GL-V9 could significantly suppress activation of FOS and sensitize colon cancer cells to 5-FU. Therefore, the flavonoid is potential to be exploited as a synergetic agent for 5-FU-resistant colon cancer.

#2116

Cariprazine is a potent chemo-adjuvant against multidrug resistant colon and lung cancer.

Noor Hussein, Angelique Nyinawbera, Haneen Amawi, Amit Tiwari. _Univ. of Toledo, Toledo, OH_.

Multidrug resistance (MDR) is considered a continuing clinical problem, reducing the efficacy of cancer chemotherapy. The over expression of the ATP - binding cassette (ABC) family G2 (ABCG2) transporter is considered to be one of the main mechanisms responsible for mediating MDR in cancer. Molecular modeling studies have shown the significant affinity and binding mode of cariprazine, a dopamine D3-preferring D2/D3 receptor partial agonist, to the drug binding cavity of ABCG2 with a Glide XP score of the -5.761. Therefore, in this in vitro study, we determined the effect of cariprazine in increasing the efficacy of MX (an ABCG2 substrate) in the ABCG2 overexpressing cancer cell lines H460-MX20 and S1M1-80. Also, studies conducted to determine whether cariprazine is a reversal compound that specifically targets the ABCG2 protein. Alone, cariprazine, at concentrations up to 20 μM, did not significantly decrease cell viability. Cariprazine, at concentrations ranging from 1 to 20 μM, did not significantly alter the cytotoxicity of mitoxantrone (MX) in the parental non-small cell cancer cell line, H460 and colon cancer cell S1. However, cariprazine (1-20 μM) significantly enhanced the efficacy of ABCG2 substrate antineoplastic drug MX in the ABCG2- overexpressing MDR cell line, H460-MX20 and S1M1-80, by reducing the resistance fold from 28 to 1 and from 93 to 1.33, respectively. The intracellular accumulation of rhodamine 123 in the ABCG2-overexpressing cells, S1M1-80 was significantly increased by the addition of cariprazine, in a concentration-dependent manner (1 - 20 μM). Interestingly, 10 or 20 μM of cariprazine significantly decreased the expression levels of the ABCG2 protein in the colon and lung cancer cell lines, suggesting that cariprazine inhibits both the function and

expression of ABCG2 transporters at non-toxic concentrations. Overall, our results suggest that cariprazine, via several distinct mechanisms, can re-sensitize resistant cancer cells to mitoxantrone.

#2117

Different sensitivities of four protein tyrosine kinase inhibitors towards drug-resistant RET mutations.

Xuan Liu,1 Tao Shen,1 Qingling Huang,2 Teng Peng,3 Frank Hilberg,4 Jianfeng Cai,3 Blaine H. Mooers,1 Jie Wu1. 1 _University of Oklahoma Health Sciences Center, Oklahoma City, OK;_ 2 _H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL;_ 3 _University of South Florida, Tampa, FL;_ 4 _Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria_.

The RET protein tyrosine kinase (PTK) is a clinically validated target of therapy in non-small cell lung cancer (NSCLC) and thyroid cancer. Mutations in the targeted PTKs is a mechanism of drug resistance in cancer therapy with tyrosine kinase inhibitors (TKIs). Mutation-sensitive secondary drugs have been used successfully to overcome acquired drug-resistance to the first line TKIs. Cabozantinib, lenvatinib, vandetanib, and nintedanib are FDA-approved multikinase TKIs with anti-RET activity. Using RET kinase-dependent BaF3/KIF5B-RET cells, we isolated thirteen mutations resistant to one of these TKIs. Cross-analysis of sensitivities of these four TKIs on these drug-resistant RET mutants and the RETM918T mutant, which is found in medullary thyroid carcinoma, revealed different TKI resistance-sensitivity profiles. In particular, the RETM918T mutant was resistant to cabozantinib, lenvatinib, and vandetanib but did not affect the potency of nintedanib. RETL881V was isolated as a vandetanib-resistant mutation. The RETL881V mutation also induced resistance to cabozantinib and lenvatinib but did not affect the nintedanib sensitivity. Examination of the RET-vandetanib co-crystal structure and the chemical structures of vandetanib and nintedanib suggested that a phenyl group in nintedanib, which corresponds to a methoxy group in vandetanib, may form hydrophobic interaction with the shorter side chain of Val881 and thus allows nintedanib to inhibit RETL881V. To test this possibility, we synthesized two nintedanib analogs (Compound 1 and Compound 2) without the phenyl group and tested their activities. The data showed that RETL881V was resistant to Compound 1 and Compound 2, supporting a role of the phenyl group of nintedanib in mediating inhibition of RETL881V. Taken together, we have identified 13 RET mutations that display different resistance-sensitivity profiles against four anti-RET TKIs. Moreover, nintedanib is effective in inhibiting RETM918T and RETL881V mutants that are resistant to the other three TKIs.

#2118

Collateral resistance trajectories following failure to antimitotic drugs.

Mark Borris D. Aldonza. _Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea_.

Secondary drug resistance stems from dynamic clonal evolution during the development of a prior primary resistance. This collateral type of resistance is often a characteristic of cancer recurrence. Yet, mechanisms that drive this collateral resistance and their drug-specific trajectories are still poorly understood. Using resistance selection in concert with small-scale pharmacological screens and data mining in a large pharmacogenomics database, we identified and validated unique collateral resistance trajectories that occur between chemically-different classes of drugs. One convincing case is the co-resistance between microtubule-targeting drugs and EGFR-tyrosine kinase inhibitors (EGFR-TKIs). With paclitaxel resistance as a proxy first-line drug failure model and EGFR-TKI resistance as a collateral, we find that a prior resistance can follow distinct trajectories towards collateral resistance, leading to formation of more stable resistant cell populations. These trajectories can be directed by mechanisms regulating the entry to a drug-tolerant persistent state during exposure to therapy. Collectively, our work unravels the alterations in cellular states defining collateral resistance trajectories to EGFR-TKIs following prior resistance to antimitotic drugs.

#2119

DCLK1 regulates ATR-DNA damage response for KRAS mutant lung cancer drug resistance and stemness.

Courtney Houchen,1 Janani Panneerselvam,1 Priyanga Mohandoss,2 Nathaniel Weygant,1 Randal May,1 Dongfeng Qu,1 Naushad Ali,1 Timothy Wang,3 Chinthalapally Rao,1 Michael Bronze,1 Parthasarathy Chandrakesan1. 1 _University of Oklahoma Health Sciences Center, Oklahoma City, OK;_ 2 _SRM Institute of Science and Technology, Chennai, India;_ 3 _Columbia University Medical Center, New York City, NY_.

Background: Lung cancer is the deadliest cancer in both men and women. About 85% of lung cancers are non-small cell lung cancer (NSCLC) and almost 25% of all types of NSCLC contain KRAS mutations, which remain as an undruggable challenge. Doublecortin-like kinase 1 (DCLK1) regulate many key oncogenes including KRAS in various solid cancers is overexpressed in lung cancer, raising the possibility to selectively target DCLK1 expression to combat the undruggable KRAS mutant NSCLC and its drug resistance.

Experimental Procedure: H460 and A549 (KRAS mutant NSCLC cell lines) and H1299 (WT KRAS NSCLC cell line) were used. Cisplatin resistance KRAS mutant NSCLC cells (CR-A549) were generated. Transfection of siRNA against DCLK1 in NSCLC cells and CR-A549 cells were carried out. Tumor cell apoptosis, DNA damage, DNA damage response (DDR) and tumor cell self-renewal were assessed. Protein and mRNA expressions by western-blot and RT-PCR, apoptosis by FACS, cell death by COMET assay and self-renewal by clonogenic assay.

Results: Here, we report that increased expression of DCLK1 is the key to develop chemoresistance and self-renewal in KRAS mutant NSCLC. We observed increased immunostaining for DCLK1 in patient lung adenocarcinoma compared to normal lung tissue. We observed an increased expression of DCLK1 protein and mRNA in KRAS mutant NSCLC cells compared to WT KRAS NSCLC cells. To investigate the role of DCLK1 in KRAS mutant NSCLC tumor progression, we knockdown DCLK1 in KRAS mutant NSCLC cells, which resulted in reduced cell proliferation/survival and self-renewal. We generated KRAS mutant NSCLC cisplatin resistance cells (CR-A549) and found that CR-A549 cells acquired higher DCLK1 expression with enhanced self-renewal capacity and display ~9fold higher IC50 for cisplatin treatment compared to parental cells. However, silencing DCLK1 in CR-A549 cells reversed the tumor cell resistance to cisplatin and increased cell death and reduced self-renewal. To further investigate mechanistically, we examine the expression of DDR signaling in CR-A549 cells. We observed an increased expression of ATR-DDR in CR-A549 cells. Furthermore, DCLK1 knockdown reduced the expression of ATR and treatment of siATR plus cisplatin (5µM) to CR-A549 cells demonstrated similar effects observed in the treatment of siDCLK1 plus cisplatin (5uM), which resulted in increased sensitivity of CR-A549 cells to cisplatin, and thus increased cell death and complete abrogation of self-renewal capacity, suggesting that DCLK1 mediated cisplatin resistance occurs via ATR-DDR dependent mechanism.

Conclusion: Our data demonstrate that the increased expression of DCLK1 is associated with enhanced cancer stem cell-like features, ATR-DDR signaling and chemoresistance in KRAS mutant NSCLC. Targeting DCLK1 alone or in combination with cisplatin may represent a novel therapeutic strategy for the effective treatment of undruggable KRAS mutant NSCLC.

#2120

Human epidermal growth factor receptor 3 might be a targetable molecule for treatment of chemotherapy resistant breast cancer.

Takeshi Kotake, Eiji Suzuki, Fengling Pu, Tatsuki Kataoka, Masahiro Hirata, Eri Hohokabe, Masakazu Toi. _Kyoto University Hospital, Kyoto, Japan_.

Introduction: Disease control of chemotherapy refractory breast cancer patients (CRBC), who do not respond to conventional treatment strategies, is important. In the setting of neoadjuvant chemotherapy (NAC), susceptibility of the chemotherapeutic agents could be confirmed with clinical relevance. Doxorubicin, docetaxel and paclitaxel have been used as conventional NAC. Eribulin and platinum may also be added in the future. When the drug sensitivity is evaluated by NAC and it is not sensitive to the drugs, there is currently no effective therapy expected to be improved. This is one of the recent unmet medical needs in breast cancer treatment strategy. Materials and Methods: In this study, in order to establish a novel therapeutic strategy for CRBC, we established chemotherapy resistant breast cancer cell lines against doxorubicin, paclitaxel, docetaxel and eribulin using BT549, Hs578t, MDA-MB231, MDA-MB468, and HCC70. We evaluated human epidermal growth factor receptor 3 (HER3) expression in these parental and resistant cell lines. We used U3-1402 (a novel anti-HER3 antibody-drug conjugate) to test if HER3 targeting treatment inhibits the cell proliferation of the chemotherapy resistant cell line with positive conversion of HER3 expression. Results: We found that the change to positive expression of HER3 in paclitaxel resistant BT549 cell line (BT549/PTX) and eribulin resistant Hs578t cell line (Hs578t/ERI) while these two cell lines initially showed negative expression of HER3. In the cell proliferation assay, as we expected, there was no inhibition of proliferation by treatment with HER3 targeting antibody agent U3-1402 in these two cell lines. However, BT549/PTX and Hs578t/ERI with positive conversion of HER3 expression were clearly inhibited their cell proliferation by treatment with U3-1402. Conclusions: It suggests that even in cases whose tumor does not express HER3 initially and show no response for chemotherapy in the NAC, U3-1402 could be effective for the patients with positive conversion of HER3 expression after NAC. We are studying for the role of positive conversion of HER3 expression in the process of acquired chemo-resistance and the elucidation of the molecular mechanism of positive conversion of HER3 expression in these unique treatment condition.

#2121

Overcoming tamoxifen resistance and inhibiting metastatic recurrence in estrogen receptor-positive breast cancer.

Pelin Gulizar Ersan,1 Ozge Saatci,2 Oguzhan Tarman,1 Rasmi Mishra,1 Nevin Belder,1 Unal Metin Tokat,1 Yasser Riazalhosseini,3 Ozgur Sahin2. 1 _Bilkent University, Ankara, Turkey;_ 2 _University of South Carolina, Columbia, SC;_ 3 _McGill University, Quebec, Canada_.

Estrogen receptor (ER)-positive breast cancer accounts for almost 75% of all breast cancers. Tamoxifen has been used for over 40 years to treat early, locally advanced and metastatic ER-positive breast cancer; however, patients develop resistance over time. Furthermore, metastatic recurrence, the major cause of death, is common in ER-positive breast cancer patients whose cancer progressed on tamoxifen and other hormone therapies. In this line, we and others reported epithelial-mesenchymal (EMT)-like changes upon acquisition of tamoxifen resistance hinting towards the co-occurrence of resistance and metastatic abilities of the cells. However, little is known about common molecular mediators of drug resistance and metastatic recurrence. Therefore, we addresses the question of how we concurrently overcome tamoxifen resistance and prevent lethal metastatic recurrence whereby eliminating the mortality associated with metastatic breast cancer.

Combining whole-transcriptome sequencing and downstream pathway analysis, we identified cyclic AMP (cAMP) signaling to be the most significantly altered pathway in acquired tamoxifen resistant breast cancer cells. We found that PDE4D (Phosphodiesterase 4D), which hydrolyzes cAMP, was significantly overexpressed in both MCF-7 and T47D tamoxifen-resistant (TamR) cells. We demonstrated that PDE4D inhibition overcomes tamoxifen resistance in cell lines and tumor xenografts via cAMP induced endoplasmic reticulum stress and cell death. Importantly, our TamR cells have partial EMT and enrichment of TGF-beta signaling, and PDE4D inhibition in combination with tamoxifen inhibited cell migration in vitro and blocked TGF-beta induced EMT. RNA-Seq experiment also identified one of the highly oncogenic long non-coding RNAs (lncRNA) to be upregulated in TamR cells, and its targeting inhibits PDE4D and leads to tamoxifen sensitization. We showed that higher expressions of both PDE4D and the candidate lncRNA predict worse survival in tamoxifen-treated breast cancer patients. Currently, we are testing the tamoxifen sensitizer and metastasis blocker functions of PDE4D and our candidate lncRNA using patient-derived xenograft (PDX) models of metastatic ER-positive breast cancer.

Overall, our results suggest that targeting PDE4D, or its upstream regulatory lncRNA, can simultaneously overcome tamoxifen resistance and prevent metastatic recurrence in ER-positive breast cancer. This is then expected to dramatically reduce mortality rates among ER-positive breast cancer patients in future.

#2122

Generation of a fully characterized preclinical PDX panel to accelerate the identification of next generation treatments for patients with acquired resistance to targeted therapies.

Olivier Déas,1 Ludovic Bigot,2 Emilie Dasse,1 Guillaume Lang,1 Yohann Loriot,2 Fabrice Andre,2 Jean-Charles Soria,2 Benjamin Besse,2 Stefano Cairo,1 Marie Tavernier,1 Katell Mevel,1 Enora Le Ven,1 Jean-Gabriel Judde,1 Luc Friboulet2. 1 _XenTech, Evry, France;_ 2 _Gustave Roussy, Villejuif, France_.

The last 20 years have witnessed the identification of an increasing number of actionable oncogenic drivers and the development and clinical use of specific inhibitors against these targets. Unfortunately, patients treated with targeted therapies consistently develop resistance and progression under treatment. Hence, important scientific, pharmaceutical and medical research efforts are directed towards understanding the mechanisms of acquired resistance to explore new therapeutic pathways.

The MATCH-R clinical trial enrolls patients with oncogene-driven cancer who have had previous clinical response to targeted therapy and subsequently experienced disease progression. In the framework of this project, Gustave Roussy and XenTech are joining forces to develop a panel of patient-derived xenografts (PDXs) derived from biopsies collected from these patients at the stage of acquired resistance. These PDX models will be fully characterized at molecular and pharmacological level and used to improve knowledge on the mechanisms underlying resistance to treatment and to evaluate response to new treatments.

In this perspective, the development of 75 PDX-AR (Acquired Resistance) models is planned over 3 years. All the models are maintained under the same therapeutic pressure the parental tumor was submitted to at the time of biopsy, and will be subjected to extensive phenotypic and genotypic characterization.

To favor successful xenograft establishment, the first two passages are performed without drug treatment, which is applied from the third passage on. When doing so, we observed 3 types of response: some models showed resistance from the first passage under treatment, some showed stabilization under treatment at the first passages and rapidly acquired resistance over passages, and others showed sensitivity to treatment, whereas the patient tumor showed progression under the same treatment. These different behaviors might be due to different mechanisms of resistance, irreversible for the former, reversible for the two latter, as well as to suboptimal correlation of the clinical dose with the one used in mice.

An example of such discrepancies has been found in two models of NSCLC PDX obtained from two metastases from a patient treated by a ROS1 and ALK inhibitor. While LCx-MR135PD2-AR PDX does not respond to the treatment, the LCx-MR135PD1 model is highly sensitive. As both metastases were progressing under treatment in the patient, molecular and pharmacological comparative analysis of these two models will investigate these discrepancy and provide important insights into the mechanisms of resistance to such inhibitors.

Overall, the MatchR PDX project will provide a unique preclinical platform to identify resistance mechanisms to current targeted therapies and to develop next generation therapeutic strategies.

#2123

A combinatorial approach using gemcitabine and bitter melon juice overcomes drug resistance impacting gemcitabine uptake and metabolism.

Deepanshi Dhar, Dileep Kumar, Komal Raina, David J. Orlicky, Chapla Agarwal, Rajesh Agarwal. _Univ. of Colorado Denver-AMC, Aurora, CO_.

Pancreatic cancer (PanC) has the lowest 5-year survival rate (<3%) of all cancer types and is projected to be the second leading cause of cancer-associated mortalities by the year 2030 in the United States. Surgery alone fails to suffice in a majority of cases; the disease relapses in 70-80% of patients and is fatal without additional therapy. Currently, of all the available treatment modalities for PanC, gemcitabine (GEM) is the standard of care frontline drug, with a moderate success rate in improving the median overall PanC patient survival by about 5.7-6.8 months. Although a modest improvement is achieved using a combination therapy approach, the patient survival still remains less than 12 months, and includes a poor quality of life due to the extensive side effects. Moreover, chemoresistance to GEM is a major contributing factor for PanC patient' morbidity and mortality resulting from dysfunctional uptake and metabolism of GEM in cancer cells. Therefore, there is an urgent need for agents that could reverse GEM resistance and allow continued chemosensitivity in PanC. Here, we employed natural agent bitter melon juice (BMJ) and GEM to examine their anticancer potential alone and in combination in PanC patient derived xenograft (PDX) - pancreatic ductal adenocarcinomas explants in nude mice. Comparing single agents versus combination in three explants PDX272, PDX261, and PDX266, both at the end of the active dosing regimen as well as following a post treatment-washout period (no drug administration) revealed enhanced efficacy of the combination treatment over failed GEM-in the post treatment termination cohort. Extensive mechanistic assessments revealed that overcoming GEM resistance was possibly due to modulation of GEM metabolism pathway molecules by the BMJ administration. While all drug exposures led to a decreased RRM1 expression during treatments in PanC-explants, at the end of treatment washout period explants displayed increased RRM1 levels in GEM-only group but a sustained decrease in BMJ and Combo groups. Investigation into hENT1 (human equilibrative nucleoside transporter 1) expression, responsible for bidirectional GEM trafficking, revealed that at the end of washout period, combination therapy was the only group that continued to express high levels of hENT1 for all three explants, whereas GEM only group showed poor hENT1 expression. Furthermore, analysis for deoxycytidine kinase (dCK), the enzyme responsible for GEM metabolism, revealed that the combination group had the most augmented dCK expression levels by study end. Taken together, the study outcomes highlight the efficacy of BMJ in combination with GEM in modifying and regulating multiple key players in GEM uptake and metabolism. These conclusions make a compelling case for future clinical investigations in PanC patients wherein BMJ is combined with GEM to target and overcome GEM resistance. 

### Molecular Classification of Tumors

#2124

**Concurrent** in vitro **and** in silico **approach for the identification of surface proteome targets on HPV-associated cancer cells.**

Hsuan-Chen Liu,1 Ivenise Carrero,1 Falguni Parikh,1 Thomas Kraus,2 Thomas M. Moran,2 Mathew J. Ellis,1 Elizabeth Y. Chiao,1 Aleksandar Milosavljevic,1 Andrew G. Sikora1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Icahn School of Medicine at Mount Sinai, New York City, NY_.

Human papillomavirus (HPV)-associated Head and neck cancer (HNSCC) are among the fastest growing cancer types. In contrast to non-viral HNSCC caused by traditional risk factors, tobacco and alcohol, HPV-HNSCC displayed distinct molecular alterations and prognosis from HPV-negative HNSCC. Currently, there is no clinically-approved therapeutic vaccine, and no targeted therapy approach for HPV-driven cancer which exploits the unique biology of HPV infection. In order to identify novel targets suitable for therapeutic and diagnostic development, we developed two parallel approaches to identification of proteins/antigens expressed on the surface of the host cells, whose expression is altered by HPV infection. One is an "antigen-agnostic" approach by using immortalized HNSCC cell membrane fractions to generate monoclonal antibodies, which does not require advance knowledge of the identities of target antigens. The second approach is bioinformatics analyses of the TCGA database using the epigenomic deconvolution tool (EDec) to identify surface proteins that are differentially overexpressed in HPV- HNSCC. Five thousand hybridoma colonies were generated by the "antigen-agnostic" approach, and were then screened by flow cytometry to test the specificity of binding to HPV-positive cancer cell lines (2 HNSCC and 2 Cervical Cancer) and HPV-negative cancer cell lines (4 HNSCC and 1 CC). After primary screening, we narrowed down to forty-four clones with preliminarily favorable binding characteristics; among these hybridoma clones, we have identified seven which preferentially bind to HPV-positive cancer cells. We then identified the binding targets of three clones via immunoprecipitation and mass spectrometry. These targets are integrin alpha6 beta4 (ITGA6, ITGB4), tissue factor (F3) and keratin 8 (KRT8) respectively. The bioinformatics-based approach identified several surface proteins that are differentially overexpressed in HPV-positive HNSCC and at levels significantly higher than found in normal control tissue. Evaluation of gene and protein expression in cancer cell lines and/or patient tissue validated several genes identified by the deconvolution approach, including: ROR2, a non-canonical WNT member not previously associated with head and neck cancer; and LY6K, a cancer-testis antigen that overexpressed in both HPV-positive and negative HNSCC. We propose targeting membrane-expressed antigens on HPV-related cancer cells as a platform for further development of novel tumor imaging and therapeutic approaches for HNSCC and other HPV-associated cancers.

#2125

Genetic analysis for classification and treatment of poorly differentiated sinonasal cancer.

Mario Hermsen,1 Cristina Riobello,1 Virginia N. Cabal,1 Rocío García-Marín,1 Sira Potes-Ares,1 Laura Suárez-Fernández,1 Blanca Vivanco,2 Alessandro Franchi,3 Reinhard Büttner,4 Fernando López,2 José Luis Llorente2. 1 _ISPA, Oviedo, Spain;_ 2 _HUCA, Oviedo, Spain;_ 3 _University of Pisa, Pisa, Italy;_ 4 _University of Cologne, Cologne, Germany_.

Introduction: The sinonasal cavities harbour a wide variety of rare tumour types, including squamous-cell carcinoma (SNSCC), intestinal-type adenocarcinoma (ITAC), olfactory neuroblastoma (ONB), neuroendocrine carcinoma (SNEC), undifferentiated carcinoma (SNUC) and malignant mucosal melanoma (MMM). Histopathological classification is related to clinical outcome, but correct diagnosis is difficult in cases with poor differentiation. Five-year overall survival ranges from 20% to 60% while therapeutic options are limited. This study aims to identify genetic alterations that may aid diagnosis and indicate targets for modern specific therapies.

Experimental procedures: DNA was extracted from 80 frozen or paraffin-embedded tumor tissues collected from three different hospital centers. Mutations were screened by next generation sequencing of a panel of 120 cancer-related genes using the SureSelect QXT Target Enrichment Kit for Ilumina Multiplexed Sequencing, and recurrent hits were confirmed by Sanger sequencing and by analyzing their effect on protein expression.

Results: Frequently mutated genes were TP53, KRAS, PI3K, NTRK1, ATM and BRCA1 and BRCA2. Several mutations were found exclusive to only one sinonasal tumor-type: APC and B-catenin in ITAC, EGFR exon 20 in SNSCC, IDH2 in SNUC and NF1 in MMM. With exception of ONB, a majority of cases showed a high mutational burden.

Conclusions: A number of tumor-specific mutations were identified that may be used for differential diagnosis. In addition, many gene mutations may serve as targets for specific antibody or small molecule inhibitors. Finally, the finding that sinonasal tumors carry a high mutational burden suggests that therapy with immune checkpoint inhibitors may be effective in clinical management.

#2126

Development of a precision chordoma research program.

Marie Groth,1 Jana Kress,2 Matea Hajnic,2 Joana Marinho,3 Kevin Mellert,4 Thomas F. Barth,4 Peter Möller,4 Birgit Dreier,3 Andreas Plückthun,3 Claudia Scholl,2 Stefan Fröhling1. 1 _National Center for Tumor Diseases Heidelberg and German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 2 _German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 3 _University Zurich, Zurich, Switzerland;_ 4 _University of Ulm, Ulm, Germany_.

Chordomas are malignant tumors of the axial skeleton that originate from remnants of the embryonic notochord. With an incidence of one per million per year, chordoma is a rare disease that accounts for 1-4% of all bone tumors and approximately 20% of primary spinal tumors. As first-line treatment, patients undergo surgical resection followed by adjuvant radiotherapy, whereas most chordomas are resistant to conventional cytotoxic drugs and there are no clinically validated molecular drug targets.

To overcome the lack of effective medical treatment and to better understand chordoma biology, we have established a chordoma precision research program. Here, whole-exome or genome and transcriptome sequencing are used to determine mutational landscapes, DNA copy number profiles, and transcriptome changes in chordoma cell lines and patient samples. Additionally, large-scale functional genomic screens and (phospho-)proteomic analyses have been performed in multiple chordoma cell lines to identify new chordoma-specific vulnerabilities. Based on these multilayered datasets, we selected a panel of previously unrecognized candidate chordoma driver genes, which are currently being validated as novel therapeutic targets using genetic and pharmacologic approaches.

In addition to this unbiased approach, we seek to inhibit the embryonic transcription factor brachyury, an established driver of chordomagenesis. Although brachyury represents, in principle, a promising therapeutic target, inhibition of transcription factors remains difficult. To circumvent this challenge, we are developing Designed Ankyrin Repeat Proteins (DARPins) that specifically prevent the binding of brachyury to DNA. These DARPins are not only a valuable tool for studying chordomagenesis, but also represent a new class of molecularly targeted agents that might be used for the treatment of chordoma patients.

In summary, using multi-omics data, functional genetics, and inhibition of an established chordoma driver, we are working towards a better understanding of chordoma biology and the development of new therapeutic approaches. Updated results of the precision chordoma research program will be presented.

#2127

Comprehensive analysis of mucinous ovarian carcinoma.

Kylie L. Gorringe,1 Dane Cheasley,1 Matthew Wakefield,2 Yoland Antill,1 Ian G. Campbell,1 Clare Scott,2 GAMuT Collaborators. 1 _Peter MacCallum Cancer Ctr., Melbourne, Australia;_ 2 _Walter and Eliza Hall Institute, Melbourne, Australia_.

Mucinous ovarian carcinoma (MOC) is a rare subtype of epithelial ovarian cancer that responds poorly to ovarian chemotherapies and has an unknown etiology. It is diagnostically challenging and can be confused with metastases from gastro-intestinal tract and other primaries. The GAMuT study is a multi-national effort to understand molecular drivers and cell of origin of this rare tumor, including identification of a genetic progression model and novel therapeutic options. We performed RNAseq (n=67), exome sequencing (n=61), SNP arrays (n=67) and whole genome sequencing (n=5) on MOC and precursor lesions. A subset of ~500 genes was further evaluated by targeted sequencing, including 129 MOC, 23 borderline mucinous tumors (non-invasive) and 23 extra-ovarian mucinous metastases. Immunohistochemistry data was collected for CK7, CK20, ER, PAX8, p53 and HER2 (n=162-256). Extensive pathology review was performed and associated clinical data obtained. Comparison with TCGA and other data sets showed that MOC are distinct from mucinous tumors from other organs, including colorectal, appendiceal and gastric cancers. They are genetically most similar to pancreatic adenocarcinomas, but a pancreatic origin is not supported by gene expression profiling. Our data provides clear support for a genetic progression model from benign and borderline precursors to both low- and high-grade MOC. TP53 mutations, ERBB2 amplifications and increasing copy number changes were key events associated with progression to invasive disease, including a novel amplicon on 9p13. Copy number aberration burden was significantly associated with poor survival in a multivariate analysis (p=0.01, HR 4.4, 95% CI 1.4-14). We identified several recurrent genetic events suggesting utility of an existing targeted therapy, including ERBB2 amplifications (26%), ERBB3 mutations (4%) and BRAF mutations (9%). MOC could be included in clinical trials for novel agents targeting TP53 missense mutations (46%), RNF43 mutations (12%), PIK3CA mutations (8%) and KRAS/NRAS mutations (66%). Other frequent events included CDKN2A inactivation (57%), ARID1A mutations (9%) and TP53 inactivating mutations (15%). Therapies exploiting homologous recombination deficiency (HRD) are unlikely to be useful in MOC, as only 1.5% had a strong HRD signature. Mismatch repair deficiency was very rare (<1%). MOC of any grade can derive from a primary ovarian tumor precursor, and is distinct from extra-ovarian metastases. MOC is genetically diverse and advanced disease should be assessed for targetable mutations which may provide novel therapeutic options. Treatment with platinum-based chemotherapy or PARP-inhibitors are unlikely to be effective in the majority of MOC. The size of this cohort for such a rare disease makes this study definitive.

#2128

Simple Western Size (SWS) in combination with precision cut cancer tissue slices: An excellent patient-derived platform to support drug development.

Nicole Grabinski, Kristina Bernoth, Mirja Piller, Moiken Petersen, Jana Krüger, Olivia Timm, Hartmut Juhl, Kerstin A. David. _Indivumed, Hamburg, Germany_.

The goal of personalized medicine is to provide individual patients with the most appropriate treatment. This approach strongly depends on extensive characterization of individual tumors and their sensitivity to therapeutics. We previously have shown that our drug testing platform based on Precision Cut Cancer Tissue Slices (PCCTS) is applicable to analyze individual responses of patient´s tumor tissue to defined compounds such as checkpoint modulators or bispecific antibodies. PCCTS from tumors of CRC and NSCLC cases were generated using a Krumdieck Slicer and incubated in triplicates with immunomodulatory compounds such as OKT3® (Muromonab) or Nivolumab with and without autologous PBMCs. After incubation in 24-well plates, tissue slices were snap frozen and subsequently lysed for analysis of protein expression by Simple Western™ Size (SWS) analysis. In addition to the tissue slices, immune cells were collected from each well and characterized by flow cytometry. Cytokines as an important read-out for immune cell stimulation were analyzed using the Meso Scale Discovery® technology. Combining the different technologies enables a comprehensive characterization of drug effects in individual patient samples. The SWS technology by ProteinSimple™, a highly standardized capillary western blot, using minute amounts of protein (ng), has become extremely useful for the low amount of protein generated from small, 500 µm tissue slices. We developed protocols for several signaling molecules such as PD-1, PD-L1, pAkt, Akt, pErk, Erk, and pS6RP running on a Peggy Sue™ instrument. In addition, a SWS assay for the detection of cleaved caspase-3 has been applied for the detection of apoptosis. All targets have been analyzed in duplicates within one run, a very efficient procedure for the limited amount of material available. PCCTS treated with OKT3® or Nivolumab showed significant changes in the expression level of pERK, pAKT, and pS6RP in the cases analyzed. An increase of cleaved caspase-3 was detectable after treatment compared to untreated PCCTS. These data are in accordance to the down-regulation of signaling molecules. The analysis of cytokines in the supernatants using a ten-plex panel from Meso Scale Discovery® confirmed the activation of T-cells after OKT3® treatment. In all patients a significant increase of IFN gamma, IL-2, and TNF alpha was measured. Treatment with Nivolumab resulted in an increase of IFN gamma.

This model of PCCTS represents a unique opportunity to test immunemodulatory compounds in a fully human, patient-derived model that is close to the in vivo situation. In combination with sensitive methods such as SWS a detailed characterization of compound induced molecular intracellular signaling pathways is possible and enables a better understanding of the mechanism of action in the process of compound development.

#2129

CPTAC: Biospecimen accrual for proteogenomics.

Mathangi Thiagarajan. _Leidos Biomedical Research Inc., Rockville, MD_.

The Clinical Proteomics Tumor Analysis Consortium (CPTAC) is a National Cancer Institute initiative that seeks to uncover the molecular basis of cancer using a proteogenomic approach to study prospective cancer specimens from treatment naïve patients. Leidos Biomed provides an infrastructure for supporting the collection of high quality biospecimens specific to proteogenomics and the corresponding clinical data, in addition to project and subcontract management for the program. The proportion of biospecimens so far from racial and ethnic minority patients are underrepresented, which preclude equitable research across all patient groups for cancer treatment. CPTAC program seeks diversity in the accrual cohort and an effort is ongoing to encourage the participation of tissue source sites serving racial and ethnic minorities that can contribute high quality biospecimens towards the collection. CPTAC applies the understanding of the molecular basis of cancer to identify biomarker candidates. CPTAC Phase II completed in 2016, collecting over 350 cases from breast, colon and ovarian patients. CPTAC Phase III that is in progress began to collect and analyze 200 cases from each of ten additional cancers. Analysis is close to completion for uterine corpus endometrial carcinoma, kidney clear cell renal cell carcinoma and lung adenocarcinoma while accruals are ongoing for 7 other cancer types. The study entails collection and pathology evaluation of biospecimens, high-quality clinical data and images from clinical sites from around the world. A biorepository evaluates and processes the biospecimens, sending nucleic acids to a sequencing center and tissues to proteomics groups. Data are combined and analyzed by analysis and translational centers. Genomic data are made available to the research community through the NCI Genomic Data Commons(GDC). Proteomic data are made available through CPTAC's Data Coordinating Center(DCC). Imaging data are made available through The Cancer Imaging Archive (TCIA). The program is expanding and initiating biospecimen collection for proteogenomic analysis of ten additional rare tumor types.

#2130

Development of a biomarker-driven patient selection strategy for AsiDNA treatment.

Wael Jdey,1 Donogh O'Brien,2 Richard Tripelon,1 Séverine Rochas,1 Marie Dutreix,3 Françoise Bono1. 1 _Onxeo, Paris, France;_ 2 _Donogh O'Brien Consulting, Saint Outrille, France;_ 3 _Institut Curie, Orsay, France_.

Purpose: Accurate evaluation and prediction of response to anti-cancer treatment remain a great challenge. Stratification biomarkers are of great value to identify responders or non-responders to a specific drug, or even to distinguish between early and delayed responses. In this study, we identified a gene signature to predict AsiDNA treatment efficacy in patients.

Experimental design: The first step of this study consisted on the analysis of gene expression profile in a set of 12 Breast cancer cell lines. Genes retrieved from correlation analysis between sensitivity to AsiDNA and gene expression were used to create a gene signature predicated on the upregulation of positively correlated genes and the down regulation of negatively correlated genes (the AsiDNA sensitivity signature - AsiSS) using the UCSC Xena platform for data visualization. The AsiSS was used to sort the TCGA Breast Cancer (BRCA) patient cohort, DNA repair gene expression and cell lines from the CCLE (1100 cell lines) into rank orders, in order to retrieve a gene set highly predictive of response to AsiDNA.

Results: A list of genes the most strongly correlated with survival to AsiDNA was compiled. 39 genes showed expression profiles positively correlated with survival to AsiDNA treatment (Spearman r > 0.67, P<0.005) and 35 genes showed a strong negative correlation with survival (Spearman r > -0.63, P<0.005). Based on these correlated genes, an AsiSS was created. In the BRCA cohort the top 25% AsiDNA sensitivity scoring patients were identified, and DNA repair gene expression levels of these patients were rank ordered and sorted according to AsiSS. The top 7 genes with significantly lower expression in the 25% top AsiDNA sensitive patient group were retrieved (XRCC2, MRE11A, POLQ, BRCA2, NBN, FANCA, RAD54B; 10-14 < p value < 10-9). To validate the predictive value of these genes, cell lines from the CCLE were rank ordered according to the AsiSS, and response to AsiDNA of 20 cell lines (10 predicted sensitive; 10 predicted resistant) as well as the expression level of the top 7 genes were analyzed. Importantly, cells predicted resistant show significantly higher survival (mean survival = 80%) compared to cells predicted sensitive (mean survival = 40%; p < 0.01). Among the 7 retrieved genes, gene expression analysis revealed a correlation between sensitivity to AsiDNA and the expression of BRCA2 (Pearson r: 0.75), NBN (r: 0.62), MRE11A (r: 0.6), RAD54B (r: 0.54), FANCA (r: 0.6) and XRCC2 (r: 0.55) genes (0.001< p <0.05). This set of genes could be used to stratify patients for future clinical trials involving AsiDNA.

Conclusion: Overall, our results highlight the interest of retrospective analysis based on patient databases to retrieve gene biomarkers predictive of drug outcome. As AsiDNA is being currently tested in a clinical trial, a potential exist for a rapid validation of our gene set in the aim to develop a Biomarker-driven patient selection strategy for AsiDNA treatment.

#2131

Significant combination benefit of anti-VEGFR antibody and oncogene-targeted agents in EGFR or ALK mutant NSCLC cells.

Hiromi Watanabe,1 Eiki Ichihara,2 Hiroe Kayatani,1 Hisao Higo,1 Go Makimoto,1 Hirohisa Kano,1 Kazuya Nishii,1 Naofumi Hara,1 Kiichiro Ninomiya,2 Toshio Kubo,3 Kadoaki Ohashi,2 Kammei Rai,2 Katsuyuki Hotta,4 Masahiro Tabata,3 Yoshinobu Maeda,1 Katsuyuki Kiura2. 1 _Department of Hematology, Oncology and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan;_ 2 _Department of Allergy and Respiratory Medicine, Okayama University Hospital, Japan;_ 3 _Center for Clinical Oncology, Okayama University Hospital, Japan;_ 4 _Center for Innovative Clinical Medicine, Okayama University Hospital, Japan_.

Background: Non-small cell lung cancers (NSCLCs) harboring driver oncogene such as epidermal growth factor receptor (EGFR) mutation or anaplastic lymphoma kinase (ALK) rearrangement are sensitive to the corresponding molecular targeted tyrosine kinase inhibitors (TKIs). However, acquired resistance inevitably develops, and more effective treatment strategies are needed. Anti-vascular endothelial growth factor (VEGF) antibody bevacizumab significantly prolongs progression free survival when added to first-generation EGFR TKIs in EGFR mutant NSCLC. However, it remains unknown whether anti-VEGF receptor (VEGFR) antibody enhances the efficacy of molecular targeted agents as well. In this study, we investigated combinations with an anti-VEGFR antibody and EGFR or ALK TKIs in preclinical models

Materials and Methods: We treated mice bearing subcutaneous tumor cell lines harboring EGFR mutation (PC-9, H3255) or ALK rearrangement (ABC-11, H3122) using combinations of anti-mouse VEGFR antibody DC101 and erlotinib (EGFR TKI) /alectinib (ALK TKI). Neovascularization of the tumors were determined by CD31 immunostaining. To determine the effects of VEGFR antibody on cancer cell itself, we also examined the efficacy of the combinations in vitro.

Results: Combination therapy demonstrated significantly better tumor inhibition compared to erlotinib or alectinib alone in all the examined xenograft tumors. CD31 positive blood vessel staining was significantly reduced in the tumors treated with combination therapy. Mutant EGFR transfection in HEK293T cells increased VEGF expression in the cell culture supernatant. Furthermore, MTT assay showed significantly enhanced cell growth inhibition when anti-human VEGFR antibody ramucirumab combined with erlotinib (against PC-9 or H3255 cells) or alectinib (against ABC-11 or H3122 cells) suggesting that anti-VEGFR antibody affects not only on tumor vasculature but cancer cell itself. Consistent with this finding, combination with siVEGFR and erlotinib also inhibited the cell proliferation significantly better than erlotinib alone in PC-9 cells.

Conclusion: A significant combination benefit was observed with anti-VEGFR2 antibody and EGFR or ALK TKI both in vivo and in vitro, suggesting anti-VEGFR antibody inhibits not only the blood vessels but may also exert direct anticancer effects. Combination therapy of erlotinib or alectinib and anti-VEGFR2 antibody could be a promising treatment strategy for oncogene addicted NSCLC.

#2132

Next generation sequencing for screening of clinically actionable mutations in intestinal-type sinonasal adenocarcinoma.

Cristina Riobello,1 Virginia N. Cabal,1 Paula Sánchez Fernández,2 Rocío García-Marín,1 Blanca Vivanco,2 Fernando López,2 José L Llorente,2 Mario Hermsen1. 1 _ISPA, Oviedo, Spain;_ 2 _HUCA, Oviedo, Spain_.

Introduction: Sinonasal ITAC is a rare tumor commonly related to occupational wood or leather dust exposure. Clinical management has improved,however, recurrences are frequent, and the overall 5-year survival rateremains poor (60%). Therefore, there is a clear need for new therapeutic options. The aim of this study was to identify genetic alterations as specific targets for personalized treatment.

Experimental procedures: Next Generation Sequencing using SureSelect QXT (Ilumina) was performed on a panel of 120 genes against which drugs have been approved by the FDA. Tumor and germline DNA of 25 patients was sequenced, for one case also the recurrence and metastasis. Pools were sequenced in a MiSeq system with a minumum coverage of 500X. To select somatic and pathogenic variants, the raw sequencing data were manually curated as follows: all silent mutations and variants with allele frequency >5% were discarded, only variants with tumor frequency >10 % of readings were considered; by comparing with the germline sample, only variants that appeared exclusively in the tumor sample were selected, as well as homozygous changes in the tumor that were heterozygous in the germline.

Results: Five tumors (20%) did not show any somatic alteration in the 120 genes tested.In the remaining 20 tumors, mutations were found in 33 genes. The most relevant were involved in the EGFR-PI3K-AKT-MTOR, the MAPK-ERK and the Wnt signaling pathways, indicating an important role in ITAC tumorigenesis. The most frequently affected genes were tyrosin-kinase receptors (44%), especially the ERBB family (20% of cases with mutations) and FGFR1 (20% of tumors with gene copy number amplifications). Thirty-six percent and 28% of tumors showed alterations in the MAPK-ERK and PI3K-AKT-MTOR cascades respectively. PI3K mutations were particulary frequent (7 different variants in 5 tumors.) With regard to the Wnt pathway, both APC and CTNBB1 genes appeared mutated in 3 cases. In one patient, the primary tumor, recurrence and lymph node metastasis were investigated. All somatic alterations observed in the primary tumor (mutations in ATM, BRC2, KMT2B and amplifications of MET, ATM and FGF1R) remained present in both recurrence and metastasis, excluding the possibility of a second primary tumor. Additional mutations in the recurrence (NTRK1, TSC2 and PIK3R2) were also seen in the metastasis, while no alterations occurred in the metastasis exclusively. Confirmatory studies are underway correlating the most relevant sequencing results with gene and protein expression data of the affected pathways.

Conclusions: By sequencing 120 clinically actionable genes in ITAC, several alterations were found that could be used as targets for new, personalized therapies. Examples include Dovitinib against FGFR1 amplification, antibody and small molecule inhibitors of ERBB, BRAF and MTOR, and tankyrase inhibitors targeting the Wnt pathway.

#2133

**24R,25(OH)** 2 **D** 3 **is differentially tumorigenic in ER+ and ER- breast cancer.**

Anjali Verma, David J. Cohen, Chandana Muktipaty, Thomas W. Jacobs, Jennifer Koblinski, Barbara D. Boyan, Zvi Schwartz. _Virginia Commonwealth University, Richmond, VA_.

Vitamin D3 (VD3) has long been associated with improved breast cancer prognoses. However, recent clinical trials examining the efficacy of VD3 supplementation on patient prognoses have been largely inconclusive. One reason for this may be that most clinical trials ignore the complex VD3 metabolome and focus solely on 25(OH)D3, the circulating form of VD3. Our study focuses on 24R,25(OH)2D3 (24,25D), a naturally-occurring metabolite of VD3. The aim of the present study was to examine the effect of 2425D on breast cancer tumorigenicity, and to determine if this effect is dependent on estrogen receptor expression in vitro and in vivo.In vivo, MCF7 (an estrogen receptor positive [ER+] breast cancer cell [BCC] line) or HCC38 (ER-) BCCs were implanted in adult female NOD SCID gamma IL2R mice. Mice were supplemented with 24,25D (0, 25, or 100 ng/ IP injection 3X/week). Tumor growth was monitored throughout the study. In vitro, both cells were treated with 0-100nM 24,25D for 15 min or 24h and assayed for phospholipase D (PLD) activity, proliferation (DNA synthesis), apoptosis (BAX, BCL2, p53, TUNEL), epithelial-to-mesenchymal transition (EMT) (SNAI1, MMP1, ERBB2), migration (scratch test), and metastasis (CXCR4/CXCL12, OPG/RANKL). Inhibitors to PLD, caveolin-1, and palmitoylation were used to examine the mechanism of 24,25D. Data were analyzed with one or two-way ANOVA with Tukey's post-tests.In vivo, mice with ER+ tumors given 24,25D showed decreased tumor burden, metastasis, and increased survival as compared to vehicle-treated controls. Conversely, mice with ER- tumors given 24,25D showed a dose-dependent increase in tumor burden compared to controls. In vitro, both ER+ and ER- cells showed increased proliferation at 24 hours after exposure to 24,25D for 15min but not after exposure for 24h. In ER+ cells, 2425D increased apoptosis and decreased EMT, migration, metastasis, and PLD activity; while in ER- cells, the opposite trend was observed, with 24,25D decreasing apoptosis and increasing EMT, migration, metastatic markers, and PLD activity. The proliferative and apoptotic effects of 24,25D in both cell lines were inhibited by pre-treatment with inhibitors to PLD, caveolin-1, and palmitolyation.24,25D had opposing effects on ER+ and ER- tumor burden in vivo, suggesting that 24,25D is differentially tumorigenic, and observed differences in tumorigenicity are dependent on the expression of ER. In vitro, this ER-associated differential effect was confirmed, with 24,25D decreasing tumorigenic markers in ER+ BCCs and enhancing them in ER- BCCs. Furthermore, 24,25D appears to act at the membrane through a PLD-dependent caveolae-associated mechanism using a palmitoylated transmembrane receptor(s) in both cell types. This study indicates the important role of 24,25D in breast cancer and suggests that ER-status may be an important prognostic marker to consider before prescribing vitamin D3 supplementation to breast cancer patients.

#2134

**Niraparib combined with abiraterone acetate inhibits the growth of BRCA2** wt **prostate tumors.**

Rajendra N. Damle, Rebecca Hawkins, Jennifer Hosbach, Georges Habineza Ndikuyeze, Jenny Driscoll, Natalie L. Fulton, David Derosa, Anna Hughes, Gerald Chu, Karl Calara-Nielsen, Denis Smirnov, Dong Shen, Linda A. Snyder. _Janssen Research & Development, LLC, Spring House, PA_.

Patients with metastatic prostate cancer may initially respond well to first- and second-generation androgen receptor (AR)-targeted therapies, but many will experience disease progression, indicating a need for alternative therapies. A subset of these patients bears prostate tumors that harbor deleterious mutations in DNA damage repair (DDR) pathway genes, such as BRCA2. These DDR mutations render the tumors sensitive to drugs that inhibit poly (ADP-ribose) [PAR] polymerase-1 (PARP1), inducing synthetic lethality. Studies indicate that there are interactions between AR signaling pathways and DDR pathways in prostate cancer cells, supporting the hypothesis that dual AR/PARP inhibition could inhibit prostate tumor growth more potently than single agents, regardless of the DDR status of the tumor. This hypothesis was tested using well-known human and mouse prostate tumor models that are sensitive to androgen deprivation. Genotyping of the models confirmed that none of them carried homozygous pathogenic mutations in DDR genes, including BRCA1, BRCA2, and ATM. When the cell lines were incubated in vitro with niraparib, a potent and highly selective PARP1/2 inhibitor, PAR formation was significantly inhibited, and there was induction of γH2AX in treated cells, indicating the drug induced DNA double-stranded breaks. In addition, the cytotoxicity IC50 values for niraparib were in the range of 1-10 µM, as expected for cells that are DDRwt. In vivo efficacy studies demonstrated that the VCaP and Myc-CaP tumor models were insensitive or modestly sensitive to single agent treatment with niraparib or abiraterone acetate (AA). In contrast, the combination treatment of niraparib plus AA significantly reduced tumor growth and increased survival (p<0.05) as compared with control or single agent arms. Studies are ongoing to understand the mechanisms of tumor growth control mediated by the combination of these two agents. The results support the hypothesis that prostate tumors may respond to a combination treatment with niraparib, a PARP inhibitor, and AA, an androgen synthesis inhibitor, even in the absence of DDR anomalies. A Phase 3 study is planned to evaluate this hypothesis and the safety and efficacy of this combination in humans.

#2135

Combined MERTK and ROCK1/2 inhibition as a potential therapeutic strategy for AML.

Dawn E. Barnes,1 Xiaodong Wang,2 Stephen V. Frye,2 H. Shelton Earp,3 Deborah DeRyckere,1 Douglas K. Graham1. 1 _Emory University and Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta, Atlanta, GA;_ 2 _Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC;_ 3 _University of North Carolina School of Medicine, Chapel Hill, NC_.

Acute myeloid leukemia (AML) has a 5-year overall survival rate under 26% in adults and current cytotoxic chemotherapies are toxic, can cause long-term side effects and are often contraindicated for elderly patients. Targeted therapies may have reduced toxicity compared to chemotherapy; however, resistance to single-agents often develops and combination therapies may provide more durable responses. MERTK is aberrantly expressed in >80% of AML patient samples and is a promising therapeutic target. MRX-2843 is a novel small molecule dual MERTK and FMS-like tyrosine kinase 3 (FLT3) inhibitor that is currently in clinical trials. Here, we report synergistic anti-leukemia activity mediated by MRX-2843 and rho-associated, coiled-coil-containing protein kinases 1 and 2 (ROCK1/2) inhibitors. Both MERTK and ROCK1/2 have been implicated in actin/microtubule dynamics and cell cycle progression and previous publications demonstrated induction of apoptosis in AML cells in response to RNAi-mediated MERTK or ROCK1 inhibition. ROCK1/2 inhibitors RKI-1447 or GSK269962A synergized with sub-therapeutic doses of MRX-2843 to reduce cell density as indicated by decreased Presto Blue staining in cultures of 4 of 6 AML cell lines tested and had an additive effect in the remaining 2 cell lines. Similar interactions were observed between RKI-1447 and UNC3997, a MERTK-selective TKI with 15-fold weaker FLT3 activity. Flow cytometric analysis of cells stained with popro-1-iodide and propidium iodide (PI) dyes revealed synergistic induction of cell death in the KG-1, OCI-AML5, and NB4 cell lines and an additive effect in Kasumi-1 cultures. In addition, cells that survived treatment exhibited subsequent defects in expansion, even when they were cultured without inhibitor(s), and these effects were significantly more pronounced in cells treated with the combination therapy compared to single agents. Flow cytometric analysis of PI-stained permeabilized cells revealed an increased fraction of cells in G2/M phase in cultures treated with MRX-2843. This effect was more pronounced in cultures treated with the combination therapy and was accompanied by accumulation of a population with slightly reduced DNA content relative to untreated G2 cells, suggestive of a defect in late S-phase. Thus, the combination therapy mediates anti-leukemia activity by multiple mechanisms, including abrogation of cell cycle progression and induction of cell death. In addition, a preliminary analysis of The Cancer Genome Atlas database revealed significantly poorer overall survival in patients with higher levels of ROCK1 expression (p=0.00147, n=172). Together, these data suggest that combination therapies targeting MERTK and ROCK1/2 may be particularly effective for treatment of AML and support further studies to test the effects of this novel strategy in animal models.

#2136

Doublecortin-like Kinase 1 (DCLK1) correlates with the cell proliferation in malignant pleural mesothelioma.

Akihiro Miura, Hiromasa Yamamoto, Hiroyuki Tao, Ken Suzawa, Syunsaku Miyauchi, Kota Araki, Yuta Takahashi, Eisuke Kurihara, Yusuke Ogoshi, Kazuhiko Shien, Junichi Soh, Kazunori Okabe, Shuta Tomida, Masakiyo Sakaguchi, Shinichi Toyooka. _Okayama University, Okayama, Japan_.

Background: Malignant pleural mesothelioma (MPM) is a refractory disease with poor prognosis despite multimodality therapy. Because standard therapy is unsatisfactory, it is expected to establish a novel strategy such as molecular-targeting therapy for MPM. Doublecortin-like kinase 1 (DCLK1), a microtubule-associated protein, is one of the tumor-specific stem cell marker and is revealed that the cell survival is reduced by inhibiting this protein in pancreatic and colorectal cancers. DCLK1 is known to have several isoforms named as isoform #1-#4, although the significance of them in MPM has not been clarified. We hypothesized that DCLK1 was a potential therapeutic target for MPM.<Materials and

Methods: Immunohistochemical staining was performed using surgically resected formalin-fixed paraffin-embedded tissue of MPM to evaluate the expression of DCLK1. Additionally, MPM cell lines were analyzed to characterize the protein and mRNA expression of DCLK1 by western blotting and RT-PCR. The expression of DCLK1 isoforms were selectively knocked down by small interfering RNA (siRNA) and cell viability was analyzed by colony formation and cell count assays.

Results: Immunohistochemical staining demonstrated that the expression of DCLK1 in MPM specimens was positive in 24 out of 27 samples (89%). Western blotting and RT-PCR revealed that DCLK1 was expressed in seven out of nine MPM cell lines. The results of RT-PCR revealed that isoform #2 and isoform #4 of DCLK1 were expressed in H2052, isoform #4 was expressed in H28, and isoform #2 was expressed in YUMC44 cell lines. Expressions of DCLK1 isoforms were selectively suppressed by isoform-specific siRNAs. Colony formation and cell count assays revealed that siRNA-mediated knockdown of both isoforms #2 and #4 suppressed cell growth compared to knockdown by negative control siRNA and knockdown of only isoform #2 or #4.

Conclusion: Our results demonstrate that simultaneous knockdown of both isoforms #2 and #4of DCLK1 results in the inhibition of cell growth and suggest its potential as a therapeutic target in MPM.

#2137

Aberrant nuclear-expressed SLC29A2 as a therapeutic target in hepatocellular carcinoma.

Roger Shen, Yuh-Shan Jou. _Academia Sinica, Taipei, Taiwan_.

Hepatocellular carcinoma (HCC) is the second most prevalent causes of death in cancer worldwide, and there is lack of promising therapeutic target for patients. Here, we demonstrate that SLC29A2 (Solute Ligand Carrier Family 29 Member 2), an equilibrative nucleoside transporter, is commonly amplified and upregulated in varieties of cancers including hepatocellular carcinoma (HCC). SLC29A2 expression is increased and accumulated in nucleus during HCC progression, accompanied by interacting with phospho-STAT3, and further regulated STAT3-downstream genes to promote tumorigenic and metastatic features in HCC cell lines and preclinical orthotopic mouse model. Oncogenic properties of nuclear SLC29A2 could be suppressed by disrupting the nuclear translocation with nuclear importing mutant RanQ69L, SLC29A2 membrane anchoring mutant, and treating the inhibitor of SLC29A2. Treating inhibitor reduces nuclear SLC29A2-driven oncogenic features by reducing STAT3 phosphorylation, interrupting SLC29A2/STAT3 interaction, and nuclear translocation of SLC29A2 while SLC29A2 is amplified or upregulated in HCC cells. Combined treatment of FDA-approved late stage HCC medicine sorafenib with SLC29A2 inhibitor further suppresses tumor progression and metastasis to prolong mice survival in preclinical orthotopic model. Together, our results suggest that SLC29A2 is a theranostic target, and provide molecular insights for targeting nuclear SLC29A2.

#2138

Complex copy number profiles in oligodendroglioma associate with anaplastic morphology and recurrence.

Steve Lowe, Scott Lindhorst, David Cachia, Adriana Olar. _Medical Univ. of SC Hollings Cancer Ctr., Charleston, SC_.

Oligodendroglioma (O) comprises approximately 6% of all diffuse gliomas (DG) and is associated with favorable outcome. It is characterized by the presence of 1p/19q full arm co-deletion and concurrent IDH mutations. Despite a general favorable outcome selected patients with Os experience repeated radiological and histological progression. Copy number aberrations in Os may highlight profiles that correlate with patient´s outcome.

Thirty-three Os from the files of our institution were profiled by immunohistochemistry, sequencing, and SNP-microarray. Pertinent clinical, pathological, and radiological data was collected. Descriptive statistics and survival analyses were performed.

Median age at initial diagnosis was 39 (range:19-80 years). Male to female ratio=1.2. All tumors were supratentorial with most common location the frontal lobe (21/33, 64%). Six tumors were anaplastic (WHO grade III). Nine patients experienced tumor recurrence, of which 5 experienced multiple recurrences. Only one patient died after 9.8 years from initial diagnosis. The median follow-up time = 2.5 years (range:0.01-23.6). 21/33(63%), 9/33(27%), and 3/33(0.09%) underwent subtotal, gross total resection, or biopsy respectively. 48%(16/33) patients received chemotherapy and/or radiation therapy at initial diagnosis. Most tumors (28; 85%) had additional copy number aberrations most commonly involving chromosomes 11q, 12q, 9p, 4q, 3p, 11p. Chromosomal deletions/full-arm losses were more common than gains. Diploidy/tetraploidy was detected in 2 cases and chromothripsis in 1. Anaplastic and recurrent tumors had more complex copy numbers aberrations, including numerous loci of loss of heterozygosity. Statistical significance however was not reached. Additional copy number aberrations characterize Os. Complex copy number profiles associate with aggressive morphology and recurrence.

#2139

Hippo pathway transcriptional coactivators YAP/TAZ in soft tissue and bone tumors.

Ilka Isfort,1 Sandra Elges,2 Magdalene Cyra,1 Danielle Brandes,1 Ruth Berthold,1 Marcus Renner,3 Gunhild Mechtersheimer,3 Olle Larsson,4 Sebastian Huss,2 Eva Wardelmann,2 Wolfgang Hartmann,1 Marcel Trautmann1. 1 _University Hospital Muenster, Gerhard-Domagk-Institute of Pathology, Division of Translational Pathology, Muenster, Germany;_ 2 _University Hospital Muenster, Gerhard-Domagk-Institute of Pathology, Muenster, Germany;_ 3 _Department of Pathology, University Hospital Heidelberg, Heidelberg, Germany;_ 4 _Department of Oncology-Pathology, Cancer Centre Karolinska, Karolinska University Hospital, Stockholm, Sweden_.

Introduction: Soft tissue and bone tumors represent a heterogeneous group of neoplasias characterized by a wide variety of genetic aberrations. Albeit knowledge on tumorigenesis in mesenchymal tumors is continuously increasing, specific findings on altered signal transduction pathways as a basis for molecularly targeted therapeutic strategies is still sparse. Given recent studies on aberrant activation of the Hippo pathway transcriptional coactivators YAP and TAZ in different cancer types, the aim of this study was to determine the involvement of YAP/TAZ-mediated signal transduction in soft tissue and bone tumors.

Experimental procedures: The expression levels of nuclear YAP and TAZ were analyzed by immunohistochemistry in a large cohort of 486 soft tissue and bone tumors. The comprehensive set of tissue specimens comprised 10 diagnostic categories: Angiosarcomas (AS; n=29), Ewing's sarcomas (ES; n=20), leiomyosarcomas (LMS; n=68), malignant peripheral nerve sheath tumors (MPNST; n=45), solitary fibrous tumors (SFT; n=36), synovial sarcomas (SySa; n=65), well-differentiated liposarcomas (WDLS; n=55), dedifferentiated liposarcomas (DDLS; n=74), myxoid liposarcomas (MLS; n=85), and pleomorphic liposarcomas (PLS; n=9). The biological effects of the small molecule YAP/TAZ-TEAD inhibitor verteporfin on sarcoma cell proliferation (MPNST, SySa and MLS) were monitored by immunoblotting and cell viability assays in vitro.

Results: Moderate to strong nuclear staining of YAP and TAZ was detected in 53% and 33% of soft tissue and bone tumor specimens, respectively. YAP nuclear expression was most prevalent in MPNST (58%), SySa (78%) and MLS (91%), whereas nuclear TAZ was predominately found in AS (55%), MLS (55%) and MPNST (71%). Immunoblotting confirmed the nuclear localization of YAP and TAZ in MPNST, SySa and MLS cell lines. Inhibition of the transcriptionally active YAP/TAZ-TEAD interaction employing the small molecular inhibitor verteporfin resulted in a significant suppression of sarcoma cell viability.

Conclusions: This study identifies elevated transcriptional activity of nuclear YAP/TAZ as specific liability of subgroups of soft tissue and bone tumors. We provide preclinical evidence that YAP/TAZ-mediated signal transduction represents a rational target for therapeutic intervention in sarcoma cell lines that warrants further investigation.

#2140

"3c-up" a new adult Philadelphia negative acute lymphoblastic leukemia subgroup: Novel molecular markers.

Anna Ferrari,1 Silvia Vitali,2 Valentina Robustelli,3 Andrea Ghelli Luserna di Rorà,3 Eugenio Fonzi,3 Simona Righi,4 Carmen Baldazzi,3 Michela Tebaldi,1 Samanta Salvi,1 Cristina Papayannidis,3 Giovanni Marconi,3 Mariachiara Fontana,3 Enrica Imbrogno,1 Antonella Padella,3 Giorgia Simonetti,1 Alessandra Santoro,5 Jesus María Hernández-Rivas,6 Maria Teresa Bochicchio,1 Fabiana Mammoli,1 Benedetta Giannini,1 Nicoletta Testoni,3 Daniele Calistri,1 Massimiliano Bonafè,1 Gastone Castellani,2 Elena Sabattini,4 Daniel Remondini,2 Giovanni Martinelli1. 1 _Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy;_ 2 _Bologna University, DIFA, Bologna, Italy;_ 3 _Bologna University, Bologna, Italy;_ 4 _Dipartimento di Medicina Specialistica Diagnostica e Sperimentale (DIMES), Bologna, Italy;_ 5 _A.O. Ospedali Riuniti Villa Sofia-Cervello,, Palermo, Italy;_ 6 _Universidad de Salamanca, Salamanca, Spain_.

Background: The genetically heterogeneous and poor survival group of Philadelphia negative (Ph-) B-ALL group that doesn't have the most recurrent adult rearrangements (t(9;22); t(1;19); t(4;11)) is collectively referred to as "triple negative" (Ph-/-/-). CRLF2 is frequently altered in adult B-ALL, especially in Ph-like pts (50-75% of cases). Alterations that lead, in the majority of cases, to a CRLF2 overexpression. Adult pts with CRLF2 upregulated have poor outcome and novel strategies are needed to improve it.

Aims: Understand the genomic background of all Ph-/-/- ALL and subsequently clustering Ph-/-/- considering CRLF2 overexpression event, in order to define new biomarkers in these subgroups.

Pts and Methods: Ph-/-/- pts were sequenced by WES (44pts/93 samples) and 92 B-Other pts were analyzed with NGS target seq (NTS) to validate the 8 most mutated genes (Fig1A). GEP were performed on 55 Ph-/-/-, 29 B-ALL Ph+ and on 7 donors. We cluster triple negative GEP data with our validated pipeline, based on CRLF2 upregulation and in a top ten-gene list. Ph-/-/- ALL samples were then characterized for the presence of gene fusions, Copy Number Alterations (CNAs) and mutations using different approaches (Pancancer and/or RNASeq; dMLPA-MRC-Holland; SNP Array; 454 Junior and PCR).

Results: WES analysis identified some recurrent mutated genes (NRAS, PAX5, KRAS, PTPN11, EP400, JAK2, TP53, CREBBP) previously reported to be involved in B-ALL, confirming the pivotal roles of these gene in ALL. For the first time we described a little known gene PKHD1L1 as highly mutated (7.2%). TP53 was the most mutated gene (Fig1A) and that between these gene is the only one associated to a worst OS (p=0.004). Combining our new Ph-/-/- GEP clustering, WES, NTS, Fusion and CNA results we identify a defined 2-clusters-subdivision (Gr1 and Gr2). The Gr2 (14.1% of all B-ALL) is characterized by CTGF, CRLF2 and CD200 (Gr2=3C-up; Fig 1B) co-overexpression. The Gr2 GEP is similar to Ph+ one (Fig1C). Gr1 represents 46.9% of all B-ALL. Fusion, CNA and mutational screening done, detected that 3C-Up group has a higher frequency of Ph-like associated lesions (primarily CRLF2, JAK2, IL7R mut or del), that mainly affect JAK-STAT pathway. Also IKZF1 and EBF1 deletions are significantly associated to Gr2 (p=0.003; p=0.016). RAS pathway genes are highly affected in Gr1. We also validated not previously described fusions. Notably p53 pathway is enriched in both groups but with different deregulated genes: CHEK2 is upreg in the group1 and CDK6 in the Gr2. Preliminary data seems to confirm an higher effect on cell viability of a TKI on Gr2 primary cell pt (vs Gr1 pt).

Conclusions: we identified a new signature, related to CRLF2 high expression, to classify Ph-/-/- ALL B. This new subclassification identifies new potential therapeutic targets with available drugs (α-CTGF, α-CD200, CDK2, CHK2 and CDK6 inhibitors; TKIs already effective on Ph+ and Ph-like) to test.

#2141

Mechanistic studies of altertoxin II: A fungal metabolite with selective activity against Ewing sarcoma.

Andrew J. Robles,1 Wentao Dai,2 Robert H. Cichewicz,2 Susan L. Mooberry,1 Peter J. Houghton1. 1 _The University of Texas Health Science Center at San Antonio, San Antonio, TX;_ 2 _University of Oklahoma, Norman, OK_.

Ewing sarcomas (ES) are bone and soft tissue tumors that affect children and adolescents. These cancers are driven by chromosomal translocations that most frequently result in expression of the abnormal EWSR1-FLI1 fusion protein, a transcriptional activator and modulator of RNA splicing, or similar fusion proteins. Despite significant advances in therapies for pediatric hematological malignancies over the past 30 years, there has been less progress in the development of curative therapies for advanced or metastatic pediatric solid tumors. Approximately 35% of ES are resistant to standard-of-care chemotherapeutic agents, and nearly 70% of ES patients with metastatic disease at the time of diagnosis die within 5 years. Therefore, there is a need to develop new, targeted therapies for ES that improve patient survival and decrease toxicity. We performed high-content phenotypic screens of natural product libraries to identify compounds with selective actions against pediatric solid tumor types and found that the fungal metabolite altertoxin II (ATXII) has highly selective cytotoxic activity against 6 ES cells lines compared to a panel of other pediatric and adult cancer cell lines. On average, the IC50 of ATXII is 94-fold higher (range 16- to 400-fold) in rhabdomyosarcoma (RMS) cells than in ES cells, indicating high selectivity for ES. Mechanistic studies were conducted to determine this compound's mechanisms of actions in ES cells, with the goal of identifying new molecular targets for the treatment of ES. We found that 100 nM ATXII, a concentration that has no effect on RMS cell viability, induces cell cycle accumulation in ES cells in the S and G2/M phases, and rapid phosphorylation of histone H2A.X (gamma-H2A.X) and checkpoint kinase 1, suggesting ATXII selectively induces cell cycle arrest and DNA damage in ES cells. To determine if ES sensitivity to ATXII is a dominant or recessive phenotype, we hybridized sensitive EW8 cells (ES) with resistant Rh30 cells (RMS) and determined the sensitivity of these hybrids to ATXII. Five single-cell subclones derived from these hybrids were resistant to ATXII, suggesting that sensitivity to ATXII is a recessive phenotype in ES cells. Ongoing studies are aimed at determining the cellular uptake and binding kinetics of radiolabeled ATXII and identifying genes that mediate sensitivity and resistance to this compound by performing a genome-wide CRISPR/Cas9 knockout screen.

#2142

CYP3A5 regulation of androgen receptor signaling: Relevance in African American prostate cancer patients and its development as novel target.

Priyatham Gorjala, Oscar B. Goodman, Ranjana Mitra. _Roseman Univ. of Health Sciences, Las Vegas, NV_.

Background: Previously we demonstrated that CYP3A5 positively regulates androgen receptor (AR) signaling and growth of prostate cancer (PC) cells. CYP3A5 inhibition decreases nuclear AR localization and downregulates AR downstream signaling (e.g. PSA and TMPRSS2), resulting in decreased PC cell growth both with and without dihydrotestosterone (DHT) induction. Our current work focusses on the effect of CYP3A5 modulation on AR signaling and therapeutic resistance, mostly in African Americans (AA) widely carrying (73%) wild type CYP3A5 (*1/*1). AAs and have higher incidence of PC and double the mortality rate compared to Non-Hispanic White Americans (NHWA) carrying mutated CY3A5 gene (*3/3, 95%) and expressing truncated non-functional CYP3A5 protein.

Methods: Immunoprecipitation was used to identify binding partners of AR. q-RT-PCR based profiler assays was used to detect changes in AR downstream signaling and effect on genes responsible for therapeutic resistance after CYP3A5 inhibition. Effect of CYP3A5 on AR nuclear translocation and induction of synthetic lethality in combination with PARP inhibitors were evaluated using cell fractionation and immunocytochemistry studies.

Results: Co-immunoprecipitation of flag-tagged CYP3A5 transfected LNCaP cell extracts showed that CYP3A5 was in a complex with HSP90, AR, HSP70 and HSP40, also confirmed with reverse immunoprecipitation using AR/ HSP90 antibodies and agarose A/G beads. AR regulated genes (SCL45A3, FKBP5, MYC, ELL2, and MT2A) were downregulated with CYP3A5 siRNA treatment in MDA PCa 2b cells (*1/*3, AA origin) in a qPCR-based profiler assay with high fold difference and a P value of ≤0.005.

PC patients are often prescribed medications for comorbidities, many of which are CYP3A modulators. Our results indicate that CYP inhibitors (amiodarone, ritonavir, fluoxetine etc.) decrease AR nuclear translocation and inducers (phenytoin, rifampicin, pioglitazone and hyperforin) increase AR nuclear translocation and activation.

CYP3A5 siRNA treatment downregulates genes involved in cancer drug therapeutic resistance (TOP2A, BRCA1/2, CCNE1, CDK2/4, DHFR, MVP, MYC, RARB and HPRT1, P ≤ 0.005 ). Loss of TOP2A, BRCA2 and BRCA1is known to impair homologous recombination (HR). In combination with topoisomerase inhibitor etoposide, CYP3A5 siRNA in PC cells increases number of gamma-H2AX foci indicating synthetic lethality.

Conclusion: A-Developing CYP3A5-AR-HSP90 interface as a target may provide a novel strategy to block PC growth. B-CYP3A5 modulation of AR signaling can affect the efficacy of androgen deprivation therapy specifically AA patients carrying wild type CYP3A5. C-Since CYP3A5 inhibition, downregulates DNA damage repair genes and impairs HR, CYP3A5 inhibition in combination with PARP inhibitors can be a novel strategy to induce synthetic lethality in PC patients.

#2143

Identification of new molecular liabilities of a subset of triple-negative breast cancers through the investigation of englerin A.

Corena V. Grant,1 Chase M. Carver,1 Shayne D. Hastings,1 April L. Risinger,1 John A. Beutler,2 Susan L. Mooberry1. 1 _University of Texas Health Science Center at San Antonio, San Antonio, TX;_ 2 _National Cancer Institute, Frederick, MD_.

Identification of new molecular liabilities of a subset of triple-negative breast cancers through the investigation of englerin A

There remains a need to identify targeted therapies for triple-negative breast cancers (TNBCs) but a major challenge has been the heterogeneity of these cancers. We initiated a screen to discover compounds that are selectively cytotoxic to cells representing distinct molecular subtypes of TNBC. The overall goal is to identify compounds with selective actions and new molecular liabilities for subtypes of TNBC. Englerin A was identified by cytotoxicity assay as a compound with a greater than 1,500-fold selectivity for BT-549 cells and 300-fold for Hs578T TNBC cells as compared to other TNBC cell lines. Interestingly, the treatment of Hs578T cells with englerin A produced a biphasic concentration response curve, which is unique among the TNBC cell lines but is seen in A-498 renal carcinoma cells where englerin A sensitivity was first noted.1 In renal cell carcinoma cell lines one strongly supported mechanism of action of englerin A is via activation of TRPC1/4/5 non-selective cation channels.2-4 In our assays, the TRPC1/4/5 antagonist Pico1455 decreases the potency of englerin A in BT-549 and Hs578T cells, but not more resistant TNBC cells, suggesting that the TRPC1/4/5 agonist activity of englerin A is likely responsible for the selective effects in the sensitive cell lines. BT-549 and Hs578T cells were found to have significantly higher expression of TRCP1 and TRPC4 subunit mRNA as compared to englerin A resistant cells. Furthermore, knockdown of TRPC4 expression in BT-549 cells decreased englerin A potency. Consistent with its function as an agonist of the TRPC1/4/5 cation channel, we found that englerin A caused a concentration-dependent increase in intracellular Ca2+ in BT-549 cells within 30 seconds of exposure, and this effect was inhibited by pre-treatment with Pico145. These studies led to the hypothesis that TNBC cells expressing high levels of TRPC4 might also be more sensitive to clinically approved classes of drugs that increase intracellular cation concentrations. Results show that BT-549 and Hs578T cells are more sensitive to digoxin than other TNBC cells. Overall, these studies suggest that a subgroup of TNBCs may be susceptible to treatments with selective targets of cation influx. Ongoing work is aimed at evaluating the sensitivity of these cell lines to other drugs that disrupt intracellular cation levels.

1. Ratnayake, R. et al. (2009).

2. Ludlow, M. J. et al. (2016).

3. Akbulut, Y. et al. (2015).

4. Carson, C. et al. (2015).

5. Rubaiy, H. N. et al. (2017).

#2144

**Mutations in** ERBB4 **may account for clinical resistance of melanomas to inhibitors of the RAS/RAF/MEK/MAPK pathway.**

Richard Lee Cullum, Taraswi Ghosh, Lauren Lucas, Damien Waits, David Riese. _Auburn Univ., Auburn, AL_.

Introduction: Despite advancements made in the treatment of metastatic melanoma, the 5-year survival rate remains about 20%. The high mortality rate is thought to be due to there being no other clinically actionable targets other than those that have been identified in the RAS/RAF/MEK/MAPK pathway. Our analyses of The Cancer Genome Atlas (TCGA) Skin Cutaneous Melanoma (SKCM) data set revealed that 15% of the cases harbor at least one nonsynonymous missense mutation in the ERBB4 gene. This mutation incidence is significantly higher than that of the other ERBB genes in the same data set. Additionally, 91% of cases that harbor a nonsynonymous missense ERBB4 mutation also harbor a known melanoma driver mutation in the RAS/RAF/MEK/MAPK pathway suggesting interplay between ErbB4 signaling and the RAS/RAF/MEK/MAPK pathway. However, unlike the validated BRAF melanoma oncogene, ERBB4 mutations in melanoma are extremely heterogenous with 76 unique ERBB4 nonsynonymous missense mutations. None of which have incidence rates of greater than 2%. Furthermore, some of these ERBB4 mutations have already been shown to be necessary for the proliferation of some human melanoma cell lines, demonstrating a need to determine which ERBB4 mutations contribute to melanoma tumorigenesis and clinical resistance.

Experimental Procedures/Summary of Data: Consequently, various in silico approaches were used to prioritize ERBB4 mutations in melanoma. We found that many TCGA-SKCM ERBB4 mutations affect highly conserved regions. Indeed, 57 ERBB4 mutations affect a residue that is conserved in at least one other ErbB receptor. Moreover, 7 ERBB4 mutations affect a residue that is conserved in another ErbB receptor and is the location of a gain-of-function mutations in the other ERBB gene. Fifteen (15) of the putative ERBB4 melanoma driver mutations are found in other tumor types, and 3 transform the growth of fibroblasts and exhibit ligand-independent signaling. And, finally, we found that the combination of the ERBB4 E542K and E872K mutations and the E452K mutation alone are necessary for the proliferation of some human melanoma cell lines.

Conclusions: Unlike genes such as BRAF or NRAS, which contain a single or a small number of "hot spot(s)" for driver mutations, the ERBB4 gene appears to harbor numerous mutations that are likely to function as drivers of melanoma tumorigenesis. Whereas some of the ERBB4 mutations found in melanoma have been identified as bona fide melanoma drivers, most remain uncharacterized. The results of these analyses indicate that many of these uncharacterized mutations are likely to contribute to the malignant phenotype of melanoma. Therefore, there remains a need to distinguish which ERBB4 mutations function as drivers and can serve as biomarkers for response to therapies that disrupt ErbB4 signaling.

#2145

Detect early tumor relapse in high-risk breast cancer.

Amy H. Tang,1 Lauren L. Siewertsz van Reesema,1 Vasilena P. Zheleva,1 Janet S. Winston,2 Rick J. Jansen,3 Emanuel F. Petricoin,4 Matthew P. Goetz,5 Matthew P. Goetz,5 Harry D. Bear,6 Richard A. Hoefer7. 1 _Eastern Virginia Medical School, Norfolk, VA;_ 2 _Sentara Norfolk General Hospital, Norfolk, VA;_ 3 _North Dakota State University, Fargo, ND;_ 4 _George Mason University, Manassas, VA;_ 5 _Mayo Clinic, Rochester, MN;_ 6 _Virginia Commonwealth University, Richmond, VA;_ 7 _Sentara Hospital Systems, Newport News, VA_.

Chemotherapy resistant breast cancer is a major health challenge, resulting in high relapse rates and poor survival. Neoadjuvant chemotherapy (NACT) is a standard treatment for women with high-risk TNBC, HER2+, and locally advanced ER+ breast cancer. A completed course of NACT results in two possible outcomes: pathologic complete response (pCR) or residual disease. While pCR is a reliable clinical prognostic biomarker associated with excellent outcomes and prolonged survival, especially for TNBC and HER2+ cancers, patients with residual disease have a higher risk of recurrence. Patients with residual disease can be further stratified clinically using the Residual Cancer Burden (RCB) classification; however, patients with similar RCB classes may experience dramatically different clinical outcomes. Thus, additional precision biomarkers to stratify patients with residual disease after NACT are needed to identify patients at highest risk of recurrence, and to develop new therapeutic strategies to eradicate multidrug-resistant tumors.

Supported by strong evidence in developmental, evolutionary and cancer biology, we found that K-RAS-SIAH pathway activation is a major tumor driver, and SIAH represents a key tumor vulnerability in breast cancer. Normal K-RAS/SIAH signaling pathway activation is indispensable for proper cell-cell communication, cell proliferation and tissue homeostasis in multicellular organisms. However, abnormal K-RAS/SIAH pathway activation is highly prevalent in high-risk and locally advanced breast cancer, and may confer chemo-resistance to these high-risk mammary tumors. Based on its evolutionary conservation and significance as the most downstream signaling module indispensable for K-RAS signal transduction, SIAHON/OFF expression is a reliable readout of K-RAS/EGFR/HER2 pathway activation/inactivation. We showed that SIAHON/OFF expression is a binary code in residual mammary tumors that can be used to stratify patients, augment RCB classification, forecast tumor relapse, and predict patient survival after 1st line NACT in a pilot retrospective study.

Multidrug-resistant high-grade breast cancer is a genetically diverse, highly heterogeneous disease that challenges our ability to individualize and optimize precision therapy. Persistent K-RAS-SIAH-EGFR pathway activation endows TNBC with therapy resistance, and increases the risk of metastasis and early relapse. As such, we developed a K-RAS/SIAH-centered biomarker discovery program and a new anti-SIAH research initiative with the intended goal of designing novel and potentially life-saving anti-K-RAS targeted strategies to control and eradicate multidrug-resistant and intractable mammary tumors.

#2146

High expression of Olfactomedin-4 correlates with chemoresistance and poor prognosis for pancreatic cancer.

Ryotaro Ohkuma,1 Erica Yada,2 Yutaro Kubota,1 Kazuyuki Hamada,1 Hiroo Ishida,1 Yuya Hirasawa,1 Hirotsugu Ariizumi,1 Etsuko Satoh,1 Junji Tsurutani,1 Kiyoshi Yoshimura,1 Tetsuro Sasada,2 Takeshi Aoki,1 Masahiko Murakami,1 Tomoko Norose,1 Nobuyuki Ohike,1 Masafumi Takimoto,1 Takuya Tsunoda,1 Satoshi Wada1. 1 _Showa University, Shinagawa, Tokyo, Japan;_ 2 _Kanagawa Cancer Center Research Institute, Yokohama, Kanagawa, Japan_.

Background: Pancreatic cancer has an extremely poor prognosis and novel therapeutic strategies are desperately needed. It is considered that suitable molecules as therapeutic targets are resistant to chemotherapy and correlate with poor prognosis. In this experiment, we aimed to identify the optimal therapeutic targets in pancreatic cancer.

Materials and Methods: We established 10 lines of patient derived xenografts (PDXs) for pancreatic cancer. Next generation sequencer (NGS) analysis was performed for PDXs which were administered chemotherapy and untreated PDXs as a control. Furthermore, we performed immunohistochemistry (IHC) analysis for the protein expression of identified molecules from PDXs and post-operative pathologic specimens from 80 patients with pancreatic cancer. When chemotherapy was added on tumor cell lines forcibly expressing the molecule, antitumor effect was compared with the control.

Results: As a result of NGS analysis, we identified Olfactomedin-4 (OLFM4) molecule which was commonly shown high expression in chemotherapy administered PDXs. In IHC analysis, OLFM4 molecule expression was shown high expression with chemotherapy administered PDXs compared to untreated PDXs. For the analysis to use tumor cell lines forcedly expressing OLFM4 in vitro, the reduction rate of the tumor cell numbers was significantly lower than control vector, even if the concentration of the chemotherapy agent was increased. In a study using tissue specimens of 80 pancreatic cancer patients, Kaplan-Meier analysis showed that low expression of OLFM4 had a better survival rate than patients with high expression (p=0.0926, log-rank test). In multivariate analysis, it was shown that the high expression of OLFM4 was an independent poor prognostic factor with a statistically significant difference (p=0.044, Cox proportional hazards).

Conclusions: It was demonstrated that high expression of OLFM4 was involved in chemoresistance and was an independent prognostic factor for pancreatic cancer. This molecule might be a candidate of therapeutic target in pancreatic cancer.

#2147

Galectin-1: A marker for claudin-low breast cancer.

Kassondra Balestrieri, Moses McDaniel, Christiana Shoopman, Nasreen Vohra, Kathryn Verbanac. _East Carolina Univ. School of Medicine, Greenville, NC_.

Metastasis and mortality among patients with triple negative breast cancer (TNBC) of the claudin-low subtype remain high because these tumors are clinically aggressive, chemoresistant, and lack targeted therapies. We have previously used proteomic approaches to analyze orthotopic and metastatic tumors from the syngeneic murine T11 tumor model, which displays gene expression profiles that mirror human claudin-low TNBC. These studies identified Galectin-1 (Gal-1), an N-acetyllactosamine-binding protein, as an overexpressed protein in cultured T11 cells, primary tumors, and lung metastases compared to normal lung and mammary fat pad tissue from healthy control mice. These results were confirmed by Western immunoblot and immunohistochemistry and were further supported by analysis of murine tumor microarray data sets, which revealed Gal-1 overexpression in the claudin-low subtype compared to other breast cancer subtypes. Here we extend our murine studies to investigate Gal-1 expression in breast cancer patients, with specific inclusion of claudin-low tumors. To compare differential Gal-1 expression across breast cancer subtypes, we initially analyzed published human microarray data sets from normal breast and primary breast tumors (total n=423, of which 51 are the claudin-low subtype): the highest Gal-1 expression was observed in claudin-low breast cancer. In order to validate these findings, we are examining breast cancer specimens from a unique cohort of ethnically diverse patients from rural Eastern North Carolina with a median follow-up of over 8 years. RNASeq data from a subset of this cohort (n=121) was used to assign subtypes (using PAM50 and the claudin-low classifier). Further studies are in progress to compare Gal-1 expression between subtypes and correlate with clinicopathologic factors and outcome. Immunohistochemical Gal-1 staining of primary tumor tissue blocks is underway to confirm expression in each subtype at the protein level. Immunohistochemistry will also define Gal-1 expression in the cell types comprising the heterogeneous tumor microenvironment (tumor, stromal, immune cells) and its subcellular localization (nuclear, cytoplasmic, membrane-associated). Through this ongoing work evaluating Gal-1 across breast cancer subtypes, we will help corroborate initial findings from the T11 claudin-low mouse model and yield further insights into the role of Gal-1 in breast cancer progression.

#2148

UHRF1 overexpression is a significant driver of tumor progression in RB-mediated cancers.

Claudia A. Benavente, Stephanie C. Wu, Loredana Zocchi. _University of California, Irvine, Irvine, CA_.

UHRF1 is an essential epigenetic reader and writer overexpressed in multiple human cancers, including retinoblastoma and osteosarcoma. For these tumors, metastasis remains the most significant fatal complication for treatment. Genetic alterations at the RB transcriptional corepressor 1 (RB1) gene are associated with initiation, increased mortality, metastasis, and poor response to chemotherapy. However, the precise mechanism(s) through which this occurs remains to be elucidated. Studies in our laboratory identified UHRF1 (Ubiquitin-like, containing PHD and RING Finger domains 1) as a gene that is upregulated and its protein overexpressed in retinoblastoma and osteosarcoma. UHRF1 is a multifunctional protein involved in epigenetic regulation and has been shown to have protein-protein interactions with RB. Further, the RB/E2F pathway directly regulates UHRF1 expression. Using genetic engineered mouse models, we found that UHRF1 is essential for retinoblastoma formation. We have also studied the role of UHRF1 in other RB-dependent malignancies, including osteosarcoma. Our results indicate that UHRF1 is overexpressed in most human osteosarcomas. Loss-of-function studies using UHRF1 shRNA knockdowns and CRISPR/Cas9 knockouts indicate that reduction of UHRF1 levels in human osteosarcoma cells results in decreased proliferation, migration and invasion. Strikingly, induction of UHRF1 overexpression in normal mesenchymal stem cells induces increased cell migration and invasion. We evaluated the role of UHRF1 in osteosarcoma development by knocking-out Uhrf1 in genetically engineered mouse models of osteosarcoma (p53 cKO: Osx-cre p53lox/lox and p53/Rb1 DKO: Osx-cre p53lox/lox Rb1lox/lox), to create p53/Uhrf1 DKO (Osx-cre p53lox/lox Uhrf1lox/lox) and p53/Rb1/Uhrf1 TKO (Osx-cre p53lox/lox Rb1lox/lox Uhrf1lox/lox) mice. Uhrf1 abrogation leads to a significant increase in survival in p53/Rb1/Uhrf1 TKO mice compared to their p53/Rb1 DKO littermate controls. While 100% of p53/Rb1 DKO mice develop tumors, 12.5% of p53/Rb1/Uhrf1 TKO mice did not, confirmed using microCT/PET imaging. Further, p53/Rb1/Uhrf1 TKO showed a significant reduction in the rate and number of lung metastasic nodules compared to p53/Rb1 DKO. Even though UHRF1 is overexpressed in osteosarcomas with wild type Rb1 (p53 cKO tumors), we found no significant difference in the median survival of p53/Uhrf1 DKO compared to p53 cKO littermate controls. These results suggest that UHRF1 overexpression is a significant contributor of the poor prognosis associated with loss of Rb1. Altogether, our data indicates that UHRF1 overexpression plays a significant role in osteosarcoma proliferation, migration, invasion, and metastasis, which translates in reduced survival mice with osteosarcoma. Our studies suggest that UHRF1 may play a role in carcinogenesis and is a putative biomarker and anticancer therapeutic target.

#2149

Association of tumor fusion burden with immune presence and androgen sensitivity in prostate cancer.

Marie Wagle,1 Kobe Yuen,1 Edward E. Kadel,1 Thomas Holcomb,1 Shrividhya Srinivasan,1 Joseph Castillo,1 Dan Halligan,2 Adrian Carr,2 Max Bylesjo,2 Julian Augley,2 Simone Daminelli,2 Mark Kockx,3 Yannick Waumans,3 Jennifer Giltnane,1 Zineb Mounir1. 1 _Genentech, Inc., South San francisco, CA;_ 2 _Fios Genomics, United Kingdom;_ 3 _Histogenex, Belgium_.

Introduction: Prostate cancer (PrCa) is the second leading cause of cancer death in men. Despite having a lower tumor mutation burden (TMB) than most tumor types (1), multiple gene fusions such as TMPRSS2-ERG have been characterized in prostate cancer and have been linked to aggressive disease (1). Individual tumor samples may contain multiple fusions and it is unknown whether these fusions could increase tumor immunogenicity. Here we investigated the effects of fusion burden on the expression of key molecular and immune effectors in prostate cancer specimens representing different stages of disease progression and androgen sensitivity (hormone sensitive vs metastatic castrate resistant prostate cancer (mCRPC)).

Methods: 187 prostate samples taken from different stages of PrCa including Early/intermediate (n:30), late (n:112) and metastatic disease (n:9) were interrogated by RNASeq and assessed for mutation burden (mutations/MB of the genome) and fusion burden (number of fusions/10,000 genes profiled in each sample). To characterize the immune microenvironment, stromal TILS, % immune infiltrate, CD8+ T-cells, and PDL-1 expression were assessed by immunohistochemistry (IHC). Key molecular and immune gene signatures derived from the expression data were clustered according to disease stage, mutation or fusion burden, and hormone sensitive vs mCRPC disease (determined by mCRPC score from (2)).

Results and Conclusion: TMB was very low across all samples analyzed (0.01-1.2 mutations per MB) while fusion burden ranged from 0 to 34 fusions/10,000 genes (up to 70 fusion(s) per sample) and was inversely correlated to TMB and not associated with disease stage. High fusion burden across samples correlated with high cell cycle progression and AR signaling (P<0.0001), ERG and ETS transcriptional activity (P<0.005) along with a modest increase in stromal TILs, % immune infiltrate and PDL-1 expression on immune cells. Fusion burden also correlated with immune signatures representing activation of M1 macrophages, checkpoint inhibitors, and T-cell activity (IFNγ-induced and T-effector signatures) (P<0.0001) whilst inversely correlating with an immune suppressor signature (P<0.01). High fusion burden also correlated with a high mCRPC score (P<0.0001), representing the most aggressive disease. Samples with a high mCRPC score also had gene signature scores suggesting high AR signaling, PI3K signaling, cell cycle progression and class I antigen presentation, and low expression of neuroendocrine tumor markers, NK cell and immune suppressor signatures. Our data suggest that high fusion burden may be associated more closely with immunogenicity and disease prognosis than TMB in PrCa and that tumors with high fusion burden could be potential candidates for immunotherapy. References(1) Chalmers et al. (2017), Genome Medicine Apr 19: 9(1):34 9:34.(2) Sharma N.L et al. (2013), Cancer Cell: Jan 14: 23(1):35-47.

### Novel Screening and Delivery Technologies

#2150

engEx: A novel exosome engineering platform enabling targeted transfer of pharmacological molecules.

Kevin Dooley, Ke Xu, Sonya Haupt, Nuruddeen Lewis, Rane Harrison, Shelly Martin, Christine McCoy, Chang Ling Sia, Su Chul Jang, Katherine Kirwin, Russell McConnell, Bryan Choi, Adam T. Boutin, Damian Houde, Jorge Sanchez-Salazar, Agata Villiger-Oberbek, Kyriakos D. Economides, John D. Kulman, Sriram Sathyanarayanan. _Codiak BioSciences, Cambridge, MA_.

Exosomes are natural and abundant nanoscale vesicles for intercellular communication, capable of transferring biological instructions between neighboring and distant cell types. Translational research efforts have focused on exploiting this communication mechanism to deliver exogenous pharmacologic payloads to treat a variety of diseases including cancer. Functionalization of the exosome surface with proteins and peptides is an important strategy to maximize the potential of exosomes as therapeutics. Comparative proteomic analysis (LC/MS) of stringently purified exosomes led to the identification of several highly enriched and unique proteins, including a transmembrane glycoprotein (Protein X, PrX), belonging to the immunoglobulin superfamily. Stable expression of PrX in a producer cell line resulted in a 200-fold increase of PrX on the secreted exosomes. Protein X was extensively characterized and the minimum structural requirements for exosome enrichment were determined. With our engExTM platform, we developed precision engineered exosome therapeutics using PrX as a scaffold to enable high-density exosome surface display of an array of structurally and biologically diverse proteins, including enzymes, antibodies, type I cytokines, and TNF superfamily members. These proteins were genetically fused to PrX and overexpressed in a producer cell. Significantly higher transgene expression on secreted exosomes was achieved compared to conventional scaffolds, including the tetraspanins CD9/CD63/CD81 and LAMP2B. Oligomerization of PrX coupled with avidity effects inherent in exosome surface display resulted in a clear activity advantage compared to free protein. Protein X-mediated display of CD40L on exosomes resulted in a 20-fold potency increase in B cell activation over recombinant CD40L. Furthermore, expression of CD40L redirected exosome uptake from phagocytic antigen presenting cells (APCs) to B cells, demonstrating exosome surface modifications can alter cellular tropism. We also evaluated the functionality of IL-12 tethered to the exosome surface and demonstrated superior tumor retention compared to free cytokine, resulting in robust anti-tumor activity in anti-PD-1 refractory B16F10 tumor models. These results demonstrate the potential of the engExTM platform to generate novel exosome therapeutics.

#2151

Monitoring AMP Concentration,the universal product of diverse enzymatic reactions.

Said A. Goueli, Kevin Hsiao, Subhanjan Mondal. _Promega Corp., Madison, WI_.

Many enzymatic reactions use Adenosine triphosphate (ATP) as a substrate and produce adenosine monophosphate (AMP) as the common product of these reactions. AMP also functions as a modulator of many enzymes and a key cellular metabolite regulating energy homeostasis and signal transduction. For example, by activating AMP-Dependent protein kinase (AMPK), it maintains the energy charge ratio at optimal level during metabolic activities; and its concentration is altered during disregulated disease conditions. Thus, monitoring the activities of these enzymes is a primary goal for developing modulators for these enzymes. Here, we demonstrate the versatility of an enzyme coupled assay that quantifies the amount of AMP produced by any enzymatic reactions regardless of its substrates in a homogenous and high through-put formatted form. We successfully implemented it to enzyme reactions that use ATP as a substrate (aminoacyl tRNA synthetase and DNA ligase) by first depleting the remaining residual ATP substrate and converting AMP produced into ATP, the later can be quantified using luciferase/luciferin and generating light. We also tested this assay to measure direct activities of AMP generating enzymes that do not require ATP as substrate, including phosphodiesterases (cAMP) and E.coli DNA ligases (NAD+). In a further elaboration of the AMP-Glo platform, we coupled it to E.coli DNA ligase, enabling measurement of NAD+ and enzymes that use NAD+ like mono- and poly-ADP-ribosyltransferases. Sulfotransferases use 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as the universal sulfo-group donor and phosphoadenosine-5'-phosphate (PAP) is the universal product. PAP can be quantified by converting PAP to AMP by a Golgi-resident PAP-specific phosphatase, IMPAD1. By coupling IMPAD1 to the AMP-Glo system we can measure the activities of sulfotransferases. In summary, we have developed a very strong platform to monitor AMP concentrations that is applicable to diverse enzymatic reactions.

#2152

A novel glycan targeting cancer therapy using lectin modified liposome.

Sota Kimura,1 Tatsuya Oda,1 Osamu Shimomura,1 Ko Kurimori,1 Tomoaki Furuta,1 Yoshihiro Miyazaki,1 Yang Yu,1 Jun Hirabayashi,2 Hiroaki Tateno2. 1 _Department of Surgery, Clinical Sciences, Faculty of Medicine, University of Tsukuba, Ibaraki, Japan;_ 2 _Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Ibaraki, Japan_.

Introduction: Since the most outer layer of cancer cells is covered with various glycans, it would be one of the most effective targets in cancer therapy. We had previously discovered the specific fucosylated glycan in pancreatic cancer and a lectin, a protein specifically binds to glycan, named "rBC2LCN" could well worked as targeting bullet (Shimomura O, Oda T, et al: Mol Cancer Ther 2018). In this study, we applied this lectin as booster bullet of anticancer nanoparticles. We originally developed a novel glycan targeting anti-pancreatic cancer nanoparticles, namely Lectin-Dox, by modifying the surface of doxorubicin encapsulating PEGylated liposome (Dox) by rBC2LCN and verified the booster anti-tumor effects of the novel agent.

Material and Methods: Lectin-Dox was prepared by the post insertion method (Ishida T, et al: FEBS Lett. 1999) from ready-made Dox using lipid linker. Experiment 1: Lectin-Dox and Dox were applied to two pancreatic cancer cell lines (Capan-1 and SUIT-2) which possess different binding affinity to rBC2LCN, thereafter the signal intensities were compared by fluorescent microscopy and flow cytometry to evaluate cellular bindings and uptakes. Experiment 2: To compare cytotoxicity in vitro of each solution, the 50% inhibitory concentration (IC50) against each cell lines were calculated. Experiment 3: Anti-tumor effects in vivo were evaluated using subcutaneous tumor bearing mouse derived from each cell lines. The solutions was injected via tail vein twice per week for 3 consecutive weeks, then tumor volumes and weights were compared.

Results: Experiment 1: Cellular bindings and uptakes were both significantly stronger in Lectin-Dox group against Capan-1 (P<0.05 and P<0.05, respectively), whereas no significant differences were observed in SUIT-2 (P=0.39, and P=0.08, respectively). Experiment 2: The IC50 in Capan-1 were 46.9µg/mL and 58.9µg/mL in Lectin-Dox group and Dox group, respectively. Although there was no significant difference between them (P=0.51), Lectin-Dox group tended to have stronger cytotoxicity than Dox group. In SUIT-2, there was no difference and tendency between two groups (P=0.83). Experiment 3: In the Capan-1 derived models, the tumor volumes at the end of study were 518mm3 and 607mm3 in Lectin-Dox group and Dox group, respectively (P=0.60). The tumor weights were 427mg and 614mg in Lectin-Dox group and Dox group, respectively (P=0.35). Lectin-Dox group tended to have stronger anti-tumor effects than Dox group. On the other hand, no difference and tendency between two groups were observed in SUIT-2 derived models (Tumor volumes; P=0.60. Tumor weights; P=0.46).

Conclusions: Modifying the liposomal surface by the rBC2LCN improved it's accumulation to the pancreatic cancer as demonstrated stronger anti-tumor effect. We regard this lectin could work as effective cancer targeting bullets, not only for small molecular drugs but also as surface decorator of nanoparticle drugs.

#2153

Ex vivo drug sensitivity testing of primary cells for precision cancer medicine.

Sergey G. Kuznetsov, Alexander Ianevski, Evgeny Kulessky, Karoliina Laamanen, Elina Lehtinen, Maria Nurmi, Swapnil Potdar, Jani Saarela, Katja Suomi, Laura Turunen, Krister Wennerberg, Päivi Tammela. _University of Helsinki, Helsinki, Finland_.

Introduction/Purpose:

Cancer therapy is increasingly moving towards individualized care and therapy, but there are still gaps between what is known and described on the molecular level about cancers and what is applied in the clinic. In an attempt to bridge the knowledge gap, we at the Institute for Molecular Medicine Finland (FIMM) have set up an Individualized Systems Medicine program that integrates clinical information, molecular profiling and functional information about individual patients' cancers (Pemovska et al, Cancer Discov, 2013). Central to this program is the Drug Sensitivity and Resistance Testing (DSRT) where we functionally profile the responses of primary cancer cells to a comprehensive clinical oncology and signal transduction inhibitor drug collection of 528 compounds.

Methods:

Acoustic dispensing platforms are integral to the success of this profiling activity. We have to date produced approximately 3000 drug sets as dose response assay ready plates. The acoustic dispensing allows for making pre-drugged single drug plate sets and/or drug combination plates within hours after sampling of the cells. The plates are also readily sent to researchers anywhere in the world for running comparable assays at other sites. The drugging reproducibility is excellent generating results with correlations of 0.98 or higher in replicate assays. We have developed in-house software solutions to aid these processes: a script for quick creation of transfer list for combination plates and automated analysis pipelines with web-based software interfaces to enable the screening biologists to analyze the screening results effectively.

Results:

The results of these assays are used to explore and understand cancer biology in terms of druggability, functional heterogeneity and mechanism of drug response and resistance. The profiling data can be used to stratify and position the relevance of specific drugs in different diseases and has been used to identify novel clinically relevant activities of existing and investigational drugs (see e.g. Pemovska et al, Nature, 2015). This information is further utilized to establish hypotheses on drug combinations selectively targeting individual cancers and their predictive biomarkers, which can be explored in the clinic by our clinical collaborators to guide the treatment of the individual patient.

Conclusions:

In summary, we describe our platform for a functional drug sensitivity testing within our individualized cancer systems medicine program, which generates consistent biological and clinically relevant data.

#2154

A novel dual-function polymeric scaffold for cancer drug screening.

Tianfu Wu, Zuan-Tao Lin. _Univ. of Houston, Houston, TX_.

Cell-based assay has been used as a conventional strategy for discovery of new anti-cancer drugs in pharmaceutical industries. Traditionally, two-dimensional (2D) cell culture based cytotoxicity assays are the most common approaches, despite the fact that they have significant limitations in simulating tissue or organ microenvironment. In comparison, 3-dimensional (3D) cell culture provides a more promising tool for new drug discovery, however, next-generation cell growth monitoring device for 3D cell culture is still challenging. Recent studies have shown the potential of metallic nanoparticle-polymeric composites in biosensing systems. We hypothesize that in a polymer-based 3D-cell culture system, cancer cell growth and the subsequent spheroid formation can cause structural change of the polymer network. With the incorporation of metallic nanoparticles into the polymeric network, electrical conductance change of the 3D-scafold may be monitored to reflect cell growth and spheroid formation. Here, we developed a dual-function polymer scaffold (DPS) by integrating thermosensitive polymers, silver flake-decorated polymers, and gNP-decorated polymers as a biosensor to monitor cancer cell growth. We found that the Triple negative breast cancer stem cell line MDA-MB-231 formed spheroid in DPS. It is notable that the increase of cancer cell numbers during the formation of spheroid was proportional to the change of electrical conductance of the 3D-scafold. Importantly, the results from DPS cell growth monitoring method correlate well with conventional cell counting during 3D cell culture of breast cancer cells. SEM imaging and AFM imaging have confirmed the integration of silver flakes and gold nanoparticles in the DPS. Finally, we demonstrated that the DPS 3D-scafold exhibited robust responses to MDA-MB-436 cells when exposed to 3 nM of paclitaxel (PTX) and docetaxel (DTX), respectively. This dual-function 3D-scaffold, for both cell growth and growth monitoring may have a good potential in drug screening in the future.

#2155

High-plex spatial profiling analysis of multidrug CIVO microdose studies in cancer patients.

Gary B. Deutsch,1 Seth M. Pollack,2 Matthew J. Thompson,3 Kenneth R. Gundle,4 Jessica A. Bertout,5 Jason P. Frazier,5 Emily Beirne,5 Marc O. Grenley,5 JingJing Gong,6 Yan Liang,6 Joseph M. Beechem,6 Richard A. Klinghoffer,5 Robert G. Maki7. 1 _Hofstra Northwell School of Medicine, Lake Success, NY;_ 2 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 3 _University of Washington, Seattle, WA;_ 4 _Oregon Health & Sciences University, Portland, OR; _5 _Presage Biosciences, Seattle, WA;_ 6 _Nanostring Technologies, Seattle, WA;_ 7 _Hofstra Northwell School of Medicine, Seattle, WA_.

Background: CIVO microdosing studies performed in patient tumors in situ allow drug developers to assess localized tumor and microenvironment responses to multiple agents without having to expose patients to high systemic drug levels. By concentrating microdoses of multiple different drugs within a living tumor in situ, it is possible to compare tumor and immune responses in spatially resolved regions of the same tumor. This type of early phase (Phase 0) clinical study represents a new path for drug developers to gain insight into drug efficacy, tumor associated immune cell modulation, biomarker discovery and validation, and microenvironment interactions for new drugs earlier in the drug development process. Traditionally, analysis of FFPE samples from these microdosing studies involve routine immunohistochemistry, immunofluorescence, and in situ assays that reveal changes in protein and RNA expression. These assays offer tissue wide protein and gene expression information but have limited multiplexing capabilities and dynamic range, as well as rapidly consume precious trial samples. Novel technologies such as NanoString's GeoMxTM Digital Spatial Profiler (DSP) enable high-plex spatially resolved analysis of proteins and RNA transcripts in single FFPE tissue sections. In this proof of principle study, we utilized GeoMxTM Digital Spatial Profiler for protein and RNA expression on single FFPE sections from patient sarcoma tumors that were microdosed with multiple FDA approved drugs.

Methods: Single FFPE sections from microdosed patient tumor samples were IF stained for DNA and CD3 and whole slide imaged. 600-micron and 100-micron diameter regions of interests (ROIs) were selected for DSP analysis within drug and control microinjection sites. Imaging and barcode counts were performed using GeoMxTM DSP and nCounter systems.

Results: DSP protein analysis highlighted phospho-S6 and phospho-ERK upregulation in response to doxorubicin compared to vehicle site. Additionally, ROIs sampled along the doxorubicin exposure gradient showed a dose-dependent reduction of phosphorylation of both S6 and ERK proteins. DSP RNA analysis revealed drug specific transcript regulation of multiple genes in microdosed tumors, including upregulation of chemokines CXCL9 and CXCL10 at sites of doxorubicin and aldesleukin injection but not to other chemotherapy agents.

Conclusions: Early phase CIVO microdosing studies combined with high-plex DSP opens the door to generating multi-omics data for multiple microdosed drugs within small patient studies. Analysis of protein and RNA expression using DSP enabled collection of targeted region of interest high density data from single FFPE sections, conserving precious patient biopsy samples. Through continued expansion of the GeoMxTM DSP analyte panels, collecting an ever-increasing depth of protein and gene expression data is possible in Phase 0 CIVO microdosing studies.

#2156

Quantifying cell subsets and heterogeneity in living cultures using real time live-cell analysis.

Nicola Bevan,1 Tim Jackson,2 Clare Szybut,1 Lauren Kelsey,1 Hinnah Campwala,1 Tim Dale,1 Nicholas Dana,2 Nevine Holtz,2 Eric Endsley,2 Dan Appledorn,2 Cicely Schramm,2 Laura Skerlos,3 Richard Lister,2 Derek J. Trezise1. 1 _Sartorius, Welwyn Garden City, United Kingdom;_ 2 _Sartorius, Ann Arbor, MI;_ 3 _Sartorius, Ann Arbor, MA_.

Considerable heterogeneity exists in the properties and activity of individual cells, even in the simplest cell system. This arises from fundamental differences in the basic cell types present, genetic or epigenetic variations, the stage of cell cycle or differentiation, and the impact of each cell's unique and dynamic local microenvironment. Such heterogeneity is mirrored by the diversity of pharmacological response at the cellular level, where even seemingly identical cells may respond differently and at different times to drug treatments and perturbagens. Accordingly, analysis at the cell-by-cell level promises valuable and additional biological insight beyond which whole population measures may deliver. Here, we describe new, enabling and industrial-scale, live-cell analysis solutions for quantifying the phenotypic biology of cell subsets in heterogeneous cultures. Time-lapse images of cultured cancer and immune cells in 96-well microplates were automatically collected using an IncuCyte S3 live analyzer (Essen Bioscience). Using new segmentation algorithms, the boundaries of individual cells (typically 300-1000 per image) were identified in each image of the sequence. Parameters and features were extracted from single cells, such as cell area, eccentricity and fluorescence (e.g. with cell labels, cytotoxicity and apoptosis probes). Populations of cells could be identified and classified over time using custom 'flow cytometry'-like visualisation and classification tools. Using this new approach, we demonstrate cell-by-cell analysis for a range of different primary and immortalised, adherent and non-adherent, living cells for up to 7 days in culture (e.g. Jurkat, A549, human PBMCs). This was coupled with a novel live-cell, fluorescent antibody-based labeling strategy (IncuCyte FabFluor-488) to probe for specific subsets within the cultures. Example data generated in PBMCs during T cell activation (anti-CD3/IL-2, 10 ng/mL) demonstrates the change in cell shape from small, spherical cells (average area 81 ± 0.5 µm2, eccentricity 0.57 ± 0.002) to larger, flatter cells (117 ± 4 µm2, 0.69 ± 0.004) over 120 h. With the addition of FabFluor-488-CD71, it was possible to show an associated, temporal increase in CD71 expression within the activated T cell subset (75 ± 1% of large cells were CD71 positive compared to 12 ± 1% of smaller cells at 48 h, increasing to >90% in the larger cells by 120 h). Other example data sets for subset analysis of proliferation, cell death and cell cycle measurements as well as immuno-phenotyping will be shared to illustrate the value of this approach.

#2157

Eliminating Edge Effect in 96-Well Plates by Controlling Thermal Conditions during Cell Plating.

Shannon Darou,1 Alicia Henn,1 Kersti Alm,2 Allayna M. Frank,1 Randy Yerden3. 1 _BioSpherix, Ltd., Parish, NY;_ 2 _Phase Holographic Imaging, Inc PHI AB, Lund, Sweden;_ 3 _Randy Yerden, Parish, NY_.

The traditional approach to avoiding the increased variability in 96-well plate edge wells has been to avoid using them. This results in a 37% loss in usable assay space, increasing time and materials needed for cell-based assays. Approaches to reducing this "edge effect" have included reducing plate evaporation during long-term incubation. However, we have preliminary data that plating cells at a constant 37 degrees C dramatically reduces edge effect. We have previously showed that in plates filled at room temp (RT), the cells in edge and corner wells experienced the most directional cell rolling during cell settling, preventing random cell deposition. In contrast, cells plated at 37 degrees C settled randomly in edge wells with less or no directional rolling. Here, we extend these findings with studies of well-specific cell settling patterns associated with plate-wide thermal changes after cell plating. Our null hypothesis was that intra-well thermal currents as plates warm to 37 degrees C from RT do not disrupt random cell deposition during settling. A549 human lung carcinoma cells were plated in warmed medium in either traditional uncontrolled RT conditions or in an Xvivo System under completely controllable conditions. In this closed chamber, everything (chamber floor, atmosphere, the pipettor, tips, fluids, gloves, and plate) was at the same temp as the incubator, where cells typically settle in the wells after plating. We used the PHI HoloMonitor M4 microscope to record holographic time-lapse images of cells settling and adhering to the well floor. Crystal violet staining was used to assess cell deposition patterns. We found that not only did constant temp conditions change well-specific cell settling patterns, but when plate-wide thermal changes were inverted, by plating cells at 37 degrees C and letting them settle at RT, we were able to alter well-specific cell rolling and cell settling patterns. We concluded that it is thermal changes that drive edge well variability and that constant temperature control during cell plating can reduce or eliminate edge effect. Plating cells under full-time control of conditions could have a tremendous impact on cell-based drug discovery and pre-clinical testing by allowing full use of 96-well plates. This could reduce assay time and materials as well as improve assay reproducibility.

#2158

Combining cell panel profiling and synthetic lethality data to efficiently screen for synergistic combinations.

Joost C. Uitdehaag, Derek W. van Tilborg, Martine B.W. Prinsen, Jeffrey J. Kooijman, Jelle Dylus, Jeroen A.D.M. de Roos, Suzanne J.C. van Gerwen, Jos de Man, Rogier C. Buijsman, Guido J.R. Zaman. _Netherland Translational Research Ctr. BV, Oss, Netherlands_.

In cancer therapy, combination drug treatment aims to improve response rate and decrease the development of drug resistance. The discovery of new effective drug combinations is constrained by the cost and effort of carrying out large unbiased screens and by poor translation of results towards the clinic. Here we describe how focusing on the biological mechanisms underlying the activity of drug candidates may aid a priori selection of promising synergy candidates and help in translate synergistic combinations towards a clinical situation.

We have previously shown [1] that curve shift analysis as developed by Straetemans et al. [2] is a better method than combination-matrix screening. Also a dose based score such as the isobologram or the CI-index more robustly assesses synergy than an effect-based score such as the Bliss-additivity [1]. On this basis, we developed a two-step synergy screening approach, called SynergyScreen™. By distinguishing separate synergy screening and synergy confirmation stages, this setup capitalizes on insights from high throughput screening to discover robust and reproducible pharmacologically synergistic pairs.

To further improve the efficiency of synergy screening, we focused on pre-selecting compounds in our screening library according to their biological mechanism. We profiled a library of more than 160 anti-cancer agents in a cell panel of 102 cell lines from diverse tumor origins [3]. Agents were clustered according to response and so-called exemplars were collected into a focused library that represents the spectrum of biological mechanisms of current cancer therapy. This synergy screening library includes many standard of care chemotherapeutic agents, approved and pre-clinical kinase inhibitors, epigenetic modulators and compounds acting by other mechanisms.

Finally, we harnassed recent insights into the biology of synergy to understand and predict synergistic pairs. A tool was developed that uses the response of a compound in a 102 cell line panel to pick potential synergistic partners from the database of preprofiled compounds. It does this by computationally assessing if pairs can pharmacologically mimick clinically validated synthetic lethal interactions [4]. We optimized prediction accuracy using the results of internal and external synergy screens. The tool was applied to specifically enrich test libraries and to predict synergies at clinically relevant doses, including the results of a SynergyScreen™ with the poly-ADP ribose polymerase (PARP) inhibitor niraparib and the BET bromodomain inhibitor JQ1.

References [1] Uitdehaag et al. (2015). PLoS ONE 10(5): e0125021. [2] Straetemans et al. (2005). Biometrical J. 47, 299-308. [3] Uitdehaag et al. (2016). Mol. Cancer Ther. 15, 3097-3109. [4] Lee et al. (2018). Nature Communications 9, 2546.

#2159

Label-free impedance analysis as a versatile tool for dynamic screening of cancer molecular therapeutics.

Fabio Cerignoli, Biao Xi, Brandon Lamarche, Leyna Zhao, Yama Abassi. _ACEA Biosciences, Inc., San Diego, CA_.

In vitro monitoring cancer cells response to treatment often involves laborious sample processing and collects single data points that are against the dynamic nature of cancer cells. Here we present the adaptation of an impedance-based methodology to dynamically monitor cancer cell behavior and therapeutic response. The technology detects cell death, proliferation or migration by measuring changes in conductance of microelectrodes embedded in 96 and 384-wells cell culture plates. It avoids sample labeling and processing and allows continuous monitoring of cell response. Our data shows validation with cancer cell lines and primary cells monitored for cell invasion/migration through transwells or after a scratch has been performed with a dedicated device, anticancer drug response and receptor signaling activation, with similar results over end point assays. Easy experiment set up and minimal sample processing make the technology ideal for applications in large screening campaigns, while label-free technology allows less artifacts and further analysis on the same samples through orthogonal assays.

#2160

Microfragmented human fat tissue is a natural scaffold for drug delivery: potential application in cancer chemotherapy.

Giulio Alessandri,1 Valentina Coccè,2 Fabio Pastorino,3 Rita Paroni,2 Michele Deicas,2 Francesco Restelli,2 Carlo Tremolada,4 Angiola Berenzi,5 Eugenio Parati,1 Anna Teresa Brini,2 Giampietro Bondiolotti,2 Augusto Pessina,2 Mirco Ponzoni3. 1 _Neurological Institute Carlo Besta, Milan, Italy;_ 2 _University of Milan, Milan, Italy;_ 3 _Institute G. Gaslini, Genoa, Italy;_ 4 _Manchester Metropolitan University, Manchester, United Kingdom;_ 5 _University of Brescia, Brescia, Italy_.

Localization of chemotherapy at the tumor site can improve therapeutic efficacy and reduce systemic toxicity. In previous studies we have shown that mesenchymal stromal cells (MSCs) isolated from bone marrow or fat tissue, can be loaded with the anti-cancer drug Paclitaxel (PTX) and kill cancer cells when localized nearby. We here investigated the capacity of human micro-fragmented adipose tissue (MFAT), used as a natural container of MSCs, to delivery PTX with the idea to improve local drug concentration and to prolong the therapeutic activity. Surprisingly, we found that both fresh but also devitalized MFAT (DMFAT) (by freezing/thawing procedure) were very effective biomaterials able to deliver and release significant amount of PTX, killing several human cancer cell lines in vitro with an impressive long lasting activity. In an orthotopic mice model of Neuroblastoma (NB) transplant, we found that DMFAT loaded with PTX prevents or delay NB relapse when placed in the surgical area of tumor resection, without any collateral toxicity. We concluded that MFAT, but also DMFAT, may work as natural biomaterials that can be used to localize and release anti-cancer molecules at the tumor site. In addition, the procedure to easily prepare DMFAT did not alter its natural scaffold structure. This suggest that such biomaterial may have a prominent role in the near future in many oncological applications to delivery anti-cancer drugs, helping to fight cancer in human.

#2161

In vitro **osteoblast assays for studying the effects of cancer therapeutics on bone biology.**

Jenni H. Mäki-Jouppila, Jussi M. Halleen, Katja Fagerlund. _Pharmatest Services, Turku, Finland_.

Bone cell activity is an important factor in regulating bone metastases from tumor cells in the skeletal environment. When cancer cells home to bone they secrete factors that stimulate osteoclast activity leading to increased bone resorption. Bone then releases stimulatory factors that in turn promote the growth and proliferation of cancer cells. This process is called the vicious cycle often leading to osteolytic lesions in bone when osteoclastic bone resorption exceeds osteoblastic bone formation. Osteolytic lesions are common in breast and lung cancer and multiple myeloma. There is a vicious cycle between cancer cells and bone cells also in the context of osteoblastic lesions. In osteoblastic metastases, the vicious cycle observed in osteolytic disease takes place but in addition to this cancer cells produce osteoblast-stimulating factors including bone morphogenetic protein (BMP), epidermal growth factor (EGF) and platelet derived growth factor (PDGF). Osteoblasts also influence osteoclasts by producing RANKL, which stimulates osteoclast differentiation. Many cancer patients with bone metastasis have both osteolytic and osteoblastic lesions. This is common for example in prostate cancer. Our aim was to establish an in vitro cell culture model to study the effects of compounds on osteoblast differentiation and activity.

A mouse osteoblast progenitor cell line KS483 was used in the study. BMP-2 was used as a test compound and was added in the beginning of culture and simultaneously with culture medium change every 3-4 days. Ascorbic acid was added into the cell culture medium at day 4. In the osteoblast differentiation assay, the activity of cellular alkaline phosphatase (ALP), a marker of osteoblast differentiation, was measured at day 8 from cell lysates. Total protein content was measured from the same samples. In the osteoblast activity assay, KS483 mouse osteoprogenitor cells were cultured for 13 days, during which N-terminal propeptide of type I procollagen (PINP) secreted into the culture medium was determined at day 11 to demonstrate effects on organic bone matrix formation. β-glycerophosphate was added to the culture at day 11. The cultures were stopped at day 13 by removing the culture media from the wells and adding hydrochloric acid. Calcium deposition, a marker of inorganic bone formation, was determined at the end of the study.

BMP-2 stimulated osteoblast differentiation and activity shown by the increase in ALP, PINP and calcium levels. The results suggest that the KS483 cell line can be used for setting up reliable in vitro models of osteoblast differentiation and activity. These osteoblast assays can be used for studying the effects of compounds on bone formation and cancer related bone events.

#2162

ActivSignal technology for multiplex analysis of signaling protein activation profiles.

Ilya Alexandrov, Malcolm Mackenzie, Irina Brandina. _ActivSignal, NATICK, MA_.

The IPAD assay technology from ActivSignal monitors activity of 26 major signaling pathways in multiplex by examining phosphorylation status or expression of 70 protein targets from canonical signaling pathways. The technology uses antibody pairs for each protein target to ensure high specificity, and these pairs are carefully selected and tested. Only if both antibodies in a pair bind to their specific target molecule will the detection take place during the analysis phase - avoiding false hits. The ActivSignal IPAD platform can be used to profile cell culture samples, tissues and different blood fractions. Human and mouse samples can be analyzed. The IPAD assay requires only a very small amount of your precious sample. Applications include:

\- Direct analysis of protein activity of the signaling cascades and not subsequent downstream events.

\- Testing physiological effects of compounds of interest.

\-- Influence on specific protein targets, as well as the signaling pathways as a whole.

\-- Secondary and/or off-target effects.

\- Similarly, analysis of signaling pathways activation status in response to different stimuli, such as: CRISPR, shRNA, hormones, stresses and other treatments.

\- Medium-throughput analysis (96-well format).

\- Tissue sample profiling from patients and animals (murine).

\- Profiling of tumor biopsies.

Our collaborators analyzed the mechanism of action of JG-98, an Hsp70 inhibitor that exhibits anti-cancer activities that affect both cancer cells and tumor associated macrophages. Using gene expression and ActivSignal protein profiling assay, the signaling pathways influenced by JG-98 were identified, which predicted potential drug combinations that can be used with JG-98 for further preclinical development. ActivSignal is a breakthrough, low-cost tool for the analysis of signaling pathway activation profiles. It enables researchers to rapidly and efficiently gain insight into function of their favorite drug molecules, genes etc.

#2163

Magnetic field stimuli improve hepatic delivery of oxaliplatin ensuing survival benefit to colorectal liver metastatic tumor bearing rats.

Venkateswara R. Gogineni,1 Dilip R. Maddirela,1 Woo Ram Park,2 Dong-Hyun Kim,2 Amit Joshi,1 Sarah B. White1. 1 _Medical College of WIsconsin, Milwaukee, WI;_ 2 _Northwestern Univeristy, Chicago, IL_.

Treatment of unresectable liver tumors with a systemic chemotherapeutic agent is severely limited by the systemic toxicities. Therefore, the aim of the current study was to site selectively release high concentration of therapeutic agents to alleviate the off target effects of the drugs. Rats orthotopically implanted colorectal liver metastases (CC-531 cells) were randomly divided into different treatment groups. The rats were treated with magnetic field sensitive and MR imageable oxaliplatin loaded liposomes or by drug alone via different routes of administration, establishing appropriate controls. Liposomes containing oxaliplatin at 6.0 mg/kg per were infused into rats via the portal vein or with free form of oxaliplatin via tail vein or with saline. Three groups were subjected to alternative magnetic fields to trigger drug release for 30 minutes. Post treatment MRI was performed to follow the tumor growth. R2* maps were constructed from the T2* MRI images acquired immediately post-infusion and one week later. ROIs were drawn within the acquired maps to measure R2* values in the tumor regions and surrounding tissues. At 8 weeks or earleir the rats were euthanized, tissues explanted and digested with nitric acid and hydrogen peroxide mix. The digestates were subjected to ICP-MS analysis of elemental platinum at the indicated time points to determine the localized delivery of Oxaliplatin. The R2* MRI maps showed changes in the tumor regions at one-week post-infusion compared to the surrounding liver parenchyma (p<.001) and to the same regions immediately after infusion (p<.001). The localized delivery of iron oxide resulted in low signal intensity immediately after infusion. Significantly higher levels of oxaliplatin was delivered to the liver tissues compared to the lungs (p<.001) and intestines (p<.001) in the animals that received site selective delivery and magnetic field triggering following liposome administration. Although, there was no significant difference between the animals treated with single or multiple cycles of magnetic field triggers, the animals tolerated 3 trigger cycles with 100% survival rates. The survival of animals treated with portal vein route delivery was significantly higher compared to tail vein delivery (p<.05). Similarly, prominent necrotic zones were observed in the primary tumors of rats which site selective delivery followed by triggering compared to those which did not get triggered and those which received systemic oxaliplatin (via tail vein). In conclusion, site selective delivery allows for high concentrations of oxaliplatin and improves survival outcomes in colorectal liver metastasis tumor bearing rats.

Key words: Triggered release, Oxaliplatin, Liposomes, Colorectal liver metastasis

#2164

In-licensing and knockout validation of monoclonal hybridomas to improve antibody reproducibility.

Sam Heaton, Sofia Koch, Jiangyan Ge, Joshua Rizzo, Michael Prater, Hanna Dreja, Bruce Hamilton, Alejandra Solache. _Abcam, Cambridge, United Kingdom_.

Antibodies are the most common tools in basic science and clinical research. Over the past several years there has been an ongoing discussion around antibody validation in light of poor experimental reproducibility between laboratories. This is in part due to the cross-reactivity between antibodies and off-target proteins and the variability between different antibody batches. The resulting experimental irreproducibility leads to wasted resources and compromises the advancement of science. Abcam partners with various research institutes and biotech companies to in-license hybridoma cell lines producing monoclonal antibodies. Through this in-licensing program, Abcam has taken steps to help ensure target-specificity of valuable clones. Our in-licensing program utilizes our ISO9001-accredited production method to ensure each batch performs in-line with previous batches. Where possible, all in-licensed antibodies are also tested in knockout (KO) material to confirm the specificity of the in-licensed clone and subsequent batches. Through our partnership with Horizon Discovery to test antibodies with KO cells, target genes are mutated via CRISPR-Cas9 within a haploid cell line, which results in a frameshift and a complete loss of gene expression. KO cell lines provide a true negative control for antibody validation that allows us to confirm the specificity of clones we in-license and produce. Alongside KO, we are making use of our expertise in recombinant antibodies to explore the conversion of selected in-licensed hybridomas to recombinant antibodies. A key issue with monoclonals is degradation of the clone over time, leading to inconsistent batches and irreproducible results. By sequencing and cloning the antibody, we expect to guarantee continued specificity and reproducibility; therefore, ensuring our licensors see long term impact and value generation for their innovations. Here we present KO data for several in-licensed published clones, validated in both western blot and immunocytochemistry. The antibodies have been tested in KO and wild-type cells to confirm their specificity. By providing researchers with reliable and specific antibodies that work as intended, we hope to minimize wasted resources and improve reproducibility. In addition to our KO data, we present data from the recombinant conversion of clone NAT105 to PD1 and show comparable, if not superior, performance when compared with the hybridoma.

#2165

Self-microemulsifying drug delivery systems (SMEDDS) containing novel compounds in combination with gemcitabine: Bioavailability enhancement and synergistic anticancer efficacy studies for pancreatic cancer management.

Sunil Prabhu, Preshita Desai, Naga Jyothi Thumma, Jeffrey Wang. _Western Univ. of Health Sciences, Pomona, CA_.

Pancreatic cancer (PC) is the third leading cause of deaths in the United States claiming approximately 45,000 lives every year and is predicted to be the second cause of cancer related deaths by 2020. Late diagnosis, poor prognosis, limited success and significant side effects with existing anticancer drugs has resulted in such high mortality rates (5 year survival rate is less than 8%). These limitations have encouraged scientists to develop chemopreventive strategies and combination drug alternatives that can serve as better therapeutics. In our previous studies, we have reported the synergistic chemoprevention potential (formulation patent pending) of lipid nanocarriers (SMEDDS) of Loratadine (LOR), H1-receptor antagonist drug; with free Sulforaphane (SFN), a neutraceutical compound from broccoli when administered at low doses in human PC cell lines (MIA PaCa-2 and Panc-1).

To confirm the hypothesis that enhanced efficacy using the SMEDDS formulation results from bioavailability enhancement, preclinical in vivo pharmacokinetic studies were performed in Sprague Dawley rats. The results confirmed that the bioavailability and Cmax of LOR increased 5-fold (2203.2 ± 293.7 ng.h/mL for plain LOR and 10266.1 ± 250.0 ng.h/mL for LOR SMEDDS) and 4-fold (120.9 ± 10.5ng/mL for plain LOR and 503.2 ± 5.8ng/mL for LOR SMEDDS) when administered as SMEDDS compared to plain drug. This corroborated the significant enhancement in bioavailbility with SMEDDS formulation.

This promising results in bioenhancement and chemoprevention activity led to an hypothesis to assess the potential of LOR SMEDDS-SFN combination as an adjuvant therapy with classical anticancer drugs like Gemcitabine (GEM) to give synergistic efficacy enhancement in PC treatment. For this, IC50 concentrations were calculated for various treatment groups [GEM alone or in addition to the LOR free drug or as SMEDDS with or without SFN] using MTS viability assay on human PC cell lines in accordance with published protocol over an incubation time of 72 h. The LOR SMEDDS-SFN-GEM combination revealed 2-fold and 2.5-fold enhancement in anticancer activity compared to free drug combination and GEM alone respectively. This confirmed the synergistic anticancer potential of the free drug combination followed by further activity potentiation using SMEDDS formulation. In conclusion, a novel SMEDDS of LOR and free SFN was invented (provisional patent applied) to successfully elicit PC chemoprevention and adjuvant treatment in combination with GEM. We anticipate that, this adjuvant therapy will lead to relatively low doses of anticancer drugs and thereby can reduce the side effects with enhanced efficacy.

#2166

A novel etoposide-bound magnetic nanoparticle for remote targeting of cancer cells.

Alexander Willis,1 Bhargava Karumudi,1 Bing Liu,2 Ashley Sough,2 Sangyeul Hwang,2 Ying Liu,1 Herbert Engelhard1. 1 _Univ. of Illinois at Chicago, Chicago, IL;_ 2 _IMRA America, Inc., Ann Arbor, MI_.

Background: Magnetic nanoparticles (MNPs) have potential for enhancing drug delivery in selected cancer patients. To date, successful clinical use of MNPs has been limited due to the: 1) limited capacity for targeting at human-sized distances, 2) route of delivery, and 3) choice of therapeutic component. Here, we present data related to the creation and in vitro use of a new two-part MNP system consisting of: 1) an AuFe base particle having streptavidin binding sites, and 2) biotinylated etoposide, which was bound to the base particle. Etoposide was chosen in part due to its previous clinical use within cerebrospinal fluid, which could serve as a delivery medium for the new particle.

Methods: Base MNPs were manufactured by IMRA America, Inc., and combined with biotinylated etoposide to create AuFe-etoposide particles (etop-MNPs), which have not been previously described. Etop-MNPs were characterized by electron microscopy, UV-vis spectroscopy, and DLS. Etop-MNPs could be moved at a distance (e.g. 10 cm) by means of a "surface walking" phenomenon, occurring in response to a rotating neodymium-boron-iron permanent magnet, kindly provided by Pulse Therapeutics (St. Louis, MO). Cell lines (including D283, U138, and normal endothelial cells) were maintained using standard tissue culture technique. Viability of the cells in response to the MNP base particles, etoposide alone, and etop-MNPs, was studied by light microscopy and MTT assay.

Results: Etoposide was successfully bound to the IMRA base particle through the streptavidin-biotin interaction. Typically, 100uM etoposide was used. Etop-MNPs could be moved remotely by the rotating magnet, which produced a tangential particle velocity of 0.15 +/- 0.026 cm/sec. With 2 hours of treatment, etop-MNPs caused morphologic changes and decreased cancer cell viability to 0.50 +/- 0.03 percent that of control values.

Conclusions: These findings indicate that our novel etop-MNPs have tumoricidal effect, and can be moved relatively rapidly at physiologic distances, using a rotating magnet. Etop-MNPs, may therefore prove to be useful for magnetic drug targeting in clinical settings, such as intrathecal use within the spinal subarachnoid space.

#2167

Antibody glycoengineering for drug delivery applications.

Drishti Navin Sehgal, Stephen Kalscheuer, Jayanth Panyam. _University of Minnesota, Minneapolis, MN_.

Antibody-drug conjugates (ADC) hold considerable promise as anticancer agents. A critical determinant of the effectiveness of ADCs is the chemistry that is used to conjugate the payload. Currently used approaches include primarily conjugation to either side-chain amine or carboxylic acid groups or conjugation to thiols. Because these reactions are not site specific and not easily controlled, these chemistries can result in reduced affinity for the target antigen. Further, these conjugation reactions lack selectivity and can result in heterogeneous mixtures of products that differ in the sites and stoichiometry of modification. We investigated a glycoengineering strategy that enables the introduction of artificial azide groups in the antibody without affecting their antigen affinity. This is based on the observation that glycosyltransferases can incorporate non-natural sugars (e.g., azido mannose) at different sites on an IgG molecule. The azide groups in these artificial sugars are then available to react with alkynes through copper-catalyzed 'click' chemistry or with strained alkynes such as dibenzyl cyclooctyne (DBCO) allowing for biorthogonal, copper-free 'click' chemistry. Because the sugars are added reproducibly and at a site that does not affect antigen binding, the glycoengineering technology would overcome problems associated with traditional conjugation strategies. Using this approach, azide groups were introduced in anti-CD133 and anti-perlecan antibodies. Further, the azide groups were available to react with various DBCO conjugates including fluorophores, drug molecules and nanoparticles. Importantly, the addition of artificial sugar and subsequent azide-alkyne reaction did not affect the affinity of the antibody for the target antigen. Conjugation of a cytotoxin to the antibody resulted in enhanced cell kill in vitro and efficacy in vivo. We plan on further improving the effectiveness of this approach by investigating cleavable linker technology in combination with the glycoengineering strategy. We expect that this strategy will prove to be a unique platform technology that will have a significant impact on antibody-based therapeutics.

#2168

Testing the electrical properties of different cell lines using 3DEP reader and compare to TTFields response.

Moshe Giladi,1 Einav Zeevi,1 Karnit Gotlib,1 Cornelia Wenger,2 Ariel Naveh,1 Zeev Bomzon,1 Eilon D. Kirson,1 Uri Weinberg,1 Adrian Kinzel,3 Yoram Palti1. 1 _Novocure, Haifa, Israel;_ 2 _Novocure, Lisbon, Portugal;_ 3 _Novocure, Munich, Germany_.

Background: Tumor Treating Fields (TTFields) are an approved anti-neoplastic treatment modality delivered via application of low intensity, intermediate frequency, alternating electric fields. The electrical properties of cells (such as permittivity and conductivity) determine the optimal frequency of TTFields that incurs the highest reduction in cell counts. Currently, there are no predictive markers for determining TTFields response or the optimal frequency to be applied for individual patient. The goal of this study is to determine the correlation between electrical properties of cells and TTFields's optimal frequency and sensitivity. The 3DEP reader (LabTech) determines the electrical properties of cells, including permittivity and conductivity, by using Dielectrophoresis (DEP) force. DEP is a physical effect that generates a force on polarizable particles experiencing a non-homogeneous electric field and can therefore be used as a technique to analyse the way cells move within electric fields at different frequencies. Methods: The baseline electrical properties (permittivity and conductivity) of 17 cell lines from different tumor types were determined using the 3DEP reader (LabTech). The curves were analyzed using 2-way ANOVA. The optimal TTFields frequency for each cell line was determined by testing the cytotoxic effect of TTFields at various frequencies using the inovitro system. The electrical properties of cells were compared with the optimal TTFields frequency and sensitivity of each cell line. Results: The results demonstrate significant differences (p<0.001) between the lower frequency range of the 3DEP curves that corresponds to membrane capacitance of cells with TTFields optimal frequency of 150kHz (9 cell lines) and cells with TTFields optimal frequency of 200kHz (8 cell lines). Membrane capacitance was also a good predictor for TTFields sensitivity based on the differences in the curves in the low frequency range. Conclusions: The results presented in this study demonstrate that cell membrane capacitance correlates with TTFields optimal frequency and sensitivity. Based on the above, there is a strong rational to further explore the potential of measuring the electrical properties of cells as a predictive marker to help determine which patient will respond better to TTFields and the optimal TTFields frequency to be applied for each patient.

#2169

Doxorubicin conjugation to reovirus enhances tumor cell-directed oncolysis.

Jameson T. Berry, Bernardo A. Mainou. _Emory University, Atlanta, GA_.

Breast cancer remains the most diagnosed type of cancer and second leading cause of cancer-related deaths in women in the United States. Triple-negative breast cancer (TNBC) constitutes 10-20% of breast cancer cases. TNBCs are characterized by lack of estrogen (ER) and progesterone (PR) receptors and growth factor receptor HER2/Neu on the cell surface. Therapies that target ER, PR, or HER2 are thus ineffective against TNBC. TNBC is primarily treated with cytotoxic chemotherapy or radiation therapy, which can cause significant damage to healthy cells and tissue. Therefore, there is need for therapies that selectively kill TNBC cells with reduced off-tumor effects. The concept of oncolytic virotherapy has existed since the early 1900s. However, oncolytic viruses have limited efficacy as single agents. Mammalian orthoreovirus (reovirus) is a non-enveloped, segmented double-stranded RNA virus with tropism for transformed cells. Reovirus is in Phase I - III clinical trials to test its oncolytic efficacy against a variety of cancers. Success has been limited and few trials have tested reovirus against breast cancer. In a high-throughput screen of small molecule inhibitors, we identified the topoisomerase II inhibitor doxorubicin hydrochloride (dox) as an enhancer of reovirus infectivity in the TNBC cell line MDA-MB-231. To better control dox delivery and enhance reovirus oncolytic potential, we chemically conjugated dox to reovirus virions (reo-dox). While reo-dox attachment to MDA-MB-231 cells is slightly impaired, viral replication is largely unaffected. Infection of MDA-MB-231 cells with reo-dox resulted in increased cytopathicity and faster induction of cell death than virus alone. MDA-MB-231 cells infected with reo-dox, but not virus alone, induced DNA double-strand breaks and activation of DNA damage response. Together, our findings show that reo-dox exhibits superior toxicity in TNBC cells than virus alone. Future studies will define the mechanism of enhanced cytopathicity of reo-dox and oncolytic efficacy of dox-conjugated reovirus in vivo. Delivery of small molecule inhibitors via conjugation to reovirus particles may provide an effective new method to directly target and kill cancer cells while minimizing toxicity to healthy cells and tissues.

#2170

Effect of 2D vs. 3D, exposure time, media and additives on growth and EGFR inhibitor response in patient-derived NSCLC lines.

Gurmeet Kaur,1 James H. Doroshow,2 Beverly A. Teicher2. 1 _NCI, Frederick, MD;_ 2 _NCI, Bethesda, MD_.

The effect of cell culture conditions on the proliferation and response to EGFR inhibitors of 4 patient-derived NSCLC lines (PDC) was determined. Variables included monolayer vs spheroid with only tumor cells or with HUVEC and MSC added, RPMI vs DMEM/F12 with or without EGF and other additives, growth time (10 days) and drug exposure time up to 4 days.

We explored the growth of 4 NSCLC PDC lines grown in RPMI1640 or in advanced DMEM/F12 media supplemented with 10% fetal bovine serum (R10 or D10) or with 5% FBS, hydrocortisone, rhEGF, adenine, penicillin-streptomycin and glutamine (complete media; RC or DC). Cells were cultured as monolayer or mixed with stromal cells HUVEC and MSC to recapitulate a tumor microenvironment. The lines form multicellular spheroids under low adhesion culture conditions. We investigated the growth inhibitory effect of EGFR inhibitors, erlotinib and osimertinib, in 4 patient-derived (one with an EGFR mutation) and 3 established NSCLC lines with known EGFR mutations. Erlotinib and osimertinib were tested in 9-point, half log concentration response starting at 30µM and 10µM, respectively. At 72h post plating, the cells were exposed to the test drugs for 72h and 96h. Cell viability was measured using luminescence by 2D CellTiter-Glo® in 2D monolayer and mixed monolayer cultures and 3D CellTiter-Glo® in 3D simple and complex spheroids. Cell viability was determined relative to a vehicle treated control and IC50s were calculated from concentration response curves.

In general, the NSCLC lines grew better in advanced DMEM/F12 media than in RPMI 1640 media with the largest effect seen in complex spheroid growth at 10-12 days. The Matrigel, hydrocortisone, rhEGF, adenine, and glutamine additives did not affect cell growth. The effect of media was more pronounced with some cell lines than others. For all 4 NSCLC lines, 3D growth in 96-well ULA culture peaked on day 8 and declined on days 10-12. Three of 4 lines cultured in monolayer or mixed monolayer were unresponsive to erlotinib (>30 μM) and osimertinib (>10 μM). The responsive line harbors a EGFR (L858R) mutation. Simple & complex spheroids formed by LG0567-F671-PDC in DMEM/F12 complete media were sensitive to erlotinib with IC50s below the clinical Cmax, indicating the importance of the 3D culture format. When tested separately as monolayers, hMSC and HUVEC were resistant to erlotinib with an IC50s of >30 and 13 μM, respectively. LG0567-F671-PDC was unresponsive to osimeritinib under all culture conditions because this PDC cell line does not harbor EGFR mutation

Patient-derived NSCLC and NSCLC cell lines response to anticancer drugs varied with culture conditions. The differential response to EGFR inhibitors in 3D simple, 3D complex spheroids, versus 2D culture, suggests that the tumor microenvironment and attachment factors may play a role in EGFR inhibitor activity and needs to be explored further using imaging techniques.

#2171

Activatable nanodelivery of high payload gemcitabine augments therapeutic efficacy in murine breast and pancreatic cancer models.

Samantha T. Tucci,1 Azadeh Kheirolomoom,2 Elizabeth S. Ingham,1 Lisa M. Mahakian,1 Sarah M. Tam,1 Josquin Foiret,2 Neil E. Hubbard,1 Alexander D. Borowsky,1 Mo Baikoghli,1 R. Holland Cheng,1 Katherine W. Ferrara2. 1 _UC Davis, Davis, CA;_ 2 _Stanford University, Stanford, CA_.

Gemcitabine (Gem) is the standard of treatment for metastatic pancreatic ductal adenocarcinoma (PDAC) and an effective anticancer drug for various solid tumors. However, clinical application of Gem chemotherapy is hampered by suboptimal delivery of the drug, mainly due to short circulation time and poor penetration of drug into the hypo-vascularized and dense fibrous stroma of PDAC. Here, we present a novel method to improve both loading and stability of Gem in temperature-sensitive liposomes (TSL). TSL have the potential to be locally activated by exposing tumor to ultrasound-mediated hyperthermia (USH) triggering release of large amounts of free drug in the tumor vasculature, which can then rapidly penetrate into the heated tumor.

Gem was passively loaded into TSL composed of DPPC:DSPC:DSPE-PEG2k (80:15:5) in the presence of copper(II) gluconate and triethanolamine at final 0.12 mg-drug/mg-lipid. Formation of a CuGem complex improved drug loading and stability and reduced systemic toxicity. Mice with bilateral invasive neu deletion (NDL) tumors (4-5 mm) were treated with an i.v. administration of CuGem-TSL at 10 mg Gem/kg body weight. USH was employed to trigger the release of drug; one tumor in the bilateral tumor model was insonified with a peak ultrasound pressure of 1.1 MPa at a frequency of 1.5 MHz at 43°C for 5 min prior to and 30 min post drug injection with a variable duty cycle.

We found that copper(II) gluconate possesses superior potential to solubilize Gem up to 150 mg/mL and augments passive drug loading. Upon loading Gem, formation of a complex with copper further improved drug loading and stability within TSL. By optimizing the lipid composition of the liposomal shell, we achieved high loading content of Gem at up to 12% by weight in TSL (CuGem-TSL), 4-fold greater than previously reported. Cryo electron microscopy

confirmed the presence of liquid crystalline structures within CuGem-TSL, which was not observed in the absence of copper (Gem-TSL). The resulting high-content CuGem-TSL displayed a rapid release of Gem (80%) within 5 min at 42°C with only 25% release over 30 min at 37°C in the presence of serum. In vitro CuGem-TSL demonstrated equivalent toxicity to free Gem against both murine breast cancer and pancreatic cancer. One hour following intravenous administration, 75% of Gem was effectively retained in circulating TSL, whereas no drug was detected in mouse plasma with free drug administration. Three repeated administrations of CuGem-TSL combined with USH over a two-week treatment course suppressed tumor growth in NDL, model of murine breast cancer, with limited toxicity, enhancing survival when compared to treatment with free Gem alone. Additionally, in a more aggressive tumor model of murine pancreatic cancer (KrasLSL-G12D/+; Trp53LSL-R172H/+; Pdx-Cre, mT4 tumor source), CuGem-TSL with USH induced cell death and regions of apoptosis and necrosis.

#2172

Quantification of ERK activity in cancer cell lysates and tumor extract using differential sensing methods.

Diana Zamora-Olivares,1 Tamer S. Kaoud,1 Lingyu Zeng,2 Mitchell Telles,1 Eric V. Anslyn,1 Kevin N. Dalby1. 1 _The University of Texas at Austin, Austin, TX;_ 2 _Wuhan University, Wuhan, Hubei, China_.

The activity of protein kinases in biological samples is typically estimated by determining the phosphorylation status of either the kinase itself, or a known cellular substrate. This is typically evaluated by multi-step western blotting, or proteomics procedures. Such procedures typically provide qualitative estimates of the modifications in question. While immunoblotting and proteomics procedures can be used in a quantitative manner, their complicated workflow diminishes reproducibility. Futhermore, modifications do not necessarily correlate closely with a protein kinase's activity. The motivation for this work was to assess the potential of a peptide array to quantify ERK activity in cancer cell lines and tumor samples, without the need to suppress related kinase activities. This work shows that a library of cross-reactive peptide-based biosensors, along with chemometric analysis can be used to profile a kinase activity in cancer cell lines. Significantly, the array is suitable for quantifying unknown levels of ERK activity in unfractionated cancer cell lysates and tumor samples using a multivariate regression model. The predicted values provided by our model were found to be comparable to those obtained by immune complex kinase assays.

#2173

Developing a high-throughput, high content screening technology to identify new anticancer drug treatments.

Andrew T. Milcarek, Kevin Daus, Mary L. Alpaugh. _Rowan University, Glassboro, NJ_.

The high attrition rate of new chemotherapeutic drugs represents the single most important challenge in anticancer drug development. With only one drug out of 10, 000 test compounds gaining FDA-approval, attrition is often attributed to failure of in vitro drug efficacy to translate to clinical success. The overall drug discovery and development pipeline can be dramatically improved with new, innovative assays of high pharmacological relevance that can shorten the discovery/preclinical phase and ensure rapid translation to clinic. We report the development of a three-dimensional phenotypic drug screening platform based on spheroidsMARY-X. The tight, compact spheroidsMARY-X form spontaneously due to innate molecular determinants with proliferative cells on the outer periphery and a dormant tumor cell core. These features mimic the intratumoral biological complexities of tumor tissue and thus provide a relevant in vitro model for drug discovery with a high degree of predictive value. Significantly, this model allows analysis of drug targeting of dormant tumor cells, the difficult to treat resistant or highly refractory cell population. Drug response can be reliably assessed using brightfield microscopy and can be quantified by measuring spheroid circularity (i.e., dissolution index). Evaluation of soluble factor concentration is performed using molecular rotors through restricted intramolecular rotation upon protein binding with a subsequent quantum yield. Overall, we present a 3-dimensional anticancer assay that provides a rapid, efficient, relevant platform for the evaluation of drug response and allows identification of small molecule hits in both proliferative and more importantly, dormant tumor cells, the major cause of relapse and recurrent metastatic disease.

#2174

A solution for simultaneous DNA & RNA extraction from single tissue without lysate splitting.

Han Wei. _Beckman Coulter, Indianapolis, IN_.

Extraction of intact and high quality gDNA or RNA is one of the most common procedures in molecular biology labs. With the advance of NGS, scientists can analyze both genomic and transcriptomic information from the same sample. Techniques that offer the ability to isolate both DNA and RNA from a single biological sample are crucial for this application. There is still great need for improved protocols in this area especially for precious and low-yield samples. Currently, most researchers use TRIzol® to isolate RNA and DNA from the same tube by extracting DNA from the organic phase and RNA from the aqueous phase. Although this method can extract high-quality RNA, the DNA yield is often low. With many biological samples in short supply, it is crucial to maximize yields. We report here using Beckman Coulter reagents, a protocol to isolate both DNA and RNA from the same fresh/frozen sample. The method utilizes size selection to separate DNA and RNA. We tested this method on 4 different frozen mouse tissues and report consistent extraction performance across all 4 tissue types. There are two advantages for this method: (1) There is no need to split a lysate for separate preparation of DNA/RNA; thus, researchers will not lose any portion of DNA or RNA. (2) Due to the different size distributions between DNA and RNA, this method offers quick and efficient extraction.

#2175

Synthetic antigen receptor mesenchymal stem cells (SAR-MSCs) targeting perlecan for drug delivery to ovarian cancer.

Susheel Kumar Nethi, Dristhi Sehgal, Shen Cheng, Jayanth Panyam, Swayam Prabha. _University of Minnesota, Minneapolis, MN_.

Mesenchymal stem cells (MSCs) can be engineered with polymeric nanoparticles for tumor-targeted delivery of small molecule drugs. Such nano-engineered MSCs have demonstrated exciting anticancer activity in multiple ovarian cancer models. Despite significantly improved delivery of chemotherapeutics to tumor tissues, non-specific accumulation of MSCs in clearance organs remains a concern. Paralleling the concept of CAR-T cells, we advance here a strategy for synthetic modification of MSCs with antibodies targeting specific antigens overexpressed on cancer cells. Our approach consists of stably incorporating recombinant protein G (PG) on the surface of MSCs, followed by binding of a full-length IgG to the PG handle. Because protein G binds to the Fc region of IgG, antigen-binding affinity of the antibody is conserved. We have previously shown the overexpression of perlecan (HSPG2) on ovarian cancer cells and its correlation to poor patient survival. In the current study, we investigated the incorporation of anti-perlecan antibody on the surface of nano-engineered MSCs. The anti-perlecan IgG antibody was first derivatized with palmitic acid (PA), which was then used to insert PG on MSCs cell membrane. We characterized the PAPG derivative by LC/Q-TOF/MS. Flow cytometry and confocal microscopy were used to confirm the incorporation of fluorescently labeled PAPG handle on MSC surface. Similarly, the binding of fluorescently labeled anti-perlecan IgG to PAPG-functionalized MSCs was confirmed by flow cytometry. We are currently investigating the anticancer efficacy of paclitaxel-loaded, anti-perlecan SAR-MSCs using in vitro and mouse models of ovarian cancer.

#2176

Topical delivery of carvedilol-loaded transfersomes for photoprotection and skin cancer prevention.

Mengbing Chen, Mengbing Chen. _Western University of Health Sciences, Pomona, CA_.

Ultraviolet (UV)-induced skin cancer is the most common cancer diagnosed in the United States and remains one of major public health concerns. Thus, there is an urgent need for the development of more effective strategies to prevent and treat skin cancer. We have previously demonstrated that carvedilol, an FDA approved β-blocker for cardiovascular diseases, could prevent UV-induced skin cancer both in vitro and in vivo. However, the β-adrenergic blockade effects of carvedilol might be a barrier for its repurposing as a cancer chemopreventive agent due to its cardiovascular side effects. In this work, we aimed to design a novel topical formulation, namely carvedilol-loaded transfersomes, which could maintain anti-cancer activities but avoid systemic cardiovascular effects. Carvedilol-loaded transfersomes were prepared using different phospholipids and surfactants at various ratios by a thin-film hydration method. Following optimization of preparation, the transfersomes were characterized in terms of particle size, zeta potential, and encapsulation efficiency. Skin permeation studies were performed using Franz vertical diffusion cells and skin dissected from SKH-1 hairless mice to examine the permeability of carvedilol. An optimal formulation was identified to compose carvedilol, Soy PC, and Tween-80 at a ratio of 1:3:0.5. The particle size, zeta potential and encapsulation efficiency for this formulation were determined to be 162.9±9.6 nm, 17.1±0.3 mV and 47.9±3.5%, respectively. Skin permeation studies showed that the specific carvedilol-loaded transfersomes could not penetrate through the skin layers in comparison with free carvedilol dissolved in acetone. Taken together, these results suggest that the transfersomal formulations of carvedilol may have desirable physical and biological characteristics for skin retention and local action. Ongoing studies are focused on further improvement of encapsulation efficiency and determination of its effectiveness against skin cancer.

#2177

Streptavidin-pHast: A readily conjugatable, pH-sensitive dye to screen for internalization.

Patrick A. Shramm, Leonardo Ancheta. _CytoLogistics, San Diego, CA_.

Quick and efficient screening of targeting agents that internalize effectively is vital for determining their suitability as potential therapeutics. Some of the most recent successes in the treatment of cancers have been from antibodies to cell surface proteins that are responsible for tumor cell proliferation. Examples are Cetuximab (target: EGFR) approved for colorectal cancer, and Trastuzumab (target: HER2) for breast cancer. These antibodies have more than one effect on the cancer cell, but one of the most important is that, upon binding to the cell surface antigen, the complex is internalized. As such, the down-regulated cell surface protein no longer plays a role in cancer cell division. Here we describe a method for determining internalization of cell surface molecules by targeting agents using a pH-dependent fluorescent reporter cross-linked to streptavidin. Streptavidin is a tetrameric protein (molecular weight 53 kDa in its recombinant form), with each subunit able to bind a single biotin molecule. The bond between streptavidin and biotin is rapid and essentially non-reversible, unaffected by most extremes of pH, organic solvents, and denaturing reagents. It is the strongest known noncovalent biological interaction (Ka = 1015 M-1) between protein and ligand. A variety of molecules, including lectins, proteins, and antibodies, can be biotinylated and reacted with streptavidin-labeled probes or other detection reagents for use in biological assays. The fluorescence from this reporter increases intensity as the pH of its surroundings becomes more acidic, as demonstrated when exposed to the environment inside a cell (thereby providing evidence of internalization). Here we describe methods that can be used to explore candidates as cancer therapeutics in a quick, reliable and reproducible manner.

#2178

Targeted delivery of chemotherapeutic agents employing nanovesicles effectively reduced intrahepatic tumors.

Joseph George, Nobuhiko Hayashi, Takashi Saito, Mikihiro Tsutsumi, Mutsumi Tsuchishima. _Kanazawa Medical University, Hepatology, Uchinada, Ishikawa 920-0293, Japan_.

Background and Aims: Currently there is no appropriate method for effective and targeted delivery of chemotherapeutic agents to tumor sites. The conventional chemotherapy is afflicted with lack of effective drug delivery to the tumor site, unwanted injury to actively dividing healthy cells, and numerous side effects due to toxicity. The present study was aimed to develop a biologically derived nanoparticle based drug delivery system for the effective treatment of primary hepatic tumors.

Methods: Intrahepatic tumors were induced in immunosuppressed mice using PLC/PRF/5 hepatocellular carcinoma (HCC) cells stably transfected with a mammalian expression vector carrying luciferase gene for in-vivo imaging. Nanovesicles ranging from 80-150 nm were isolated from bovine skim milk using ultracentrifugation and characterized using nanoparticle tracking analysis (NTA) and electron microscopy. Purified nanovesicles were loaded with chemotherapeutic agents and antisense oligonucleotides for grossly deregulated microRNAs specific to HCC. Animals with uniform intrahepatic tumors were injected with nanovesicle loaded anticancer agents through tail vein. Tumor regression was monitored weekly using in-vivo imaging system and the animals were maintained up to 8 weeks.

Results: Bioluminescence based in-vivo imaging demonstrated significant reduction of tumor size in treated animals compared to untreated controls. There was increased survival rate and complete absence of metastasis in all treated animals. Untreated mice developed large intrahepatic tumors, which also metastasized to other organs. There were no side effects or immunological reactions in animals injected with plain nanovesicles without loading of any anticancer agents.

Conclusions: The present study demonstrated that milk derived nanovesicles can be effectively used for successful delivery of anticancer agents into intrahepatic tumors. Nanovesicle based targeted drug delivery system could be an effecient method for the treatment of primary hepatic tumors.* Presenting author E-mail: georgej@kanazawa-med.ac.jp 

### Novel Therapeutics and Pathways

#2179

Antitumor activity of JNJ-63576253 (TRC253), a small molecule antagonist of F877L mutant and wild-type androgen receptor.

Tammy L. Bush,1 Georges Habineza Ndikuyeze,1 Gilles Bignan,1 Jonathan Branch,1 Janine Ondrus,1 Yifan Shi,1 Leopoldo Luistro,1 James Hastings,1 Joseph Erhardt,1 Ian Hickson,2 Shefali Patel,1 Peter Connolly,1 Zhuming Zhang,1 James Bischoff,3 Brent Rupnow,1 Marco Gottardis,1 Kathryn Packman1. 1 _Janssen Research and Development, Spring House, PA;_ 2 _Newcastle University, Newcastle upon Tyne, United Kingdom;_ 3 _Roche, Grenzacherstrasse, Basel, Switzerland_.

Androgen receptor (AR) antagonists have transformed prostate cancer patient care by targeting a key nodal point in tumor cell signaling. However, despite the impressive clinical activity of first- and second-generation antiandrogens, acquired resistance frequently emerges. Point mutations in the ligand-binding domain of AR, such as phenylalanine to leucine at position 877 (ARF877L), account for 10-20% of resistance. Such mutations are characterized by receptor activation, rather than inhibition, by first- and second-generation antiandrogen therapeutics.

JNJ-63576253 is a potent, high affinity competitive binder of wild type and mutant AR, including F877L. JNJ-63576253 blocks AR nuclear translocation, AR binding to DNA, and AR-dependent transcription. JNJ-63576253 inhibits the proliferation of androgen receptor driven prostate cancer cell lines, including those bearing ARF877L.

In the Hershberger assay in male Sprague Dawley rats, oral administration of JNJ-63576253 inhibited androgen sensitive organ (ASO) development in a dose-dependent manner. In male SHO mice bearing LNCaP xenografts with either wild-type or ARF877L, daily treatment with 30 mg/kg JNJ-63576253 treatment resulted in statistically significant antitumor activity, whereas second-generation antiandrogen enzalutamide had no antitumor efficacy in the LNCaP ARF877L mutant model.

Janssen and Tracon Pharma have entered a strategic licensing collaboration, whereby Tracon possesses exclusive rights for clinical development of JNJ-63576253 (now called TRC253). Tracon has entered TRC253 into Ph1/2A clinical evaluation in metastatic castration-resistant prostate cancer patients.

#2180

AMG 176 exhibits robust antitumor activity in combination with standard of care agents in models of acute myeloid leukemia.

Sean Caenepeel, Brian Belmontes, Tao Osgood, Elaina Cajulis, Angela Coxon, Jude Canon, Paul E. Hughes. _Amgen, Thousand Oaks, CA_.

Acute myeloid leukemia (AML) is a heterogeneous malignancy involving the clonal expansion of abnormal myeloid progenitor cells. The anti-apoptotic BCL-2 protein family member MCL1 has been implicated in AML pathogenesis. AMG 176 is a potent and selective MCL1 inhibitor currently being evaluated in an AML Phase I clinical trial. Standard of care (SOC) treatment for AML involves "7+3" induction therapy with cytarabine and an anthracycline (i.e. doxorubicin). In patient's ineligible for conventional induction therapy, the hypomethylating agent decitabine is a well-tolerated alternative. Here we investigate the activity of AMG 176 and AM-8621, a closely related analog of AMG 176 with similar potency and selectivity, in combination with SOC therapies in preclinical models of AML. We profiled a panel of AML cell lines (MOLM-13, MV-4-11, GDM-1 and EOL-1), testing AM-8621 in combination with cytarabine, doxorubicin and decitabine in 3-day viability assays. Evidence for synergistic activity was detected with each of the combinations across subsets of the cell lines. A time course of AM-8621 treatment revealed a rapid induction of caspase 3/7 activity, with significant increases observed within the first 2-4 hours of treatment while longer duration treatment (>8 hours) was required to activate caspase 3/7 with SOC agents. Combined treatment with AM-8621 and SOC agents exhibited significant improvements in caspase 3/7 activity beyond either single agent alone. To provide mechanistic insight into the ability of SOC agents to sensitize AML cell lines to AM-8621 treatment, signaling experiments were performed to characterize changes in BCL-2 protein family members following treatment with SOC agents. Clear changes in the levels of anti-apoptotic BCL-2 family members (MCL1, BCL-2 and BCL-XL), BH3-only domain containing proteins (BIM, NOXA, PUMA) and the executioner protein BAK were observed, providing insight into potential mechanisms of priming cells to MCL1 inhibition. Combinations of AMG 176 and each of the SOC therapies were also tested in a MOLM-13 orthotopic xenograft model of AML. Mice were treated with combinations of AMG 176 and cytarabine, decitabine or doxorubicin and compared against the single agent therapies. Significant reductions in tumor burden compared to single agent treatments were observed with the AMG 176 + doxorubicin (30% regression) and AMG 176 + decitabine (98% tumor growth inhibition) combinations. These data highlight the promise of combining AMG 176 with SOC therapies in the setting of AML.

#2181

**A novel CXCR4 antagonist strongly mobilizes hematopoietic stem cells** in vivo **.**

Xiao Fang,1 Qian Meng,1 Xiong Fang,1 Yujia Mao,1 Yan Xu,1 Jing An,2 Ziwei Huang1. 1 _Tsinghua University, Beijing, China;_ 2 _University of California, San Diego, La Jolla, CA_.

Introduction: The transplantation of hematopoietic stem cells (HSCs) has been the standard of care for the treatment of chemotherapy sensitive relapsed acute leukemia. HSCs mainly reside in the bone marrow with only few HSCs circulating in the peripheral blood (PB), due to homing effect mediated by the interaction of chemokine receptor CXCR4 expressed on HSCs with chemotactic ligand SDF-1α rich in the bone marrow. It is clinically desirable to enhance the number of HSCs in the PB for collection during the transplantation procedure. Since SDF-1α/CXCR4 axis and interaction mediates the homing and retention of HSCs in the bone marrow, our laboratory has recently discovered and developed a novel small molecule, HFX51116, that is capable of binding CXCR4 and blocking its interaction with SDF-1α, raising the possibility that this compound can be used for releasing HSCs from the bone marrow to the PB useful for the transplantation of HSCs.

Method and Results: In vitro, there was almost no toxicity of HFX51116 at high concentrations in bone marrow stromal cells that were originally extracted from rats. In addition, HFX51116 was stable in rat serum. In vivo, we tested the short-term and long-term mobilization efficacy of HFX51116. For the short-term efficacy study, we determined the dynamic change of efficacy at different times and doses of HFX51116 in HSC mobilization assays. Furthermore, different strains of mice, such as C57/BL6, C3H/HEJ and DBA/2, were sensitive to HFX51116. HFX51116, when used in combination with G-CSF, had higher efficacy than G-CSF alone. For the long-term repopulation study, we employed the CD45.1/CD45.2 competitive assay to demonstrate that the HSCs mobilized by G-CSF and HFX51116 had the repopulation ability when compared with the bone marrow cells.

Conclusion: Our studies have demonstrated high HSC mobilization efficacy of HFX51116 in vivo and the promise of this new lead compound for further preclinical and clinical studies.

#2182

Preclincial in vitro and in vivo combination therapies for mutant IDH1R132 tumors with BAY 1436032.

Stefan Kaulfuss,1 Julia Zaman,2 Anuhar Chaturvedi,3 Holger Hess-Stumpp,1 Jennifer Höde,1 Ricarda Biber,1 Renan Borowicz,1 Michael Heuser,3 Andreas von Deimling,4 Stefan Pusch2. 1 _Bayer AG, Berlin, Germany;_ 2 _Ruprecht-Karls-University, Heidelberg, Germany;_ 3 _Hannover Medical School, Hannover, Germany;_ 4 _German Cancer Research Center (DKFZ), Heidelberg, Germany_.

Background: Isocitrate dehydrogenase (IDH) mutations are drivers of different tumors. The IDH1R132 mutation is frequently found in glioma (80%), chondrosarcoma (CS, 45%), intrahepatic cholangiocarcinoma (ICC, 20%), acute myeloid leukemia (AML, 7%), and with low incidences in other solid tumors. Although clinical therapies of mutant IDH inhibition were recently approved for the treatment of AML, few patients benefit from a single therapy with a mutant IDH inhibitor and most of them relapse. Here we report preclinical in vitro and in vivo data on combination therapies of the mIDH1R132 inhibitor BAY 1436032 with standard of care chemotherapies.

Methods: in vitro: The sarcoma cell line HT1080 (IDH1R132C), together with two IDH1 wildtype sarcoma cell lines, the SW1353 (IDH2R172S) and SW872 (IDHwt) cell lines, were treated with BAY 1436032 alone and in combination with different standard of care drugs. in vivo: The patient derived xenograft (PDX) ICC model LIXFC2084 (IDH1R132L), the melanoma PDX model MEXF1341 (IDH1R132C) (MEXF1341), the PDX AML model 13PB020 (IDH1R132C) and a sarcoma cell line derived xenograft model HT1080 (IDH1R132C) were included. All models were treated with BAY 1436032 alone and in combination with different standard of care (SOC) drugs.

Results: The in vitro treatment with BAY 1436032 unexpectedly increased growth in HT1080 (IDH1R132C) cells with no effect to the control cell lines SW1353 and SW872. In contrast, in vivo, combination of BAY1436032 with everolimus had additive anti-tumor efficacy and was synergistic with cisplatin or the MEK inhibitor refametinib in the treatment of HT1080 xenografts. The combination of BAY 1436032 and refametinib was also additive in the treatment of a melanoma xenograft with IDH1R132C mutation. Another additive efficacy of BAY 1436032 and gemcitabine or cisplatin could be observed in an ICC xenograft model and further in the combination with everolimus in the same model. The combination of BAY 1436032 together with azacitidine in a PDX AML model was also synergistic.

Conclusion: These results highlight the combinatorial potential of the mIDH1R132 inhibitor BAY 1436032 with standard of care therapy and the importance of microenvironmental factors, which has to be taken into account when dealing with mutant IDH tumors.

#2183

Protective autophagy elicited by RAF®MEK®ERK inhibition suggests a treatment strategy for pancreatic cancer.

Conan G. Kinsey, Soledad A. Camolotto, Amelie M. Boespflug, Katrin P. Guillen, Mona Foth, Amanda Truong, Sophia S. Schuman, Jill E. Shea, Michael T. Seipp, Jeffrey T. Yap, Lance D. Burrell, David H. Lum, Jonathan R. Whisenant, G. Weldon Gilcrease, Courtney C. Cavalieri, Kaitrin M. Rehbein, Stephanie L. Cutler, Kajsa E. Affolter, Alana L. Welm, Bryan E. Welm, Courtney L. Scaife, Eric L. Snyder, Martin McMahon. _University of Utah, Salt Lake City, UT_.

Pancreatic ductal adenocarcinoma was responsible for ~43,000 deaths in the USA in 2017 and is the epitome of a recalcitrant cancer driven by an, as yet, pharmacologically intractable oncoprotein, KRAS. Although the clinical picture remains grim, the mechanisms by which key alterations in tumor suppressors and proto-oncogenes contribute to PDA have been dissected. Downstream of KRAS, the RAF→MEK→ERK signaling pathway plays a central role in pancreatic carcinogenesis. However, to date, pharmacological inhibition of this pathway has provided no clinical benefit to PDA patients. Here we show that inhibition of KRAS→RAF→MEK→ERK signaling in PDA cell lines elicits autophagy, a process of cellular recycling that protects pancreatic cancer cells from the potentially cytotoxic effects of KRAS pathway inhibition. Furthermore, combined inhibition of MEK1/2 plus autophagy displays synergistic anti-proliferative effects against PDA cell lines in vitro, and promotes regression of xenografted patient-derived PDA tumors in mice. Finally, treatment of a PDA patient with the combination of trametinib plus hydroxychloroquine resulted in a partial, but nonetheless striking disease response. These data suggest that this combination therapy may represent a novel strategy to target RAS-driven cancers such as PDA.

#2184

Preclinical verification of the efficacy of targeting peptides linked liposomal nanoparticles for therapy of hepatocellular and nasopharyngeal carcinomas and breast cancer.

Chin-Tarng Lin,1 Cheng-Der Wu,1 Jen-Chien Lee,1 Han-Chung Wu,2 Chung-Wei Lee,1 Ming-Chieh Hsu1. 1 _National Taiwan Univ. College of Medicine, Taipei, Taiwan;_ 2 _Academia Sinica, Taipei, Taiwan_.

The efficacy of systemic cytotoxic chemotherapy has been widely assessed in patients with advanced hepatocellular carcinoma (HCC). For example, doxorubicin is the most commonly studied chemotherapeutic agent for HCC. However, it has been shown to have a response rate of only 10-20% in clinical trial. In addition, its potential benefit has been reduced by the related adverse effect. So far, the multikinase inhibitor, sorafenib, is considered to provide survival benefit over supportive care. However, the long term prognosis of those cancer patients still remain poor. Therefore, in the present experiment, we proposed to use the so-called peptide targeting chemotherapy to overcome the adverse event in the conventional targeted chemotherapy. In order to perform this experiment, we have construct some specific peptides which can bind specifically to the cancer cells and cancer vascular endothelia by using a phage displayed 12-mer random peptide library. We have obtained 3 different peptides and one control peptide. Each contains 12 amino acids: a. L-peptide: RLLDTNRPLLPY (anti-different cancer cell membrane); b. control peptide: RLLDTNRGGGGG; c. SP-94-peptide: SFSHHTPILP (anti-NPC tumor cell and hepatoma cell membranes) and d. PC5-52-peptide: SVSVGMKPSPRP (anti-tumor endothelia). Those L-peptide (L-P), SP-peptide (SP-P), PC5-52-peptide and a control peptide (C-P) were linked to liposomal iron oxide nanoparticles; and also used those peptides to link liposomal doxorubicin (L-D). Using peptide linked liposomal iron oxide, we can localize the peptide targeted tumor cells and tumor endothelia, and then we used those peptides linked liposomal doxorubicin to treat SCID mice bearing different cancer xenografts. Our results showed that when L-P-L-D containing 2mg/kg of SCID mouse body weight was used to treat xenografts bearing SCID mice, the tumor could be well controlled, and no specific adverse event was seen. However, when the control peptide was used to replace the specific peptide, the xenograft size was also shown decrease, but the visceral organs revealed marked apoptotic change. In conclusion, the specific peptides linked liposomal doxorubicin nanoparticles can be used for treatment of SCID mice bearing cancer xenografts with minimal adverse event, especially in the SCID miceγspecies (NGS), which show a remarkable tumor suppression.

#2185

Efficacy of Rac/Cdc42 inhibitors in multiple cancer models.

Jean F. Ruiz Calderon,1 Linette Castillo-Pichardo,2 Irmaris Lopez-Lopez,3 Suranganie Dharmawardhane1. 1 _UPR-Medical Science Campus, San Juan, PR;_ 2 _Universidad Central del Caribe, Bayamon, PR;_ 3 _University of Puerto Rico Rio Piedras Campus, San Juan, PR_.

The RhoGTPases Rac and Cdc42 are excellent therapeutic targets for inhibiting metastasis because these homologous proteins regulate numerous cellular functions that promote tumorigenesis, angiogenesis, invasion and therefore, metastasis. Also, Rac1, Cdc42, and their effector p21-activated kinase (PAK) are overexpressed in multiple cancer types, including breast, gastric, and pancreatic, and are associated with higher mortality rates. Furthermore, Rac and Cdc42 are activated through the signaling of oncogenic cell surface receptors such as EGFR and HER2, therefore hyperactivating their signaling pathways. Our laboratory has developed two small molecules targeting Rac and Cdc42, Ehop-016 and MBQ-167, that inhibit Rac1 and Cdc42 with IC50s in the low micro- and nano-molar concentrations, respectively (Montalvo-Ortiz et al. 2012, J Biol Chem; Humphries-Bickley et al. 2017, Mol Cancer Therap), making them the most potent Rac/Cdc42 inhibitors developed to date. We hypothesize that targeting Rac1, 3, and Cdc42 is a rational strategy to inhibit aggressive TNBC, pancreatic and gastric cancer. To test this hypothesis, we treated the TNBC cell lines MDA-MB-231, MDA-MB-468 and 4T1 (murine breast cancer), gastric cancer cells NCI-N87, and pancreatic cancer cells MIA-PaCa-2 with 4uM and 500nM of Ehop-016 and MBQ-167 respectively, and measured cell viability. Both TNBC cell lines and the pancreatic cancer cell line exhibited a drastic reduction in cell viability (>60%) after 48hrs of treatment Ehop-016 or MBQ-167. Additionally, experimental and spontaneous metastasis assays were performed with TNBC and gastric cancer xenografts in immunocompromised and immunocompetent mice. Treatment with 1 mg/kg BW MBQ-167 3X week resulted in reduced tumor growth by 60-90% in TNBC and gastric cancer models, and metastasis inhibition by ~90% in the MDA-MB-231 model. In the syngeneic experimental metastasis study with 4T1, both Ehop-016 and MBQ-167 reduced lung metastasis in comparison to the vehicle treated group. Moreover, these compounds were not toxic to both immunocompromised and immunocompetent mice, as observed by no significant changes in weight and liver enzymes. Therefore, both Ehop-016 and MBQ-167 are promising targeted therapeutics against cancers that overexpress Rac, Cdc42, and PAK.

#2186

The novel nitric oxide and hydrogen sulfide releasing anti-cancer agent, NBS-1120, protects the GI tract from the ulcerogenic activity of aspirin and naproxen.

Jan J. Scicinski,1 Mark El-Miniawi,2 Tasneem Zahran,2 Khosrow Kashfi2. 1 _Avicenna Therapeutics, Inc, Raleigh, NC;_ 2 _City University of New York School of Medicine, New York, NY_.

NBS-1120 is a member of the novel 'enhanced NSAID' class of anti-cancer agents that release nitric oxide and hydrogen sulfide targeting tumor inflammatory pathways while inducing ROS, caspases and apoptosis and inhibiting proliferation, beta catenin and FOXM1, ultimately impacting the regulation of NF-kB. NBS-1120, a derivative of aspirin that is devoid of GI side effects, demonstrated potent anti-cancer activity in vitro and in vivo while maintaining all the classical pharmacological/therapeutic activities of aspirin. Cancer pain takes many forms and is managed by a variety of pain medications that include NSAIDs. Because of their anti-inflammatory activity, NSAIDs may be particularly helpful in moderate and severe pain arising from bone cancer or metastatic lesions. A patient may be on a low-dose aspirin regimen as an antithrombotic but may need an NSAID such as naproxen for moderate to severe cancer pain. In these situations, GI side effects are a major concern. Commonly, a proton pump inhibitor (PPI) is co-administered to minimize GI injury. While significantly reducing upper GI side effects, recent animal studies suggest that suppression of acid secretion leads to exacerbation of NSAID-induced small intestinal injury and bleeding. Here, we investigated whether NBS-1120, itself protective against GI injury, could also protect the GI tract from the ulcerogenic activity of aspirin and naproxen. In an acute study, male rats (n=4/gp), were treated with naproxen; aspirin then naproxen; or NBS-1120 then naproxen. 6 h post naproxen, rats were euthanized, stomachs removed, cut along the greater curvature and the number and length (mm) of all hemorrhagic lesions (ulcer index, UI) recorded. In a chronic study, animals were pretreated with Lansoprazole then with aspirin and naproxen or NBS-1120 and naproxen. UI was determined for stomach and jejunum as above. Results: Acute studies UI: naproxen = 35.5±7.2 mm; aspirin + naproxen = 47.8±8.8 mm; NBS-1120 + naproxen = 4.5±10 mm. Chronic studies: PPI alone = 0 mm; PPI + aspirin + naproxen = 10±5 mm; PPI + NBS-1120 + naproxen = 0 mm. Jejunum ulceration: PPI alone = 0 mm; PPI + aspirin + naproxen = 15.8±6.8 mm; PPI + NBS-1120 + naproxen = 2±3 mm. Aspirin + naproxen result in significant upper GI damage; this was mitigated to a considerable degree by pretreatment with Lansoprazole. However, this regimen moved the problem to the jejunum, causing bleeding. In both acute and chronic studies NBS-1120 protected the GI tract against naproxen and/or aspirin injury. Significantly, in a chronic study with Lansoprazole, NBS-1120 reduced naproxen and aspirin induced jejunum ulceration. Molecular targets affected by these treatment regiments are currently under investigation. These data suggest that the anti-cancer agent, NBS-1120, can be combined safely with other NSAIDs and protects against GI injury.

#2187

Tryptophan Metabolism Plays a Central Role in Immunosuppression.

Sandaruwan Geeganage,1 Lillian Sams,1 James Henry,1 Frank Dorsey,1 Kenneth Roth,1 Alexander Nikolayev,1 Karim Benhadji,1 Raymond Gilmour,1 Ana Cerezo,2 Sandra Peregrina,2 Gloria Martínez-del Hoyo,2 Ramón Campos-Olivas,2 Juan Manuel Funes,2 Laura Diezma,2 Susana Velasco-Miguel2. 1 _Eli Lilly and Company, Indianapolis, IN;_ 2 _Spanish National Cancer Research Center, Spain_.

Tryptophan metabolism plays a central role in immunosuppression through the local depletion of tryptophan with the concomitant production and accumulation of kynurenine, both of which are immunosuppressive. Indoleamine 2,3-dioxygenase 1 (IDO1), a heme-dependent enzyme, catalyzes the initial and rate-limiting step of the kynurenine pathway. Tumor cells selectively upregulate IDO1 as an immune-evasion mechanism either through intrinsic expression of IDO1, or in response to IFN-γ, a cytokine secreted by immune cells during an active immune response.

In addition to the immuno-suppressive role of IDO1 and kynurenine, we investigated their additional roles in tumor cells. Here we describe an NMR-based readout assay for IDO1 activity, in which we are able to trace the extra- and intracellular destination of the single tryptophan carbon groups. This assay allowed us to detect contribution of the tryptophan catabolism to purine synthesis in tumor cells, and suggested further roles of tryptophan catabolism in tumors, apart from kynurenine production, that contribute to the modulation of tumor growth. We detected the incorporation of labeled tryptophan carbon units in intracellular purine pools. Our highly potent, orally available, and CNS penetrant small molecule inhibitor of IDO1 (LY3381916) inhibits this incorporation of tryptophan-derived one carbon units into purines in vitro and in vivo. This activity leads to pre-clinical efficacy and may be an added advantage of inhibiting IDO1 in cancer.

#2188

Comparing effects of small molecular weight compounds on proliferation and chemotaxis of pancreatic cancer cells.

Masahiro Yamamura,1 Akira Yamauchi,1 Naoki Katase,2 Masakiyo Sakaguchi,3 Yosuke Katata,1 Hiroaki Tanioka,1 Makoto Okawaki,1 Takeshi Nagasaka,1 Yoshiyuki Yamaguchi1. 1 _Kawasaki Medical School, Kurashiki, Japan;_ 2 _Nagasaki University, Graduate School of Biomedical Sciences, Nagasaki, Japan;_ 3 _Okayama University, Graduate School of Medicine, Okayama, Japan_.

Background & Aims: The new therapy for pancreatic cancer which has the poorest prognosis among malignant tumors is high demand nowadays. The overall 5-year survival rate in all stages of this type of cancer is less than 5%, even with the multimodality therapy, including surgery, chemotherapy and radiotherapy. Although gemcitabine (GEM) is a key drug in the treatment of advanced pancreatic cancer, the therapeutic effect is limited because of drug resistance. Heat shock protein 90 (HSP90) is known to be overexpressed in several types of cancer cells, and its inhibition has shown promise in the treatment of solid malignancies. Src kinases work as a signal switch for multiple molecular signal transduction pathways. The objective of this study is to evaluate an antitumor effect of three kinds of small molecular weight compounds (antimetabolite Gemcitabine, HSP90 inhibitor NVP-AUY922, and Src kinase inhibitor Dasatinib) on tumor growth and chemotaxis in pancreatic cancer cell lines. Methods: Pancreatic cancer cell lines were treated with each of the 3 compounds to examine the effect on proliferation assay, apoptosis assay, Western blotting, and chemotaxis assay. Results: Of these compounds, HSP90 inhibitor inhibited cell proliferation in pancreatic cancer cells best in a dose- and time-dependent manner. Growth inhibitory effect of HSP90 inhibitor for pancreatic cancer cells were highest of the three. Further, HSP90 inhibitor inhibited the growth of gemcitabine sensitive and resistant pancreatic cancer cell lines. With flowcytometry and immunostaining, HSP90 inhibitor had higher apoptosis-inducing effect than gemcitabine. In the chemotaxis assay, HSP90 inhibitor inhibited the migration of pancreatic cancer cell best in the three compounds. In western blotting, HSP90 inhibitor suppressed the expression of multiple receptor tyrosine kinase proteins as well as the signal transduction in PI3K-Akt and RAS-MAPK pathways. Conclusion: In this study, HSP90 inhibitor was the best of 3 small molecular compounds and has proven effective in gemcitabine sensitive and resistant pancreatic cancer. The HSP90 inhibitor is useful as an antitumor drug by inhibiting proliferation and chemotaxis of pancreatic cancer cells through multiple kinase pathways.

#2189

Evaluating efficacy of repurposed drugs in treatment of glioblastoma.

Tristan Neal, Nanyun Tang, George Reid, Sara Byron, Harshil Dhruv, Michael Berens. _Translational Genomics Research Institute (TGen), Phoenix, AZ_.

Glioblastoma (GB) is the most aggressive adult brain tumor with a devastating median survival time of about fourteen months post-surgery and standard of care therapy with radiation and temozolomide. The low incidence of GB is a dis-incentive to develop novel therapies. To overcome that obstacle, we investigated the efficacy of repurposing four FDA approved drugs known to cross the BBB, minocycline, propranolol, chlorpromazine, and metformin, to inhibit signaling and metabolism in GB cells. Minocycline is a tetracycline class broad spectrum antibiotic commonly used to treat severe acne and other skin infections. Propranolol is a beta blocker type heart medication primarily used to treat high blood pressure and irregular heartbeat. Chlorpromazine is a phenothiazine antipsychotic usually used for schizophrenia. Metformin is the most widely used first-line oral treatment for type-2 diabetes. Based on a literature survey, minocycline is expected to prevent the phosphorylation of STAT3, a transcription factor downstream of EGFR; propranolol is expected to disrupt EGFR trafficking; chlorpromazine is expected to target the mTOR/AKT pathway; metformin is believed to exploit vulnerabilities in cancer cell metabolism. Efficacy of minocycline in inhibiting EGFR-driven STAT3 activation was investigated using western blot analysis. Our results demonstrate that Minocycline effectively inhibits activation of EGFR-driven STAT3 in U373 glioma cells at 100μM. The ability of chlorpromazine to inhibit the mTOR/AKT pathway was similarly tested via western blot, which showed inhibition of phosphorylated Akt and S6 at 10μM. Efficacy of propranolol in perturbing EGFR trafficking was evaluated using flow cytometry and immunofluorescence, which depicted altered membrane-associated EGFR abundance. Finally, concentration-dependent inhibition of colony formation was tested for all four drugs. Propranolol and minocycline showed potential stimulatory effects at 10μM, but all drugs inhibited cell growth at 50μM and higher. Efficacy of these drugs in treatment of GB is being further evaluated using in vitro neurosphere cultures from patients identified as having the cellular vulnerabilities potentially targeted by these drugs. Successful completion of this project will lead to in vivo efficacy testing of these four drugs in orthotopic GB PDX models.

#2190

Repurposing the FDA-approved antiviral drug ribavirin as targeted therapy for nasopharyngeal carcinoma.

Andy S. Ding, Joshua Casaos, Sakibul Huq, Henry Brem, Nicolas Skuli, Betty Tyler. _Johns Hopkins University School of Medicine, Baltimore, MD_.

Purpose: Nasopharyngeal carcinoma (NPC) is a squamous cell carcinoma that is often diagnosed at an advanced stage, leading to poor disease-free and overall survival. Accumulating literature suggests that elevated protein expression of EZH2, eIF4E, and IMPDH correlates with poor prognosis in NPC. These three proteins are modulated by the FDA-approved antiviral drug ribavirin, which has recently been repurposed by our laboratory and others as a promising anti-cancer agent. Based on this intersection of molecular signature and drug targets, we investigated the potential use of ribavirin as a therapeutic agent for NPC.

Experimental Design: We assessed anti-neoplastic efficacy of ribavirin on human NPC cell lines in vitro using cellular growth assays, flow cytometry, and scratch wound assays. Mechanistic pathways involved were investigated using genomic expression datasets, western blots, and enzymatic activity assays. The effects of combining ribavirin with radiation were assessed via clonogenic assays and flow cytometry. Finally, we evaluated the effects of ribavirin on tumor growth in vivo using human cell line-derived xenograft models.

Results: Ribavirin significantly decreased NPC cellular proliferation and migratory capacity in addition to promoting cell cycle arrest and cell death. Modulation of the EZH2, Snail, eIF4E, and IMPDH pathways was observed in response to ribavirin treatment. Ribavirin significantly enhanced the cytotoxic effects of radiation therapy in NPC. Most importantly, ribavirin significantly reduced flank tumor growth in NPC xenograft models.

Conclusions: Our work suggests that ribavirin has potent anti-cancer effects in NPC and could represent a safe and promising addition to current NPC treatment regimens.

#2191

**Reduced clonogenic potential of patient-derived lung adenocarcinoma brain metastasis cells after** in vitro **application of Tumor Treating Fields (TTFields).**

Sharon K. Michelhaugh, Sam Kiousis, Sandeep Mittal. _Wayne State University, Detroit, MI_.

Up to 40% of all non-small cell lung cancer (NSCLC) patients will develop brain metastases during their disease, with ~25% of patients presenting with brain metastases at the time of their initial lung cancer diagnosis. For the latter patient population, median survival is a dismal 8 months. Systemic therapies are typically ineffective at treating the brain metastases, even in cases when the extracranial disease responds to the therapy. Clinical trials utilizing TTFields therapy in patients with lung cancer brain metastases are underway. For these trials, TTFields are delivered at 150 kHz, as that is the frequency that has been determined for the treatment of primary lung cancer. In contrast, for the FDA-approved treatment of the most common malignant primary brain tumor, glioblastoma, TTFields are delivered at 200 kHz. In our previous study, we found that 150 kHz TTFields decreased proliferation of patient-derived lung adenocarcinoma brain metastasis cells to a greater degree than 200 kHz. In this study, patient-derived lung adenocarcinoma brain metastasis cells were assessed for clonogenic potential after in vitro TTFields application at either 150 or 200 kHz and compared to untreated control cells. Methods: Use of patient tumor tissue for this study was approved by the Institutional Review Board and written informed consent was obtained from the patient. A lung adenocarcinoma brain metastasis tumor was collected immediately following microsurgical resection. A single-cell suspension from the tumor tissue was prepared by enzymatic and mechanical disruption. After 3-6 passages in vitro, cells were plated on plastic coverslips (5x104 each) in DMEM/F12 media supplemented with 10% fetal bovine serum. TTFields were applied at ~1.6 V/cm at 150 or 200 kHz (groups n=3-6) with one control group. Culture media was replaced every day. After 14 days of in vitro TTFields application, cells were assayed for lactase dehydrogenase (LDH) release into the media and cells were harvested and replated for the clonogenic assay. Control vs. TTFields-treated groups were compared with one-way ANOVA. Results: TTFields application reduced LDH in the media ~15% at 150 kHz and ~30% at 200 kHz (p=0.0011). In the clonogenic assay, cell counts were markedly reduced after TTFields in both the 150 kHz (142.4±120.1) and 200 kHz (457.2±431.9) compared to the controls (9136±2735; p=0.0007) although the post-hoc Tukey analysis found no statistical difference between the two TTFields-treated groups. Conclusions: Application of TTFields in vitro at 150 and 200 kHz reduced LDH release into the media as well as the clonogenic potential of the patient-derived lung adenocarcinoma brain metastasis cells.

#2192

**Concurrent Tumor Treating Fields (TTFields) and dexamethasone decrease proliferation and clonogenicity of patient-derived glioblastoma cells** in vitro **.**

Sharon K. Michelhaugh, Sam Kiousis, Sandeep Mittal. _Wayne State University, Detroit, MI_.

Background: TTFields are FDA-approved for treatment of newly-diagnosed and recurrent glioblastoma and extend progression-free survival and overall survival in newly-diagnosed glioblastoma patients. Dexamethasone is administered perioperatively to GBM patients to reduce tumor-associated edema and increased intracranial pressure. In addition, high dose corticosteroids may be required during TTFields therapy. In this study, a patient-derived GBM cell line from a newly-diagnosed glioblastoma patient was treated in vitro with TTFields ± dexamethasone followed by proliferation and clonogenic assays.

Methods: This study was approved by the Institutional Review Board and written consent obtained from the patient. A single-cell suspension was generated from tumor tissues obtained from the newly-diagnosed patient. Cells were cultured in DMEM/F12 media with 10% fetal bovine serum and gentamicin. Prior to TTFields initiation, cells were plated on plastic coverslips (1×104cells/coverslip; n=7-8/group) and incubated overnight. Then, TTFields were applied at 200 kHz with a field intensity of ~1.6 V/cm for 14 days. Cells were treated with TTFields with or without 50 µM dexamethasone. At the end of the TTFields application, proliferation was assessed with XTT assay and cells were harvested and replated for the clonogenic assay (n=3/group). XTT data and clonogenic assay cell counts were analyzed by one-way ANOVA with Tukey's post-hoc test.

Results: In patient-derived glioblastoma cells treated with TTFields and dexamethasone for 14 days, proliferation was significantly decreased compared to the dexamethasone-only and control groups (0.483±0.30; 1.33±0.18; 1.42±0.11, respectively, ANOVA p<0.001). Similarly, cell counts in the clonogenic assay were markedly decreased in the TTFields ± dexamethasone group compared to dexamethasone-only (1184±425.9; 46464±4580; p<0.05).

Conclusions: In glioblastoma cells from a newly-diagnosed patient, concurrent TTFields with dexamethasone produced a ~three-fold reduction of proliferation and reduced clonogenicity compared to control groups.

### Tyrosine Kinase and Phosphatase Inhibitors 2

#2193

AXL inhibitor TP-0903 attenuates AXL-TGFbeta Hippo signaling axis in lung adenocarcinoma cells.

Josephine Amalia Taverna,1 Chia-Nung Hung,1 Chun-Lin Lin,1 Pawel Osmulski,1 Meizhen Chen,1 Chiou-Miin Wang,1 Nicholas L. L. Lucio,1 Nameer Kirma,1 Chih-Wei Chou,1 Maria E. Gaczynska,1 Alia Nazarullah,1 Mark Wade,2 Lars Mouritsen,2 Tim Huang1. 1 _University of Texas Health Science Center at San Antonio, San Antonio, TX;_ 2 _Tolero Pharmaceuticals, Salt Lake City, UT_.

Background: Non-small cell lung cancer (NSCLC) is a molecularly heterogeneous disease with a high propensity for drug resistance and metastasis. AXL, a member of the Tyro3-AXL-Mer family of receptor tyrosine kinases, is a central regulator of epithelial-to-mesenchymal transition (EMT) and enables tumor cells to invade and acquire drug resistance. AXL is overexpressed in lung tumors, correlates positively with tumor invasion, drug resistance, and negatively predicts overall survival. We mechanistically interrogate the effects of AXL inhibitor TP-0903 on EMT in lung adenocarcinoma cells using transcriptomic and proteomic profiling.

Methods: Atomic force microscopy, Western blot analysis, RNA sequencing and mass cytometry (CyTOF) were all used to scrutinize the biomechanical properties, phenotypic, transcriptomic and proteomic profiles of A549 cells treated with 40nM TP0903 or shAXL knockdown.

Results: TP-0903 attenuates total AXL/AXL phosphorylation and blunts transcriptional responses to TGFβ-Hippo signaling by disrupting the transcriptional complexes formed by SMAD2/3, SMAD4, YAP1 and TAZ. AXL knockdown or TP-0903 reverses EMT phenotype and reduces migration potential in A549 and H2009 adenocarcinoma cell lines. CyTOF data also identified resistant clones that overexpress TGFβ receptor II, TAZ protein and display hybrid EMT phenotypes.

Conclusions: We are the first to report the interplay between AXL and TGFβ-Hippo signaling axis. TP-0903 study agent has excellent therapeutic promise in NSCLC and we speculate that TP-0903 drug can target epithelial to mesenchymal transitional states in lung cancer cells possibly through the inhibition of the AXL-TGFβ-Hippo signaling axis.

#2194

Preclinical pharmacological profiling of ACP-5862, the major metabolite of the covalent BTK inhibitor acalabrutinib, displays intrinsic BTK inhibitory activity.

Allard Kaptein,1 Terry Podoll,2 Gerjan de Bruin,1 Maaike Emmelot-van Hoek,1 Anouk de Jong,1 Bart van Lith,1 Niels Hoogenboom,1 Tjeerd Barf,1 Joseph Ware,3 J. Greg Slatter4. 1 _Acerta Pharma, Oss, Netherlands;_ 2 _IV/PO LLC, Seattle, WA;_ 3 _Acerta Pharma, South San Francisco, CA;_ 4 _Kartos Therapeutics, Bellevue, WA_.

Acalabrutinib is a potent and highly selective, oral covalent inhibitor of Bruton tyrosine kinase (BTK) that received accelerated approval for relapsed/refractory mantle cell lymphoma by the United States Food and Drug Administration in October 2017.

ADME studies in humans, rat and dog, revealed extensive metabolism of acalabrutinib. The major circulating metabolite (M27, ACP-5862) was produced by CYP3A oxidation, resulting in a pyrrolidine ring-opened metabolite, with the butynamide electrophile still present. We investigated the on-target BTK inhibition and kinase selectivity profile of ACP-5862 using the same biochemical and cellular assays previously employed to profile acalabrutinib.1

The apparent BTK IC50 was determined over time using the LanthaScreen assay. Results indicate that ACP-5862, like acalabrutinib, is a covalent inhibitor of BTK. Binding kinetics derived from IC50 over time data, indicated that acalabrutinib and ACP-5862 have similar affinity (KI), however the BTK inactivation rate (kinact) for ACP-5862 was half that relative to acalabrutinib. This indicated a covalent BTK inhibition potency for ACP-5862 that was 2-fold lower than acalabrutinib.

The overall kinome inhibition profile at 1 µM using KINOMEscan (Eurofins DiscoverX), as well as IC50 determinations on closely related kinases with a Cys in the same position as Cys481 in BTK (ITK, TXK, TEC, BMX, EGFR, ERBB2, ERBB4, BLK, JAK3), both revealed that the kinase selectivity profiles of acalabrutinib and ACP-5862 were similar.

On-target inhibition in B cells was investigated measuring inhibition of B-cell antigen receptor-mediated activation of CD69 cell surface expression on human peripheral B cells using human peripheral blood mononuclear cells and human whole blood (hWB). The EC50 for anti-IgD-induced CD69 expression in hWB was 64 ± 6 nM for ACP-5862, compared to 9.2 ± 4.4 nM for acalabrutinib. The hWB EC90, representing near complete inhibition of BTK by acalabrutinib and ACP-5862 was observed at 72 ± 20 nM and 544 ± 376 nM, respectively.

In conclusion, the results indicate that ACP-5862, the major metabolite of acalabrutinib, has intrinsic BTK inhibitory activity and a similar kinase selectivity profile as acalabrutinib. The contribution of ACP-5862 to on-target covalent inhibition of BTK in humans is unclear at present, but is most likely limited, since the EC90 in hWB for ACP-5862 approximated observed plasma Cmax in humans dosed with 100 mg acalabrutinib. The relative contribution of acalabrutinib and ACP-5862 to BTK pharmacodynamics is under further investigation.

Terry Podoll and J. Greg Slatter are former Acerta Pharma employees.

Reference

1. Barf T, Covey T, Izumi R, et al. Acalabrutinib (ACP-196): A covalent Bruton tyrosine kinase inhibitor with a differentiated selectivity and in vivo potency profile. J Pharmacol Exp Ther. 2017;363(2):240-252.

#2195

Real-world experience with afatinib as first-line treatment in advanced non-small cell lung cancer in a university hospital.

Taewon Jang, Jaehoon Kim. _Kosin Univeristy Gospel Hospital, Pusan, Republic of Korea_.

Purpose: In patients with non-small cell lung cancer (NSCLC), mutations in the epidermal growth factor receptor (EGFR) have been associated with sensitivity to EFGR-Tyrosin Kinase Inhibitors (TKIs). The aim of this study was to analyze clinical efficacies of first line afatinib treatment.

Experimental Design: We collected patients with EGFR mutation positive NSCLC who started first-line afatinib treatment between Jan 2015 and Sep 2017. We reviewed clinical characteristics of the patients, laboratory finding, tumor response rate, side effects, progression free survival (PFS), 2nd biopsy result, and overall survival (OS), retrospectively.

Results: Forty six patients were available for analysis among all 62 patients (1st to 8th line chemotherapy). At the time of data analysis in October 2018, nine patients were still on afatinib. Median age was 67.4 years-old. 20 (44%) patient had EGFR 19 deletion, 25 (54%) EGFR L858R mutation, 1 (2%) S768I. 10 patients (21.7%) had brain metastasis at baseline study. Tumor response was 58.7% with partial response, no change 19.6%, progressive disease 2%, and non-evaluable 19.6%. Mean follow-up time was 21.6 months (13.5 to 42.5 month) from start of afatinib treatment. Twenty-eight progression or death events were observed. Median PFS was 13.5 months (95% CI: 10.5 to 16.5 months). There were no differences in smoking, gender, EGFR types. 37 patients (80.4%) had dose reduction from initial dose. 6 patients (13.1%) discontinued due to afatinib toxicity. 6 patients (13%) had G3 toxicity, but usually manageable by dose reduction or supportive care. 1 case of doxycycline induced pneumonitis occurred. The PFS of dose reduction group due to adverse events was longer than standard group (14.9 months vs 10.1 months, p=0.023). In dose reduction group, body weight was low (53.1 kg vs 68.4 kg, p=0.002), the ratio of never smoker (87.9% vs 37.5%, p=0.001) and female (87.4% vs 47.4%, p=0.048) were higher than standard group. After progression, 2nd biopsy was done in 17 patients. T790M mutation was detected in 8 patients, so detection rate was 47.1%. 6 patients received oximertinib treatment. Median OS was 38.6 months.

Conclusions: Afatinib is one of appropriate first-line treatment option for patients with advanced EGFR positive NSCLC. The dose reduction due to adverse events was common in low-body weight female never smoking patients, but the PFS was increased. With use of new 3rd-gen EGFR TKIs in the first-line setting, optimal sequencing of EGFR TKIs needs to be validated in maximizes treatment outcomes.

#2196

Strong anti-tumor activity of MEK inhibitor GDC-0623 and determination of predictive biomarkers.

Vincent Vuaroqueaux,1 Hoor Al Hasani,1 Gerhard Kelter,2 Hans R Hendriks,3 Heinz-Herbert Fiebig1. 1 _4HF Biotec GmbH, Freiburg, Germany;_ 2 _Charles River, Discovery Services, Freiburg, Germany;_ 3 _Hendriks Pharmaceutical Consulting, Purmerend, Netherlands_.

MEK inhibitors emerged as a promising class of anti-cancer agents to inhibit KRAS/RAF driven tumors like melanoma and colorectal cancer. GDC-0623 is a potent ATP-noncompetitive allosteric MEK1 inhibitor. The compound forms a strong hydrogen bond with Ser212 of MEK1, blocking MEK feedback phosphorylation of wild-type RAF. Preclinical studies showed efficacy in vitro and in vivo and in a phase I clinical study, suggesting that the compound has clinical potential. In the present work, we aimed to define candidate tumors to GDC-0623 treatment and gain insight into biomarkers and predictive models of response.

GDC-0623 was tested in vitro in a 2D monolayer assay (260 human tumor cell lines of 36 histological subtypes). Next, activity was tested in vivo in a human tumor cell line engrafted subcutaneously in nude mice. Drug sensitivity results were then bound to molecular information of the tumor models, including whole exome mutations, gene copy number variations and gene expression profiles, to identify determinants and build a predictor of response.

In vitro, GDC-0623 showed a strong anti-tumor activity (abs. IC70 <1 µM) in a wide range of blood and solid tumors. IC50 values varied from 0.001 to >30 µM (Geometric mean: 2.95 µM). The majority of leukemia CML and AML cell lines were sensitive (4/7 and 6/11, respectively) whereas lymphoma cell lines were mainly resistant. In solid tumors, strong activity was seen in almost all melanoma cell lines (10/12) and similar activity was observed in 5/8 bladder, 10/16 colon, 7/12 head and neck, 5/12 pancreas, 4/12 gastric and 4/12 breast cell lines. Anti-tumor activity of GDC-0623 was confirmed in vivo in the human colon HTX model COLO-205 (BRAF V600E mutant). An oral dose of 40 mg/kg/day caused a strong inhibition of the tumor growth (T/C = 6%) without impacting animal body weight.

Screening of cell lines for molecular features associated with response showed BRAF V600E, NRAS Q61K, Q61R and DUSP27 gene mutations among the strongest predictors of response (Fisher test, p <0.05). Furthermore, cell lines with EGFR and MET amplifications were almost all sensitive (4/5 and 3/4, respectively, Fisher p<0.03). At transcriptome levels, a total 2108 genes were significantly associated with response to GDC-0623 (intersection of Spearman and Limma tests, adjusted p values <0.05). Integrative analysis with significant genomic and transcriptomic parameters is currently ongoing to develop a molecular predictor of response to GDC-0623. Extending evaluation of the compound in vivo and validating predictors of response are needed to complete these preclinical investigations.

#2197

The AXL kinase inhibitor, TP-0903, demonstrates efficacy in preclinical models of colorectal cancer independent of KRAS mutation status.

Ryan Mangelson, Peter Peterson, Jason M. Foulks, Yuta Matsumura, Lars Mouritsen, Clifford J. Whatcott, David J. Bearss, Steven L. Warner. _Tolero Pharmaceuticals, Inc., Lehi, UT_.

Colorectal cancer (CRC) is the third leading cause of cancer-related mortality in the US (2017). Most patients present with stage II or III disease at diagnosis, with the 5-year survival rate between 53-89%. Survival in patients with stage IV CRC, however, is a discouraging 11%. Metastasis in CRC is linked to a mesenchymal phenotype, which is associated with chemoresistance. AXL, a receptor tyrosine kinase, promotes the mesenchymal phenotype in cancer cells and its expression is associated with drug resistance and poor outcomes. TP-0903, a clinical-stage, investigational small molecule inhibitor of AXL has been shown to reverse the mesenchymal phenotype and restore drug sensitivity in cells that no longer respond to standard agents in preclinical models. TP-0903 is hypothesized to be an active agent in CRC through reversal of the mesenchymal phenotype. In cell viability assays of CRC lines, TP-0903 treatment resulted in IC50 values ranging from 4.5 – 123 nM. Notably, cell growth inhibition by TP-0903 was independent of KRAS mutation status; the KRAS mutant HCT-116 line was the most sensitive CRC cell line tested. Mesenchymal markers, including Snail, were suppressed by 7.6-fold (mRNA) and 4.9-fold (protein) in the HCT-116 line at 500 nM. TP-0903 activity was also assessed in vivo using two KRAS mutant CRC models: HCT-116 and a patient-derived xenograft (PDX) model. In the HCT-116 xenograft model, single agent TP-0903 treatment achieved 69% tumor growth inhibition (%TGI) with an oral dosing schedule at 40 mg/kg. In a KRAS-mutant PDX model, TP-0903 achieved 44% TGI when mice were dosed at 40 mg/kg. Pharmacodynamic analyses were performed on tissues from the HCT-116 and PDX models. The ligand for AXL, GAS6, was significantly upregulated in tissues after TP-0903 treatment in both CRC in vivo models while soluble AXL and GAS6 were significantly downregulated in plasma in the PDX model. Furthermore, Axin2, a Wnt/β-catenin regulated gene, was downregulated by TP-0903 in tumor tissue from the PDX model, suggesting inhibition of the Wnt/βcatenin pathway. These data support a potential role for AXL in the promotion of the mesenchymal phenotype in CRC, and showed that AXL inhibition by TP-0903 suppressed the mesenchymal phenotype and was effective against CRC cells regardless of KRAS mutation status. These observations support further clinical investigation of TP-0903 as a potential therapeutic agent in metastatic CRC. A Phase I trial with this investigational agent is ongoing, including patients with KRAS mutant CRC (clincaltrials.gov, NCT02729298).

#2198

PBI-200: A novel, brain penetrant, next generation pan-TRK kinase inhibitor.

Anthony Regina,1 Aram Elagoz,1 Vincent Albert,1 Jonathan Boudreault,1 Marc Ouellet,1 Nicolas Brunei-Latour,1 Edith Bellavance,1 Peter White,1 Stephane Ciblat,1 W. Robert Bishop,2 Kollol Pal2. 1 _Paraza Pharma, Montreal, Quebec, Canada;_ 2 _Pyramid Biosciences, Needham, MA_.

Fusions of NTRK 1, 2 and3 genes encoding the TRK family of receptor tyrosine kinases (TrkA, B and C, respectively) have been reported in 1-3% of all human cancers including lung and breast cancers. NTRK gene fusions are also reported in lung and breast tumors metastatic to the brain as well as in primary brain tumors including glioblastoma (3%), astrocytoma (3%) and pediatric glioma (40%). These gene fusions result in constitutive TRK kinase activity and act as oncogenic drivers of disease. First generation TRK kinase inhibitors have demonstrated clinical proof of concept in patients with tumors bearing NTRK gene fusions. PBI-200 is a novel, selective pan-TRK kinase inhibitor. In biochemical assays it inhibits TrkA, B and C with IC50 values of 0.45, 2.2 and 1.9 nM, respectively. This molecule is highly selective against a panel of 122 other protein kinases tested with the exception of Ros1 (IC50= 31 nM). Importantly, PBI-200 retains potency against resistance mutations reported in patients receiving first generation TRK kinase inhibitors. Biochemical IC50 values are 3.4 nM for the TrkA G595R gatekeeper mutation and 10 nM for the TrkA G667C mutant. PBI-200 potently inhibits proliferation of human tumor cell lines expressing TRK fusions including the KM-12 colorectal cancer cell line (TPM3-NTRK1 fusion; EC50 value = 22 nM) and the MO-91 acute myeloid leukemia cell line (ETV6-NTRK3 fusion; EC50 value = 3.5 nM). It also potently inhibits proliferation of rat BaF3 cells expressing oncogenic NTRK fusions and their resistant variants. In a subcutaneous xenograft model of KM-12 colorectal cancer, PBI-200 induces tumor stasis when dosed intraperitoneally at 15 or 30 mg/kg twice daily (93 and 100% tumor growth inhibition, respectively). The activity of PBI-200 was comparable or superior to that seen with the first generation TRK kinase inhibitors used as comparators in this model. PBI-200 has oral bioavailability in both rat and mouse. Importantly, PBI-200 demonstrates excellent brain penetration in rodent species, with brain/plasma AUC ratios of 3.9 in the mouse and 3.2 in the rat. This is in contrast to the poor brain penetration observed with the first generation TRK kinase inhibitors, larotrectinib and entrectinib.

Based on the activity of PBI-200 against clinically-relevant resistance mutations and its excellent brain penetration, this molecule has potential to be a next-generation TRK kinase inhibitor especially for primary brain tumors or brain metastatic lesions that harbor a NTRK gene fusion. PBI-200 is currently undergoing further characterization in IND enabling studies.

#2199

BOS172738: A novel highly potent and selective RET kinase inhibitor in Phase 1 clinical development.

Mitchell Keegan, Keith Wilcoxen, Peter T. Ho. _Boston Pharmaceuticals, Cambridge, MA_.

Background: RET gene alterations including point mutations and gene fusions have been detected in multiple cancers, including colorectal cancer, NSCLC and various thyroid cancers. RET fusion events involve the kinesin family member 5B (KIF5B-RET), as well as a number of other fusion partners such as CCDC6, TRIM33, and NCOA, and are considered to be the driver mutations in NSCLC and other tumors harboring RET fusions. Selective inhibitors of RET kinase recently has been associated with durable objective responses in patients with RET gene-altered NSCLC and papillary thyroid carcinoma. BOS172738 (formerly DS-5010) is an orally available small-molecule RET kinase inhibitor that has previously been shown to have potent in vitro RET inhibitor activity and in vivo potency against transfected allograft and xenograft models. In this study, we discuss additional in vitro characterization and in vivo efficacy in patient derived xenograft (PDX) models driven by RET fusions and emergent resistant mutations.

Results: An expanded kinase profile was conducted with BOS172738 against over 450 kinases, and demonstrated exquisite potency for RET and RET mutations with Kd values ≤ 1 nM for RETwt, RET(M918T), RET(V804L) and RET(V804M). BOS172738 also demonstrated high selectivity over KDR (>300 fold). To confirm this selectivity in vitro, BOS172738 was compared to ponatinib, a multikinase inhibitor with potent activity against both KDR and RET. While BOS172738 demonstrated similar potency to ponatinib (IC50 0.5 µM vs. 0.3 µM) in inhibiting the in vitro proliferation of a NCO4-RET human CRC cell line (CR1520), it resulted in significantly less anti-proliferative activity in HUVEC normal endothelial cells (IC50 2.9 µM vs 0.2 µM). BOS172738 was also evaluated in a number of PDX models of RET fusion cancers, including three CRC PDX models (CR2518:CCDC6-RET, CR1520: NCOA4-RET and CR2545: CCDC6-RET with a V804M mutation) and one NSCLC PDX model (CTG-0838:KIF5B-RET). In all cases tested, BOS172738 demonstrated potent and durable tumor regression at doses of 30 mg/kg, with tumor growth inhibition at lower doses. BOS172738 also demonstrated potent tumor regression on larger, established tumors (>1000 mm3).

Conclusion: These results support that BOS172738 is a potent and selective RET kinase inhibitor, with strong antitumor activity against a range of RET fusion proteins, as well as resistant RET active site mutations. BOS172738 is currently being evaluated in a Phase 1 clinical study.

#2200

Potent in vitro activity of Tarloxotinib for EGFR C797S and other mutations refractory to current EGFR tyrosine kinase inhibitors.

Kenichi Suda,1 Masaya Nishino,1 Takamasa Koga,1 Toshio Fujino,1 Yoshihisa Kobayashi,1 Tetsuya Mitsudomi,1 Avanish Vellanki,2 Vijaya G. Tirunagaru2. 1 _Kindai University Faculty of Medicine, Osaka-Sayama, Japan;_ 2 _Rain Therapeutics Inc., Fremont, CA_.

Introduction: EGFR tyrosine kinase inhibitors (TKIs) are key drugs in the treatment of lung cancers with activating EGFR mutations. However, inherent resistance such as exon 20 insertions and acquired resistance such as T790M / C797S hamper further improvement of treatment outcomes in lung cancer patients with EGFR mutations. In this study, we evaluated efficacy of a novel hypoxia-activated EGFR-TKI, tarloxotinib, for these resistant EGFR mutations using Ba/F3 models.

Methods: Ba/F3 cells with either of exon 20 insertions (A763insFQEA, V769insASV, D770insSVD, or H773insNPH) or Del 19/T790M/C797S in cis (triple mutations) were generated as previously described (Nishino, et al. Lung Cancer, in press). Ba/F3 cells with Del 19 alone was used as sensitive control. Growth inhibitory assays against these Ba/F3 cells were performed for tarloxotinib-E (activated form), tarloxotinib (pro-drug before activation), afatinib, poziotinib, and osimertinib. In vitro kinase assays for various EGFR and HER2 mutations at ATP Km were used to test the potency of Tarloxotinib-E.

Results: Tarloxotinib-E was highly potent across various EGFR and HER2 mutations, including C797S double mutants in the in vitro kinase assays with an IC50 of <0.38 nM. Low nM potency was observed for EGFR C797S triple mutants (d746-750/T790M/C797S, T790M/C797S/L858R). Tarloxotinib showed about 100 times higher IC50 values compared with tarloxotinib-E in all tested Ba/F3 cells, indicating that tarloxotinib-E can selectively inhibit cell growth in lung cancers with EGFR mutations. Poziotinib and tarloxotinib-E were effective against all Ba/F3 cells with exon 20 insertions (IC50 < 10 nM), while afatinib and osimertinib effectively killed only the Ba/F3 cells with A763insFQEA mutation. For Ba/F3 cells with Del19/C797S mutation, afatinib, poziotinib and tarloxtinib-E showed potent activity with an IC50 of 2.8, 1.6 and 5.1 nM respectively. For Ba/F3 cells with Del 19/T790M/C797S, tarloxotinib-E also exhibited activity (IC50 198 nM), while the triple mutations conferred resistance to the other TKIs.

Conclusion: As the hypoxia-activated fragmentation of tarloxotinib into tarloxotinib-E broadens the therapeutic window versus competing small molecule EGFR TKIs, direct IC50 comparisons across agents may not be relevant as tarloxotinib may be dosed higher. These data highlight potential opportunity for tarloxotinib in the treatment of lung cancers with inherent or acquired resistance to current available EGFR-TKIs.

#2201

**Preclinical development of novel pyrazolo[3,4-** d **]pyrimidines structure-based TKIs for the treatment of glioblastoma.**

Arianna Mancini,1 Anna Lucia Fallacara,1 Claudio Zamperini,2 Giulia Iovenitti,1 Alessio Molinari,1 Enrico Rango,1 Adriano Angelucci,3 Silvia Schenone,4 Maurizio Botta1. 1 _Università degli Studi di Siena, Siena, Italy;_ 2 _Lead Discovery Siena S.r.L, Italy;_ 3 _Università degli Studi dell'Aquila, L'Aquila, Italy;_ 4 _Università degli Studi di Genova, Genova, Italy_.

The aim of this study was the preclinical development of a set of pyrazolo[3,4-d]pyrimidine structure-based small molecules ATP-competitive SRC inhibitors for the therapy of glioblastoma (GBM), a rare but extremely aggressive and infiltrative glial tumor with a very limited response to therapies. Ongoing preclinical and clinical studies of tyrosine kinase inhibitors (TKIs) for the therapy of GBM identified SRC family inhibitors as a significant and effective therapeutic strategy (i.e. dasatinib, bosutinib, PP2).

In vitro Screening: From the wide in-house synthesized pyrazolo[3,4-d]pyrimidines' library, nine compounds have been selected to be formulated and tested in in vitro and in vivo GBM pharmacological models. Among all the examined formulations, the prodrug approach has been applied to the candidate drugs, introducing the N-methylpiperazine moiety by a carbamate-based linker (in vivo enzymatically hydrolysable). Drugs and corresponding prodrugs have been screened on the basis of their SRC Ki and in vitro ADME properties. GBM U87 cell assays showed IC50 in the sub-micromolar range (1.5 - 15.0 µM) for almost all compounds and improved cellular uptake of the prodrugs in respect to the corresponding drugs.

PK and Toxicity: After that, the most promising compound has been selected for further in vitro assays and in vivo tests. In vivo pharmacokinetics and pharmacodynamics have been evaluated for the compound by various formulations, routes of administration and dosages. In all cases investigated, the compound has been proved to reach the brain. As expected, prodrug plasmatic MRT and AUC confirmed the realization of an extended release formulation that allows a higher overall exposure to the drug. In addition, considering an estimated oral bioavailability of 40.4% and a 11-fold higher Cmax in respect to i.v. and i.p. administration, the oral route can be identified as the most advantageous for the compound for GBM treatment. Moreover, an acute toxicity study revealed that the LD50 for i.v. toxicity is more than 100 mg/kg with a good tolerability.

In vivo Efficacy Tests: The selected compound and its prodrug were orally administered in GBM orthotopic mouse model and results indicated a median survival rate 10% greater for prodrug-treated mice in respect to drug-treated mice and 35% greater in respect to control group. The combination effect of the compound and radiotherapy was also investigated in subcutaneous U87 xenografted mice model and resulted in a reduction of the tumor growth close to 80% in respect to the control growth.

Conclusions: Taking into account all the data collected regarding their pharmacological properties, pyrazolo[3,4-d]pyrimidines SRC inhibitors class of compounds deserve further investigation to show its potential to become a new therapy for GBM.

#2202

Tarloxotinib exhibits potent activity in NRG1 fusion and rearranged cancers.

Vijaya G. Tirunagaru,1 Adriana Estrada-Bernal,2 Hui Yu,3 Chris Rivard,3 Fred Hirsch,3 Matthew Bull,4 Maria Abbatista,4 Jeff Smaill,4 Adam V. Patterson,4 Robert C. Doebele,5 Avanish Vellanki1. 1 _Rain Therapeutics Inc., Fremont, CA;_ 2 _University of Colorado Anschutz Medical Campus, Aurora, CO;_ 3 _University of Colorado, Aurora, CO;_ 4 _The University of Auckland, Auckland, New Zealand;_ 5 _University of Colorado Anschutz Medical Campus, Fremont, CA_.

Background: NRG1 fusions and rearrangements are oncogenic drivers that have been observed in a variety of tumor types and enriched in invasive mucinous adenocarcinomas (IMA) of the lung. The oncoprotein binds HER3-HER2 heterodimers and activates downstream signaling, supporting a therapeutic paradigm of ERBB3/ERBB2 inhibition. While patient responses have been observed with Afatinib, durable responses have largely been absent. Tarloxotinib, a clinical-stage prodrug that releases a potent, irreversible EGFR/HER2 inhibitor (Tarloxotinib-E) selectively in severely hypoxic regions of tumours has been shown to overcome the intrinsic resistance of EGFR exon 20 insertion mutations and HER2 activating mutations to existing TKIs in vitro and in vivo.

Results: NRG1 altered cell lines (MDA-MB-175 with DOC4-NRG fusion, HCC95 with NRG gene amplification and H1793 with increased NRG1 mRNA) showed high sensitivity to Tarloxotinib-E whereas Tarloxotinib prodrug was >45 fold less potent under normoxic conditions, consistent with lower sensitivity of the prodrug and the requirement of hypoxia for activation. Tarloxotinib-E inhibited HER2, HER3, Akt and ERK phosphorylation between 10-100 nM whereas afatinib was 10-fold less potent. In vivo activity of Tarloxotinib was evaluated using the CLU-NRG1 patient-derived xenograft model. Nude mice bearing OV-10-0050 tumors implanted subcutaneously were treated with vehicle, the human equivalent dose (HED) of afatinib (6 mg/kg, qd, p.o.) and two doses of tarloxotinib (26 and 48 mg/kg, qw, i.p.) corresponding to Tarloxotinib HED of 75 and 150 mg/m2. Afatinib showed initial activity that reduced over time and was ineffective. In contrast, Tarloxotinib elicited a profound, durable and dose dependent anti-tumor response. Tarloxotinib dosed at 48 mg/kg showed dramatic tumor regression in all the mice while Tarloxotinib dosed at 26 mg/kg showed significant dose dependent tumor regression. Efforts are underway to define the PK/PD correlation by measuring Tarloxotinib and Tarloxotinib-E exposure in plasma and tumor along with the evaluation of hypoxia and the expression of STEAP4 reductase. Tarloxotinib mediated on-target and signaling effects in OV-10-0050 tumors will be presented at the meeting.

Conclusions: Tarloxotinib, a prodrug of a potent irreversible inhibitor of EGFR/HER2 in clinical development demonstrated significant activity in CLU-NRG1 patient-derived xenograft model. NRG1 fusions and rearrangements represent an emerging actionable driver alteration in a variety of cancers. Clinical development of Tarloxotinib in NRG1 altered cancers presents an attractive opportunity.

#2203

WSD1227: A BBB penetrable VEGFR small molecule for the treatment of primary and metastatic brain tumors.

Jinqiang Zhang, Wei Zhong, Zhihua Mu, Claire Sun. _Wayshine Biopharm (Shanghai), Shanghai, China_.

Anti-angiogenesis has been proved to be a good strategy for cancer treatment (such as in GBM, NSCLC, mBC, CRC, OC, etc.) with satisfactory performance to extracranial lesions, but not in intracranial lesions. Insufficient penetration across BBB could be one of factors limiting intracranial activity for those approved anti-VEGF agents. There is a need to develop a BBB penetrable VEGFR inhibitor to clinic to assess the efficacy and safety. Here we report that WSD1227, a novel, selective VEGFR inhibitor, displays potent activity, excellent CNS penetration and preclinical safety and efficacy. The IC50 of WSD1227 against VEGFR1/2/3 at 0.69/0.35/0.41nM versus against other targets such as PDGFRα IC50 22.9nM, PDGFRβ IC50 19.4nM, cKit IC50 383nM, FLT3 IC50 555 nM and CSF1R IC50 1062nM. WSD1227 treatment resulted in significant anti-tumor activity in VEGF related extracranial and intracranial mice models, with superior efficacy than bevacizumab. There is no increased risk of CNS hemorrhage in intracranial tumor bearing mice treated with WSD1227. In addition, WSD1227 is predicted to be CNS penetrable with decent PK in human. Taken together, our data provide a good rationale for WSD1227 to be developed toward clinic to investigate anti-angiogenetic therapies for management of patients with primary or metastatic brain tumors.

#2204

Novel FAK/ALK/ROS1 inhibitor APG-2449 synergizes with osimertinib in preclinical xenograft models of EGFR-mutant NSCLC.

Guangfeng Wang, Douglas D. Fang, Ping Min, Ran Tao, Chunyang Tang, Shoulai Gu, Li Rui, Jiajun Li, Jingwen Wang, Miaoyi Wu, Yingfeng Li, Dingxiong Chen, Fei Zhang, Kejie Lian, Feifei Zhang, Lvcheng Wang, Rongcheng Xu, Dajun Yang, Yifan Zhai. _Ascentage Pharma, China_.

Focal adhesion kinase (FAK) plays an important role in cell migration, growth factor signaling, cell cycle progression and cellular survival. It had been shown that FAK and SRC family kinases were able to sustain downstream AKT and MAPK signaling under continuous EGFR inhibition driven by osimertinib (AZD9291), a mutant-selective third-generation EGFR inhibitor. Incomplete inhibition of AKT and MAPK was consistently observed even under increased concentrations of osimertinib and led to acquired resistance to osimertinib, implicating the need of combination therapy with FAK/SRC inhibitors to enhance antitumor activity of osimertinib and overcome its resistance. APG-2449 is a novel oral active small-molecular inhibitor that targets FAK, ALK and ROS1. In this study, we investigated the effect of combination treatment with APG-2449 and osimertinib using NSCLC NCI-H1975 cells carrying EGFRL858R/T790M mutation and an osimertinib-resistant patient-derived xenograft (PDX) model carrying EGFRT790M/19del/C797S mutation. In vitro, the combination treatment with APG-2449 and osimertinib synergistically inhibited the proliferation of NCI-H1975 cells. In vivo, APG-2449 significantly enhanced antitumor activity of osimertinib in NCI-H1975 xenograft models, leading to complete or partial tumor regression. More profound antitumor activity of this combination was also demonstrated in the osimeritinib-resistant PDX model in comparison with single agents. In term of mechanism, the combination arm significantly suppressed the on-target phosphorylation of EGFR, FAK and SRC, as well as AKT and ERK in NCI-H1975 xenografts. Collectively, these results suggest that addition of APG-2449 to osimeritinib may enhance antitumor activity and suppress the development of resistance in NSCLC.

#2205

CBT-102, an oral small molecule multi-kinase inhibitor, demonstrates favorable CSF-1R activity, offering means of controlling tumor associated macrophages.

Elaine Liu,1 Lan Yang,1 Gavin Choy,2 Xiaoling Zhang,2 Tillman Pearce,2 Mamatha Reddy,2 Sanjeev Redkar,2 Qian Shi1. 1 _Apollomics, Xiaoshan, Hangzhou, China;_ 2 _Apollomics, Pleasanton, CA_.

Background: The role of the tumor microenvironment (TME) in fostering the development of malignancies is prompting the pursuit of anticancer therapies that target its components as to opposed to the tumor itself. As part of their immune surveillance duties, immune cells form part of this microenvironment, and yet, cancer cells have devised means to downplay their tumoricidal capabilities. Colony stimulating factor 1 receptor (CSF-1R) may offer such a means of controlling tumor associated macrophages in the tumor microenvironment.

Methods: The IC50 of CBT-102 against CSF1R was evaluated in both cell free system and cell based assays, where enzyme activity was evaluated with radiometric assay format, and cell growth inhibition was evaluated in the Ba/F3 hCSF1R cell line by the CellTiter-Glo (CTG) method. Further evaluation of CBT-102 IC50 was undertaken in M-NFS-60 syngeneic cell lines and human monocytes. In vivo studies of CBT-102 in combination with anti-PD1 antibody were performed in H22 and MC38 syngeneic models.

Results: CBT-102 demonstrated a reproducible activity against CSF1R in a radiometric enzyme activity assay. Using engineered BaF3 hCSF1R cell lines, we demonstrated the IC50 value of CBT-102 was 0.588 µM compared to 1.333 µM of sulfatinib, a similar multi-kinase inhibitor, and 0.279 µM of GW2580, a more specific CSF1R kinase inhibitor. Similar studies conducted in M-NFS-60 cell lines and human monocyte also showed sub-micromolar activities of CBT-102. In vivo study combining CBT-102 with anti-PD1 antibody in syngeneic models demonstrated a favorable combination effect compared to each of the single agents.

Conclusions: We demonstrated previously that CBT-102 effectively inhibits VEGFR and angiogenesis. The newly revealed mechanism of CBT-102, inhibiting CSF1R and macrophages, offers a promising dual mechanism of action in addition to targeting VEGFR and angiogenesis. Rational combination with check-point inhibitors (CPIs) may improve the efficacy of CBT-102 and broaden the impact of CPIs. Further studies may be needed to delineate the interplay among CBT-102's different mechanisms and its impact in combination with CPIs.

#2206

Anti-tumor activity of infigratinib, a potent and selective inhibitor of FGFR1, FGFR2 and FGFR3, in FGFR fusion-positive cholangiocarcinoma and other solid tumors.

Gary Li,1 Melanie Krook,2 Sameek Roychowdhury,2 Francesca Avogadri,1 Yining Ye,1 Susan Moran1. 1 _QED Therapeutics, San Francisco, CA;_ 2 _The Ohio State University, Columbus, OH_.

Abnormal expression and constitutive activation of receptor tyrosine kinases, such as ALK, ROS1 and TRK, as a result of gene rearrangements have been clinically validated as therapeutic targets for cancer. Recently, fusions involving the FGFR family, especially FGFR1, FGFR2 and FGFR3, have been identified in diverse solid tumors such as cholangiocarcinoma, glioblastoma, bladder, lung, breast, thyroid and prostate cancers. FGFR fusions are oncogenic drivers that ligand-independently activate the receptor kinases and their downstream signaling pathways, leading to uncontrolled cell proliferation and invasion.

Infigratinib (BGJ398) is an oral and selective inhibitor of FGFR. At biochemical and cellular levels, infigratinib selectively inhibits the activity of FGFR1, FGFR2 and FGFR3 with low nM potency, while sparing FGFR4, VEGFR2 and other kinases. In FGFR fusion-positive xenograft models, including cell line-derived and patient-derived models representing diverse tumor histologies, infigratinib treatment leads to significant tumor growth inhibition and regression at clinically achievable exposure levels. The anti-tumor effect of infigratinib is accompanied by inhibition of downstream signaling pathways and induction of tumor apoptosis.

Clinically, in an open-label phase II trial, infigratinib demonstrated a cORR of 39.3% in FGFR2 fusion-positive cholangiocarcinoma patients who had received one or fewer prior chemotherapy regimens. Additionally, RECIST responses were achieved in non-cholangiocarcinoma solid tumors tested positive for FGFR fusions.

In conclusion, the available preclinical and clinical data demonstrate the potential of infigratinib as an effective treatment for patients with FGFR fusion-positive tumors, regardless of the fusion partners or the origin of tissue. On the basis of these data there is a clear rationale for performing tumor-agnostic clinical trials in molecularly defined cancers to maximally benefit patients with serious and life-threatening diseases.

#2207

The dual role of BI 2536, a small-molecule inhibitor that targets PLK1, in induction of apoptosis and attenuation of autophagy in neuroblastoma cells.

Zhiheng Li. _Soochow University, Soochow, China_.

Neuroblastoma(NB) is the most common extra-cranial solid tumor in childhood with the overall survival less than 40%. Polo-like kinase 1 (PLK1) is a serine/threonine-protein kinase expressed during mitosis and overexpressed in multiple cancers, including neuroblastoma. We found the PLK1 expression related to the outcome of NB patients. BI2536, a small molecule inhibitor against PLK1, significantly reduced cell viability in a panal of NB cell lines, with IC50 less than 100 nM. PLK1 inhibition by BI 2536 treatment induced cell cycle arrest at G2/M phase and cell apoptosis in NB cells. Realtime PCR array revealed the PLK1-regulated genes, such as BIRC7, TNFSF10, LGALS1 and DAD1 et al. Moreover, autophagy activity was investigated in the NB cells treated with BI 2536. BI 2536 treatment in NB cells increased LC3-II puncta formation and LC3-II expression. Formation of autophagosome induced by BI 2536 was observed by TEM. However, BI2536 abrogated the autophagic flux in NB cells by reducing SQSTM1/p62 expression and AMPKαT172 phosphorylation. These results provide new clues for the molecular mechanism of cell death induced by BI 2536 and suggest that BI 2536 may act as new candidate drug for neuroblastoma.

#2208

Targeted inhibition of FGF/PDGF/VEGF signaling enhances nanoparticle taxane response in preclinical gastric cancer models.

Niranjan Awasthi,1 Kate Crawford,1 Erin Bontrager,2 Sazzad Hassan,1 Urs von Holzen,1 Margaret A. Schwarz,1 Roderich E. Schwarz1. 1 _Indiana Univ. School of Medicine, South Bend, IN;_ 2 _Goshen College, Goshen, IN_.

Background: Gastric adenocarcinoma (GAC) remains the third most common cause of cancer-related deaths worldwide. Most available systemic chemotherapy options for advanced GAC have limited efficacy. Nab-paclitaxel (NPT), a nanoparticle albumin-bound taxane formulation, has shown significant activity in preclinical GAC studies. Overexpression of multiple growth factors and their receptors such as FGF/FGFR, PDGF/PDGFR and VEGF/VEGFR, promote angiogenesis in several cancers including GAC that leads to tumor progression and metastasis. Dovitinib is a small molecule RTK inhibitor that potentially inhibits the activity of FGFR, PDGFR and VEGFR. We evaluated the antitumor efficacy of dovitinib and its ability to enhance nab-paclitaxel response in preclinical GAC models.

Methods: In vitro cell proliferation and protein expression were measured by WST-1 assay and Immunoblotting. In vivo tumor growth and animal survival studies were performed in NOD/SCID mice using human GAC MKN-45 cells.

Results: Nab-paclitaxel and dovitinib had in vitro growth inhibitory effect on several GAC associated cell lines tested, with additive effects in combination. Immunoblot analysis revealed that dovitinib treatment caused a decrease in the expression of phospho-FGFR, phospho-AKT, phospho-ERK; and an increase in the expression of apoptosis-related proteins cleaved PARP-1 and cleaved caspase-3. Nab-paclitaxel activity correlated with the increased expression of phospho-stathmin. In subcutaneous GAC xenografts, NPT and dovitinib demonstrated inhibition in tumor growth, while NPT+dovitinib had an additive effect. Net tumor growth was 533.7 mm3 in controls, 394.8 mm3 after oxaliplatin, 135.8 mm3 after NPT, 128 mm3 after dovitinib, 98.9 mm3 after oxaliplatin+dovitinib and -41.8 mm3 (tumor regression) after NPT+dovitinib. Tumor tissue analysis revealed that dovitinib reduced tumor vasculature, while nab-paclitaxel reduced tumor cell proliferation. In a peritoneal dissemination model, animal survival compared to controls (23 days) remained unchanged after monotherapy with oxaliplatin (24 days) or dovitinib (25 days) but increased significantly after NPT monotherapy (42 days, a 83% increase). Combination of dovitinib with NPT exhibited a further increase in animal survival (66 days, a 187% increase), while the combination of dovitinib with oxaliplatin had no survival benefit (25 days).

Conclusion: These findings demonstrate that the antitumor effect of nab-paclitaxel can be significantly enhanced by the FGFR/PDGFR/VEGFR pathway inhibitor dovitinib in preclinical GAC models. The data support the clinical relevance of this therapeutic combination for advanced GAC patients.

#2209

Novel therapeutic agents for RET-driven cancers, which is highly potent and selective inhibitor to RET kinase.

Hua Li,1 SeoHyun Jo,1 Woomi Do,1 Hyunkyung Kim,1 Hwan Kim,1 Kyung-ah Seo,1 Ji-hoon Oh,1 Jieun Choi,2 Jaeyoung Ahn,2 Jung Beom Son,1 Nam Doo Kim1. 1 _NDBio Therapeutics, Incheon, Republic of Korea;_ 2 _Voronoi, Incheon, Republic of Korea_.

The rearranged during transfection (RET) tyrosine kinase is mostly altered by fusion and it makes RET as a attractive therapeutic target for medullary thyroid cancer and non-small cell lung cancer (NSCLC). Multi-kinase inhibitors (mKIs) such as cabozantinib and vandetanib expressed poor tolerability and limited efficacy due to off-target kinase inhibitory activity in academia and clinical cases. As for selective RET inhibitor, compounds such as BLU-667 and LOXO-292 are under development, currently in clinical trials. We are currently developing potent and selective RET inhibitors to address unmet medical needs for RET-driven cancers. Our team has validated its high potency and selectivity via kinase profiling and in-vitro assay results. We also anticipate our compound to overcome certain mutation, such as V804M. Our compounds strongly suppress phosphorylation of RET in tumor cells with various RET fusions and mutations by inhibiting proliferation at below 10 nM. This potency result is higher than those of other mKIs with RET inhibitory activity. The inhibitory concentration of our compounds to VEGFR2 is more than dozens of fold compared with RET. In addition, our compounds reveal low cytotoxicity in Ba/F3 naïve cells in the 1 µM concentration. These findings lead us to conclude that our compounds can be potent and highly selective inhibitors of RET kinase, a potential novel therapeutic agents for RET-driven cancers.

#2210

Discovery of a novel third generation EGFR TKI and identification of its potential mechanism of resistance.

Tao Zhang,1 Rong Qu,1 Chengbin Chen,2 Linjiang Tong,1 Fang Feng,1 Yi Chen,1 Meiyu Geng,1 Ke Ding,2 Hua Xie,1 Jian Ding1. 1 _Shanghai Institute of Materia Medica, Shanghai, China;_ 2 _School of Pharmacy, Jinan University, Guangzhou, China_.

Non-small cell lung cancer (NSCLC) patients with activating EGFR mutations initially respond to first generation EGFR inhibitors (such as gefitinib and elortinib), however, clinical efficacy is limited by acquired resistance, frequently driven by the EGFR T790M mutation. In this work, we identified a pyrimidine-backbone compound D7 as a novel irreversible inhibitor of EGFR T790M mutation and sensitizing mutation, with selectivity over the wild type EGFR (WT EGFR). D7 exhibited potent in vitro anti-proliferation activity in tumor cells harboring EGFR T790M (NCI-H1975 cells) and sensitizing mutations (PC-9 and HCC827 cells), whereas it showed moderate or weak inhibition in cells expressing WT EGFR. Oral administration of D7 induced dramatic tumor regression in EGFR mutated NSCLC tumor xenograft models as well as in patient-derived xenograft (PDX) model harboring EGFR T790M mutation. To further explore potential mechanisms mediating drug resistance, we constructed D7-resistant cells by prolonged exposure of parental NCI-H1975 cells to gradually increasing concentration of this compound. Resistant cells exhibited signs of apoptosis-resistance to D7 treatment and demonstrated increased sensitivity when combined with Src kinase inhibitors both in vitro and in vivo. Mechanistic investigation revealed that hyperphosphorylation of Src and subsequent activation of anti-apoptotic signaling via Akt pathway contributed to D7 resistance. These results support D7 as a promising third generation EGFR inhibitor deserving further development, and identify potential combination strategies with potential biomarkers for NSCLC tumors that is resistant to this compound.

Keywords: EGFR inhibitor, lung cancer, drug resistance, combination therapy

#2211

Discovery of orally available and potent MPS1(TTK) kinase inhibitors for anti-cancer drugs.

Younho Lee,1 Juhee Kang,1 Sein Kang,1 Hyunkyung Kim,1 Kyung-ah Seo,1 Jieun Choi,2 Jaeyoung Ahn,2 Jung Beom Son,1 Nam Doo Kim1. 1 _NDBio Therapeutics, Incheon, Republic of Korea;_ 2 _Voronoi, Incheon, Republic of Korea_.

There are many cell cycle regulating kinases such as CDKs, Polo, Aurora, Bub, NEK/NimA, etc. Dual specificity protein kinase, MPS1(TTK) is one of them. The role of MPS1 is to regulate spindle assembly checkpoint(SAC) during cell mitosis. The inhibition of MPS1 activity causes cell death by premature mitotic exit. Some of MPS1 inhibitors (NMS-P153, BOS-172722, CFI-402257, BAY-1217389, and BAY-1161909) are in clinical trials. These compounds are treated for solid tumors with or without Paclitaxel. We discovered new orally available and potent inhibitors of MPS1 kinase (MPS1 enzyme IC50 < 10nM / MDA-MB-231 GI50 < 50nM, MDA-MB-468 GI50 < 50nM / HCT116 GI50 < 50nM, F% > 30%). The compounds selectively inhibit MPS1 based on kinase profiling and decrease phosphorylation of MPS1 and Phospho-HH3 signaling, effectively. Some of compounds were selected for further preclinical studies. 

## CLINICAL RESEARCH

### Circulating and Cell-free Biomarkers for Diagnosis and Monitoring of Cancer 3

#2212

Biomarker screening of surface epitope expression on exosomes using high throughput flow cytometry.

Christian Manganti, Mary Patton, Ajay Singh, Mohamed Khushman, Steven B. Mcclellan. _Univ. of South Alabama Mitchell Cancer Inst., Mobile, AL_.

Biomarker discovery is an active field of research for pancreatic cancer. These tumors are often diagnosed in very late stages of progression, when there is little hope of treatment. Unique biomarker signatures could lead to tests to detect these cancers at a much earlier stage when treatment might be successful. Many approaches have been used, proteomic analysis of body fluid, cell-free DNA from blood and exosomes in blood. We have developed a highly sensitive method using an acoustic focusing flow cytometer to analyze individual exosomes for the expression of surface markers using multi-color staining. Plasma samples were obtained from IRB consented patients with stage IV pancreatic cancer or normal healthy donors. Exosomes were isolated from the plasma via differential ultracentrifugation or size exclusion chromatography. Exosome preparations were measured by DLS to confirm correct size. We performed flow cytometric analysis using the lipophilic styryl dye FM 1-43 to identify true lipid bi-layer vesicles from debris, as well as directly labeled monoclonal antibodies to markers such as CD9, CD63 and CD81 to confirm that true exosomes were isolated. We have developed a robust workflow based in 96 well plates to screen over 300 epitopes via high throughput flow cytometry, using a 4 color assay. We have also validated the ability to sort exosomes using the OnChip Biotechnologies microfluidic cell sorter. Our group has previously shown that exosomes containing miR 155 can confer gemcitabine resistance to cells by down regulating DCK. Exosomes derived from cancer patient plasma displaying unique surface epitopes (compared to healthy samples) were sorted and tested via PCR for the presence of miR 155. We are in the process of refining our biomarkers of interest and will be presenting proprietary information at the conference.

#2213

Investigation of treatment resistance with DNA-damage agents in patients with triple negative breast cancer by ctDNA.

Rafael R. Brianese, Giovana T. Torrezan, Marina De Brot, Maria Nirvana Formiga, Vladmir de Lima, Fabiana B. Makdissi, Dirce Maria Carraro. _AC Camargo Cancer Center, São Paulo, Brazil_.

Loss of function (LOF) germline mutation in BRCA1 increases the risk of breast cancer, especially the triple-negative breast cancer (TNBC) subtype, a very aggressive tumor. TNBC exhibits high variability at both molecular and clinical aspects. BRCA impairment is associated to deficiency in homologous recombination repair (HRD) and tumor with HRD has benefit from treatment with drugs that induces DNA damage and also with PARP inhibitor. We previously detected that a significant fraction of TNBC diagnosed in young Brazilian exhibits BRCA1 impairment by both mechanisms - germline pathogenic mutation and promoter hypermethylation - and that this group of tumor presented better overall survival (Brianese et al 2018).

In the current study our aim is to comprehensively characterize the resistance to DNA-damage agents in patients with TNBC associated or not with HRD by investigating somatic mutations in circulating plasma DNA (cDNA). Thus, are also investigating 6 serial cDNA samples of patients during neoadjuvant and adjuvant chemotherapy treatment. Patients are subjected to genetic testing using a 26-gene panel including the homologous recombination (HR) predisposing genes for classifying the TNBC in hereditary or sporadic. The somatic mutations identified in the tumor biopsy by using a gene panel containing frequently mutated genes in breast tumor cancer have been screened in serial plasma samples to check allele frequency of the somatic mutation in circulating DNA and to correlate to therapy response. Results: We have enrolled 32 TNBC patients of which 28 were tested by germline variants. Based on 18 samples we had 50% of pathological complete response. Pathogenic mutations were identified only in BRCA1 in 5 out of 28 (17.8%) patients. Additionally, variants of uncertain significance (VUS) were identified in 18 out of the 26 genes (64.2% - 18/28) patients, being ATM the most affected gene by VUS. In terms of somatic variants, tumor mutation burden (TMB) analysis showed that 25% has high and 75% low TMB, with no association with BRCA1 germline status. Also, we found an average three somatic variants per tumor (range 1-7) and used as tumor marks in the screening of circulating DNA in plasma

(cDNA). Somatic mutations in TP53 were identified in all tumor biopsy samples. In cDNA in plasma before treatment, confident detection of at least one tumor mutation was observed in 6 out of 8 patients (75%), including somatic mutations in TP53 and SAMD9 genes. Serial plasma cDNA samples were completely investigated for two patients until now and the results showed great association with the clinical response data suggesting that the chemotherapy-resistance mechanisms can be investigated by ctDNA in TNBC. Supported by FAPESP, CNPq and Capes.

#2214

Development and validation of PredicineATLAS, a 600-gene liquid biopsy panel for simultaneous detection of TMB and genomic alterations in Immuno-Oncology.

Zhixin Zhao, Amy Chang, Tak Cheung, Feng Xie, Carlos Montesinos, Amy Xiaohong Wang, Kemin Zhou, Shidong Jia, JIanjun Yu, Pan Du. _Predicine, Hayward, CA_.

Introduction: Tumor mutation burden (TMB) is a measurement of the number of somatic mutations carried by tumor cells. Microsatellite instability (MSI) reflects the genetic hypermutability. Both TMB and MSI have been shown to be effective genomic biomarkers for predicting response to anti PD-1/PD-L1 immunotherapy. Whole exome sequencing (WES) is a traditional means to estimate TMB and MSI from tumor tissue, however, it is challenging to translate it into blood or urine due to its high expense and low tumor cell percentage. In the present study, we developed a 600-gene liquid biopsy TMB panel, PredicineATLAS, to measure genomic alterations and estimate TMB and MSI using human body fluids.

Methods: The PredicineATLAS panel contains 600 genes and covers 2.4Mb genomic regions (1.4 Mb of exonic coding regions). It was specifically designed for immuno-oncology, covering comprehensive immune- and cancer-related genes, biomarkers and MSI sites that are associated with anti PD-1/L1 therapy response. Reference materials with known genomic alterations and tumor cell lines were titrated with different concentrations to mimic different tumor contents for TMB and MSI measurement. Matched tissue and blood were used in validation study.

Results: PredicineATLAS panel-based TMB strongly correlated with the WES-TMB using TCGA dataset. Analytical validation showed that PredicineATLAS NGS assay is highly robust and sensitive for detection of genomic alterations. Using matched tissue and blood sample set, blood-based PredicineATLAS panel-TMB and MSI correlate with its counterpart derived from tissue WES.

Conclusions: PredicineATLAS liquid biopsy assay was developed and validated using reference materials and clinical samples. It detects genomic alterations and accurately estimate TMB and MSI, providing valuable genomic information to guide immuno-oncology therapy and clinical drug development.

#2215

SLFN11 and EZH2 protein expression and localization in circulating tumor cells to predict response or resistance to DNA damaging therapies in small cell lung cancer.

Lauren Averett Byers,1 Allison Stewart,1 Carl Gay,1 John Heymach,1 Luisa Fernandez,2 David Lu,2 Robin Rich,2 Lincy Chu,2 Yipeng Wang,2 Ryan Dittamore2. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Epic Sciences, San Diego, CA_.

Background: A recent phase II trial in recurrent small cell lung cancer (SCLC) demonstrated that patients with high SLFN11 expression in tissue biopsies had improved survival when treated with PARP inhibition (PARPi). EZH2 is also highly expressed in SCLC tissue and is associated with chemo-resistance through epigenetic silencing of SLFN11. Here we developed a liquid biopsy test to explore the clinical feasibility of utilizing SLFN11 and EZH2 expression on circulating tumor cells (CTCs) to aid in the prediction of drug response/resistance of DNA damaging chemotherapy or PARPi in SCLC.

Material and methods: 26 blood samples (19 baseline and 7 on-therapy) from SCLC patients treated at MDACC were sent to Epic Sciences for CTC enumeration (intact DAPI, CK+, cancer cell morphology) and characterization with SLFN11 and EZH2 immunoassays. Nuclear localization and expression of SLFN11 and EZH2 were quantified.

Results: 17/19 (89%) of baseline and 5/7 (71%) post-therapy samples had detectable CTCs. 19/26 (73%) samples had SLFN11+ CTCs and 8/26 (31%) samples had nuclear localized SLFN11. For samples with SLFN11+ CTCs detected, the percentage of SLFN11+ cells ranged from 2.4 to 100%. 10/26 (38%) samples had EZH2+ CTCs and 9/26 (35%) samples had nuclear localized EZH2. For samples with EZH2+ CTCs, the percentage of EZH2+ cells ranged from 19 to 83%. 6 samples had both nuclear localized SLFN11 and EZH2 CTCs detected.

Conclusions: Inter- and intra-sample heterogenous SLFN11 and EZH2 protein expression was observed in SCLC patients. Use of these assays is underway in SCLC patient samples to assess correlation with PARPi and platinum agent response.

#2216

Circulating tumor cell isolation based on both physical & biological properties.

Hugh Fan, Kangfu Chen, Pablo Dopico, Thomas George. _Univ. of Florida, Gainesville, FL_.

Isolation and detection of circulating tumor cells (CTCs) in peripheral blood have been used for cancer prognosis and treatment monitoring. Most CTC isolation methods are based on either physical property (e.g. size) or biological property (e.g. immunoaffinity) of CTCs. However, it is well known that CTCs are very heterogeneous and their properties are not uniform. For immunoaffinity-based approaches such as the FDA-approved CellSearch® that employs antibodies against epithelial cell adhesion molecule (EpCAM), they cannot detect those CTCs expressing little or no EpCAM (due partially to epithelial-to-mesenchymal transition). For size-based methods such as those using microfilters, CTCs with a smaller size are not detected while a large number of normal blood cells are retained by the filters. To address the challenge, isolation methods based on both physical and biological properties of CTCs have been explored.

We report here a microfluidic device that contains a serpentine main channel and an array of lateral microfilters. The key difference of the device from the conventional microfilter devices is that our filters are not in the direction of the main flow. The unique design of the device layout produces a two-dimensional flow that allows the majority of a sample to pass by while all cells have opportunities to interact with filters, resulting in a larger throughput, reduced cell clogging, and increased purity of cells isolated. The device is further functionalized by immobilizing antibodies on the surfaces of microfilters to achieve a platform that combines filtration-based CTC isolation with immunoaffinity-based isolation.

We first tested the device for the isolation of L3.6pl cells (pancreatic cancer cells) spiked in a buffer or blood, and compared the antibody-immobilized device with the same device containing filters only. Without antibody, the device has a capture efficiency from 69.8% to 83.9%, depending on the flow rate we used. With antibody, the device with combined CTC isolation mechanisms can accomplish a capture efficiency of (98.7 ± 1.2)% at a flow rate of 1.8 mL/h. Other tumor cells with different sizes have also been evaluated in the device.

Further we employed the device for enumerating CTCs from blood samples of pancreatic and colorectal cancer patients. We compared this integrated device with a previously reported device containing herringbone-based micromixers. We found that the new device generally detected a higher number of CTCs.

In summary, a microfluidic device has been developed to integrate filtration-based CTC isolation with immunoaffinity-based isolation. The device offers better performance than those based on one isolation mechanism only, with a potential to address CTC heterogeneity and detect CTCs with different size (single versus cluster) and/or various expression level (epithelial versus mesenchymal).

#2217

Serum metabolic profiling identified a distinct metabolic signature in bladder cancer smokers: A key metabolic enzymes associated with patient survival.

Chandra S. Amara,1 Chandrashekar R. Ambati,1 Venkatrao Vantaku,1 Piyarathna W b Danthasinghe,1 Sri Ramya Donepudi,1 Shiva S. Ravi,1 James M. Arnold,1 Vasantha Putluri,1 Gurkamal Chatta,2 Khurshid A. Guru,2 Hoda Badr,1 Martha K. Terris,3 Roni Bollag,3 Arun Sreekumar,1 Andrea B. Apolo,4 Nagireddy Putluri1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Roswell Park Comprehensive Cancer Center, Buffalo, NY;_ 3 _Augusta University, Augusta, GA;_ 4 _Center for Cancer Research, Bethesda, MD_.

Urinary bladder cancer (BLCA) is the 9th most common malignant disease and the 13th most common cause of cancer death worldwide. Occupational exposure to carcinogens has been long associated with increased BLCA risk. Tobacco smoke contains more than 60 carcinogens causing at least 18 different types of cancer including BLCA. Reliable biomarkers for accurately predicting survival in Bladdder cancer (BLCA) smokers is lacking due to complex genomic and transcriptomic heterogeneities associated with the disease. We performed liquid chromatography-mass spectrometry (LC-MS) based targeted metabolomic analysis for >300 metabolites in serum obtained from two independent cohorts of BLCA never smokers, smokers, healthy smokers, and healthy never smokers. 40 metabolites (FDR <0.25) were identified to be differential between BLCA never smokers and smokers. Increased abundance of amino acids (tyrosine, phenylalanine, proline, serine, valine, isoleucine, glycine, asparagine) and taurine levels were observed in BLCA smokers. Integration of differential metabolomic gene signature and transcriptomics from TCGA cohort resulted in intersecting 17 gene signature that showed significant correlation with patient survival in BLCA smokers. Importantly Catechol-O-Methyltransferase (COMT), Iodotyrosine Deiodinase (IYD), and Tubulin Tyrosine Ligase (TTL) showed a significant association with patient survival in publicly available BLCA smokers datasets and did not have any clinical association in never smokers.

#2218

Exosomal PD-L1 and T lymphocyte status predict the effect of anti-PD-1 therapy.

Xiujuan Qu, Chaoxu Zhang, Yibo Fan, Xiaofang Che, Yunpeng Liu. _The First Hospital of China Medical University, Shenyang, China_.

Background: Anti-PD-1 therapy is approved to be remarkable for cancer treatment. However, the response rate is dissatisfactory. PD-L1 expression in tumor tissue, which affected by chemotherapy, radiotherapy and activity of signal transduction pathways, is unreliable to predict treatment response. Recent studies suggest that exosomal PD-L1 not only exerts immunosuppressive effects besides tumors, but also plays a significant role in development of tumor microenvironment in the distance. A deeper research of exosomal PD-L1 assessment is needed to improve the predictive value and treatment efficacy.

Method: We enrolled 28 patients with advanced tumor, including 8 lung adenocarcinoma, 7 lung squamous carcinoma, 4 esophageal carcinoma, 2 colorectal carcinoma, 2 cholangiocarcinoma, 1 small cell lung cancer, 1 gastric carcinoma, 1 duodenal adenocarcinoma, 1 nasopharyngeal carcinoma and 1 tongue squamous cell carcinoma, who were treated by anti-PD-1 therapy followed the standard regimens. Exosomes were collected and purified from plasma with exosome isolation kit. Exsomal PD-L1 was detected by ELISA. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll gradient. Exhausted T lymphocyte were stained for cytometry.

Result: Compared with responder, exosomal PD-L1 of non-responders is significantly higher than responder (P=0.036) before treatment. Moreover, the CD4+ and CD8+ T lymphocyte count is higher and the percentage of exhausted T lymphocyte rate is lower in responder than the non-responder cohort before treatment.

Although there is no statistical difference, the level of exosomal PD-L1 decreased in responder cohortat on 6 to 8 weeks after treatment, on the contrary, increased in non-responder cohort after anti-PD-1 therapy. Concurrently, exosomal PD-L1 and tumor burden were decreased when the therapy was effective.

Conclusion: Low levels of exosomal PD-L1 and exhausted T lymphocyte rate on baseline and decreasing expression of exosomal PD-L1 after treatment could screen out the potential benefit populations of anti-PD-1 therapy in a variety of solid tumors. These findings suggest that expression of exosomal PD-L1 and exhausted T lymphocyte cell rate could be attractive and predictive biomarkers for clinical responses to anti-PD-1 treatment.

#2219

Evaluation of PD-L1 expression on circulating tumor cells (CTCs) in patients with advanced urothelial carcinoma of the bladder.

Sonja Bergmann, Anja Coym, Gunhild von Amsberg, Melanie Janning, Armin Soave, Michael Rink, Klaus Pantel, Sabine Riethdorf. _Univ. Medical Ctr. Hamburg-Eppendorf, Hamburg, Germany_.

Introduction: Immune checkpoint therapies with PD-1/PD-L1 inhibitors show durable responses in a subset of patients with metastatic urothelial carcinoma of the bladder (UCB). However, PD-L1 expression in tumor biopsies does not correlate with response to PD-1/PD-L1 inhibitors in all patients. In addition, determination of PD-L1 expression in tumor tissue has not been harmonized to date and biopsies from metastatic sites are often hardly accessible. Thus, a reliable predictive biomarker is still urgently needed. In this study, we perform detection of PD-L1 on circulating tumor cells (CTCs) in blood samples of patients with advanced UCB.

Experimental procedures: Blood samples of 93 non-metastatic (M0) and 33 metastatic (M1) UCB patients were analyzed using the CellSearch® system. Following EpCAM-based enrichment, CK+/DAPI+/CD45- cells were identified as CTCs using the CTC-Kit. For PD-L1 detection, the CellSearch® CXC-Kit (optional fluorescence channel: PE) proved more suitable than the CellSearch® CTC-Kit (optional fluorescence channel: FITC). After enumeration of CTCs by the CTC-Kit, for nine CTC-positive patients (M1) a second blood sample was further analyzed for PD-L1 protein expression using the CXC-Kit. Specificity of the PE-labelled anti-PD-L1-antibody (clone E1L3N) was validated by Western blot analysis, IF and FACS.

Results: Enumeration of CTCs by the CellSearch® CTC-Kit revealed significantly higher CTC-positivity in M1 than in M0 patients (51.5% vs. 22.6%, p=0.004 for ≥1 CTCs/7.5 mL blood; 30.3% vs. 3.2%, p<0.001 for ≥5 CTCs/7.5 mL blood). Also mean absolute CTC numbers were significantly higher in M1 than M0 patients (p<0.001). PD-L1 detection in the CellSearch® system was established using five UCB cell lines with different PD-L1 expression, according to Western blot analysis and FACS, which were spiked into whole blood of healthy donors. Using the CXC-Kit, ≥1 PD-L1+ CTCs were detected in 77.8% (7/9) of CTC-positive M1 patients. Thus, PD-L1 expression levels presented with intra- and inter-patient heterogeneity. The follow-up analysis of one patient who received anti-PD-L1 therapy showed substantial increases in CTC numbers as well as in the ratio and intensity of PD-L1+ CTCs in conjunction with disease progression under immunotherapy. The patient died within one month after the follow-up blood draw.

Conclusion: This is the first study demonstrating the feasibility to detect and monitor PD-L1 expression on CTCs of advanced UCB patients in the CellSearch® system. The established assay is readily available for clinical application and should be implemented in future clinical trials to evaluate its relevance for predicting response to immune checkpoint inhibitors.

#2220

Benchmarking of somatic variant calling algorithms for detection of circulating tumor DNA.

Preetida J. Bhetariya,1 Sabine Hellwig,2 David A. Nix,3 Gabor T. Marth,1 Mary P. Bronner,1 Hunter R. Underhill1. 1 _University of Utah, Salt Lake City, UT;_ 2 _ARUP Laboratories, Salt Lake City, UT;_ 3 _Huntsman Cancer Institute, Salt Lake City, UT_.

Purpose: Establishing a reliable bioinformatics variant calling workflow is crucial for untargeted searches of low-frequency variants in plasma circulating cell-free DNA (ccfDNA). We sought to benchmark the performance of nine variant calling algorithms on simulated data and cancer patient ccfDNA sequencing samples.

Methods: A simulated tumor dataset was constructed by in-silico introduction of pathogenic variants (303 SNVs and 91 INDELs; VAF of 0.1-12%) into a healthy control sequencing sample (128 gene capture panel; 128kb; 4200X depth). An unspiked replicate served as a control. Tumor and control datasets were analyzed by nine somatic variant calling algorithms available in the public domain (Table 1). Sensitivity and specificity were calculated for each caller. Variant calling algorithms that proved most successful in synthetic data (Lofreq, Mutect) were applied to 24 EGFR-mutant lung cancer patient ccfDNA library samples captured and sequenced under conditions identical to the benchmarking data set. Droplet digital PCR (ddPCR) was used to independently determine the VAFs of two EGFR SNVs (T790M and L858R) and exon 19 deletions in all lung cancer libraries (detected range: 0.1-48.5%).

Results:

Table 1: Sensitivity (Sens.) and false discovery rate (FDR) as determined for 9 variant callers. For synthetic data, results are shown for 1.6% VAF (NC = no call)

Caller | Synthetic data (1.6% VAF) | Lung cancer ccfDNA

---|---|---

|

SNV | INDEL | SNV | INDEL

|

% Sens. | % FDR | % Sens. | % FDR | % Sens.

<1% VAF | % Sens.

>1% VAF | % Sens.

<1% VAF | % Sens.

>1% VAF

Lofreq | 87.1 | 5.7 | 92.3 | 5.6 | 75.0 | 94.4 | 66.7 | 100

Mutect | 47.5 | 2.2 | 91.2 | 6.7 | 62.5 | 88.9 | 16.7 | 87.5

Mutect2 | 35.6 | 3.2 | 96.7 | 1.1

|

Strelka | 17.8 | 3.5 | 7.6 | 9.2

|

Freebayes | NC | NC | NC | NC

|

Varscan | NC | NC | NC | NC

|

Vardict | NC | NC | NC | NC

|

Lumpy | 13.9 | 86.1 | 7.7 | 92.1

|

Delly | NC | NC | NC | NC

|

Conclusion: Performance of variant callers differed greatly, with Lofreq providing the best sensitivity at low VAF. In cancer ccfDNA libraries Lofreq detected most but not all low-frequency variants, indicating that ccfDNA diagnostics require optimized variant calling workflows to improve detection of variants with a VAF <1%.

#2221

Chemotherapy sensitivity of tumor cells from ascites of ovarian cancer patients: Relationship with immune status and clinical response.

Guido Zaman,1 Judith E. den Ouden,2 Jelle Dylus,1 Antoon M. van Doornmalen,1 Rogier C. Buijsman,1 Astrid Eijkelenboom,2 Leon F. Massuger,2 Anne M. van Altena2. 1 _Netherlands Translational Research Center, Oss, Netherlands;_ 2 _Radboud Univeristy Medical Center, Nijmegen, Netherlands_.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. Standard treatment of advanced EOC is surgery in combination with chemotherapy, consisting of a platinum-based drug (carboplatin or cisplatin) and paclitaxel. Although most women show a good response to first line treatment, tumors do not respond in 15-20% of patients, whereas in 80% of cases of advanced EOC, the disease recurs within three years. Patients who have responded to first line treatment are referred to as 'platinum sensitive', and are treated with a PARP inhibitor in second line. Several other new targeted inhibitors are investigated in clinical trials, including PI3-kinase and bromodomain inhibitors. There is great need of new, more effective therapies with improved long-term treatment outcome, and diagnostic assays and biomarkers to predict chemotherapy response in the clinic. We have determined whether tumor cells isolated from ascites of patients with EOC can be used to develop in vitro cell proliferation assays. We also characterized the immune status of ascites by cytokine analysis and used flow cytometry to analyze the immune cell types in ascites. In a pilot study, ascites was gathered from 18 patients with advance stage EOC. Cells were isolated by centrifugation and tumor cells were separated from immune cells by adhering them to tissue culture plates. Sensitivity of tumor cells to platinum compounds, paclitaxel, PARP inhibitors, and other second line and investigational drugs, as well as drug combinations, was determined in cell proliferation assays, using intracellular ATP content as an indirect read-out of cell number [1]. The mutant status of a number of known oncogenes and tumor suppressor genes in the patient-derived cell samples was determined by DNA sequence analysis. Mutation status was related to histopathological data and in vitro drug response. We demonstrate that ascites from EOC patients contains many tumor cells and that these tumor cells can be used to perform drug sensitivity assays in vitro. In an ongoing study, in which ascites from hundred EOC patients will be included, the in vitro sensitivity to standard-of-care chemotherapy will be related to the immune status of the ascites and to the clinical outcome of the patient, which is defined by platinum sensitivity. An update of this study will be presented at the conference. [1] Uitdehaag et al. (2014) PLoS ONE 9(3): e92146

#2222

Progastrin a new biomarker for hepatocellular cancer patient follow-up.

Alexandre Prieur,1 Marie Dupuy,2 Dominique Joubert,1 Eric Assenat2. 1 _ECS Progastrin, Prilly, Switzerland;_ 2 _St-Eloi University Hospital, Montpellier, France_.

Background and aims: Alpha-fetoprotein (AFP) is the most widely used biomarker for hepatocellular carcinoma (HCC) patient follow-up, even though around 30% of HCC do not express AFP and its capacity to reliably monitor treatment efficacy is questioned. Interestingly, progastrin, a direct target of the oncogenic Wnt/beta-catenin pathway that is commonly activated in HCC, is secreted as such from tumor cells of various origin, from the very first steps of tumorigenesis. Here, we show the first study evaluating the potency of blood progastrin levels to monitor treatment efficacy and compared it with AFP.

Methods: Progastrin was quantified in the blood of 87 patients with HCC (focal (n=23), locally advanced (n=49) or metastatic (n=19). AFP was available in 79 patients. The cancerREAD technology (CE marked since 2017) specifically designed to detect progastrin with a high sensitivity and not any maturation products such as gastrin or gastrin glycine extended was used to assay progastrin in the blood. Patients received a systemic (nevaxar, tepotinib, regorafenib, nivolumab, anti FGR, or carbozantinib) and/or a local treatment. Variations in the levels of progastrin and AFP were analyzed in relationship with evolution of the disease: progression, decrease, stable, in remission, without taking into account the different treatment regimens.

Results: Progastrin was detected in 80% of the patients (cut-off value 1 pM, range 0 to 75.9 pM, mean value +/- SE 16.29 +/-1.97 pM). Patients in remission had a significantly lower blood progastrin compared to focal (p=0.019), locally advanced (p=0.0001) or metastatic HCC (p=0.015) (Focal 16.96 +/- 4.82 pM; locally advanced 18.57 +/- 2.75 pM; metastatic 17.81 +/- 4.9 pM; in remission 4.89 +/- 1.05 pM). AFP was above 10 ng/ml (a common threshold value) in 59.4% of the samples; levels of progastrin were not statistically correlated with those of AFP. Blood samples for follow-up of treatment were available for 48 patients. Compared to AFP, progastrin was a better biomarker of the evolution of the disease in 35.1% of the patients. Both biomarkers were equivalent in 32.4% and none were good in 10.8%. AFP was better than progastrin only in 21.6%.

Conclusion: Thus, and for the first time, we showed that not only progastrin is a better blood biomarker than AFP to detect HCC but also is better for the follow-up of HCC patients, whatever the treatment of the patient. This is the first study that shows the potential of progastrin (alone or in combination with AFP) as a tool for follow-up of cancer treatment efficacy.

#2223

High-purity isolation method of rare cells for molecular analyses using microfluidic chip type cell sorter.

Yasuhiro Koh,1 Mio Ikeda,1 Shunsuke Teraoka,1 Masayuki Ishige,2 Yuu Fujimura,2 Kazuo Takeda,2 Nahomi Tokudome,1 Yuichi Ozawa,1 Hiroki Ueda,1 Nobuyuki Yamamoto1. 1 _Wakayama Medical University, Wakayama, Japan;_ 2 _On-chip Biotechnologies Co., Ltd., Tokyo, Japan_.

Background: Liquid biopsy using noninvasive sampling of blood is rapidly advancing in cancer diagnostics. Circulating tumor cells (CTCs) are intact tumor-derived cells and are potentially utilized for further molecular analyses. Capturing rare CTCs with high purity, however, remains technically challenging. Here we report the results of preclinical evaluation of the multistep sorting method using On-chip Sort system (On-Chip Biotechnologies) for high-purity capture and isolation of rare cells in the blood for further molecular analyses.

Materials and Methods: Non-small-cell lung cancer cell lines PC-9, NCI-H1975 and A549 were used for this preclinical study. Cells were spiked into 4 mL of peripheral blood form healthy donors. After hemolysis and fixation, the samples were labeled with PE/Cy7-CD45 and FITC-cytokeratin (CK) antibodies and Hoechst for nuclei staining followed by three multiple sorting steps based on positive staining for CK and negative staining for CD45. For evaluating the potential of recovered tumor cells for genome sequencing, recovered NCI-H1975 cells, known to harbor EGFR L858R and T790M mutations, from spike-in samples were subjected to next-generation sequencing after whole genome amplification. To further establish the rare cells detection using the preserved blood samples, PC-9 cells were spiked the peripheral blood from healthy donors and transferred to the preservative tubes (Streck, Omaha, NE) and processed and sorted between 24 and 48 hours after the sample preparation.

Results: Up to 50 PC-9 and A549 cells were spiked into 4 mL of peripheral blood and recovery rate of both cells were as high as 70% and recovered white blood cells were as low as 20% after three cycles of sorting steps, suggesting the sufficient recovery and purity of tumor cells for further analyses. One NCI-H1975 cells were also spiked into 4 mL of peripheral blood and recovered cells were subjected to next-generation sequencing after whole genome amplification. Both EGFR L858R and T790M mutations were successfully called and mutant allele frequencies of these gene mutations were as high as those called from the control DNA which were extracted from 1000 NCI-H1975 cells. PC-9 cells (0, 10, 100, 500 cells) were spiked into 20 mL of peripheral blood and split into two preservative tubes and the recovery rate ranged from 55.0 to 61.3%, suggesting the robustness of the multistep sorting method when preservative tubes were used. Good correlation was also observed between the observed number and the expected number of cancer cells demonstrating the linearity with a correlation coefficient (R2) of 0.99.

Conclusions: We confirmed that rare cancer cells in the blood can be captured and isolated by the multistep sorting method using On-chip Sort system and can be utilized for further molecular analyses Results also suggest that this method allows us to deal with the preserved

#2224

A panel of autoantibodies against multiple tumor-associated antigens in the early immunodiagnosis of lung cancer.

Di Jiang,1 Tingting Wang,2 Lu Pei,3 Peng Wang,4 Hua Ye,4 Chunhua Song,4 Kaijuan Wang,4 Jianying Zhang,1 Liping Dai1. 1 _Institute of Medical and Pharmaceutical Sciences & Henan Key Laboratory for Tumor Epidemiology, Zhengzhou University, Zhengzhou, Henan Province, China; _2 _Department of Clinical Laboratory, Fuwai Huazhong Cardiovascular Hospital, Zhengzhou, Henan Province, China;_ 3 _Department of Clinical Laboratory, Zhengzhou Hospital of Traditional Chinese Medicine, Zhengzhou, Henan Province, China;_ 4 _Department of Epidemiology, College of Public, Zhengzhou University, Zhengzhou, Henan Province, China_.

Lung cancer is the most common cancers worldwide and with very low 5-year survival rate due to lack of effective early diagnostic methods. In our previous study, nine autoantibodies against tumor-associated antigens (TAAs) were identified to be potential biomarkers in detection of lung cancer and this study aimed to find the optimal panel of TAAs to detect lung cancer in the early stage.

Methods A total of 1,130 serum samples were included in this study, including patients with lung cancer, normal control and benign lung diseases patients. Autoantibodies against PSIP1,TOP2A,ACTR3,RPS6KA5,HMGB3,MMP12,GREM1,ZWINT and NUSAP1 were detected by enzyme-linked immunosorbent assay. Logistic regression models were generated from the training set and further validated in another independent set. We also evaluated the ability of the model to detecting benign lung diseases and early-stage lung cancer.

Results Two greater models were selected. One model including 5 autoantibodies panel (GREM1,HMGB3,ZWINT,TOP2A and PSIP1) and another model including 3 autoantibodies panel (GREM1,HMGB3 and PSIP1). Finally, the panel of 3 autoantibodies showed high diagnostic accuracy with areas under the curve (AUC) of 0.711(95 % CI 0.674-0.746) in training cohort and 0.891(95% CI 0.845-0.927) in validation cohort, respectively. The AUC of the model of 3 autoantibodies panel was 0.948(95% CI 0.912-0.972) in discriminate lung cancer and benign pulmonary disease. This model could discriminate early-stage lung cancer patients from normal controls, with AUC of 0.686(95 % CI 0.633-0.735) in training cohort and AUC of 0.911(95 % CI 0.849-0.954) in validation cohort, and the overall AUC for early-stage lung cancer was 0.779(95 % CI 0.738-0.816) when the two cohorts were combined.

Conclusions In summary, it would be more cost-effective and more efficient to use the model with 3 autoantibodies panel which has a high diagnostic performance for lung cancer detection, especially for early-stage lung cancer. And the model may apply a new help for the discrimination for benign pulmonary disease and lung cancer.

#2225

Exosomes in plasma for protein based cancer biomarkers: A clinical research tool using UHPLC-MS.

David A. Sarracino,1 Xiaolei Xie,1 Courtney E. Martel,2 Shen Luan,1 Kevin Rosenblatt3. 1 _ThermoFisher Scientific, Cambridge, MA;_ 2 _The Angiogenesis Foundation, Cambridge, MA;_ 3 _Consultative Genomics, Bellaire, TX_.

Introduction:

Exosomes have been used as a source of biomarkers in diagnostics for many diseases, including cancer, and are now being used as therapeutic delivery systems. To date, the most efficient method for isolating exosomes from serum or plasma samples has been to use a size exclusion, chromatography (SEC) based isolation. The conditions of the SEC separation can be modified to yield extracellular vesicles of different sizes and diagnostic value. Apoptotic bodies, 50-5000nm, microvesicles, 50-1000nm, and exosomes, 30-120nm, all have different diagnostic and targeted applications. Here, we show a routine and robust method for the isolation and monitoring of proteins in exosomes isolated from plasma samples. The methods and conditions used here are focused on rapidly providing a high yield of exosome vesicles in a monodisperse 40nm size range from a plasma sample.

Materials and methods:

K2EDTA plasma samples from donor disease and control groups were collected with IRB approval. All SEC columns and reagents for exosome isolation were obtained as a kit (NX Prenatal, Houston, TX). The 40nm exosome fraction from the column was lysed, the proteins reduced, alkylated and digested with trypsin; the subsequent peptides were analyzed by ultra-high performance liquid chromatography (Vanquish Horizon, ThermoFisher Scientific), tandem mass spectrometry (Q-Exactive, ThermoFisher Scientific), to quantify and identify greater than 500 proteins from exosomes, isolated from a single plasma sample, in a 1 hour method. Subsequent isolations of 40nm exosome fractions were labeled with a sulfo-NHS-biotin linker, followed by lysis and enrichment with streptavidin columns, and an identical LC-MS analysis to obtain quantitative analysis of the surface membrane protein contents on the exosomes.

Results:

Raw data was analyzed using the label-free quantitation module in Proteome Discoverer (Thermofisher Scientific). Peptide peak areas from three technical replicate runs were used to ascertain the reproducibility of the method across both normal and disease samples. Membrane protein extracts LC-MS data was further grouped by Protein Center (ThermoFisher Scientific) by cell type and tissue of origin.

Conclusions:

The methods shown here provide a robust and efficient platform for 40nm exosome isolation from human plasma samples, and quantitative analysis of the protein content thereof for the purposes of biomarker discovery, quantification and screening purposes.

#2226

Validation of a liquid biopsy tool to identify point mutations in pediatric brain tumor patients.

Liana Nobre, Michal Zapotocky, Monique Johnson, Jonathan Wasserman, Oussama Abla, Jim Whitlock, Uri Tabori, Cynthia Hawkins. _SickKids Hospital, Toronto, Ontario, Canada_.

The use of liquid biopsies to diagnose and monitor tumors has been widely explored especially in adult cancers. In pediatric cancers, the feasibility of this method as a clinical tool is still to be established. Pediatric low grade gliomas (PLGG) are characterized by mutations in the MAPK pathway, and 20% harbour BRAFV600E mutation, which can also be found in paediatric patients with Langerhans cell histiocytoma (LCH), thyroid carcinoma and melanomas. Specific histone mutations have been described in pediatric high grade gliomas(HGG) and diffuse midline pontine gliomas(DIPG). Midline gliomas are not amenable to a gross total resection; however, a biopsy is needed for pathological and molecular diagnosis. In this scenario, liquid biopsy is of utmost importance and has the potential to spare the risk of morbidity with surgical procedures, determine diagnosis and prognosis, as well as serve as a tool to guide and monitor response to therapy with targeted agents. The objective of this study is to evaluate the use of droplet digital PCR (ddPCR) for identification of point mutations in cerebro-spinal fluid (CSF) or plasma of BRAF V600E or H3K27M positive pediatric patients. CSF was collected from a total of 51 patients, and plasma from 55 patients. ctDNA was extracted from 3 ml of plasma or 2 ml of CSF, and pre-amplified prior to ddPCR, which was conducted on the RainDance system. For BRAFV600E: Forty-five patients had CSF samples available for analysis, 8 had known positive brain tumors, 32 negative brain tumors, 5 normal controls; 5/8 positive cases had BRAFV600E mutation in ctDNA from CSF, one of the patients with negative CSF and positive tumor was on treatment with BRAF inhibitors. Plasma was analysed in 17 patients with known positive brain tumors and additionally 10 patients negative for BRAFV600E, all of the plasma samples were negative for the mutation. For H3K27M: Thirty patients had CSF samples for analysis 1 was a known positive tumor, 27 negative and 2 unknown status (radiological diagnosis of DIPG). Of the 3 DIPG samples 2 were positive, one with unknown status of the primary tumor. Plasma was also analysed for H3K27M mutation in 7 patients with known positive brain tumors, one DIPG with unknown status and 7 negative samples. All plasma samples tested negative for the mutation. Sensitivity and Specificity for this assay was respectively 62% and 97% for BRAF and 66% and 98 % for H3K27M in CSF. The sensitivity in plasma for brain tumors is poor, however seems better for non-CNS lesions. In summary, we show that liquid biopsy with analysis of CSF ctDNA is feasible with high specificity, important in the context of a diagnoses tool. Moving forward larger cohorts need to be validated towards the goal of implementing ddPCR as a clinical tool for diagnosis of specific point mutations in pediatric patients with CNS tumors.

#2227

Identifying 14,15-DHET, a metabolite of soluble epoxide hydrolase (sEH), as an early biomarker to predict cancer therapy-induced cardiotoxicity and screening of sEH inhibitors for medical intervention.

Hyesook Kim, Julia M. Santos, Aby Joiakim, David J. Kaplan, David A. Putt. _Detroit R &D, Inc., Detroit, MI_.

Approximately 1.7 million new cancer cases and 610,000 cancer-related deaths are estimated for 2018 in United States. Chemo and radiation therapy are the most common treatments given to cancer patients. However this treatment may induce cardiac events such as hypertension, left ventricular dysfunction, cardiomyopathy and heart failure. Chronic cardiotoxicity from anthracyclines, e.g., doxorubicin (DOX), occurs in ~2-5% of patients at least 1 year after completion of chemotherapy. Detection of early biomarkers, which predict cardiac dysfunction before any damage occurs, offers an opportunity to adjust an individual's dosage and protocol during chemo and radiation therapy and provide targeted medical intervention. Epoxyeicosatrienoic acids (EETs) have antihypertensive properties and attenuation of the conversion of EETs to dihydroxyeicosatrienoic acids (DHETs) by inhibiting the activity of soluble epoxide hydrolase (sEH) has been tested as a therapeutic approach for treating various cardiovascular diseases. Aims of this study are to find whether 14,15-DHET and an oxidative stress biomarker, 8-isoprostane, are early biomarkers to predict DOX-induced cardiotoxicity using DOX-treated H9c2 rat cardiomyocytes with and without recovery periods and to screen inhibitors, which may prevent the sEH-dependent cardiotoxicity. H9c2 cells were incubated with and without 1 µM DOX for 2 hr and cells and media were collected after 2, 6 and 26 hr recovery periods. Cardiomyocyte hypertrophy, cell area, ANP and BNP mRNA expressions of the H9c2 cells were measured. The 14,15-DHET and 8-isoprostane levels of cell media were measured using ELISA kits. No cellular hypertrophy was detected after a 2 hr recovery period following 2 hr DOX treatment. However, cellular hypertrophy was detected after 6 and 24 hr recovery periods. Levels of 14,15-DHET, but not 8-isoprostane, increased after a 2 hr recovery period and prior to detection of cellular hypertrophy. 14,15-DHET remained elevated after 6 and 24 hr of recovery periods. To confirm the presence of sEH enzyme in the cells, 14,15-DHET levels were measured in cell media after treatment of the cells with and without 1 µM and 5 µM EET (substrate) for 30 min and it was found that 14,15-DHET levels increased 2.6- and 4.5-fold, respectively. Honokiol, a component of magnolia bark, and AUDA, a synthetic sEH inhibitor, inhibited sEH activity in a reconstituted system with recombinant sEH. The results suggest that 14,15-DHET is an early serum biomarker to predict cardiotoxicity. Early diagnosis of patients to predict cardiac dysfunction is necessary to adjust the anti-cancer drug treatment protocol. Moreover, inhibition of activity of the target enzyme, sEH, may ameliorate the chemotherapy-induced cardiotoxicity. Supported by NHLBI SBIR Phase I Contract HHSN261201600028C.

#2228

Comparison of the Epic CTC HRD assay vs. tumor DDR mutations in metastatic castration-resistant prostate cancer (mCRPC) patients (pts).

Joseph Schonhoft, Adam Jendrisak, Jerry Lee, Angel Rodriguez, Ramsay Sutton, Yipeng Wang, Ryan Dittamore, Mark Landers. _Epic Sciences, San Diego, CA_.

Background: PARPi have recently demonstrated clinical activity in a subset of mCRPC pts with defects in homologous recombination (HRD). Predicting PARPi response with HRD gene mutations have struggled to show clinical specificity. Previously, we described a novel circulating tumor cell (CTC) based method (CTC-HRD) that identifies a specific cell morphology associated with genomic scarring [e.g. large scale transitions (LST)] and demonstrated CTC-HRD's clinical utility in identifying pts likely to respond to PARPi and likely to resist to standard of care (SOC) therapies (Tx), including AR signaling inhibitors. We also observed some discordance between CTC-HRD and DNA damage response (DDR) mutational status from tissue sequencing. Here we compare the prevalence of CTCs, # LSTs/CTC and the % of CTCs with BRCA2 loss in mCRPC patient samples with matched tumor sequencing DDR status.

Materials and methods: 302 CTCs from 26 mCRPC pts collected prior to Tx were collected from pts with matched tissue biopsy sequencing. CTCs were identified, assayed for CTC-HRD status, and low pass whole genome sequenced for LSTs and gene copy number alterations (CNA). We compared the prevalence of CTCs, HRD properties of each identified cell including BRCA2 copy loss, # of LSTs/CTC and their prevalence in cells derived from pts vs. CTC-HRD score and tissue DDR mutational status.

Results:

CTC-HRD | DDR Tumor | # Pts | Median CTC/mL | Median LST/CTC | Median % CTC BRCA2 Loss per Pt

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

+ | + | 9 | 13.8 | 32 | 59.00%

+ | - | 10 | 10.4 | 23 | 47.20%

- | + | 7 | 2.4 | 3 | 0.00%

CTC-HRD positive pts, independent of tissue DDR status had higher CTC/mL, higher median # LST/CTC and higher percentages of BRCA2 loss in their CTCs vs. those pts who were CTC-HRD negative. Notably, we identified 10 pts that were negative for tissue DDR mutation but were CTC-HRD+ with a high frequency of CTC BRCA2 loss.

Conclusions: These results highlight the concordance of the Epic CTC-HRD assay with known markers of HRD and demonstrate differences in CTC-HRD status from tissue DDR mutations which have struggled to achieve the required clinical specificity required for PARPi patient selection.

#2229

Molecular profile of histological and mutational heterogeneity of adenocarcinoma of the stomach in tumor burden monitoring using circulating tumor DNA.

Noriyuki Sasaki,1 Takeshi Iwaya,1 Takehiro Chiba,1 Masashi Fujita,2 Fumitaka Endo,1 Mizunori Yaegashi,1 Ryo Sugimoto,3 Tamotsu Sugai,3 Doris Siwak,4 Lance Liotta,5 Yilling Lu,4 Gordon Mills,6 Hidewaki Nakagawa,2 Satashi S. Nishizuka7. 1 _Department of Surgery, Iwate Medical University School of Medicine, Morioka, Japan;_ 2 _Laboratory for Genome Sequencing Analysis, RIKEN Center for Integrative Medical Science, Tokyo, Japan;_ 3 _Department of Molecular Diagnostic Pathology, Iwate Medical University School of Medicine, Morioka, Japan;_ 4 _Department of Systems Biology, The University of Texas, MD Anderson Cancer Center, Houston, TX;_ 5 _Center for Applied Proteomics and Molecular Medicine, George Mason University, Fairfax, VA;_ 6 _Department of Cell, Development & Cancer Biology, Oregon Health Science University School of Medicine, Portland, OR; _7 _Division of Biomedical Research and Development, Institute of Biomedical Sciences, Iwate Medical University, Morioka, Japan_.

PURPOSE: This study aimed to perform molecular profiling of the heterogeneous histological and mutational background of primary adenocarcinoma of the stomach for pre- and post-operational tumor burden monitoring using circulating tumor DNA (ctDNA).

METHODS: Ten patients with gastric adenocarcinoma who underwent surgical resection for clinical Stage IB or higher were included. Multi-region samples (three sites) per resected specimen were used for cancer cellulality estimation, a 151-gene ClearSeq panel sequencing on Next Generation Sequencer (NGS), and proteomic profiling with 294 proteins using reverse-phase protein arrays (RPPAs). The ctDNA level was evaluated by digital PCR (dPCR) using 25 originally-designed sets with the Hypercool Primer & ProbeTM technology based on mutations of individual tumors. With the median follow-up of 808 days, a total of 187 serial plasma samples were examined for ctDNA.

RESULTS: A total of 103 mutations were detected in 30 regions from 10 tumors. Twenty founder mutations (i.e., mutations found in all three regions) were observed in eight tumors whereas two tumors had only non-founder mutations. Twenty-three and 60 non-founder mutations were detected in two regions and one region per tumor, respectively. Variant allele frequencies (VAFs) of founder mutations were higher than those of non-founder mutations (31.2% vs 14.5%; p < 0.01) in primary tumors. With sample regions in which mutations were not detected in trio by NGS, non-founder mutations could be detected by dPCR at low VAFs (ranging from 0.02 to 2.2%) in 95% (19/20) regions. In preoperative patient plasma, ctDNA was detected in 30% (3/10) patients (with a mean VAF of 0.59%). The rate of preoperative ctDNA detection was lower than that of esophageal squamous cell cancer (24/26, 92.3%) and colorectal cancer (10/12, 83.3%) patients. With respect to the tumor stromal effect to ctDNA, no ctDNA was detected in three cases of scirrhous type cancers (0/3, 0%), whereas three of six cases of the other stromal types (3/6, 50%) showed detectable ctDNA (VAF>0.03%). In two relapsed cases with peritoneal dissemination, the elevation of ctDNA was not apparent even at the time of diagnosis of the relapse by CT scan. Among 42 gene-protein matched pairs, the level of proteins did not seem to predict the coding gene mutation. However, tumors with TP53 mutations had significantly higher levels of p53 than those with the wild-type, which was likely due to protein stabilization (p = 0.0004).

CONCLUSIONS: Detecting ctDNA in gastric cancer patients may not be as feasible as in other gastrointestinal cancer patients likely due to the heterogeneous histological and mutational background. If applicable, founder mutations are the most suitable marker for detection and monitoring ctDNA. Mutations in the primary tumor itself did not appear to predict protein levels except for TP53.

#2230

Diversity of peripheral CD8+PD-1+ T cells is a novel predictive biomarker for response to anti-PD-1 antibody treatment in lung cancer patients.

Seiji Matsumoto,1 Takaji Matsutani,2 Yoshiko Fujita,1 Kazutaka Kitaura,2 Tohoru Nakamichi,1 Akifumi Nakamura,1 Ayumi Kuroda,1 Masaki Hashimoto,1 Nobuyuki Kondo,1 Ryuji Suzuki,2 Seiki Hasegawa1. 1 _Hyogo College of Medicine, Nishinomiya, Japan;_ 2 _Repertoire Genesis Incorporation, Ibaraki, Japan_.

Background: Anti-PD-1 antibodies (nivolumab) are effective in the treatment of many cancers, including malignant melanoma, non-small cell lung cancer (NSCLC), renal cell cancer, and head and neck squamous cell carcinoma. Immune checkpoint inhibitors (ICIs) are only effective in around 20% of patients, which saw a demand for the development of biomarkers that predict a therapeutic response before administering treatment. As the biomarkers intratumoral PD-L1 expression and tumor mutation burden (TMB) both require tumor tissue biopsy, there is now a demand for safer and less invasive biomarkers. Peripheral and tumor CD8+PD-1+ T cells share neoantigen-specific T-cell receptors (TCRs), and are presumed to act as effector T cells with an antitumor effect at the tumor site. We analyzed the diversity in terms of TCR α and β repertoires on peripheral CD8+PD-1+ T cells and examined the relationship between this diversity and therapeutic effect of nivolumab.

Methods: This study used patients administered nivolumab after exhibiting no response to chemotherapy for recurrence following surgery. Peripheral blood mononuclear cells were collected from patients before administration of nivolumab. CD8+PD-1+ T cells were subjected to FACS sorting, NGS-based TCR repertoire analysis was performed by Repertoire Genesis Inc., and TCR diversity was evaluated statistically. CT scan was performed during week 12 of treatment and used to determine response to nivolumab. This study was approved by the Ethical Committee of Hyogo College of Medicine.

Results: Six of 12 patients responded to treatment. Upon comparing these responders (CR, PR) with non-responders (SD, PD), there were no differences in the proportion of PD-1+ in CD8+ T cells and the proportion of CD8+PD-1+ T cells in gated lymphocytes. TCR α diversity was significantly higher among responders than non-responders based on Shannon index, Simpson index and DE50 (P < 0.05, P < 0.05, P < 0.01, respectively). TCR β diversity was also significantly higher among responders than non-responders based on Shannon index, Simpson index and DE50 (all P < 0.01). Progression-free survival (PFS) was 371 days for responders and 148 days for non-responders. Overall survival (OS) was 633 days for responders and 308 days for non-responders, showing a significant difference between the groups.

Conclusion: TCR repertoire analysis was performed on CD8+PD-1+ T cells in easily-obtainable peripheral blood before nivolumab treatment in patients with NSCLC, and nivolumab was observed to be effective in patients with high TCR diversity. This result indicates the TCR diversity of peripheral CD8+PD-1+ T cells is effective as a predictive biomarker for response to ICI therapy. In the future, we intend to analyze TMB and neoepitopes using surgical specimens of these patients and determine the efficacy of this biomarker for cancers other than NSCLC.

#2231

A simple blood base test for predicting clinical benefit of cancer immunotherapy.

Luhui Shen,1 Jianfen Chen,2 Stephen Albert Johnston,3 David Hong,2 Jianjun Gao,2 Aung Naing2. 1 _The Biodesign Inst. at Arizona State Univ., Tempe, AZ;_ 2 _UT MD Anderson Cancer Center, Houston, TX;_ 3 _The Biodesign Inst. at Arizona State Univ. and Calviri, Inc., Tempe, AZ_.

Immunotherapy with immune check point inhibitors (CPI) has dramatically transformed cancer therapy. However, only ~25% cancer patients have a positive clinical response to CPI. Moreover, the cost of the treatment and the risk of severe side effects makes it necessary to develop biomarkers to predict the benefit of the treatment.

It has been shown that the tumor neoantigen load correlates with a positive response to treatment. This indicates that pre-existing anti-tumor immune responses to neoantigens can be used for the CPI response prediction. We have discovered a new source of frameshift (FS) neoantigens created by errors in RNA production in tumor cells, including the insertion and deletion (INDEL) of microsatellite regions during the RNA transcription and the mis-splicing of exons. These errors can generate FS neoantigens, which are highly immunogenic and can elicit both T cell and B cell immune response in cancer patients. We have shown that, although most antibody reactivity to FS peptides (FSPs) are personal, there are common antibodies reactive in different cancer patients, even across different cancer types. The FSPs with positive reactive antibodies can offer protection in mouse tumor models as vaccines.

We thus hypothesize that antibodies reactive to FSPs in cancer patients can be used for predicting the clinical benefit of cancer immunotherapy. There is a total of~ 220,000 potential FS neoantigens that can be generated by INDELs of transcription and mis-splicing of genes. These neoantigens can be represented by ~400,000 FSPs, 15-amino acids peptides. We have created arrays of by in-situ synthesis of these FSPs. We used these array to test our hypothesis with pre-treatment serum of 40 cancer patients, from 26 different cancer types in clinical trials with CPI treatments. A total of 13 patients had a clinical response to CPI treatment. Similar to ELISA, diluted serum were applied to the FSP array, and total IgG were detected by fluorescent labeled antibody. IgG reactive to each FSP was measured by the fluorescent intensity and then median normalized within each array for the analysis. As predicted, there are common IgG antibodies reactive to FSPs in the response patients. By selecting 100 to 500 most significantly different reactive FSPs between two group patients, and trained with prediction models, such as SVM, our FSP array can reach up to 96% accuracy in the prediction of clinical response with leave-one-out validation. We hypothesize that the FSPs with positive IgG reactive in response patients may be related to anti-tumor immune response, which is need to be further investigated. We also showed that the FSP array can potentially predict the patients who may have high grade immune related adverse events with the CPI treatment.

Our preliminary data indicates that the FSP array is a promising technology for predicting the clinical benefit of immunotherapy. We will expand our sample size to further evaluate this technology.

#2232

The assay of thymidine kinase 1 in sera from subjects with hematological and solid tumors with AroCell TK 210 ELISA: Comparison with TK-liaison assay and its clinical implications.

Jagarlamudi Kiran Kumar,1 Staffan Eriksson,1 Kerstin Hamberg Levedahl,2 Martin Höglund,2 Mojca Zupan,3 Joško Osredkar,,4 Diana Cviič,4 Urska Furar4. 1 _AroCell AB, Uppsala, Sweden;_ 2 _Uppsala University, Uppsala, Sweden;_ 3 _Blood transfusion centre, Ljubljana, Slovenia;_ 4 _Institute of clinical biochemistry, Ljubljana, Slovenia_.

Background: Thymidine Kinase 1 (TK1) is an ATP dependent enzyme involved in DNA precursor synthesis. It is released into the blood from cells undergoing un-controlled proliferation where it forms stable aggregates. Serum TK1 activity has been used as a biomarker for diagnosis and monitoring of hematological malignancies. Several commercial assays are available for measuring serum TK1 activity but they require radioactive materials or special instruments. To overcome these, AroCell has developed the TK 210 ELISA for TK1 protein measurements. In this study, we compared the performance of the TK 210 ELISA to that of the TK-Liaison assay on sera from subjects with hematological and solid tumors

Experimental Procedures Sera from patients with hematologic malignancies (N=51) were collected at the University Hospital, Uppsala. Samples from solid tumors [breast cancer (n=60); subjects with benign prostate hyperplasia or prostate carcinoma (n=60)] along with blood donors (N=102) were collected at the University Medical Centre, Ljubljana. TK1 protein levels were determined with AroCell TK 210 ELISA and TK1 activity levels by the TK-Liaison assay.

Results In blood donors, the TK1 protein levels were in the range of 0.1 to 0.35 µg/L with a median value of 0.2. Men had a higher median value TK1 protein levels compared to women (0.21 vs 0.19) with no significant difference. Both the TK 210 ELISA and the TK-Liaison assays showed significantly higher TK1 levels in hematological and solid tumors compared to blood donors (P<0.0001). ROC curve analysis demonstrated that across all malignancies, at 96% specificity, TK 210 ELISA (cut-off value of 0.34 µg/L) gave a higher sensitivity of 0.43 compared to TK-Liaison assay (cut-off value of 10 U/L) of 0.36. The performance of the assays differed depending on the type of malignancy, TK 210 ELISA and TK-Liaison had a similar sensitivity (0.65) for hematological malignancies. However, in the case of solid tumors, TK 210 ELISA showed a higher sensitivity (0.35) compared to TK-Liaison (0.26) at a specificity of 0.96. A regression analysis of the TK 210 ELISA (y) and TK-Liaison (x) assays across all three groups gave an equation of y = 0.15 + 0.017x (rs = 0.80, n = 273). The correlation value in hematological malignancies was higher than in blood donors and solid tumors (0.95 vs. 0.67 and 0.50 respectively).

Conclusions This study showed that the AroCell TK 210 ELISA has a similar sensitivity and specificity to the TKLiaison assay for hematological malignancies. However, the AroCell TK 210 ELISA demonstrated a higher sensitivity for TK1 in sera from subjects with solid tumors and this may facilitate the application of TK1 as a biomarker for solid tumors. Furthermore, the robustness and convenience of the ELISA not only overcomes the limitations of TK-Liaison but also widens the clinical applications of TK1 in cancer management.

#2233

Correlation between mutations found in FFPE tumor tissue and paired cfDNA samples.

Lauren Saunders, Antonia Hur, Brittany Niccum, Asmita Patel. _Beckman Coulter Life Sciences, Indianapolis, IN_.

Cell free DNA (cfDNA) consists of small (150 - 500 bp) DNA fragments that circulate in the blood. cfDNA levels tend to be low in healthy, non-pregnant patients, and increase in patients with cancer, pregnancy, or extensive damage to tissue. cfDNA is believe to derive mostly from apoptotic cells, and biomarkers for a variety of diseases have been found in cfDNA. As cfDNA is extracted from blood, it is a non-invasive way to detect disease; however, there is some concern that cfDNA does not contain the same biomarkers as tumor tissue. This study measures the efficacy of cfDNA as a biomarker detection medium by comparing mutations found in both FFPE tumor samples and paired cfDNA samples. This study determines whether the same biomarkers are found in each sample type, and which of those can be used as biomarkers in both and which are preferred biomarkers for only a single sample type. Trends in mutation detection with the two different sample types are discussed.

#2234

Monitoring of genetic change in circulating tumor DNA isolated from Her2 positive advanced gastric cancer patients.

Woo Sun Kwon,1 Seung-Hyun Jung,2 Yeun-Jun Chung,2 Joong Bae Ahn,3 Hyun Cheol Chung,3 Sun Young Rha3. 1 _Songdang Institute for Cancer Research, Yonsei University College of Medicine, Seoul, Republic of Korea;_ 2 _Integrated Research Center for Genome Polymorphism, Precision Medicine Research Center, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea;_ 3 _Songdang Institute for Cancer Research, Brain Korea 21 PLUS Project for Medical Science, Division of Medical Oncology, Department of Internal Medicine, Yonsei Cancer Center, Yonsei University Health System, Seoul, Republic of Korea_.

Gastric cancer is the common solid cancer and a leading cause for cancer related mortality worldwide. Despite developments and improvement in diagnosis, surgical technique, chemotherapy and radiotherapy, median survival time of advanced/metastatic gastric cancer (AGC) patients who received systemic chemotherapy is between 15-18 months. Tumor heterogeneity is associated with heterogeneous genetic profiling, which lowers diagnostic precision and consequently becomes an obstacle to determining the appropriate therapeutic strategies for individual cancer patients. Therefore, predictive biomarkers for determining potential treatment strategy are needed to improve the prognosis of patients. Personalized medicine has emerged as the future of cancer care to ensure that patients receive individualized treatment specific to their needs. In order to provide such care, molecular techniques that enable oncologists to diagnose, treat, and monitor tumors are necessary. Liquid biopsy by genotyping circulating tumor DNA (ctDNA) has provided a non-invasive and real-time liquid biopsy approach in assessing tumor genomic alterations in clinical oncology. This ctDNA have been associated with poor prognosis and clinical outcome in various cancer including lung, breast and colorectal. In this study, we evaluated utility and genetic profiling of ctDNAs in AGC. Under IRB approval, blood samples were collected in Cell-Free DNA BCT® streck tubes from 14 Her2-positive. Depending on the treatment schedule, blood can be collected 2 to 5 times per patient sequentially. The isolated ctDNAs were analyzed to genetic profiling by OncoChase (95 genes) based on AmpliSeq. And tumor tissue genotyping by CANCER MASTER and compared different sample preparation conditions. Validation of blood-based tumor genomic profiling in additional multicenter outcome studies is necessary; however, ctDNA monitoring can provide clinically important actionable information for precision oncology approaches.

#2235

Circulating microRNA-205 as a potential prognostic biomarker for recurrent ovarian cancer.

Meghan W. Kusch, Julia A. Chapman, Carl P. Weiner, Katherine F. Roby, Dineo Khabele, Helen H. Zhou. _University of Kansas School of Medicine, Kansas City, KS_.

Background:

Ovarian cancer is the most lethal gynecological malignancy and the fifth most common cancer in women in the U.S. Patients diagnosed in advanced-stage ovarian cancer have 70-95 % chance of relapse. Additional non-invasive approaches to monitor response to treatment and to predict recurrent disease are urgently needed. Aberrant expression levels of circulating microRNAs are diagnostic, prognostic, and predictive biomarkers in ovarian cancer. We recently published that microRNA-205 (miR-205) expression is elevated in ovarian cancer and correlates with advanced-stage disease. Here, we investigate circulating microRNA-205 as a potential biomarker for monitoring disease in patients diagnosed with advanced-stage ovarian cancer.

Methods:

Plasma samples from normal controls (n=12) and recurrent stage III/IV ovarian cancer patients (n=5) in our biorepository were used. Samples were collected 1 day before surgery operation and at 1 month, 2 months and 6 months after the primary surgery. All samples were from serous ovarian carcinomas. All the patients went through the standard chemotherapy and CA-125 levels were decreased to the normal range (<35U/ml) at 6 months after the surgery. All recurrent disease was detected within 2 years after primary surgery. Circulating microRNAs were extracted from plasma of ovarian cancer patients and normal controls using miRNeasy Mini Kit (Qiagen). The reverse transcriptions were conducted using qScrip microRNA cDNA Synthesis Kit. (VWR). The circulating miR-205 expression was confirmed by quantitative real-time polymerase chain reaction (qPCR). Results are reported as mean ± S.E. Student's t-test was applied.

Results:

Our results confirmed that the expression of circulating miR-205 was significantly elevated in stage III/IV ovarian cancer plasma samples compared with normal controls (5.91 fold; p<0.05). Compared with levels in paired pre-operative plasma, circulating miR-205 expression was significantly lower in the post-operative plasma at 1 month (p<0.01), gradually rose to pre-operative levels at 2 months, and significantly increased by 6 months post-operation (p <0.05). Circulating miR-205 expression levels were significantly increased at 6 months in post-operative samples when CA-125 levels were at normal range, suggested the circulating miR-205 can potentially be used as a biomarker for ovarian cancer recurrence.

Conclusion:

Dynamic changes of the circulating miR-205 expression in pre- and post-operative plasma samples of recurrent stage III/IV ovarian cancer patients demonstrated that circulating miR-205 levels could be used as non-invasive biomarkers for monitoring disease in patients diagnosed with advanced stage ovarian cancer. Further investigation is warranted in larger cohorts of patients.

#2236

Development and validation of urine cfDNA preservative kit for detection of genomic alterations in cancer.

Zheng Li, Haoran Tang, Tiantian Zhang, Chunxiao Liu, Shengnan Zhu, Ying Zhang, Binggang Xiang, Zhixin Zhao. _Predicine, Shanghai, China_.

Introduction: cfDNA (circulating cell-free DNA), short DNA fragments shredded from dying normal and tumor cells, has been proved capable of serving as molecular biomarkers for clinical decision making, including but not limit to tumor diagnosis, treatment and monitoring. Urine, by its non-invasive nature and unique biological characteristics, is far superior comparing to other liquid biopsy specimens, such as blood and cerebrospinal fluid, and has broad clinical implications for genitourinary cancers. The challenge for urine cfDNA test, is the instability of cfDNA, caused by nuclease, high content of urea, and contamination from genomic DNA released from nucleated cells and microbials in urine. Here, we have developed a robust and user friendly urine preservative collection Kit to maintain integrity of cfDNA in urine and to facilitate transportation of urinary samples to clinical laboratory.

Methods: Urine samples from 5 donors collected using the developed urine collection kit were tested after incubation under 4°C and 37°C, mimicking extreme storage or transporting environments, and were compared to untreated urine and treated urine by another commercial preserve buffer. cfDNA was extracted after incubation of 0, 4, 7 days and then quantified by ddPCR with 3 markers: EGFR as an endogenous DNA marker, PSA (synthetic oligos containing intron-deleted sequence, pre-incubation spike-in) as an exogenous DNA marker, ARv7 (synthetic oligos containing intron-deleted sequence, post-incubation spike-in) as an inner control for the extraction and detection assays.

Results While untreated urine showed significant decrease after incubation under 37°C, cfDNA derived from urine stored in developed urine collection tubes remained stable till day 7. There is no significant change on normalized copies of EGFR and PSA in the treated urine samples under 4°C for up to 7 days.

Conclusion: the developed Urine Collection Kit demonstrated great urine cfDNA preserving capacity under different temperatures for at least 7 days, on par with commercially available preserve buffer and even outperforming it under certain circumstances. The developed kit provides an easy-to-use solution of obtaining stable urine cfDNA for clinical use.

#2237

**Detection of** CK-19 **mRNA positive CTCs, isolated with a size-based microfluidics platform, in NSCLC patients under osimertinib therapy.**

Aliki Ntzifa,1 Athanasios Kotsakis,2 Vassilis Georgoulias,2 Evi Lianidou1. 1 _University of Athens, Athens, Greece;_ 2 _Hellenic Oncology Research Group (HORG), Athens, Greece_.

Background: Patients with EGFRmt non-small cell lung cancer (NSCLC) and resistant to first and second generation EGFR tyrosine kinase inhibitors (EGFR TKIs), receive therapy with osimertinib (third generation EGFR TKI). However, some patients experience resistance to osimertinib and disease progression. Molecular characterization of circulating tumor cells (CTCs) could offer great advantages as a non-invasive approach for disease monitoring. In this study, we isolated CTCs using a label-independent microfluidic-based platform to elucidate resistance mechanisms in these NSCLC patients.

Materials and methods: Peripheral blood (PB) (15mL) was obtained at different time points from 27 patients with advanced NSCLC under osimertinib therapy. CTCs were isolated using the ParsortixTM system (ANGLE, Plc) and further harvested in Trizol reagent, followed by extraction of total RNA and cDNA synthesis. RT-qPCR was performed for CK-19 and B2M (used as a reference gene) mRNA in the COBAS z480 system (Roche Diagnostics).

Results: PB samples were obtained at baseline (n=25), after one cycle of treatment with osimertinib (n=23), every 3 months (n=83) and at disease progression (PD) (n=16). CK-19 positive CTCs were detected in 3/25(12%) baseline samples, 24/83(28.9%) during treatment at different time points; 5/23(21.7%) after one cycle of treatment, 8/23(34.8%) after 3 months, 3/17(17.6%) after 6 months, 4/12(33.3%) after 9 months, 1/2(50%) after 12 months, 3/5(60%) after 15 months; and 5/16(31.2%) at PD.

Conclusions: CK-19 mRNA-positive CTCs were isolated using an epitope-independent enrichment microfluidic device. Further molecular characterization of these cells at the gene expression, DNA mutation, DNA methylation and miRNA level will provide biomarkers for the elucidation of resistance mechanisms in NSCLC patients treated with osimertinib.

#2238

Characterization of T cell clonality and tumor mutation burden utilizing the TCRSeq NGS and the comprehensive NGS assays.

Hua C. Gong, Hua Gong, Andreas Papoutsis, Wen Wu, Christian Laing. _Navigate BioPharma, a Novartis Subsidiary, San Diego, CA_.

Background: Somatic mutations are the basis for the generation of potential neoantigens recognized by antitumor T lymphocytes, which may contribute to increase T cell diversity [1]. Next-generation sequencing of expressed T cell receptor (TCR) transcripts allows for quantification and assessing clonality of T cell diversity [2]. Tumors that exhibit a low TCR diversity and potentially having a high clonality have been shown to be lower responders to immunotherapy treatments.

Methods: Utilizing the Shannon entropy to arrive at a clonality readout, we sought to gain mechanistic insights and demonstrate a link between clonality and total mutation load. We conducted the analysis using a diverse set of solid tumors and hematological malignancies across three different specimen types (bone marrow (BM), peripheral blood (PB), formalin-fixed paraffin embedded (FFPE tissue)). Specifically, we used the beta TCRSeq NGS and the Comprehensive NGS assays (internally developed) to characterize sample clonality and mutation load respectively.

Results: The TCRSeq NGS assay sequences the V(D)J domains of the variable region of the TCR heavy chain (involved in epitope recognition), while the hybrid capture Comprehensive NGS assay detects variants across a 236 gene panel optimized for SNV and INDEL detection. Initial analysis across the different specimen types resulted in average clonalities of BM = 0.255, PB = 0.149, FFPE = 0.092, with average tumor mutation loads of 7.6, 7.6, and 21 respectively. Categorization of all samples by increasing clonality, independent of specimen type, established a clear differentiator with samples harboring a clonality of less than 0.10 to exhibit an average mutation load of 26.33. This was statistically significant from samples harboring clonalities between 0.10 and 0.19, which had an average mutation load of 7.86, and samples with clonalities equal to or greater than 0.20, having an average mutation load of 7.67.

Conclusions: Based on two different categorical analyses, lower T cell clonality readouts appear to be associated with solid tumors harboring greater genetic diversity/neoantigen burden than hematological malignancies with higher clonalities. As tumor mutation burden has been correlated with clinical outcomes of varying efficacy, this study provides further insight into the utility of T cell clonality as a "molecular tag" for immune response and resistance in oncological therapeutics.

#2239

Analysis of nucleosomal DNA as an extraction control for plasma-based circulating tumor DNA assays.

Mahrukh M. Syeda,1 George Karlin-Neumann,2 Iman Osman,1 David Polsky1. 1 _New York Univ. School of Medicine, New York, NY;_ 2 _Bio-Rad Laboratories, CA_.

Purpose: Challenges to accurate quantitative measurements of circulating tumor DNA (ctDNA) include variable contamination with large DNA fragments released during mononuclear cell lysis, and potential variability in extraction efficiency. We investigated optimal procedures for plasma processing and the potential utility of a DNA spike-in as a process control for extraction efficiency.

Methods: Initial experiments analyzed the potential benefits of different centrifugation protocols (1600 x g vs. 1600 x g followed by 16000 x g) for plasma separation by analyzing the size distribution of extracted DNA using a BioAnalyzer. In subsequent experiments we used droplet digital PCR (ddPCR) to compare differences in extraction efficiency between nucleosomal DNA (nDNA) and genomic DNA (gDNA) spiked into healthy donor plasma and extracted using the QIAamp DSP Circulating NA Kit (Qiagen). To evaluate the properties of the purification columns with respect to the yield of different DNA sizes, we compared the plasma results to experiments extracting purified nDNA and gDNA spiked into PBS instead of plasma. We investigated the potential competitive effect of large DNA on the extraction efficiency of small DNA by mixing nDNA with commercially available human gDNA in different concentrations. To assess if nDNA, when spiked into patient plasma, would track with endogenous ctDNA, we spiked a pool of plasma from patients with BRAFV600E mutations with known quantities of nDNA purified from NRASQ61K mutant cells (CHP212). The spiked plasma pool was divided and extracted using multiple separate columns. Copies/ml and mutant fraction were determined by ddPCR assays for BRAFV600E and NRASQ61K. Coefficients of variation were also calculated.

Results: Comparison of different centrifugation speeds for plasma processing before and after freeze-thaw indicated that a 2nd high-speed centrifugation (16000 x g), either before or after a freeze-thaw cycle, minimizes large DNA in plasma samples prior to ctDNA extraction. In initial spike-in experiments extraction efficiency for nDNA was 87.7% compared to 42% for gDNA. We obtained similar results when purifying the nDNA and gDNA from PBS instead of plasma. We observed greater variability in nDNA extraction efficiency when increasing amounts of gDNA were added as a contaminant. Finally, we found that the slight, paralell variations in extraction yields of externally spiked NRASQ61K mutant nDNA and endogenous BRAFV600E ctDNA in 7/9 replicate extractions. The overall coefficients of variation (total copies/mL) for 9 extractions for BRAFV600E and NRASQ61K were 3.9 and 4.2 respectively.

Conclusion: A second centrifugation step helps reduce potential contamination of ctDNA with large DNA, and may improve the recovery of ctDNA from plasma samples. Nucleosomal DNA can potentially be used as a process control for extracting ctDNA from plasma samples.

#2240

Redefining personalized medicine by drug response profiling of patient-derived spheroids.

Stephen Shuford, Christine Wilhelm, Ashley M. Smith, Melissa Rayner, Jeremy Stuart, Lillia Holmes, Matt Gevaert, Howland E. Crosswell, Teresa M. DesRochers. _KIYATEC, Inc., Greenville, SC_.

Personalized medicine in cancer typically refers to the use of genetics and/or biomarkers to direct the use of targeted therapy or predict overall prognosis based on statistical probability. Therapy selection and predictions are not based on any physical interaction between a patient's tumor cells to clinically relevant therapies based on their disease indication. We have developed an assay using 3D cell culture that exposes a patient's tumor cells to standard of care chemotherapies for the purpose of predicting their clinical response to potential treatment options prior to treatment. We have analytically validated this assay enabling its performance under CLIA regulations as a Laboratory Developed Test (LDT) and prospectively validated it against clinical patient outcome in ovarian cancer. The test utilizes excess fresh patient tissue acquired during standard of care surgical debulking or biopsy and returns results within 7 business days of tissue receipt, typically well before the start of chemotherapy. Previously, we have shown in newly diagnosed ovarian cancer, the test has an accuracy of 87% with a specificity of 100% and a sensitivity of 84% in the prediction of standard first-line carboplatin/taxol combination therapy using the biomarker CA-125 and CT imaging as clinical readouts. We have similar results for the prediction of response to neoadjuvant therapy following laparoscopic biopsy using RECIST criteria. We have now analytically validated the assay in glioblastoma (GBM) and rare tumors. Preliminary clinical validation in GBM has shown the ability of the test to accurately predict response to standard first-line temozolomide using RANO criteria as the clinical readout. Rare tumor validation has included a panel of 12 drugs covering those used as standard of care for most rare tumors. Aspects of validation have included examining inter- and intra-assay variability and drug panel response in a defined number of rare tumors including sarcomas, neuroendocrine, and other tumors such as Sertoli-Leydig. In breast cancer, we have validated the assay for the use of a single diagnostic biopsy core as the tissue source and established preliminary clinical validation against standard of care such as doxorubicin and paclitaxel with pathologic complete response (pCR) as the clinical readout. We are further validating the predictive ability of the test in newly diagnosed and relapsed ovarian cancer and GBM patients (clinical trial NCT03561207). With demonstrated accurate prediction of patient specific response, the transition to cancer therapy selection based on physical evidence vs statistical probability would significantly improve patient outcomes and benefit economic stakeholders.

#2241

Molecular characterization of circulating tumor cells in head and neck squamous cell carcinoma: Direct comparison of a label-independent size-based microfluidic device with EpCAM-based CTC enrichment.

Martha Zavridou,1 Sophia Mastoraki,1 Areti D. Strati,1 George Koutsodontis,2 Apostolos Klinakis,3 Amanda Psyrri,2 Evi S. Lianidou1. 1 _Univ. of Athens, Athens, Greece;_ 2 _Medical School, Athens, Greece;_ 3 _Biomedical Research Foundation Academy, Athens, Greece_.

Background: Circulating tumor cells (CTCs) heterogeneity is highly affecting the efficiency of their isolation and thus the reliability of downstream analysis. Especially in Head and Neck Squamous Cell Carcinoma (HNSCC) epithelial mesenchymal transition (EMT) is highly affecting CTC isolation and downstream analysis. We directly compared two different approaches used for CTC isolation, a label-independent size-based microfluidic-based system versus an EpCAM-based positive selection for downstream molecular characterization of CTC both at the gene expression and DNA methylation level in HNSCC.

Methods: Peripheral blood (PB) in EDTA (20mL) was collected from 50 HNSCC patients and 18 healthy donors (HD). A size-based microfluidic device (Parsortix, ANGLE) and an EpCAM-based positive immune-magnetic isolation procedure were applied in parallel, using 10mL PB in each case. Total RNA was isolated from enriched CTCs and RT-qPCR was used to study the expression levels of CK-19, PD-L1, EGFR, TWIST1, CDH2 and B2M. Real time methylation specific PCR (MSP) was used to study the methylation status of SOX17, RASSF1A and MLL3 genes in DNAs isolated from the same enriched CTCs.

Results: In identical blood draws, the label-free size-based CTC-isolation system was superior in terms of sensitivity when compared to the EpCAM-based CTC enrichment, since a significantly higher percentage of identical PB samples was found positive for all genes tested both at the gene expression and DNA methylation level, while the specificity was not affected.

Conclusions: In HNSCC CTC molecular characterization at the gene expression and DNA methylation level should be based on a label-free size-based isolation system.

### Combination Immunotherapies 1

#2242

Roles of FGFR inhibition on murine RCC cells in combination antitumor activity of lenvatinib with anti-PD1 antibody via regulatingIFN gamma signaling pathway.

Yusuke Adachi, Kenji Ichikawa, Yu Kato, Yasuhiro Funahashi. _Eisai Co., Ltd., Tsukuba-shi, Ibaraki, Japan_.

Lenvatinib mesilate (LEN) is an oral multiple receptor tyrosine kinase (RTK) inhibitor that selectively inhibits the kinase activities of VEGFR1-3, in addition to other proangiogenic and oncogenic pathway-related RTKs including FGFR1-4; PDGFRα; KIT; and RET. LEN plus everolimus for advanced renal cell carcinoma (RCC) after one prior anti-VEGF therapy was approved in the US and EU in 2016. Currently, Phase1b/2 clinical trial of LEN in combination with pembrolizumab with selected solid tumors including RCC and Phase3 study of LEN in combination with everolimus or pembrolizumab for metastatic RCC patients are in progress. In this study, we investigated the antitumor and immunomodulatory activities of LEN alone and in combination with anti-PD1 Ab and explored the mechanism of action underlying the activities of LEN and PD1 blockade in preclinical syngeneic RCC models.

We examined antitumor activity of combination treatment of LEN at 10 mg/kg (po, qd) and anti-PD1 Ab at 10 mg/kg (ip, twice weekly) in the murine subcutaneous RCC model using RAG cell line. Immune cell population analyses in tumor tissues were performed by flow cytometer. Immune-related gene expression profile in tumor tissues or cultured cells was analyzed by qPCR and RNA seq. Effects on FGFR and IFN gamma (IFNγ) signaling pathways and their downstream molecules in cultured cells were analyzed by western blot.

In the murine syngeneic RAG tumor model, the combination treatment of LEN and anti-PD1 Ab showed tumor shrinkage, and some of tumors were regressed to nonpalpable sizes with long duration of response. LEN alone and in combination with anti-PD1 Ab significantly decreased the population of tumor associated macrophage in tumor tissues by FCM analysis. Gene expression analysis for immune cell responses in tumor tissues revealed the upregulation of IFNγ and its downstream genes including PD-L1 with combination of LEN and anti-PD1 Ab. Western blot analysis elucidated that FGFR signaling pathway inhibited the activation of IFNγ signaling pathway and the induction of PD-L1 expression in murine RCC cells, and LEN-treatment reactivated the IFNγ signaling pathway by inhibition of FGFR signaling.

Combination of LEN and anti-PD1 Ab showed greater antitumor activity including tumor shrinkage with long duration of response in the murine RCC model. Modulation of immune microenvironment by LEN and combination of LEN with PD1 Ab may underlie the strong antitumor activity in preclinical RCC model. Inhibitory activity of LEN against FGFR reactivated the IFNγ pathway and PD-L1 expression suppressed by activation of FGFR signaling pathway, and this inhibitory activity of LEN may potentiate the antitumor activity of combination treatments with anti-PD1 Ab. These preclinical results provide one of the unique mechanisms of combinational effect of LEN with PD1 blockade in the preclinical RCC model.

#2243

Pharmacologically modified pluripotent stem cell-based cancer vaccines demonstrate major anti-tumor activity and anti-metastatic potential.

Masae Kishi,1 Afag Asgarova,1 Diana Chaker,1 Christophe Desterke,1 Marie-Ghislaine de Goër de Herve,2 Ali G. Turhan,1 Annelise Bennaceur Griscelli,1 Frank Griscelli3. 1 _Paris Sud University, Villejuif, France;_ 2 _Paris Sud University, Bicetre, France;_ 3 _Université Paris Descartes, Villejuif, France_.

Cancer stem cells (CSCs) are difficult to isolate and to eradicate due to their resistance to conventional therapies. They represent a major target in order to achieve cure and to prevent metastatic spread. Their presence has been shown in several cancers with "stemness" signature and EMT/mesenchymal-like phenotype. Moreover, CSC have been shown to express some of the critical pluripotency genes shared with embryonic stem cells (ESC) and induced pluripotent stem cells (IPSC). The goal of thus work was to determine the feasibility of developing a novel universal cancer vaccine strategy, combining an allogenic pluripotent stem cell (PSC)-based vaccine and epigenetic drugs that will target selectively CSCs compartment. To test the feasibility of this strategy, we have used an aggressive triple negative breast cancer model using 4T1-GFP-Luc cell line. Upon transplantation into mammary fatpads of immuno-competent Balb/C mice, 4T1 cells generated a population of CD44+/CD24-/low stem-like cells with induction of a massive metastatic spread to lungs. Transcriptome analysis of these stem-like cells revealed more than 1000 pluripotency genes, commonly expressed with murine ESC. To determine if the vaccination with mESC was able to prevent the tumorigenicity of 4T1 cells, Balb/C mice pretreated with intradermal injection of irradiated murine ESC (mESC), murine IPSC or human ESC were challenged with 5.104 4T1 cells. A drastic reduction of tumor volume (73%) was observed after vaccination with murine ESC / IPSC as well as with hESC. To enhance the immunogenicity of ESC, hESC were primed with HDAC inhibitors (HDACi) to enhance the level of MHC class I expression. Combined treatment by primed hESC-based vaccine and HDACi induced a significant reduction of metastatic spread to the lung (more than 12-fold) as compared to controls. This synergistic effect and the superior anti-tumor response was accompanied by highly increased levels of tumor infiltrating lymphocytes (TILs) with recruitment of CD4+ and CD8+ lymphocytes into the tumor and with reduced levels of PD-1 expression on spleen CD4+ and CD8+ T cells. In addition, this anti-tumor effect was associated with reduction of T-Regs and MDSC both in the tumor and the spleen. Thus, these results show for the first time the possibility of using PSC as allogeneic anticancer vaccines. They also demonstrate for the first time that the methodology that we developed is able to drastically reduce the metastatic spread and to profoundly modify the tumor microenvironment. Finally they also strongly suggest that the observed conversion of immunologically "cold" tumors to "hot" tumors should improve the response rates to checkpoint inhibitor therapies with synergistic effects. Overall, these experiments pave the way for the future off-the shelf, universal pan-cancer vaccines strategies based on PSC in a broad range of cancers.

#2244

DeepIL-15 primed T cells synergize with PD-L1 blockade to overcome resistance to checkpoint immunotherapy.

Elena Geretti, Xiaoyan Liang, Jesse Lyons, Philip Bardwell, Santina Caruso, Becker Hewes, Jonathan Fitzgerald, Karsten Sauer, Thomas Andresen. _Torque Therapeutics, Cambridge, MA_.

Introduction Interleukin-15 (IL-15) activates and expands both CD8+T cells and NK cells but not immunosuppressive Tregcells. While IL-15 is an attractive asset for cancer immunotherapy, its systemic administration is limited by toxicities. To limit IL-15 systemic exposure, we have developed DeepTM IL-15, a multimer of chemically crosslinked IL-15/IL-15 Rα/Fc heterodimers (IL15-Fc). Deep IL-15 is surface anchored to tumor reactive T cells prior to adoptive cell transfer (ACT). This novel therapeutic approach enables Deep IL-15 loading onto cells at concentrations unachievable with systemic IL15-Fc, causes autocrine T cell activation and expansion, yet limits systemic exposure and associated toxicities. The anti-tumor activity of T cell therapies has been limited by insufficient T cell expansion and by checkpoint immunosuppression. Here, we combined Deep IL-15 primed T cells with PD-L1 blockade to overcome these limitations.

Methods Deep IL-15 was synthesized by incubation of IL15-Fc with a crosslinker. PMEL CD8+T cells (PMEL) were isolated from B6.Cg-Thy1a/Cy Tg(TcraTcrb)8Rest/J mice,activated, expanded, and Deep IL-15 was anchored to PMEL to generate Deep IL-15 Primed PMEL (Deep-15 PMEL). B16-F10 tumor-bearing mice were treated with cyclophosphamide (4 mg/mouse) one day prior to injection of 107Deep-15 PMEL (carrying ~ 58 ug of IL15-Fc)± anti-PD-L1 antibody (10 mg/kg q2w). We monitored mouse weight, tumor volumes, transferred and endogenous T cells, IL15-Fc systemic exposure, and cytokine release.

Results Deep-15 PMEL showed significantly improved expansion and anti-tumor activity compared to PMEL, and provided long-term protection against tumor re-challenge. Importantly, the toxicity profile of Deep-15 PMEL was better than that of IL15-Fc co-injection with PMEL, yielding lower systemic exposure to IL15-Fc, less IFN-γinduction and no expansion of endogenous CD8+T and NK cells. Deep-15 PMEL resulted in 2/10 partial regressions and a statistically significant tumor growth inhibition compared to both vehicle and PD-L1 blockade alone. Co-administration of anti-PD-L1 further improved the anti-tumor activity of Deep-15 PMEL, resulting in 10/10 Complete Regressions (CR) and 6/10 Tumor Free Survivors. Conclusions By loading tumor-reactive T cells with Deep IL-15 before ACT, Torque's proprietary Deep Priming technology leverages the immune-stimulating power of IL-15 while avoiding immunotoxicity. Deep IL-15 Priming promotes engrafted T cell-specific activation and proliferation, and a favorable toxicity profile. In vivo transfer of Deep-15 PMEL combined with checkpoint inhibition was well tolerated and elicited remarkable anti-tumor activity in the PD-L1 refractory B16-F10 model, with 10/10 CRs. Torque is initiating a Phase I trial for TRQ15-01 (Deep IL-15 Primed multi-targeted human T cells) and planning a combination arm with checkpoint blockade in solid tumors.

#2245

Improving the effectiveness of immunotherapy in breast cancer by targeting the tumor microenvironment.

Hasan Korkaya,1 Eunmi Lee,1 Raziye Piranioglu,1 Maria Ouzounova,1 Ahmet Korkaya,1 Jason Gestwicki,2 Max S. Wicha,3 Esteban Celis1. 1 _Augusta Univ., Augusta, GA;_ 2 _UCSF, San Francisco, CA;_ 3 _University of Michigan, Ann Arbor, MI_.

Large cohorts of recent clinical studies have firmly established that increased levels of tumor-infiltrating lymphocytes (TILs) in TNBC and HER2+ subtypes predicted better clinical outcome compared to the luminal subtype. These observations led to the hypothesis that women with TNBC or HER2+ subtypes may respond to a checkpoint blockade. However, early results from these trials using check point inhibitors alone or in combination with chemotherapy have shown very little promise in breast cancer patients, despite the remarkable long-lasting responses in other hard to treat malignancies such as non-small cell lung and melanoma. Although the outcome falls short of the expectation, it has suggested that the combinations of check point blockade with therapeutics that target immunosuppression may potentiate its efficacy. TNFα exhibits paradoxical roles; it may fuel tumor cell growth, invasion and metastasis in some tumor types, while in others it induces cytotoxic cell death. We recently demonstrated that TNFα distinctly induces A20 in TNBC subtype and protects these cells from TNFα-induced cytotoxic cell death by upregulating HSP70 protein and maintaining EMT/CSC phenotype. In contrast, luminal MCF7 or ZR75-1 cells display approximately 70% apoptosis when treated with TNFα. Overexpression of A20 in luminal cells not only protected them from TNFα-induced cytotoxicity by upregulating HSP70 and EMT/CSC phenotype, but also exhibited aggressive metastatic properties in mouse xenograft models. Furthermore, we show that A20/HSP70 pathway attracts tumor-infiltrating lymphocytes (TILs) while inducing the accumulation of immunosuppressive MDSCs in syngeneic mouse models. Interestingly, pulmonary DTCs as well as the immune infiltrates from 4T1 tumor-bearing mice exhibited significantly higher HSP70 expression. Therefore, we proposed that targeting HSP70 may potentiate the efficacy of immunotherapy in preclinical models of breast cancer. As previously reported, murine 4T1 tumors do respond to check point inhibitors. We reasoned that this may be an appropriate model to test the efficacy of HSP70 inhibitor, JG-231. Expectedly, there was no difference in tumor growth and metastasis between control and anti-PDL1 treated animals, however, combination of anti-PDL1 antibody ed with JG-231 and chemotherapy (cyclophosphamide-CTX) significantly reduced primary tumor growth (>10 fold) and eliminated metastasis. Collectively, our pilot experiments provide a strong rationale for testing our hypothesis and may lead to a rapid translation into the clinical utility.

#2246

Targeting co-inhibitory checkpoint signals of PD-1, LAG3 and Tim3 mediates syngeneic cell killing.

Huajun Yang,1 Zegen Wang,1 Fei Wang,1 Kaiyue Hao,1 Weibin Tan,2 Xiaoyan Fu,2 Henry Li,3 Frank Xing1. 1 _CrownBio, Taicang, Jiangsu, China;_ 2 _Taicang Blood Center of Suzhou, Taicang, Jiangsu, China;_ 3 _CrownBio, San Diego, CA_.

Cancer immunotherapy, with immune checkpoint inhibition in particular, has provided substantial clinical benefit in a significant number of patients with advanced diseases. The overall response rate to such therapies, however, remains low, and the effectiveness of immunotherapies for many tumors has been disappointing. Since it is known that recognition of a single antigen is insufficient for T cell activation, we propose to target co-inhibitory checkpoint signals in connection to screening of a large panel of syngeneic murine cell lines for improved in vitro therapeutic efficacy. Here we sought to examine the pharmacological consequence of co-inhibiting PD-1/LAG3 and PD-1/Tim3 signals upon T cell activation and their killing effect over a panel of 20 frequently used syngeneic cell lines. The goal of this work is to provide an in vitro system to evaluate the effectiveness of targeting multiple checkpoint targets in preclinical studies and discover biomarkers associated to the in vitro responses of murine tumor cells to activated T cell. To this end, we first investigated mutation and gene expression profiles of 20 mouse cancer cell lines for 50 well defined cancer-related mutations among 30,690 variants in exonic regions called by RNAseq (Illumina HiSeq X10). The oncogenic mutations include ALK (3 - frequency, same for the rest), BRAF (4), BRCA1 (7), BRCA2 (12), EGFR (3), ERBB2 (6), EGFR3 (2), FBXW7 (10), FLT3 (12), HRAS (1), KRAS (8), NRAS (1), PDGFRA (11), PTCH1 (9), PIK3CA (2), PTEN (6), RET (3), SETD2 (5), SMAD4 (3), SMO (13), TRP53 (13), TSC1 (3), and TSC2 (10). The same set of genes were also subject to mRNA expression change analysis. We next developed an in vitro co-culture system to include activated mouse splenocyte primed in murine tumor-bearing mouse spleen, which were then further activated by CD3/CD28, and co-cultured with each of the 20 syngeneic cell lines. The co-cultures were treated against anti-PD-1, anti-LAG3 and anti-Tim3 mAbs as single agents, and the combination of the three therapeutic agents, anti-PD-1 and anti-LAG3, and anti-PD-1 and anti-Tim3. Tumor cell viability was measured at the end of the assay, and immune cell markers were analyzed before and after the treatments as well. The data were analyzed for biomarker discovery. The results and clinical implications will be discussed.

#2247

Platinoid-induced MHCI antigen presentation enables checkpoint inhibitor responsiveness.

Ingmar Niels Bastian,1 Weihua Li,1 Qui Phung,2 Kathleen Fisch,1 Jennie Lill,2 Michael Karin,1 Shabnam Shalapour1. 1 _UCSD, La Jolla, CA;_ 2 _Genentech, SSF, CA_.

Many cancers are refractory to immune checkpoint therapy (ICT), but the causes of ICT resistance remain unknown. Despite low mutational burden, hepatocellular carcinoma (HCC) responds to PD-1 blockade, but prostate cancer (PCa) does not. RNA-seq analysis revealed that, unlike HCC, PCa cells poorly express MHC-I molecules and components of the MHC-I antigen processing and presentation machinery. We found that treatment of PCa cells and other tumor types with platin-based drugs, especially low dose Oxaliplatin, led to a pronounced upregulation of MHC-I molecules and their antigen generating and loading machinery. Most importantly, Oxaliplatin synergistically potentiated the effect of exogenous IFNγ; a conclusion based on two cumulative observations. First, ATAC-seq analyses revealed that Oxaliplatin activates MHC machinery through different pathways compared to well-known IFNγ signaling. Second, Oxaliplatin upregulates IFNγR2 expression in cancer cells. Induction of IFNγR2, which was needed for Oxaliplatin-potentiation of ICT-induced tumor rejection in vivo, depended on NF-κB signaling. Induction of IFNγR2, which was needed for Oxaliplatin-potentiation of ICT-induced tumor rejection in vivo, depended on NF-κB signaling. Moreover, we found that Oxaliplatin activates histone acetyltransferases (HATs) which alter chromatin structure of genes encoding MHC-I complex components, thereby enabling recruitment of a diverse array of activating transcription factors.

Our data indicates that combining ICT with an appropriate low dose platinoid chemotherapeutic agent is useful in reinvigorating the immune response to tumors that have escaped immune destruction due to MHC-I downregulation and do not respond to single agent therapy.

#2248

Generation and formatting of a bispecific Affimer® biotherapeutic for the inhibition of the LAG-3 and PD-L1 pathway.

Amrik Basran, Emma Jenkins, Estelle Adam, Michele Writer, Floriane Laurent, Assa Oumie, Jyrki Sivula, Jennifer Hillman, Lisa Strong, Maureen West, Emma Stanley. _Avacta Life Sciences, Cambridge, United Kingdom_.

Programmed Cell Death-Ligand 1 (PD-L1) binds to its receptor, Programmed Cell Death protein (PD-1) and is expressed on both a range of activated immune cells and tumor cells. Lymphocyte Activation Gene-3 (LAG-3) is a member of the Ig superfamily and is expressed on activated T cells, NK cells, DCs and B cells. Both the PD-1/PD-L1 and the LAG-3 pathway are involved in immune suppression, leading to the down-regulation of T cell activity and the generation of exhausted T cells, allowing tumor escape and metastasis.

Despite the impressive success of PD-L1 targeted immunotherapy, there remains a large proportion of patients with many tumor types that fail to benefit or acquire resistance during therapy. Consequently, it is necessary to consider future combinations of immunotherapies to enhance the clinical outcomes and reduce tumor escape. LAG-3 is upregulated on T-cells following PD-1/PD-L1 pathway blockade, indicating that targeting of both pathways as a combination may lead to more durable antitumor responses. To reach the full potential of LAG-3/PD-L1 dual blockade the development of a bispecific immunotherapy may further augment T-cell activation.

Affimer biotherapeutics are a new protein scaffold with great potential for the generation of biotherapeutics. Based on the human protease inhibitor Stefin A, the scaffold is small (14kDa), lacks any post-translational modifications such as disulfide bonds and expresses at high levels. Affimers against a target are generated through the use of proprietary, highly diverse phage display libraries that have been created by engineering in two nine amino acid peptide loops into the scaffold backbone.

We have identified a range of potent Affimer biotherapeutic antagonists to both human PD-L1 and LAG-3. When formatted as a bispecific Fc fusion to both the N and C-terminal of a human IgG1 Fc protein the Affimers demonstrate pM to nM binding affinities and binding to both target antigens simultaneously without compromising the affinities. The bispecific Fc fusion has been shown to block the interaction of LAG-3 with its ligand, MHC class II and PD-L1 with both PD-1 and CD80 by competitive ELISA. Functionally, the LAG-3/PD-L1 bispecific enhanced T-cell activation in vitro in both SEB-driven and mixed lymphocyte reaction assays. The Fc fusion format expresses over 500 mg/L from Expi293F mammalian transient transfections and can be purified to over 95% purity. The pharmacokinetic (PK) properties showed half-life extension of the Fc fusion format in mouse by using the IgG Fc-FcRn recycling pathway, dosing at 10 mg/kg resulted in a half-life extension of over 90 hours.

We have demonstrated that the Affimer scaffold protein has all the necessary attributes to be developed as a bispecific LAG-3/PD-L1 immunotherapy. The Affimer scaffold was formatted as a bispecific Fc fusion molecule with ease, had good biophysical properties and shown to be well tolerated in vivo.

#2249

Checkpoint inhibitor therapy after dendritic cell vaccination elicits tumor response in mesothelioma patients.

Robin Cornelissen, Robert Belderbos, Joachim Aerts. _Erasmus MC, Rotterdam, Netherlands_.

In mesothelioma most patients do not have functional T cells present in the tumor. Therefore, PD-1/PD-L1 checkpoint inhibitor (CI) therapy is likely to induce an anti-tumor response in only a small portion of patients. Activated autologous dendritic cells (DCs) that have been directed against mesothelioma tumor cells have been shown to reinvigorate T cell responses. This T cell response that is induced is prone to be blocked by the tumor using the PD-1/PD-L1 mechanism.

Earlier, we published the results of three phase I/II trials in which patients were treated with DC immunotherapy as first line therapy of after completion of first line chemotherapy. 9 patients were subsequently treated with checkpoint inhibitor therapy in several trials and compassionate use programs. Six patients were treated with nivolumab, two with pembrolizumab and one patient was treated with combination treatment of nivolumab and ipilimumab. In all patients, the initial biopsy was stained for PD-L1 and TIM3, in five patients, repeat biopsy was done at time of progression. Radiological response was measured on CT scan using modified response evaluation criteria in solid tumors (RECIST). A radiological partial response was seen in three patients after PD-1 inhibition, stable disease was seen in five patients and one patients had progression of disease as best response on CI therapy, giving a disease control rate of 88%. No CTCAE grade 3-4 adverse events were seen. Updated adverse events, median PFS and OS will be presented at the meeting, as well as correlation of response with PD-L1 and TIM3 status.

We conclude that CI treatment after DC immunotherapy is safe and feasible. In addition, after induction of a T cell response, disease control could be achieved in all but one treated patient on subsequent CI therapy. This report is a proof of concept that the induced T cell response from DC immune therapy may be blocked by PD-L1 upregulation from the tumor which in turn can be targeted by CI.

#2250

Combinational markers of neoantigen intratumor heterogeneity and neoantigen burden predict progression-free survival for lenvatinib plus PD-1 blockade in patients with intrahepatic cholangiocarcinoma.

Jianzhen Lin,1 Yiying Liu,2 Xu Yang,1 Yi Bai,1 Junyu Long,1 Junyu Long,1 Yongchao Li,2 Jiaqian Wang,2 Dongxu Wang,1 Xiaobo Yang,1 Mei Guan,1 Li Huo,1 Jie Pan,1 Ke Hu,1 Zhibo Gao,2 Haitao Zhao1. 1 _Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College (CAMS & PUMC), Beijing, China; _2 _YuceBio, Shenzhen, China_.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignant neoplasm derived from biliary epithelial cells. Although many targetable gene alterations have been identified, there has no molecular targeted drug available for these patients at present, which may be due to the high intratumor heterogeneity (ITH) of ICC. Previous studies have demonstrated that some ICC patients could be benefited from immune checkpoint inhibitors (ICIs), but the predictive biomarkers are still unknown. We used whole exome sequencing to evaluate the impact of neoantigen ITH and tumor neoantigen burden (TNB) in 45 ICC patients, in which 15 patients were treated by anti-PD1 inhibitor combined with lenvatinib. The median neoantigen and clonal neoantigen load of these patients were 0.65 and 0.29 per Mb, respectively. The median neoantigen ITH index was 0.45, with a range from 0.02 to 1. Survival analysis of those patients who received the combined treatment revealed significantly longer progression-free survival (PFS) in patients with higher TNB and low neoantigen ITH (9.5 vs 3.5 months, p=0.00583). Among the 30 patients without anti-PD1 combined therapy, there were 36.7% (11/30) ICC patients with high TNB and low neoantigen ITH, indicating these patients might be benefited from this combined therapy. One of these 45 patients had the highest TMB and TNB with high MSI and dMMR has the longest PFS (12 months) after the combined treatment. Besides, we found that the clonal KRAS mutation (G12D) occurred exclusively in patients with shorter PFS and OS and lower TMB and TNB, while the FLG mutation occurred only in patients with longer PFS and OS and higher TMB and TNB. These results indicated the potential relationship between mutations in KRAS and FLG and the levels of TMB and TNB, which needs to further investigation. Therefore, these data revealed that the high TNB and lower neoantigen ITH might be a promising marker for predicting the efficacy of the combination therapy of anti-PD1 inhibitor and lenvatinib in ICC patients.

#2251

Nanocage therapeutics FHSIRPα mediating immune checkpoint blockade awakens immunity against cancer with an albumin-binding caspase-cleavable doxorubicin prodrug EMC-DEVD-S-DOX.

Na Kyeong Lee,1 Jeong Uk Choi,1 Jung Je Park,2 Ji-Hyun Seo,2 Mi Ra Kim,3 Hyo Won Chang,4 Sang Yoon Kim,4 In-San Kim,5 Youngro Byun1. 1 _Seoul National University, Seoul, Republic of Korea;_ 2 _Gyeongsang National University School of Medicine, Seoul, Republic of Korea;_ 3 _Inje University College of Medicine, Seoul, Republic of Korea;_ 4 _University of Ulsan College of Medicine, Seoul, Republic of Korea;_ 5 _Korea Institute of Science and Technology, Seoul, Republic of Korea_.

Clinical efficacy of immune checkpoint blockade is often enhanced by their co-administration with immunogenic chemotherapy. Here a novel chemo-immunotherapy combination is reported that successfully overcomes the immunosuppressive tumor microenvironment to elicit an effective anti-tumor immune response. The CD47-SIRPα innate immune checkpoint was blocked by surface engineering human ferritin with a SIRPα variant, and this FHSIRPα efficiently inhibited the "don't-eat-me" signal of cancer cells, thereby enhancing cancer cell phagocytosis in a colon carcinoma (CT26) model in immunocompetent mice. Doxorubicin prodrug, EMC-DEVD-S-DOX, that binds to circulating albumin following intravenous administration showed enhanced half-life, and the active drug was released via caspase-3 at tumor site following radiation treatment. Radiation-induced apoptosis of cancer cells resulted in release of active drug doxorubicin and upregulation of CD47 on cancer cell surface, sensitizing cancer cells to FHSIRPα treatment. The induction of immunogenic cell death (ICD) by the prodrug was confirmed in vitro by upregulation of calreticulin, translocation of HMGB1 and increased release of chaperone proteins in CT26.CL25 mouse colon cancer cells by flow cytometric analysis and confocal microscopy. The ICD effect of the prodrug correlated well in vivo and increased phagocytosis of tumor cells by antigen-presenting cells (APCs) in immunocompetent mice as analyzed by flow cytometry of tumor tissue and draining lymph nodes. Tumor infiltrates analyzed by flow cytometry also showed enrichment of APCs; and T cell-mediated elimination of immunogenic tumors was confirmed by the tumor-specific activation of T cells ex vivo as detected by secretion of IFNγ. Whereas FHSIRPα monotherapy showed modest tumor growth inhibition, combination with a doxorubicin prodrug resulted in effective inhibition of tumor growth by 95.2% (p<0.001) compared to the control group, in an established tumor model. The combination therapy resulted in tumor free mice (57% complete response), also in an established tumor model, compared to none in monotherapy groups and controls.Taken together, we demonstrated that the synergistic anti-cancer effect and tumor targetability of the combination therapy FHSIRPα and EMC-DEVD-S-DOX successfully led to the linkage of innate to adaptive immunity to elicit a lasting anti-cancer immune response.

#2252

YIV-906 (PHY906) enhanced the anti-tumor activity of immune checkpoint blockade therapy (Anti-PD1) against liver cancer by changing the tumor micro-environment associated with M1 macrophages infiltration.

Wing Lam,1 Xiaochen Yang,1 Zaoli Jiang,1 Xue han,1 Fulan Guan,1 Rong Hu,1 Chang-Hua Xu,1 Wei Cai,1 William Cheng,1 Shwu-Huey Liu,2 Yuping Cai,1 Nicholas Rattray,1 Caroline Johnson,1 Lieping Chen,1 Yung-chi Cheng1. 1 _Yale Univ. School of Medicine, New Haven, CT;_ 2 _YIVIVA, New York, NY_.

YIV-906 (PHY906) is inspired by a traditional 1800-year Chinese herbal formulation, "Huang Qin Tang", which is commonly used for treating diarrhea. Following chemotherapy and radiation, preclinical and clinical results suggest that YIV-906 has the potential to improve the patient's quality of life and prolonging survival. Consistent preparations of PHY906 could be manufactured apart 15 years. The effects of YIV-906 were studied on the anti-tumor activity of anti-PD1 using BDF1 mice bearing Hepa 1-6 tumors. Results indicated that anti-PD1 alone had moderate effects on tumor growth however YIV-906 plus anti-PD1 eradicated all tumors in all tumor bearing mice. Further re-implantation of Hepa 1-6 cells did not grow in the "cured" mice, but implanted CMT167 (non-small lung carcinoma) cells or Pan02 (Pancreatic Ductal Adenocarcinoma) cells did grow; suggesting that YIV-906 plus anti-PD1 created a tumor-specific vaccine-like effect. The combination treatment exhibited a highly inflamed tumor microenvironment with more M1-like macrophage expression over M2. In culture YIV-906 could potentiate the action of IFNg (interferon gamma) to polarize bone marrow-derived macrophages (BMDM) into M1 macrophages while inhibiting IL4 action for M2 macrophage polarization. YIV-906 potentiated IFNg action through: 1) stimulating IFNg secretion, 2) phosphorylation of JAK1/2 and STAT1 and 3) increasing IRF1 protein expression. Scutellaria baicalensis Georgi (S) and its flavonoids of YIV-906 were responsible for potentiate the IFNg to polarize macrophage into M1. In conclusion, YIV-906 enhanced the anti-tumor activity of anti-PD1 by enhancing inflammation in the tumor microenvironment and enriching M1-like macrophages. This suggests the potential use of combination YIV-906 and anti-PD1 in cancer treatment. This work was supported by grant (1PO1CA154295-01A1) from National Cancer Institute (NCI), NIH, USA. Dr. Yung-Chi Cheng is a fellow of National Foundation for Cancer Research (NFCR), USA.

#2253

Rapid personalized selection of immune checkpoint blockage agent combinational treatment for an adenocarcinoma patient.

Kang-Yun Lee,1 Chien-Chung Chen,2 Won-Ting Huang2. 1 _Taipei Medical University, Taipei, Taiwan;_ 2 _Taipei Medical Univ., Taipei, Taiwan_.

The combination of immune checkpoint blockage (ICB) agent and chemotherapy for cancer treatment is gaining lots of attention lately. However, there is no clear biomarker or index for such treatment, particularly after tumor gaining resistance to ICI. In this study, an improved hollow fiber assay (HFA) was utilized based upon a novel substrate of microtube array membrane (MTAM) to test the possible patient response towards the combined treatment of immunotherapy and chemotherapy. In this case study, biopsy of a stage 4 adenocarcinoma (cT2N3M1b) patient, with the disease progression after a durable response to prior pemetrexed and Nivolumab, was retrieved and treated with enzyme for a homogenous cell suspension. Pieces of MTAM preloaded with 106 of cancer cell along with patient's peripheral blood mononuclear cell (PBMC) were implanted subcutaneously in bulb C mice. These mice were grouped and subjected to two different ICB combinational treatments (1) Nivolumab with Paclitaxel and (2) Nivolumab with Pemetrexed. 10 days later MTAMs were explanted and cancer cells were subject to MTT assay for their viability. The results of two treatments clearly demonstrated the better efficacy of Nivolumab and Paclitaxel towards this patient cancer cells. Clinically, CT images taken before and after a two-month treatment show positive response towards the combinational treatment of Nivolumab and Paclitaxel. This case study suggests a rapid clinical regiment prediction/selection of ICB combinational treatment is possible in two weeks using MTAM/HFA and provides reasonable evident for the corresponding treatment.

#2254

Solid tumor activity with a selective PI3Kdelta inhibitor as a single agent and in combination with anti-PD-1.

Joey L. Methot, Hua Zhou, Meredith A. McGowan, Benjamin W. Trotter, Michael Altman, Xavier Fradera, Charles Lesburg, Chaomin Li, Stephen Alves, Craig P. Chappell, Renu Jain, Ruban Mangado, Elaine Pinheiro, Sybil M. Williams, Peter Goldenblatt, Armetta D. Hill, Lynsey Shaffer, Dapeng Chen, Robbie L. McLeod, Hyun-Hee Lee, Sanjiv Shah, Jason D. Katz. _Merck & Co., Boston, MA_.

Inhibitors of PI3Kdelta are approved as monotherapy for the treatment of hematologic malignancies such as follicular B-cell non-Hodgkin lymphoma, chronic lymphocytic leukemia, and small lymphocytic lymphoma. Recent reports suggest that inhibition of PI3Kdelta in mice is effective against solid cancers as well. Mechanistic studies by several groups have suggested a possible adaptive immune-mediated anti-tumor response involving immunosuppressive Tregs and/or MDSCs in the tumor microenvironment. Our structure-guided discovery program led to the identification of a novel heterocycloalkyl purine inhibitor class with excellent isoform and kinome selectivity, as well as good solubility. Optimization included potency enhancements, addressing metabolism and increasing half-life in preclinical species to provide a PI3Kdelta inhibitor with a low once-daily predicted human dose. In FoxP3 reporter mice harboring MC38 tumors treated with inhibitor, we observed reduced peripheral Treg function and an increased ratio of CD8 to Treg in both spleen and tumor-infiltrating lymphocytes. In three-week efficacy studies, modest tumor growth inhibition was found in 4T1, MC38 and CT26 syngeneic tumor models when our inhibitor was dosed as a single agent and we demonstrated a trend towards better efficacy when our inhibitor was dosed in combination with an anti-mPD-1 antibody in CT26 tumor-bearing mice as compared to anti-mPD-1 alone. These preliminary studies confirm the impact of PI3Kdelta inhibition on T-cell subpopulations, and support continued exploration of PI3Kdelta inhibitors for the treatment of solid tumors, either as monotherapy or in combination with other agents.

#2255

Combination treatment of cancer cell-targeted photoimmunotherapy plus anti-PD1 leads to synergistic anticancer activity in an immunocompetent mouse model.

Stephanie M. Okamura, Michelle A. Hsu, Daniele M. Bergeron, Estela Solis, Deepak Yadav, Jerry J. Fong, Roger Heim, Miguel Garcia-Guzman. _Rakuten Aspyrian, San Diego, CA_.

INTRODUCTION: Photoimmunotherapy (PIT) is a new cancer-targeted platform technology that utilizes a photoactivatable dye (IRDye 700DX) conjugated to a cancer-targeting antibody. Binding of the antibody- dye conjugate to cancer cells followed by photoactivation with nonthermal red light elicits rapid necrosis of the cancer cells bound to the antibody conjugate. In previous studies using immunocompetent mouse models, intratumoral innate and adaptive immune cell activation was observed after PIT treatment. We hypothesized that combination therapy with PIT plus anti-PD1 may enhance the activity of anticancer immune activation elicited by PIT treatments and in this study we evaluated the effects of the combination treatment in an immunocompetent mouse model.

METHODS: Immunocompetent mice implanted with CT26 tumor cells engineered to overexpress specific recombinant mouse antigens, received PIT treatment alone, anti-PD1, or combination therapy and were assessed for tumor growth inhibition and complete response rate. In animals achieving a complete response, the specificity of the response was evaluated by tumor re-challenge experiments. Animals with residual disease had their tumors excised and assessed for intratumoral immune activation. Splenocytes from treated animals were harvested and evaluated for cytotoxic activity against the cancer cells.

RESULTS: Of the mice that received combination treatment, 7 of 14 achieved complete response compared to 0 of 14 and 1 of 14 when treated with anti-PD1 monotherapy and PIT monotherapy, respectively. All tumor-free animals from combination treatment also rejected formation of new tumors following inoculation with the same tumor type (CT26), but not tumors from a different cancer cell type (4T1). Splenocytes derived from animals that received combination treatment had the greatest cytotoxic activity against CT26 cells when compared to single agent controls. In addition, splenocytes derived from animals that received combination treatment had increased total CD3+ CD8+ cells, as well as increased CD62L-CD44+ effector memory CD8+ cells, in comparison to splenocytes derived from animals that received single agent therapies.

CONCLUSION: In this study, combination cancer-targeted PIT plus anti-PD1 treatment in immunocompetent mice resulted in synergistic anticancer activity due to enhanced adaptive immune activation, as demonstrated by tumor rejection and increased systemic, tumor-specific cytotoxic T cells, and effector memory CD8+ cells. The results of this study warrant the clinical evaluation of combination treatment of cancer-targeted PIT plus anti-PD1 therapies.

#2256

Combination of neoantigen DNA plasmid vaccine VB10.NEO and NKTR-214, a CD122-biased immunostimulatory cytokine, induces strong neoantigen-specific T cell responses and sustained tumor regression in pre-clinical models.

Stine Granum,1 Helene Zell-Flagstad,1 Audun Bersaas,1 Lise Skullerud,1 Elisabeth Müller,1 Mads Axelsen,1 Karoline Schjetne,1 Jonathan Zalevsky,2 Agnete Fredriksen1. 1 _Vaccibody, Oslo, Norway;_ 2 _Nektar Therapeutics, San Fransisco, CA_.

BACKGROUND: VB10.NEO is a highly potent DNA plasmid vaccine with intrinsic adjuvant effect designed for efficient delivery of personalized neoepitopes and is currently being tested in combination with CPI in patients with advanced solid tumors. NKTR-214, a CD122-biased agonist that targets the IL-2 pathway, provides sustained signaling through the heterodimeric IL-2 receptor pathway (IL-2Rβɣ) to preferentially activate and expand NK and effector CD8+ T cells over T-regulatory cells, and is currently in multiple phase I, II and III clinical trials in combination with nivolumab. Here we explored the combination of VB10.NEO with NKTR-214 in the presence or absence of anti-PD-1 therapy in different pre-clinical syngeneic tumor models.

METHODS: Mice were vaccinated with VB10.NEO (i.m.), NKTR-214 (i.v) and anti-PD-1 (i.p.) at different intervals to study efficacy and optimal dosing schedule before spleens were harvested to analyze CD8+ and CD4+ neoantigen-specific T cell responses in IFN-γ ELISpot in two different tumor models (B16 and CT26). The tumor protective effect of the combination therapy of VB10.NEO with NKTR-214 in the presence or absence of anti-PD-1 therapy was investigated in CT26 colon carcinoma model.

RESULTS: Combination of VB10.NEO and NKTR-214 greatly synergizes, resulting in 5-fold increase in the number of neoantigen-specific T cell responses compared to VB10.NEO alone. Dosing of VB10.NEO vaccination relative to NKTR-214 administration was found to impact the extent of the synergetic activity. Mice treated with VB10.NEO containing 10 neoepitopes from B16 in combination with NKTR-214 showed both a stronger response to each neoantigen, but also to increased numbers of neoantigens, showing that both the breadth and depth of the immune response were elevated. The VB10.NEO and NKTR-214 combination showed an even more evident effect on CD8+ T cell responses as the combination elicited a neoantigen-specific CD8+ T cell response against 10 neoantigens compared to 5 neoantigens with VB10.NEO alone, further strengthening the combination's ability to induce strong neoantigen-specific CD8+ T cell responses. In the therapeutic tumor model, increased number of complete responders was observed in mice treated with the triple combination of vaccine, NKTR-214 and CPI demonstrating the strong rational for bringing together these unique and non-overlapping mechanisms that create an effective treatment of established tumors.

CONCLUSION: VB10.NEO in combination with NKTR-214 with or without anti-PD-1 therapy synergizes to elicit greater breadth and depth of neoantigen-specific T cell responses and provide durable complete tumor regression in pre-clinical models supporting the rationale for examining the combination clinically.

#2257

Phase II study of dual immune checkpoint blockade (ICB) with durvalumab (Durva) plus tremelimumab (T) following palliative hypofractionated radiotherapy (SBRT) in patients (pts) with microsatellite-stable (MSS) metastatic colorectal cancer (mCRC) progressing on chemotherapy: NSABP FC-9.

James J. Lee,1 Greg Yothers,2 Thomas J. George,3 Marwan G. Fakih,4 Atrayee Basu Mallick,5 Edith P. Mitchell,6 James L. Wade,7 John C. Krauss,8 Omar R. Kayaleh,9 Dwight E. Heron,1 Carmen J. Allegra,3 Corey Lipchik,10 Huichen Feng,10 Marion Joy,10 Ashok Srinivasan,10 Katherine L. Pogue-Geile,10 Peter C. Lucas,11 Sarah E. Warren,12 Alessandra Cesano,12 Samuel A. Jacobs10. 1 _NSABP Foundation, Inc, and UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, PA;_ 2 _NSABP Biostatistical Center, and The University of Pittsburgh, Pittsburgh, PA;_ 3 _NSABP Foundation, Inc, and University of Florida Health, Gainesville, FL;_ 4 _NSABP Foundation, Inc, and The City of Hope Comprehensive Cancer Center, Duarte, CA;_ 5 _NSABP Foundation, Inc, and Thomas Jefferson University Hospital, Philadelphia, PA;_ 6 _NSABP Foundation, Inc, and The Kimmel Cancer Center at Thomas Jefferson University Hospital, Pittsburgh, PA;_ 7 _NSABP Foundation, Inc, and Decatur Memorial Hospital, Decatur, IL;_ 8 _NSABP Foundation, Inc, and The University of Michigan, Ann Arbor, MI;_ 9 _NSABP Foundation, Inc, and UF Cancer Center at Orlando Health, Orlando, FL;_ 10 _NSABP Foundation, Inc, Pittsburgh, PA;_ 11 _NSABP Foundation, Inc, and The University of Pittsburgh School of Medicine, Pittsburgh, PA;_ 12 _NanoString Technologies, Inc., Seattle, WA_.

Background

Monotherapy with ICB has not been effective in MSS mCRC. Preclinical data demonstrate that immunomodulators synergize with radiotherapy (RT) resulting in tumor regression at irradiated sites and rarely at non-irradiated sites. Anecdotal reports in pts have confirmed an abscopal effect of RT and ICB. This phase II, open-label, single-arm study is testing dual ICBs following SBRT in pts with MSS mCRC who have progressed on chemotherapy. We report here efficacy and safety results.

Methods

Eligible pts have: MSS mCRC, progressed on prior oxaliplatin and irinotecan-based regimens, measurable lesions with at least one amenable to SBRT and another for core biopsy, and performance status <2. Following 3 doses of SBRT at 9 Gy daily (D −2, −1, and D 0 prior to Cycle 1), pts received the combination of T (75 mg IV infusion) and Durva (1500 mg IV infusion) on D 1 of Cycles 1-4. Beginning with Cycles 5-12, pts received Durva alone on D 1 q 28d. Pts were considered evaluable using RECIST 1.1 if a tumor response at non-irradiated target lesions was measured at baseline and after 2 cycles. Subsequent scans were performed every 8 wks. Toxicity was graded according to NCI CTCAE v4.0. Primary aim is to determine overall objective response rate (ORR) in non-irradiated metastasis. Secondary aims are clinical benefit rate, duration of response, tolerability, and correlates of response. Archived and fresh tumor biopsies were obtained at study entry and after cycle 2, blood samples were collected before treatment, at cycle 1 D 15, and on D 1 of cycles 2, 4, and 6.

Results

FC-9 is open at six academic centers. From October 2017 to November 1, 2018, 33 pts enrolled with 20 pts evaluable. Median age was 58 y (range, 37-71). Toxicity was assessed without regard to attribution. One pt had grade 5 unexplained sudden death (3%); two had grade 4 (6%, colitis, dehydration, pneumonitis); and 12 had grade 3 (38%, primarily GI, other than colitis, and nutrition). Partial responses were seen in 2 pts lasting 44 wks and 44+ wks; two pts had stable disease of 12 and 16 wks duration. Correlative studies will be presented including PD-L-1 expression, CD8+ T cell infiltration into tumor tissues, and gene expression of immune pathways.

Conclusions

The combination of SBRT and dual immunotherapy was safe and well tolerated following immunotherapy standard guidelines. In this refractory group of MSS mCRC pts, we observed 2 partial responses of 44 and 44+ wks among 20 evaluable pts. Correlative analyses will be presented.

Support: Astra-Zeneca; NSABP Foundation, Inc.

#2258

Erymethionase (methionine-gamma-lyase encapsulated into red blood cells) potentiates anti-PD-1 therapy in TNBC syngeneic mouse model.

Karine Sénéchal,1 Sylvie Maubant,2 Marie Leblanc,2 Séverine Ciré,1 Fanny Gallix,1 Aurély Andrivon,1 Olivier Duchamp,2 Fabrice Viviani,2 Françoise Horand,1 Alexander Scheer,1 Vanessa BOURGEAUX1. 1 _Erytech Pharma, Lyon, France;_ 2 _Oncodesign, Dijon, France_.

Background : Anti-PD-1 immunotherapy, although efficient in several cancer indications; has shown some limitations due to the appearance of resistance mechanisms. More specifically, the overexpression of A2A receptor (adenosine receptor) on the surface of infiltrated CD8 T cells following anti-PD-1 treatment blunts the immune response in an adenosine-rich tumor microenvironment (TME). In addition, increased evidence of immune escape has been described through gene hypermethylation processes. Erymethionase (methionine-gamma-lyase encapsulated into red blood cells) is an innovative experimental therapy designed to directly reduce systemic levels of L-methionine, an amino-acid nutrient needed for tumor growth and metastasis. Erymethionase is expected to indirectly decrease adenosine levels in TME and to reduce hypermethylation reactions. This bimodal action makes of erymethionase a promising agent to combine with immune checkpoint inhibitors. The benefit of such combination (erymethionase and PD-1 blockade agent) was investigated in a TNBC-like syngeneic mouse model.

Methods : Mice bearing orthotopic EMT-6 syngeneic breast carcinoma were intravenously injected once weekly for 4 consecutive weeks with vehicle or mouse erymethionase at 30 or 60 U/kg alone or in combination with anti-mouse PD-1 antibody (RMP1-14 clone, intraperitoneal route, 10 mg/kg, twice weekly for 3 consecutive weeks) from D7 (D0 referring to injection of tumor cells). Erymethionase treatment was associated to daily oral vitamin B6 (pyridoxine, PN) uptake to maintain enzyme activity. The body weight, the length and width of the tumor were measured twice a week. Animal behavior was checked every day. Satellite animals receiving 60 U/kg of erymethionase or vehicle were sacrificed to collect 500-1000 m3 size-tumors to perform immunophenotyping, measure metabolites and/or assess biomarkers.

Results : Analysis of health parameters revealed that all treatments were well tolerated along the experiment. A delayed start of exponential growth was reported for the combination at the highest dose of erymethionase vs single agents leading to a significant tumor growth inhibition and an increased survival (median survival time of 35 days for the combination vs 23 days for anti-PD-1 or erymethionase 60 U/kg alone; statistically significant). The antitumor effects were less pronounced at the lower dose level of erymethionase in combination with anti-PD1. Analysis of collected tumors to investigate on the mechanism(s) of action is ongoing.

Conclusion : To our knowledge, this is the first in vivo demonstration of anti-PD-1 therapy potentiation using a L-methionine-restricting agent. The data are supportive of the planned erymethionase first-in-human study.

#2259

Tumor abscopal responses induced by the TLR9 agonist, SD-101, are strongly potentiated by a HDAC class I inhibitor, domatinostat.

Émilie Degagné, Jose Romo, Marilena Gallotta, Shravan Kannan, Robert L. Coffman, Cristiana Guiducci. _Dynavax Technologies Corporation, Berkeley, CA_.

In cancer settings severe T cell exhaustion is one major road block limiting immunotherapy efficacy. Epigenetic changes happening during T cell effector differentiation lock T cells in a non-programmable state which limits the efficacy of checkpoint blockade. In addition, histone modification has been reported to critically modulate MHC I and II expression on tumor cells and influx of T cells into tumor.

We have previously shown that intratumoral administration of a TLR9 agonist, SD-101, induces T cell responses capable of attacking tumor lesions throughout the body. SD-101, combined with the PD-1 inhibitor pembrolizumab, is currently being investigated in patients with metastatic melanoma and head and neck cancer with encoraging results. Domatinostat (4SC AG) is a well-tolerated orally available selective class I histone deacetylase inhibitor HDACi) with demonstrated anti-tumor activity in hematological cancers as well as in numerous animal models of solid tumors as monotherapy and in combination with checkpoint blockade. Domatinostat is currently being investigated in patients with metastatic melanoma and gastrointestinal cancers in combination with PD(L)-1 antibodies.

This study addresses whether the addition of domatinostat to the SD-101 regimen would potentiate the T cell response induced by SD-101. Studies were conducted in multiple syngeneic models in which tumors grow in multiple site of the body but only one is treated intratumorally with SD-101 and domatinostat is given systemically (oral). SD-101 and domatinostat were given alone or in combination, at clinically relevant dosages. The combination of intratumoral SD-101 and domatinostat induced substantial regression of the primary tumor (injected) and distant site cutaneous tumors, as well as lung metastases

The combination was superior to either agent given alone and led to increased tumor specific cytotoxic CD8 T cells (CTL) infiltrating distant site lesions. CTL's were characterized by a low exhaustion phenotype, increased proliferative capacity and increased co-expression of cytolytic markers, CD107 and GranzymeB. Gene expression profiling was performed on abscopal tumors using the PanCancer immune profiling panel from Nanostring.

The combination of SD-101 and domatinostat led to upregulation of multiple immune related signatures indicating increased T, NK and dendritic cell functions. Notably, the combination of SD-101 and domatinostat was more efficacious than SD-101 combined with various other epigenetic compounds. The addition of PD-1

blockade to SD-101 and domatinostat further boosted T cell responses leading to rejection in mice with high metastatic burdens. These results suggest that domatinostat acts on SD-101 primed tumor specific CD8+ T cells to expand them, render them more cytolytic and facilitate their infiltration in abscopal tumors.

#2260

Targeting PP2A with LB100 enhances efficacy of CAIX CAR-T cells against GBM.

Jing Cui,1 Qi Zhang,1 Qi Song,1 Herui Wang,1 Pauline Dmitriev,1 Mitchell Sun,1 Jared S. Rosenblum,1 Kaiyong Yang,2 John Kovach,3 Mark R. Gilbert,1 Zhengping Zhuang1. 1 _National Cancer Institute, National Institutes of Health, Bethesda, MD;_ 2 _Oasis Biotechnology Inc., Bethesda, MD;_ 3 _Lixte Biotechnology Holdings, Inc, New York, NY_.

The need for novel therapies for glioblastoma multiforme (GBM) is well established. Chimeric antigen receptor (CAR)-engineered T cells represent a promising cancer treatment modality for treating GBM. However, CAR-T therapy has limitations, such as low penetration into solid tumors and short duration of CAR-T survival. Protein phosphatase 2A (PP2A) inhibition has been implicated in enhancing T cell anti-tumor activity. LB-100 is a novel, first-in-class, small molecule inhibitor of PP2A recently shown in a Phase I trial to be well-tolerated at doses resulting in stabilization of progressive solid tumors. We set out to overcome limitations of CAR-T therapy by activating the mTOR signaling pathway through inhibition of PP2A by LB-100. GBM highly expresses CAIX due to increased hypoxia signaling. Thus, antitumor CAIX-specific CAR T cells were developed and tested against GBM in vitro and in vivo. CAR-T cells displayed GBM cells cytotoxicity and increased IFN-γ, TNF-α, and IL-2 production. Intra-tumor injection of the CAR-T cells into an intracranial GBM xenograft mouse model efficiently suppressed the tumor growth. We found that PP2A inhibition by LB-100 further enhanced CAIX CAR-T activity and significantly prolonged mouse survival. Herein we demonstrate that CAIX represents a viable target for CAR-T cells and inhibition of PP2A enhances CAR-T efficacy against GBM. Targeting PP2A via inhibition with LB-100 may be a promising therapeutic strategy to improve CAR-T efficacy in other solid tumors.

#2261

Understanding the mechanism underlying resistance to immunotherapy in TNBC.

Ann C. Anselme,1 Wei Qian,2 Anthony Kozielski,2 Roberto R. Rosato,2 Jenny C. Chang2. 1 _Texas A &M College of Medicine and Houston Methodist Research Institute, Houston, TX; _2 _Houston Methodist Research Institute, Houston, TX_.

Triple Negative Breast Cancer (TNBC) accounts for 15-20% of all breast cancer cases. TNBC patients have the worst outcome amongst all breast cancers; and do not respond well to conventional therapies. Although TNBC patients have a high amount of tumor infiltrating lymphocytes, and express higher level of PD-L1 compared to other breast cancer subtypes, they do not all respond to PD-1 blockade therapy. IL-12 is a cytokine produced by dendritic cells (DCs), and other APCs, known to induce the activation of natural killer cells, cytotoxic T-cells, and induce a Th1 phenotype. Although the immune activating potential of Il-12 are known, its systemic uses have been limited due to high toxicity. Using three different tumor models (PDXs: BCM 4913, MC1 in humanized mice and E0771 syngeneic model C57/Bl), we are investigating the response of combining intratumoral injection of Adenovirus IL-12 (ad.Il-12) and anti-PD1 therapy.

Materials/Methods: E0771, a mice TNBC cell line was injected in the mammary fat pad of C57BL/6 mice. The same procedure was followed using MC1 and BCM 4913, in humanized NSG-MGM3 mice. The mice received weekly intratumoral injection of ad.IL-12; a replication defective adenoviral vector containing mIL-12 (mouse) and hIL-12 (PDX) cDNA under the transcriptional control of Rous sarcoma virus long terminal repeat (received from Dr. Chen). Anti-PD1 (InVivoMab anti-mouse PD-1 CD279) was administered 3 times a week, Pembrolizumab (once a week). Post treatment, INF-gamma level were measured from blood and tumor samples, in addition, tumor sizes were compared between treatment groups (Control/ad.IL-12/anti-PD1/ad.IL-12 + anti-PD1), as well as survival curves. Immunohistochemistry was used to assess levels on TILs in the tumors.

Results: Ad.Il-12 and PD-1 blockade therapy combination was more effective at reducing tumor growth in BCM4913, while in E00771 and MC1 combination treatment showed no difference compared to single agent. In all the models, levels of INF-gamma in the tumor were significantly upregulated in the combination group compared to control and single agents, while no difference was found between treatment groups from blood samples. Tumor growth in the combination group and the treated group were slower compared to the controls in BCM 4913. IHC data indicate a higher level of TILs in the treatment groups, with a statistically significant difference in the combination group compared to the others.

Conclusion: Our preliminary data suggest that intratumoral injection of IL-12 confine the release of INF-gamma in the tumor microenvironment, and mitigate the toxicity associated with systemic admission of ad.IL-12. Treatment response is not achieved in every model, suggesting an underlying mechanism which confers resistance to the treatment. We are currently investigating different models in the hope to identify markers of responders versus non responders.

#2262

Chemotherapy, checkpoint inhibition, and MGMT-modified adoptive gamma-delta (γδ) T cell-based therapy to treat post-resection, primary glioblastomas.

Sailesh Pillai, George Y. Gillespie, Louis B. Nabors, Samantha Langford, Catherine P. Langford, Lawrence S. Lamb. _Univ. of Alabama at Birmingham, Birmingham, AL_.

Introduction: Recent evidence supports the crucial contribution of innate immunity to chemotherapy-based cancer treatments. Our previous in vitro modeling shows improved killing of glioblastoma (GBM) cell lines when γδ T cells are combined with alkylating agents such as Temozolomide (TMZ), which cause DNA damage but are also lymphodepleting. We now report findings from combined TMZ therapy with MGMT-modified γδ T cells (Drug Resistant Immunotherapy - DRI) in a xenograft (PDXT) mouse model of primary and recurrent GBM.

Methods: Effectorγδ T cells were manufactured from apheresis product in media containing 5mM Zoledronic Acid and 50U/mL rhIL-12. Cultures were transduced with a P140K-MGMT lentivector, maintained for up to 14 days, harvested, and evaluated in vitro for PDXT lysis +PD-1, CTLA-4 and PD-L1 by flow cytometry. Primary (P) and recurrent TMZ-resistant (T) GBM were modeled orthotopically using classical JX12P and mesenchymal JX59P intracranial (IC) xenografts versus JX12T, JX22T and JX59T. After placement of IC xenografts, mice received concurrent intraperitoneal TMZ 60mg/kg and 1 x 106IC DRI γδ T cells on post-tumor days 6, 8,13 and 15. Control mice received either γδ T cells, TMZ, or no therapy. Survival was assessed by Kaplan-Meier analysis.NKG2DL and PD-L1 expression was assessed on FFPE sections of tumor during therapy and at termination.

Results: DRI-treated JX12P mice showed significant improvement in median survival (MS) over TMZ alone (p=0.0001) with 80% tumor-free survivors. DRI also improved survival for JX59P-bearing mice with 60% survivors as compared to 30% for mice receiving TMZ alone (p=0.044). TMZ reduced tumor growth in primary but not TMZ-resistant PDXT-bearing mice while γδ T cells alone had no survival benefit over untreated controls for any model. For JX12T, DRI improved MS from 27 to 38 days (+41%) over TMZ alone (p=0.017). JX22T and JX59T tumors were resistant to DRI (p=0.0966 and 0.1390). DRI γδ T cells upregulated PD-1 and CTLA-4 over baseline and showed improved function against TMZ-resistant xenolines after blockade of PD-1 and/or CTLA-4 by as much as 200%. TMZ induced upregulation of NKG2DL (MIC-A/B, ULBP-4), and PD-L1 on both primary and TMZ-resistant PDXT following TMZ injection and for several days thereafter.

Conclusions: Primary GBM PDXT are significantly more sensitive to DRI than TMZ-resistant GBM suggesting that DRI would be more effective if administered as adjuvant therapy in the TMZ-maintenance phase following primary resection and chemo/radiotherapy. Additionally, TMZ increases GBM immunogenicity by upregulating NKG2DL expression but also PD-L1. Checkpoint blockade improves γδ T cell function. These findings show the potential to augment the effect of DRI and improve immune effectiveness against more resistant tumors.

#2263

Ceramide mimetics regulation of tumor cell response to immune checkpoint inhibitor immunotherapy.

Priscilla S. Redd, Chunwan Lu, John D. Klement, Mohammed Ibrahim, Dafeng Yang, Daniela Payne, Iryna Lebedyeva, Kebin Liu. _Augusta University, Augusta, GA_.

Programmed cell death-1 (PD-1) is an immunosuppressive receptor expressed on T cells upon activation. Once interacting with its ligand, programmed death ligand-1 (PD-L1), the PD-1:PD-L pathway curbs T cell activation and thereby serves as a natural protection mechanism against autoimmunity. However, this pathway has been hijacked by cancer cells in order to evade the immune system. This interface has already been recognized as an effective target for cancer therapy leading to the development of immune checkpoint inhibitor (ICI) immunotherapy. Blocking the PD-1:PD-L axis unleashes T cells from suppression to maintain their effector function against tumor cells. ICI has durable efficacy in many types of cancer; however, colorectal cancer, except for the approximately 4% microsatellite-instable (MSI) colorectal cancer, stands out as one of the few human cancers that does not respond to ICI immunotherapy. One potential mechanism that these tumor cells may be using is resisting cell death, making them insensitive to apoptosis induction by T cells. Ceramide, the central metabolite of the sphingolipid metabolism pathway, plays a critical role in many cell signaling processes, and one of its most prevalent roles can be seen in cell death. Our lab has developed five novel ceramide mimetics (IG4, IG7, IG14, IG17, and IG19) which function to sensitize tumor cells to apoptosis and enhance T cell-mediated cell death. We have previously shown that these ceramide mimetics significantly increase FasL-induced apoptosis by perforin-deficient cytotoxic T lymphocytes. Using the murine colon carcinoma cell line CT26, we show that the ceramide mimetics induce tumor cell death in a dose-dependent manner. Interestingly, the ceramide mimetics appear to be functioning by different mechanisms in vitro. Still, we report here that these five ceramide mimetics exhibit tumor suppression activity in a colon tumor mouse model in a dose-dependent manner in vivo. Also, ceramide mimetic IG19 significantly increased the efficacy of anti-PD-1 mAb immunotherapy in suppressing colon tumor development in vivo. Our data thus indicate that ceramide mimetics are potential effective enhancers for ICI immunotherapy in colon cancer.

#2264

Synergistic combination of oncolytic virotherapy and immunotherapy for glioma.

Bingtao Tang,1 Zong Sheng Guo,2 David L. Bartlett,2 David Yan,1 Claire P. Schane,1 Jia Liu,3 Grant McFadden,4 Joanna L. Shisler,1 Edward J. Roy1. 1 _University of Illinois at Urbana-Champaign, Urbana, IL;_ 2 _University of Pittsburgh School of Medicine, Pittsburgh, PA;_ 3 _University of Arkansas for Medical Sciences, Little Rock, AR;_ 4 _Arizona State University, Tempe, AZ_.

Oncolytic viral immunotherapy is a novel approach to cancer treatment. Viruses can directly kill cancer cells, provide antigens to dendritic cells to stimulate a T cell response, and make cancer cells express genes of immune-enhancing cytokines locally within the tumor microenvironment. We hypothesized that the combination of oncolytic virus with a local stimulus for activating tumor-specific T cells and an anti-immunosuppressant would improve treatment of gliomas. An oncolytic virus encoding IL15-IL15Rα (the T cell activating stimulus) and the prostaglandin synthesis inhibitor celecoxib (the anti-immunosuppressant) were combined with adoptive transfer of tumor-specific T cells. Two oncolytic poxviruses, vvDD vaccinia virus and myxoma virus, were each engineered to express the fusion protein IL15-IL15Rα and a fluorescent protein (YFP or tdTomato Red). Viral gene expression was tested in the murine glioma line GL261 in vitro and in vivo. Orthotopic GL261 tumors in immunocompetent C57BL/6 mice were treated with vvDD-IL15-Rα vaccinia virus or vMyx-IL15Rα-tdTr combined with other treatments, including vaccination with GARC-1 peptide (a neoantigen for GL261), rapamycin, celecoxib, and adoptive T cell therapy. We found that vvDD-IL15-Rα and vMyx-IL15Rα-tdTr each infected and killed GL261 cells in vitro. In vivo, NK cells and CD8+ T cells were increased in the tumor due to the expression of IL15-IL15Rα. Each component of a combination treatment contributed to prolonging survival: an oncolytic virus, the IL15-IL15Rα expressed by the virus, a source of T cells (whether by pre-vaccination or adoptive transfer), and prostaglandin inhibition all synergized to produce total elimination of gliomas in a majority of mice. vvDD-IL15-Rα occasionally caused encephalitis, but vMyx-IL15Rα-tdTr was safe and effective, causing a strong infiltration of tumor-specific T cells and eliminating gliomas in 83% of treated mice. All these facts suggest that IL15-IL15Rα-armed oncolytic poxviruses provide potent antitumor effects against brain tumors when combined with adoptive T cell therapy, rapamycin and celecoxib.

#2265

Localized nanodelivery combined with immunotherapy promotes curative anti-tumor responses in a murine breast cancer model.

Azadeh Kheirolomoom,1 Matthew T. Silvestrini,1 Elizabeth S. Ingham,1 Lisa M. Mahakian,1 Sarah M. Tam,1 Spencer K. Tumbale,2 Josquin Foiret,2 Neil E. Hubbard,1 Alexander D. Borowsky,1 William J. Murphy,1 Katherine W. Ferrara2. 1 _UC Davis, Davis, CA;_ 2 _Stanford University, Stanford, CA_.

Optimal pairing of chemotherapy with immunotherapy can harness potential synergies and augment tumor immunity in solid tumors with low mutation burden by i) reducing tumor bulk, ii) releasing tumor antigen for cross-presentation and cross-priming, iii) releasing cancer-suppressive cytokines/chemokines, and iv) retaining circulating immune cell populations. Temperature-sensitive liposomes (TSL) have the potential to accomplish these goals as ultrasound (US) insonation or other treatment modalities can release the chemotherapy within bulky solid tumors in response to hyperthermia to maximize the local response and minimize off-target effects. Here we used Doxorubicin (Dox), stabilized within the core of TSL by forming a drug complex with copper (CuDox), to mediate immunogenic cell death (ICD) and release of type I interferons (IFNs). This activatable chemotherapy (CuDox-TSL+US) was combined with immunotherapy using CpG-ODN, a toll-like receptor 9 agonist, and a PD-1 antibody (αPD-1) for checkpoint blockade. We assessed the schedule and sequence of the chemo-immunotherapy protocol to maximize therapeutic outcome.

CuDox-TSL were made of DPPC:MPPC:DSPE-PEG2k, 86:10:4 in the presence of copper (II) gluconate and triethanolamine at 0.2 mg-Dox/mg-lipid. Mice with bilateral invasive neu deletion (NDL) tumors (4 mm) were treated with an i.v. administration of CuDox-TSL at 6 mg Dox/kg body weight. One tumor was insonified with a peak ultrasound pressure of 1.1 MPa at a frequency of 1.5 MHz at 42°C for 5 min prior to and 20 min post drug injection with a variable duty cycle. Upon completion of US-hyperthermia, 100 µg of CpG-ODN 1826 was injected intratumorally to the insonified tumor. Three days later αPD-1 (200 µg, i.p.) was administrated.

We found that Dox in combination with hyperthermia induced release of ICD markers and type I IFNs in cell culture of various murine cancer cell lines. Following intravenous administration

of CuDox-TSL, ultrasonic activation of liposomes in the treated tumors promoted release and cross-presentation of a model tumor antigen by antigen-presenting cells in distant tumors. While a variety of chemo-immunotherapy protocols achieved a complete response in more than 50% of treated mice, the complete response rate was greatest (90%) when one week of immunotherapy priming preceded activatable chemotherapeutic administration. Repeated chemotherapeutic delivery, while effectively reducing viable tumor, also reduced efficacy and the fraction of viable CD8\+ T-cells as indicated by flow cytometric analysis. Taken together, the results suggest that a single-dose administration of activatable chemotherapy after immune priming enhances survival in a murine model of breast cancer. In clinical practice, however, assessment of macrophage and T-cell populations over the course of treatment could help inform dose timing and number of treatment repetitions.

#2266

Efficacy of CEACAM1-targeting immunoglobulin in combination with pembrolizumab in lung cancer.

Jae-Hwan Kim,1 Kyoung-Ho Pyo,1 Min-Jee Jung,1 Minkyu Hur,2 Jonghwa Won,2 Byoung Chul Cho1. 1 _Yonsei Univ., Seoul, Republic of Korea;_ 2 _Mogam Institute for Biomedical Research, Republic of Korea_.

Background: Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) as an immune check point molecule is expressed in lung cancer cells. The homophilic interaction of CEACAM1 inhibits killing effect of T cells by co-inhibitory signal transduction. Thus, up-regulated CEACAM1 in the tumor microenvironment is considered as a promising therapeutic target for cancer. In this study, anti-cancer effect of anti-CEACAM1 monoclonal antibody (MG1124, from GC Pharma, Yongin, South Korea) was investigated with lung cancer-xenograft humanized mice.

Materials & Methods: Multispectral image analysis using Vectra polaris system was performed in 49 of human lung cancers (36 adenocarcinoma; 13 squamous cell carcinoma) to evaluate the expressions of CEACAM1, PD-L1 and CD8. Four tumors (YHIM-01, YHIM-02, YHIM-03 and YHIM-04) with high CEACAM1 expression were subcutaneously implanted into humanized mice (hu-CD34 NSG) matched with HLA-A. Efficacy of MG1124 was performed by the single mouse trial format. Humanized mice were treated with MG1124 (q2w, 20 mpk, i.p. injection) with or without pembrolizumab (q2w, 10 mpk, i.p. injection).

Findings: Among 49 lung cancers evaluated, proportion of tumor cells expressing CEACAM1 and PD-L1 with any intensity ranged 0.1- 89.2% (median, 15.4%) and 0.2% - 95.6% (median, 6.8%) respectively, and TIL counts ranged 0 - 1,200 cells/mm2 (median, 129 cells/mm2). Four tumors with high CEACAM1(14.5% in YHIM-01, 81.2% in YHIM-02, 23.8% in YHIM-03 and 36.7% in YHIM-04) were selected to establish humanized mouse models. MG1124 as a monotherapy demonstrated antitumor activity and also was superior to pembrolizumab in 1 (YHIM-02, TGI=63.4±1.7% for combination vs. TGI=58.7±9.1% for pembrolizumab alone) out of 4 humanized mouse models. Combination of MG1124 with pembrolizumab also demonstrated synergic anti-tumor effect in 3 models (YHIM-01, TGI= 39.6±19.7% vs. 0.6±11.4% for pembrolizumab alone, vs. 16.5±33.3% for MG1124 alone; YHIM-02, TGI=58.7±9.1% vs. 38.2±14.9% for pembrolizumab, vs. 63.4±1.7% for MG1124 alone; YHIM-03, TGI= 35.6±13.2% vs. TGI=0.0±13.2% for pembrolizumab alone, vs. TGI=3.1±13.3% for MG1124 alone). PD-L1 expression was not associated with MG1124 alone or in combination with pembrolizumab (PD-L1, 2.5% in YHIM-01, 0.5% in YHIM-02 and 14.0% in YHIM-03 respectively). Interestingly, however, combinational effects of MG1124 and pembrolizumab were shown only in 3 models showing high CD8+ T cell infiltration in the tumors (209, 432, 362 vs. 46 in a model without combination synergy).

Conclusion: CEACAM1-targeting monoclonal antibody, MG1124, exhibits promising anti-cancer effects in CEACAM1-expressing lung cancers. Tumoral CEACAM1 expression and high intratumoral CD8+ T cell infiltration could serve as a predictive biomarker to MG1124 monotherapy or in combination with pembrolizumab and need to be further investigated.

#2267

Therapeutic synergy of the chimeric, oncolytic virus VSV-GP with immune modulators.

Philipp Mueller,1 Patrik Erlmann,2 Monika Petersson,2 Tobias Nolden,2 Carles Urbiola,3 Liesa-Marie Schreiber,3 Bart Spiesschaert,2 Klaus Erb,1 Birgit Stierstorfer,1 Maria Antonietta Impagnatiello,4 Eric Borges,1 Knut Elbers,5 Guido Wollmann,3 John Park1. 1 _Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany; _2 _ViraTherapeutics GmbH, Innsbruck, Austria;_ 3 _Medical University of Innsbruck, Christian Doppler Laboratory for Viral Immunotherapy of Cancer, Innsbruck, Austria;_ 4 _Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria; _5 _C. H. Boehringer Sohn AG & Co. KG, Ingelheim, Germany_.

Introduction: Oncolytic viruses (OVs), which selectively replicate in and destroy tumor cells, represent a highly promising class of cancer therapeutics. In recent years, the focus in the OV field has significantly shifted from a pure oncolytic mode of action to their strong immune stimulatory potential. Tumor cells lysed by the viruses release pro-inflammatory signals, tumor associated antigens as well as display hallmarks of immunogenic cell death. VSV-GP, a chimeric VSV pseudotyped with the glycoprotein of the lymphocytic choriomeningitis virus (LCMV) is a potent tumor cell-lysing agent and capable of jump-starting α-tumor immunity. The current study explores the immune promoting properties of VSV-GP and investigates clinically relevant combination therapies with checkpoint inhibition (α-PD-1) as well as a "second mitochondria-derived activator of caspases" mimetic (SMACm).

Experimental Procedures: Immune competent mice bearing established LLC1-IFNARKO, B16-F1-OVA or CT26.CL25-IFNARKO tumors were subject to treatments comprising VSV-GP, α-PD-1, a SMACm or combinations of VSV-GP with either of the latter therapies. α-tumor effects were determined by tumor growth inhibition and/or changes in overall survival. VSV-GP´s tumor cell selective replication, resulting in immune activation, was assessed using FFPE based IHC staining for the viral N-protein, PD-L1 and different immune cell subsets as well as the NanoString platform (nCounter Pan Cancer Immune Profiling Panel).

Data Summary: Here we present data on VSV-GP, which displays potent α-tumor efficacy and upon systemic delivery increases the local T-cell infiltration, expression of chemokines, T-cell and myeloid cell specific activation markers as well as expression of immune checkpoints, such as PD-1 and PD-L1 within the tumor microenvironment. Building on the observed therapeutic potential of VSV-GP as monotherapy, its immune promoting properties and the delineated compensatory mechanisms, such as PD-1/-L1 upregulation, we explored therapeutic combinations with a PD-1 blocking antibody, which resulted in a strong improvement of VSV-GP-mediated α-tumor efficacy as well as overall survival. Cured, tumor free animals were protected from tumor rechallange indicating immunological memory formation. We further explored the therapeutic combination of VSV-GP with a SMACm, which also resulted in a strongly improved therapeutic benefit and increased overall survival.

Conclusion: In summary VSV-GP induces a pro-inflammatory microenvironment within infected tumors, increases immune cell infiltration and synergizes with α-PD-1 as well as a SMACm. VSV-GP is a potent OV with a strong α-tumor immunity promoting potential. Preclinical data justify further testing of VSV-GP and the combinations with PD-1 blockade or a SMACm in clinical trials.

#2268

Agonistic T cell non depleting ICOS antibody strongly enhances anti-tumor activity with CTLA4 blocking monoclonal antibody without exacerbating colitis.

Clélia Coutzac,1 Jean Jouniaux,1 Aurore Vozy,1 Marc Ballas,2 Sara Brett,2 Sapna Yadavilli,2 Eric Angevin,1 Axel Hoos,2 Nathalie Chaput1. 1 _Gustave Roussy, Paris, France;_ 2 _GSK, Philadelphia, PA_.

Background: ICOS is a member of the CD28 superfamily mainly expressed on activated T cells. An up-regulation of ICOS+ T cells is observed in patients treated with ipilimumab. These findings prompt further studies examining the synergy between CTLA-4 blockade and ICOS stimulation in generating optimal anti-tumor T cell immunity.

Methods: Peripheral blood mononuclear cells (PBMC) were collected from French patients with metastatic melanoma and treated with ipilimumab at baseline (V1) and after 1-2 ipilimumab infusion and restimulated in vitro with anti-CD3 plus anti-human non-depleting agonist ICOS IgG4 isotype antibodies. T cell proliferation and cytokine secretion was determined after 24 and 48h incubation. For in vivo experiments, C57Bl/6 and BALB/c mice were inoculated subcutaneously with MC38 and CT26 tumor cell lines respectively. Mice received intraperitoneal injections of anti-CTLA4 or its isotype control along with anti-mouse ICOS or corresponding isotype at D7, D10 and D13 with tumor growth assessments three times a week.

Results: PBMC collected from patients that respond to anti-CTLA-4 mAb exhibited stronger activation of T cells at baseline compared to patients with poor benefit. Treatment with a non-depleting agonist ICOS IgG4 mAb was able to rescue T cell activation in PBMC from patients with poor benefit. Furthermore, ICOS IgG4 with concurrent TCR engagement favored IL-10 secretion in patients that do not develop colitis during ipilimumab treatment suggesting that these patients may be protected from colitis due to a stronger capacity to secrete IL-10 after ipilimumab blockade particularly when ICOS is stimulated. In both mouse tumor models, combination with agonist non-depleting ICOS mAb increased the anti-CTLA-4 tumor activity without any colitis development.

Conclusion: This study provides evidence in mice and in humans that non-depleting agonistic ICOS mAb may increase anti-CTLA-4 tumor activity particularly in patients that do not benefit from anti-CTLA-4 alone without exacerbation of colitis.

#2269

ATR inhibitor M6620 enhances anti-tumor efficacy of the combination of the anti-PD-L1 antibody avelumab with platinum-based chemotherapy.

Marat Alimzhanov,1 Patricia Soulard,1 Astrid Zimmermann,2 Andreas Schroeder,2 Keyvan Tadjalli Mehr,2 Christiane Amendt,2 Geok Choo Sim,2 Andree Blaukat,2 Joern-Peter Halle,2 Frank T. Zenke2. 1 _EMD Serono R &D Institute, Billerica, MA; _2 _Merck KGaA, Darmstadt, Germany_.

Recently, combination of checkpoint inhibitors with chemotherapy demonstrated significant improvement of overall survival (OS) and progression-free survival (PFS) compared with chemotherapy alone in patients with metastatic non-squamous and squamous non-small cell lung cancer (NSCLC). However still a larger number of patients do not benefit of the combination treatment. Ataxia telangiectasia mutated and Rad3 related kinase (ATR) is one of the key players in the DNA damage response (DDR) to replication stress. M6620 is a potent and selective ATR inhibitor currently in Phase I clinical studies in combination with multiple DNA-damaging drugs. M6620 has been shown to sensitize cancer cells to the lethal effects of DNA-damaging chemotherapeutic agents. Here we tested the hypothesis that ATR inhibitor M6620 could further enhance anti-tumor efficacy of chemotherapy combinations with avelumab, an FDA approved human IgG1 anti-PD-L1 monoclonal antibody, in vivo.

We profiled a panel of murine tumor cell lines to identify models sensitive to combination treatment of M6620 with cisplatin, carboplatin or oxaliplatin. Addition of M6620 to the chemotherapeutic agents in vitro potentiated growth inhibition effect and led to the increase in markers of immunogenic cell death (ICD) in the sensitive tumor cell lines. Based on these data we investigated anti-tumor efficacy of carboplatin or cisplatin in combination with M6620 and avelumab in MC38 murine colorectal tumor model. The triple combination regimens demonstrated a statistically significant better control of tumor growth, more frequent tumor regressions and increased overall survival compared to the double combination treatment groups. The triplet combination regimens were well tolerated as assessed by changes in body weight and clinical signs. Animals with complete tumor responses were re-challenged with MC38 tumor cells and were found refractory to the second tumor inoculation, indicating that durable, protective anti-tumor immunity was established in complete responders.

Taken together, robust anti-tumor efficacy and acceptable toxicity of selected triplet combinations in preclinical models provide strong rationale for combining avelumab with M6620 and carboplatin in patients with PARP inhibitor resistant recurrent ovarian cancer.

#2270

**Recruitment of CD103** + **DCs via tumor-targeted chemokine delivery enhances efficacy of checkpoint inhibitor immunotherapy.**

John-Michael Williford, Jun Ishihara, Ako Ishihara, Aslan Mansurov, Melody A. Swartz, Jeffrey A. Hubbell. _University of Chicago, Chicago, IL_.

Checkpoint inhibitor antibody (CPI) therapy has demonstrated significant clinical benefit in a number of tumor types. Unfortunately, certain tumor characteristics, such as the lack of immune cell infiltration, often correlate with poor responses to CPI therapy. Studies have identified C-C Motif Chemokine Ligand 4 (CCL4) as a key molecule necessary for the recruitment of cross-presenting, CD103+dendritic cells (DCs) to the tumor; tumors lacking CCL4 expression exhibit a "cold tumor" phenotype and respond poorly to immunotherapy (Spranger and Gajewski, Nat. Rev. Cancer, 2018). Based on these results, we hypothesized that tumor-targeted CCL4 could enhance immune cell infiltration into the tumor and synergize with CPI therapy. We generated a fusion protein comprised of CCL4 and a collagen binding domain (CBD) derived from von Willebrand factor. Utilizing exposure of collagen in leaky tumor vasculature due to its disordered structure, we observed that that i.v. infusion of CBD-CCL4 fusion proteins, but not native CCL4, can enhance infiltration of CD103+DCs, CD8+T cells, and natural killer cells and slow B16F10 tumor growth when combined with CPI therapy consisting of anti-cytotoxic T-lymphocyte antigen 4 antibody (αCTLA4) + anti-programmed death-ligand 1 antibody (αPD-L1). Further analysis showed strong correlations between the presence of CD103+DCs and CD8\+ T cells and tumor regression. Similarly, in the EMT6 breast cancer model, tumor-targeted CCL4 in combination with CPI, but not native form CCL4, enhanced recruitment of CD103\+ DCs, CD8\+ T cells, and led to a reduction in tumor growth. To confirm the importance of CD103+DCs in mediating anti-tumor responses, we utilized Batf3 knockout mice bearing B16F10 tumors; in this instance, anti-tumor efficacy of CPI + CBD-CCL4 was completely lost. Preliminary results also showed that CBD-CCL4 can synergize with αPD-1 antibody therapy, slowing tumor growth in CT26 and MC38 colon cancer models compared to αPD-1 therapy alone. Importantly, CBD-CCL4 treatment did not lead to any detectable levels of systemic cytokine elevation or serum liver damage markers, suggesting that this therapy was well-tolerated following systemic administration. These results highlight the utility of recruiting CD103+DCs to the tumor to improve the efficacy of CPI therapy. This engineered chemokine delivery strategy demonstrates significant translational potential by targeting the tumor stroma following systemic administration.

#2271

Development and characterization of SL-115154 (CSF1R-Fc-CD40L) for cancer immunotherapy.

Taylor H. Schreiber, George Fromm, Suresh De Silva, Arpita Patel, Kellsey Johannes, Kyung Jin Yoo, Kaiwen Huang. _Shattuck Labs, Inc., Durham, NC_.

SL-115154 (CSF1R-Fc-CD40L) is a first-in-class biologic derived from the Agonist Redirected Checkpoint (ARC™) platform developed by Shattuck Labs. The ARC platform was developed to solve a fundamental challenge in cancer immunotherapy, which was to develop combination therapeutics that could block immune checkpoints (such as PD-1), while simultaneously activating Tumor Necrosis Factor (TNF) SuperFamily Receptors (such as OX40, 4-1BB, GITR and CD40).

Pre-clinical development of SL-115154 has been completed, and demonstrated that the CD40L end of the ARC binds immobilized CD40 at 0.8 nM affinity and was found to saturate physiological levels of soluble CSF1 at molar ratios of approximate 1 CSF1 to 2-3 ARC molecules; levels which are easily achievable in the clinic. CSF1R-Fc-CD40L binds CD40 on plate bound recombinant receptors and on cells, and in NHP tox immunophenotyping studies, was found to deplete specific populations of CD20+ B cells. Sequestrations of CSF1 and depletion of B cells demonstrated on-target activity culminating in the secretion of cytokines specific to this ARC; including IL-7 & IL-15. SL-115154 was also shown to be highly potent in a series of in potency assays including: CD40 dependent NFkB reporter assays, TCR-independent proliferation/cytokine ELISpot assays, and TCR-dependent SEB super antigen cytokine release assays. In addition, the anti-tumor efficacy of the mouse surrogate CSF1R-Fc-CD40L, was superior to monotherapy or combination treatment with anti-CSF1R and anti-CD40. Interestingly, anti-tumor activity and the rejection of primary and 're-challenged' secondary tumors, was significantly enhanced when CSF1R-Fc-CD40L was sequenced with or following anti-CTLA4 or anti-PD1 therapy. A PD1-resistant in vivo model was established in the mouse to examine CSF1R-Fc-CD40L mono- and combo- efficacy in the molecular context to which most available patients will belong during the phase I study.

These data demonstrate feasibility and functional activity of a novel chimeric fusion protein platform, providing cytokine sequestration of CSF1 by CSF1R, along with APC co-stimulation via the CD40/L axis. This strategy may uniquely inhibit immunosuppressive signaling / DC differentiation into pro inflammatory phenotypes, and instead promote an M1 phenotype and co-stimulation of CD40 positive cells, increasing cell activation, antigen presentation/processing, and enhanced anti-tumor response. 

### Current Developments in Non-invasive Biomarkers for Assessment of Cancer 3

#2272

Exploratory analysis of T cell repertoire dynamics upon systemic treatment with the oncolytic virus pelareorep in combination with pembrolizumab and chemotherapy in patients with advanced pancreatic adenocarcinoma.

Grey A. Wilkinson,1 Devalingam Mahalingam,2 Sukeshi Patel Arora,3 Paul A. Fields,4 Patrick Raber,4 Karol Cheetham,1 Matt Coffey1. 1 _Oncolytics Biotech Inc., Calgary, Alberta, Canada;_ 2 _Northwestern University, Chicago, IL;_ 3 _UT Health San Antonio, San Antonio, TX;_ 4 _Adaptive Biotechnologies, Seattle, WA_.

Background: Pelareorep is an immuno-oncolytic virus that induces an inflamed tumor phenotype in metastatic adenocarcinoma of the pancreas (MAP). Systemically delivered pelareorep in combination with chemotherapy achieves 1 & 2 year-survival rates of 46% & 24% in MAP pts, respectively. Analysis of tumor tissue from patients (pts) treated with pelareorep, chemotherapy and anti-PD-L1 have shown reovirus RNA and protein replication, T-cell infiltration, and upregulation of PD-L1, highlighting that effective T-cell recognition of tumor antigens may be critical to success for this combination therapy. Thus, we hypothesized that pelareorep in combination with chemo and pembrolizumab in pts with MAP would alter the peripheral T-cell repertoire, creating new T-cell clones via the release of novel neoantigens in addition to expanding existing T-cell clones.

Methods: A phase 1b study enrolled 11 MAP pts who progressed after first-line treatment. Pts received pelareorep (4.5e10 TCID 50 IV, D1 & D2), plus pembrolizumab (2mg/kg IV, D8) plus either 1) 5-FU (LV (200 mg/m2 /5-FU 200 mg /m2 IV bolus, 5-FU 1200mg/m2 continuous IV infusion D1) or 2) gemcitabine (1000 mg/m2 IV, D1), or 3) irinotecan (125 mg/m2 IV, D1) q3w, until disease progression/unacceptable toxicity. DNA from peripheral blood mononuclear cells from nine patients at cycle 1 day 1 (C1D1) & C2D1 (approx. 3 weeks later) was analyzed using the immunoSEQ® Assay (Adaptive Biotechnologies, Seattle) sequencing the T-cell receptor beta chain region to interrogate changes in the T-cell repertoire.

Results: The median Morisita index between C2D1 and C1D1 was 0.83 with 3 samples below 0.6, indicative of significant peripheral repertoire turnover. The median number of expanded clones equated to 45.7 per 100,000 cumulative temples; normal variation over 4 weeks is ~ 5-10 expanded clones. Strikingly, most (median: 86%) peripheral clonal expansion occurred in clones below the limit of detection at C1D1. Cox regression analysis revealed that high peripheral clonality correlates with progression free survival at C1D1 (p=0.01, HR=0.053). Moreover, high clonality correlates with overall survival at both C1D1 (p=0.013, HR=0.124) and C2D1 (p=0.010, HR=0.079).

Conclusions: High levels of peripheral T-cell repertoire turnover occur between C1D1 and C2D1. Repertoire turnover is accompanied by significant clonal expansion, mostly by expansion of new clones (i.e. undetected in C1D1). Higher peripheral clonality is associated with better progression free survival at C1D1, and overall survival at C1D1 and C2D1. This research highlights the potential utility of T-cell clonality as a predictive and prognostic biomarker to pelareorep therapy and warrants further clinical investigation.

#2273

A machine learning approach to identify a peripheral prognostic cytokine signature via nivolumab clearance in patients with advanced melanoma.

Rui Wang, Xiao Shao, Junying Zheng, Abdel Saci, Max Qian, Irene Pak, Amit Roy, Akintunde Bello, Jasmine Rizzo, Fareeda Hosein, Rebecca A. Moss, Megan Wind-Rotolo, Yan Feng. _Bristol-Myers Squibb, Princeton, NJ_.

Background: Data suggest that peripheral factors such as circulating cytokines could function as predictive indicators of clinical response to specific therapies or as general prognostic indicators of outcome in patients with cancer. Previous studies have focused on evaluating the association between individual cytokines and clinical outcome. There is no clear consensus on how or which of these cytokines should be selected for prediction of treatment outcome. Clearance of immunotherapeutic agents such as the anti-PD-1 nivolumab (NIVO) has been shown to be a predictor of best overall response and overall survival (OS) across multiple indications. Determination of clearance requires post-treatment pharmacokinetic (PK) samples which negates its utility as a baseline prognostic factor. We investigated a novel machine learning approach to identify a baseline composite cytokine signature that correlates with the established efficacy response relationship for NIVO clearance in advanced melanoma. This approach, which is capable of integrating multivariate factors into a composite feature, may provide a more accurate reflection of underlying biologic factors that contribute to outcome following treatment.

Methods: Peripheral serum PK (NIVO clearance) and pharmacodynamic (Myriad Rules Based Medicine-customized inflammatory cytokine panel) data from 2 phase 3 studies (NCT01721772; NCT01844505) of NIVO monotherapy in patients with melanoma (n = 471) were used for machine learning model development (training data set). A third phase 3 study of NIVO in advanced melanoma (NCT01721746) was included in model application (test data set; n = 158). Random forest, a tree-based ensemble learning method, was used for feature selection and classification (low vs high) of NIVO clearance. Kaplan-Meier analyses and log-rank p values were used to assess statistical difference in long-term OS between predicted high- vs low-clearance groups.

Results: A panel of ~20 baseline inflammatory cytokines related to immune cell modulation were selected by the machine learning model to predict clearance of NIVO monotherapy in patients with advanced melanoma (area under the curve of receiver operating characteristic: 0.79). The predicted clearance value from the cytokine signature was significantly associated with OS across all 3 studies, including patients treated with NIVO or dacarbazine (control) (p<0.01).

Conclusions: We developed a machine learning approach to identify a serum prognostic cytokine signature based on the relationship between NIVO clearance and response. The strong association of the defined cytokine signature with OS from NIVO and dacarbazine suggests a prognostic role for drug clearance. This cytokine signature has the potential to be a prognostic biomarker in melanoma and may be used for balancing and randomization of patients in clinical studies.

#2274

Whole-genome bisulfite sequencing of cell-free DNA (cfDNA) in muscle-invasive bladder cancer patients to predict the risk of recurrence.

YI-TSUNG LU,1 Gerald Gooden,2 Ben Y. Tew,2 David Buckley,2 Kimberly Siegmund,3 Siamak Daneshmand,4 Amir Goldkorn,1 Bodour Salhia2. 1 _Division of Medical Oncology, Department of Medicine, Keck School of Medicine and Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA;_ 2 _Department of Translational Genomics, Keck School of Medicine and Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA;_ 3 _Department of Preventive Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA;_ 4 _Institute of Urology, Keck School of Medicine, University of Southern California, Los Angeles, CA_.

Background: Muscle invasive bladder cancer (MIBC) is a lethal disease with high recurrence rate despite standard radical cystectomy with neoadjuvant chemotherapy. Informative new biomarker strategies are needed to accurately assess the risk of recurrence and guide treatment. Epigenetic marks play a key regulatory role in cancer initiation and progression, and methylation patterns in the tissue have been correlated with prognosis in multiple cancer types. We hypothesized that whole-genome bisulfite sequencing (WGBS) can be used to generate a cell-free DNA (cfDNA) based methylation signature prognostic of bladder cancer recurrence after curative cystectomy.

Methods: We identified a retrospective MIBC cohort with annotated clinical outcome more than 10 years after curative cystectomy. In the cohort, we selected a total of 46 patients who did not recur after 3 years of follow-up, and 40 patients who relapsed within 3 years of surgery. cfDNA was extracted from the archival plasma, and samples were pooled for WGBS based on their pathologic stages (T2a, T2b, T3a, T3b, and T4/N+) and recurrence status (recurrence vs no recurrence). A paired-end 150bp sequencing was performed on an Illumina NovaSeq platform. Sequences were aligned using the bismark tool and DNA methylation differences between groups were identified using metline.

Results: CfDNA was extracted with a median cfDNA concentration 10.6 ng/ml (range 0.97-121.5 ng/ml). After WGBS, differential methylation regions (DMRs) for each stage were called by comparing the recurrence (R) and non-recurrence (NR) groups. We identified 6412 to 9532 DMRs across stages, and greater overall hypomethylation in cfDNA before systectomy in R groups was observed in all stages. While most of the DMRs are in the gene bodies, 10.3 to 11.7% of the DMRs were in the promoter regions. DMRs were also compared with publically available datasets using Basespace correlation engine. The cfDNA methylation in R groups across all stages was positively correlated with tumor tissue methylation in a bladder cancer dataset. Similarly, the cfDNA methylation in R groups was inversely correlated with bladder cancer tissue expression in TCGA dataset. We also called the DMRs by comparing all R and NR groups, and 729 DMRs were found to be shared in all stages. Similar patterns of global hypomethylation and positive correlation to the bladder cancer tissue methylation were observed in R group.

Conclusions: cfDNA methylation in MIBC patients was concordant with multiple reported datasets. The overall hypomethylation in the cfDNA prior to the cystectomy was observed in patients who later recurred. Additional data filtering and annotation is ongoing, and these DMRs will be validated as potential biomarkers for predicting the risk of recurrence in MIBC patients.

#2275

Detection of IDH1 mutations in plasma cell-free circulating tumor DNA (ctDNA) from patients with cholangiocarcinoma.

Elia Aguado-Fraile,1 Sung Choe,1 Camelia Gliser,1 Lori Steelman,1 Liewen Jiang,1 Bin Fan,1 Kha Le,1 Maeve A. Lowery,2 Susan Pandya,1 Bin Wu1. 1 _Agios Pharmaceuticals, Inc., Cambridge, MA;_ 2 _Trinity College, Dublin, Ireland_.

Somatic mutations in the isocitrate dehydrogenase 1 (IDH1) gene are detected in 13-15% of cholangiocarcinoma (CC) cases overall and up to ~25% of intrahepatic CC cases. Ivosidenib (AG-120) is a first-in-class, oral, potent, reversible, targeted inhibitor of the mutant IDH1 (mIDH1) protein, and AG-881 is an oral, potent inhibitor of both mIDH1 and mIDH2. Phase 1 studies for both molecules in patients with mIDH1/2 solid tumors, including CC, have completed enrollment, and analyses are ongoing (AG120-C-002 [NCT02073994]; AG881-C-002 [NCT02481154). Patients with mIDH1 CC were enrolled into the clinical studies based on local tissue testing for the IDH1 mutation. In this work, we examined the feasibility of mIDH1 detection in ctDNA from patients with mIDH1 CC, and explored the correlation with detection by tissue-based next-generation sequencing (NGS) assays and with plasma levels of the oncometabolite D-2-hydroxyglutarate (2-HG). Baseline plasma samples were collected from a total of 41 patients, including 28 from AG120-C-002 and 13 from AG881-C-002. A BEAMing digital PCR platform (Sysmex) was used for the detection and quantification of five mIDH1 alleles, including R132C, R132H, R132L, R132S, and R132G, with 0.02% analytical sensitivity (0.04% for R132H). Tumor tissues were available from 35 of 41 patients and analyzed using the FoundationOne and/or Personalis ACE cancer research targeted panel. Additionally, baseline plasma levels of 2-HG were measured and correlated with plasma mIDH1 ctDNA. Detection of mIDH1 in plasma ctDNA was concordant with IDH1 mutation status by central testing of tissue in 32 of 35 patients (91.4%), with 31 mIDH1-positive results from both plasma and tissue (88.6%). One patient was mIDH1-negative in both plasma and tumor. In all 31 mIDH1-positive cases, the mIDH1 allele detected was concordant. In general, mIDH1 variant allele frequency (VAF) was lower in plasma than in tumor tissue (mean VAF 2.9% in plasma, 26.2% in tumor). Additionally, the level of plasma ctDNA mIDH1 VAF was significantly correlated with baseline plasma 2-HG concentration (Spearman R=0.535, p=0.0009). Correlation with clinical outcome is currently being explored and will be presented. Taken together, our results demonstrate the feasibility of IDH1-R132 mutation detection in plasma from CC patients, with a 91.4% concordance rate with detection in tumor tissue. These results provide a rationale for exploring liquid biopsy-based testing methods when the feasibility of repeated biopsies or sample exhaustion limits the ability to detect actionable mutations through tissue-based NGS panels, which can be a major challenge for trial participation in this indication. Confirmatory liquid biopsy studies will be conducted with baseline and longitudinal samples collected from the ongoing phase 3 study of ivosidenib in mIDH1 CC (NCT02989857).

#2276

Performance and cost comparison of circulating tumor DNA detection platforms.

Daan C. Vessies,1 Marjolein J. Greuter,2 Karlijn L. van Rooijen,3 Theodora C. Linders,1 Mirthe Lanfermeijer,1 Kalpana L. Ramkisoensing,1 Flore E. Grijseels,4 Boris van Doorn,4 Gerrit A. Meijer,1 Miriam Koopman,3 Veerle M. Coupé,2 Geraldine R. Vink,3 Remond J. Fijneman,1 Daan van den Broek1. 1 _Netherlands Cancer Institute, Amsterdam, Netherlands;_ 2 _Amsterdam University Medical Centers, Amsterdam, Netherlands;_ 3 _University Medical Center Utrecht, Amsterdam, Netherlands;_ 4 _Merck B.V., Schiphol-Rijk, Netherlands_.

Purpose

The field of circulating cell free DNA (cfDNA) testing is quickly developing. Multiple platforms to detect hotspot mutations in ctDNA are available, including Bio-Rad droplet digital PCR (ddPCR), BioCartis Idylla, Roche COBAS z480 and Sysmex BEAMing. These platforms vary in the amount of plasma required, the method of ctDNA isolation, the number of hotspots analyzed, and the costs per sample. These factors can impact the applicability of a specific platform in the analysis of ctDNA.

In this study we compared these platforms in terms of sensitivity and total costs per sample.

Methods

The platform comparison was performed as follows:

1. Plasma from six metastatic colorectal cancer (mCRC) patients with known tissue KRAS mutation status was analyzed according to the manufacturers' protocols.

2. Sensitivity was tested using six constructed reference samples with seven KRAS hotspot mutations at various total input (10ng, 50ng) and mutant allele frequencies (0%, 0.04%, 0.50%); each in four replicates.

3. Twelve mCRC patients were analyzed using equal amounts of ctDNA for each platform.

4. Total costs per sample were evaluated, including costs for consumables, technician hands-on time, equipment and maintenance.

Results

1. In six mCRC samples BEAMing (3ml plasma) detected 5/6 mutations, ddPCR and Idylla (both 1ml plasma) 4/6 mutations and COBAS z480 (2ml plasma) 3/6 mutations.

2. In constructed reference samples ddPCR (65%) and BEAMing (46%) yielded the highest sensitivity. With 10ng input BEAMing and COBAS z480 produced "Too little DNA" errors in 60% and 100% of cases, respectively.

3. In twelve mCRC samples, eight had more than 10ng ctDNA and four had less. All platforms were fully concordant for samples with more than 10ng input. Detection rate across twelve samples: Idylla detected 7/11 detectable mutations, COBAS z480 and BEAMing both detected 5/11, ddPCR detected 4/10.

4. BEAMing has the highest cost per sample (€486-€821) whereas ddPCR has the lowest cost per sample (€39-€298).

Conclusions

A direct comparison of ctDNA mutation detection platforms is complex and should take into account the differences in input and output specifications of the platforms.

Factors such as complexity of analysis (Idylla is a low complexity platform, whereas BEAMing requires more specialized training), total costs (varying from ddPCR to BEAMing), sensitivity (ddPCR and BEAMing yielded the highest sensitivity) and the number of mutations evaluated vary greatly between the platforms. All these factors influence ctDNA analysis and will have to be considered when choosing a specific platform for a specific (clinical) question, or when comparing results between studies.

Our data provide insight in the comparative performance of four commercial ctDNA analysis platforms, allowing future users to make an informed decision regarding a platform.

Acknowledgement

Powered by Health~Holland, Top Sector Life Sciences & Health, grant LSHM16047-H005.

#2277

Workflow evaluation for molecular characterization of single circulating tumor cells in blood samples of patients with gynecological malignancies.

Janina Levermann,1 Norbert Hochstein,2 Paul Buderath,1 Benjamin Franken,2 Siegfried Hauch,2 Charline Bemmann,2 Rainer Kimmig,1 Sabine Kasimir-Bauer1. 1 _University Hospital of Essen, Essen, Germany;_ 2 _QIAGEN GmbH, 40724 Hilden, Germany_.

Background: We recently presented the feasibility of single circulating tumor cell (CTC) analysis using the QIAscout single cell isolation platform (QIAGEN), followed by whole transcriptome amplification (WTA) and targeted Next Generation Sequencing (NGS). We here applied this workflow in blood samples of patients (pts) with metastatic breast cancer (MBC) and primary ovarian cancer (POC) and compared the result with a new workflow for single cell transcriptome analysis applying the QIAseq UPX 3' Transcriptome Kit (QIAGEN) for high throughput gene expression analysis.

Methods: 5 ml blood of 7 MBC and 3 POC pts were analyzed for CTCs using the AdnaTest EMT2/StemCell SelectTM, followed single cell isolation. Adhesion of CTCs to the QIAscout array microrafts was supported by pre-treating the array with Corning® Cell-TakTM Adhesive. Single CTCs have been identified utilizing an inverted microscope, and isolated with the QIAscout single cell isolation platform. Subsequently, mRNA of single cells was amplified by WTA (REPLI-g WTA Single Cell Kit, QIAGEN) and cDNA of the cells was further analyzed by targeted NGS using a QIAseq™ Targeted RNA Panel (Human Cancer Transcriptome Panel, 395 genes). Applying the QIAseq UPX 3' Transcriptome Kit, each single cell was tagged with an individual cell ID-sequence and each RNA molecule was simultaneously tagged with an unique molecular index (UMI) during reverse transcription. Tagging of each individual cDNA molecule enabled pooling of all samples for subsequent amplification and library preparation steps, followed by high-throughput NGS. Methods were validated in spike-in experiments using different cell lines spiked into blood of healthy donors.

Results: Using the QIAscout in combination with the WTA-workflow, we have already demonstrated an absent leukocyte contamination. However, only a very few of the detected cells in MBC pts (15%) were finally applicable for NGS. Nevertheless, analyzable CTCs expressed genes involved in angiogenesis (ITGB3), cell cycle (CCND2) and apoptosis (FASLG & SKP2). Applying the UPX 3' Transcriptome-workflow, the number of analyzable CTCs could be markedly improved as documented in spike-in experiments as well as in blood samples of MBC and POC pts. Up to now, in the latter group, we were able to isolate a total number of 84 CTCs [median 25 CTCs (range 24-35 cells)] with 44/84 (52%) ID-sequences detected, being applicable for NGS. Experiments and detailed transcriptome profiling of CTCs are ongoing to be presented at the meeting.

Conclusion: Although both described workflows have to be further evaluated in more detail, the QIAscout single cell isolation platform allows subsequent transcriptome analysis followed by NGS to get insights into single cell heterogeneity for further therapeutic strategies.

#2278

A high throughput method for the optimization of digital PCR assays for personalized circulating tumor DNA detection.

Maya M. Arce,1 Christina Wood-Bouwens,1 Derrick Haslem,2 Billy T. Lau,3 John Bell,1 Alison Almeda,1 Matt Kubit,1 Bryce Moulton,2 Robin Romero,2 Robert P. St. Onge,3 Lincoln Nadauld,2 Hanlee P. Ji3. 1 _Stanford University, Palo Alto, CA;_ 2 _Intermountain Healthcare, UT;_ 3 _Stanford University, CA_.

Single color digital PCR (sc-dPCR) is a robust approach for the quantitation of low allelic fraction mutations in clinical oncology samples. More recently this technology has been employed to identify mutations from circulating tumor DNA (ctDNA) that has been extracted from the blood samples of cancer patients. The use of digital PCR has great potential for non-invasive longitudinal monitoring via liquid biopsies. However, this application requires low input DNA volumes and relies on a single nucleotide variant (SNV) to distinguish between normal and ctDNA, necessitating that sc-dPCR primer binding is both highly efficient and specific. These stringent requirements make assay optimization a tedious process that greatly limits the rate at which personalized detection panels can be generated. We have developed a high throughput method to optimize sc-dPCR assays utilizing Next Generation Sequencing (NGS) technology to assess amplification more quickly and with more flexibility than traditional gel based analysis.

Using our assay optimization approach, a segment of each gene containing a tumor specific SNV was incorporated into the genome of Saccharomyces cerevisiae. These renewable positive control colonies were cultured in a 96 well plate format and pooled to mimic the low allelic frequency conditions of ctDNA. The presence of each tumor specific SNV was confirmed by preparing and sequencing a library containing the unique barcode region of each colony. Using bulk PCR, up to 96 primer sets were tested at one annealing temperature in a singleplex format. Alternatively, we multiplexed up to 11 primers in each well, greatly increasing the number of assays that can be developed per plate. Using this multiplexed format, we introduced a thermal gradient across the plate to identify the optimal annealing temperature of each primer set in a single run. A parallel experiment with identical PCR conditions was run using NA18507 human DNA to act as a negative control for primer specificity.

All amplicons in each PCR condition were uniquely indexed and sequenced using an NGS platform. Using a ratio of the number of reads associated with on target and non-mutation specific amplicon sequences for each primer set, the success of each assay was determined. This method was also used to identify specific mismatches incorporated in the primer sequence that increased binding specificity. Using a sequencing based analysis method, we have observed that sc-dPCR assays can be optimized rapidly across multiple mutations, making them more accessible for personalized monitoring.

#2279

Serial changes in tumor-derived whole-genome cfDNA fraction to identify early disease progression prior to imaging.

Andrew A. Davis,1 Wade T. Iams,2 David Chan,3 Michael S. Oh,1 Robert W. Lentz,1 Neil Peterman,4 Alex Robertson,4 Abhik Shah,4 Rohith Srivas,4 Nicole Lambert,4 Tim Wilson,4 Peter George,4 Becky Wong,4 Ayse Tezcan,4 Ram Yalamanchili,4 Ken Nesmith,4 John C. Spinosa,4 Haluk Tezcan,4 Young Kwang Chae1. 1 _Northwestern University Feinberg School of Medicine, Chicago, IL;_ 2 _Vanderbilt University Medical Center, Nashville, TN;_ 3 _Cancer Care Associates TMPN, Redondo Beach, CA;_ 4 _Lexent Bio, Inc., San Francisco, CA_.

Background:

Response to cancer treatment is usually determined by clinical exam and imaging assessment. Here, we analyzed changes in tumor-derived whole-genome cell-free DNA (cfDNA) at baseline and after treatment initiation to determine response to treatment prior to routine imaging.

Methods:

We prospectively enrolled and serially collected blood from 54 patients with metastatic malignancies (21 lung, 20 breast, 13 other tumor types). Baseline blood samples were drawn prior to initiation of a new treatment and at one or two additional time points, after the first cycle (median 21 days) and the second cycle (median 42 days). 4 mL of plasma was separated from peripheral blood collected in Streck Cell-Free DNA Blood Collection Tubes, and cfDNA was isolated from plasma aliquots using Qiagen QIAmp extraction kits. To prepare sequencing libraries, a method optimized for whole genome sequencing (WGS) was used based on the Kapa HyperPrep chemistry. WGS was performed at approximately 25X depth on the Illumina HiSeq X. Based on a patient-specific profile of whole genome features, changes in the fraction of tumor-derived cfDNA were quantified over the initial course of treatment. Imaging was performed per standard practice with treatment response determined by RECIST.

Results:

Median number of prior treatment lines was 1 [range 0-6]. Patients were treated with the following therapies: chemotherapy (27), immunotherapy (14), hormone therapy (7), or targeted therapy (6). For the entire cohort, patients with predicted progression by cfDNA (n=11), indicated by an increase in tumor fraction at either post-treatment blood collection, had worse event-free survival compared to patients that did not show an increase (n=43) (hazard ratio 8.0, [95% CI 3.4-19.2], log-rank p=4.5 x 10-8). For the patients who were predicted to progress, the cfDNA assay preceded clinical evaluation by a median of 39 days. Median progression-free survival was 62 days for patients with predicted progression versus 232 days for others. All patients with predicted progression were later confirmed to progress at the first follow-up evaluation (11/11, 100% positive predictive value). For the remaining patients, 32 of 43 did not progress (74% negative predictive value). Therefore, sensitivity for the assay was 50% and specificity was 100%.

Conclusions:

Analyzing tumor-derived cfDNA early in the course of a new therapy holds promise to identify patients with early disease progression across a variety of tumor histologies and types of treatment. Early identification of patients who are not benefitting from treatment will enable initiation of other potentially effective therapies, and reduce unnecessary side effects and cost associated with these treatments. Further studies are warranted to validate these findings in larger cohorts and to confirm the histology and treatment-independent nature of the approach.

#2280

Co-detection of circulating tumor DNA and RNA in the plasma of patients with breast cancer increases the detectable number of mutated molecules.

Yu-Hsiang Chen, Bradley A. Hancock, Jeffrey P. Solzak, Milan Radovich. _Indiana Univ. School of Medicine, Indianapolis, IN_.

Introduction: Circulating tumor DNA (ctDNA) has been shown in several studies to be a biomarker to predict tumor relapse and prognosis in breast cancer. However, the sensitivity of detection using ctDNA is modest in early-stage breast cancer and in patients with low-burden metastatic disease. Unlike ctDNA, which is released into circulation through necrosis or apoptosis, circulating tumor RNA (ctRNA) can be released into the circulation from living cancer cells. Therefore, detecting ctRNA along with ctDNA could be a potential solution to increase the sensitivity of detection. This pilot study is aiming to investigate the contribution of ctRNA in increasing the level of mutation detection in patients with breast cancer.

Methods: Plasma samples were isolated from six patients with metastatic breast cancer for this proof of concept study. Circulating nucleic acids (DNA+RNA) were extracted from plasma for each patient and split into 2 groups: ctDNA+ctRNA OR ctDNA alone. In the ctDNA/ctRNA co-detection group, circulating nucleic acids underwent reverse transcription, followed by library preparation using the Roche Avenio ctDNA Expanded Kit, a capture-based target enrichment sequencing methodology. For the control group, the same amount of circulating cell-free DNA underwent the same library preparation (with no reverse transcription) for ctDNA detection. Sequencing was performed on an Illumina NextSeq to obtain at least 45 millions reads per sample.

Results: All six patients had at least one somatic mutation detected. TP53 mutations were detected in three patients, followed by PIK3CA mutations in two patients and ERBB2 mutation in one patient. In total 14 mutations were detected across the 6 patients. The distribution of allele frequency among all mutations was from 0.41% to 11.1%. Given that the molecular barcode methodology was applied, we were able to determine the number of unique mutated molecules. Notably, the numbers of mutant molecules in the ctDNA+ctRNA co-detection group were all higher than the corresponding mutations in ctDNA-only group. We observed an average percentage increase of 67% (range=2.8%-385%) in the number of mutated molecules when mutations from ctRNA were co-detected with ctDNA.

Discussion: Compared to ctDNA only, incorporation of ctRNA into the sequencing assay demonstrated increased detection of mutated molecules. This study has shown that the enhancement of mutation detection by adding ctRNA could be a potential method to increase the sensitivity of circulating tumor mutations in breast cancer. A validation study in patients with early-state triple-negative breast cancer to predict relapse using ctDNA/ctRNA co-detection is currently in progress.

#2281

The role of baseline and early dynamics of ctDNA in predicting response and prognosis of early and advanced gastroesophageal adenocarcinomas.

Ali Abdulnabi Mohamed,1 Mark R. Openshaw,1 Barbara Ottolini,1 David Guttery,1 Daniel Fernandez Garcia,1 Cathy J. Richards,2 Jacqui A. Shaw,1 Anne L. Thomas1. 1 _University of Leicester, Leicester, United Kingdom;_ 2 _University Hospitals of Leicester NHS Trust, Leicester, United Kingdom_.

Background: Circulating tumor DNA (ctDNA) is emerging as a valuable less-invasive adjunct to tissue biopsy for real time monitoring and personalization of cancer treatment. This study aimed to determine the prognostic value of baseline ctDNA in both early and advanced gastroesophageal adenocarcinoma (GEA) and establish whether dynamic changes in ctDNA provides useful response and prognosis information. Patients and Methods: Formalin-fixed, paraffin-embedded (FFPE) tissue DNA and serial plasma cell-free DNA (cfDNA) were obtained from 36 patients (23 early stage (63.9%), 13 advanced stage (36.1%)) undergoing treatment for GEA. Tumor DNA was analyzed by targeted next generation sequencing (NGS) (custom ampliseq six gene panel) and Nanostring™ nCounter® technology (87 gene panel). In each patient, selected mutations and gene amplifications were profiled in serial cfDNA samples using a combination of NGS, droplet digital PCR and real-time quantitative PCR. Results: Mutations and/or gene amplifications were identified in tumor DNA of 33/36 patients (91.7%). Patient specific profiling detected ctDNA in 19/33 patients (57.6%) at baseline: 9/22 with early stage disease (40.9%) and 10/11 with advanced stage disease (90.9%). Objective Response Rate (ORR) by RECIST 1.1 criteria was 71.4% (10/14) for patients who were ctDNA negative at baseline (group A) and 52.9% (9/17) for patients with detectable ctDNA at baseline (group B). Multivariate Cox regression analysis, adjusted for stage of disease and patient performance status, showed presence of ctDNA at baseline was associated with both reduced progression free survival (PFS) and overall survival (OS) [hazard ratio (HR) of 6.2 (95% CI 1.9-19.8, P = 0.002) and 7.3 (CI 1.9-28.2, P = 0.004), respectively]. The median PFS was 34.7 months and 12.1 months and median OS was not reached and 14.5 months for group A and B, respectively (Mantel-Cox Log Rank P = 0.008 and P < 0.001, respectively). ctDNA was detected before relapse in 3/22 (13.6%) early stage patients and before progression in 5/11 (45.5%) advanced stage patients, with an overall median lead-time of 6.3 weeks before radiological evidence of relapse or progression. Conclusion: This pilot study suggests that presence of ctDNA at baseline and recurrence during treatment are poor prognostic biomarkers both in early and advanced GEA.

#2282

Progress toward non-invasive oncology diagnostics: Evaluation of cfDNA next-generation sequencing assays.

Nathan Riccitelli, Nancy Valencia, Ashley Beams, Sarah Johnson, Reinhold Pollner. _Navigate BioPharma Services, a Novartis subsidiary, Carlsbad, CA_.

Introduction

Surgical biopsy or excisional resection is a standard practice in oncology diagnosis that requires a detectable tumor mass and can be highly invasive, limiting its role in the very early detection, as well as ongoing monitoring efforts, of genetic aberrations relevant to tumor biology. In contrast to surgical biopsies, liquid biopsies provide a readily accessible source of circulating tumor DNA, albeit in a highly diluted state that necessitates a very sensitive and robust method of detection. With recent advents in molecular barcoding and bioinformatics, next-generation sequencing (NGS) has emerged as a platform for probing this liquid oncology space. Herein, we evaluate the ability of the Oncomine™ cell-free DNA (cfDNA) Breast, Colon, and Lung NGS panels to reliably detect clinically relevant genomic alterations in a large background of non-mutated genetic material.

Methods

A total of 197 individual libraries were prepared across all three cfDNA panels according to the manufacturer protocols. Libraries were manually normalized, pooled, and loaded onto the ThermoFisher IonChef/S5xL system for bead-templating and sequencing. Samples included a mix of commercially available cfDNA controls, plasma samples, and cell lines spiked into healthy donor plasma, and were selected to cover a wide range of clinically relevant mutations and mimic the conditions inherent to real patient specimens. Isolation of cfDNA was performed on both Qiagen and Kingfisher platforms. Raw variant call files were generated using IonReporter and processed through a custom filtering pipeline to generate the final results.

Results

Nucleic acid isolations yields were comparable for cfDNA samples isolated using the Qiagen or Kingfisher extraction methods, with both yielding a 40-60% DNA recovery rate when known DNA quantities were spiked into plasma background. Library yield mapped to NGS panel size, and the greatest variability was observed for the Colon cfDNA panel. The sensitivity for all Oncomine™ cfDNA assays exceeded 92%, and was highly dependent on sample input, with robust detection at the 0.1% allele frequency requiring up to 50 ng of input cfDNA. A single false positive was present in one HapMap sample analyzed with the breast cfDNA panel.

Conclusion

The Oncomine™ cfDNA panels provide accurate detection of variants in cfDNA down to 0.1% variant allele frequency. However, increased DNA inputs are required to achieve this highly-sensitive level of detection. Although sensitivity is reduced at lower inputs, the ability to generate high-quality libraries from as little as 10 ng DNA ensures sufficient material can be obtained for sequencing from even low-yielding samples. Overall, these results indicate that the Oncomine™ cfDNA assays can aid in the very early diagnosis and monitoring of tumor populations via minimally-invasive blood-based diagnostics.

#2283

Accurate and efficient Next-Generation Sequencing approach for liquid biopsy detection and monitoring.

Cai Chen, Christopher Kasbek, Yang Song, Si Chen, Nouran Almalki, Jiangyu Li, Yifei Wan, Jun Huang, Wei Ding. _Admera Health LLC, South Plainfield, NJ_.

Liquid biopsy detection using ctDNA has great potential in cancer diagnosis, monitoring, and predicting survival. However, detection sensitivity and specificity remain major challenge at the current stage. Here we utilized a targeted next-generation sequencing (NGS) approach to develop a noninvasive test to identify single nucleotide variations (SNVs), indels, fusions, copy number variations (CNVs) and microsatellite instability (MSI) present at low frequencies in different solid cancer patients. Target regions of interest were sequenced using tagging of individual molecules, followed by amplicon library generation and massive parallel sequencing using an Illumina platform. The variants were called and annotated using our in-house Actionable Genomic Interpretation System (AGIS) bioinformatics pipeline. This CLIA-approved NGS panel (LiquidGx™) detects more than 170 variants in 17 genes which were chosen mostly based on NCCN clinical actionable guidelines (AKT1, ALK, BRAF, EGFR, ERBB2, HRAS, KIT, KRAS, MAP2K1, MET, NRAS, PDGFRA, PIK3CA, PTEN, RET, ROS1 and TP53) with detection limit of 0.1%. Mutations are detected with 94% sensitivity and 99.8% specificity in a short turnaround time of 3-5 business days. To assess the performance in a clinical setting, accurate sequencing was performed on 210 lung cancer patients; the frequency of mutations identified of each gene closely mirrored the published lung cancer tissue biopsy data. Fusions are detected from exosomal RNA, which could increase the sensitivity and/or concordance to tumor. For spike-in data validation, the limit of detection (LOD) of CNV can reach 2.2 -2.5 total gene copies. In addition to actionable mutations for targeted therapy, LiquidGx™ provides MSI status, which can predict a predisposition to mutations as a result from impaired DNA mismatch repair (MMR) and help predict if a tumor will respond to anti-PD-1 therapy. The LOD of our MSI detection is 2-4%. In summary, we have developed a potent liquid biopsy platform for highly sensitive genomic biomarker test. Our technology is also available as an assay development service and can be easily adaptable to the diagnosis and monitoring of different cancer types.

#2284

Patient-specific circulating tumor DNA monitoring using esophageal squamous cell cancer gene panel and digital PCR.

Takeshi Iwaya,1 Fumitaka Endo,1 Yasushi Sasaki,2 Noriyuki Sasaki,1 Mizunori Yaegashi,1 Yuji Akiyama,1 Akira Sasaki,1 Mari Masuda,3 Tesshi Yamada,3 Takashi Tokino,4 Satoshi S. Nishizuka5. 1 _Iwate Medical Univ. School of Medicine, Morioka, Japan;_ 2 _Sapporo Medical University, Sapporo, Japan;_ 3 _National Cancer Center Research Institute, Tokyo, Japan;_ 4 _Research Institute for Frontier Medicine, Sapporo Medical University, Sapporo, Japan;_ 5 _Institute of Biomedical Sciences, Iwate Medical University, Morioka, Japan_.

Background: Esophageal squamous cell carcinoma (ESCC) is belligerent cancer with poor prognosis. At present, no specific ESCC biomarkers have been established for therapeutic monitoring. This study aimed to monitor ESCC therapeutic response using circulating tumor DNA (ctDNA) using digital PCR (dPCR) with individual ESCC tumor-specific mutations as identified from the originally-designed ESCC-specific next generation sequencing (NGS) panel.

Methods: Thirty-five ESCC patients were enrolled in our study (stage I/II/III/IV: 8/3/19/5, respectively). First-line therapies included surgery, chemotherapy with cisplatin/5-FU, and docetaxel/cisplatin/5-FU for 8, 3, and 24 patients, respectively. Mutation screening of primary ESCCs was performed with amplicon sequencing using ESCC panel, targeting 31 genes. The level of ctDNAs was evaluated by dPCR using 58 originally-designed sets by Hypercool Primer & ProbeTM based on mutations of the individual patient. A total of 458 blood samples were examined for ctDNA with a median follow-up time of 392 days. NGS screening of TP53 gene in plasma DNA was also performed.

Results: Among mutations whose variant allele frequency (VAF) was higher than 5%, the ESCC panel identified 5.1 mutations per sample. The most frequent mutated gene was TP53 (33 of 36, 92%). Among the 58 mutations identified from primary tumors analyzed by dPCR in pretreatment blood, 44 (76%) were detectable as ctDNA. The VAFs of plasma DNA in advanced stages were significantly higher than those of early stages (p < 0.0001). The rate of positive ctDNA was 12.5% (1/8) in stage I, whereas that of stage II or higher was 92.3% (24/26). In stage II or higher, the average tumor volume was 65,572 mm3 (range 3,816-521,818 mm3). There was a low correlation between tumor volume and ctDNA-VAF (r=0.51, 95%CI: 0.13-0.76). Sensitivity/specificity and positive/negative predictive value of ctDNA in plasma were 73.5%/93.5% and 96.2%/70.0%, respectively. These data suggest that ctDNA has higher credibility than conventional serum tumor makers, such as CEA, SCC, and CYFRA. In chemotherapy patients, those with a detectable level of ctDNA (n = 12) showed poorer survival than those with an undetectable level of ctDNA (n = 13) (hazard ratio: 0.12, 95% CI, 0.03-0.49, p = 0.001, log-rank). The VAFs of primary tumor mutations by both NGS and dPCR were strongly correlated (n = 59, r = 0.97), whereas the correlation between NGS and dPCR for VAFs of ctDNA for TP53 mutations in pretreatment plasma was relatively modest (r = 0.71), likely because of low VAFs (<1%). In a relapsed patient, ctDNA elevation was observed six months before the lymph node metastasis was diagnosed by CT scan 1.5 years after resection with curative intent for stage II ESCC.

Conclusion: With the ESCC panel and dPCR, patient-specific ctDNA monitoring readily indicates the disease stage and may facilitate an early relapse detection of ESCC.

#2285

Comparison of the nCyte Dx and CellSearch systems for CTC enumeration.

Josefa Andrade,1 Ramsey Mohl,2 Stephanie Thomas,1 John Kearney,1 Phillip Bernard,3 Hillary S. Sloane1. 1 _Axon Dx, Earlysville, VA;_ 2 _ARUP Laboratories, Salt Lake City, UT;_ 3 _University of Utah/Huntsman Cancer Institute, Salt Lake City, UT_.

Introduction: Circulating Tumor Cells (CTCs) have clinical utility for cancer prognosis and monitoring disease. The goal of this study was to compare CTC enumeration by the FDA-approved CellSearch technology to a new technology, the nCyte Dx system by Axon Dx, LLC.

Methods: Duplicate peripheral blood samples from 24 patients with advanced prostate cancer were collected in CellSave tubes as standard of care and submitted for CTC quantification using the CellSearch system at ARUP Laboratories. Following CellSearch testing, a paired sample was sent from ARUP to Axon Dx for nCyte Dx analyses. For every pair of samples, the blood was collected at the same time, but the sample tested by Axon Dx was aged 24 to 72 hours beyond the sample tested with CellSearch. CellSearch identifies CTCs by immunomagnetic capture of EpCAM-postive cells, while nCyte Dx does not incorporate specific CTC enrichment. With nCyte Dx, all PBMCs are isolated via density-based separation in a CPT tube and stained with Axon's immunofluorescent cocktail (nPac). The cells are drawn down onto semi-porous membranes and placed onto microscope slides. Slides are imaged with the nCyte, a rapid fluorescent scanning instrument, and Axon's advanced cell finding and analysis software (nAble) facilitates automated CTC detection. Due to the high-quality optical properties of the nCyte, the detected material can be characterized and categorized based on morphology and cellular structure.

Results: CellSearch-derived CTC counts were compared with the enumeration results from the nCyte Dx system. Results were reported per 7.5mL blood for both methods. Bland-Altman analysis demonstrates that CellSearch and nCyte Dx results agreed in 22 of the 24 patient sample sets tested within a 95% confidence interval (CI). For both discrepant samples, the CellSearch count was found to be higher. The nCyte Dx method generally detected more CTCs than CellSearch in the lower enumeration range (<10 CTCs). Because previous data from Axon Dx shows no CTCs detected in healthy patients, this finding may be attributable to increased sensitivity rather than decreased specificity. Differences between the results may also be attributable to the natural variation between duplicate blood draws, best characterized by the Poisson distribution model.

Summary and Conclusions: A comparison of CTC enumeration results using the CellSearch and nCyte Dx platforms was performed using duplicate blood samples from 24 prostate cancer patients. The results reveal high similarity between the two technologies, with 22 of 24 patients (92%) having enumeration results that agreed within a 95% CI. While both technologies deliver similar results, there are notable advantages of the nCyte Dx system over CellSearch, including a lack of bias toward EpCAM positive material. Additionally, the superior image quality of nCyte Dx may limit subjectivity in CTC identification and offer the ability to characterize CTC heterogeneity.

#2286

Liquid biopsy for early cancer detection.

Hyunji Kim, Fehmi Civitci, Josiah Wagner, Pavana Anur, Matthew Rames, Xiaolin Nan, Terry Morgan, Thuy Ngo. _Oregon Health & Science University, Portland, OR_.

Introduction: Early cancer detection remains as a critical challenge to improve patient's survival and clinical outcome. A non-invasive liquid biopsy permits the analysis of multiple circulating biomarkers including cell free nucleic acids and extracellular vesicles that facilitate the discovery of disease. We hypothesize that cancer-derived circulating biomarkers contain heterogeneous surface membrane proteins and/or nucleic acids representing the original tumor, requiring tools which can identify multiple biomarkers to be detected simultaneously. However, challenges including a lack of established isolation standards and determining sufficiently sensitive detection platforms remain for clinical implementation. Our goal is to develop a multiplexed biomarker based assay which can be leveraged to capture the molecular heterogeneity of distinct subpopulations of tumor derived circulating biomarkers.

Materials and Methods: Materials and methods vary greatly by project, each project design will be briefly summarized: 1) Single molecule imaging of ctDNA was conducted using PEG-coated surface whereby biotin streptavidin linked DNA reference strands were exposed to fluorophore conjugated mutant DNA strands for FRET imaging of mutant specific sequences. 2) Extracellular vesicle imaging and profiling was conducted using antibody based surface capture and high-resolution flow cytometry for high throughput exosome characterization. 3) RNA-biomarkers were identified by converting RNA into cDNA using target gene panels selected from data bases at each cycle during a PCR.

Results and Discussion: RNA profiling of initial sample cohort distinguished cancer from healthy, while detecting early cancer patients with high sensitivity. Differentiating genes with biological relevance were identified which can classify pancreatic cancer patients from healthy donor. cfDNA imaging method had nonspecific signal < 0.1-1%, wherein 1-10% mutant fraction can be genotyped. High resolution flow cytometry identified distinct subpopulations of plasma exosomes and revealed their molecular heterogeneity and cancer specific marker candidates can be screened against purified exosomes.

Conclusions: Extracellular vesicle imaging and profiling co-validated by high resolution flow cytometry, exemplifying the utility of a multi-platform detection scheme to characterize plasma-derived extracellular vesicle populations. Cell free RNA biomarkers of cancer by cell-free RNA sequencing were analyzed that can improve early cancer detection outcome, this proof of concept was done on pancreatic patient plasma and is being expanded into other cancer cohorts.

#2287

Extracellular vesicle particle concentration, size and associated DNA levels in plasma fluctuate with therapy in cancer patients.

Bella H. Nguyen,1 Katie Meehan,1 Carlos Salomon,2 Si Hong Lim,1 Connull Leslie,3 Anitha Thomas,3 Chady Sader,4 Peter Friedland,4 Andrew Lindsay,5 Camile Farah,6 Colin Tang,4 Michael Millward,7 Annette M. Lim4. 1 _University of Western Australia, Perth, Australia;_ 2 _University of Queensland, Brisbane, Australia;_ 3 _PathWest QEII Medical Centre, Perth, Australia;_ 4 _Sir Charles Gairdner Hospital, Perth, Australia;_ 5 _Hollywood Private Hospital, Perth, Australia;_ 6 _University of Western Australia Dental School, Perth, Australia;_ 7 _University of Western Australia School of Medicine, Perth, Australia_.

Introduction Extracellular vesicles (EVs) are actively secreted nanovesicles from all cell types including cancer and have the potential to be a novel biomarker. The aim of this study was to explore the use of tumour-derived EV particle concentration, EV particle size and EV-associated DNA as novel pan-cancer biomarkers in a series of patients with metastatic solid malignancies receiving therapy.

Method A minimum of two serial blood samples were collected prior to and during therapy at approximately 6-weekly intervals from patients with metastatic solid tumours. EV were isolated using differential ultracentrifugation. EV suspensions were examined using a NanoSight NS500 instrument (Malvern Panalytical), equipped with a 405 nm laser and a sCMOS camera. Sample analysis was performed at a camera level of 10, gain of 250, and with a detection threshold of 10 pixels, with videos recorded for 60 seconds in triplicates. Particle movement was analyzed using NanoSight NTA software v3.0. EV-associated DNA was extracted using Qiagen Microkit® and quantified using Qbit dsNDA HS Assay Kit (ThermoFisher Scientific). EV particle concentrations (nanoparticles/ul), EV-associated DNA concentrations (ng/ul), EV particle size (nm) were correlated with patients' clinical progress, using serial full-body computed tomography (CT) scans.

Result From a cohort of 16 patients, EV particle concentrations was assessed in a total of 45 serial blood samples (11 sets of three, 2 sets of two, 2 sets of four) collected from 10 patients. EV-associated DNA was isolated from a total of 65 serial blood samples (17 sets of three, 2 sets of four, and 3 sets of two) collected from all 16 patients. All patients had malignancies including heterogeneous head neck cancers, gastric and cutaneous cancer and received palliative radiotherapy, chemotherapy or immunotherapy. EV nanoparticle concentration levels paralleled imaging responses in 25 time points out of 45 collected, and EV-associated DNA levels paralleled in 59 time points out of 65 collected, such that the levels rose with radiographical progression and declined with stability or regression. However, there was no consistent correlation between treatment response and nanoparticle concentrations, or EV-derived DNA levels. There was also no consistent correlation between treatment response and nanoparticle size.

Conclusion Although EV particle concentrations, and EV-associated DNA concentrations were observed to vary with therapy, these were not consistent with CT scan assessments in a heterogeneous cohort of patients.

#2288

Cell-surface vimentin as a novel biomarker for detecting circulating tumor cells from advanced gastric cancer patients.

Heming Li, Yuting Liu, Zhi Wang, Shanshan Liang, Xiangyu Kong, Wenzhi Liu, Tong Zhao, Xuening Ji, Gang Wang, Fuguang Wang, Ruoyu Wang. _Zhongshan Affiliated Hospital of Dalian University, Dalian, China_.

Background: Aberrant activation of epithelial-mesenchymal transition (EMT) is closely associated with gastric cancer (GC) progression and metastasis. However, biomarkers specifically capturing EMT-circulating tumor cells (EMT-CTCs) that can be used to noninvasively determine the disease status and prognosis in GC patients have been lacking.

Patients and Methods: To fill this gap, we performed a head-to-head comparison clinical study between CSV+CTCs and EpCAM+CTCs using proprietary cell-surface vimentin (CSV) antibody 84-1 from MD Anderson and the epithelial tumor markers EpCAM and CK based on previous established CTCs detection method from 100 resectable and unresectable GC patients and 10 healthy donors. Also, freshly procured GC primary tissues and metastatic lymph nodes were obtained from five patients. Additionally, in vitro experiments were performed to investigate the role of CSV as a specific EMT marker in GC cell lines.

Results: Using 84-1 antibody, we detected CSV more exclusively expressing on lymph node metastasis than primary tumor of GC. Subsequent experiments in vitro showed that the expression level of CSV was correlated with EMT phenotype and migration ability in GC cells. A dramatic increase in the levels of vimentin bound to the surface of GC cells after IGF-I stimulation using flow cytometry which indicated CSV as a specific marker in GC cell populations undergoing EMT. We further enumerated relative higher number of CSV+CTCs in unresectable advanced GC patients and EpCAM+CTCs in resectable population with early stage; none was detected from healthy donors. From our observations, we defined a cutoff of < five or ≥ five CTCs as optimal threshold with respect to therapeutic response using ROC curves. CSV+CTC counts were significant in differentiating disease stable and progression populations in comparison to the EpCAM+CTC. Notably, patients with unfavorable CTCs at baseline had a significantly shorter median PFS and OS in advanced GC populations. These results also indicated that a summation of CTCs detected from both methods increased the sensitivity and specificity of CTCs in monitoring therapeutic response and prognosis prediction compared to individual test.

Conclusions: Our data demonstrate that CSV is a highly EMT-CTCs specific marker that may assist in assessing treatment efficacy and prognosis in advanced GC patients; however, an independent prospective study with a large population will be necessary to fully validate the efficiency of this method.

#2289

Prognostic impact of progastrin levels in blood compared to MSKCC based clinical prognosis in metastatic renal cell cancer patients.

Manish Kohli,1 Winston Tan,1 Lea Payen,2 Carole Langlois-Jacques,3 Pierre Liaud,4 Delphine Maucort-Boulch,3 Dominique Joubert,5 Alexandre Prieur6. 1 _Mayo Clinic, Rochester, MN;_ 2 _Laboratoire de Biochimie et Biologie Moleculaire; Institut de Cancérologie des Hospices Civils de Lyon (IC-HCL); CITOHL; Centre Hospitalier, Lyon, France;_ 3 _Hospices Civils de Lyon, Service de Biostatistique et Bioinformatique, Lyon, France;_ 4 _Eurobiodev, Montpellier, France;_ 5 _ECS-Progastrin, Lausanne, Switzerland;_ 6 _ECS-Progastin, Lausanne, Switzerland_.

Background and Aims: Progastrin is a tumor promoting peptide which is detectable in the blood of patients with different cancers (Prieur et al. AACR 2017, Prieur et al. ASCO 2017). Progastrin gene is a direct target of the WNT/ß-catenin oncogenic pathway involved in tumorigenesis of many organs, but it is unknown if it has any prognostic significance in metastatic renal cell cancer (mRCC) patients. We evaluated progastrin as a prognosis marker along with known markers of prognosis in mRCCC.

Methods: 145 patients with mRCCC were enrolled in this study and blood samples were drawn after consent. Progastrin was measured using the ELISA cancerREAD®. Progastrin concentrations in the 145 mRCCC patients (test set) was assessed against 213 samples from asymptomatic volunteers from the French blood establishment (control set). The prognostic impact of progastrin levels was determined with overall survival (OS) using Cox proportional hazards and also compared to MSKCC based clinical prognosis (good;intermediate; poor). Statistical significance was considered at P≤0.05.

Results: The median follow-up of the cohort was 5.45 years (IQR: 1.87-9.95) and at the time of analysis 98/145 patients had died from disease progression. Plasma progastrin was detected in 95% of the patients (cut-off value 1 pM, range 0 to 272 pM, median value of 7.2 pM; IQR 3.20-19.71) compared to the control set (median value=0.61 pM; IQR 0.00-1.58). The Receiver Operating Characteristic analysis indicated an area under the curve of 0.92 (p<0.0001; 95% CI 0.89 to 0.94). At the univariate level, MSKCC scores (good; intermediate; poor categories) in this cohort was associated with OS and was prognostic (p<0.0001). We detected progastrin levels were higher with MSKCC score poor prognostic (p<0.0002) (median 30.39 pM; IQR 9.31-57.20). Elevated progastrin was also independently associated with poor survival (p<0.0001) and a multivariate model of MSKCC taken with progastrin levels remained significantly associated with poor survival (p<0.0001).

Conclusion: Elevated progastrin levels in mRCCC is correlated with poor survival and further refines clinically used MSKCC prognostic scores. Progastrin assay is a simple and inexpensive blood test that might define subsets of mRCCC patients with poor survival who need to be identified for aggressive treatments.

#2290

Expression pattern of androgen receptor (AR), splice variant 7 (AR-V7) and splice variant 567 (AR-567) in circulating tumor cells and paired plasma-derived exosomes in metastatic castration resistant prostate cancer.

Areti D. Strati,1 Martha Zavridou,1 Evangelos Bournakis,2 Sophia Mastoraki,1 Evi S. Lianidou1. 1 _Univ. of Athens, Athens, Greece;_ 2 _Medical School, Athens, Greece_.

INTRODUCTION: Androgen-receptor splice variant 7 (AR-V7)is a highly promising liquid biopsy predictive biomarker indicating primary or acquired resistance to novel androgen receptor signaling inhibitors in metastatic castration resistant prostate cancer (mCRPC). We present for the first time the expression pattern of AR-FL, AR-V7, and AR-567es at a quantitative level in circulating tumor cells (CTCs) and paired plasma-derived exosomes in mCRPC.

METHODS: We first developed and analytically validated a novel multiplex RT-qPCR assay for AR full length (AR-FL), AR-V7, AR-567es and AR-total. We then quantified the expression levels of AR-splice variants, CK-19 (epithelial marker) and B2M (reference gene) in EpCAM+ CTCs and paired plasma-derived exosomes isolated from peripheral blood (20mL) of 62 mCRPC patients and 10 healthy donors.

RESULTS: In CTCs AR-FL was detected in 57/62(92%),AR-V7 in 32/62(52.0%),AR-567es in15/62(24.2%) and AR-total in 56/62(90.0%). In paired plasma-derived exosomes, AR-FL was detected in 45/62(72.5%), AR-V7 in 4/62(6.5%), AR-567 in 2/62(3.2%) and AR total in 47/62(75.8%).CK-19 expression was detected in 18/62(29.0%) of CTCs and in 28/62(45.2%) of exosomes. In all cases AR splice variants were expressed at higher levels in CTCs than in paired exosomes, while AR-V7 was detected at higher percentages than AR-567es.

CONCLUSIONS: Our results reveal for the first time a remarkable heterogeneity on the expression levels of AR-FL, AR-V7 and AR-567es in EpCAM+ CTCs and paired exosomes between individual mCRPC patients. The clinical significance of this finding will be further investigated in large patient cohorts in respect to therapy response.

#2291

Single cell capture and molecular analysis of live CTCs using integrated microwells and single cell aspirator.

Charlotte N. Stahlfeld,1 Jake J. Tokar,1 David Quigley,2 David Niles,1 Jamie M. Sperger,1 Felix Feng,2 Joshua M. Lang1. 1 _University of Wisconsin, Madison, WI;_ 2 _University of California San Francisco, San Francisco, CA_.

Resistance to targeted therapies can be caused by acquired genomic alterations in genes that code for the targeted protein. Circulating tumor cells (CTCs) provide an accessible source of tumor cells from diverse metastatic lesions from which to evaluate the frequency of genomic alterations that can be matched to distinct phenotypes, including therapeutic resistance. Successful analysis of the diversity of molecular mechanisms of therapeutic resistance requires isolation of live tumor cells for nucleic acid extraction. We have developed a semi-automated single cell aspirating platform to enrich and isolate live, rare cell populations for downstream molecular analysis. Using immortalized cell lines, we were able to enrich a target population of cells from a mixed population resulting a high purity sample (71.7-83.8%) to interrogate. Target cells were captured for nucleic acid extraction for downstream gene expression to validate enrichment. We demonstrate the ability to achieve high quality whole genome and transcriptome endpoints from low cell numbers in cell lines: the lower threshold for cellular input required to attain whole genome amplification (WGA) products reliable for downstream sequencing applications is now being validated. We also isolated CTCs from patient blood samples and showed phenotypic heterogeneity of EpCAM expression among individual CTCs. EpCAMhigh and EpCAMlow CTCs were detected, with a broad range of fluorescent intensities (141.2-5804.56 MFI). Using the phenotypic characterization of EpCAM expression, individual live CTCs were identified and captured for evaluation of their genomic heterogeneity. We have identified molecular heterogeneity in these cells that may reflect heterogeneity driving treatment resistance in CRPC. Early identification of treatment resistant clones may help develop intervention strategies that prolong the efficacy of molecular targeted therapies.

#2292

Bile cell-free DNA as a powerful liquid biopsy of the somatic variants in biliary tract cancer.

Ningjia Shen,1 Dadong Zhang,2 Lei Yin,1 Yinghe Qiu,1 Jian Liu,1 Wenlong Yu,1 Xiaohui Fu,1 Bin Zhu,1 Xiaoya Xu,2 Anqi Duan,1 Zishuo Chen,2 Xiang Wang,1 Xinkai Cao,2 Teng Zhao,1 Zisong Zhou,2 Lianghe Yu,1 Hao Qin,2 Zheng Fang,1 Jing-Yu Li,2 Yuanjin Liu,1 Lei Xiong,2 Bo Yuan,1 Fugen Li,2 Yongjie Zhang1. 1 _Shanghai Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China, Shanghai, China;_ 2 _3D Medicine Inc., Shanghai, China_.

Purpose: Biliary tract cancers (BTCs) are less common, but rapidly lethal tumors. Despite recent improvements in multimodal therapy, the 5-year survival rate is less than 10% in patients with advanced or metastatic BTC. BTC patients lack personalized treatment options, partly because tissue samplings are often inadequate for molecular characterization. Thus, this study examined the use of bile cell-free DNA (cfDNA), which is released by tumor and normal cells, as a potential liquid biopsy able to provide genomic information for the whole tumor.

Materials and Methods: Ten BTC patients, four with gallbladder carcinomas and six with cholangiocarcinomas, were enrolled in this study August 2017 to March 2018. To investigate the use of bile cfDNA as a liquid biopsy in BTC patients, targeted deep sequencing, with a panel of 150 tumor-related genes, was used to analyze paired bile cfDNA and tumor DNA for mutational variants individually and then compared. The sensitivites and specificities of detecting mutations including SNV/Indel and copy number variation (CNV) in bile cfDNA were analyzed.

Results: This study is the first to show that bile cfDNA is predominantly comprised of long DNA fragments, which is not the case in plasma cfDNA. Herein, paired bile cfDNA and tumors from ten BTC patients were examined using targeted deep sequencing. The mutational profiles of bile cfDNA showed that of the SNV/Indel mutations, the highest variation was seen in TP53, followed by KRAS, NOTCH1, NOTCH2 and KMT2A. Among the CNV mutations, low recurrent amplified genes, such as CCNE1, ERBB2, CDKN1B, ZNF217, and CDK6, were identified; with CDKN2A shown to have a deep deletion. Among these genes, NOTCH1, NOTCH2, ERBB2 and CDK6 are of interest as potential drug targets. When comparing bile cfDNA and tumor SNV/Indel detection results using targeted deep sequencing, a high sensitivity (94.7%) and specificity (99.9%) were obtained. Additionally, the sensitivity of detecting a copy number variation (CNV) was 75.0%, with a specificity of 98.9%. When comparing two bile extraction methods, including percutaneous transhepatic cholangial drainage and operation, no significant difference in SNV/Indel or CNV detection sensitivity was noted. Moreover, when examining the tumor stage and incidence site, AJCC stage II and the distal bile duct both had significantly decreased CNV detection sensitivities.

Conclusions: This study shows that long DNA fragments are prevalent in bile cfDNA samples and targeted deep sequencing can reliably detect mutational variants within bile cfDNA obtained from BTC patients. This is the first time that a mutational landscape, including SNVs, Indels and CNVs, has been obtained for bile cfDNA from BTC patients. These preliminary results maybe shed light on bile cfDNA as a promising liquid biopsy for BTC patients.

#2293

Is a single driver gene mutation sufficient for monitoring early response in advanced colorectal cancer.

Pashalina Kehagias,1 Caroline Vandeputte,1 Lieveke Ameye,1 Hakim El Housni,2 Amélie Deleporte,1 Karen Geboes,3 Thierry Delaunoit,4 Gauthier Demolin,5 Marc Peeters,6 Lionel D'Hondt,7 Jos Janssens,8 Javier Carrasco,9 Maria Gomez Galdon,1 Pierre Heimann,2 Marianne Paesmans,1 Patrick Flamen,1 Alain Hendlisz1. 1 _Institut Jules Bordet, Bruxelles, Belgium;_ 2 _Hôpital Erasme-ULB, Bruxelles, Belgium;_ 3 _Universitair Ziekenhuis Gent, Gent, Belgium;_ 4 _Hôpital de Jolimont, La Louvière, Belgium;_ 5 _Centre Hospitalier Chrétien St-Joseph, Liège, Belgium;_ 6 _Universitair Ziekenhuis Antwerpen, Antwerpen, Belgium;_ 7 _Centre Hospitalier Universitaire, UCL Namur, Namur, Belgium;_ 8 _AZ Turnhout, Turnhout, Belgium;_ 9 _Grand Hôpital de Charleroi, Charleroi, Belgium_.

Purpose: Circulating tumor DNA (ctDNA) monitoring based on an individual mutation profile during therapy is under intense investigation in modern oncology. We previously reported that the increase of ≥50% of at least one somatic mutation among multiple monitored mutations per patient is associated with a significantly worse outcome1. This study investigates whether the ctDNA monitoring of one driver gene mutation, provides enough information as compared to multiple mutations to assess response to regorafenib in advanced chemorefractory colorectal cancer (aCRC) at an early timepoint.

Experimental procedures: Archival tumor tissue and plasma samples (PL) at baseline (BL) and 14 days (D14) after treatment initiation in aCRC pts (n=141) were prospectively collected in the RegARd-C multicenter clinical trial (NCT01929616). Somatic mutations were identified based on a CRC-oriented targeted gene sequencing of tumor tissue. All available (median 2 (1-4)) driver gene mutations were monitored per patient in PL at BL and D14 via droplet digital PCR (Bio-Rad QX200 ddPCR system) to assess ctDNA dynamics.

Results: In 96 evaluable patients, the most frequently monitored mutated genes were APC (73%), TP53 (72%), KRAS (66%), and PI3KCA (23%). Among patients with ≥2 monitored mutations (73/96), one was selected at random and compared to previous methodology taking in account dynamics of all followed mutations. Optimal cutoff (CO) evaluation (Contal & O'Quigley method) separated patients (n=96) according to a ctDNA increase of ≥50% versus an increase of <50% or a decrease. The concordance of ctDNA dynamics based on one randomly selected mutation and multiple monitored mutations was 91%. Our data demonstrated that a ctDNA increase based on one single mutation taken at random is significantly associated with a worse clinical outcome in terms of progression-free survival (HR 2.42, 95% CI (1.56-3.74), P<0.001) and overall-survival (HR 2.17, 95% CI (1.41-3.34), P<0.001). In addition, when combining patients' ctDNA dynamics to BL ctDNA levels (≥ or < 5 ng/mL optimal CO) or BL cell-free DNA (cfDNA) levels (≥ or < 50 ng/mL optimal CO), we could distinguish 4 patients' subgroups with different prognosis. However, when performing a multivariate analysis including clinical parameters, BL ctDNA and BL cfDNA levels, BL ctDNA was not relevant in the presence of BL cfDNA.

Conclusion: The monitoring of ctDNA dynamics based on only one randomly selected driver gene mutation versus multiple is equally informative to describe adequately aCRC patients' outcome under regorafenib after 14 days of treatment onset. Especially, combined with pre-treatment ctDNA levels, this simplifies a personalized patient monitoring. 1 P. Kehagias, et al. Circulating tumor DNA detects early response to regorafenib in advanced colorectal cancer [abstract]. AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-221

#2294

Plasma progastrin level as a predictive and prognostic biomarker in advanced prostate cancer.

Manish Kohli,1 Winston Tan,1 Lea Payen,2 Carole Langlois-Jacques,3 Pierre Liaud,4 Delphine Maucort-Boulch,3 Dominique Joubert,5 Alexandre Prieur5. 1 _Mayo Clinic, Rochester, MN;_ 2 _Laboratoire de Biochimie et Biologie Moleculaire; Institut de Cancérologie des Hospices Civils de Lyon (IC-HCL); CITOHL; Centre Hospitalier, Lyon, France;_ 3 _Hospices Civils de Lyon, Service de Biostatistique et Bioinformatique, Lyon, France;_ 4 _Eurobiodev, Montpellier, France;_ 5 _ECS-Progastrin, Lausanne, Switzerland_.

Background and Aims: Progastrin is a tumor promoting peptide which is detectable in the blood of patients with different cancers. Progastrin gene is a direct target of the WNT/ß-catenin oncogenic pathway involved in tumorigenesis and possibly tumor progression/ treatment efficacy. Since WNT/ß-catenin oncogenic pathway is dysregulated in advanced prostate cancer we evaluated plasma progastrin in metastatic prostate cancer as a predictive and prognostic biomarker.

Methods: Metastatic hormone sensitive prostate cancer (mHSPC) and metastatic castration resistant prostate cancer (mCRPC) states were enrolled in a cohort study of blood sample collection and follow-up for outcomes between 9/2009 and 11/2013. Patients were enrolled in mHSPC unique sub-cohorts before initiating androgen deprivation therapy (ADT); during ADT; at the time of failure of ADT and before starting chemotherapy. Plasma progastrin was measured using the ELISA cancerREAD®. Progastrin concentrations in the cancer patients (test set) was assessed against 213 samples from asymptomatic volunteers from the French blood establishment (control set) and prograstin levels were also compared for each of the above four mHSPC and mCRPC cohorts as well as for association with time to failure on AA for the mHSPC cohort and overall survival for both mHSPC and mCRPC subcohorts. We also determined progastrin levels in patients with two serial samples to evaluate if changes were predictive for overall survival.

Results: Of the 523 mHSPC+mCRPC patients 96 were in mHSPC state enrolled before starting ADT; 101 mHSPC patients were enrolled while receiving ADT; 143 mHSPC patients were enrolled at the time of ADT failure and 143 were clinically progressive mCRPC. All cohorts were unique and 246/523 patients had two serial samples collected and analyzed across all subcohorts. The median time of follow up of the whole cohort was 8.34 years (IQR: 4.53-12.97) and 371/523 had died at the time of the analysis. Plasma progastrin levels was detected in 87.6% of all the patients (cut-off value 1 pM, range 0 to 311 pM, median value of 4.7 pM) compared to the control set (median value=0.61 pM; IQR 0.00-1.58). The Receiver Operating Characteristic analysis indicated an area under the curve of 0.84 (p<0.0001; 95% CI 0.81 to 0.87). Of these, 106 patients had a decrease and 140 patients an increase of progastrin levels. Patients with a serial increase of progastrin had a worst overall survival compare to the other group (p=0.019).

Conclusion: Progastrin is a blood based biomarker elevated in advanced prostate cancer patients serial increases in progastrin levels during treatment are predictive of poor survival. Progastrin assay might be useful for monitoring therapeutic interventions like androgen deprivation therapy effects as well for advanced prostate cancer patients.

#2295

Influence of endocrine therapy on the ratio of androgen receptor (AR) to estrogen receptor (ER) positive circulating epithelial tumor cells (CETCs) in breast cancer.

Monika Pizon, Dorothea Schott, Daniel Lux, Ulrich Pachmann, Katharina Pachmann. _Transfusion Center Bayreuth, Bayreuth, Germany_.

Background: The androgen receptor (AR) is expressed in the majority of breast cancers and across the main breast cancer subtypes. Despite the high frequency of AR expression in breast cancer its appraisal remains controversial because its role is complex, dependent on the hormonal milieu. The aim of the current study was to investigate the frequency of AR and ER positive CETCs in breast cancer patients.

Material and Methods: The number of vital CETCs was determined from blood of 66 patients suffering from breast cancer and the expression of AR and ER on these cells was investigated using the maintrac method.

Results: Numbers of CETCs/ml blood were significantly higher in patients with advanced disease as compared to patients with early stage disease. The fraction of AR positive CETCs was significantly higher than the fraction of ER positive CETCs (90% vs. 50%; p<0.001). Patients with positive lymph nodes had less AR positive CETCs as compared to patients with negative lymph node status. The AR:ER ratio was higher in patients receiving tamoxifen therapy as compared to patients without tamoxifen therapy whereas treatment with aromatase inhibitor had no influence on AR:ER ratio.

Conclusions: The ratio of AR to ER positive CETCs, obviously, is influenced by endocrine therapy, more specifically therapy with tamoxifen. Since AR expression seems to be responsible to resistance to endocrine therapy this may provide a new biomarker to select patients who might benefit from combination treatment of ER and AR inhibitors.

#2296

Validation of a novel one-step digital PCR platform with precision circulating cell-free DNA standards.

Megan E. Dueck,1 Robert Lin,1 Andrew Anfora,2 Andrew Zayac,1 Steve Gallagher,1 Omo Clement,2 Dana Ruminsky-Lowe,2 Paul Hung1. 1 _COMBiNATi, Burlingame, CA;_ 2 _SeraCare, Gaithersburg, MD_.

In this study we demonstrate precision quantification of Seraseq ctDNA EGFR T790M mutation mix at AF0.1% (mutant/wild-type ratio) using a novel one-step digital PCR (dPCR) platform. This novel yet simple workflow has the potential to make cancer liquid biopsy a clinical application. EGFR is an important drug target for the treatment of non-small cell lung carcinoma (NSCLC). During the treatment of NSCLC with tyrosine kinase inhibitors (TKIs), there is typically a significant response initially, followed by a secondary mutation as the carcinoma develops resistance. Early detection of cancer can better inform patient treatment and guide drug selection. One key EGFR mutation that leads to TKI resistance is the T790M mutation. Only a few clinical assays have been approved as companion diagnostics in patient biopsies (FFPE or plasma), while a larger number of laboratory developed assays (LDTs) under CLIA/CAP guidance are finding routine use in cancer disease diagnosis or treatment monitoring. Herein we describe the validation of a novel, fully integrated dPCR platform for the detection and absolute quantification of EGFR T790M. The integrated dPCR platform consists of a patented micro-molded plastic consumable and a fully-integrated instrument combining consumable sample loading, thermal cycling and 5-color fluorescence detection. The platform was designed to have a simplified, single-step workflow and provide results in less than one hour. In addition, developments in the micro-molded plastic consumable allow for near-zero dead volume. The novel integrated instrument is 100% dry and contamination-free, making it attractive for the transition to clinical applications. Seraseq ctDNA EGFR T790M mutation mix AF1% and AF0.1% reference standards in combination with a commercially available EGFR T790M dPCR assay were used to validate the novel integrated platform. In conclusion, we highlight the capability to precisely quantify samples as low as 0.1% T790M EGFR in a background of wild-type EGFR with high reproducibility and high accuracy.

#2297

Anti-labyrinthin monoclonal antibody reduces human adenocarcinoma circulating tumor cells in the blood of patient-derived xenograft models and inhibits the growth of adenocarcinoma cell cultures.

Natalia Saleev*,1 Netta R. Blondheim-Shraga*,1 Evgeny Solomonov,2 Shiran Shapira,3 Izhak Haviv,1 Solomon M. Stemmer,4 Hanoch Slor5. 1 _The Azrielyi Faculty of Medicine, Bar Ilan University, Safed, Israel;_ 2 _Ziv Medical Center and Azrieli Faculty of Medicine, Bar Ilan University, Safed, Israel;_ 3 _Integrated Cancer Prevention Center, Tel Aviv Sourasky Medical Center, affiliated to the Sackler Faculty of Medicine, Tel Aviv University, Tel-Aviv, Israel;_ 4 _Institute of Oncology, Davidoff Centre, Rabin Medical Centre, Petah Tikva, and Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel;_ 5 _Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel_.

Labyrinthin is a 255-amino acid protein antigen that is expressed on the cell-surface of most adenocarcinoma tumors and is not expressed on the surface of most normal cells, which makes it an attractive candidate for targeted therapy. Originally, labyrinthin had been deduced to be a product of a large deletion in the gene coding for junctate. We found that the junctate gene in labyrinthin-expressing adenocarcinoma cells does not have any deletion, and we propose that the labyrinthin protein is a splice variant of the junctate gene. This study aimed to determine the effects of an anti-labyrinthin monoclonal antibody, on adenocarcinoma cells in vitro and on patient-derived xenograft (PDX) models in vivo. Lung, breast and colorectal adenocarcinoma cell lines, as well as a primary-cell culture of a colorectal adenocarcinoma patient, were assessed for cell surface expression of labyrinthin. The cells were then incubated in the presence of the anti-labyrinthin antibody and growth rate was monitored by live imaging methods. Untreated primary-cell cultures of the colorectal adenocarcinoma patient were also grafted to immunodeficient mice to produce PDX tumors. When establishment of palpable tumors was observed, the mice were injected with the antibody. Peripheral blood was sampled from the mice and circulating tumor cells (CTCs) were quantified by flow cytometry. A significant reduction of approximately 60% was detected in the number of CTCs in the peripheral blood of the antibody-treated PDX mice compared to untreated control PDX mice. Additionally, a significant reduction was measured in the growth-rate of cultured primary cells and in lung, breast and colorectal adenocarcinoma cell lines in the presence of the antibody. We conclude that the anti-labyrinthin antibody has an inhibitory effect on the formation of adenocarcinoma CTCs in vivo as well as on the growth rate of cultured adenocarcinoma tumor cells.

#2298

Monitoring of circulating tumor DNA in non metastatic gastric cancer.

Luc Cabel,1 Charles Decraene,1 Ivan Bieche,2 Jean-Yves Pierga,1 Mostefa Bennamoun,3 David Fuks,3 Jean-Marc Ferraz,3 Marine Lefevre,3 Sylvain Baulande,2 Virginie Bernard,2 Pascale Mariani,2 Paul Cottu,2 Charlotte Proudhon,2 François-clément Bidard,1 Christophe Louvet3. 1 _Curie, St Cloud, France;_ 2 _Curie, Paris, France;_ 3 _Mutualiste Montsouris, Paris, France_.

Introduction Perioperative chemotherapy significantly improves the survival of patients with non-metastatic gastric adenocarcinoma (GA), yet some patients develop local and/or distant relapse during follow-up. The present study was designed to monitor circulating tumor DNA (ctDNA) levels during perioperative chemotherapy in patients with a non-metastatic GA.

Patients and Methods As part of a prospective study (NCT02220556), plasma samples were prospectively collected in patients undergoing perioperative chemotherapy for non-metastatic GA. Plasma was collected prior to the initiation of perioperative chemotherapy, before surgery and after surgery. In each patient, mutations retrieved by panel NGS on tumor sample were then tracked in circulating cell-free DNA from 4ml of plasma by droplet digital PCR (ddPCR, Biorad). Circulating tumor DNA detection was correlated with the prospectively collected patient characteristics and outcome.

Results Thirty-two patients with a diagnosis of non-metastatic GA were included in this study from 06.2014 to 10.2016. Tumor panel sequencing retrieved a mutation deemed to be trackable in plasma for 20 patients, of which 7 experienced a relapse during follow-up (median follow-up: 26.4 months). Among the 19 plasma samples available prior to perioperative chemotherapy, ctDNA was detectable in four patients (N=4/19, sensitivity: 21%; 95%CI=[2.7-39%]), with a median allelic frequency of 1.6% (range=0.8-2.3%). ctDNA detection prior to perioperative chemotherapy was not correlated with any patient characteristic and had no significant prognostic effect. Before surgery, no patient with available plasma sample (N=0/18) had detectable ctDNA levels. After surgery, one patient (N=1/13) had detectable ctDNA level with a low allelic frequency (0.7%); that patient experienced a very short-term distant relapse only 3 months after surgery. The other four patients who experienced a relapse with available plasma sample after surgery had no ctDNA detected after surgery.

Conclusion This study is the first to report the ctDNA detection rate before and during perioperative chemotherapyfor non-metastatic GA. Despite the use of large volume of plasma, the detection rate at baseline was lower than expected, knowing that the same technical approach (tumor NGS followed by customized ddPCR on plasma) yielded a 75% detection rate in non-metastatic triple negative breast cancer (Riva et al, Clin Chem 2017). Mechanisms underlying this low detection rate are still to be characterized. In regards to ctDNA kinetic during therapy, preoperative chemotherapy was associated with a marked decrease of ctDNA levels - which became undetectable in all patients. Our data showed a very short lead time between rising ctDNA detection after curative surgery and the occurrence of a metastatic relapse, suggesting that postoperative ctDNA detection can uncover growing metastases.

#2299

**Development of a novel next-generation sequencing (NGS)-based assay for measurable residual disease (MRD) in** FLT3 **-ITD acute myeloid leukemia (AML) and its potential clinical application in patients.**

Wenge Shi, Christian Laing, Wei Ding, Marc Mycoco, Jelveh Lameh, Reinhold Pollner. _Navigate BioPharma Services, Inc., a Novartis subsidiary, Carlsbad, CA_.

Introduction The presence of MRD in patients with AML who are in morphologic remission has been shown to be a powerful predictor of eventual relapse. FMS-like tyrosine kinase 3 (FLT3) internal tandem duplications (ITD) confer a negative prognostic impact by increasing risk of relapse. The ability to detect FLT3-ITD mutations in remission bone marrow specimens is hampered by the limited sensitivity at 1% of PCR-based assays. To address such limitations, we developed a novel NGS-based MRD assay for the detection of FLT3-ITD mutations.

Method Genomic DNA was isolated from bone marrow (BM) aspirates or peripheral blood (PB) samples. PCR was performed to amplify exons 13 to 15 of the FLT3 gene. Highly diverse NGS libraries were then generated and sequenced using Illumina's sequencer. Using a custom bioinformatics approach, unique FLT3-ITD mutations of varying lengths were identified and mutant allelic frequency calculated. The assay was validated using clinical samples and contrived samples. For accuracy assessment, 30 samples (including PB or BM remnant patient DNA and cell line DNA, above DNA diluted in normal DNA) were included. Data were compared with a Fragment Size Analysis by Capillary Electrophoresis assay. To assess precision, the assay was validated at multiple levels evaluating intra-assay, inter-assay, inter-operator, inter-instrument and inter-reagent lot precision. DNA samples from selected mutant cell lines representing different FLT3-ITD lengths were spiked into normal DNA to evaluate assay sensitivity and linearity.

Summary The ideal DNA input range was established as 300 ng to 500 ng. In all FLT3-ITD-positive cell line samples covering diverse FLT3-ITD lengths (6 bp to 156 bp), the FLT3-ITD MRD NGS assay showed 100% concordance with the reference assay. The assay is also capable of detecting ITDs as expected after the samples were diluted in normal DNA to mimic samples from patients in remission. All acceptance criteria for the different precision parameters were met. The lower limit of detection of 0.013% regardless of ITD length was established but the assay was capable of detecting FLT3-ITD mutations at a level as low as 0.003% without false-positive results. The assay was linear (R2 = 0.958) down to FLT3-ITD allele frequency levels of 0.035% or the lower limit of quantitation.

Conclusion Analytical validation results established the role of this NGS-based MRD assay for the clinical management of FLT3-ITD AML. The FLT3-ITD MRD NGS assay demonstrated high sensitivity and high specificity by detecting the unique length of each patient's mutation with no false-positive results in expected negative samples. This assay might be helpful in defining the depth of remission, identifying persistent disease, and helping to guide decision making in the use of FLT3 inhibitors as continuation therapy.

#2300

**Validation of highly sensitive ctDNA assays for ESR1 resistance mutations with the NGS TargetSelector** TM **enrichment technology.**

Shan-Fu Wu, Lyle J. Arnold, Jolly Shrivastava, Jason C. Poole. _Biocept, Inc., San Diego, CA_.

Background: Acquired resistance to anti-hormonal therapy is a common reason for treatment failure in advanced and metastatic breast cancer (mBRC). Mutations in the ESR1 gene, encoding the estrogen receptor (ER) protein, are often associated with this drug resistance. Accurate and timely ESR1 mutation detection may enable early identification of progression and prediction of treatment failure. In contrast to using difficult and painful tissue biopsies, "liquid biopsy" offers a non-invasive and systemic approach to identify solid tumor mutations in peripheral blood by assessing circulating tumor DNA (ctDNA). Biocept has developed the highly sensitive TargetSelectorTM NGS assay to detect low frequency mutant alleles in ctDNA using a patented blocker that suppresses excess WT alleles, while still allowing amplification of tumor derived mutant sequences. Here, we focus on a five-codon ESR1 mutation hotspot that imparts tumor cell resistance to anti-hormonal therapy in mBRC.

Methods: The TargetSelectorTM assay applies a specific blocker on a short stretch of target DNA (up to 13 bp). The ESR1 assay consists of two blockers in the region of codons 534-538 of the ESR1 gene. We validated the TargetSelectorTM assays by spiking low level g-block DNA fragments (IDT Inc.) carrying point mutations for each of the targeted ESR1 sites, into human placental DNA. Mutant sequences were enriched via PCR and mutations were subsequently confirmed on the Illumina MiSeq down to a limit of detection (LOD) of 0.01-0.05% minor allele frequency (MAF). For ctDNA testing, whole blood was collected in Biocept CEE-SureTM Blood Collection tubes, and total nucleic acid extraction from plasma was performed on the QIAsymphony.

Results: In total, we tested >600 samples for ESR1 TargetSelectorTM ctDNA assays, with ESR1 WT as the background reference. The assay shows >99% analytical sensitivity (at 0.05% MAF) and >97% analytical specificity. Based on practical and theoretical estimates and using ddPCR as an orthogonal verification method, the ESR1 assays demonstrate single mutant copy detection for most tested positions, and in all cases showed an LOD of at least 0.05% (5 mutant copies in a background of 10,000 WT copies). Samples tested from 20 healthy donors (40 tests in total) showed high clinical specificity, indicating the test is competent for use in a CLIA certified, CAP accredited laboratory setting.

Conclusions: Biocept's TargetSelectorTM ctDNA assays are validated both analytically and clinically, showing single mutant copy detection and LOD at 0.05% or better in a background of excess WT DNA. Implementation of the Illumina MiSeq platform into our TargetSelectorTM assays leads to higher sample throughput, greater sensitivity and a faster turnaround time than Sanger sequencing. These factors enable a sensitive and efficient test crucial for guiding treatment decisions and patient care.

#2301

Antiglycan antibodies as predictors of therapeutic outcome in cervical cancer patients.

SHARAD PUROHIT,1 Paul M. Tran,1 Lynn K. Tran,1 Xuezheng Song,2 Ferris G. Ferris,1 John J. Wallbillich,1 Boying Dun,1 Jin-Xiong She1. 1 _Augusta University, Augusta, GA;_ 2 _Emory University, Atlanta, GA_.

Cervical cancer is a significant cause of cancer related death among women, with the highest mortality rates in developing countries. We aim to identify serum biomarkers for predicting outcome of radio therapy alone or combination of radiotherapy and brachytherapy. We measured natural anti-glycan IgG antibodies in the serum of stage II (n=276) and III (n=292) cervical cancer patients treated with radiation alone or in combination with brachytherapy on a suspension glycan array containing 184 glycans. Overall and progression free survival was used to determine the treatment outcome by Kaplan-Meier and Cox proportional hazards ratio. Significant protective effects were observed for Blood Group H antigens (HR=0.642, p=0.0013), Lewis Antigens (0.72, 0.015; 0.654, 0.003), Galili antigens (0.671, p=0.004), and other glycans belonging to Isoglobo (0.671,0.004) and 3fucosyllactose series (0.687,0.004). The protective effects remained significant after adjusting for stage and treatment. Our data suggest that measurement of natural anti-glycan IgG antibodies in serum can predict outcome of therapy in cervical cancer patients.

## IMMUNOLOGY

### Adoptive Cell Therapy 2

#2302

Natural killer cell cancer therapy in mouse model.

Shih-Wen Huang, Yein-Gei Lai, Jen-Qi Wu, Yae-Huei Liou, Nan-Shih Liao. _Academia Sinica, Taipei, Taiwan_.

Natural killer (NK) cells exert anti-tumor function through direct killing of tumor cells and indirect modulation of the function of other immune cells, such as T cells, dendritic cells and macrophages. However, the efficacy of adoptive NK cell transfer in cancer treatment has been limited, and only found in certain myeloid leukemia under allogeneic setting. The functional heterogeneity of NK cells and the immunosuppressive tumor microenvironment likely contribute to the limited efficacy. We establish a method to condition ex vivo expanded murine NK cells with anti-tumor activity. The conditioned NK cells are EOMES+CD127-DX5-CD49a+and CD11b+CD27+. Encountering with solid tumor cell lines in vitro enhances the production of IFN-γand triggers killing of the tumor cells by conditioned NK cells. In a syngeneic orthotopic EO771 breast cancer model, adoptive transfer of conditioned NK cells into tumor-bearing mice slows tumor progression and alters the composition and function of intra-tumor immune cells. To better imitate clinical condition, we transfer conditioned NK cells into tumor-resected mice, and found the therapy improved survival rate. We are investigating the anti-tumor mechanism of the conditioned NK cells.

#2303

In vitro **evaluation of TCR efficacy and toxicity using 3D spheroid models.**

Maja Buerdek, Kathrin Mutze, Kai Pinkernell, Dolores J. Schendel. _Medigene Immunotherapies GmbH, Martinsried, Germany_.

Clinical studies using TCR-transgenic T cells for adoptive T cell therapy revealed the efficacy of this therapeutic approach but also uncovered possible toxic effects against healthy tissues, such as neuronal or cardiac cells, caused by on-target/off-tumor or off-target toxicity (Morgan et al., J Immunother. 2013; Linette et al., Blood 2013). Due to the lack of adequate in vivo models for prediction of TCR efficacy or potential TCR-mediated toxicity against healthy tissues, physiologically relevant in vitro models need to be developed. The usage of 3D spheroids as targets for TCR-transduced T cells represents a powerful strategy to test killing capacity or possible safety issues of TCRs, which could fill the gap between 2D co-cultures and animal models. Compared to 2D cell layers, cells within a 3D spheroid structure more closely resemble the physiological in vivo situation regarding cell-cell-interactions, proliferation rates, hypoxia, and gene expression. Here we describe the use of 3D spheroids generated from tumor cell lines for assessment of TCR efficacy or healthy primary cells/iPSC-derived cells for evaluation of potential TCR-derived toxicity. HLA-A2-positive cells served as target cells in functional co-culture assays with CD8+ T cells transduced with an HLA-A2-restricted TCR. 3D spheroids have been generated using ULA (ultra-low attachment) plates or via the hanging-drop culture method. Spheroids were co-cultured with TCR-transduced T cells and killing of spheroids was analyzed in real-time using the IncuCyte live cell imaging system. For better visualization of spheroid killing, tumor cell lines were transduced using a lentivirus encoding a nuclear-restricted red fluorescent protein or by adding Annexin V red fluorescent reagent for detection of apoptotic cells. Thereby, spheroid killing could be quantified via decrease or increase in red fluorescence over time. Killing of 3D spheroids derived from healthy cells was analyzed via phase contrast and evaluation of spheroid morphology. TCR-transduced T cells showed efficient killing of tumor cell spheroids, confirming the results generated in a 2D system. Furthermore, 3D spheroids generated from healthy cells (e.g. normal human lung fibroblasts) or iPSC-derived cells (e.g. cardiomyocytes or neurons) were not killed via TCR-transduced T cells, whereas exogenous loading of the spheroids with the specific peptide led to efficient killing, showing the general susceptibility to TCR-mediated killing of healthy cell 3D spheroids.In summary, we showed that functional in vitro assays using 3D spheroid structures using tumor cell lines or healthy cells represent an elegant approach to assess both efficacy and potential toxicity of a given TCR in vitro. The analyzed TCR showed potent efficacy as well as a favorable safety pattern in 3D in vitro cultures.

#2304

Expanded and engineered NK cells upregulate expression of activation and survival genes associated with increased cytotoxicity and persistence.

Mira Tohme, Sasha Lazetic, Kate Jamboretz, Luxuan Buren, Chao Guo, James Trager. _Nkarta Therapeutics, South San Francisco, CA_.

Background: Natural Killer (NK) cells expanded using irradiated K562 cells engineered to express membrane-bound IL-15 and 4-1BBL (K562-mb15-4-1BBL) have been employed in the clinic [1]. Expansion upregulated activating NK cell receptors and increased NK cytotoxicity against tumor cells. Expanded NK cells can be engineered to express chimeric receptors to further improve their cytotoxicity. Chimeric receptors commonly incorporate an antigen binding domain, a costimulatory domain (i.e. 41BB, CD28) and a CD3ζ signaling domain. Receptor structure can bias engineered cells to chronic activation and exhaustion [2,3], and can regulate metabolic pathways that influence cell phenotype and function. Characterization of NK cells expressing activating NK receptor (aNKr)-expressing NK cells may enable assessment of their clinical potency. We therefore evaluated the phenotypic characteristics of expanded and engineered NK cells and investigated signatures of activation or exhaustion upon antigen stimulation.

Material and Methods: NK cells from peripheral blood mononuclear cells were expanded on irradiated K562-mbIL15-41BBL and transduced with NKG2D based chimeras (NKG2D.aNKr) and mbIL15. The expression of relevant receptors on expanded and transduced NK cells was assessed by flow cytometry. To evaluate the impact of tonic aNKr signaling versus antigen-driven stimulation on NK activation, we analyzed gene expression after stimulation with either isotype or anti-NKG2D coated beads using a Nanostring immune function panel.

Results: Ex vivo expansion generates potent, activated NK cells with high expression of NKG2D, Nkp30, Nkp44, Nkp46, CD69, CD25, and DNAM-1 activation markers and decreased expression of KLRG1 and CD158b inhibitory receptors. Expanded NK cells also upregulated TIGIT and TIM-3 expression, markers associated with functional exhaustion. However, expanded NK cells retained enhanced tumor cell cytotoxicity and cytokine production.

NKG2D.aNKr construct expression induced tonic signaling in engineered NK cells characterized by the upregulation of genes involved in cell survival (Bcl-2), cell activation (CD25) and cell mediated cytotoxicity (TRAIL).

NKG2D engagement further enhanced expression of anti-apoptotic genes, and genes encoding cytokines and chemokines required to control cell cytotoxicity (INF-γ, TNF-α) and recruitment to tumor sites (XCL1, CCL3, CCL4). Lastly, NKG2D stimulation of engineered cells led to a significant increase in the expression of TNF family members (41BB, Ox40L, Ox40, TRAIL). Increased 41BB, Ox40L and TRAIL expression was confirmed by flow cytometry and correlated with enhanced

cytotoxicity. Our data suggest that, upon stimulation, NKG2D.aNKr cells are highly activated with greater longevity. Moreover, our data allow the identification of biomarker candidates that reflect NK.aNKr potency.

#2305

Tumor MUC1 glycoprotein-highly specific CAR T cells control triple-negative breast cancer.

Ru Zhou,1 Mahboubeh Yazdanifar,1 Lopamudra Das Roy,1 John Maher,2 Pinku Mukherjee1. 1 _Univ. of North Carolina at Charlotte, Charlotte, NC;_ 2 _King's College London, London, United Kingdom_.

Antibody-derived Chimeric Antigen Receptor (CAR) T cells have great success in reduction of liquid form of tumor, but not solid tumor. Our novel antibody TAB004 can specifically recognize the tumor form of MUC1 (tMUC1) while sparing the normal MUC1 in several subtypes of breast cancers including >95% of triple negative breast cancer (TNBC). In vivo delivery of TAB004 specifically accumulates in the xenograft TNBC tumor only. Therefore, we hypothesize that TNBC can be specifically targeted with TAB004-derived tMUC1-specific CAR T cells. A panel of 45 human breast cancer cell lines were tested for tMUC1 level, and 11 out of 13 TNBC cell lines showed higher frequency of tMUC1 expression compared to that on normal cells. We engineered a 2nd generation human CAR using TAB004 that was coupled to the CD28-CD3zeta, named as MUC28z CAR. MUC28z was well expressed on activated human T cells. Compared to their mock control, MUC28z CAR T cells retained higher CD25 and CD11c, particularly on CD8 T cells. The susceptibility of TNBC cells to MUC28z CAR T cell cytolysis was dramatic while still corresponding to their tMUC1 level. Only the tMUC1-specific MUC28z CAR T cells expressed and released large amount of IFN-gamma after antigen recognition on TNBC cells. However, IFN-gamma was only involved in the tumor killing by MUC28z CAR T cells at lower E:T ratio. The in vivo treatment of TNBC tumor by MUC28z CAR T cells dramatically controlled HCC70 TNBC tumor growth in a xenograft model. Taken together, our TAB004-derived MUC28z CAR T cells are very potent at killing TNBC tumor cells both in vitro and in vivo, which is very promising to provide a safe, effective and tumor antigen-specific novel immunotherapeutic intervention.

#2306

CAR-T cell therapies targeting aspartyl β-hydroxylase (ASPH).

Thomas Thisted, Kanam Malhotra, Michael S. Lebowitz, Hossein Ghanbari. _Sensei Biotherapeutics, Gaithersburg, MD_.

Aspartate β-hydroxylase (ASPH) is a unique cancer target that is expressed exclusively on the surface of cancer cells. It is a type II transmembrane protein predominantly expressed during embryogenesis where it promotes cell migration for organ development, but has very low expression in healthy adult tissue, and is localized to the intracellular compartment of the endoplasmic reticulum. However, re-expression and translocation to the tumor cell surface has been detected in more than 20 different types of cancers, with expression levels inversely correlated with disease prognosis. Promising phase I clinical trial results have been achieved with our ASPH-targeted vaccine, SNS-301, in the treatment of biochemical recurrence of prostate cancer (reported previously: Nordquist, et al. (2018) J Clin Oncol. 36, suppl; abstr e15166; Nordquist, et al. Annals of Oncology, 29, suppl_8, 1 October 2018, mdy279.404). This study demonstrated reductions in disease-specific biomarkers (i.e. PSA) which correlated to the breaking of immune self-tolerance as indicated by stimulation of both cellular and humoral ASPH-specific immune responses. Importantly the vaccine was well tolerated with minimal numbers of adverse events or off-target effects. Thus, ASPH represents a promising target for immunotherapeutic approaches to human cancers. Here we present studies that we have initiated towards the development of ASPH-targeted CAR (chimeric antigen receptor)-T cell therapies. A series of ASPH-targeting chimeric antigen receptor constructs have been produced which combine various ASPH-specific scFv domains and either the 4-1BB or CD28 co-stimulatory domains. The ASPH-specific scFv explored include several scFv that we previously isolated from a fully human non-immune scFv library displayed on the surface of yeast and targeted at different epitopes within the extracellular domain of ASPH as well as several affinity matured variations of these scFv (Yeung, et al. (2007) Human Antibodies 16, 163-76). All constructs were incorporated into a lentiviral vector which was used to transfect human CD4+ and CD8+ T-cells. CAR expression and affinities were determined by flow cytometry using a fluorescently labeled antigen. Cell killing efficacy and cytokine responses of the resulting CAR-T cells were observed in co-culture experiments with a series of ASPH-expressing human cancer cell lines allowing for the ranking of the CAR constructs in terms of overall extent of immune stimulation ex vivo. It is our expectation that the unique features of the ASPH tumor specific antigen will help overcome one of the major hurdles in T-cell therapy: the identification of antigens that permit effective targeting of tumors in the absence of non-tolerable and/or off-target toxicities to essential tissues and organs. Experiments to further characterize ASPH-targeted CAR-T cells are ongoing with the goal of moving these promising therapeutics into clinic.

#2307

Preclinical evaluation of TC-210, a mesothelin-specific T cell receptor (TCR) fusion construct (TRuC™) T cells for the treatment of solid tumors.

Jian Ding,1 Holly Horton,1 Seema Shah,1 Adam Zieba,1 Janani Krishnamurphy,1 Thomas Ashhurst,2 Ashley V. Menk,3 Patrick Baeuerle,1 Nicholas J. King,2 Gregory Delogoffe,3 Robert Hofmeister,1 Daniel R. Getts1. 1 _TCR2 Therapeutics, Cambridge, MA;_ 2 _University of Sydney, Sydney, Australia;_ 3 _University of Pittsburgh, Pittsburgh, PA_.

Despite success in treating hematological malignancies, T cells expressing chimeric antigen receptors (CARs) have shown poor efficacy in solid tumor indications. The failure to initiate and elicit a complete TCR response is arguably a primary underlying factor preventing CAR-T cell success in solid tumor indications. Here, we present a novel T cell engineering platform: T Cell Receptor Fusion Constructs (TRuC™s), which target tumors independent of MHC. Unlike CARs, the constructs integrate into the TCR complex, harnessing the full potential of natural T cell activation, effector function and regulation. Here we describe preclinical evidence underscoring the efficacy of TRuC™-T cells re-programmed to target the solid tumor antigen mesothelin (TC-210).

TC-210 has shown robust anti-tumor activity in cellular assays and animal models of lung, ovarian and MPM cancers. In these studies, TC-210 was compared head-to-head against mesothelin-targeting CAR-T cells (MSLN CAR-T cells) bearing the same mesothelin binder expressed on TC-210. Mesothelin-dependent T cell activation, expansion and tumor clearance by TC-210 was faster than that observed with MSLN CAR-T cells. In a study using Renilla-luciferase-labelled MSLN-CAR T cells and TC-210, TC-210 migrated and accumulated at a faster rate in mesothelioma tumors than MSLN-CAR T cells. This migration pattern correlated with an increased level of chemokine receptors and number of TC-210 expressing these receptors, including CXCR3. TC-210 T cells also showed long-term functional persistence capable of preventing relapse in a mesothelioma model. Metabolic profiling of TC-210 versus MSLN-CAR T cells showed that the observed persistence may be related to the metabolic profile of TC-210 T cells. Unlike MSLN-CAR-T cells, TC-210 showed increased levels of oxidative phosphorylation and mitochondrial reserve, attributes associated with long-term memory T cells. Finally, systemic cytokine levels in animals treated with TC-210 were lower than those observed in MSLN CAR-T cell treated animals.

Together, these findings warrant the investigation of TC-210 in clinical trials as effective treatment for mesothelin-expressing tumors with potentially lower rates of adverse events.

#2308

The secondary immune selection is the dominant mechanism for acquired resistance against adoptive cell therapy.

Takahiro Kamada, Yosuke Togashi, Yoshihiro Ohue, Hiroyoshi Nishikawa. _National Cancer Center, Kashiwa, Chiba, Japan_.

Background: Adoptive cell therapy (ACT) has shown a promising result in the treatment of various types of cancers. However, as treated patients sometimes experience recurrences, understanding the mechanism(s) of the recurrences is important to maximize the clinical effect of ACT. Although tumor antigen loss has been reported as a major cause of acquired resistance in ACT, how and when these antigen loss variants are developed remains unclear. Objective:To clarify the mechanism(s) developing tumor antigen loss variants by ACT.

Results: BALB/c mice were inoculated subcutaneously with CMS5a cells (CMS5aP) harboring a neoantigen derived from mutated ERK2 (mERK2). Splenic CD8+ T cells from DUC18 mice (TCR specific for mERK2 transgenic mice) were transferred intravenously into CMS5a tumor-bearing mice. Although all tumors were totally eradicated by ACT, tumors were recurred in 4/12 treated mice. Cell lines were established from recurrent tumors (CMS5aR) and were subjected to further analyses. Whole exome sequence revealed a loss of mERK2 genes due to the deletion of chromosome 16 which contained mERK2 gene in resistant CMS5aR cells, but not in CMS5aP cells. The deletion of chromosome 16 was also confirmed with spectral karyotyping. We therefore addressed whether CMS5aR which lost chromosome 16 were newly developed by ACT or expanded from minor population which had exited at the initiation of ACT. Single cell clones (CMS5aS) were generated from the CMS5aP cells and confirmed to harbor mERK2. Mice were inoculated with these single clones and treated with ACT. Tumors were completely eradicated and no relapses were observed. When ACT was delayed, the frequency of tumor recurrence was increased, indicating that a resistant clone is newly developed in the course of tumor progression and selected during ACT. Then, the sensitive CMS5aS cells and resistant CMS5aR cells were labeled with DsRed-Express and GFP, respectively. Resistant CMS5aR cells were mixed in various proportions with sensitive CMS5aS cells, and the mixture was inoculated into BALB/c mice and treated with ACT. Tumor relapses were observed even in the mixture of resistant CMS5aR cells with the sensitive CMS5aS cells in 1/10000. These relapsed tumors were composed of only resistant CMS5aR tumors labeled with GFP. Based on the experimental data, a mathematical model for the prediction of recurrence was established using tumor-cell growth rates and tumor sizes, which completely followed the outcome of tumors in vivo model.

Conclusion: We propose that the acquired resistance clone against ACT is secondarily induced in the course of tumor progression and selected during the ACT.

#2309

Chimeric antigen receptor T-cell therapy targeting glypican-1 in pancreatic cancer.

Nan Li,1 Dan Li,1 Hua Ren,2 Madeline Torres,1 Mitchell Ho1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _East China Normal University, Shanghai, China_.

Pancreatic cancer is the fourth most common cause of death from cancer in the US, with a 5-year overall survival of 8.5%. While immunotherapy with chimeric antigen receptor (CAR) T cells has shown promise in certain hematological malignancies, their efficacy for solid tumors including pancreatic cancer remains elusive. Thus, laboratory research is urgently needed to identify and validate a new target of CAR T-cell therapy for patients with pancreatic cancer. In this study, we made CAR T cells that recognize glypican-1 (GPC1), a cell surface heparan sulfate proteoglycan protein that is overexpressed in pancreatic cancer. We isolated two GPC1-specific antibodies; the mouse monoclonal antibody HM2 that binds the C-lobe of GPC1 close to the cell surface, and the camel single domain antibody D4 that recognizes a conformational epitope in the protein core of GPC1. The transduction efficiencies of CAR (HM2) and CAR (D4) in primary T cells were 86% and 58%, respectively. GPC1-overexpressing KLM1 cells (2B9) and GPC1-overexpressing A431 cells (H8) were efficiently lysed by both CAR T cells in dose-dependent manners. By contrast, minimal cell lysis was observed in GPC1-negative cell line A431. In the presence of GPC1-positive tumor cells, greater amount of TNF-α, IFN-γ and IL-2 were produced by GPC1-specific CAR T cells than mock-transduced T cells. Although both CAR T cells showed similar cytolytic activity in vitro, CAR (D4) T cells induced 2-4 times more cytokines than CAR (HM2) T cells when co-cultured with GPC1-postive tumor cells. Notably, both GPC1-targeted CAR T cells demonstrated potent antitumor efficacy in a peritoneal dissemination xenograft mouse model. Our findings indicate that GPC1-targeted CAR T cell therapy could be a new approach for treating pancreatic cancer.

#2310

Functional demonstration of CD19 chimeric antigen receptor (CAR) engineered Epstein-Barr virus (EBV) specific T cells: An off-the-shelf, allogeneic CAR T-cell immunotherapy platform.

Rhine R. Shen, Christina D. Pham, Michelle Min Wu, Daniel J. Munson, Blake T. Aftab. _Atara Biotherapeutics, Inc., Thousand Oaks, CA_.

Chimeric antigen receptor (CAR) T cells have transformed the treatment of advanced B cell malignancies, but broad application has been limited due to technical and operational challenges in the autologous approach. Virus-specific T cells maintain native T cell receptors and have an established clinical profile and potential utility as an off-the-shelf, allogeneic CAR T-cell immunotherapy platform.

Tabelecleucel (tab-cel®) is an investigational off-the-shelf, allogeneic T-cell immunotherapy that utilizes endogenous T cell receptors targeting EBV antigens associated with select lymphomas and solid tumors. Tab-cel® functions through endogenous TCRs restricted to EBV antigens and has been shown to be generally well tolerated with low incidence of GvHD and cytokine release syndrome, as well as efficacy in EBV+ post-transplant lymphoproliferative disorders (PTLD). Introduction of CAR transgenes into EBV-specific T cells provides an appealing approach for developing off-the-shelf, allogeneic CAR T immunotherapies.

To evaluate feasibility for this platform, we engineered EBV-specific T cells to express second-generation CD19 CARs, utilizing CD28 or 4-1BB co-stimulatory domains. Resulting allo-EBV.CD19.CAR T cells exhibit high expression of both the CD19 CAR and EBV TCR. Additionally, allo-EBV.CD19.CAR T cells demonstrate an enriched central memory phenotype with higher frequency expression of CD62L, CCR7, and CD45RO. Allo-EBV.CD19.CAR T cells also show increased frequency of activation markers CD25 and 4-1BB, and produce activation-associated levels of IFN-γ comparable to EBV-specific T cells. Allo-EBV.CD19.CAR T cells exert potent and specific cytotoxicity against CD19-positive Nalm6 and Raji cells but have limited activity against CD19-negative K562 cells. Both CD19.CAR T and allo-EBV.CD19.CAR T-mediated cytotoxicity displayed rapid and comparable kinetics. Stimulation of allo-EBV.CD19.CAR T cells with either EBV- or CD19-positive targets also induced robust proliferation over several days of antigen challenge. Like unmodified EBV-specific T cells, allo-EBV.CD19.CAR T cells retain the ability to kill B-lymphoblastoid cell lines (BLCL) but spare autologous and allogeneic PHA-blast targets lacking CD19 and EBV antigen expression. This specificity in cytotoxicity is further supported by inflammatory cytokine profiles of effector-target cultures.

From these results, allo-EBV.CD19.CAR T cells demonstrate efficient and effective lysis of antigen-positive target cells, while maintaining low allo-cytotoxic potential. These findings establish feasibility for engineering EBV-specific T cells by leveraging next-generation CAR technologies, and support further development as an off-the-shelf, allogeneic CAR-T immunotherapy platform to generate IND clinical candidates.

#2311

Analysis of glypican 3-targeted chimeric antigen receptor T cells in hepatocellular carcinoma cell and mouse models.

Dan Li,1 Nan Li,1 Yifan Zhang,1 Haiying Fu,1 Zhijian Duan,1 Alissa Hummer,1 Hongjia Yang,1 Madeline B. Torres,1 Xiaoling Luo,1 Ling Su,2 Hu Zhu,3 Josh Kramer,4 Jinqiu Chen,1 Xiaolin Wu,2 Matthew Hall,3 Qun Wang,5 Stephen Hewitt,1 Tim Greten,1 Mitchell Ho1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _National Cancer Institute, Frederick, MD;_ 3 _National Center for Advancing Translational Sciences, Rockville, MD;_ 4 _Leidos Biomedical Research, Bethesda, MD;_ 5 _East China Normal University, Shanghai, China_.

Background: Glypican-3 (GPC3) is a cell surface protein that is highly expressed in hepatocellular carcinoma (HCC).

Methods and Results: In this report, we analyzed two chimeric antigen receptors (CARs) targeting GPC3, CAR (HN3) recognizes the N-lobe of GPC3 while CAR (hYP7) targets the C-lobe that is close to the cell surface. In a real-time cell cytolytic assay, we found that CAR (hYP7) T cells showed higher cytolytic activity than CAR(HN3) T cells at an E:T ratio of 2:1 when co-cultured with HepG2 cells for up to 140 hours. The Luminex analysis showed that CAR (hYP7) T cells produced significantly more Th1/Th2 cytokines and chemokines than CAR (HN3) T cells following exposure to Hep3B and HepG2 cells. In the Hep3B peritoneal dissemination xenograft mouse model, a single intraperitoneal infusion of CAR (hYP7) cells exhibited sustainable antitumor efficacy, and all of the mice survived without recurrence 10 weeks post treatment. Strikingly, we demonstrated that single injection of CAR (hYP7) T cells derived from either healthy donor or HCC patient significantly regressed tumor growth in the orthotopic HCC mouse model. Using a highly sensitive droplet digital PCR (ddPCR), we detected high frequencies of CAR T cells in the spleen and tumor microenvironment of mice in the CAR (hYP7) group. By contrast, CD19 CAR T cells showed no sign of gene integration in either tissue. More importantly, we found that CD8+ CAR (hYP7) T cells recovered from mouse spleens exhibited much higher lytic activity against HCC cells than CD4+ CAR (hYP7) T cells. Finally, we found that the GPC3-targeted CAR T cells downregulated the Wnt signaling in HCC cells.

Conclusion: The hYP7 antibody-derived GPC3-specific CAR is a promising therapeutic that could be tested for the treatment of advanced stage liver cancer.

#2312

**Anti-CS1 x anti-CD3 bispecific antibody (BiAb)-armed anti-CD3 activated T cells (CS1-BATs) specifically kill CS1** + **myeloma cells and release type-1 cytokines.**

Manley Huang, Archana Thakur, Lawrence G. Lum. _University of Virginia, Charlottesville, VA_.

Although advances in multiple myeloma (MM) therapy such as stem cell transplantation and newly approved biological agents have shown promising results, MM remains a terminal disease. Thus, new strategies to eliminate or control MM cells are needed. Elotuzumab, a humanized monoclonal antibody approved for treatment of MM, targets myeloma cells expressing CS1/SLAMF7. We heteroconjugated anti-CD3 (OKT3) and elotuzumab to develop a BiAb, CS1Bi, to target activated T cells (ATC) to CS1+ MM cells. To examine the potential therapeutic activity of BATs against MM, we tested the ability of ATC armed with CS1Bi to bind to MM cell lines, specifically kill MM cells lines expressing different levels of CS1, and produce Th1 cytokines upon engaging MM tumor targets. Initial studies showed that CS1-BATs bound to MM cells by flow cytometry and specifically killed CS1+ targets as measured in a 6 hour 51Cr-release assay. Cytokine secretion by CS1-BATs vs. unarmed ATC (n=2) of interferon-γ (IFN- γ: 512 pg/mL vs. 260pg/mL), tumor necrosis factor-α (TNF- α: 450pg/mL vs. 110pg/mL), granulocyte-macrophage colony-stimulating factor (GM-CSF: 217 pg/mL vs. 135 pg/mL), macrophage inflammatory protein-1α (MIP-1α: 8250 pg/mL vs. 400 pg/mL), macrophage inflammatory protein-1β (MIP-1β: 14750 pg/mL vs. 600 pg/mL), and RANTES (3550 pg/mL vs. 2600 pg/mL) were induced when CS1-BATs were co-cultured with CS1+ cell line, L-363 for 24h at E:T of 25:1. A more sensitive quantitative flow cytometry-based cytotoxicity assay was developed based on comparing of the number of eFluor 450-labeled target cells pre- and post-incubation with either CS1Bi-BATs or unarmed ATC prepared from healthy donors, or culture media alone. The number of eFluor450+/7AAD- viable cells/unit volume post-incubation were compared to the number of viable cells in untreated wells to calculate the percent cytotoxicity. This assay reproducibly demonstrated significant killing of five MM cell lines by CS1-BATS (armed with 50ng CS1Bi/106 ATC at an E:T ratio of 1:1) ranging up to 60%, depending on the target line, (n=8). Between 78- 95% of MM.1S cells were eliminated at an E:T ratio of 3:1 (n=2). These data demonstrate the clinical relevance of this anti-MM strategy for use in a Ph1/II clinical trial in patients with relapsed and/or refractory MM.

#2313

Enhanced activity of second-generation MAGE-A4 SPEAR T-cells through co-expression of a CD8α homodimer.

Victora Anderson, Anika M. Weber, Guy E. Wiedermann, Anette Pachnio, Sumaya Dauleh, Tina Ahmed, Roslin Y. Docta, Adriano Quattrini, George Pope, Laura Quinn, Thomas M. Ashton, Helen M. Tunbridge, Joseph P. Sanderson, Andrew B. Gerry. _Adaptimmune, United Kingdom_.

Affinity enhanced T-cell receptors (TCRs) have shown promise in the clinic. Second-generation strategies that enhance T-cell function alongside the TCR may improve the depth and durability of anti-tumor responses. In this second-generation TCR study, we added a CD8α homodimer to our MAGE-A4c1032TCR, a first-generation TCR currently being tested in a clinical trial (NCT03132922). Transduction of HLA class I-restricted, specific peptide enhanced affinity receptor (SPEAR) TCRs into peripheral blood lymphocytes creates both cytotoxic (CD8+) and helper (CD4+) T-cells of the same specificity; however, the lack of CD8 co-receptors on CD4+ T-cells may affect binding avidity of the engineered TCR. The addition of CD8α co-receptor into CD4+ T-cells alongside the engineered TCR (CD8α_MAGE-A4c1032) is anticipated to increase TCR binding avidity and enhance the polyfunctional response of CD4+ T-cells against tumor antigens, thereby widening the immune response to the tumor through dendritic cell (DC) activation and enhanced cytotoxicity. The effect of co-expressing the CD8α co-receptor on the MAGE-A4c1032TCR was assessed by in vitro assays addressing proof of concept for increased potency, focusing on CD4+ function, in parallel with assessment of potential safety issues. In assays involving antigen-positive tumor cell lines co-cultured with T-cells, we demonstrated improved T-cell engagement, as measured by increased CD40L on the T-cell surface in response to antigen. There were modest improvements in T-cell proliferation and cytokine production in response to tumor cells, particularly when isolated CD4+ cells were analyzed. However, when immature DCs were added to the co-culture, a more marked improvement with the second-generation T-cells was seen. DCs and T-cells in conditions containing CD8α_MAGE-A4c1032T-cells produced higher levels of cytokines and chemokines (e.g. IL-12, MIG for DCs, IFNγ, IL-2 for T-cells) than in conditions that contained MAGE-A4c1032T-cells without the CD8α co-receptor. Flow cytometry analyses illustrated T-cell-driven maturation of the DCs during the course of the co-culture. We also saw that second-generation CD4+ T-cells expressing the CD8α homodimer were able to kill antigen-expressing 3D tumor line microspheres, an additional benefit to the improvement of CD4+ helper functions. No changes in TCR specificity, sensitivity, or aberrant cytokine release arose from co-expressing the CD8α co-receptor in T-cells transduced with the MAGE-A4c1032TCR, suggesting no change to the existing safety profile. These data illustrate improved engagement and function in the CD4+ T-cells transduced with CD8α_MAGE-A4c1032, without additional off-target reactivity. The second generation CD8α_MAGE-A4 SPEAR T-cells are expected to improve long term T-cell functions as well as immediate anti-tumor activity in vivo.

#2314

Pharmacology studies of chimeric antigen receptor (CAR) T cells in luminescent xenograft and 3D in vitro tumor models.

Ruyi Li, Shanshan Gan, Leixin Zhang, Thomas Broudy, Yong Hu. _BioDuro LLC, San Diego, CA_.

Chimeric antigen receptor (CAR) T cell therapy is a type of immunotherapy that uses a patient's T cells, engineered with chimeric antigen receptors, to attack the patient's cancer cells. The U.S. Food and Drug Administration (FDA) has recently approved two CAR T therapies: Yescarta® to treat adults with lymphoma and Kymriah® to treat pediatric and young adult patients with acute lymphoblastic leukemia. With significant efficacy shown in patients, more CAR T cells are being developed at pre-clinical phases with new targets or chimeric antigen designs. This work shows that the efficacy of CAR T cells could be evaluated using xenograft mouse models.

Two investigative studies on CAR T cells were performed, in a lymphoma orthotopic model and an ovarian cancer subcutaneous model. Luciferase-labeled Raji (Raji-luc) cells were injected into immuno-deficient B-NDG mice (Biocytogen), and 9 days after tumor inoculation, CAR T-1 and CAR T-2 were given through single IV injections. About one week later, significant tumor growth inhibition was observed in both CAR T cell treated groups. At Day 37 of post-tumor inoculation, all animals in the control group died, while the CAR T treated group had a 75% survival rate.

The same batch of CAR T cells were tested using BioDuro's proprietary 3D culture method which is aligned to human tumor microenvironmental conditions (3D-hTME). Raji-luc cells were mixed with CAR T cells at Effector: Target (E: T) ratios of 1:0.5, 1:2 and 1:8, then seeded in semi-gel form 3D culture in 96-well plates. The luminescent signals were read by imager after 4- or 24-hours of incubation at 37oC. Consistent with our in vivo results, the CAR T cells had an increased killing effect on Raji-luc cells, with higher E:T ratio and increased incubation time.

To test CAR T cells in solid tumors, a similar study was done in luciferase-labeled SK-OV-3 (SK-OV-3-luc) subcutaneous xenografts. SK-OV-3-luc cells were inoculated subcutaneously into B-NDG mice and the CAR T treatment was given 9 days later by single IV injection. Tumor volumes were measured with calipers or by luminescent signal using an in vivo imager. The results showed that starting from Day 20, the animals treated with CAR T cells had significantly smaller tumors in comparison to the control animals.

In summary, xenograft models with luciferase-labeled cancer cell lines can be used to investigate the efficacy of experimental CAR T therapies to treat liquid or solid tumors. Additionally, the use of in vitro 3D-hTME culture shows promising results as a potential earlier-stage screen for preclinical assessment of CAR T activity.

#2315

Alteration of tumor metabolism by CD4+ T cells leads to TNF-α-dependent intensification of oxidative stress and tumor cell death.

Zhichun Ding, Tsadik Habtetsion, Gang Zhou. _Augusta University, Augusta, GA_.

The inhibitory effects of cancer on T cell metabolism have been well established, but the metabolic impact of immunotherapy on tumor cells is poorly understood. Here, we developed a CD4+ T cell-based adoptive immunotherapy protocol that was curative for mice with implanted colorectal tumors. By conducting metabolic profiling on tumors, we show that adoptive immunotherapy profoundly altered tumor metabolism, resulting in glutathionedepletion and accumulation of reactive oxygen species (ROS) in tumor cells. We further demonstrate that T cell-derived tumor necrosis factor alpha (TNF-α) can synergize with chemotherapy to intensify oxidative stress and tumor cell death in an NADPH (nicotinamide adenine dinucleotide phosphate hydrogen) oxidase-dependent manner. Reduction of oxidative stress, by preventing TNF-α-signaling in tumor cells or scavenging ROS, antagonized the therapeutic effects of adoptive immunotherapy. Conversely, provision of pro-oxidants after chemotherapy can partially recapitulate the antitumor effects of T cell transfer. These findings imply that reinforcing tumor oxidative stress represents an important mechanism underlying the efficacy of adoptive immunotherapy.

#2316

Systemic Control of CAR T Cell Activity Using a Secret Passageway Fusion Receptor.

Boning Zhang, John V Napoleon, Philip Low. _Purdue University, West Lafayette, IN_.

Chimeric antigen receptor (CAR) T cells are modified T cells that have been genetically engineered to recognize and kill any cancer cell expressing a specific cell surface tumor antigen. Despite the remarkable success of CAR T cell technologies to date, adoption of the technologies has been limited by severe side-effects such as a potentially fatal cytokine release syndromes (CRS), neurologic toxicities, and on-target off-tumor toxicities, etc. Moreover, chronic exposure of CAR T cells to tumor antigen can lead to CAR T cell exhaustion, which results in inactivation of the CAR T cell therapy. Therefore, an ability to sensitively control CAR T cell fate in vivo, either to suppress its activity when confronted with a cytokine storm, or to rejuvenate its activity when encountering CAR T cell exhaustion, would be highly desirable.

We have developed a CAR T cell regulatory platform that enables delivery of any desired modulator selectively into an engineered CAR T cell without simultaneously permitting any modulator uptake by other cells. For this purpose, a fusion receptor that contains two subdomains, a ligand binding module and a membrane-anchoring/internalizing module, is co-expressed with the classical CAR construct in the desired T cell. In the embodiment described here, the ligand binding domain is comprised of human FK506 binding protein (FKBP), while the membrane-anchoring/internalizing domain is comprised of sequences from human folate receptor alpha (FRα). Because the fusion construct is human, it is not expected to be highly immunogenic. Moreover, because FRα is a GPI-anchored cell surface protein that constitutively internalizes, the fusion construct is predicted to constantly enter cells by endocytosis throughout the lifespan of the CAR T cell.

We show here that this fusion construct can be co-expressed with an anti-CD19 CAR in the same human T cell with a T2A linker in between in one lentiviral vector. We then demonstrate that any CAR T cell modulator can be selectively targeted to CAR T cells in vivo by linking the desired modulator to FK506; i.e. the ligand that binds FKBP. Upon binding FKBP, the FK506-modulator conjugate is shown to rapidly internalize into the targeted CAR T cell via standard FRα endocytosis. Furthermore, when the linker connecting FK506 to the desired modulator is designed to be cleaved within early endosomes, the modulator is demonstrated to be released following entry into the CAR T cell, where it becomes immediately available for modulation of CAR T cell properties. In the panels shown in this poster, examples of the use of this universal CAR T cell regulatory strategy to control the activities of CAR T cells following their infusion into animal models are provided.

#2317

Dose control of CAR-like T cell activity through post-translational covalent loading of ligands to a universal immune receptor.

Nicholas G. Minutolo, Prannda Sharma, Andrew Tsourkas, Daniel J. Powell. _University of Pennsylvania, Philadelphia, PA_.

The use of chimeric antigen receptor (CAR) T cells for the treatment of CD19+ hematologic malignancies has shown great success in the clinic, leading to FDA approval for various forms of leukemia. However, their broad use is limited since a CAR targets a single tumor associated antigen (TAA), which is not effective against tumors with heterogeneous TAA expression or emerging antigen loss variants. Further, stably engineered CAR T cells can continually proliferate and activate in response to antigen, potentially causing fatal cytokine release syndrome (CRS) or neurotoxicity without a means of quantifiable control. These toxicities and limitations have prompted the development of an alternative CAR format called universal immune receptors (UIRs). UIRs allow multi-antigen targeting with a single receptor to prevent and address antigen escape due to heterogeneity and loss, targeting ligand (TL) dose dependent control of CAR activity, control of CRS, and normal B cell reconstitution following CD19 targeting. However, all current formats rely on non-covalent engagement of the TL which can lead to issues with sub-optimal affinity and specificity.

We have developed a next generation UIR system that allows for post-translational covalent attachment of TLs to the receptor. This is accomplished through the use of the SpyCatcher-SpyTag protein system. When combined at physiologic pH and temperature, these two proteins form a covalent bond. The SpyCatcher immune receptor (Spy-IR) contains the SpyCatcher protein as its extracellular domain attached to standard second-generation CAR intracellular signaling domains. Addition of TAA-specific TLs labeled with SpyTag allows for on-demand CAR generation through covalent bond formation between the TL and the receptor at the T cell surface. Here, primary human T cells expressing the Spy-IR are quantitatively loaded with targeting ligands, allowing for dose-titratable control of redirected T cell effector function and tumor cell lysis. Even as a single cell product, Spy-IR T cells can recognize an array of tumor antigens, (e.g. Her2, EGFR, EpCAM, and CD20) either simultaneously or sequentially to address the evolving tumor antigen landscape. Further, dose-dependent arming of Spy-IR T cells with TL allows for controlled immune recognition of cells expressing either high or low level antigen to distinguish between tumor and normal cells, thus providing an additional means of safety. Finally, preclinical efficacy of the Spy-IR system was achieved in NSG mouse models of aggressive solid tumor and intraperitoneal disease. In conclusion, the SpyCatcher immune receptor is the first UIR designed for post-translational covalent attachment of targeting ligands that permits timed "on demand" redirection of T cells against a diverse array of antigens, to address limitations of safety and antigen diversity by conventional CAR T cell therapy.

#2318

Combinatorial IGK-CD19 CAR primarily targets IgK+ malignant B-cells and is less prone to serum IgG inhibition.

Hakan Köksal, Pierre Dillard, Sólrún M. Maggadóttir, Gunnar Kvalheim, Erlend B. Smeland, June H. Myklebust, Else Marit Inderberg, Sébastien Walchli. _Oslo University Hospital, Oslo, Norway_.

The first Chimeric antigen receptor (CAR) T-cell therapies have been approved for treatment of B-cell malignancies. This is mainly due to the success of CAR T cells targeting B-lymphocyte antigen CD19, which has led to astonishing results in clinical trials. Since CD19 is a general B-cell antigen, CAR-T cells eliminate all B-lineage cells, including non-malignant B cells. Therefore, the patients suffer from the impaired humoral immune response, increasing susceptibility to severe infections.Since B-cell lymphoma and chronic lymphocytic leukemia cells have a clonally restricted expression of Immunoglobulin (Ig) light chains, either Ig-kappa or Ig-lambda, Ig-kappa positive tumor cells can be targeted while sparing normal Ig-lambda positive B-cells. In this respect, we isolated the sequence encoding the antigen-binding parts of an anti-Ig kappa antibody and designed a second-generation CAR construct (IGK CAR). Expression of IGK CAR in expanded peripheral blood T cells and subsequent testing of the CAR T cells in various in vitro assay with target cells, demonstrated cytokine production and potent killing of Ig-kappa expressing B-cell lines such as BL-41, whereas no response was observed against Ig lambda positive B-cell lines such as Granta-519. We compared IGK CAR with a clinical CD19 CAR (fmc63) and observed similar potency in target cell killing. Previous reports have shown that the presence of free immunoglobulins present in human serum could inhibit IGK CAR T cells, and our tests confirmed this. To improve IGK CAR T cells in the presence of IgGs while maintaining the specificity, we utilized a combinatorial CAR system, where the signaling domains were split. Our design demonstrated efficient killing of Ig-kappa positive cells and was less sensitive to free IgG as compared to IGK CAR T cells. Additionally, we observed a trade-off between specificity and cytotoxic potential. Increasing one individual component of the combinatorial system made the cells less prone to serum IgG inhibition but demonstrated somewhat higher cytotoxic activity against Ig-kappa negative targets. Our fully adjustable design, therefore, brings another perspective to the field by regulating the individual expression levels according to the treatment needs. Hence, enabling T cells to be either more aggressive or specific depending on the treatment efficiency and on the on-target toxicity in patients. Taken together, our in vitro data demonstrate that IGK-CD19 CAR combination is as potent as IGK or CD19 CAR T cells, and provides an alternative by combining their benefits into one design and thus reduces on-target toxicity.

#2319

Accumulation and anti-tumor effect of chimeric antigen receptor (CAR) NK cells in metastasis uveal melanoma.

Bao Quoc Lam,1 Takahito Sugase,1 Mizue Terai,1 Meggie Danielson,1 Nadezhda Anikeyeva,2 Melissa A. Wilson,1 Yuri Sykulev,2 Takami Sato1. 1 _Sidney Kimmel Medical College of Thomas Jefferson University, Philadelphia, PA;_ 2 _Thomas Jefferson University, Philadelphia, PA_.

Introduction: Metastatic uveal melanoma (MUM) is resistant to currently available treatments and overall prognosis is poor. MUM is an "immune cold" tumor and immune checkpoint blockades have only a marginal effect on this tumor. To bring immune cells to the tumor site and change the tumor microenvironment, we developed Chimeric Antigen Receptor (CAR)-NK cells targeting high molecular weight melanoma associated antigen (HMW-MAA/CPSG4).

Methods: NK92 cell line derived from NK cell lymphoma was obtained from the American Type Culture Collection (ATCC). NK92 cells were transfected with retroviral pBMN-I vector co-expressing Green Fluorescent Protein (GFP) and anti-HMW-MAA-CD28-CD3z CAR construct. The efficacy of CAR construct transfection was confirmed by expression of His-Tag and F(ab)2 by using BD FACS Celesta flow cytometry. MUM cell line, TJU-UM001, was established from a hepatic metastasis of a uveal melanoma patient in our institution. TJU-UM001 cells have approximately 37,000 HMW-MAA molecules on their surface and are sensitive to NK92 cell killing. Tdtomato-transfected TJU-UM001 cells (106 cells) were intra-hepatically implanted into NOD-scid IL2Rgammanull (NSG) mice. Once establishment of hepatic tumor was confirmed by IVIS 200 imaging system (PerkinElmer) at Red Fluorescent Protein (RFP) excitation 535nm, 3 x 106 CAR-NK92 cells or NK92 cells without CAR constructs were injected into the spleen. Accumulations of GFP+ NK92 cells in the TJU-UM001 tumors in the liver were analyzed under fluorescence microscopy. In a separate experiment, 107 TJU-UM001 cells were subcutaneously implanted into NSG mice. The size of subcutaneous tumor was measured by Caliper. Once tumor became palpable (7-10 mm), 3 x 106 CAR- or non-CAR NK92 cells were directly injected to the MUM tumors. Forty thousand units of human IL-2 were intra-peritoneally injected into mouse once daily for 4 consecutive days after NK92 cell injections.

Results: There were expressions of both His-tag and F(ab)2 on CAR-GFP NK92 cells. GFP and CD54 (ICAM-1) were expressed on 95% and 90% of CAR-GFP NK92 cells, respectively. There was no cross-expression of GFP or human CD54 on mouse cells. Therefore, GFP and CD54 were confirmed to be reliable markers for tracking CAR-GFP NK92 cells in mice. Both CAR- and non-CAR NK92 cells exhibited suppression of TJU-UM001 cell proliferation in in vitro assay. Direct injections of both CAR- and non-CAR NK92 cells showed anti-tumor growth effects on subcutaneously implanted TJU-UM001 tumors. More importantly, via splenic injection, CAR-NK92 cells accumulated selectively in the MUM tumors in the liver, while non-CAR NK92 cells were randomly distributed to normal liver tissue and the MUM tumors. These results support the rationale for hepatic arterial infusion of CAR-NK cells as treatment for uveal melanoma patients with hepatic metastasis.

#2320

Avadomide (CC-122) increases effector function and reverses exhaustion in chronically stimulated lisocabtagene maraleucel anti-CD19 CAR T drug product.

Heidi K. Jessup, Evan P. Thomas, Jim S. Qin, Yue Jiang, Oleksandr Baturevych, Neha Soni, Aye T. Chen, Ruth A. Salmon, Melissa G. Works, Michael O. Ports. _Juno Therapeutics, a Celgene Company, Seattle, WA_.

Lisocabtagene maraleucel (liso-cel) is an investigational drug product composed of autologous CD8+ and CD4+ T cells expressing a CD19-specific chimeric antigen receptor (CAR), being evaluated in clinical trials for treatment of B cell malignancies. The CELMoD CC-122 is being evaluated in non-Hodgkin lymphoma (NHL) and can augment T cell function through Cereblon-mediated degradation of Ikaros and Aiolos. Combination approaches aimed at enhancing CAR T cell function may increase the rate, depth, and durability of clinical responses. Here we report pre-clinical results supporting the potential combination of CC-122 an liso-cel.

To determine potentiating effects of CC-122 following acute activation (anti-idiotypic Ab or CD19+ targets), T cells expressing the CAR of liso-cel were cultured with CC-122 and assessed for intracellular signaling, cytolytic activity, cytokine production, and activation marker expression. CC-122 significantly increased NFκB, Nur77, and STAT5 as well as effector cytokine production and activation marker expression.

Activation-induced exhaustion of CAR T may decrease durable responses in patients. We developed a chronic stimulation assay to render CAR T cells hypofunctional (reduced cytolysis and IL-2 secretion) and examined whether enhanced activation mediated by CC-122 would exhaust the CD19-directed CAR T cells more rapidly. CC-122 was added in one of two regimens: during chronic stimulation ("concurrent") or during re-challenge ("rescue"). Effects on exhaustion in "concurrent" or "rescue" cultures were evaluated, including by RNAseq and measuring of effector function in re-challenge assays with CD19+ targets. Surprisingly, "concurrent" CC-122 partially reversed a gene signature associated with CAR T cell hypofunctionality and preserved more effector function relative to controls. In "rescue" assays, CC-122 dose-dependently increased cytokine production and restored cytolytic function of exhausted CAR T cells upon re-challenge with CD19+ tumor spheroids.

In a disseminated tumor xenograft model, concurrent or delayed dosing of CC-122, in combination with two sub-curative doses of liso-cel, significantly increased median survival by 30 to 40 days. In some cases, survival endpoints were not reached with the combination.

Taken together, CC-122 treatment of CD19-directed CAR T cells was shown to enhance CAR T activation in acute stimulation assays; limit CAR T exhaustion onset during chronic stimulation as assessed by gene expression; and reverse an exhausted, hypofunctional CAR T state induced by chronic stimulation. The data suggest that liso-cel pharmacologic activity may both be enhanced and extended by this combinatorial approach. These findings support an ongoing clinical investigation evaluating the addition of CC-122 to liso-cel in patients with R/R, aggressive NHL (NCT03310619).

#2321

Dual-function of CD27-CD70 costimulatory signal in CAR T cell therapy.

Dongrui Wang, Alfonso Brito, Darya Alizadeh, Renate Starr, Brenda Aguilar, Behnam Badie, Stephen J. Forman, Christine E. Brown. _City of Hope, Duarte, CA_.

Purpose: To investigate the function of co-stimulatory molecule CD27 on the antitumor efficacy of chimeric antigen receptor (CAR) T cells.

Experimental Design: IL13 receptor α2 (IL13Rα2)-targeted CAR T cell products from glioblastoma (GBM) patients were characterized for CD27 surface expression and proliferation potential against recursive tumor challenge. Isolated CD27+ and CD27- fractions of CAR T cells were then evaluated for their antitumor potency against orthotopic GBM xenografts, as well as their molecular characteristics before and after tumor stimulation. Further, T cells were engineered to express a CAR construct together with a constitutively-expressed CD27 to investigate the role of continuous CD27 costimulation on CAR T cell maintenance and effector potency.

Results: CD27 expression on CAR T cell products was correlated with their proliferation capacity against recursive tumor stimulation. Isolated CD27+ CAR T cells outperformed CD27- and unsorted cells in mediating long-term tumor eradication. CD27+ CAR T cells exhibited memory-associated genetic signatures, and were less exhausted following in vitro and in vivo tumor stimulation, both phenotypically and functionally. Moreover, CD27 also sensitized CAR T cells to target low antigen-expressing tumors. When targeting tumor cells expressing CD70 (CD27 ligand), CD27 on CAR T cells became rapidly down-regulated, suggesting a transient CD27-CD70 interaction. The advantageous effector function of CD27+ CAR T cells was diminished by blocking the CD27-CD70 interaction with CD70-targeting antibodies. CD70 is also expressed on CAR T cells, displaying a mutually exclusive expression pattern with CD27. Extended in vitro culture resulted in a phenotypic switch of CAR T cells from CD27+CD70- to CD27-CD70+. Surprisingly, constitutive expression of CD27 (CD27-cons) greatly impaired the effector activity of CAR T cells. These cells were able to eliminate tumor cells in vitro, but failed to mediate potent antitumor effect in vivo. Further characterization showed an early exhaustion phenotype for CD27-cons CAR T cells during in vitro expansion. Additionally, CD27-cons CAR T cells were also prone to activation-induced T cell apoptosis especially against high antigen-expressing targets.

Conclusion: These studies demonstrate a dual-function of CD27 costimulation for CAR T cell antitumor activity. In the enriched CD27+ CAR T cell subset, the transient CD27-CD70 interaction with tumor cells resulted in enhanced antitumor potency. By contrast, constitutively expressed CD27 drove CAR T cell exhaustion and induced apoptosis. These results illustrate the critical role of CD27 in mediating potent CAR T cell therapeutic efficacy, and suggest the potential of CD27 as a marker for the functional quality of CAR T cell products.

#2322

ICAM-1 targeting CAR T cell therapy for triple negative breast cancer.

Yanping Yang, Yogindra Vedvyas, Jaclyn E. McCloskey, Irene M. Min, Moonsoo M. Jin. _Weill Cornell Medical College, New York, NY_.

Background: Triple negative breast cancer (TNBC) is an aggressive disease with a poor prognosis. TNBC patients do not respond to hormone receptor or HER2-targeted therapies owing to low expression of these cell surface biomarkers. The lacking of targeted therapy leaves conventional combination of surgery, chemotherapy and radiation therapy as the standard-of-care treatment options for TNBC. But even this combination usually fails to prevent disease recurrence. Intercellular adhesion molecule-1 (ICAM-1) was recently discovered to be upregulated in TNBC and could serve as an attractive molecular target (Guo et al., PNAS 2014). Chimeric antigen receptor (CAR) T cell therapy has shown remarkable success against hematological malignancies, however there has been little success in the treatment of solid tumors. Here, we developed an ICAM-1 targeting CAR T cell-based immunotherapeutic strategy to redirect T cell to kill solid TNBC.

Methods: Patient-derived xenograft (PDX) models of TNBC were utilized to determine ICAM-1 expression by immunohistochemistry. ICAM-1 surface protein expression in TNBC cell line MDA-MB-231 was measured by flow cytometry. Primary T cells (n = 4 donors) were isolated and transduced with ICAM-1 CAR lentivirus twice at 24 and 48 hours after activation with anti-CD3/CD28 Dynabeads. In vitro killing ability was determined by co-incubation of GFP-Firefly Luciferase (GFP/Fluc) transduced target cells (HeLa, MDA-MB-231, and 293T) with transduced and non-transduced T cells. NSG mice bearing MDA-MB-231 xenografts were treated with CAR T cells to test the in vivo efficacy. Bioluminescence was used to quantify cell lysis in vitro and to track tumor growth in vivo.

Results: 4 out of 8 TNBC PDX models (JAX, tumor markers: TER−/PR−/HER2−) showed strong IHC ICAM-1 staining. We validated by flow cytometry that ICAM-1 is highly expressed on TNBC cell line MDA-MB-231. These results provide further evidence supporting ICAM-1 as a molecular target for TNBC by CAR T cell therapy. Primary T cells were transduced to express ICAM-1 targeting CAR at approximately 50%. Co-incubation of CAR T cells or non-transduced T cells with ICAM-1 positive cell lines (HeLa and MDA-MB-231) or negative control cell line (293T) showed that CAR T cell-mediated killing was ICAM-1 expression dependent. After 48 hours, 70% of HeLa and 85% of MDA-MB-231 cells were specifically lysed at effector to target ratio of 5:1, while no obvious killing of 293T cells was observed. Additionally, non-transduced T cells produced little killing with less than 20% of target cell lysis. Ongoing in vivo studies will determine the efficacy of this ICAM-1 targeting CAR T cell against TNBC.

Conclusions: We developed an ICAM-1 targeting CAR T cell with the ability to induce potent and specific killing of TNBC cells. Preclinical studies are being conducted to evaluate the feasibility of ICAM-1 specific CAR T cells as a potential therapy for patients with ICAM-1 positive TNBC.

#2323

First-in-human CAR T for solid tumors targets the MUC1 transmembrane cleavage product.

Cynthia C. Bamdad, Nelson D. Glennie, Andrew K. Stewart, Pengyu Huang, Benoit J. Smagghe, Tyler E. Swanson, Erin K. Hanahoe, Gregory L. Riley. _Minerva Biotechnologies, Waltham, MA_.

Purpose: To develop a CAR T for solid tumors that hits a wide range of cancers, is effective and has little or no effect on normal tissues.

We developed a MUC1* targeting CAR T that is scheduled for a 1st-in-human clinical trial for metastatic breast cancers at the Fred Hutchinson Cancer Research Center in Q1 2019.

Unlike previous attempts at making an anti-MUC1 cancer therapeutic, huMNC2-CAR44 targets MUC1*, which is the transmembrane cleavage product. MUC1* is a growth factor receptor that is activated when onco-embryonic growth factor NME7AB dimerizes its truncated extra cellular domain. The binding site for NME7AB is masked in full-length MUC1; anti-MUC1* antibody huMNC2 binds to the same site. huMNC2 does not bind to full-length MUC1 nor other cleaved forms of MUC1 that are on some healthy tissues that need to rapidly divide.

Monoclonal antibodies against the truncated MUC1* extra cellular domain were generated and screened by IHC for reactivity to cancerous tissue micro arrays (TMAs) and lack thereof on normal tissues. The best monoclonals were incorporated into CARs, transduced into human T cells and tested in vitro for specific killing of MUC1* positive but not MUC1* negative cells. We developed a novel cell line in which MUC1 is not cleaved. By adding specific cleavage enzymes, we identified antibodies that recognized conformational epitope that were created by specific cleavage enzymes. The final selection of an anti-MUC1* antibody for the targeting head of our CAR was based on its widespread binding to breast cancer tissues, low cross reactivity to normal tissues, and its recognition of a conformational epitope created when MUC1 is cleaved to MUC1* by a specific cleavage enzyme that is overexpressed in many cancers, especially breast cancers. In vivo we showed that injecting this cleavage enzyme near a MUC1/MUC1* breast tumor dramatically accelerated tumor growth, which was stopped by simultaneous injection of the cleavage enzyme and our CAR T cells.

IHC studies of thousands of normal vs. cancerous human tissue specimens show that huMNC2-scFv almost exclusively binds to tumor tissues, hitting over 90% of breast, 83% ovarian, 78% pancreatic and 71% of lung cancers. Recognition of breast cancer specimens appears not to be limited by cancer sub-type.

In vivo experiments of human tumors in NSG mice (n>300), show that huMNC2-CAR44 T cells inhibited or completely obliterated a variety of MUC1* positive solid tumors. Dual tumor experiments showed that adequate MUC1* density is required for a CAR T response, further supporting the idea that huMNC2-CAR44 T cells will selectively kill MUC1* positive tumors, while sparing normal tissues.

Conclusion: MUC1* is the predominant form of MUC1 present on cancers. Antibodies that target conformational epitopes produced by specific subsets of cleavage enzymes make anti-MUC1* CAR T cells highly tumor selective. CARs could be patient specific based on which cleavage enzymes their tumors express.

#2324

Preclinical safety, biodistribution, and tumor infiltration analysis of CAR T cell targets using in situ hybridization technology.

Helly Pimentel,1 Helen Jarnagin,1 Hailing Zong,1 Courtney Todorov,1 Kenneth Ganley,2 Fay Eng,2 Kevin Friedman,2 Molly Perkins,2 Shannon Grande,2 Courtney M. Anderson,1 Bingqing Zhang,1 Christopher Bunker,1 James B. Rottman,2 Xiao-Jun Ma1. 1 _Advanced Cell Diagnostics, Newark, CA;_ 2 _Bluebird Bio, Cambridge, MA_.

Chimeric antigen receptor (CAR) T cell therapy has proven to be highly effective in treating hematologic malignancies, and major efforts are being made to achieve similar efficacy in solid tumors. These efforts face multiple challenges, including off-tumor target expression and checkpoint inhibition of CAR T cell activity in the tumor microenvironment. CAR T cells are much more potent compared to antibody therapeutics, therefore there is a need for more stringent CAR T target safety assessment to avoid adverse events resulting from "on-target/off-tumor" activity. Furthermore, it is critical to track and monitor CAR+ T cells within the context of intact tissue and tumor to understand the mechanisms underlying off-tumor toxicity and efficacy in tumor killing. In this study we employed the RNAscope in situ hybridization (ISH) technology to assess target expression specificity and to track CAR T cell distribution and activation in xenograft and host tissues using the RPMI-8226 xenograft mouse model, which expresses both BCMA and ROR1. The anti-ROR1 CAR T cells used in this study recognized both the mouse and human proteins whereas the anti-BCMA CAR T cells recognized only the human protein. RNA ISH revealed that BCMA was only expressed in the xenograft tumor and in no mouse organs, while ROR1 was found to be expressed in the xenograft tumor as well as at low levels in mouse lung and liver. Duplex RNA ISH assay with probes targeting the CAR 3' UTR and either IFNG or GZMB mRNA allowed highly sensitive and specific detection of CAR T cells and their activation state in both tumor and normal tissues from vehicle, anti-ROR1 CAR T cell, or anti-BCMA CAR T cell treated mice. Activated anti-BCMA CAR T cells expressing GZMB and IFNG were found only in the xenograft tumor, where BCMA was expressed. In contrast, activated anti-ROR1 CAR T cells were found almost exclusively in mouse lung and liver with very few anti-ROR1 CAR T cells being found in the xenograft tumor, consistent with the previously observed pulmonary and hepatic toxicity of anti-ROR1 CAR T cells that was not predicted by IHC analysis of ROR1 protein presumably due to lack of antibody sensitivity. Lastly, we employed a multiplex ISH-IHC approach to confirm the presence of activated anti-BCMA CAR T cells in the xenograft tumor through simultaneous detection of the BCMA CAR 3' UTR, IFNG, GZMB, and CD3, validating the anti-tumor activity of anti-BCMA CAR T cells. These data thus demonstrate how the RNAscope ISH assay can be utilized for CAR T efficacy and safety/toxicity assessment in preclinical models by detecting very low levels of target antigen expression in off-tumor tissues and monitoring CAR T cell pharmacodynamics and activation in tumor models. This technology has equal utility in understanding both CAR T and TCR-T cell activity in patient tumors.

#2325

Long term time-course monitoring of NK cell-mediated ADCC using the Celigo Image Cytometer.

Leo L. Chan,1 Lucas Ferrari De Andrade,2 Charles A. Thomas,3 Dmitry Kuksin,1 Kai Wucherpfennig2. 1 _Nexcelom Bioscience LLC, Lawrence, MA;_ 2 _Dana Farber Cancer Institute, Boston, MA;_ 3 _Dana Farber Cancer Institute, Lawrence, MA_.

Antibody-dependent cell-mediated cytotoxicity (ADCC) assay has been widely performed for immuno-oncological research. Traditionally, ADCC assays are conducted by measuring the amount of released Chromium, calcein AM, or Lactate dehydrogenase (LDH) molecules after the target cancer cells are killed by effector/antibody pair. These methods can be inaccurate due to indirect measurement of supernatant at the end point of 4 hour, however, there is a need to characterize the effects of antibody-dependent cytotoxicity for longer than 72 hours. Previously, we have demonstrated an image-based ADCC detection method using the Celigo Image Cytometer, where the target cells are stained with calcein AM and other tracer dyes to monitor cell count or viability. However, the fluorescence of these dyes can significantly diminish within a 48 hour time frame. In this work, we demonstrated the time-course monitoring of NK cell-mediated ADCC of A375 cells in the presence or absence of IL2 and antibodies for 76 hours. First, A375-ZsGreen cells were seeded into a 96-well plate with and without target antibodies. Next, NK cells with and without IL2 were added to the wells at 10:1, 5:1, 2:1, and 1:1 E:T ratios. The co-culture was allowed to incubate for 76 hours and the image cytometer was used to scan and analyze at times 0, 3, 4, 6, 24, 27, 30, 51, 74, and 76 hours. The software was able to count individual cells as well as confluence % at each time point by segmenting highly fluorescent objects in the images. We were able to show time-dependent ADCC killing with decrease in ZsGreen positive cells as the read-out. In addition, the uniformity of cell killing can be observed in the Celigo obtained whole well images. One of the most important findings was that there was regrowth of target cells after the initial 30 hours of cell killing, indicating the activated NK cells without antibodies did not eradicate the cancer cells. Therefore, the ability to perform long term time-course monitoring of ADCC is highly important to better characterize the effects of target antibodies on cell killing function.

#2326

**Allo CAR T** TM **targeting CD70 for the treatment of renal cell carcinoma.**

Siler Panowski,1 Surabhi Srinivasan,1 Silvia Tacheva-Grigorova,1 Nguyen Tan,1 Thomas Van Blarcom,1 Tao Sai,2 Jonathan Heyen,3 Niranjana Nagarajan,2 Mathilde Dusseaux,4 Roman Galetto,4 Barbra Sasu1. 1 _Allogene Therapeutics. Inc., South San Francisco, CA;_ 2 _Pfizer Inc, South San Francisco, CA;_ 3 _Pfizer Inc, La Jolla, CA;_ 4 _Cellectis, Inc., Paris, France_.

Renal Cell Carcinoma (RCC) represents a substantial patient population, with 65,340 new cases estimated in the US in 2018. Patients with localized tumors undergo nephrectomy, however 30-40% of patients develop metastasis, with a 5-year median survival rate of only 11.6%. Current treatment for advanced disease improves overall survival, but disease relapse is common and additional treatments are needed. RCC is a highly T-cell infiltrated tumor type with responsiveness to Immuno-Oncology (IO) agents and thus it may be amenable to a T-cell based therapy. T cells can be genetically modified to express chimeric antigen receptors (CARs), and adoptive transfer of CAR T cells is showing great promise in hematologic malignancies. To translate this approach for RCC treatment, expression data were mined and CD70 was identified as an antigen expressed in a high proportion of patients with RCC, with limited normal tissue expression on a fraction of activated lymphocytes and subsets of dendritic cells. Since CD70 expression is present on activated T cells, targeting it with a CAR could lead to fratricide and T cell exhaustion. For this reason, screens were specifically designed to identify CARs that were less impacted by these issues and used to triage a large panel of CD70 CARs. Optimal CARs were successfully manufactured and demonstrated robust in vitro and in vivo anti-tumor activity against target cells expressing relevant levels of CD70. A large panel of single chain fragment variables (scFvs) that bind to CD70 were generated and formatted into second-generation 41BB-CD3zeta CARs. CARs were first screened in a Jurkat cell CD70-knockout assay and clones showing tonic signaling were eliminated. CD70 CAR T cells were generated with various scFvs and ranked based on transduction efficiency, phenotype, activation status and expansion. A subset of CD70 CAR T cells were moved into in vitro short and long-term cytotoxicity assays. Target cells expressing high, medium, and low levels of CD70 were utilized. Ability to kill cell lines with CD70 expression similar to that seen in primary tumors was used as criteria for candidate selection. CAR T were evaluated in multiple in vivo xenograft models and robust anti-tumor activity was observed. Some candidates performed better with CD70 knockout and some worked irrespective of knockout. To profile potential toxicity, a cyno toxicity study was conducted with one clone formatted as a CD70-CD3 bispecific antibody. No unexpected findings were observed. In the event of unexpected toxicity, CAR T cells were further engineered to contain an off-switch, by which CAR T are eliminated upon administration of Rituximab. Multiple off-switch CAR formats were evaluated, and optimal formats determined independently for each CAR. One CD70 CAR T was also successfully manufactured in a large-scale process. In summary, multiple CD70 CAR T have been profiled and a subset selected for further investigation as potential clinical candidates.

#2327

**Same day transduction and** in vivo **expansion of chimeric antigen receptors and synthetic driver constructs for adoptive cellular therapy.**

Frederic Vigant,1 Jianfang Hu,1 Laurence Jadin,1 Benjamin Lopez,1 Tiffany Lam,1 Hiba Shaban,2 Anirban Kundu,3 Timothy Mayall,1 Gregory Schreiber,2 Farzad Haerizadeh,1 James Onuffer,1 Gregory Frost2. 1 _F1 Oncology, Inc, San Diego, CA;_ 2 _F1 Oncology, Inc, West Palm Beach, FL;_ 3 _Exuma Biotechnology SEZC, Georgetown, Cayman Islands_.

Adoptive cellular therapy (ACT) using ex vivo expanded chimeric antigen receptor (CAR) modified T cells to target cancer cells expressing CD19 has been very successful in the treatment of hematologic malignancies and the clinical application of this technology for solid tumor malignancies is a major focus of several research and development programs. Despite the clinical success of these products, there are several obstacles that currently limit widespread deployment of CAR-T. Current autologous CAR-T manufacturing approaches are complex, requiring centralized manufacturing facilities and extensive logistical control over the receipt and delivery of individually patient matched products. Several weeks are required to prepare and release CAR-T cells using current manufacturing processes from apheresis to genetic modification and expansion before the cell product is reinfused into the patient. However, recent studies suggest that limiting the ex vivo expansion time results in less differentiated CAR-T products with enhanced effector function. Successful engraftment and persistence for current autologous CAR-T cell products require the depletion of normal lymphocytes in patients with cytotoxic drugs (primarily cyclophosphamide, fludarabine or combinations) prior to administration of CAR-T cells. Although the use of non-myeloablative lymphodepleting regimens prior to CAR-T infusion significantly enhances the successful in vivo homeostatic expansion and persistence of administered CAR-T cells, non-myeloablative chemotherapy also requires significant supportive care. The development of a point of care approach to ACT has the potential to reduce the complexity of CAR T-cell immunotherapy and broaden access to a substantially greater number of cancer patients and address many of the limitations discussed above. The most ideal system would allow for rapid genetic modification of patient's cells next to the patient, thereby eliminating chain of custody risks, combined with successful in vivo expansion and engraftment of cells in the patient to achieve therapeutic cellular levels without preconditioning through lymphodepletion. Here we describe and provide data demonstrating initial proof of concept for a novel point of care approach for CAR-T using engineered lentivirus vectors and resting human PBMC. Resting human PBMC were isolated from blood and successfully transduced within a four hour exposure to engineered lentivirus particles encoding a synthetic lymphoproliferative element. These modified cells expanded in vivo upon administration in mice. The entire process of PBMC isolation, genetic modification and dosing was completed within twelve hours vein to vein and represents a significant step forward in advancing the development of CAR-T therapies with point of care potential expanding upon patient accessibility and deployment.

#2328

BiTE-T cell: A novel design for solid tumor.

Feifei Luo,1 Yuedi Wang,2 Jie Liu,1 Yiwei Chu3. 1 _Department of Digestive Diseases of Huashan Hospital, and Biotherapy Research Center, Fudan University, Shanghai, China;_ 2 _Department of Immunology of School of Basic Medical Sciences, Fudan University, Shanghai, China;_ 3 _Department of Digestive Diseases of Huashan Hospital, and Biotherapy Research Center, Department of Immunology of School of Basic Medical Sciences, Fudan University, Shanghai, China_.

Genetically engineered T cells therapy is a promising strategy in cancer immunotherapy. Chimeric antigen receptor (CAR)-T cells therapy, as one successful strategy, has achieved the breakthrough and been approved in non-solid tumor but not solid tumor, which might be limited partially by CARs-restricted on cell surface and side effects of retrovirus. Meanwhile, bispecific T cell engager (BiTE), as another successful strategy, has been approved in non-solid tumor, the therapeutic limitation of which in solid tumor might be the short half-life of antibodies, the lack of endogenous effector T cells in patients with advanced cancer, and severe adverse effects. In this study, we developed a novel glypican-3 (GPC3)-targeting BiTE, αGPC3/CD3. These GPC3-BiTE engineered human T cells persistently produced GPC3-BiTE fusion proteins, which were released extracellularly to help both engineered T cell and bystander T cells to exhibit GPC3-specific activity. As a result, GPC3-BiTE-T cells could possess the capability of both CAR-T cells and BiTE, and even aid more than conventional CART cells in the CART-like function. In addition, GPC3+ tumor-bearing mice treated with the secretable GPC3-BiTE-T cells got a significant tumor growth inhibition and prolonged survival without observed adverse effect. Compared with control groups, more GPC3-BiTE-T cells with less PD-1 expression were detected at tumor site, which was similar to the GPC3-CART cell therapy. And the accumulation of GPC3-BiTE-T cells was positively related with the inhibition of tumor growth. Moreover, GPC3-BiTE-T cells assisted bystander T cells accumulated into tumor site in vivo, which was lack in GPC3-CART cell therapy. Thus, the novel GPC3-BiTE-T cells have the characteristics of high potency, long term and low toxicity, which might supply an attractive GPC3-targeting immunotherapy for solid tumors.

#2329

Clinical manufacturing of CAR T cells targeting ICAM-1 for a phase I study against advanced thyroid cancer therapy.

Yogindra Vedvyas,1 Jaclyn E. McCloskey,1 Yanping Yang,1 Irene M. Min,1 Thomas J. Fahey,1 Yen-Michael S. Hsu,1 Jing-Mei Hsu,1 Koen V. Besien,1 Ian Gaudet,2 Ping Law,2 Joon Kim,2 Eric V. Hofe,3 Moonsoo M. Jin1. 1 _Weill Cornell Medical College, New York, NY;_ 2 _Miltenyi Biotec Inc., Sunnyvale, CA;_ 3 _AffyImmune Therapeutices, Inc., New York, NY_.

Background: Patients with anaplastic or poorly differentiated recurrent thyroid carcinomas have a poor prognosis, with a median survival of < 1 year. Previously, we have shown a significant correlation between ICAM-1 overexpression and malignancy in thyroid cancer and have pioneered the use of ICAM-1 targeted CAR T cells as a novel treatment modality. For clinical translation, we designed CAR T cells possessing micromolar affinity to ICAM-1 to avoid cytotoxicity in normal cells with basal levels of ICAM-1 expression. Here, we report the automated process of CAR T cell manufacturing with CliniMACS Prodigy (Miltenyi Biotec) using cryopreserved peripheral blood leukopak cells as starting material.

Methods: We have chosen to use CliniMACS Prodigy system for a closed, automated, and robust CAR T cell manufacturing process. Using a cryopreserved leukopak, TCT v2.0 protocol began with rapidly thawing the leukopak while directly welded to the Prodigy. T cell lentivirus transduction was performed 2x, 24 and 48 hr post activation. CAR T cells were released after ~10 days of culture and cryopreserved in ready-to-infuse formulation (AIC100). Products were then subject to in vitro cytotoxicity, in vivo efficacy and safety in xenografted mice with ATC cells (8505C), and PET/CT imaging using 18F-NOTA-octreotide to detect T cell dynamics.

Results: Prodigy manufactured CAR T cells were subjected to qualification and functional assays (n=7): cell viability, cell number, CAR expression, virus copy number (VCN), T cell subset and phenotype, and E to T assays. Consistent with reported values for Prodigy, our manufacturing protocol produced 55±9% transduction rate, 96±1.8% viability, and 2.9±0.7x10^9 final cell number. The VCN was in the range of 1.0-2.1, below the criteria of < 5 copies per cell. In NSG mice xenografted with 8505C ATC cells and treated with CAR T cells (1X= 1 million live CAR T maximum tolerable dose (MTD)= 10 million live CAR T) or non-transduced (NT) T cells, tumor burden was quantitatively evaluated by whole body luminescence imaging. Compared to cohorts with no treatment (NoT) or NT, the cohorts of AIC100 exhibited complete or near-complete tumor elimination lasting variable times before tumor relapse was seen in some subjects. Median survival time for NoT and NT groups were similar (38.5 vs. 42 days), while it was significantly longer for AIC100 1X and MTD groups (72 vs. >100 days).

Conclusions: Selective anti-tumor activity in the absence of toxicity provides proof-of-concept that micromolar affinity tuned CAR T cells can be used to target tumors expressing high levels of antigen while avoiding normal tissues expressing basal levels of the same antigen. These studies support the initiation of a phase I study to examine the safety and potential efficacy of micromolar affinity tuned CAR T cells against newly diagnosed anaplastic and refractory or recurrent thyroid cancers.

#2330

Preclinical evaluation of TC-110: CD19-specific T cell receptor (TCR) fusion construct (TRuC™) T cell for the treatment of hematologic malignancies.

Holly Horton,1 Jian Ding,1 Seema Shah,1 Jessica Gierut,1 Niko Thorausch,2 Anna Morath,2 Wolfgang Shamel,2 Marcela Maus,3 Patrick Baeuerle,1 Robert Hofmeister,1 Daniel R. Getts1. 1 _TCR2 Thearpeutics, Cambridge, MA;_ 2 _University of Freiburg, Freiburg, Germany;_ 3 _Massachusetts General Hospital, Boston, MA_.

T cells expressing chimeric antigen receptors (CARs) have demonstrated clinical benefit in certain hematological malignancies albeit with disease relapse as well as side effect such as cytokine release syndrome. To overcome potential hurdles of CAR-T cells and other T cell receptor modalities, we have designed a novel T cell engineering platform. Specifically, we have generated T Cell Receptor Fusion Constructs (TRuC™) that fuse a binder domain to subunits of the T cell receptor. These constructs when introduced using lentiviral technology, integrate into endogenous TCR, reprograming T cells to target tumor antigen in a non-major histocompatibility complex (MHC) restricted fashion and harnessing the full spectrum of TCR signaling.

Here, we present the results of the preclinical evaluation of TC-110, a CD19-specific ε-TRuC™ variant. In vitro, TC-110 T cells are equally potent as CAR T cells in eliminating tumor cells, while producing less inflammatory cytokines. In preclinical mouse models of ALL and Burkitt's lymphoma, TC-110 T cells demonstrate better efficacy than 2nd generation CAR-T cells bearing CD28 or 41BB co-stimulatory domains. Of note, the efficacy of TC-110 T cells, both in vitro and in vivo, is not dependent on co-stimulatory signal. Overall, we demonstrate the efficacy of TC-110 T cells both in vitro and in preclinical mouse models, indicating the clinical potential of TRuC™ platform for treating hematologic malignancy.

#2331

Intratumor adoptive transfer of IL-12 mRNA transiently engineered anti-tumor CD8+ T cells.

Inaki Etxeberria,1 Elixabet Bolaños,1 Alvaro Teijeira,1 Arantza Azpilicueta,1 Jose Ignacio Quetglas,1 Alfonso Rodriguez Sanchez-Paulete,1 Itziar Otano,1 Uxua Mancheño,1 Sandra Hervas-Stubbs,1 Susana Inoges,2 Saray Garasa,1 Maite Alvarez,1 Pedro Berraondo,1 Ignacio Melero3. 1 _Center for Applied Medical Reseach (CIMA), Pamplona, Spain;_ 2 _Navarra University Clinic (CUN), Pamplona, Spain;_ 3 _Center for Applied Medical Research (CIMA) and Navarra University Clinic (CUN), Pamplona, Spain_.

Adoptive T-cell therapy with TILs, CAR-T cells, and TCR-engineered T-cells is achieving remarkable clinical efficacy. Retroviral gene transfer of IL-12 into the T cell cultures markedly enhances efficacy but has shown unacceptable severe side effects in the clinic. To ensure transient gene transfer, tumor specific CD8 T cells were engineered by electroporation of mRNA encoding single chain IL-12 into OT1 and Pmel-1 TCR transgenic lymphocytes. Engineered T cells were injected intratumorally and achieved complete rejections not only of the injected lesion but also of distant concomitant tumors. Efficacy was further enhanced if a small dose of agonist anti-CD137 mAb was co-injected or if CD137-ligand mRNA was co-electroporated. Importantly, intratumor T-cell administration was more efficacious that the intravenous or subcutaneous routes. Therapeutic effects were dependent on IFNɣ, cDC1 dendritic cells and endogenous T cells. Interestingly, treatment induced epitope spreading of the immune response to antigens not recognized by the adoptively transferred lymphocytes. Efficacy was also achieved when mouse TILs cultures were used and human TIL cultures can also be transiently IL-12 mRNA engineered. Intratumor release, transient IL-12 mRNA-engineering and repeated administration jointly constitute a safe, feasible and powerful cancer immunotherapy strategy.

### Cancer and Inflammation

#2332

Tumor infiltrating lymphocytes, immunoSeq, and CMS classification in the molecular epidemiology of colorectal cancer study.

Marilena Melas,1 Charalampos Lazaris,1 Stephanie L. Schmit,2 Asaf Maoz,3 Rebeca Sanz Pamplona,4 Chenxu Qu,1 Joel K. Greenson,5 Rork Kuick,5 Flavio Lejbkowicz,6 Hedy S. Rennert,6 Christopher P. Walker,1 Chase M. Bowen,1 Diane M. Da Silva,1 W. Martin Kast,1 Gregory E. Idos,1 Kevin J. McDonnell,1 Victor Moreno,4 Gad Rennert,6 Stephen B. Gruber1. 1 _USC Norris Comprehensive Cancer Center, Los Angeles, CA;_ 2 _Moffitt Cancer Center, Tampa, FL;_ 3 _Boston University School of Medicine, Boston, MA;_ 4 _Catalan Institute of Oncology, Barcelona, Spain;_ 5 _University of Michigan, Ann Arbor, MI;_ 6 _Carmel Medical Center and Technion, Haifa, Israel_.

Background: Tumor infiltrating lymphocytes (TILs) are prognostic and predictive biomarkers in colorectal cancer and are associated with improved prognosis and response to immunotherapy. While TILs are routinely assessed by pathologists, a standardized technique (immunoSEQ, Adaptive Biotechnologies) that leverages targeted next-generation sequencing can also be used to quantify and characterize the T-cell receptor (TCR) repertoire of individual colorectal cancers. In a large, population-based study of incident colorectal cancer, the host immune responses were measured by an expert pathologist and ImmunoSEQ to understand the relationships between TILs, TCRs/cell and specific subgroups of colorectal cancer. Methods: Incident cases of adenocarcinoma of the colon or rectum from the Molecular Epidemiology of Colorectal Cancer (MECC) study included 1,000 cancers that were uniformly evaluated for TILs and other histopathologic features by one pathologist. FFPE-derived DNA from microdissected tumor tissue was extracted and sequenced using ImmunoSEQ analysis for the same 1,000 individuals. A resulting quantitative metric from this assay includes TCRs/cell, a measure of rearranged T cell quantity relative to all nucleated cells in a tumor sample. Gene expression in snap-frozen tissue available from 342/1,000 MECC colorectal cancers was measured with Affymetrix Human Genome U133 Arrays (U133A and U133 Plus2.0) as previously described. CMS classification was performed using the R package 3.5.1, CMS classifier, randomForest 4.6-14. Multivariate analysis assessed CMS by age, gender, TILs/HPF, TCRs/cell, MSI status, BRAF and KRAS mutational status. Results: TILs/HPF and TCRs/cell were significantly correlated among all 1000 cases (r=0.5, p<0.001). Among the 342 cases with available expression profiles, CMS1 constituted 12.0% of all CRC, with CMS2 (41.8%), CMS3 (8.5%), and CMS4 (13.7%) and unclassified (24%) representing the remaining distribution. There were statistically significant differences in the molecular and histopathologic features of colorectal cancers by CMS subgroups. MSI-H tumors were most frequently observed within CMS1 cancers (56.6% of CMS1 were MSI-H), with lower representation among CMS2 (1.5%), CMS3 (10%), CMS4 (3.5%), and unclassified CRC (9.5%) (p<0.0001). In addition, BRAF positive tumors were more frequently observed within the CMS1 group (12.2%, p =0.0065) and KRAS positive tumors within the CMS3 group (31%, p<0.0001). Consistent with prior reports, TILs/HPF were significantly higher in the CMS1 group (mean=7.7, p<0.0001). Similar statistically significant trends were observed across classes for TCRs/cell (mean=0.16, p=0.04). Conclusions: Subtypes of CRC have distinct histopathologic and molecular features that can be distinguished by expression profiles and immunoSEQ.

#2333

Aspirin may extend biliary tract cancer survival: Results from population-based cohort.

Sarah S. Jackson, Bin Zhu, Ruth Pfeiffer, Zhiwei Liu, Shahinaz Gadalla, Jill Koshiol. _National Cancer Institute, Bethesda, MD_.

Background: Biliary tract cancers (BTCs) are rare, but lethal cancers with a median survival of <12 months. Because of this poor prognosis, there is a critical need for treatments that extend survival. Cyclooxygenase-2 (COX-2) is an enzyme responsible for producing prostaglandin E (PGE). PGEs increase mucin production and reduce bile flow in the gallbladder, leading to gallstones, a precursor to gallbladder cancer. Overexpression of COX-2 due to chronic inflammation from cholangiocarcinoma has been found to enhance PGE production and promote tumor growth. Previous research suggests that COX-2 inhibitors, such as aspirin, may slow cancer cell growth. We investigated aspirin use following diagnosis and overall survival in a large cohort of patients with BTCs.

Methods: All patients diagnosed with incident biliary tract cancer between 1990 to 2017 were selected from the population-based United Kingdom's Clinical Practice Research Datalink. Site-specific associations between aspirin use after BTC diagnosis and overall survival was analyzed using Cox proportional hazards models with aspirin use modeled as time-dependent, adjusting for age at diagnosis, sex, comorbidities, history of aspirin use, and use of statins, using follow-up time as the time scale.

Results: We identified 3,211 biliary tract cancer cases in which 2,694 (84%) deaths occurred after an overall median follow-up time of 5 months. The table presents the number of cases by cancer subtype, deaths, and median survival time. Aspirin use following a diagnosis of BTC was associated with a reduction in mortality for each of the BTC sites: gallbladder, HR 0.41 (95% CI: 0.28, 0.61); cholangiocarcinoma, HR 0.46 (95% CI: 0.36, 0.60); ampulla of Vater, HR 0.24 (95% CI: 0.11, 0.51); and mixed, HR: 0.51 (95% CI: 0.33, 0.78).

Conclusions: The use of aspirin after BTC diagnosis was associated with a lower risk of overall mortality. These results warrant additional studies to understand the mechanisms underlying improved survival with aspirin use.

Table. Association between post-BTC diagnosis aspirin use versus no use and survival in the UK CPRD dataset

---

Cancer site | BTC patients

N (%) | Mortality

N (%) | Median survival time in months (interquartile range) | HR (95% CI)*

Gallbladder | 668 (23) | 555 (83) | 3.9 (2 - 11) | 0.41 (0.28, 0.61)

Cholangiocarcinoma | 1,560 (53) | 1321 (85) | 4.8 (2 - 12) | 0.46 (0.36, 0.60)

Ampulla of Vater | 224 (8) | 142 (63) | 9 (4 - 21) | 0.24 (0.11, 0.51)

Mixed | 484 (16) | 401 (83) | 5 (2 - 13) | 0.51 (0.33, 0.78)

*Adjusted for age at diagnosis, comorbidities, prior aspirin use, and use of statins.

Abbreviations: CPRD, Clinical Practice Research Datalink; CI, confidence interval; HR, hazard ratio; N, number; and UK, United Kingdom.

#2334

Intratumoral classical complement pathway promotes tumor growth in renal cancer.

Marie Daugan, Lubka Roumenina, Rémi Noé, Florent Petitprez, Catherine Sautès-Fridman, Wolf H. Fridman. _Inserm UMRS1138, Paris, France_.

Objective: The complement system has been considered for long as anti-tumoral effector in the context of monoclonal antibody therapy. Nevertheless, recent studies highlighted a pro-tumoral effect of complement, acting on angiogenesis, tumor cells proliferation and immunosuppression. The kidney is capable to produce a whole spectrum of complement components. Nevertheless, the role of complement activation for the progression of renal cancer remains poorly understood. In the present work we address the presence of complement components and their impact on the clinical outcome of patients with clear cell renal carcinoma (ccRCC). In addition, we evaluate the tumor growth of complement proteins-expressing tumor cell lines in complement-deficient mice.

Methods: The production of complement proteins by RCC lines was measured by RT-qPCR and by western blot (WB). Their expression in the tumor tissue was evaluated by immunohistochemistry and multicolor staining by immunofluorescence (IF) on sections from paraffin embedded tumors. The capacity of RCC cells-produced proteins to activate complement was evaluated by ELISA and western blot. The presence of complement was evaluated by immunohistochemistry on tumor sections from 3 retrospective cohorts composed by primary ccRCC patients (over 300 cases total). Complement producing tumor cell lines were grafted in WT and classical complement pathway deficient mice and the tumor growth was followed.

Results: We found that C1q is expressed in the ccRCC tumors by the infiltrating macrophages, having a particular phenotype. The remaining complement proteins are produced by the tumor cells. A dynamic interaction between the complement proteins produced by the tumor cells and the immune microenvironnement led to a local activation of complement revealed in vitro and in situ. The high level of C1q and complement activation correlated with a poor prognosis for patients. Moreover, the tumor growth was reduced in C1q-/- and C4-/- mice.

Conclusion: Early steps of the classical complement pathway can be activated locally and are associated with a pro-tumoral effect in mice models and in patients with renal cancer.

Keywords: renal cancer; C1q; classical pathway

#2335

Breast cancer cell-derived DNA activates STING pathway in macrophages.

GaYoung LIM, Na-Lee Ka, Mi-Ock Lee. _SEOUL National University , Seoul, Korea, Republic of, Seoul, Republic of Korea_.

Cell-free DNA (cfDNA) has been studied to be a diagnostic and prognosic marker. In breast cancer, however, there have been few studies on the biological role of cfDNA in the tumor microenvironment. Thus, investigating a role of cfDNA released from cancer cells to the tumor microenvironment is important in order to understand cancer biology. In this study, we showed that free DNA was a mediator that elicited inflammation and activated stimulator of interferon genes (STING) signaling, an essential pathway in cytosolic DNA-associated innate immune reponses in breast cancer. First, we found that mRNA levels of pro-inflammatory cytokines were increased in THP-1 macrophage cells co-cultured with MDA-MB-231 breast cancer cells. Second, the amount of free double strand DNA derived from MDA-MB-231 cells culture media was much more than THP-1 cells. Third, there was little induction of STING pathway in response to exogenous DNA in MDA-MB-231 cells, suggesting that cancer cells may not be sensitive to exogenous DNA for activating STING pathway. In contrast, STING pathway was activated in response to exogenous DNA in THP-1 cells. Taken together, cancer cell-derived DNA could be a factor to induce inflammation through activating STING pathway in tumor-associated macrophages.

#2336

Clinical-pathological characterization of the immunomodulator subgroup of triple negative breast cancer as a promising step in the selection of patients in personalized immunotherapies.

Ruben R. Bautista,1 Oscar Gerardo A. Rodríguez,1 Alfredo H. Miranda,2 Alejandro Z. Dehesa3. 1 _National Cancer Institute of Mexico, Mexico City, Mexico;_ 2 _National Institute of Genomic Medicine, Mexico City, Mexico;_ 3 _National Institute of Medical Sciences and Nutrition Salvador Zubirán, Mexico City, Mexico_.

Introduction: Triple Negative Breast Cancer (CMTN) represents 23.1% of breast cancer cases in Mexico, is defined as a tumor that is absent of hormone receptors (estrogen and progesterone) and low HER2 overexpression and clinically is characterized by its agressive tumoral behavior.

Lehmann et al. described an immunomodulatory (IM) group which is composed of genes for immune antigens and transduction pathways of immune signals and cytokines. Recent studies have shown that patients with the IM phenotype have a better prognosis. So it is important to continue with the description of this subgroup as the first step for personalized therapies in this heterogeneous tumor.

Methodology: Retrospective clinical study, patients with diagnosis of CMTN, attended at the National Cancer Institute of Mexico (Total: 69 patients) were included. Following, the extraction of RNA from the primary tumor tissue of CMTN paraffin blocks was performed to subsequently generate labeled cDNA for hybridization in Affymetrix® Human Gene 2.0 microarrays, to later carry out the scanning and analysis of the data. For the analysis of microarrays we used the free software R and Bioconductor.65. And then the classification of patients using the platform "TNBCtype (http://cbc.mc.vanderbilt.edu/tnbc/)". And finally the analysis of survival, univariate and multivariate through SPSS.

Results: We observed some clinical variables with statistical difference such as radiotherapy (p<0.001), metastasis to lung (p=0.009), liver (p=0.013), and central nervous system (p=0.0029), as well as the Lehmann classifier showed significant difference between patients classified as IM vs. No IM (p=0.04).

Conclusions: the IM classifier could not only serve as an excellent prognostic classification in CMTN but as a predictor of immunological treatments.

#2337

Changes in GDF15 (growth/differentiation factor 15) expression and M2 macrophages during prostate carcinogenesis.

Sudha M. Sadasivan,1 Yalei Chen,1 Nilesh Gupta,1 Sean Williamson,1 Dhananjay Chitale,1 Xiaoxia Han,1 Kevin Bobbitt,1 Andrew Rundle,2 Deliang Tang,2 Benjamin A. Rybicki1. 1 _Henry Ford Health System, Detroit, MI;_ 2 _Columbia Mailman School of Public Health, New York, NY_.

GDF15 (growth/differentiation factor 15), also known as MIC-1 (Macrophage inhibitory cytokine 1), is a divergent member of the TGFβ superfamily of cytokines and is highly expressed in prostate tumors, but its role in prostate carcinogenesis and utility as a prognostic biomarker is unclear. We studied 91 prostate cancer cases that underwent surgery as their primary treatment and were then subsequently followed for biochemical recurrence (BCR). These cases also had a benign prostate biopsy at least one year before their prostate cancer diagnosis. In both the benign biopsy and tumor specimens, we quantified the intensity of GDF15 expression and characterized the presence of tumor associated macrophages by measuring the density of CD68-positive stained cells, and the M2 macrophage marker CD204 by immunohistochemical analysis. Marker expression was measured in prostate a) benign biopsy (BS), b) tumor-adjacent benign (BGS) and c) tumor tissue (MGS) using an automated multi-image processing macro developed in the ImageJ software. A Cox proportional hazards model was used to test the association of time to BCR of the low expression group compared with medium or high biomarker expression. The risk of BCR differed with the extent of GDF15 expression in the prostatic epithelial tissue of benign and tumor regions of the prostate. For instance, in BGS, high expression of GDF15 was associated with increased risk of BCR (hazard ratio (HR)=2.58, p=0.07). After controlling for PSA at diagnosis, Gleason grade and pathologic stage, high expression of GDF15 in BGS had a stronger association with BCR (HR=3.30, p=0.04). The risk of BCR increased with increasing level of infiltration of CD204/CD68 positive macrophages in BGS (HR=4.19, p=0.02) and MGS (HR=4.16, p=0.01) even after controlling for PSA at diagnosis, Gleason grade and pathologic stage. The combined expression levels of GDF15 and CD204/CD68 showed that prostate cancer cases with small GDF15 expression changes between BGS and MGS and high CD204/CD68 expression in BGS were at 3-fold higher risk of BCR (HR=3.67, p=0.05). In summary, expression levels of GDF15 and CD204 M2 macrophages in different regions of the prostate can change as the disease progresses from benign to a tumorigenic state and serve as markers for aggressive disease. Further evaluation of these dynamic differences in the prostate immune cellular profile in the pre-malignant and malignant state may offer additional insight into inflammatory-mediated prostate carcinogenesis.

#2338

Checkpoint inhibitor-induced colitis shares mucosal histopathological features with inflammatory bowel and graft-versus-host disease.

Sara Hone Lopez, Gursah Kats-Ugurlu, Liesbeth De Vries, Marco De Groot, Marijn Visschedijk, Mathilde Jalving, Jacco de Haan. _Univ. Medical Ctr. Groningen, Groningen, Netherlands_.

Background: Checkpoint inhibitor-induced colitis (CIC) is a severe side-effect of immune-checkpoint inhibitors (ICI). Its pathophysiology is poorly understood, impairing improvement of prevention and treatment strategies. CD4+ and CD8+ T cells and CD68+ macrophages are thought to be critical in colitis development. Studies suggest that CIC is histopathologically similar to inflammatory bowel disease (IBD) and acute graft-versus-host disease (aGVHD) colitis. However, the histopathology of these entities has not been directly compared. We aimed to improve knowledge on CIC by performing a direct immunohistochemical comparison of CIC, IBD and aGVHD.

Methods: Archival formalin fixed paraffin-embedded colon biopsies obtained during routine diagnostic procedures from consecutive, eligible, treatment-naïve patients with CIC, IBD and aGVHD (all n=20) were studied. Biopsies from patients without histopathological abnormalities served as controls (n=20). Immunohistochemical staining was performed for CD4+ and CD8+ T cells and CD68+ macrophages. In each biopsy 3 mucosal areas with highest immune cell density were selected for cell counting. The number of cells/hotspot were classified as 0-10, 10-50, 50-100, 100-150 and >150. Next, the distribution of these cells was studied and compared between patient groups. We used descriptive statistics to depict and compare cell counts and infiltration patterns among patient groups.

Results: In CIC higher CD4+ and CD8+ T cell density (100-150 cells/hotspot in most patients) was observed compared to IBD, aGVHD and controls (50-100 cells/hotspot in most patients in each group). In contrast to controls, some IBD (12%) and aGVHD (15%) patients had a CD4+ T cell count of 100-150 cells/hotspot and some IBD (20%) and aGVHD (21%) patients had a CD8+ T cell count of 100-150 cells/hotspot. CIC, IBD aGVHD and controls shared a similar CD68+ macrophage count (50-100 cells/hotspot in most patients in each group). During cell counting we noted that cells infiltrated the superficial (subepithelium) and deep (lamina propria and muscularis mucosae) layers of the mucosa with either a scattered or patchy distribution. We recognized 4 distribution patterns: A) superficial diffuse, deep scattered/patchy; B) superficial scattered/patchy, deep diffuse; C) superficial and deep diffuse; D) superficial and deep scattered/patchy. The most prevalent infiltration pattern of CD4+ T cells was D in CIC patients (74%), C in IBD patients (47%), B in aGVHD patients (54%) and A in control patients (83%). The most prevalent infiltration pattern of CD8+ T cells was D in CIC (45%), and A in IBD (85%), aGVHD (63%) and all control patients. In most patients, CD68+ macrophages occurred in pattern A (56% of CIC, 58% of IBD, 59% of aGVHD, 100% of controls).

Conclusion: CIC has a distinct immunohistopathological pattern that shares key elements with both IBD and aGVHD.

#2339

High fat diet modulates host immunity and impairs the efficacy of combined radiation and immunotherapy.

Gregor Manukian, Brittany A. Simone, Tiziana DeAngelis, Kevin Ko, Charles Kivolowitz, Sylvester Jusu, Nicole L. Simone. _Sidney Kimmel Medical College of Thomas Jefferson University Hospital, Philadelphia, PA_.

Introduction: Obesity is associated with a higher incidence of cancer, increased cancer mortality, impaired responses to radiation therapy (RT), and worse toxicity after RT. Paradoxically, recent studies suggest that obese patients may actually have improved outcomes with immunotherapy (IO), such as programmed death (PD)-1 inhibition, highlighting a link between the host's metabolic state and immune homeostasis. We therefore hypothesized that a high fat diet could be used to alter the host's immune response to improve the efficacy of combined RT and IO.

Materials/Methods: 12-week old female Balb/c mice were injected with 5x104 syngeneic, luciferase tagged, triple-negative, murine breast cancer cells (4T1). Upon development of a palpable tumor, the mice were randomized to either an ad lib (AL) or high fat (HF), defined as 45% calories from fat, diet. Additionally, each cohort was treated with either diet alone (D), diet plus RT (4 Gy in 3 daily fractions), diet plus PD-1 inhibitor, or a combination of all three (Combo). On day 26, the mice were injected with luciferin and tumor growth was measured using the IVIS bioluminescence in vivo imaging system. At time of sacrifice, primary tumors and spleen were harvested for downstream analysis of immune markers via RNA microarray and flow cytometry.

Results: The median overall survival (OS) for animals fed either an HF or AL diet was 36 and 39 days, respectively (NS). The difference in OS however became much more apparent between the individual treatment arms: HF+αPD1 vs AL+αPD1 (37d vs 41.5d, p=0.041); HF+RT vs AL+RT (36d vs 45d, p=0.039); HF+Combo vs AL+Combo (39d vs 48d, p=0.037). HF fed mice also developed metastases sooner than AL fed mice across all arms (D: 22d vs 30d; RT: 24d vs 32d; αPD1: 24d vs 34d; Combo: 27d vs 37d, p=0.047). Microarray analysis of primary tumor tissue revealed an at least 1.5 fold increase expression of cytokines associated with inflammation including IFN-y, TNF-α, IL-17F, IL-22 and IL-1β in the HF diet groups while AL fed mice demonstrated increased expression of the anti-inflammatory cytokines IL-10 and TGF-β. Consistent with the increase in inflammatory markers after a HF diet, flow cytometric analysis of splenocytes revealed a significant decrease in the number of CD4+CD25+Foxp3+ regulatory T cells in the HF group compared with AL fed mice (3.3% versus 7.68%, respectively; p = 0.006). Notably, these Tregs also produced less IL-10 compared with those from AL mice (24.4% vs 54.5%, p=0.037).

Conclusions: We demonstrate that a HF diet impairs the efficacy of RT, IO, and combined therapy. This is associated with the development of a pro-inflammatory state demonstrated by increased expression of pro-inflammatory cytokines and a decrease in the number and activity of regulatory T cells. Based on these findings, we conclude that diet can directly impact treatment efficacy and should be considered as a variable in future clinical studies.

#2340

Differentiation of pro- and anti-inflammatory features of monocyte-derived dendritic cells.

Ragnhild Maukon Bakke,1 Waqas Azeem,2 Silke Appel,1 Karl-Henning Kalland,1 Anne Margrete Oyan2. 1 _University of Bergen, Bergen, Norway;_ 2 _Helse Bergen, Bergen, Norway_.

Background: Dendritic cells (DCs) are the most potent antigen-presenting cells of the immune system and are consequently exploited in cancer immunotherapy in order to present tumor-associated antigens to lymphocytes. The ability of tumor cells to induce tolerogenic DCs and avoid the immune system challenges DC-based immunotherapies. Utilizing buffycoats of healthy blood donors we have investigated the activation of gene expression programs conventionally associated with either pro- or anti-inflammatory features of monocyte-derived DCs.

Methodology: Following isolation of monocytes from buffycoats, various culture conditions were tested to generate immature DCs and to mature and activate them in vitro. Genome-wide gene expression, secretion of cytokines and tryptophan metabolites, surface expression of maturation and activation markers, and functional abilities were analyzed. Specifically, beta-catenin and indoleamine 2,3-dioxigenase 1 (IDO-1) activation were examined using both agonists and inhibitors of those pathways.

Results: A considerable variability among buffycoats of different healthy blood donors was observed regarding DC yield and cytokine secretion. Induction time to generate immature DCs with interleukin-4 and granulocyte-macrophage colony-stimulating factor affected DC differentiation. Several maturation conditions induced strong IDO-1 expression. Activation of beta-catenin during DC maturation induced anti-inflammatory features that could be competed by adding beta-catenin inhibitors during cultivation of the cells. Pro- and anti-inflammatory features often co-existed in mature DCs.

Conclusions: Beta-catenin and IDO-1 activation are prevalent according to different conditions of monocyte-derived DC maturation and associated with anti-inflammatory features. Both pathways can be inhibited by specific antagonists. More robust pro-inflammatory DC generation is desirable for more effective cancer immunotherapy.

#2341

Indoleamine 2,3 dioxegenase 1 (IDO1) and T-cell infiltration in esophageal cancer.

Ari J. Rosenberg, Derek A. Wainwright, Angela J. Fought, Lijie Zhai, Kristen Lauing, Victoria M. Villaflor. _Northwestern University Feinberg School of Medicine, Chicago, IL_.

Background: Immune checkpoint blockade is emerging as an important immunotherapeutic strategy in esophageal cancer, yet response rates remain low. IDO1 is a rate-limiting immunosuppressive enzyme that may contribute to resistance to immune checkpoint blockade in human cancer, and may be an important immunotherapeutic target. The expression pattern of IDO1 across esophageal cancer histologies remains poorly understood. Here, we utilize immunohistochemistry to characterize the expression pattern and relevance of IDO1 in esophageal cancer.

Methods: Tumor microarray containing samples of surgically resected esophageal carcinoma and normal esophageal tissue were stained for IDO1 and CD3ε (marker of T-cell infiltration). Demographic and staging data were collected. Light microscopic immunoscoring analysis was performed. Correlations were analyzed using Fisher's exact test.

Results: 192 samples were assessed (180 esophageal carcinoma, 12 normal esophageal tissue.) Median age was 60 years (range 34-78). 69.3% of patients were male. Of the 180 esophageal carcinoma samples, 150 (83.3%) squamous cell carcinoma, 16 (8.9%) adenocarcinoma, 13 (7.2%) small cell carcinoma, 7 (3.9%) undifferentiated carcinoma, 2 (1.1%) carcinoid, 2 (1.1%) adenosquamous carcinoma, and 2 (1.1%) adenoid cystic carcinoma. IDO1 was positive in 81 samples (45.0%) overall with 52.0% in squamous cell carcinoma and 46.7% in adenocarcinoma. Positive IDO1 was associated with higher stage and nodal positivity (P=0.04). T-cell infiltration in esophageal carcinoma specimens was present in 133 samples (73.9%). IDO1 positivity correlated with increased T-cell infiltration (P<0.01).

Conclusions: Here our study suggests that expression of IDO1 is positive in almost half of esophageal tumors, and is associated with T-cell infiltration. Additionally, positive IDO1 was associated with higher likelihood of nodal involvement and more advanced stage. These data suggest that IDO1 may play a role in adaptive immune resistance in T-cell inflamed tumors, and that incorporating IDO1 inhibition into an immunotherapeutic strategy in esophageal cancer may enhance T-cell mediated anti-tumor effect.

#2342

Experimental modeling of acute- and chronic-GvHD by xenotransplanting human donor PBMCs or cord blood CD34+ cells (HSC) into NSG mice.

Ann E. Lin,1 Annie X. An,1 Mingfa Zang,1 Derron Yu,1 Eunmi Hwang,1 Israel Romero,1 Linda Quirino,1 Marybeth George,2 Pirouz Daftarian,2 Wenqing Yang,3 Henry Li1. 1 _Crown Bioscience, La Jolla, CA;_ 2 _JSR Micro, Inc., Sunnyvale, CA;_ 3 _Crown Bioscience, Taicang, China_.

Graft vs. host diseases (GvHD) accounts for 15-30% of deaths following allogenic hematopoietic stem cell transplantation (allo-HSCT) for treatment of malignant diseases. Acute GvHD (aGvHD) typically involves skin, gastrointestinal and hepatic inflammation, and occurs within 100 days of transplantation; Chronic GvHD (cGvHD) involves multiple organs and occurs beyond 100 days. aGvHD is largely due to the rapid activation of donor T cells (Th1, CD8+ biased), causing tissue damage and often leading to mortality; in contrast, cGvHD is Th2 biased, typically display autoimmune-like syndrome, involving both T- and B-cell. Currently, GvHD models are mostly allo-transplantations between mice, which are criticized for poor physiological relevance. Here, we present Xenotransplantation of human donor into NSG mice, which will address some of the limitation of current models. Human peripheral blood mononuclear cells (PBMC) derived from normal donors were transplanted into NSG or NSG-like strains for modeling aGvHD. Cord blood derived HSCs (hCD34+) NSG (Jackson) were transplanted for modeling cGvHD. Hosts were monitored twice weekly, including clinical observations (e.g. animal postures, activity, fur texture, and skin integrity), body weight changes, gross pathology and histopathology upon termination, along with human-immunological phenotyping of peripheral blood, spleen, lung, liver and skin by flow cytometry, histopathology and/or immunohistochemistry. In addition, GvHD novel biomarkers Elafin, IL-18, REG3α and ST2, measured by ELISA (MBL International) at baseline and post cells engraftment. NSG mice engrafted with human PBMC, or purified T-cells, from normal donors rapidly developed typical symptoms of aGvHD, as early as 4 weeks post transplantation, including severe body loss, reduced activity, hunched posture, loss of fur, severe ruffling and overall poor grooming. Significant engraftment of human CD45+ cells were detected up to 6 weeks post-engraftment dominated with CD3\+ human T-cells of single positive CD4+ or CD8+ T-cells. The corresponding kinetics of clinical symptoms in parallel with the degree of engraftment of T cells suggests xenografting were responsible for aGvHD. As for the cGvHD, NSG-mice engrafted with CD34+ cells derived from cord blood with cGvHD high- risk HLA haplotypes C*0602 and C*0401. Manifestation of cGvHD was observed 18 weeks to 39 weeks post-engraftment; symptoms were similar to aGvHD, but also include facial/full body alopecia, and scaly skin. Interestingly, in correlation of this, human engraftment of CD45+, particularly T-cells including CD4+ and their CD30+ subsets, are kinetically increased in blood and spleen with similar timeline, suggesting their roles in the observed cGvHD. The xenograft murine model using adult human PBMC and cord blood derived CD34+ HSCs could be alternative experimental systems to model human aGvHD and cGvHD for investigating disease mechanisms and evaluating treatment strategy.

#2343

Immunogenetics of cancer and aging.

RMichael Williams,1 Edmond J. Yunis2. 1 _Northern California Cancer Research Institute, Beverly Hills, CA;_ 2 _Dana Farber Cancer Institute Harvard Medical School, Boston, MA_.

Antigen altered idiotype regulates specific immune responses (Cell Immunol. 1978: 4). Mendelian segregation of a given +/- phenotype defines an immune response gene (Benacerraf, Nobel). Autoimmunity producing anti-self clones must be eliminated or specifically suppressed, so a large portion of the antigenic repertoire resembles allogeneic MHC molecules (Medawar, Nobel). We proposed that exposure to many different allogeneic HLA molecules could cause specific immune suppression within the repertoire of epitopes and that these blank spots or holes in the universe of target antigens eventually would overlap with specificities including targets for eliminating pathogens such as viruses or tumor antigens. Some "holes" become large enough to result in an acquired immunodeficiency syndrome (AIDS) The opposite is also possible (Allogeneic Effect, Transplant Rev. 1972, 12; 141). This requires viable cells and cytokines released by host and/or donor. IR-genes for tumor resistance localize to the MHC. Class I/II MHC genes (Cancer Res. 1982; 35:1586) and NK cell receptor ligands (J. Stem Cell Res. 2007), regulate adaptive and innate immunity. We examined the combination of HLA specificities and the alleles of ligands of receptors of NK cells in breast cancer patients, HIV non progressors, and centenarians in Mexico. Immunological evaluation (CD4+,CD8+,CD19+ & NK cells and quantitative IgM, IgG, & IgA in breast cancer (BCA) patients in California. Overall survival (OS) depended on specificities (idiotypes) and the regulation of innate and adoptive immunity. The BCA patients and centenarians did not have HIV infection. People with more CD4 cells, more NK cells and more IgG, survive longer than those with the opposite immune profile. The immune specificity repertoire has evolved to avoid fatal autoimmunity, all orchestrated by T regs via T-cell antigen receptors and variable regions of Ig. The antigen receptors (CAR-T) and Ig variable region antigen receptors (Jerne, Nobel) are idiotypes, Single amino acid (AA) changes become altered "antigens" of the history of the patient: environment (e.g., microbiome), vaccination (e.g., BCG), and virus infections (Sendai or CMV in mice, HIV and/or others like HPV in man). This is true for single AA changes in both mice (H-2 mutants) and man; NK cell receptor ligands (Thr v Met). The results show profound effects on survival. Data, including MHC and NK receptor ligand phenotypes, OS and immunological evaluation of patients/people will be presented. Immunogenetic profiles of cancer survivors and long lived individuals are similar to those of HIV long term non progressors. Our data support our Immunogenetic Theory of Cancer and Aging.

#2344

The cGAS-STING pathway is a therapeutic target in a preclinical model of hepatocellular carcinoma.

Martin K. Thomsen. _Aarhus University, Denmark_.

Activation of the STING pathway is currently being explored in cancer immune therapy. The first STING agonist tested was DMXAA, but failed in clinical trial, since it does not bind human STING. The second generation of STING agonists are cyclic di-nucleotides (CDNs), either the naturally produced 2'3' cyclic GMP-AMP or derivatives, and many of these has proven to have high affinity for all common human variations of STING. In our work, we have focused on 3'3'-cyclic 3'3'-cAIMP (Invivogen) for analysis of anticancer activity in mice.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer, and the incidence of HCC is increasing. We have used a mouse model of mutagen-induced HCC to explore the therapeutic usefulness of targeting the DNA-activated STING pathway in HCC. STING-deficient mice exhibited unaltered initial development of HCC, but had higher number of large tumors at late stages of disease. In the liver of STING-deficient HCC mice, we observed reduced levels of phospho-STAT1, autophagy, and cleaved caspase 3. These responses were activated in the liver by treatment with a cyclic dinucleotide (CDN) STING agonist. Importantly, CDN treatment of mice after HCC development efficiently reduced tumor size. Initiation of CDN treatment at an even later stage of disease to allow HCC detection by MR scanning, revealed that the majority of tumors regressed in response to CDN, but new tumors were detected, which were unresponsive to CDN treatment. Conclusion: modulation of the STING pathway impacts on development of HCC, and holds promise for use in treatment of this disease, most likely in combination with other immunomodulatory treatments such as PD1 inhibitors or with standard of care.

#2345

Colorectal peritoneal metastases: Exploring the immune landscape and the potential of immunotherapy using organoid models.

Vignesh Narasimhan,1 Toan Pham,1 Kasmira Wilson,1 Susan Woods,2 Daniel Worthley,2 Jeanne Tie,1 Michael Michael,1 Alexander Heriot,1 Robert Ramsay1. 1 _Peter MacCallum Cancer Centre, Melbourne, Australia;_ 2 _SAHMRI, Adelaide, Australia_.

Background: Colorectal cancer (CRC) is the third most common cancer worldwide. Up to half the patients with CRC will develop metastatic disease, with an associated 5-year survival of 13.5%. The peritoneum is a common site for metastases, but carries the worst prognosis among all sites. Most patients with colorectal peritoneal metastases (CRPM) are inoperable, leaving systemic chemotherapy as the only treatment option. With systemic therapy alone, only 4% of patients with CRPM are alive at 5 years. Immunotherapy, while effective in a number of cancers, remains unexplored in peritoneal disease. Here, we aim to firstly explore the immune landscape of CRPM, and subsequently evaluate the role of immunotherapy in CRPM using organoid models.

Methods: Fresh microsatellite stable CRPM tissue from 25 patients was used for flow cytometry and multi- spectral immunohistochemistry (OPAL) to assess the immune cell infiltrate and density in the tumour microenvironment. Remaining tissue was used to develop organoid models, and to culture and expand patient matched tumour-infiltrating lymphocytes (TILs). To evaluate the immune profile further, organoids were exposed to interferon gamma for 48 hours and stained for MHC-I and PD-L1. Organoids were co-cultured in a cytotoxic assay with TILs to assess the cytotoxic ability of the lymphocytes. Organoids were then co-cultured with TILs with the addition of an anti PD-1 antibody to assess the role of checkpoint blockade. To assess TIL specificity, organoids were co-cultured with TILs with MHC-I antibody, and FACS sorted populations of CD8+, CD4+, CD56+ and CD3+CD56+ cells.

Results: FACS analysis revealed a heterogenous population of immune cells in CRPM with a significantly higher T cell infiltrate compared to a predominantly myeloid cell infiltrate in normal peritoneum (T cell 30% v 11%, p <0.05; myeloid cells 20% v 64%, p<0.05). There were high levels of T cell memory and activation markers such as CD45RO, CD69 and HLA-DR. PD-1 expression on T cells, whilst variable, was upto 75% in some patients, suggesting a possible role for anti PD-1 therapy in selected cases. OPAL demonstrated a significant immune infiltrate in the tumour microenvironment in many cases, with a strong stromal immune infiltrate. Co-culture of organoids and matched TILs demonstrated successful organoid killing by the TILs. In patients with high T cell PD-1 expression on FACS, addition of an anti PD-1 antibody revealed significantly increased cytotoxic killing of the organoids.

Conclusion and future direction: There appears to be a significant immune infiltrate in many cases of CRPM that can be potentially harnessed with the use of checkpoint blockade. These highly promising results need to be expanded with a larger cohort

#2346

Curcumin suppresses PhIP induced inflammatory protein expression in breast epithelial cells.

Ashok Jain. _Albany State Univ., Albany, GA_.

PhIP (2-Amino-1-methyl-6-phenylimidazo [4,5-b] pyridine), a prominent mutagen/carcinogen is a heterocyclic amine found in meat cooked at high temperature and in tobacco. PhIP is known to induce liver, colon, skin, prostate, and breast cancer as evident from several in vivo and in vitro studies. Our previous studies have demonstrated that curcumin significantly inhibits PhIP-induced DNA adduct formation and DNA double-strand breaks with the concomitant decrease in reactive oxygen species (ROS) production via multiple molecular targets. We hypothesize that PhIP induced ROS production induces inflammatory proteins and co-treatment with curcumin would suppress the induced inflammation. Therefore, human breast epithelial cells (MCF 10A) were used to test the expression of inflammatory proteins. MCF 10A cells were treated with PhIP alone and in presence of curcumin. Untreated cells and treated only with curcumin served as control. Human Inflammation array (Ray Biotech) was used to analyze the expression of forty inflammatory proteins. Results show that the expression of twenty inflammatory proteins is induced in MCF 10 A cells when treated with PhIP as compared to the controls. Presence of curcumin reduces the induced expression of these twenty inflammatory proteins. However, three inflammatory proteins (IL-8, IL 12p40 and ICAM-1) showed a significantly higher level of expression with PhIP and curcumin co-treated cells as compared to cells treated with PhIP alone. It is shown that higher expression of IL-8, IL 12p-40 and ICAM-1 is involved in chemotactic factor involve in attracting the neutrophils, basophils; activation of T and NK cells which might be helping in coordinating the inflammation process. Hence the present study suggests that curcumin is a promising natural compound to revert the effect of PhIP induced inflammation. 

### Molecular Mechanisms in the Immune Response to Cancer

#2347

NOD-like receptor NLRP12 suppresses hepatocellular carcinoma via regulation of JNK signaling.

Hasan Zaki. _UT Southwestern Medical Center, Dallas, TX_.

Hepatocellular carcinoma (HCC) is a deadly human cancer associated with chronic inflammation. The cytosolic pathogen sensor NLRP12, a member of NOD-like receptor, has emerged as a negative regulator of inflammation, but its role in HCC is unknown. Here we investigated the role of NLRP12 in HCC using mouse models of HCC induced by carcinogen diethylnitrosamine (DEN) or DEN plus hepatotoxin carbon tetrachloride (CCl4). Livers collected from wild-type and Nlrp12-/- mice at 10 and 5.5 months following DEN or DEN plus CCl4 treatment, respectively, were examined for tumor burden, histopathological changes, expression of inflammatory and tumor-promoting genes, and activation of signaling pathways. Primary hepatocytes from healthy mice were isolated, cultured, and used to assess cell signaling, apoptosis, and proliferation. Cancer genomics database analysis showed that NLRP12 expression is significantly reduced in human HCC and 2-3% HCC patients carry mutations in NLRP12. Nlrp12-/- mice were highly susceptible to DEN or DEN plus CCl4-induced HCC with increased inflammation, hepatocyte proliferation, and tumor burden. Consistently, Nlrp12-/- tumors showed higher expression of proto-oncogenes cJun and cMyc and downregulation of tumor suppressor p21. Interestingly, antibiotics treatment dramatically diminished tumorigenesis in Nlrp12-/- mouse livers. Signaling pathway analyses demonstrated higher JNK activation in Nlrp12-/- HCC and cultured hepatocytes during stimulation with microbial pattern molecules. Inhibition of JNK activation or NLRP12 overexpression reduced proliferative and inflammatory responses of Nlrp12-/- hepatocytes. In summary, this study demonstrated for the first time that NLRP12 negatively regulates HCC pathogenesis via downregulation of JNK-dependent inflammation and proliferation of hepatocytes.

#2348

BRD4 regulates the expression of Livin and thereby confers resistance to Fas-mediated immune cytotoxicity in aggressive B-cell lymphoma.

Sugihara Eiji,1 Norisato Hashimoto,2 Satoru Osuka,3 Sayaka Ueno,4 Takatsune Shimizu,5 Shogo Okazaki,6 Taka-aki Sato,1 Shinichiro Okamoto,4 Hideyuki Saya4. 1 _University of Tsukuba, Ibaraki, Japan;_ 2 _Keio University, Tokyo, Japan;_ 3 _School of Medicine and Winship Cancer Institute, Emory University, Atlanta, GA;_ 4 _Keio University School of Medicine, Tokyo, Japan;_ 5 _Hoshi University, Tokyo, Japan;_ 6 _Tokyo University of Science, Chiba, Japan_.

Aggressive types of non-Hodgkin's lymphoma (NHL) include Burkitt's lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL), which are germinal center (GC) B-derived lymphomas. The death receptor Fas is expressed in GC B-lymphocytes and contributes to the elimination of nonspecific and autoreactive B cells. Fas has been implicated in lymphomagenesis on the basis of its pro-apoptotic function and in autoimmune lymphoproliferative syndrome, with germline mutations in the Fas gene having been found to predispose individuals to the development of lymphoma. However, the possible mechanisms underlying the impairment of Fas-mediated apoptosis induction during human lymphomagenesis have remained unknown. In the present study, we established a mouse model of mature B-cell lymphoma based on transplantation into recipients of ex vivo-induced GC B-like cells derived from Ink4a/Arf-/- mice and transduced with MYC. Overexpression of MYC and deletion or silence of INK4A/ARF (also known as CDKN2A) are frequently found in both BL and DLBCL. The downregulation of Fas was necessary for both lymphomagenesis and lymphoma cell survival in vivo, suggesting that GC-derived lymphomas require persistent Fas downregulation to evade antitumor immunity by Fas ligand (FasL)-expressing cells. CD40 signal activation in lymphoma cells significantly restored the expression of Fas and thereby induced apoptosis following FasL treatment. Similarly, the majority of human BL and DLBCL cell lines exhibited downregulated Fas, and its restoration by CD40 signal activation. While half of the lymphoma cell lines exhibited high sensitivity to Fas-mediated apoptosis, the remaining cell lines exhibited resistance to it. We found that an inhibitor of the apoptotic protein Livin was responsible for the resistance to Fas-mediated apoptosis and that a high expression of Livin is a poor prognostic factor for patients with BL and DLBCL. We further demonstrated that a bromodomain and extra-terminal (BET) protein BRD4 (bromodomain 4) drives the expression of Livin through its direct binding to the proximal region of the Livin gene promoter, suggesting that the expression of Livin is epigenetically regulated in refractory human lymphoma cells to protect them from immune cytotoxity. Accordingly, the combination of CD40-mediated Fas expression with the targeting of Livin or BET inhibitors is a potential immunotherapeutic strategy for treating aggressive B-cell NHLs.

#2349

Modulation of inflammation in the tumor microenvironment by ER and PPARγ in an NNK-induced lung carcinogenesis model.

Erika Louiselle, Oshin Miranda, Huiyu Li, MiKayla Boeder, Zhijie Li, Jill M. Siegfried, Mariya Farooqui. _University of Minnesota, Minneapolis, MN_.

Lung cancer is the number one cause of cancer-related fatalities, and to date, there are no FDA-approved chemoprevention options for lung cancer prevention. NNK, the principal carcinogen in cigarette smoke can induce mutations in oncogenes and can also act as an inflammatory mediator of the tumor microenvironment (TME) by promoting macrophage infiltration into the lungs. Macrophages play a key role in regulation of the lung TME. Pioglitazone, a synthetic peroxisome proliferator-activated receptor gamma (PPARγ) agonist belonging to the thiazolidinediones (TZDs) drug class has been used in preclinical studies to mitigate lung tumorigenesis, progression, and metastasis. A retrospective analysis found that diabetics using TZDs experienced a 33% reduction in lung cancer incidence. Cross-talk between PPARγ and estrogen receptor (ER) signaling has been previously reported, and ER has already been implicated as a driver of lung cancer severity. Both ER and PPARγ are also known to be functional in macrophages. Agents that block ER signaling such as fulvestrant, a complete ER antagonist, have shown activity against lung cancer. We therefore tested the ability of pioglitazone and fulvestrant in combination to reduce lung tumor formation and to modulate the TME. Preliminary results from a co-culture simulation of the TME in vitro using human macrophages and lung adenocarcinoma cells show pioglitazone and fulvestrant together can significantly suppress inflammatory modulators such as IL-1β, IL-10, Amphiregulin (AREG), and VEGF compared to single treatments. Similar effects were observed in a mouse model system of the TME. This drug combination also reduced number of tumor cell colonies in a soft agar assay, suggesting a direct effects on tumor cells. In vivo use of combined pioglitazone and fulvestrant in an NNK female mouse model of former smokers showed a significant reduction in tumor size by 40% compared to placebo (p= 0.0010), 13% by pioglitazone alone and 5% by fulvestrant alone. Inflammatory cytokine array analysis of the bronchio-alveolar lavage fluid (BALF) revealed maximum down-modulation of pro-tumor inflammatory mediators with combination treatment compared to single treatments. Fulvestrant alone did not suppress tumor-promoting inflammatory pathways, suggesting possible activation of compensatory signaling pathways. Further analysis of the cell cultures and the BALF confirmed EGFR compensatory activation through release of AREG with fulvestrant single treatment, that is suppressed with the addition of pioglitazone. Thus, the strong combination effect of pioglitazone and fulvestrant on lung tumor growth and macrophage function, and the ability of the combination to suppress compensatory signaling pathways, supports the hypothesis of ER/PPARγ cross-talk in lung cancer and provides a rationale for further investigation of chemopreventive effects of this combination.

#2350

Type I interferon suppresses tumor growth through activating the STAT3-granzyme B pathway in tumor-infiltrating cytotoxic T lymphocytes.

Chunwan Lu, John D. Klement, Mohammed L. Ibrahim, Priscilla S. Redd, Gang Zhou, Kebin Liu. _Augusta University, Augusta, GA_.

Type I interferons (IFN-I) have recently emerged as key regulators of tumor response to chemotherapy and immunotherapy. However, IFN-I function in immune cells in the tumor microenvironment is largely unknown. We determined that IFNα13 and IFNβ are selectively expressed in colon carcinoma tissues in vivo. Although IFNAR1 KO mice exhibited increased susceptibility to carcinogen and inflammation-induced sarcoma and colon tumorigenesis, IFNAR1-sufficient tumors grow significantly faster in IFNAR1 KO mice and in mice with IFNAR1 deficiency only in T cells, suggesting that IFN-I functions in T cells to enhance host cancer immunosurveillance. Surprisingly, unlike in an anti-viral immune response where IFN-I regulates T cell activation and clonal expansion, IFNAR1-deficient T cells exhibit no deficiency in generation of antigen-specific CTLs and IFNAR1-deficient CTLs infiltration level is similar as compared to that of WT CTLs in colon tumor-bearing mice. Gene expression profiling identified Gzmb as an IFN-I target gene in tumor-infiltrating CTLs and in antigen-specific CTLs in vivo. Mechanistically, we determined that IFN-I activates STAT3 that directly binds to the Gzmb promoter to activate Gzmb transcription in CTLs. IFN-I also regulates GZMB expression in human T cells, but IFNAR1 is not only significantly down-regulated in human colon carcinomas but also down-regulated in CTLs of human colon cancer patients as compared to normal colon and CTLs from healthy donors. Our data determined that IFN-I plays a more dominant role in CTLs than in tumor cells to execute its anti-tumor function, and doing so through activating the STAT3-granzyme B pathway in tumor-infiltrating CTLs.

#2351

AXL and c-MET cross-talk in the regulation of PD-L1 expression on renal cancer cells.

Murugabaskar Balan,1 Toni Choueiri,2 Soumitro Pal1. 1 _Boston Childrens Hospital, Boston, MA;_ 2 _Dana Farber Cancer Institute, Boston, MA_.

Purpose: The purpose of this research is to study the cross-talk between receptor tyrosine kinases (RTKs) c-MET and AXL in renal cancer cells; and to explore if the blockade of AXL-c-MET pathways can restrict PD-L1-mediated growth and immune escape of renal cancer cells.

Background: c-MET and AXL are overexpressed in renal cell carcinoma (RCC). Upon binding with their respective ligands hepatocyte growth factor (HGF) and GAS6, c-MET and AXL activate a wide range of pathways, including tumor promoting events. Renal tumors show increased T cell infiltration, yet the T cells are rendered incapable of tumor rejection. The co-inhibitory molecules PD-1 and PD-L1 belong to cell surface glycoprotein B7 family that governs T cell function and proliferation. In RCC, tumor-infiltrating T cells express PD-1 and are associated with poor outcomes for patients. Similarly, PD-L1 expression on renal cancer cells hinders prognosis and indicates tumor aggressiveness. Therefore, PD-L1 in renal tumors may play a significant role in down-regulating host anti-tumor immune responses. Our recent report suggests that c-MET activation induces overexpression of PD-L1 in renal cancer cells; and the blockade of PD-L1 promotes immune-mediated killing of renal cancer cells. However, the cross talk between AXL and c-MET in the regulation of PD-L1 has not been studied in RCC.

Results: We have confirmed that both AXL and c-MET are overexpressed in human renal cancer cells (786-O and ACHN) when compared with normal renal tubular epithelial cells. We performed co-immunoprecipitation assays with c-MET antibody and found that c-MET forms a complex with AXL in renal cancer cells. We utilized siRNA-mediated gene silencing approach and found that the knock-down of AXL increased the sensitivity of 786-O and ACHN cells to apoptosis following treatment with XL-184 (a small molecule inhibitor of c-MET, AXL and VGFRs, obtained from Exelixis) as measured by flow cytometry in Annexin V/PI stained cells. We also found that GAS6 treatment enhanced HGF-induced expression of cytoprotective molecule Heme oxygenase-1 (HO-1) and anti-apoptotic molecules Bcl-2 and Bcl-xL in renal cancer cells. In both 786-O and ACHN cells, c-MET activation increased the cell surface expression of PD-L1 as measured by flow cytometry. Interestingly, GAS6 treatment further increased the expression of PD-L1, suggesting a synergistic action of RTKs AXL and c-MET in renal cancer cells. We also utilized a PD-L1 promoter-luciferase plasmid to measure PD-L1 promoter activity and found that GAS6 treatment enhanced HGF-induced effects on PD-L1 transcription. In mouse renal cancer cells (RENCA), which express very high levels of PD-L1, we observed a significant decrease in the expression of PD-L1 when AXL was knocked down in cells.

Conclusion: Together, our results demonstrate that AXL-mediated signaling enhances c MET-induced cytoprotection and PD-L1 expression on renal cancer cells.

#2352

Epigenetic modulation— unlocking the potential of checkpoint inhibition in advanced breast cancer.

Christine Rafie, Kayla Cruz, Skylar Woolman, Todd Armstrong, Elizabeth Jaffee, Evanthia Roussos Torres. _Johns Hopkins University, Baltimore, MD_.

Immune checkpoint inhibition (ICI) has revolutionized treatment in immunogenic cancers by enabling infiltration of T cells into the tumor microenvironment (TME) and promoting cytotoxic signaling pathways. Tumors with complex immunosuppressive TME's such as breast cancer present unique therapeutic obstacles as response rates to ICI remain low. Such tumors often recruit myeloid-derived suppressor cells (MDSCs) whose functioning prohibit both T cell activation and infiltration. Our current work aims to uncover the efficacy of ICI in advanced HER2 positive (HER2+) disease and to enhance response rates to these promising therapies by altering the metastatic TME epigenetically. Using a HER-2/neu transgenic mouse model, we syngeneically tumor challenge the NT2.5LM metastatic cell line to evaluate survival outcomes and metastatic burden upon treatment with combinations of the HDAC inhibitor entinostat (ENT), and the checkpoint inhibitors anti-PD-1 and anti-CTLA-4. We show that in the HER2+ mouse model of advanced disease, combining ENT + ICIs improves survival, and ENT + anti-CTLA-4 most significantly improves survival and decreases metastatic burden among responders. By investigating immune changes in sites of metastases, we show that treatment with ENT + ICIs significantly increases infiltration and proliferation of CD8+ T cells, increasing effector T cell infiltration, cytokine production, and markers of activation in cytotoxic T cells in the lung. Flow cytometry, ex vivo co-culture assays, western blots, and other functional assays performed on MDSCs and TIL elucidate further mechanisms behind response. We have found that the metastatic sites of animals treated with ENT + ICIs have significantly decreased infiltration of granulocytic-MDSCs and increased infiltration of monocytic-MDSCs, leading to the apparent cytotoxic anti-tumor response. In mouse models of early stage disease, ENT + ICI therapy alters MDSC infiltration and function in primary tumors, allowing for a more robust adaptive immune response. A significant anti-tumor effect is also seen in the metastatic state, though the function of MDSCs is not consistently altered, suggesting a key mechanism of ENT synergy with ICI to incite immune response and survival benefit that remains to be elucidated. In summary, addition of ICIs to ENT is beneficial in models of advanced disease by altering the recruitment of suppressive cells into the metastatic microenvironment, changing the dynamic interaction of T cells and tumor cells causing a robust anti-tumor response. These novel findings provide insight into how these combination therapies may function in patients with advanced stages of HER2+ breast cancer and suggest that responses are linked to stage of disease and likely follow different mechanisms of action within the different tumor microenvironments.

#2353

EZH2 Inhibition sensitizes prostate cancer to PD-1 blockade.

Leigh Ellis. _Dana-Farber Cancer Institute, Boston, MA_.

Prostate cancers are considered immunologically 'cold' tumors as they have demonstrated poor response to check-point inhibitor therapy (CPI). Recently, enrichment of interferon response genes suggests a favorable response to CPI across various disease sites. The enhancer of zeste homolog-2 (EZH2) is over-expressed in prostate cancer and is known to negatively regulate IFN response genes. Here, we demonstrate that inhibition of EZH2 catalytic activity in prostate cancer models increases expression of double-strand RNA (dsRNA), that is associated with upregulation of genes connected with antigen presentation, Th-1 chemokine signaling, and interferon (IFN) response genes, including PD-L1. Likewise, application of a novel EZH2 derived gene signature and TMA analysis indicated an inverse correlation between tumor EZH2 activity/expression with, T-cell inflamed and IFN gene signatures, and PD-L1 expression in human prostate cancer samples. Elevated PD-L1 tumor expression resulted in protection from splenocyte-mediated cytotoxic tumor elimination, which is rescued upon CPI. In vivo, EZH2 inhibition combined with PD-1 CPI to significantly enhance anti-tumor response associated with significant reprogramming of both tumor and immune infiltrating cells. Our study identifies EZH2 as a potent inhibitor of antitumor immunity and responsiveness to CPI. This data, suggests EZH2 inhibition as a novel therapeutic direction to enhance prostate cancer response to PD-1 CPI.

#2354

Targeted IRAK-M degradation as a novel and efficacious cancer-immunotherapy overcoming innate-driven immunosuppression.

Kanae Gamo, Naomi Kitamoto, Yoshihide Tomata, Yusuke Tominari. _FIMECS, Inc., Fujisawa, Japan_.

IRAK-M has an important role in tightly controlling innate immune responsiveness to preserve homeostasis, mediating immune tolerance, and acts as a negative feedback regulator of TLR/IL-1R signaling pathway. Targeting IRAK-M, which expression is restricted to myeloid cells, would be potentially limiting adverse events against non-target tissues. From supporting evidence for the role of IRAK-M in innate immunosuppressive capacity of tumor-associated macrophages (TAMs) or dendritic cells (DCs), we have developed compounds targeting IRAK-M as an effective cancer-immunotherapy strategy. Since IRAK-M is a pseudokinase which is characterized by the lack of conserved motifs involved in catalytic kinase activity, drug design for inhibition of IRAK-M has been challenging because the conventional small molecule could not modulate its function in cells. Therefore, we conceived and optimized heterobifunctional for degradation comprising IRAK-M-binding moiety linked to the our proprietary XIAP binders to eliminate the IRAK-M protein via ubiquitin-proteasome system. We demonstrated a concentration-dependent degradation of IRAK-M protein in THP1 human monocytic leukemia cells with degraders, and this degradation activity was confirmed to be proteasome-dependent by rescue of depletion following pre-incubation with the proteasome inhibitor epoxomicin. In addition, to estimate the cellular degradation selectivity of our degrader to IRAK-M in an unbiased manner, we carried out whole proteome mass spectrometry in THP1 cells and exhibited good selectivity against identified quantifiable proteins including IRAK-4. As functional studies, we performed MDSC suppression assays in a co-culture model with T cells and found IRAK-M degrader could release the suppressive function of MDSC. Lead optimization steps for IRAK-M degraders have conducted by applying RaPPIDSTM which is a proprietary divergent degrader synthetic platform and already identified multiple drug candidates within a year. Finally, in LLC Lewis lung carcinoma syngeneic model, treatment with candidate resulted in significant tumor growth inhibition. Pre-clinical toxicological studies have been entered and backup compounds are also in development. Taken together, these results highlight the promise of combined IRAK-M silent binder and targeted protein degradation technology as a novel therapeutic strategy for the cancer-immunotherapy that shifts the balance between tolerance and immunity by releasing immunosuppressive activity and in turn by unleashing tumor-specific T-lymphocytes.

#2355

IKKa serves as a molecular switch linking miRNA biogenesis and inflammation in skin tumorigenesis.

Feng Zhu,1 Jian Zhang,1 Jami Willette-Brown,1 Ling Su,2 Xiaolin Wu,3 Yinling Hu1. 1 _NCI, NIH, Frederick, MD;_ 2 _Leido Biomedical Research, Inc, Frederick National Laboratory for Cancer Research, Frederick, MD;_ 3 _Leido Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD_.

Our previous studies have shown that the absence of tumor suppressor IKKα leads to development of spontaneous mouse squamous cell carcinomas (SCCs) in the skin, lungs, and esophagus, which is partially mediated by aberrant epigenetic alterations and inflammation. However, it remains unknown whether there is a crosstalk between the above-mentioned mechanisms. As a part of the epigenetic machinery, miRNAs regulate many genes post-transcriptionally. Here we show that IKKα deletion in the mouse epidermis results in impaired miRNA processing with markedly reduced expression of ribonuclease Dicer, an enzyme required for miRNA maturation. We found that the lack of IKKα causes Dicer gene silencing through DNA methylation. As a result, the maturation of miR-203-3p, which is the most abundant miRNA in keratinocytes, is blocked in Ikkα-null keratinocytes. Next, we identified Myd88, an important component of innate immunity and inflammation, as one of miR-203-3p targets. The level of Myd88 expression is robustly increased in Ikkα-null keratinocytes due to the lack of miR-203-3p. We also observed increased activity of caspase 1 cleavage, an indicator of inflammasome activation, in Ikkα-null keratinocytes. Interestingly, Myd88 deletion or 5-aza-2'-deoxycytidine (DNA methyltransferase inhibitor) treatment rescued the survival of Ikkαf/f;K5Cre mice, which usually die within three weeks after birth. Ikkαf/f;Myd88f/f;K5Cre mice also develop much fewer spontaneous skin tumors. By analyzing a cohort of human head and neck SCCs from TCGA database, we found that Dicer expression is concurrently downregulated among 32% of patients with low IKKα expression. Taken together, our finding illustrated how IKKα loss mediates Dicer gene silencing, which turns off miRNA processing, including miR-203-3p maturation, and eventually triggers Myd88-dependent inflammation. Thus, our study revealed a novel IKKα/Dicer/miR-203-3p/Myd88 signaling loop, in which IKKα serves as a molecular switch linking microRNA biogenesis and inflammation in skin tumorigenesis.

#2356

Estrogen medicates sex specific function of epithelial STAT3 in K-ras mutant lung tumorigenesis by reprogramming lung tumor microenvironment.

Shanshan Deng, Marco Ramos-Castaneda, Walter Velasco Torrez, Oscar Noble, Neha Daga, Mauricio Caetano, Seyed Javad Moghaddam. _MD Anderson Cancer Center, Houston, TX_.

Activating mutations of K-ras are one of the most common molecular alterations associated with lung cancer but have not yielded to therapeutic attack so far. We have recently shown a sex-specific role for epithelial Stat3 signaling in the pathogenesis of K-ras mutant lung cancer. We specifically found that deletion of STAT3 in K-ras mutant lung epithelial cells (LR/STAT3Δ/Δ mice) significantly reduced lung tumor burden in female mice which was associated with an enhanced anti-tumor immune response but, surprisingly, caused a dramatic enhancement of lung tumorigenesis and induction of a NF-κB driven pro-tumor immune response in male mice. To further dissect the mechanism of STAT3 dependent sex disparity in the pathogenies of K-ras mutant lung cancer, we performed loss- and gain- of function studies to manipulate estrogen signaling in both female and male mice. In LR/Stat3Δ/Δ males, implantation of estrogen pellet led to significantly reduced lung tumor burden and decreased neutrophils in the lung. This was accompanied by decreased Il6, and Cxcl1 expression explaining reduced neutrophil numbers. We also detected reduced expression of immunosuppressive markers; Arg1, Nos2, Tgfb, Il10, indicating to an attenuated immunosuppressive pro-tumor microenvironment. On the other hand, bilateral oophorectomy in female LR/Stat3Δ/Δ mice led to higher tumor burden, higher lung neutrophil counts, and increased Il6, and Cxcl1 expression. We also found decreased expression of Gzmb, and Ifng, and increased expression of T regulatory markers Foxp3 and Il10, suggesting an attenuated cytotoxic immune response but a stronger immune-inhibitory response. Hormonal replacement therapy by implanting estrogen pellet in oophorectomized LR/Stat3Δ/Δ females rescued the tumor-promoting effect of estrogen depletion characterized by reduced tumor number and neutrophil infiltration, decreased expression of Il6, Cxcl1, Foxp3 and Il10, and increased Gzmb and Ifng expression. Taken together, our study suggests that there is a differential regulation of NF-κB activation in K-ras mutant lung epithelium within sex, and estrogen plays a protective role (anti-tumor) by suppressing the activation of NF-κB pathway and reformatting the lung tumor microenvironment toward an anti-tumor phenotype. Our finding could lead to development of personalized (e.g. sex-based) immunotherapeutic and/or preventive modalities for K-ras mutant lung cancer.

#2357

Identification of microRNAs associated with melanoma immunity and immunotherapy outcome.

Robert A. Szczepaniak Sloane, Miles C. Andrews, Guangchun Han, Lauren Haydu, Elizabeth Burton, Gershenwald E. Jeffrey, Michael A. Davies, Linghua Wang, Scott Woodman, Jennifer Wargo. _UT M.D. Anderson Cancer Center, Houston, TX_.

Introduction:Resistance to immunotherapy is a significant clinical challenge, preventing or limiting response to PD1 checkpoint blockade in approximately 60% of melanoma patients. We sought to identify microRNAs (miRs) that modulate the tumour immune microenvironment and potentially influence outcome to immunotherapy.Methods:To identify miRs associated with immune infiltration and exclusion in melanoma, we first compared miR expression in the human skin cutaneous melanoma (SKCM) dataset from The Cancer Genome Atlas (TCGA) with a selection of immune correlates including histologic assessment of tumour infiltrating lymphocytes (TIL), gene expression-based cytolytic score using Granzyme A and Perforin 1, and transcriptomic estimation of immune cell populations using CIBERSORT. In parallel, we performed miR sequencing of 22 pre-PD-1 inhibitor treatment melanoma tumors to identify differentially-expressed miRs according to PD-1 inhibitor response. We then tested the immune effects of candidate immunomodulatory miRs using microRNA mimics in a panel of human melanoma cell lines, evaluating immunomodulatory cytokine secretion using a bead-based immunoassay (LEGENDplex) and cell surface expression of HLA-A and PD-L1 by flow cytometry.Results:We identified 14 microRNAs associated with immune infiltration or exclusion in the TCGA melanoma dataset. In vitro characterisation of these miRs showed that they regulate secretion of IL-6 and VEGF, and expression of HLA-A in human melanoma cell lines. Furthermore, we identified 25 downregulated and 8 upregulated miRs in responder vs non-responder melanoma lesions (log2 fold change >=1, padj<0.1), including several miRs with high-confidence targets involved in oncogenesis, cancer germline antigens and modification of the extracellular matrix that may impact upon susceptibility to immune attack.Conclusions:This study identified several immunomodulatory miRs associated with immune infiltration in melanoma tumors of TCGA. Several of these candidate immunomodulatory miRs show evidence of activity relevant to the efficacy of PD-1 checkpoint blockade, whilst additional response/resistance-associated miRs were identified. These miRs represent strong candidates for ongoing in vitro and in vivo studies in combination with immunotherapy.

#2358

Porphyromonas gingivalis promotes colorectal cancer development by regulating NLRP3 inflammasome signaling.

Zhi Wang, Xi Wang, Yiqun Jia. _Sun Yat-sen Univ. School of Stomatology, Stomatological Hospital, Guangzhou, China_.

Metagenomic analyses indicate that Porphyromonas spp. is associated with human colorectal carcinoma (CRC), however, whether this is an indirect or causal link remains unclear. We found that Porphyromonas gingivalis (P. gingivalis) were enriched in human stool or FFPE samples from colorectal carcinoma patients compared to colorectal adenoma or healthy subjects. Two colorectal cohort studies demonstrated that P. gingivalis infection was associated with poor prognosis of CRC. Moreover, in the ApcMin/+ mouse model of colorectal tumorigenesis, P. gingivalis increases tumor counts and in the orthotopic rectal MC38 carcinoma model increases tumor growth. Furthermore, tumors from orthotopic MC38 model exposed to P. gingivalis exhibited a tumor-infiltrating myeloid cells recruiting signature that was confirmed in human P.gingivalis-positive colorectal carcinomas. We next evaluated the role of NLRP3 in P. gingivalis promoting on CRC progression. When comparing with wild type mice, the increased tumor growth was subtracted in Nlrp3-/- mice by the same orthotopic MC38 model exposed to P. gingivalis. To determine relevant cell compartment responsible for NLRP3 function, we generated Nlrp3-/- chimeric mice with adoptive bone marrow transplantation by using the same orthotopic tumor model. The results showed the effect of NLRP3 on P. gingivalis pathogenesis was from both hematopoietic and non-hematopoietic sources. Additionally, we performed expression profile to show that NLRP3 attenuates multiple pathways associated with P. gingivalis promoting colorectal cancer development. Collectively, these data suggest that, through recruitment of tumor-infiltrating immune cells by regulating NLRP3 inflammasome activity, P. gingivalis generates a proinflammatory microenvironment that is conductive for colorectal neoplasia progression.

#2359

The MCPyV st-ag and IL33/ST2 axis: critical role in merkel cell carcinoma.

Kashif Rasheed,1 Silje Fismen,2 Øystein Grimstad,2 Baldur Sveinbjørnsson,1 Ugo Lionel Moens1. 1 _The Arctic University of Norway, Tromso, Norway;_ 2 _University Hospital of Northern Norway, Tromso, Norway_.

Merkel Cell polyomavirus (MCPyV) is a causal factor in Merkel Cell Carcinoma (MCC). The oncogenic potential is mediated through its viral oncoproteins large T-antigen (LT-ag) and small t-antigen (st-ag). Many cancers arise from sites of infection, chronic irritation and inflammation, and inflammatory signaling pathways are often activated by oncogenic mutations. Chemokines are a family of cytokines that regulate leukocyte trafficking in immunity and inflammation. The aberrant expression of chemokines and chemokine receptors in tumors may regulate the trafficking of leukocytes into the tumor microenvironment. IL33, a member of IL-1 family cytokines, is involved in the response to viral infection and plays important roles in type-2 innate immunity, tumorigenesis and tumor immune evasion.

We assessed IL33/ST2 expression in MCPyV-positive (MKL2) and –negative (MCC13) MCC cell lines in order to examine a possible role of IL33/ST2 pathway in MCC.

Using the human RT2 Profiler PCR Inflammatory Cytokines and Receptors Array, we did a PCR array analysis of 84 different human cytokines and receptor genes. Expression of IL-33 and ST2 was determined by qRT-PCR, immunohistochemistry and western-blot analysis. Luciferase analysis was done to monitor the effect of MCPyV early proteins on IL33 promoter activity and the effect of IL33 on MCPyV early and late promoter activity. Furthermore, mitogen-activated protein kinase (MAPK) and the NF-κB pathways were also assessed after stimulating MCC13 cells with recombinant human (rh) IL33 protein. Finally, proliferation activity after rhIL33 stimulation was assessed by MTT-assay.

PCR array analysis showed higher transcript levels of IL33 in MCC13 cells transfected with st-ag compared to control cells. Western blot analysis confirmed increased IL33 expression by st-ag. Luciferase analysis showed higher IL33 promoter activity in st-ag transfected MCC13 cells. Interestingly, IL33 also increased MCPyV early and late promoter activity. Furthermore, rhIL33 activated both MAPK and the NF-κB pathways and blocking ST2 receptor using polyclonal goat ST2 antibody abolished IL33 activity. Dose-dependent effect of rhIL33 showed higher proliferation activity of MCC13 cells. Immunohistochemical analysis of IL33 and ST2 showed a significant stronger expression in MCC tissues compared to normal skin.

Our study unveiled a novel st-ag dependent IL33 function in MCC. Therefore, neutralizing the IL-33/ST2 axis may thereby represent a therapeutic target in MCPyV-positive MCC patients.

#2360

Targeting IL-4R alpha on tumor-associated macrophages as a therapeutic strategy for prostate cancer.

Amber E. De Groot, Kayla Myers, Sarah R. Amend, Kenneth J. Pienta. _Johns Hopkins Univ. School of Medicine, Baltimore, MD_.

Prostate cancer tumor growth and disease progression are highly influenced by non-cancerous host cells within the tumor microenvironment including immune cells. High prevalence of tumor-associated macrophages (TAMs) in tumor biopsies is associated with poorer outcomes. TAMs comprise as much as 50 percent of prostate cancer tumors making them an abundant and critical target for treating prostate cancer.

Macrophages adopt different functions in response to signaling molecules in their environment. The majority of prostate cancer TAMs resemble alternatively-activated M2 macrophages which, in healthy individuals, are primarily involved in wound healing. M2 TAMs behave similarly to wound-healing M2s by promoting cancer progression and immune suppression.

Macrophages are converted to M2s when the signaling molecules interleukin-4 (IL-4) or IL-13 bind IL-4 Receptor Type I or II. Both of these receptor complexes contain the IL-4 receptor alpha subunit and signal through STAT6 to regulate downstream gene transcription and conversion to M2. Due to the central role of IL-4R alpha in tumor-promoting M2s, we hypothesize that disrupting IL-4R alpha signaling will undermine the tumor-promoting capabilities of M2-like TAMs.

Using human macrophages cultured in vitro, we detect IL-4R alpha expression in non-malignant human macrophages by qPCR and immunoblot, critical data that is absent in the current literature. Additionally, using the FDA-approved antibody dupilumab, we show that antagonizing IL-4R alpha prevents STAT6 phosphorylation both before and after macrophages have been converted to M2s. Additionally, we show that dupilumab mitigates the M2 characteristics of these macrophages by nanostring analysis of M2-associated genes and ELISA of immunosuppressive cytokines. These data provide evidence that targeting IL-4R alpha on human macrophages impairs tumor-promoting M2 functions.

To assess IL-4R alpha expression on TAMs in vivo, we used the HiMyc and TRAMP transgenic mouse models. These mice develop spontaneous prostate cancer tumors and are more accurate representations of the primary tumor microenvironment than injected tumor models (i.e. subcutaneous and orthotopic models). Using nanostring analysis and immunohistochemistry of prostates from 8-week-, 6-month-, and 12-month-old mice, we assessed immune cell infiltration levels, M2 characteristics of TAMs, and IL-4R alpha expression of TAMs. This data provides evidence for the M2-like characteristics of TAMs and IL-4R alpha as a viable TAM target.

Subsequent experiments will include assessing IL-4R alpha expression on TAMs in human tumors using prostate cancer tissue microarrays. Moreover, we will continue to investigate the therapeutic potential of antagonizing IL-4R alpha through in vivo studies. In conclusion, these data implicate targeting IL-4R alpha as a promising therapeutic strategy for targeting TAMs in prostate cancer.

#2361

Progesterone receptor promotes immunosuppressive microenvironment in the mammary gland.

Merit Goodman, Gloria Trinca, Katherine Walter, Katelin Gibson, Prabhakar Chalise, Mary Markiewicz, Christy R. Hagan. _Univ. of Kansas Medical Ctr., Kansas City, KS_.

Why are some early neoplastic lesions identified through immunosurveillance and subsequently destroyed, whereas other tumors evade this clearance step and progress to clinically-relevant tumors? The cancer immunoediting hypothesis highlights that the innate and adaptive immune responses work together to "flag" early neoplastic lesions for immune-mediated elimination. An early mediator of this "elimination" process is activation of type I interferon signaling. Thus, suppression of type I interferon signaling may help developing tumors evade the critical early steps of immune recognition and clearance. Preliminary data from our lab suggest that progesterone, working through the progesterone receptor (PR), may be a key player in tumor immune evasion through downregulation of interferon signaling. In breast cancer cells, we have shown that presence/activation of PR can attenuate interferon-signaling at multiple steps within the signaling cascade: decreased STAT1 phosphorylation, decreased STAT1/STAT2/IRF9 transcriptional complex formation (called ISGF3, required for interferon-alpha signaling), decreased recruitment of ISGF3 to ISG promoter sequences, and decreased transcriptional activation of interferon-stimulated genes (ISG, end point of interferon signaling). These data suggest a concerted effort aimed at reducing interferon-signaling through activation of PR. These data translate to the immune microenvironment in the murine mammary gland. In mice treated with progesterone for 21 days, we see immunosuppressive changes in the mammary gland: increased T-regulatory cells (immunosuppressive), decreased frequency of antigen-presenting cells (decreased ability to present potential tumor-associated antigens to CD8 T-cells), and a decrease in MHC class I presentation in non-lymphoid cells (decreased ability for epithelial cells to present potential tumor-associated antigens). We observe similar phenotypes in mice with transgenic overexpression of PR. These changes in the immune system suggest that PR can promote immunosuppressive changes in the mammary gland. Moreover, 80% of these mice in a multiparous cohort develop mammary gland tumors. Therefore, early changes in the immune microenvironment in the mammary gland may translate to the development of mammary gland tumors in PR transgenic mice. Cumulatively, these data suggest a mechanism through which PR aids early breast cancer lesions in escaping immune surveillance. These data have significant implications for the use of progesterone-containing hormone replacement therapy, as well as underscore the importance of studying anti-progestins as novel chemo-preventative agents for breast cancer.

#2362

**NF-κB (p65) mediates interleukin-1 (IL-1) repression of androgen receptor (AR) in AR** + **prostate cancer (PCa) cell lines.**

Shayna Elizabeth Thomas-Jardin,1 Haley Dahl,1 Monica Bautista,1 Freedom Ha,1 Joan Jacob,1 Afshan Fathima Nawas,1 Mohammed Kanshwala,2 Chao Xing,2 Nikki Delk1. 1 _UT Dallas, Richardson, TX;_ 2 _UT Southwestern, Dallas, TX_.

Inflammation promotes cancer initiation and progression. The Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B cells (NF-κB) family of transcription factor proteins, p65 (RelA), RelB, c-Rel, p105/p50 (NF-κB1), and p100/52 (NF-κB2), form homo- and heterodimeric complexes that mediate inflammatory cytokine signaling. We previously reported that the inflammatory cytokines, interleukin-1 alpha (IL-1α) and IL-1 beta (IL-1β), repress androgen receptor (AR) levels and activity in AR+ prostate cancer (PCa) cell lines. AR drives PCa tumorigenicity and, thus, AR is a therapeutic target. Our data suggests that IL-1 selects for ARlow/- PCa cell subpopulations that can evade AR-targeting therapy. Using RNA sequencing, we identified an IL-1-regulated gene signature predicted to supported AR-independent tumorigenicity in PCa cells. Pathway analysis of the RNA sequencing data predicted that AR is a target molecule of p65 (RELA). p50:p65 heterodimers are known to mediate IL-1 signaling, therefore, we investigated if NF-κB (p50:p65) signaling mediates IL-1 repression of AR. We found that siRNA silencing of p65 is sufficient to attenuate IL-1 repression of AR mRNA and protein levels and AR activity in AR+ PCa cell lines, while siRNA silencing of p50 had no effect on IL-1 repression of AR. Our data suggests that p65 represses AR levels, directly or indirectly, in response to IL-1. Thus, therapeutic inhibition of p65 activity could prevent the accumulation of inflammation-induced treatment-resistance ARlow/- PCa cell subpopulations.

#2363

Targeting squamous lung tumor heterogeneity with EZH2 inhibition to improve immunotherapy responses.

Tanner J. DuCote, Christine Fillmore Brainson. _University of Kentucky, Lexington, KY_.

Immunotherapies, including those targeting the PD1/PD-L1 immune checkpoint, have become popular treatment options, but they are effective for only 20% of squamous lung cancer patients. Work in our laboratory has suggested that inhibition of the histone methyltransferase EZH2 drives a basal-like transcriptional state which includes expression of the immunotherapy target PD-L1, and work from others has demonstrated that EZH2 inhibition could influence the tumor microenvironment. Therefore, we hypothesize that combining EZH2 inhibition with anti-PD1 therapy could boost response in squamous lung cancers by targeting both the tumor and the microenvironmental heterogeneity. Our laboratory uses genetically engineered mouse models of lung cancer and patient-derived cell lines and organoids to dissect the mechanistic effects of EZH2 inhibition on tumor cells and immune cells. First, we treated a panel of squamous and adeno-squamous human NSCLC cell lines with EZH2 inhibition and observed increased expression of PD-L1 and another basal cell marker, NGFR, by flow cytometry. Similar results were obtained by treating two patient-derived organoids of squamous lung cancer with EZH2 inhibitor, suggesting that EZH2 inhibition drives squamous tumor cells into a more basal-like state which may be more susceptible to anti-PD1 immunotherapy. In our mouse models of squamous lung cancer, we noted abundant tumor-associated neutrophils, which we believe may be inhibiting efficacy of immunotherapy by suppressing CD8 T cell function. In support of the idea that inhibiting EZH2 will ameliorate this phenotype, lung neutrophils isolated from EZH2 conditional knock-out mice had significantly lower levels of the T cell suppressive protein Arginase 1 than WT lung neutrophils or Lkb1/Pten TANs. Lastly, we treated mice harboring autochthonous squamous lung tumors with EZH2 inhibitor, and observed a striking increase in the proportion of tumor cells expressing PD-L1 but now defect in tumor growth. In contrast, two squamous lung tumor bearing mice treated with EZH2 inhibitor and anti-PD-1 immunotherapy had tumor regression that was durable up to 4 weeks post-treatment initiation. We plan to expand the cohorts of mice and test this promising combination therapy in a variety of lung cancer mouse models. Completion of these studies will solidify the efficacy of a promising therapeutic combination and uncover mechanisms by which tumor hierarchies and microenvironments are changed by EZH2 inhibitors in squamous lung cancers. Given that one arm of a Phase 1/2 clinical trial combining EZH2 inhibition with anti-PDL1 just began recruiting late stage non-small cell lung cancer patients, learning the phenotypes and mechanisms of responders and non-responders will be extremely timely for any Phase 2/3 trials that ensue. Funded by V Foundation Scholar Award, K22 CA201036 and KY LCRP to CFB

#2364

**1α, 25 dihydroxyvitamin D (1,25(OH)** 2 **D) inhibits the T cell suppressive function of myeloid derived suppressor sells (MDSC).**

James C. Fleet, Grant N. Burcham, Ryan Calvert, Timothy L. Ratliff. _Purdue University, West Lafayette, IN_.

MDSC within the tumor microenvironment are antagonists to immune surveillance and T-cell based immunotherapies because they can suppresses the ability of cytotoxic T cells to attack and clear tumor cells from the body. The active, hormonal form of vitamin D, 1,25(OH)2D, regulates many components of the immune system and previous research shows that in mouse models 1,25(OH)2D reduces the number of CD34+ cells, an MDSC precursor, in tumors and can reduce metastasis when coupled to adoptive immunity. We tested whether MDSC are vitamin D target cells by examining granulocytic- (G-MDSC) and monocytic (M-MDSC) MDSC from tumors, spleen, and bone marrow. Messenger RNA levels of the vitamin D receptor (VDR), the mediator of 1,25(OH)2D molecular action, are low in MDSC from marrow and spleen but are 20-fold higher in tumor MDSC. In addition, at all sites the M- subtype cells have 4-fold higher VDR mRNA expression than G- subtype cells. Next, we tested whether MDSC are responsive to changes in 1,25(OH)2D signaling. Bone marrow MDSC were isolated by flow cytometry and induced to differentiate into tumor MDSC-like cells using treatment with a cytokine cocktail of IFN-γ, IL-13, and GM-CSF for up to 72 h. This treatment significantly elevated Arg1 and Nos2 levels (48 h) and activated the ability of the bone marrow MDSC to suppress T cell proliferation in a 16 h assay. 48 h of cytokine treatment also increased VDR expression 100-fold and made the bone marrow MDSC responsive to 1,25(OH)2D treatment i.e. 10 nM for 24 h increased CYP24A1 and VDR mRNA in wild-type (WT) cells. Importantly, 1,25(OH)2D treatment reduced the T cell suppressive capacity of cytokine-induced total MDSC and M-MDSC by ≥70% in a 16 h assay. We next examined the impact of VDR deletion on the development of tumor M- and G-MDSC function. WT and VDR KO mice were injected ip with 106 RM-1 cells and tumor MDSC were isolated by FACS 7 days later. As expected, the T cell suppressive function of WT M-MDSC was 4X greater than G-MDSC. However, T cell suppression was significantly greater in both VDR KO M-MDSC (2X vs WT at 1:4 MDSC:T-cell ratio) and G-MDSC (4X vs WT, 1:4 ratio). The major finding of this study is that MDSC are novel targets of 1,25(OH)2D. The effect of 1,25(OH)2D is to decrease the immunosuppressive capability of MDSC. In addition, expression of VDR is necessary to attenuate the immunosuppressive capabilities of MDSC that are recruited to the tumor microenvironment. Collectively, this data suggests that activation of vitamin D signaling could be used to suppress MDSC function and release a constraint on T-cell mediated clearance of tumor cells.

#2365

Selective Stat3 inhibition in the tumor microenvironment restricts gastrointestinal tumor growth.

Mariah Grace Alorro,1 Frederick Masson,2 Moritz Eissmann,1 Matthias Ernst1. 1 _Olivia Newton-John Cancer Research Institute, Heidelberg, Australia;_ 2 _Centre of Physiopathology Toulouse-Purpan, ISERM UMR, Toulouse, France_.

The transcription factor Stat3 plays essential roles in biological processes involving development, immunity and inflammation. Stat3 is known to regulate the expression of a large repertoire of genes which when deregulated, as in the context of tumorigenesis, can lead to the development of some of the well-known hallmarks of cancer, such as resistance to cell death and sustained proliferation. Indeed the aberrant activation of Stat3 has been characterized across a number of different cancers including gastrointestinal cancers, presenting Stat3 and its signalling pathway a promising therapeutic target. Although intrinsic Stat3 signalling in tumor cells is well characterized, the effect(s) of Stat3 signalling among the infiltrating cells of the tumor microenvironment is still poorly understood. With this project we aim to elucidate the role of Stat3 in the non-tumoral compartment, and explore the therapeutic value of Stat3 inhibition for gastric and colon cancer.

We generated the CAGsrtTA3;STAT3.1348 (shStat3) mouse that utilizes short-hairpin RNAi technology allowing for conditional and reversible Stat3 reduction. To study the effects of systemic Stat3 inhibition, the shStat3 was crossed with the gp130F/F mutant mouse that spontaneously develops gastric cancer. To assess the effects of Stat3 suppression in the non-tumoral compartment alone, the shStat3 mice were sub-cutaneously injected with MC38 murine colon cancer cells. Tumors and other tissues of these mice were excised and analysed.

We found that shStat3-mediated systemic Stat3 suppression resulted in significant tumor reduction in the gp130F/F gastric cancer mice. Similarly, isolated Stat3 reduction in the non-tumoral compartment of the shStat3 mice, restricted the growth of Stat3-proficient MC38 tumor allografts. Immunophenotyping of excised tumors highlighted an increase of a monocytic Ly6C+Ly6G\- myeloid population.

Our data provides evidence for the therapeutic value of specific Stat3 targeting in gastrointestinal cancers. Interestingly, we have shown that Stat3 suppression in the non-tumoral compartment alone was enough to result in significant tumor reduction, indicating a prominent role of Stat3 in creating a pro-tumorigenic microenvironment. We have also identified a monocytic population as candidate cell type that drives this Stat3 suppression mediated anti-tumor effect.

#2366

HDAC inhibition improves immune checkpoint inhibitor efficacy in renal cell carcinoma.

Justin Budka, Nur Damayanti, Roberto Pili. _Indiana University School of Medicine, Indianapolis, IN_.

Background: Immune checkpoint inhibitors have shown clinical benefit in solid tumors, including renal cell carcinoma (RCC); however, the rate of clinical response remains modest and improved therapeutic approaches need to be tested. Growing evidence suggests that epigenetic modifying agents may have an immunomodulatory effect that improves the efficacy of immune checkpoint inhibitors. Our group has previously demonstrated that entinostat, a histone deacetylase (HDAC) inhibitor, decreases the function of regulatory T cells (Treg) and myeloid derived suppressor cells (MDSC), synergizing with PD-1 blockade. Here we assessed the combination of PD-1 blockade with pan-HDAC inhibition in a RCC model.

Methods: To test the efficacy of combined PD-1 inhibition, mDX-400 (10 and 20 mg/kg I.P) (Merck & Co, Inc) with pan-HDAC inhibition, vorinostat (100 and 150 mg/kg I.P) (Merck & Co, Inc), we utilized a syngeneic mouse model of metastatic RCC following orthotopic implantation of RENCA cells in immunocompetent mice. Antitumor activity was assessed by measuring bioluminescence, end point tumor weights, and survival times. Immune profiling of tumor infiltrating lymphocytes (TILs) was performed by flow cytometry, immunohistochemistry, and immunofluorescence. Peripheral blood mononuclear cells (PBMC) were assessed for differential chromatin accessibility by Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq).

Results: Significant reductions in tumor weight and lung metastases were observed in mice treated with the combination of vorinostat and mDX-400. Combination therapy significantly increased the survival of the mice (median survival = 60) compared to treatment with mDX-400 alone (median survival=42 days). Immune landscape profiling demonstrated an increase in natural killer cell infiltration (P=0.048) and decrease of exhausted T cells (P=0.049, CD8\+ PD1+) in the combination group. Furthermore, decreased immunosuppressive Treg (CD4\+ FOXP4+) and MDSC (CD11b\+ Gr1+) populations were identified in the combination group. Analysis of the mouse PBMC ATAC-seq data in the combination and mDX400 alone conditions demonstrated numerous regions of differential chromatin accessibility. Pathway analysis of genes associated with increased accessibility in the combination treatment identified enrichment of cell cycle and immune activation pathways.

Conclusions: Our results demonstrate that pan-HDAC inhibition augments the antitumor effect of immune checkpoint inhibitors, prolonging survival in our preclinical mouse model. This antitumor effect was achieved by changing the immune landscape in TILs and was associated with higher chromatin accessibility near genes involved in cell cycle progression and immune cell activation. Taken together, our results support the clinical testing of pan-HDAC inhibitors in combination with anti-PD-1.

#2367

Enhancing the efficacy of liver cancer immunotherapy by specific inhibition of histone deacetylase 8.

Wei-Qin Yang, Jingying Zhou, Yu Feng, Ka Wing Cheung, Alfred Sze-Lok Cheng. _School of Biomedical Sciences, Hong Kong, Hong Kong_.

The heterogeneous responses to immune-checkpoint blockade (ICB) therapy e.g. anti-programmed death-ligand 1 (PD-L1) antibody are attributable to the complex interplay between a range of cancer-cell-autonomous cues and the immunosuppressive tumor microenvironment. Two recent phase I/II trials of checkpoint inhibitors in patients with advanced hepatocellular carcinoma (HCC) have produced promising results, yet the objective response rates were relatively low (17-20%) as compared to other solid tumors such as melanoma and lung cancer (~40-60%). Accumulating evidence is underscoring the fundamental importance of epigenetic regulation in tumor immune evasion. We have previously elucidated a critical role of histone deacetylase 8 (HDAC8) in hepatic carcinogenesis (Cancer Research 2015;75:4803-16). Here, we aim to investigate the therapeutic potential of a HDAC8-specific inhibitor PCI-34051 in preclinical hepatocellular carcinoma (HCC) models. PCI-34051 significantly reduced HCC tumorigenicity in immunocompetent but not immunodeficient mice. Immune profiling revealed specific reduction in tumor-infiltrating regulatory T cells, which was associated with significant increase in CD8+ T cells. Notably, combined PCI-34051 and anti-PD-L1 treatment resulted in complete tumor eradication in all of the co-treated mice. Moreover, the combination therapy promoted long-term survival, which was associated with elevated CD8+ T effector and central memory cells. Our data suggest that selective chromatin modifications by HDAC8 alter the tumor immune surveillance program and demonstrate the potential of rational combinatorial epigenetic immunotherapy to fully unleash T-cell responses, leading to long-term remission of HCC. Acknowledgement: This work is supported by the RGC CRF (C4017-14G) and the Focused Innovations Scheme-Scheme B from the Chinese University of Hong Kong (1907309).

#2368

Domatinostat increases apoptosis, G2M cell-cycle arrest and immunogenicity of Merkel cell carcinoma.

Lina Song,1 Anne Catherine Bretz,2 Jan Gravemeyer,1 Ivelina Spassova,1 Svetlana Hamm,2 Shakhlo Muminova,1 Rene Bartz,2 Juergen C. Becker1. 1 _German Cancer Consortium (DKTK) Partner Site Essen, Essen, Germany;_ 2 _4SC, Munich, Germany_.

Merkel cell carcinoma (MCC) is a rare, highly aggressive skin cancer prevalent in elderly and immunocompromised patients. MCC is highly immunogenic as it is either associated with Merkel cell polyoma virus (MCPyV) integration or UV-associated mutations in non-viral cases. Indeed, immune checkpoint inhibitors (CPI) like PD-1/PD-L1 blocking antibodies exert a strong clinical activity; however, primary or secondary resistance often occurs. Domatinostat (4SC-202) is an orally available small molecule inhibitor targeting histone deacetylases (HDAC) class I currently in clinical evaluation to improve response to CPI (SENSITIZE, NCT03278665). In syngeneic tumor mouse models domatinostat treatment demonstrated immune-modulatory effects by increasing the intra-tumoral infiltration of cytotoxic CD8+ T cells (CTLs) and enhancing gene expression of a CPI response signature.

Immune escape mechanisms described for MCC include low intra-tumoral levels of CTLs and reduced expression of antigen-presenting MHC class I molecules. Our previous data suggest that low MHC-I levels are reversible and involve epigenetic silencing of genes encoding the antigen-processing machinery (APM). Here, we present novel preclinical data about the efficacy and mode of action of domatinostat in MCC. Global gene expression by single cell RNA-seq revealed regulation of genes involved, among others, in apoptosis and antigen presentation. Importantly, domatinostat additionally inhibited proliferation of MCC cell lines by induction of a G2M cell-cycle arrest and apoptosis. Moreover, expression of MCPyV-encoded transforming early genes, particularly during the G1-phase, was inhibited by domatinostat. Thus, it exerts also a direct anti-tumoral effect. In viable cells, domatinostat increases their susceptibility to immune responses, as qPCR and immunoblot analysis confirmed the induction of APM and MHC-I expression by domatinostat. APM and MHC-I expression has been reported to be restored by an epigenetic drug combination (vorinostat plus mithramycin). In contrast, we now provide evidence that the single agent treatment with domatinostat alone is sufficient for increasing immunogenicity of MCC cells.

In summary, domatinostat counteracts immune escape of MCC in many aspects suggesting a combination treatment of the HDACi domatinostat with CPI as a promising therapeutic strategy. Prospective clinical trials are needed to confirm this hypothesis.

#2369

HPRT overexpression may contribute to the immunosuppressive tumor microenvironment.

Michelle H. Townsend, Zachary D. Ewell, Claudia M. Tellez Freitas, Dallas J. Larsen, Eliza L. Bitter, Kelsey B. Bennion, Stephen R. Piccolo, K Scott Weber, Richard A. Robison, Kim L. O'Neill. _Brigham Young University, Provo, UT_.

The purpose of this study was to examine the possible effects that Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) may have on the immune response in the tumor microenvironment. HPRT is a salvage pathway enzyme responsible for recycling guanosine throughout the cell cycle, and we have shown that it is significantly upregulated in numerous solid malignancies. As a necessary component of the S phase in the cell cycle, guanosine has also been regarded as a potential neuroprotectant in the central nervous system (CNS). We thus hypothesized that there may be a correlation between the observed upregulation of HPRT and the relative downregulation of the immune system mediated by the differential regulation of guanosine. Using data from The Cancer Genome Atlas (TCGA), we analyzed over 200 cytokine genes and found a negative correlation between the level of HPRT expression in cancer and the general expression level of both pro-inflammatory and anti-inflammatory cytokine expression. We further analyzed data showing the relative infiltration level of individual immune cell subsets in solid tumors compared with elevated HPRT levels. We found the most significant negative correlations between HPRT expression and immune cell infiltration for B-cells (p < 0.001), CD8+ T-cells (p < 0.01), CD4+ T-cells (p < 0.001), macrophages (p < 0.001), neutrophils (p < 0.001), and dendritic cells (p < 0.001) in lung squamous cell carcinoma. Other significant negative correlations were found in kidney renal clear cell carcinoma, prostate adenocarcinoma, and breast invasive carcinoma. To further analyze this reduction in infiltration, we examined the relationship between HPRT expression and both costimulatory and coinhibitory molecules and found a significant negative correlation. Because of these observed associations between HPRT levels and cytokine expression, we hypothesized that the increased guanosine production was the mechanism by which HPRT was affecting the immune system in the tumor microenvironment. To test this, we examined the level of Ca2+ influx using Fluo-4 assays of B-cell (Raji), T-cell (Jurkat), and monocyte (THP-1) cell lines when treated with guanosine as a measure of overall cell activation. We then compared this activation to untreated cells and cells treated with adenosine, a known anti-inflammatory agent. When treated with guanosine, a significant reduction in activation was found in THP-1s (p < 0.01) and Rajis (p < 0.01), but not in Jurkats. However, under hypoxic and hypoglycemic conditions similar to those commonly found within the tumor microenvironment, Jurkat activation was significantly reduced (p < 0.05) when treated with guanosine. From this data, we conclude that guanosine levels significantly reduce immune cell activation in the tumor microenvironment and that the upregulation of HPRT in malignant tissue may be one of the contributing factors of the immunosuppressive nature of the microenvironment.

#2370

Synergistic effect of carboplatin on farletuzumab (FAR) tumor cell killing via downregulation of the humoral immunosuppressive CA125 protein and enhanced antibody dependent cellular cytotoxicity (ADCC).

J. Bradford Kline,1 Keiji Furuuchi,1 James Fulmer,1 Jennifer McDonough,1 Luigi Grasso,2 Nicholas C. Nicolaides2. 1 _Morphotek, Inc., Exton, PA;_ 2 _Navrogen, Inc., Glen Mills, PA_.

Farletuzumab (FAR) is a humanized monoclonal antibody (mAb) that targets folate receptor alpha (FRA), a cell surface protein highly overexpressed on a large number of cancers. Clinical evidence suggests that FAR enhances the anti-tumor effects of carboplatin and taxane in ovarian cancer patients whose tumors express low CA125 levels. In light of the recent evidence that CA125 has a direct effect on suppressing the humoral immune mechanisms [antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC)] of FAR (1, 2) and the findings that chemotherapeutic agents can enhance the ADCC activity of certain mAbs, we tested the potential synergistic effects that carboplatin may have on FAR immune effector function. Here we show the immunosuppressive effects that CA125 has on FAR-mediated tumor cell killing in vivo and demonstrate that carboplatin is able to suppress cell surface CA125 expression leading to enhanced FAR ADCC on FRA-expressing ovarian tumor cells. CA125 has been previously found to suppress FAR-mediated tumor cell killing in vitro. To determine its effect in vivo, we tested single agent FAR against isogenic tumor cells in mouse xenografts. OVCAR-3 is a FRA positive ovarian tumor cell line that expresses high levels of CA125 on its cell surface. A CA125 shRNA knockdown line (OV-KD) has been previously generated and shown to be more susceptible to FAR ADCC than its parental counterpart. Xenograft studies using these lines found that FAR had significant single agent tumor killing in the OV-KD xenografts in contrast to OVCAR-3 xenografts. We next tested FAR in vivo killing when used in combination with carboplatin. These data showed that carboplatin had a synergistic effect when combined with FAR treatment as compared to single agent treatment alone. To determine if this mechanism involved enhanced ADCC, we conducted similar studies in vitro whereby OVCAR-3 cells were treated with a sublethal concentration of carboplatin for 72 hrs, washed and then tested for FAR ADCC killing. These data found that FAR had enhanced ADCC activity on carboplatin treated cells as compared to untreated cells. Finally, to explore the potential mechanism by which this effect may occur, we examined the steady-state levels of both the mannose-6-phosphate receptor (M6PR), reported to be upregulated by chemotherapeutic drugs leading to enhanced ADCC via higher granzyme B uptake and the CA125 immunosuppressive factor. Our data found that both M6PR and CA125 expression were significantly suppressed following carboplatin exposure. These data support a mechanism by which carboplatin may enhance the humoral immune response for FAR and potentially other anti-tumor mAbs via downregulation of the immunosuppressive CA125 protein. 1. OncoTarget 8:66747-57, 20172. 2. EJI 201847707, 2018

#2371

Breast cancer induces tolerogenic state of healthy activated CD4+ lymphocytes, characterized by reduced PI3K, NFκB, JAK-STAT, Notch, and increased TGFβpathway activity.

Anja Van De Stolpe,1 Wim Verhaegh,1 Arie van Doorn,1 Grégory Noël,2 Chunyan Gu-Trantien,2 Karen Willard-Gallo2. 1 _Philips Research, Eindhoven, Netherlands;_ 2 _Institut Jules Bordet, Brussels, Belgium_.

Tumor cells can induce immunotolerance, which is reversed by checkpoint blockade immunotherapy in some patients, although response prediction remains a challenge. CD4+ T cells play an important role in activating adaptive immune responses with their conversion to a suppressed state impairing anti-tumor immune responses. CD4+ T cells function by activating and controlling various signal transduction pathways. Over the past decade we have developed tests that quantitatively measure functional activities of signal transduction pathways (e.g. Hedgehog, Wnt, TGFβ Notch, NFκB, PI3K, JAK-STAT 1/2 and 3, and MAPK). They are based on Bayesian computational model inference of pathway activities from measurements (expression microarray, qPCR) of mRNA levels of target genes of the transcription factor associated with the respective signalling pathways1,2. These tests were extensively biologically validated, including on immune cells, and can be used to characterize their functional activity status. In the present study, this approach was used to investigate cellular mechanisms underlying breast cancer-induced immunosuppression of CD4+ T cells.

Method: Generation of Affymetrix gene expression data has been previously described (J Clin Invest 2013;123(7):2873-92) and data is publically available (GEO dataset GSE36766). Briefly, breast cancer tissue sections from fresh surgical specimens were mechanically dissociated in X-VIVO 20. Following activation with anti-CD3/CD28, CD4+ T cells from healthy donor blood were incubated with primary tumor supernatants (SN) and compared to controls. Signaling pathway activities were measured using Affymetrix expression data from the individual CD4+ T cell treatment groups.

Results: CD4+ T cell activation resulted in induction of PI3K, NFkB, JAK-STAT1/2, JAK-STAT3, Notch, and parallel decrease in TGFβ pathway activities. Incubation with primary tumor SN did not affect pathway activity in non-activated CD4+ T cells, but reduced activity of PI3K, NFκB, JAK-STAT1/2, JAK-STAT3, Notch, while increasing TGFβ pathway activity in activated CD4+ T cells.

Conclusion: A soluble factor(s) from breast tumor tissues increases TGFβ and reduces effector immune pathway activity in activated CD4+ T cells and thereby can induce an immunotolerant state. Investigation into the nature of this soluble factor(s) is in progress. These data demonstrate that signaling pathway assays can be used to quantitatively measure the functional state of immune responses in CD4+ lymphocytes. The ultimate goal is to apply this approach for predicting and monitoring immunotherapy responses and identifying novel drug targets that can reverse tumor-induced immunosuppression. Ref: 1. Verhaegh W, et al. Cancer Res 2014;74(11):2936-45; 2. Ooijen H. van, et al. Am J Pathol 2018;188(9):1956-1972.

#2372

Tumoral p53 mutations differentially mediate poor T-cell infiltration and autologous T-cell killing in preclinical models.

Deborah A. Silverman,1 Emily Ashkin,1 Benjamin Whitfield,1 Simone Punt,1 Soraya Zorro Manrique,1 Yunfei Wang,1 Anil Korkut,1 Leila Williams,1 Minying Zhang,1 Eran Kotler,2 Moshe Oren,2 Anirban Maitra,1 Patrick Hwu1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Weizmann Institute of Science, Rehovot, Israel_.

Harnessing the immune system through the attenuation of endogenous immune checkpoints on T-cells has led to dramatic, durable tumor rejection in multiple solid tumors; however, most cancer types remain resistant to immunotherapy. It is imperative to understand the unique mechanisms by which these lethal malignancies evade the immune system in order to design efficacious therapies. Research by several groups is elucidating how overall mutational burden, tumor stroma, and patient microbiome predict response to immunotherapies in immune-resistant cancers. However, the frequency and distribution of driver mutations in the tumors themselves differ between immunogenic and non-immunogenic cancer types and may play a role in immune escape. Whereas p53, the most commonly mutated gene in cancer, is mutated in 70-90% of non-immunogenic tumors like pancreatic adenocarcinoma (PDAC), it is mutated in only 10-15% of immunogenic skin cutaneous melanoma cases. Loss of p53 through truncating mutations mediates tumor escape from apoptosis and senescence. Furthermore, many p53 missense mutations (mtp53) not only lose wild-type activity (LOF), but acquire novel gain-of-function (GOF) activities which promote oncogenesis and resistance to therapy. Pre-clinical data suggest that mtp53 differentially mediates tumor escape from immune surveillance by altering the innate immune response, including NK cell function and macrophage phenotype, thereby allowing tumorigenesis through chronic local immunosuppression. Few studies have been completed, however, to demonstrate the role of mtp53 in regulating the adaptive immune response. Understanding the role of p53 and its mutants in the regulation of T-cell function in cancer would provide a novel framework by which to understand and overcome resistance to cancer immunotherapy in many deadly cancer types. We hypothesize that mtp53 mediates evasion of T-cell anti-tumor activity, and that gain-of-function pathways downstream of mtp53 drive this process. Here we elaborate on previously presented work, elucidating how GOF vs. LOF mtp53 influences T-cell infiltration and killing using both novel model systems. Ultimately, these results may define a new role for mtp53 in influencing the immune system, and provide a rationale for developing effective combination strategies to improve response to immunotherapy.

#2373

The mechanism study of propranolol in suppressing the genesis and development of gastrointestinal cancer through simultaneously activating autologous CD8+T cells and inhibiting tumor AKT/MAPK pathway.

Ping Liao. _Central South University, Changsha, China_.

Background: Propranolol may exert the anti-tumor effect in cutaneous and acral melanoma by suppressing AKT and MAPK pathways. Reduced tumor growth following propranolol results from enhanced CD8+T-cell-mediated antitumor immune response.

Objective: To clarify the efficacy and mechanism of propranolol in suppressing the genesis and development of gastrointestinal cancer through simultaneously activating autologous CD8+T cells and inhibiting tumor AKT/MAPK pathway.

Methods: MTS assay was performed to detect gastrointestinal cancer cells viability, TUNEL staining was used to measure apoptosis in cell lines. IHC was used to detect the expressions of Ki67/p-AKT/p-MEK/p-ERK in tumor tissue. Flow cytometry was performed to explore the immune status of the tumor microenvironment. Clinical study has been performed by Case-control design. Subjects who had not been received neoadjuvant were randomly divided into propranolol or placebo group. Then the primary foci of gastrointestinal cancer subjects were removed by D2 gastrectomy. IHC was used to observe the immune status, tumor proliferation in tumor tissue.

Results: We found propranolol reduced cell viability in CT26WT and MFC (mice derived melanoma cell line). Furthermore, propranolol could shrink the tumor size in CT26WT and MFC xenografts (P<0.0001). The frequency of CD3+CD8+ T cells in spleen was significantly elevated in propranolol-treated mice compared with controls. The IFNg and T-bet was significantly increased in the CD8+ T-cell population of propranolol treated mice tumor tissue. Propranolol reduced cell viability and induced apoptosis in AGS, HGC, SW480 and HCT116 cell line. But We did not observe differences in NCM460 cell line (human intestinal epithelial cells). Western Blot showed propranolol reduced the expression of p-AKT, p-MEK and p-ERK. In this prospective clinical study, the frequency of CD8+ was significantly elevated in tumor microenvironment. The expression of p-AKT, p-MEK and p-ERK was decreased in propranolol-treated subjects compared with controls.

Conclusion: The potential of propranolol to suppress the genesis and development of gastrointestinal cancer through simultaneously activating autologous CD8+T cells and inhibiting tumor AKT/MAPK pathway.

#2374

Non-canonical TGF-beta signaling modulates pancreatic ductal adenocarcinoma progression.

S. Mazher Hussain, Gustavo Miranda-Carboni, Marcus A. Alvarez, Abul Elahi, Leah E. Hedrick, David Shibata, Evan S. Glazer. _University of Tennessee Health Science Center, Memphis, TN_.

Introduction: While the genomic and proteomic characterization of pancreatic ductal adenocarcinoma (PDAC) is extraordinarily complex, the transforming growth factor-beta (TGF-β) pathway is critically important. TGF-β has a context dependent role- it is tumor suppressive in early PDAC and tumor promotive in metastatic PDAC. Our hypothesis is that non-canonical TGF-β signaling induces metastatic PDAC. Non-canonical signaling may help explain the paradoxical nature of TGF-β in PDAC.

Methods: Two cell lines were utilized- Panc-1, a cell line derived from primary PDAC, was compared to Capan-1, a cell line derived from metastatic PDAC- in order to model an early PDAC (Panc-1) and a more aggressive PDAC (Capan-1). We investigated the gene and protein expression of markers of epithelial to mesenchymal transition (Snail and Twist), Akt, β-catenin, and PD-L1 upon treatment with TGF-β and the Akt inhibitor MK-2206.

Results: While TGF-β increased Snail gene and protein expression in Panc-1 cells, it had nil effect on Capan-1 cells where TWIST1 protein expression increased with TGF-β. TGF-β increased pAkt expression in both cell lines. TGF-β increased active and total β-catenin expression in Capan-1 but not Panc-1 cell lines. We then found that PD-L1 expression was generally diminished by Akt inhibition in both cell lines but this was reversed with TGF-β receptor inhibition and gemcitabine treatment in the Capan-1 cells.

Conclusions: Our data suggests that modulation of the non-canonical TGF-β pathway may lead to increased epithelial to mesenchymal transition through multiple mechanisms. PD-L1 expression is a response to chemotherapy in metastatic PDAC when multiple proteins in that pathway are inhibited. Our data suggests that (1) personalized therapy based on the degree of non-canonical TGF-β pathway activation will be critical, (2) patients with uncontrolled non-canonical TGF-β pathway activation may have lower survival, and (3) there is an opportunity to utilize anti-PD-L1 based therapy in combination with other therapy in selected patients based on their response to chemotherapy.

#2375

Developing double knock-in models of human immune checkpoint targets (dKI HuGEMM) for efficacy assessment of combinatorial therapeutic antibodies.

Lei Zheng,1 Daniel Xianfei He,1 Ruilin Sun,2 Annie Xiaoyu An,1 Jian Fei,2 Henry Qixiang Li,1 Davy Xuesong Ouyang1. 1 _Crown Bioscience, Inc., Taicang, Jiangsu, China;_ 2 _Shanghai Model Organisms Center Inc., China_.

Immune checkpoint inhibitors (ICIs), i.e. PD1, PDL1 and CTLA4 therapeutic antibodies, have led to long-term survival in many late stage solid tumor patients. Despite their revolutionary clinical impact, the overall response rate is still low. One major roadblock is that compensatory immune inhibitory pathways are turned on to protect tumor cells from being attacked by T cells. Various combinatory ICI treatments have now been investigated in clinical trials to tackle this challenge. We have previously reported the development of immune checkpoint target humanized mice (HuGEMM) through CRISPR-Cas9 knock-in. We have generated PD1 and CTLA4 HuGEMM mice and used them for efficacy assessment of corresponding therapeutic antibodies. We have also generated human PDL1 expressing MC38 cells and PDL1 HuGEMM, and demonstrated robust efficacy of several PDL1 therapeutic antibodies. Here we report the establishment of double knock-in (dKI) mice of ICI targets, i.e., PD1/PDL1 and PD1/CTLA4 dKI HuGEMM, and use them as tools to test immunotherapies with PD1/PDL1 and PD1/CTLA4 combos. Similar to clinical benefit of PD1 and CTLA4 combo shown in the melanoma and NSCLC patients, we found combined treatment of Nivolumab and Ipilimumab antibody leads to 103% TGI in the corresponding PD1/CTLA4 dKI-HuGEMM, with complete tumor remission in 80% of treated mice. These mice remained disease free throughout a tumor re-challenge study for over 40 days. We have also developed PD1/PDL1 dKI HuGEMM and tested combined treatment of Atezolizumab and Nivolumab. Interestingly, we observed improved efficacy in the combo treatment group despite the two ICIs are thought to target the same PD1/PDL1 axis. Monotherapies may not fully release the PD1/PDL1 blockade due to complicity of multiple compatible ligands & receptors, including PDL2 and B7-1. We have also tested PD1/OX40 combo treatment with dKI HuGEMM, which is reported separately. Taken together, Our dKI HuGEMM models offer robust tests on ICI combinations, as well as combos of ICIs with other anti-tumor therapeutic modalities.

#2376

Comprehensive multiplexed protein analysis of biomarkers of hypoxia and the immune microenvironment of diffuse large B-cell lymphoma with clinical outcome analysis.

Vladislav V. Makarenko, Karen Dresser, Benjamin J. Chen. _University of Massachusetts Medical School, Worcester, MA_.

Background: The host immune response has been shown to play an important role in the pathogenesis and clinical outcome of diffuse large B-cell lymphoma (DLBCL). Immune checkpoint molecules, such as PDL1, TIM3, and LAG3, can modulate the immune microenvironment by downregulating T-cell activity. Hypoxia inducible factor (HIF) is a transcription factor that regulates genes involved in responses to hypoxia and can induce PDL1 expression. PDL1, TIM3, and HIF1α expression in DLBCL tumor cells have been associated with inferior survival. We performed a comprehensive multiplexed analysis of hypoxia and immune-related proteins to better understand the DLBCL microenvironment.

Design: Immunohistochemical (IHC) analysis was performed on 49 DLBCL cases and correlated with clinicopathologic data. A microarray with an additional 36 DLBCL cases was evaluated by IHC and by using the NanoString nCounter Digital Spatial Profiling (DSP) platform with a 30-protein immuno-oncology panel. This study was approved by the UMass IRB.

Results: Subsets of tumors were positive by IHC in tumor cells and/or infiltrating non-tumor cells for PDL1 (12%), PD1 (39%), TIM3 (73%), LAG3 (18%), HIF1α (31%) and HIF2α (47%). A positive correlation was found between IHC expression of tumor-associated PDL1, PD1, and TIM3 (r>0.6, p<0.001) but not LAG3 (r=0.27, p>0.05). HIF1α, but not HIF2α, expression positively correlated with PDL1 IHC expression (p=0.011). Pairwise comparison between proteins using data from the DSP analysis showed positive correlations between PDL1/LAG3 (r=0.71, p<0.001), PDL1/B7-H3 (r=0.78, p<0.001), PDL1/TIM3 (r=0.46, p<0.01), and VISTA/LAG3 (r=0.46, p<0.01). Levels of pSTAT, AKT, CD44, β-catenin, β-2-microglobulin, and PTEN were also found to correlate with PDL1 expression. HIF1α IHC expression correlated with PDL1, LAG3, B7-H3 levels, but not with PD1, VISTA and TIM3. Patients with tumors expressing HIF1α, but not HIF2α, demonstrated inferior OS upon long term follow up (p=0.045 and 0.059, respectively).

Conclusions: A comprehensive protein-level analysis of the immune microenvironment of DLBCL reveals coordinated expression of several therapeutically-relevant immunomodulatory molecules. HIF1α expression strongly correlated with PDL1 and other immune checkpoint molecules and was associated with inferior patient outcome. Further studies are required to elucidate causative links between hypoxia and the immune environment of lymphoma, as well as potential clinical and therapeutic implications of these findings.

### Therapeutic Antibodies 3

#2377

Development and characterization of scFvs against TIP1/PDZ protein for cancer targeting.

Abhay Singh, Vaishali Kapoor, Philipp Diebolder, Aaron Rhee, Buck Rogers, Dinesh Thotala, Dennis Hallahan. _Washington University in St. Louis School of Medicine, St. Louis, MO_.

We identified TIP1 as a potential target to treat various cancers. TIP1 is over-expressed in glioblastoma, lung, head and neck and breast cancer. It is also a radiation inducible neo-antigen. TIP1consists of a single PDZ domain that binds the c-terminus of various cellular proteins like β-catenin, Rho, FAS, etc. It plays an important role in cell signaling, cancer development, and progression. TIP-1's involvement in various survival pathways makes it an attractive target for anticancer therapeutics. Cancer can be targeted by different agents like antibodies (monoclonal, scFv, bi-specific, diabodies), peptides and small molecules. We will be targeting TIP1 using single-chain variable fragment (scFv) antibodies. scFv is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a peptide linker. We generated a phage-displayed scFv library (WASHU 2) from patient samples. For library screening, we cloned and purified the recombinant TIP1 protein. We used the TIP1 protein as bait for biopanning to pulldown the TIP1-binding scFvs from the library. Three rounds of biopanning were performed to enrich the specific TIP1 binding scFvs. Phage ELISA was performed to confirm the binding of the identified clones to the TIP1 protein. Approximately fifty-one clones showing positive binding to TIP1 protein were identified.We have sequenced the identified clones to verify the full length of scFv sequence. Unique clones are being characterized and developed further as targeting agents of various cancers. We found cancer specific binding of our lead scFvs in mouse models of cancer. The lead scFvs will be expressed as scFv-Fc to enhance their biological activity. We will characterize the scFv-Fcs for their binding affinity to TIP1, cancer cell surface binding, in vivo tumor targeting and tissue cross-reactivity. The scFvs having the potential to internalize will be developed as immunotoxins.

#2378

Differential exhaustion on cytokine release (DECREASE) by ERY974, a novel T-cell-redirecting antibody targeting glypican-3: A new type of T-cell exhaustion.

Mayumi Hoshino, Yuji Sano, Yasuko Kinoshita, Yumiko Azuma, Toshiaki Tsunenari, Yoko Kayukawa, Mizuho Noguchi, Takahiro Ishiguro, Shohei Kishishita, Noriaki Sawada, Mika Endo, Junichi Nezu. _Chugai Pharmaceutical Co., Ltd., Japan_.

Background: T cell-redirecting antibody (TRAB), which bispecifically binds to CD3 and tumor antigen, is a key player in next-generation cancer immunotherapy (CIT). Because TRAB can redirect T cells regardless of their TCR specificity, it is expected to be efficacious in immune checkpoint inhibitor-resistant tumors. The CD19-targeting bispecific T cell engager, blinatumomab, has been used for the treatment of blood cancers, and CEA-TCB and IMCgp100 have shown promising clinical efficacy in solid tumors as well. ERY974 is another promising TRAB targeting glypican-3 (GPC3) and a phase 1 study is in progress (Ishiguro et al., Sci Transl Med 2017). On the other hand, cytokine release syndrome (CRS) has been recognized as a common side effect of TRAB. Thus, mitigation of CRS is an urgent issue. Intra-patient step-up dosing regimens have been incorporated in clinical trials to reduce cytokine release, but the mechanism behind this phenomenon remains elusive. In this study, we pre-clinically explored the phenomenon and its mechanism by using ERY974.

Method & Results: GPC3-expressing human cancer cells were incubated with human PBMCs and ERY974. The incubated PBMCs were harvested and mixed with newly prepared GPC3-expressing cancer cells and higher doses of ERY974. The pre-treated PBMCs showed reduced cytokine production compared to that without pre-treatment, while maintaining the same level of T cell-dependent cellular cytotoxicity (TDCC). Comprehensive gene expression analysis of the pre-treated PBMCs was also conducted. A murine cancer cell line expressing human GPC3 was established and implanted into immune competent mice. A mouse surrogate version of ERY974 (mERY974) was first administered at a low dose expected to elicit weak anti-tumor activity, and mice were then treated with a higher dose of mERY974. Anti-tumor activity was similar regardless of the pre-treatment, but plasma cytokine levels were shown to be reduced in the pre-treated mice upon administration of the higher dose of mERY974.

Conclusion: Cytokine production following the high-dose ERY974 treatment was mitigated by the low-dose pre-treatment in vitro and in vivo. Cytotoxic activity did not decrease in this setting, thus demonstrating that the pre-treatment selectively suppressed only cytokine production. It is well known that repeated stimulation of TCR leads to T-cell exhaustion. The phenomenon we observed here can also be characterized as a sort of exhaustion, but is quite unconventional. Thus, we propose a new type of exhaustion and suggest calling it 'Differential exhaustion on cytokine release (DECREASE)'. Management of CRS is critical for the clinical use of TRAB. A detailed analysis of the mechanism of DECREASE currently in progress, is expected to contribute to the development of desired dosing regimens for this new type of CIT agent.

#2379

In vivo **drug screening platform accelerated 4-1BB agonistic antibody development.**

Benny(Yi) Yang,1 Jian Ni,2 Yanan Li,3 Yuelei Shen,1 Qingcong Lin3. 1 _Biocytogen, Beijing, China;_ 2 _Eucure, Beijing, China;_ 3 _Biocytogen, Wakefield, MA_.

4-1BB (TNFRSF9/CD137) belongs to the TNFR superfamily. 4-1BB is expressed by activated CD4 helper T cells, B cells, natural killer cells, natural killer T cells, dendritic cells, and activated endothelium. A majority of regulatory T cells (Tregs) also express 4-1BB, but it remains unclear whether agonistic antibody treatment exerts pro- or anti-suppressive effect on these cells. 4-1BB enhances tumor rejection because it is upregulated on T cells following activation and its engagement increases T cell proliferation and pro-inflammatory cytokine production. The clinical development of 4-1BB targeting therapy was slow due to the toxicity associated with overt immune activation. Fortunately, new therapeutic modalities using 4-1BB targeting aptamers as well as therapeutic combinations with other immuno-modulatory and traditional anti-cancer treatments have revived excitement for the use of 4-1BB agonists in the clinic.

We focused on discovering novel 4-1BB therapeutic antibodies for cancer patients. Candidate molecules obtained through the classical hybridoma technology were screened by high-throughput flow cytometry. Then, we took the advantages of Biocytogen's humanized mice and screened the purified antibodies in B-h4-1BB mice bearing MC-38 tumor cells. We found some antibodies that worked as equivalent as BMS's Urelumab. To determine which isotype should be adopted, we engrafted the antigen binding domain onto various human Fc backbones. Interestingly, we found that the human IgG1 isotype appeared to be more effective against tumors than other isotypes in B-4-1BB mice bearing MC-38 tumors. This study provides a reliable basis for our follow-up mechanism research and clinical evaluation for this target.

#2380

Preclinical safety and efficacy of a tumor-directed T cell activating 4-1BB x 5T4 ADAPTIR™ bispecific antibody.

Anna Dahlman,1 Michelle Nelson,2 Jeannette Bannink,2 Starrla Johnson,2 Doreen Werchau,1 Anneli Nilsson,1 Lill Ljung,1 Gabriele Blahnik-Fagan,2 Robert Bader,2 Adnan Deronic,1 Peter Ellmark,1 Maria Askmyr,1 Gabriela Hernandez-Hoyos,2 Cathy McMahan,2 Sara Fritzell1. 1 _Alligator Bioscience AB, Lund, Sweden;_ 2 _Aptevo Therapeutics Inc, Seattle, WA_.

The ability to induce potent anti-tumor activity by stimulating 4-1BB (CD137), a key co-stimulatory receptor, makes 4-1BB an attractive immunotherapeutic target. However, a clinically tested, 4-1BB targeting monospecific antibody has been hampered by dose-limiting hepatic toxicities. To improve safety of 4-1BB targeting therapies we have developed a 4-1BB x 5T4 bispecific antibody designed to direct tumor-specific T cell responses to the tumor by stimulating 4-1BB only when co-engaged with 5T4, a tumor-associated antigen. The preclinical dataset presented here provides an overview of the mechanism of action and the efficacy and safety profile of ALG.APV-527, supporting its advancement into the clinic. ALG.APV-527 was built using the ADAPTIR™ platform with binding domains from the ALLIGATOR-GOLD® human scFv library. Its 5T4-dependent agonistic function was assessed using primary CD8+ T cells or NK cells in the presence of 5T4-expressing cells. Secretion of IFN-γ or granzyme B was measured at 72 hrs using ELISA. To measure proliferation, PBMCs were labelled with Cell Trace™ and gated CD8+ T cells were analyzed using flow cytometry. For tumor inhibition studies, the human 5T4-expressing colon carcinoma HCT116 xenograft model was used. 5T4 expression was evaluated in normal human tissues and different human tumors by immunohistochemistry. The preclinical safety profile of ALG.APV-527 was evaluated in a single and repeated dose, dose-range finding toxicology study in non-human primates (NHP). The study design included all the standard repeated dose toxicity parameters and in addition, pharmacokinetics, immunogenicity, and pharmacodynamic end-points. ALG.APV-527 induces a 5T4-dependent increase in IFN-γ and granzyme B production and enhances proliferation of T cells and NK cells. Furthermore, ALG.APV-527 inhibits tumor growth in a human 5T4-expressing colon carcinoma xenograft model. 5T4 is overexpressed in multiple solid tumors, potentially directing the activity of 4-1BB induced by ALG.APV-527 to 5T4-expressing tumors, improving the risk/benefit profile. Four doses (administered once weekly) did not cause any adverse events in the NHP toxicity study. In conclusion, ALG.APV-527 induces potent CD8+ T cell and NK co-stimulation but only in the presence of 5T4. Based on its efficacy and preclinical safety profile, ALG.APV-527 is a promising anti-cancer therapeutic for the treatment of multiple 5T4-expressing solid tumors.

#2381

Blinatumomab enhanced anti-tumor activity against rituximab sensitive and resistant Burkitt Lymphoma (BL) and Primary Mediastinal B-cell Lymphoma (PMBL).

Aradhana Awasthi Tiwari, Dina Edani, Janet Ayello, Mitchell S. Cairo. _New York Medical College, Valhalla, NY_.

Background: Childhood, Adolescent and Young Adult (CAYA) B-NHL represents the third most common malignancies in children under the age of 15yrs (Hochberg/Cairo et al, BJH 2009; Miles/Cairo, BJH. 2012).The prognosis of mature-B-NHL (which include BL/PMBL) has significantly improved over the last 40 years through the use of short and intense multi-agent chemo-immunotherapy, however; a subset of patients with relapsed/refractory disease has chemoimmunotherapy resistant disease and a dismal prognosis (≤ 20% 5 yr. EFS, Cairo et al.Blood. 2007; Cairo et al. JCO.2012 ,Goldman/Cairo et al. Leukemia, 2013, Gerrard/Cairo et al. Blood. 2013). It is therefore critical to investigate and develop targeted translational strategies in BL/PMBL to reduce acute morbidities, decrease late effects, and provide new options for those with recurrent disease. Blinatumomab, targeting CD19, has demonstrated encouraging clinical activity against pre-B-ALL (Topp et al, Leukemia, 2018) and highly expressed in BL/PMBL.

Objectives: To determine in-vitro activity of blinatumomab against rituximab sensitive/resistant BL and PMBL cell lines.

Methods: BL; Raji, Raji-4RH, and PMBL: Karpas1106p/MedB-1 were cultured in RPMI with 10 or 20% FBS. Tumor cells were incubated with/without blinatumomab (generously supplied by Amgen) for 4 hr. with T-cells. CD3+ isolated and expanded T-cells were used for cytotoxicity assay. Cytotoxicity was determined by DELFIA® cytotoxicity assay at 10:1 E: T ratio and cytokines secretion was measured by multiples ELISA kit. CD3+/CD107a+, granzyme b and perforin T-cell expression level were measured by flow-cytometry.

Results: Blinatumomab+T-cells compared to T-cells only elicited a significant increased cytotoxicity against, Raji 58.18±7.6% vs 27.3.81±11.2% (p=0.007), Raji-4RH 67.4±8.0% vs 27.6±2.5%, (p=0.004), Karpas1106p, 75.7±3.06 % vs. 17.86± 4.82% (p=0.003) and MedB1, 65.17±13.3% vs18.1±2.03%, respectively (p=0.05). Blinatumomab treated T-cells also increased IFN-γ secretion compared to IL2-T cells, Raji 1 (p=0.002) and Raji-4RH (p=0.02) respectively.

Furthermore, CD107a, granzyme b and perforin expression were significantly enhanced with blinatumomab treated/activated T-cells against; Raji (p=0.03, p=0.003 & p=0.009), Raji-4RH (p=0.03, p=0.02 & p=0.03) and Karpas1106p (p=0.006, p=0.03 & p=0.02) respectively.

Conclusion: Blinatumomab significantly enhances T-mediated in-vitro cytotoxicity and cytokine secretion against BL/PMBL. Further, blinatumomab treated and activated T-cells significantly enhanced CD107a, granzyme b and perforin expression. These preliminary studies demonstrate that BiTE (CD3/CD19) would be a novel agent to investigate as immunotherapy therapy in patients with relapse refectory BL/PMBL.

#2382

Development and testing of the first in class immunotherapy targeting immuno-suppressive δ1 containing γδ T cells for the treatment of pancreatic ductal adenocarcinoma and other solid tumors.

Tatyana Panchenko,1 Wei Wang,1 Eric Denbaum,1 Takamitsu Hattori,1 Akiko Koide,1 Aleksandra Filipovic,2 George Miller,1 Shohei Koide1. 1 _NYU Langone, New York, NY;_ 2 _PureTech Health, Boston, MA_.

Background: Targeting and engineering γδ T cells has recently emerged as an orthogonal therapeutic approach in oncology with capacity to effectively modulate both innate and adaptive immune properties. We and others have shown that in solid tumors such as pancreatic ductal adenocarcinoma (PDA), melanoma, glioblastoma, breast cancer etc., γδ1 T cells express immunosuppression-related molecules and possess a pro-tumorigenic capacity. Previously, we have shown that intra-tumoral γδ T cells from patients bearing PDA, colorectal cancer (CRC) and hepatocellular carcinoma (HCC) potently inhibit patients' αβ T cells, rendering them immunosuppressed. We hypothesized that anti-δ1 monoclonal antibody would be a potent and effective, novel therapeutic. In order to harness the therapeutic potential of γδ1 T cell blockade we have developed a set of highly specific, fully human anti-δ1 T cell receptor (TCR) antibodies.

Methods: We have sequenced δ chains from 19 cancer patients with primary PDA, primary CRC and gastric cancer (GC) to identify tumor-specific δ1. A proprietary synthetic, human antibody library was screened via phage display to identify high-affinity antibodies. Surface plasmon resonance and bead-based assays were used to measure binding affinity. We used a panel of ex-vivo experiments to assess the immunosuppressive features of γδ T cells. Antibody efficacy was assayed using patient-derived organotypic tumor spheroids (PDOTS) which recapitulate complex tumor architecture. PDOTS of n = 20 patients (PDA, CRC and liver metastases, HCC) were treated with the antibodies and resulting immune profiles analyzed by flow cytometry.

Results: Because the δ1 chains in patients showed diverse CDR3 sequences, we used a selection strategy to identify antibodies that bind diverse δ1 TCRs. Our first-in-class anti-δ1 antibodies have low nanomolar affinity to human δ1 TCRs and show no binding to δ2 TCRs. Furthermore, we chose the lead clinical candidate that showed no preference for the γ chains of the TCR, as patients may harbor a diverse set of γδ heterodimers. Importantly, we show that our lead anti-δ1 antibody achieves reproducible and robust efficacy in the PDOTS system as shown by the up-regulation of pro-inflammatory T cell markers (IFNγ, TNF-α, CD44). Ongoing experiments in γδ knockout mice will investigate the efficacy of combination of γδ1 T cell targeting and existing checkpoint inhibitors (anti- PD1, PDL1, and CTLA4).

Conclusion: We have defined a novel therapeutic immuno-oncology strategy and translated it to develop and characterize a lead clinical candidate anti-δ1 monoclonal antibody. Overall, our results demonstrate that we have an

efficacious, novel immunotherapy that has the potential to be transformative for the treatment of cancers where γδ1 T cells drive a pro-tumorigenic, immunosuppressive environment.

#2383

The molecular binding mechanism of tislelizumab, an investigational anti-PD-1 antibody, is differentiated from pembrolizumab and nivolumab.

Yingcai Feng, Yuan Hong, Hanzi Sun, Bo Zhang, Hongfu Wu, Kang Li, Xuesong (Mike) Liu, Ye Liu. _BeiGene, Ltd., Beijing, China_.

Programmed cell death protein 1 (PD-1) is an immune checkpoint receptor expressed by activated T, B, and NK cells, which interacts with its ligand PD-L1/L2 to inhibit T-cell proliferation and effector functions such as tumor cell killing and cytokine production. Two anti-PD-1 antibodies approved by the FDA, pembrolizumab and nivolumab, have shown efficacy in many cancer types, nevertheless there are some indications where limited efficacy is observed. Tislelizumab (BGB-A317), an investigational anti-PD-1 antibody, has demonstrated significant clinical activity (85.7% ORR, including 61.4% CR) in relapsed/refractory classical Hodgkin's lymphoma (R/R cHL). Additionally, tislelizumab is being studied in global pivotal trials in a number of malignancies, including non-small cell lung cancer, hepatocellular carcinoma, and esophageal squamous cell carcinoma. However, how tislelizumab binds to PD-1 has yet to be shown, particularly in comparison to pembrolizumab and nivolumab. Here we report the co-crystal structure of PD-1 extracellular domain and the Fab of tislelizumab. Tislelizumab interacts with IgV-like domain of PD-1 with an interface area of 1112 Å2. Structure-guided mutagenesis of PD-1 and surface plasmon resonance were performed to compare the binding of tislelizumab, pembrolizumab and nivolumab to mutant and wild type PD-1. The dissociation rate (kd) of tislelizumab from wild type PD-1 is about 100-fold and 50-fold slower than that of pembrolizumab and nivolumab, respectively. Gln75, Thr76, Asp77 and Arg86 on PD-1 are critical epitopes for tislelizumab, but mutation of them showed little effect on binding of PD-1 to pembrolizumab and nivolumab. Both the co-crystal structure and mutagenesis study identified the unique epitopes of tislelizumab that correlate to the extremely slow-off property of tislelizumab after binding to PD-1. In conclusion, we observed that tislelizumab is differentiated from pembrolizumab and nivolumab by its unique binding epitopes as well as binding kinetics.

#2384

TAK-169, an exceptionally potent CD38 targeted engineered toxin body, as a novel direct cell kill approach for the treatment of multiple myeloma.

Erin K. Willert,1 Garrett L. Robinson,1 Jack P. Higgins,1 Jensing Liu,1 Janice Lee,2 Sakeena Syed,2 Yuhong Zhang,2 Dan Tavares,2 Anya Lublinsky,2 Nibedita Chattopadhyay,2 Haiqing Wang,2 Laura Packer,2 Pu Shi,2 Carole Harbison,2 Sanjay Patel,2 John Newcomb2. 1 _Molecular Templates Inc., Austin, TX;_ 2 _Takeda, Cambridge, MA_.

Advances in the treatment of multiple myeloma have been driven by the development of new modalities, including proteasome inhibitors, immunomodulatory agents and monoclonal antibodies. TAK-169 is an engineered toxin body (ETB) comprising a proprietarily engineered, deimmunized form of the ribosome inactivating Shiga-like toxin A-subunit (SLTA) genetically fused to an antibody fragment that specifically targets the CD38 cell surface receptor. Although CD38 is a poorly internalizing receptor, TAK-169 is able to efficiently internalize and directly kill CD38-expressing cells through the enzymatic and irreversible inhibition of protein synthesis. This novel mechanism of action may be relevant in patients who have progressed after or are unlikely to respond to CD38-targeted antibody therapy. A library of unique CD38-targeting antibodies fused to deimmunized SLTA was screened in a variety of efficacy and safety models to identify TAK-169. TAK-169 demonstrated potent cytotoxicity across a range of myeloma cell lines with a range of CD38 expression in vitro as well as in patient-derived samples including with previous exposure to daratumumab. TAK-169 retains activity in whole blood and PBMCs, indicating that the presence of red blood cells is not restrictive to cytotoxicity. Furthermore, TAK-169 retains activity in the presence of excess approved, CD38 targeted therapeutic daratumumab. TAK-169 is efficacious in several myeloma xenograft models, both subcutaneous and disseminated. Complete regressions were observed using both a once-weekly and bi-weekly schedule. In addition, a syngeneic model cell line expressing human CD38 was shown to be responsive to TAK-169 treatment in an immunocompetent mouse model, suggesting the possibility of immune mediated cell death induced by TAK-169. Tolerability studies in non-human primates demonstrate that repeat administration is tolerated at doses expected to be efficacious. Takeda Pharmaceuticals and Molecular Templates jointly discovered TAK-169 and are co-developing the molecule for the treatment of multiple myeloma. TAK-169 is expected to enter clinical studies in 2019.

#2385

Discovery of highly specific claudin 6 monoclonal antibodies.

L Joe Stafford. _Integral Molecular, Philadelphia, PA_.

Claudin 6 (CLDN6) is an oncofetal tight junction protein involved in the cell-to-cell adhesion of epithelial and endothelial cell sheets. Although silenced in healthy adult human tissues, CLDN6 expression has been found in ovarian, gastric, pediatric, and other cancer tissues and can lead to a poor prognosis. Monoclonal antibody (MAb) discovery against CLDN6 has been encumbered by the high homology of endogenously expressed claudin 9 (CLDN9), which varies from CLDN6 by only 3 amino acids in the extracellular domain. Our MPS Antibody Discovery platform utilizes proprietary technologies to overcome key barriers to MAb discovery, enabling the isolation of MAbs against multipass membrane proteins such as CLDN6. Using the MPS platform, we have successfully isolated and characterized panels of highly specific MAbs to CLDN6. Nine chickens were immunized with the target antigen, and titer was achieved in all but one animal. Following 2 rounds of phage panning, we screened 2,000 clones for CLDN6 specificity, identifying 788 hits and 68 unique VH CDR3 families. From these clones, 72 antibodies were selected for binding to CLDN6 and CLDN6/9. Two lead antibodies, IM-171 and IM-136, showed high specificity and high affinity to CLDN6 but not to claudins 3, 4, or 9. Using high-resolution Shotgun Mutagenesis Epitope Mapping, we confirmed that our lead candidate MAbs bind distinct residues on CLDN6 compared to previously described MAbs. Specificity analysis with our Membrane Proteome Array revealed no significant binding of either of our lead MAbs to any other membrane protein in the human proteome, including any of the other 20+ claudin proteins. Both of our lead MAbs bound endogenously expressed CLDN6 on ovarian carcinoma (PA-1) cells. Our preclinical CLDN6 antibodies are now being developed for use in various formats, including bispecifics, antibody-drug conjugates, and CAR-T applications.

#2386

TROP2-targeted photoimmunotherapy in experimental human pancreatic cancer.

Takashi Nishimura, Makoto Mitsunaga. _Jikei University School of Medicine, Japan_.

Background: Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer therapy that employs a selective monoclonal antibody (mAb) conjugated to a photosensitizer phthalocyanine dye, IR700. The complex mAb-IR700 binds to the specific antigen on the cellular membrane, and then NIR light is delivered, leading to rapid and target-selective cell death. The type I trans-membrane protein, TROP2, is found to be expressed at high levels in many epithelial cancers, including pancreatic carcinoma. Further, TROP2 overexpression correlates with poor prognosis and tumor aggressiveness. In this study, we investigated the antitumor effect of TROP2-targeted PIT in an experimental human pancreatic cancer model using anti-TROP2 mAb-IR700 conjugate.

Methods: The experiments were performed on TROP2-overexpressing human pancreatic cancer cells (PK-59 and KP-3L) and 3T3/HER2 cells were used as the negative control. Anti-TROP2 antibody (Chiome Bioscience, Tokyo, Japan) was conjugated with IR700 and purified (TROP2-IR700). The expression and localization of TROP2 were determined by fluorescence microscopy and flow cytometry. LIVE/DEAD assay was performed to determine cytotoxicity exerted by NIR light irradiation in vitro. Subsequently, for in vivo studies, PIT was tested on a mouse tumor xenograft model (PK-59 cells). Specifically, tumor-bearing mice were intravenously injected with TROP2-IR700 and tumors were irradiated with NIR laser light, 1 day after the injection. Distribution of TROP2-IR700 was then monitored with small animal imaging system and the antitumor effects were determined by measuring the tumor diameter using a caliper. Additionally, the expression patterns of TROP2 and EGFR in human pancreatic cancer were analyzed by immunohistochemistry, using tissue microarray samples.

Results: We found that TROP2-IR700 specifically localized on the cellular membrane as well as on lysosomes, in TROP2-overexpressing PK-59 and KP-3L cells, whereas no specific signals were observed in TROP2-negative 3T3/HER2 cells. Moreover, TROP2-IR700-mediated PIT was able to induce a strong cytotoxic effect in PK-59 and KP-3L cells, which was NIR-light dose dependent. The localization of TROP2-selective IR700 fluorescence was observed in the mouse xenograft, 1 day after intravenous injection with TROP2-IR700, and the tumor growth was significantly inhibited by NIR light irradiation compared to the control groups. Moreover, pancreatic cancer tissue microarray analysis revealed that 80% of the tumors were positive for TROP2 and 50% were positive for EGFR and the expression patterns were independent from each other.

Conclusions: We demonstrated that TROP2-IR700-targeted PIT exerts an antitumor effect against TROP2 positive pancreatic cancer, both in vitro and in vivo, and could be a promising therapeutic option for human pancreatic cancer.

#2387

NKG2A-antagonizing monoclonal antibodies identified through phage display.

Jing Gao, Lili Hu, Ruirui Sui, Cuicui Guo, Lina Wang, Teddy Yang, Jack Han, Qing Duan, Louis Liu. _ChemPartner, Shanghai, China_.

The inhibitory receptor NKG2A-CD94 is mainly expressed on NK cells and some T cell and mediates an inhibitory signal through association with a non-classical MHC class I molecule HLA-E, which is commonly over-expressed in human cancers. Targeting NKG2A with monoclonal antibody holds potentials in immunotherapy of cancer. However, proteins with homologous sequences such as NKG2C/CD94 or NKG2E/CD94 complexes generally deliver an activating signal upon ligand binding. NKG2A-specific antagonizing antibodies would be needed to further explore therapeutic potentials of targeting NKG2A for immunotherapy of cancer.

We have developed and characterized panels of monoclonal antibodies (mAbs) that specifically recognize NKG2A. Balb/c or SJL mice had been immunized with recombinant NKG2A/CD94 protein, recombinant plasmids encoding hNKG2A or stable cell line with ectopic expression of hNKG2A. Splenocytes from such immunized animals were collected for construction of phage-displayed antibody library in a single chain variable fragment (scFv) format. The NKG2A-specific antibodies, that had no binding to NKG2C, NKG2E or CD94, were isolated through multiple rounds of phage panning and ELISA-based screening. Such antibodies also block HLA-E ligand binding and enhanced cytotoxicity of NK cell lines and primary NK cells on cancer cells.

In summary, isolation and characterization of a panel of NKG2A-specific antagonizing antibodies enable us to further determine the feasibility of NKG2A-targeted immunotherapy of cancer.

#2388

In vitro assessment of tumor associated I.O. markers in cancer cell lines following Vicinium treatment.

Shilpa Chooniedass, Rachelle L. Dillon, Arjune Premsukh, Glen C. MacDonald, Jeannick Cizeau, Gregory P. Adams. _Sesen Bio, Winnipeg, Manitoba, Canada_.

Vicinium is a fusion protein that comprises a scFv fragment specific for the Epithelial Cell Adhesion Molecule (EpCAM) genetically fused to a truncated form of Pseudomonas exotoxin A, ETA, via a flexible linker. Vicinium is used for the treatment of loco-regionally accessible tumors and is currently in a phase III clinical trial (VISTA) for the treatment of high-grade non-muscle invasive bladder cancer and a phase I study in combination with durvalumab for the same indication. In a different phase I study in late stage squamous cell carcinoma of the head and neck, direct injection of Vicinium led to shrinkage of the principal injected tumor as well as non-targeted tumors in some patients suggesting the activation of a T cell-mediated anti-tumor response through cross-priming. Supporting this hypothesis, in vitro studies have demonstrated that Vicinium mediated tumor cell killing elicits biological features of an immunogenic cell death (ICD). Tumor cells' ability to evade the innate and adaptive immune response plays a major role in cancer development and progression. Altered expression of immune modulators is a key mechanism responsible for tumor escape. Recent studies have shown that exposure to chemotherapeutic drugs results in the selection of tumor cells expressing immunosuppressive markers such as PD-L1, CD47 and CD39. Following in vitro treatment with Vicinium, the enrichment of tumor cell population expressing I.O. markers was examined and compared to chemotherapeutic agents. Unlike small molecule chemotherapies, Vicinium treatment did not lead to an enrichment of CD47 or CD39 positive tumor cell populations. Taken together, the data suggests that Vicinium does not induce an immunosuppressive environment.

#2389

Role of Midkine in molecular targeted therapy to TIP1 for lung cancer treatment.

Vaishali Kapoor, Abhay K. Singh, Dinesh Thotala, Dennis Hallahan. _Washington University in St. Louis School of Medicine, St. Louis, MO_.

Non-small cell lung cancer (NSCLC) ranks among the highest cancer-related mortalities world-wide. Targeted therapy has been a growing topic of investigation to improve therapeutic efficacy for NSCLC. A few targeted therapies that exploit aberrant protein expression profiles have been approved for NSCLC. However, marginal improvement in efficacy observed with these therapeutic approaches highlights the need for discovery of additional therapeutic targets. We identified Tax interacting protein 1 (TIP1) which is overexpressed in various cancers as a molecular targets. We focused on antibody-based targeting of the chaperone protein TIP1. The functional domain of TIP1 (PDZ domain) caps the C-terminus of many cellular proteins that regulate important cellular functions. Knocking down TIP1 revealed that it plays an important role in cell signaling, cancer development, and progression. Comparing antibodies targeting different epitopes of TIP1, we found that antibodies against the PDZ domain of TIP1 were most effective in inducing cytotoxicity of lung cancer cells but not normal cells. Anti-PDZ/TIP1 antibodies injected into mice bearing lung cancer bind specifically to cancer and substantially enhance tumor control. A mass spectrometry-based approach identified Midkine (MDK) as a putative protein that modulates this cytotoxicity by anti-TIP1 antibodies. Additional studies suggested that the β-catenin/Wnt signaling may be involved in induction of MDK after blocking of TIP1. These studies led to the hypothesis that MDK is upregulated by the anti-PDZ/TIP1 antibody via the β-catenin/Wnt signaling pathway, which subsequently modulates downstream signaling mechanisms. This project will systematically characterize the mechanisms of cytotoxicity by the anti-PDZ/TIP1 antibodies. To achieve this, a number of novel methodologies including genome-wide CRISPR knockout, proteomics and mass cytometry to characterize the link between TIP1 and MDK will be applied. The project will guide development of combination therapies to optimize efficacy of NSCLC treatment. One strategy proposed, is dual-targeting of TIP1 and MDK for enhanced efficacy. This project will lead to identification of novel molecular targets for NSCLC treatment that have never been considered before.

#2390

FcgR co-engagement by anti-TIGIT monoclonal antibodies enhances T cell functionality and antitumor immune responses.

Dhan Chand,1 Jeremy D. Waight,1 Elena Paltrinieri,1 Sylvia Dietrich,1 Mark Bushell,1 Mathew Costa,1 Randi Gombos,1 Nicholas S. Wilson,2 Jennifer S. Buell,1 Robert B. Stein,1 Alexander Duncan,1 David A. Savitsky1. 1 _Agenus Inc., Lexington, MA;_ 2 _Gilead Sciences Inc., Foster City, CA_.

T-cell immunoreceptor with Ig and ITIM domains (TIGIT) has emerged as an important regulator of the cancer-immunity cycle. Preclinical studies have demonstrated that TIGIT antibodies can enhance T and NK cell anti-tumor immunity, and therapeutic antibodies targeting TIGIT are advancing in the clinic. Nonetheless, questions on the optimal format for TIGIT therapeutics remain unresolved. Preclinical studies have demonstrated that anti-TIGIT antibodies enhance anti-tumor immunity by (1) blocking inhibitory signaling downstream of TIGIT/PVR and TIGIT/PVRL2 receptor-ligand interactions and (2) redirecting PVR/PVRL2 ligand binding to the co-stimulatory receptor CD226. Here we describe a novel and unanticipated mechanism of action of anti-TIGIT antibodies in which interactions between the antibody Fc domain and select FcγRs, particularly FcγRIIIA, dramatically improve T cell activation and effector function. In mouse tumor models, TIGIT antibodies that promote FcγR interactions enhance anti-tumor responses, whereas Fc-inert TIGIT antibodies show no such benefit. Moreover, TIGIT antibodies that enhance binding to human FcγRIIIA or mouse FcγRIV promote superior single agent activity and combination activity with other checkpoint modulators in antigen-stimulation assays and mouse tumor models respectively. Notably, this mechanism of action is independent of regulatory T cell depletion. Our findings support a dependence on Fc-FcγR interaction for promoting T cell responsiveness and effector function upon TIGIT antagonism. We further demonstrate that this novel mechanism also extends to anti-CTLA-4, but not anti-PD-1 or anti-LAG-3 antibodies. Altogether, our data describe a novel FcγR-dependent mechanism of action that may enhance the therapeutic activity of anti-TIGIT antibodies, deepen our understanding of this class of therapies, and inform on the optimal design for a new class of Fc-engineered antibodies that could be leveraged to potentially enhance antitumor immune responses.

#2391

**DR5 agonist activity of HexaBody** ® **-DR5/DR5 (GEN1029) is potentiated by C1q and independent of Fc-gamma receptor binding in preclinical tumor models.**

Marije B. Overdijk,1 Kristin Strumane,1 Antonio Ortiz Buijsse,1 Claudine Vermot-Desroches,2 Thessa Kroes,1 Bart de Jong,1 Naomi Hoevenaars,1 Frank J. Beurskens,1 Rob N. de Jong,1 Andreas Lingnau,1 Paul W. Parren,1 Ulf Forssmann,1 A Kate Sasser,1 Janine Schuurman,1 Esther C. Breij1. 1 _Genmab, Utrecht, Netherlands;_ 2 _IDD Biotech, Lyon, France_.

Hyperclustering of Death Receptor 5 (DR5) after binding of its ligand TRAIL induces apoptosis. Targeting DR5 with agonistic antibodies has been evaluated for the treatment of cancer, however clinical efficacy of conventional DR5-targeting monoclonal antibodies (mAbs) has been disappointing. We applied the HexaBody® technology to improve antibody-mediated DR5 clustering on cancer cells. This technology is based on the natural concept that, upon binding to antigens on a cell surface, immunoglobulin G (IgG) molecules can organize into ordered hexamers through intermolecular Fc-Fc interactions. HexaBody molecules are IgG1 molecules with a single point mutation in the Fc domain that enhances these Fc-Fc interactions upon binding to membrane-bound targets, while retaining solution-monomericity.

HexaBody-DR5/DR5 (Hx-DR5-01/05) is a 1:1 mixture of two humanized non-competing DR5-specific mAbs, each carrying an E430G hexamerization-enhancing mutation. We previously demonstrated that both dual epitope targeting and enhanced hexamerization through Fc-Fc interactions are required for DR5 agonist activity of Hx-DR5-01/05 in vitro.

Here, we confirmed that Hx-DR5-01/05 showed superior anti-tumor activity compared to the single HexaBody molecules or a 1:1 mixture of their wild type counterparts in vivo, using a mouse xenograft model. Furthermore, we screened the potency of Hx-DR5-01/05 in vitro in a broad panel of human tumor cells lines using a cell viability assay, and in vivo in xenograft models. As IgG hexamers are known to provide an optimal docking site for complement component C1q, we studied if there was a role for C1q in Hx-DR5-01/05-dependent DR5 agonist activity.

Hx-DR5-01/05 induced potent cytotoxicity in 104 tumor cell lines and in more than ten xenograft models representing many solid cancer lineages. For optimal cytotoxicity in vitro, Hx-DR5-01/05 required the presence of serum or purified C1q. In contrast, polyclonal IgG crosslinking, a mimic for FcγR-mediated antibody crosslinking, did not enhance potency. These data were confirmed in vivo. A Hx-DR5-01/05 variant deficient for both C1q and FcγR binding, showed significantly reduced anti-tumor activity in a colon cancer xenograft model, while a Hx-DR5-01/05 variant deficient in FcγR but not C1q binding showed anti-tumor activity comparable to Hx-DR5-01/05.

In summary, Hx-DR5-01/05 is a mixture of two DR5-specific HexaBody molecules that shows potent DR5 agonist activity in a multitude of preclinical models through enhanced IgG hexamerization upon binding to two different DR5 epitopes on the cell surface. Cytotoxicity of Hx-DR5-01/05 was most optimal in the presence of C1q and completely independent of FcγR-mediated antibody crosslinking or effector functions in vitro and in vivo. A clinical trial to assess clinical safety of Hx-DR5-01/05 in patients is currently ongoing.

#2392

CDX-527: A novel bispecific immune-modulating antibody targeting CD27 and PD-L1.

Laura A. Vitale,1 Lawrence J. Thomas,2 Thomas O'Neill,1 Jenifer Widger,1 Laura Mills-Chen,1 Andrea Crocker,1 Colleen Patterson,1 Anna Wasiuk,1 Eric Forsberg,2 James Boyer,2 Crystal Sisson,1 Jeffrey Weidlick,1 Shannon Renn-Bingham,3 Ioannis Papayannopoulos,3 Russ Hammond,2 Joel Goldstein,1 Henry C. Marsh,2 Tibor Keler,1 Li-Zhen He1. 1 _Celldex Therapeutics, Hampton, NJ;_ 2 _Celldex Therapeutics, Needham, MA;_ 3 _Celldex Therapeutics, Fall River, MA_.

CD27 is a costimulatory molecule that provides a complementary target to the PD-1/PD-L1 axis on T cells. Combining a CD27 agonist monoclonal antibody (mAb) with PD-1/PD-L1 blockade has been shown to promote synergistic antitumor activity in preclinical models. In advanced cancer patients, the combination of the CD27 agonist mAb, varlilumab, with the PD-1 mAb, nivolumab, was generally well tolerated, induced strong biological effects, and led to sustained clinical responses in some patients that generally have low response rates to checkpoint inhibitors. We theorized that combining CD27 costimulation with PD-1/PD-L1 blockade in a bispecific antibody (bsAb) may provide greater immune activating properties than combining the individual mAbs due to enhanced CD27 activation by crosslinking through PD-L1 in addition to Fc receptors. To test this approach, we developed CDX-527, a tetravalent human anti-CD27/PD-L1 IgG1 bsAb. CDX-527 inhibits PD-1 signaling in vitro in a manner similar to clinically approved PD-1/PD-L1 antibodies. CDX-527 also elicits potent T cell costimulation through PD-L1 crosslinking as measured by cytokine induction and cell proliferation, whereas the PD-L1 or CD27 parental antibodies show no significant activity. In a mixed lymphocyte reaction assay, CDX-527 is more potent than the combination of the parental antibodies, suggesting that cross-linking through both Fc receptors and PD-L1 results in enhanced CD27 agonist activity. In human CD27 transgenic mice, we observed that antigen-specific T cell responses to a vaccine in vivo are significantly more enhanced with a surrogate CD27/PD-L1 bsAb than with either individual antibody. Furthermore, the surrogate bsAb exhibits greater antitumor activity than the combination of the parental antibodies in a syngeneic lymphoma model. Taken together, these results suggest that the enhanced activity of CDX-527 can be attributed to more efficient cross-linking of the bispecific antibody acting on the CD27 receptor, resulting in stronger T cell activation, combined with efficient PD-1/PD-L1 blockade, providing a novel approach to immunotherapy of cancers. We have initiated development activities for CDX-527 including a pilot study in cynomolgus macaques to investigate the PK and PD properties and provide guidance for the design of a GLP toxicology study.

#2393

**Bridging MOA-based reporter bioassays with PBMC based ADCC for immunotherapy drug** d **evelopment.**

Mei Cong, Pete Stecha, Aileen paguio, brock binkowski, Zhi-Jie Cheng, Frank Fan. _Promega Corp., Madison, WI_.

Having a functional bioassay that is MOA-based, accurate, precise, robust and reproducible is critical for the development of antibody-based biologics. We have developed reporter bioassays that meet these criteria for a broad range of antibody modalities including Fc effector function, immune checkpoint modulation, bispecific antibody engagement, cytokine modulation, and others. Here we will present the latest technology advancements and bridging of the PBMC based ADCC cell killing assay with reporter based ADCC Bioassay.

#2394

Discovery of small anti-PD-L1 peptides for cancer immunotherapy.

Hao Liu, Zhen Zhao, Yuanke Li, Kun Cheng. _University of Missouri-Kansas City, Kansas City, MO_.

Immunotherapy using checkpoint inhibitors, especially PD-1/PD-L1 inhibitors, has now evolved into the most promising therapy for cancer patients. However, most of these inhibitors are monoclonal antibodies, and their large size may limit their tumor penetration, leading to suboptimal efficacy. As a result, there has been a growing interest in developing low-molecular-weight checkpoint inhibitors. Using a phage display peptide library, we discover small peptide-based anti-PD-L1 inhibitors to block the PD-1/PD-L1 interaction. These peptides exhibit high affinity and specificity to human PD-L1 as well as PD-L1-positive human cancer cells. Molecular docking studies indicate that the CLP002 peptide specifically binds to PD-L1 at the residues where PD-L1 interacts with PD-1. Compared to anti-PD-L1 antibody, the anti-PD-L1 peptides exhibited better tumor penetration in a 3D tumor spheroid model. The CLP002 peptide restores proliferation and prevents apoptosis of T cells that are co-cultured with cancer cells. The CLP002 peptide also inhibits tumor growth and increases survival of CT26 tumor-bearing mice, suggesting that the CLP002 peptide represents a promising low-molecular-weight checkpoint inhibitor for cancer immunotherapy.

#2395

A bispecific T- cell engager antibody (BiTE) against TAG-72/ CD3 for targeted cancer immunotherapy.

Fatema Khambati, Hatem Soliman. _Moffitt Cancer Center, Tampa, FL_.

Background: Immunotherapies involving cytotoxic T lymphocytes (CTL's) have been central to cancer immunotherapy. Bi-specific T cell engager antibody (BiTE) therapy has emerged as an effective immunotherapy by redirecting cytotoxic T cells against cell surface protein on tumor cells. Our BiTE antibody targets human tumor associated glycoprotein 72 (TAG-72). TAG-72 is a pan-carcinoma marker expressed on the surfaces of many cancer tissues including breast, prostate, ovary, endometrium, stomach, esophagus, and pancreas. It is not expressed in normal tissues making it an ideal target therapy for cancer. Our BiTE antibody can bind CD3 to activate the T-cell's while simultaneously binding TAG-72 on the surface of the cancer cell leading to perforin induced cancer cell death. The in vitro and in vivo effects of TAG-72/ CD3 BiTE antibody on different cancer cells are presented here.

Methods: We studied in vitro and in vivo effects of TAG-72/ CD3 BiTE antibody on different cancer cells; breast (DOX resistant MCF-7, SKBR3, MDA-MB-175 VII), ovarian (OVCAR-3), endometrium (KLE) and colon (Ls174T). In vitro cytotoxicity assay was performed by flow cytometry and immunofluorescence staining using live/ dead cell labelling fluorescent dyes. The cells were co-cultured with naïve human CD8 T cells with/ without BiTE antibody (1 µg, 5 µg and 10 µg) for different time intervals and assayed. In vivo study was carried out in NSG mice implanted with MCF-7 breast cancer cells + naïve human CD8 T cells and treated with a total of 4 doses of BiTE antibody (200µg/ dose).

Results: Different tumor cells showed varying (%) of TAG-72 expression determined by flow cytometry, viz; DOX (55%), SKBR3 (16%), MDA 175 VII (8%), OVCAR-3 (27%), KLE (27%), Ls174T (30%). The BiTE antibody also showed dose dependent binding to these cells. Flow cytometry analysis showed that addition of the BiTE antibody to naïve CD8 T-cells and incubation for 4 days significantly increases tumor cell death as indicated by the Annexin V/ PI+ve signal from DOX (52.4% vs. 41.4% CD8 alone), SKBR-3 (47.9% vs. 30% CD8 alone), MDA 175 VII (23% vs 11% CD8 alone) , OVCAR-3 (31.1% vs. 23.8% CD8 alone), KLE (27% vs 13% CD8 alone) and Ls174T (54% vs 29% CD8 alone) respectively (p<0.05). Further, a significant level of IFN-γ secreted by activated T-cells was only detected in the bispecific T-cell engager Ab + CD8 conditions. In vivo data showed good tumor regression at day 45 with tumors measuring (8 mm2) in group treated with BiTE antibody as compared to untreated group (31 mm2) (p<0.01).

Conclusion: Our in vitro and in vivo data indicates that the TAG-72/ CD3 BiTE antibody can effectively redirect cytotoxic T cells against different cancer cells expressing TAG-72 glycoprotein and may be effective in the treatment of breast, ovarian, endometrium and colon cancers.

#2396

Development of a new generation of dimeric, highly cytotoxic fusion proteins containing active GrB and VEGF targeting tumor neovasculature: Incorporating IgG heavy chain Fc fragments improves cytotoxicity, stability and pharmacokinetics.

Khalid A. Mohamedali, Lawrence H. Cheung, Ana Alvarez-Cienfuegos, Michael G. Rosenblum. _UT MD Anderson Cancer Ctr., Houston, TX_.

VEGF121 is a naturally-occurring splice variant that binds to VEGF receptors R-1 and R-2, which are over-expressed on the endothelium of tumor vasculature but not normal vasculature. The serine protease granzyme B (GrB) is capable of inducing intense cellular apoptosis through both caspase-dependent and caspase-independent multiple-cascade mechanisms. We have previously reported the expression of a fusion protein composed of Granzyme B and VEGF121 (GrB/VEGF121) and its subsequent in vitro and in vivo characterization. Here we report on the development and characterization of GrB-Fc-VEGF, which fuses the two domains via an IgG heavy chain Fc-fragment. We hypothesized that this modification, resulting in a relatively high molecular weight (133 kDa vs 80 kDa) would result in improved in vivo circulation and efficacy. VEGF inter-chain dimerization domains were modified such that dimerization occurred only through the Fc domain. GrB-Fc-VEGF121 was expressed in HEK-293E cells under serum-free conditions and purified from the media by immobilized metal affinity chromatography (25 mg/L). Western blotting confirmed incorporation of both VEGF121 and GrB moieties into the construct. Comparison of GrB-Fc-VEGF121 with its expected molecular weight suggests significant glycosylation when produced in HEK293E cells. The enzymatic activity of GrB in GrB-Fc-VEGF121 was comparable to that of human GrB. In vitro studies of GrB-Fc-VEGF121 suggests improved cytotoxicity against cell lines expressing high levels of VEGFR-1 or VEGFR-2, unlike GrB/VEGF121, which targeted VEGFR-2+ cells more efficiently. Thus, the GrB-Fc-VEGF121 produced in HEK293E cells is active and has significant potential as a targeted therapeutic. In vitro cytotoxicity against an expanded panel of cells, internalization, and in vivo pharmacokinetics and efficacy against various tumor models are currently ongoing and will be presented. Research conducted, in part, by the Clayton Foundation for Research.

#2397

Pre-clinical evaluation of PE0116: A potent cross-linking depednet 4-1BB agonist antibody.

Ningning Song,1 Zhengwu Li,2 Shan He,1 Teddy Yang,1 Qing Duan2. 1 _Shanghai ChemPartner Co., Ltd., Shanghai, China;_ 2 _Jiangsu HyaMab Pharmathetical Co, Shanghai, China_.

4-1BB is a member of the tumor necrosis factor superfamily and plays a critical role in activation of cytotoxic T cells to promote anti-tumor immunity. As a co-stimulatory molecule, it is ideally suited for activation via agonist therapeutic antibodies in order to reactivate the immune system and promote cytolytic activity through CD8+ T cells. Clinical trials of two agonist antibodies (Urelumab and Utomilumab) are ongoing. Urelumab has shown clinical efficacy but been hampered by inflammatory liver toxicity at doses above 1 mg/kg, suggesting potential safety concerns at higher doses. Utomilumab has a superior safety profile, but is a less potent 4-1BB agonist. Hence, it may be necessary to achieve potent immune activation while avoiding limiting immune-related adverse events. PE0116 is a fully human monoclonal antibody generated from immunization of Harbour H2L2 human transgenic mice with recombinant human 4-1BB protein. It blocks the binding of 4-1BB to its ligand 4-1BBL and its activity in T cell activation is crosslinking dependent. After crosslinking, PE0116 strongly increases IFNg secretion from T cells in the presence of anti-CD3 antibody and its activity is superior to Utomilumab analog. More importantly, PE0116 has demonstrated robust anti-tumor activity and better potency than both benchmark antibodies in MC38 tumor model with human 4-1BB KI mice. PE0116 has also demonstrated good safety profile in a non-GLP safety study in cyno monkey. Currently the antibody is under late stage IND-enabling study. Its safety and efficacy will be further tested in patients.

#2398

Dual agonist bispecific antibody targeting OX40 and CD137 mediates anti-tumor immunity and synergizes with PD-1/PD-L1 blockade to improve survival in a syngeneic mouse model.

Edmund Poon, Miguel Gaspar, Leonor Nunes Rodrigues, John Pravin, Anne Skogø Lind, Alexander Koers, Emma McConnell, Mihriban Tuna, Michelle Morrow, Neil Brewis. _F-star Biotechnology Limited, Cambridge, United Kingdom_.

Despite recent advances in cancer patient treatment with checkpoint inhibitors (CPI), clinical benefit is limited to a sub-population of patients. This may be explained by inadequate co-stimulation provided to tumor-specific T-cells to elicit and sustain an effective anti-tumor immune response. CD137 (4-1BB) and OX40, members of the TNF receptor superfamily, are key mediators of costimulatory signals and they play important roles in driving anti-tumor immunity. Combination of CPI with costimulatory agonists represents a promising approach with multiple clinical trials currently underway. However, to date, limited clinical activity has been observed with OX40 or CD137 agonists. We hypothesize that the activity of monospecific Fcγ receptor-dependent agonists may be limited by (1) suboptimal co-stimulation of CD8+ T-cells by OX40 agonists, or (2) insufficient 'help' from CD4+ T-cells for optimal CD137 agonist activity, and (3) inadequate level of clustering via Fcγ receptors at the relevant tissue compartments. To overcome these limitations, we have developed FS120, a dual agonist bispecific antibody (mAb2) with minimized FcγR binding, that drives potent activation of CD4+ and CD8+ T-cells in vitro dependent on co-engagement of CD137 and OX40.

To evaluate the pharmacology and mechanism of action (MOA) of anti-OX40/CD137 mAb2 in the CT26 mouse tumor model, as monotherapy or in combination with PD-1 blockade, a murine surrogate was generated. This surrogate has comparable properties in mouse assay systems compared to FS120 in human systems in relation to binding affinities, potency in primary T-cell-based assays in vitro, and the requirement for OX40/CD137 co-engagement for activity. Here we report the relationship between dose, anti-tumor activity, pharmacodynamic (PD) response and the FcγR-binding independent MOA of an anti-mouse OX40/CD137 mAb2 in vivo. Anti-tumor activity was dose-dependent from 0.1 to 1 mg/kg. Surrogate mAb2 induced substantial increases in proliferating CD4+ and CD8+ T-cells in the blood, the magnitude of effect was inversely correlated with dose between 1 to 30 mg/kg. In a depletion study, anti-mouse OX40/CD137 mAb2 driven increases in peripheral proliferating CD8+ T-cells was partially inhibited by CD4+ T-cell depletion. Combination with anti-PD-1 antibody showed significant improvement in long-term survival resulting in complete tumor regression in 47% of animals compared to 7% and 0%, following single agent therapy with surrogate mAb2 or anti-PD-1 antibody, respectively. In addition, we show data that indicates PD-1/PD-L1 pathway plays a role in limiting the activity of an anti-OX40/CD137 mAb2. Taken together, these results demonstrate the potential for FS120, as a single agent or in combination, to drive an effective anti-tumor immune response in cancer patients.

#2399

**LAG-3/PD-L1 mAb** 2 **can overcome PD-L1-mediated compensatory upregulation of LAG-3 induced by single-agent checkpoint blockade.**

Mustapha Faroudi, Matthew Kraman, Natalie Fosh, Claire Reader, Daniel Gliddon, Claire Seal, Christa Lucas, Alexander Koers, Mateusz Wydro, Michelle Morrow, Neil Brewis. _F-star Biotechnology Ltd, Cambridge, United Kingdom_.

FS118, currently tested in Phase I clinical trial in patients with advanced malignancies (NCT03440437), is a first-in-class bispecific antagonistic antibody (known as mAb2) targeting LAG-3 (Lymphocyte-Activation Gene 3) and PD-L1 (Programmed Death-Ligand 1), two immune checkpoint molecules that promote tumour escape from immune surveillance. Resistance to anti-PD-1 treatment is associated with upregulation of other checkpoint inhibitor receptors such as LAG-3. Dual targeting in immune checkpoint blockade (ICB) using bispecific monoclonal antibodies could potentially overcome this resistance, further increase the clinical benefit of ICB therapy and prevent relapse or resistance after immunotherapy. In a syngeneic model we used a surrogate mAb2 of FS118 (blocking both mPD-L1 and mLAG-3 binding to their receptors) to determine the effect of dual checkpoint blockade on tumour growth and to elucidate the modulation of the underlying immune mechanisms following treatment with the mAb2. Dual blockade of LAG-3 and PD-L1 with the surrogate mAb2 in the MC38 tumour-bearing mice model resulted in an increased anti-tumour activity comparable to a combination of the single agents targeting LAG-3 and PD-L1. Moreover, the mAb2 and single agent combination resulted in distinct modulations of LAG-3 and PD-L1 cell surface expression within the spleen and tumour microenvironment (TME). While both the mAb2 and combination therapy significantly reduced the number of free PD-L1 binding sites on CD4+ and CD8+ T cells in spleen and TME, total LAG-3 cell surface expression increased following treatment with the combination, whereas it reduced with mAb2 treatment. In addition, analysis of serum samples collected at various timepoints confirmed evidence of drug target engagement with increasing levels of both soluble LAG-3 (sLAG-3) and soluble PD-L1 (sPD-L1) in the mAb2 treated animals. Modulation of sPD-L1 levels in the serum of treated animals was also observed in cynomolgus monkeys treated with FS118. These data provide a strong rationale for investigating both cell surface and soluble LAG-3 and PD-L1 levels as potential pharmacodynamic biomarkers in further clinical development.

#2400

K01401-020 is a novel anti-VISTA antibody for cancer treatment: Pharmacodynamic impact on non-human primates and human immune system.

Noureddine Loukili, Isabelle Vandenberghe, Olivier Delfour, Françoise Fraboul, Eric Chetaille, Francisco Cruzalegui, Nathalie Corvaïa, Pierre J. Ferré. _Inst. de Recherche Pierre Fabre, Toulouse Cedex 01, France_.

VISTA (V-domain Ig suppressor of T cell activation), also known as PD-1 homolog (PD-1H), is a transmembrane protein which is highly expressed in myeloid cells such as neutrophils, monocytes, macrophages, and dendritic cells, and at low levels on naive CD4, CD8 and regulatory T-cells. VISTA is also expressed in cancer cells in some patient subsets (Villarroel-Espindola, 2017; Böger, 2017; Liao, 2018). VISTA is functionally described as a negative checkpoint regulator of immune response. Hence, VISTA blockade increases proliferation when T cells are stimulated with VISTA expressing myeloid antigen presenting cells (Wang et al, 2011). Immune suppression by VISTA-positive cells has been hypothesized in the primary or acquired resistance to cancer immunotherapy (Kakhavand, 2017); and even a negative impact of VISTA on immune response has been functionally demonstrated in prostate cancer samples from patients having received prior immunotherapy (Gao, 2017). Here we show that VISTA RNA and protein are expressed in lung cancer samples. We also show by using bioinformatics, that VISTA expression is linked to the higher myeloid infiltrated tumors and can also be found in tumor cells. K01401-020 is novel monoclonal, humanized IgG antibody that binds to cynomolgus monkey and human-VISTA (hVISTA) with a high affinity (Kd ~0.1 nM). The antibody binds to VISTA extracellular domain and blocks its associated function. The pharmacology of K01401-020 was studied in a series of in vitro (ex vivo) experiments with human blood cells, and in vivo studies in non-human primates. Peripheral Blood Mononuclear cells (PBMCs) from several couples of human donors were mixed together in a Mixed Lymphocyte Reaction (MLR) setting, which showed that K01401-020 stimulated IFNg release after 5 days of culture. K01401-020 stimulates NK cells to proliferate and induces activating cytokines by NK cells and monocytes, contributing to T cell activation. PDL1 expression is also clearly induced on myeloid cells by K01401-020, suggesting that combination therapy with anti-PD1/L1 may be required to optimize the anti-tumor efficacy. In non-human primates, preliminary results show that both K01401-020 monotherapy, given weekly at 3 dose levels for 4 weeks and anti-VISTA antibody combination with anti-PD1 antibody (pembrolizumab) were well tolerated. Pharmacodynamic exploration using peripheric blood sampling demonstrated full VISTA occupation by the antibody, and notably, dendritic cell activation. K01401-020 antibody drug is thus expected to increase global and specific anti-tumor immune response in situations where VISTA is expressed in cancer patients and may be synergistic with anti-PD1/L1 antibodies.

#2401

Selection of first-in-human clinical dose range for the tumor-targeted 4-1BB agonist MP0310 using a pharmacokinetic/pharmacodynamics modeling approach.

Alexander Link,1 Laurent Juglair,1 Heïdi Poulet,1 Guy Lemaillet,1 Christian Reichen,1 Patricia Schildknecht,1 Ivana Tosevski,1 Joanna Taylor,1 Niina Veitonmäki,1 Jörg Herbst,1 Keith Dawson,1 Christof Zitt,1 Camila de Almeida,2 Rik de Greef,3 Victor Levitsky,1 Michael T. Stumpp,1 Hong Ji,1 Elmar Vom Baur1. 1 _Molecular Partners AG, Schlieren, Switzerland;_ 2 _Certara Strategic Consulting, London, United Kingdom;_ 3 _Certara Strategic Consulting, Oss, Netherlands_.

Following the clinical success of checkpoint inhibitors, cancer immunotherapy is rapidly expanding into combination treatments to enhance response rates and duration. Engagement of co-stimulatory molecules from the tumor necrosis factor receptor (TNFR) superfamily, including 4-1BB, may be a promising approach to enhance the benefits of cancer immunotherapy. Agonistic antibodies against the costimulatory receptor 4-1BB (CD137) have been shown to effectively enhance the anti-tumor activity of checkpoint inhibitors and other agents in preclinical animal models. However, the clinical development of 4-1BB agonistic antibodies has met with limited success thus far. Anti-4-1BB monoclonal antibodies have either been reported to cause significant dose-limiting hepatotoxicity or demonstrated limited efficacy as single agent therapeutics. We generated a DARPin® therapeutic candidate, MP0310, which comprises domains binding to 4-1BB and fibroblast activation protein (FAP). MP0310 triggers 4-1BB activation only if bound and clustered via FAP which is abundantly expressed by cancer associated fibroblasts present in many solid tumors. In vitro functional assays indicate that MP0310 is a potent T cell co-stimulator in the presence of FAP-expressing cells. In vivo activity of tumor-targeted 4-1BB agonism was assessed in the HT-29 colon carcinoma xenograft model in PBMC humanized mice in combination with a T cell engager. Consistent with tumor targeting, MP0310 enhanced intra-tumoral CD8 T cell expansion while showing only limited systemic activity. Moreover, in a non-human primate GLP toxicity study, no adverse effects or systemic immune cell activation were detected at any tested dose (up to 80 mg/kg twice a week for four weeks). We used a translational pharmacokinetic-pharmacodynamic (PK-PD) modeling approach to integrate in vitro and in vivo data to support the estimation of a minimal anticipated biological effect level (MABEL) and the relevant dose range for first in human (FIH) studies. In addition to a PK model describing the MP0310 concentrations over time in mouse and monkey, direct and indirect response models were used to describe receptor occupancy (RO), intra-tumoral CD8 T cell infiltration and peripheral CD8 count kinetics. Predictions from the combined PK and PD models using average intra-tumoral and peripheral drug concentrations (Cav) provide a MABEL dose with minimal expected systemic PD effects at 20% RO as well as the anticipated therapeutic dose range in humans. In conclusion, our PK/PD-modeling approach provides support for the selection of a safe starting dose for MP0310 in patients in the absence of non-clinical safety signs that could otherwise educate starting dose selection. It provides a rationale for selecting the maximum tested dose, and may help reduce the number of cancer patients receiving sub-therapeutic doses.

#2402

GITR cancer immunotherapy: Epitope swapping of anti-GITR TRX518 to inform functional translatability from mouse to human.

Roberta Zappasodi,1 Heidi Heath,2 Yang Zhang,3 Michael Haas,2 Min Yang,2 Christopher Mirabelli,2 Jedd D. Wolchok,1 Cyndi Sirard,2 Walter Newman,2 Taha Merghoub1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Leap Therapeutics, Cambridge, MA;_ 3 _University of Michigan, Ann Arbor, MI_.

Introduction: At 52.5% identity in their extracellular domains (ECDs), mouse and human GITR (CD357) sequences are only modestly conserved. Thus transitioning promising murine anti-tumor activity data with the anti-GITR DTA-1 and 2F8 Mabs to clinical trials with the non-crossreactive anti-human GITR Mab TRX518 poses a considerable limitation. Here we investigated three elements of the GITR mouse and human antibodies to address this limitation: 1) the functional epitopes targeted by anti-human GITR (TRX-518) and anti-murine GITR (DTA-1 and 2F8) antibodies; 2) their biological activities on primary T cells; and 3) the role of anti-GITR Fc receptor engagement.

Results and Conclusions: Alanine scanning, amino acid substitutions and flow cytometry experiments were performed to define critical residues important for the binding of all three Mabs to their respective GITR targets. Swaps of 5-8 of these critical residues showed both the loss of murine and the gain of human directed Mab binding when residues critical for TRX18 binding were substituted into murine GITR. Likewise, the substitution of residues critical for DTA-1/2F8 binding into human GITR resulted in loss of TRX518 binding and the gain of 2F8/DTA-1 binding. We then mapped these critical residues to 3D structural models of the murine and human GITR ECDs built by contact-map guided fragment assembly simulations. Regions defined by these critical residues showed a high degree of overlap, indicating structural conservation even without amino acid identity. Thus the epitopes defined by both DTA-1 and 2F8 are homologous to those defined on human GITR by TRX518. In in vitro standard proliferation/suppression assays with effector T cells (Teff) and regulatory T cells (Tregs) isolated from human healthy donors and naïve mice respectively, TRX518, similarly to DTA-1, significantly increased proliferation, activation and pro-inflammatory cytokine production of human and murine Teff cultured alone or in the presence of Tregs. These observations indicate that Mabs directed to similar mouse and human GITR ECD epitopes result in similar biological activities. Lastly, we questioned the importance of a functional Fc domain in anti-GITR Mabs in order to translate these in vitro effects into anti-tumor activity in vivo. To this end, we compared the anti-tumor activity of DTA-1 and its aglycosyl version in the aggressive and poorly immunogenic B16 melanoma model. Both Fc and non-Fc binding DTA-1 were able to control tumor growth. This suggests that the aglycosyl-anti-GITR Mab TRX518 has the potential to be effective in the clinic despite the lack of Fc receptor engagement. These results align with our preliminary results in patients treated with TRX518 monotherapy showing reductions in Treg frequencies both in tumor biopsies and in the periphery. TRX518 would therefore appear to be well-poised for use in the treatment of solid tumor malignancies.

#2403

Targeting 4-1BB in tumors enhances anti-tumor immune responses.

Amy Mackay Weber, Patrick Innamarato, Jennifer Morse, Scott Kidd, Sarah Asby, Amod Sarnaik, Shari Pilon-Thomas. _H. Lee Moffitt Cancer Center, Tampa, FL_.

4-1BB (CD137) is a co-stimulatory receptor expressed on activated T cells and other immune cells and is associated with enhanced T cell proliferation, survival, and increased cytokine production upon engagement with its ligand (4-1BBL). We have previously shown that addition of agonistic 4-1BB antibody to tumor fragments increased tumor infiltrating lymphocyte (TIL) expansion in human melanoma and pancreatic tumors. Furthermore, agonistic 4-1BB antibody led to increased expression of maturation markers in resident dendritic cells (DCs) present within tumor fragments. While the role of 4-1BB in T cell biology is well described, little is known about 4-1BB signaling in tumor-resident DCs. We hypothesized that 4-1BB agonism in tumor-resident DCs would promote anti-tumor T cell immune responses. To evaluate the role of 4-1BB signaling on dendritic cells, murine bone marrow derived-DCs were cultured with agonistic 4-1BB antibody and loaded with OVA protein. Treatment with agonistic 4-1BB in vitro led to increased OVA-FITC uptake by DCs (32.6% compared to 15.9% in DCs treated with control antibody). DCs treated with agonistic 4-1BB antibody demonstrated increased T cell stimulatory capacity upon co-culture with OVA-specific OT-I and OT-II T cells. We next compared systemic and intralesional (IL) treatment with agonistic 4-1BB antibody. For the systemic model, mice were injected subcutaneously (SC) with B16 melanoma or Panc02 pancreatic tumor cells and treated by intraperitoneal injection of control or anti-4-1BB antibodies. Mice treated systemically with agonistic 4-1BB antibody exhibited a minimal reduction in tumor growth. In contrast, IL injection of agonistic 4-1BB antibody led to dramatic decreases in tumor growth. Next, we wanted to determine whether IL treatment with agonistic 4-1BB antibody would improve DC stimulatory capacity. Mice bearing B16 or Panc02 tumors were treated IL with normal control or anti-4-1BB antibody. After 7 days, tumor-resident CD11b+ DCs exhibited enhanced in vivo antigen uptake. In addition, an increased number of CD8\+ T cells was measured within tumors treated with agonistic 4-1BB antibody (28.1% compared to 20% in NrIgG-treated Panc02OVA tumors and 19.45% compared to 7.3% in NrIgG treated B16-OVA tumors; p=0.0059). Together, these studies demonstrate that agonistic 4-1BB can enhance the ability of DCs to stimulate T cells and IL administration of agonistic 4-1BB antibody can significantly reduce tumor growth. This suggests that IL treatment of agonistic 4-1BB may yield greater efficacy than systemic therapy in patients with melanoma or pancreatic cancer.

#2404

Mechanistic insight into anti-tumor activity of DKN-01, a therapeutic antibody with immune modulatory activity.

Michael Haas,1 Heidi Heath,1 Franziska Schurpf-Huber,1 Yinyuan Wu,2 Xinjun Zhang,2 Xi He,2 Min Yang,1 Walter Newman,1 Michael Kagey1. 1 _Leap Therapeutics, Cambridge, MA;_ 2 _Boston Children's Hospital, Harvard Medical School, Boston, MA_.

Wnt signaling is a fundamental pathway that is dysregulated in oncology. The Wnt signaling modulator DKK1 is expressed in a variety of tumor types and elevated levels frequently correlate with poor survival. DKK1 promotes tumor growth by stimulating proliferation, metastasis, and angiogenesis, and has been implicated in contributing to an immune suppressive tumor microenvironment. DKN-01 is a humanized monoclonal therapeutic antibody that binds DKK1 with high affinity and selectivity. It is currently being evaluated clinically as a monotherapy and in combination with chemotherapies and other immunotherapies in a variety of solid tumors. Here we demonstrate that efficacy from a murine version of DKN-01 (mDKN-01) in a syngeneic tumor model requires NK cells. mDKN-01 also reduces lung metastasis, further supporting a model where DKK1 neutralization stimulates a NK mediated anti-cancer response. In addition, DKN-01 has efficacy in a human prostate xenograft model that expresses high levels of DKK1 and retains functioning NK cells. Taken together, these data support an innate immunotherapeutic mechanism of action of DKN-01 and suggest that tumor expression of DKK1 may predict response. As such, evaluation of peripheral blood and tumor biopsies from patients undergoing treatment with DKN-01 in combination with chemotherapy or other immunotherapies are underway to demonstrate innate immune mediated anti-tumor activity and validate DKK1 expression as a biomarker.

#2405

B7H4 as a T cell inhibitory regulator in bladder cancer.

Joshua Meeks,1 Alexander P. Glaser,2 Damiano Fantini,3 Yanni Yu,1 Valerie Eaton,1 Joseph R. Podojil,1 Stephen D. Miller1. 1 _Northwestern Univ. Feinberg School of Medicine, Chicago, IL;_ 2 _NorthShore University Health System, Chicago, IL;_ 3 _Northwestern Univ. Feinberg School of Medicine, IL_.

Despite a high number of neoantigens and T cell infiltration, most locally advanced and metastatic bladder cancers (BC) (>70%) do not respond to anti-PD1 treatment. Thus, targeting additional checkpoints may play an important role in treatment of BC. We sought to identify negative regulators of T cell activity and evaluate their function using a previously validated pre-clinical carcinogen-induced mouse model of BC.

To identify regulators of T cell activity, we first investigated the timing and infiltration of T cells during tumor development in the BBN mouse BC model. We found that numbers of CD4+ and CD8+ T cell increased dramatically within the first 1-2 weeks, but then decreased over the next six weeks decreasing to the lowest levels at 8 weeks before increasing again coordinating with increasing tumor volume. During the decrease in T cell numbers, macrophages increased and by transcriptome profiling we identified increased levels of the T cell inhibitory protein B7-H4. B7-H4+CD11b+ macrophages increased over 4 weeks with opposite expression of CD4+ and CD8+ T cells by FACS. By IHC we localized B7-H4 expression in macrophages adjacent to tumor. A bioinformatic analysis of the human TCGA identified highest B7-H4 expression in luminal infiltrated subtypes of MIBCs, but did not correlate with CD4, CD8 or overall tumor mutation burden. MIBCs with elevated B7-H4 expression had significantly worse survival. In vitro, addition of anti-B7-H4 significantly increased proliferation and IFN-γ production by human T cells co-cultured with B7-H4 expressing APCs. To determine the role of B7-H4 in BC, we treated mice with overt BC (beginning at 4 months of carcinogen administration) with vehicle, anti-PD1 or anti-B7-H4 antibody. Compared to vehicle treated animals, anti-B7-H4 treated mice had a reduction in the number of pT3 tumors (50% vs. 73%), but less than anti-PD1 treated tumors (27%). However, anti-B7-H4 treated mice had significantly increased tumor infiltrating CD8+ T cells per histological field (77.1+34.3 vs. 11.5+7.8; p=0.0121), as well as a significant increase splenic CD8+IFN-γ+ T cells and levels of secreted IFN-γ upon anti-CD3 stimulation. Transcriptome profiling of tumors that responded to anti-B7-H4 antibody by RNA-Seq identified a decrease in mitotic cell cycle, cell cycle checkpoints, RHO GTPases and mitotic spindle checkpoint pathways in responsive tumors.

In summary, we confirm the expression of B7-H4 in macrophages found in a murine model of BC and correlate expression to poor survival in the TCGA of MIBC. Inhibition of B7-H4 results in lymphocyte proliferation in vitro and inhibition of B7-H4 in mice resulted in decreased tumor stage and increased CD8+ TILs. This data suggests that B7-H4 may be a candidate alternative checkpoint that can be targeted in humans with BC unresponsive to PD1 therapy.

## EPIDEMIOLOGY

### Early Detection and Risk Prediction

#2406

A general probabilistic algorithm to predict de novo mutations in familial diseases as demonstrated in Li-Fraumeni Syndrome.

Xuedong Pan,1 Fan Gao,2 Elissa B. Dodd,1 Jasmina Bojadzieva,1 Phuong L. Mai,3 Valen E. Johnson,4 Kristin Zelley,5 Kim E. Nichols,5 Judy E. Garber,6 Sharon A. Savage,3 Louise C. Strong,1 Wenyi Wang1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Rice University, Houston, TX;_ 3 _National Cancer Institute, Bethesda, MD;_ 4 _Texas A &M University, College Station, TX; _5 _Children's Hospital of Philadelphia, Philadelphia, PA;_ 6 _Dana-Farber Cancer Institute, Boston, MA_.

Purpose:

Germline variants (mutations) occurring in the carrier but not in the carrier's parents are defined as de novo mutations (DNMs). DNMs have been increasingly recognized as causal factors for rare diseases. Given this important role, the ability to identify DNM carriers, scarcely distributed among all mutation carriers, will allow researchers to study the likely distinct molecular mechanisms of DNMs in these diseases. However, testing the mutation status of the parents is often impractical in retrospective studies due to lack of blood samples.

Methods:

To fill this need, using Li-Fraumeni syndrome (LFS) as a representative of a rare inherited syndrome, we developed a method called Famdenovo to predict the de novo status (de novo or familial) of germline mutations, such as in TP53. We have collected a total 324 LFS family pedigrees with confirmed TP53 germline mutations from four medical centers in the US, with 186 pedigrees having TP53 genetic testing results available in at least one full trio in the pedigree, serving as a validation set. We further apply Famdenovo to the remaining 138 families (discovery set) to identify individuals who may carry a de novo mutation in TP53.

Results:

In the validation set, the area under the ROC curve (AUC) of Famdenovo is 0.95 (95% CI: [0.92, 0.98]), suggesting excellent ability for discrimination, and the ratio of the observed to expected (OE) is 1.32 suggesting good concordance. In the discovery set, we predict that an additional 40 individuals (95% CI: [30, 50]) are DNM carriers, which increases the total number of DNM carriers to 82. The corresponding predicted DNM rate is 28.9%, also consistent with what is observed in the validation set. Across the validation and discovery sets, we observed similar distributions of ages-of-onset for DNM carriers at specific cancer sites: breast, brain, leukemia, osteosarcoma, soft tissue sarcoma, lung, and adrenal cortical carcinoma. Interestingly, Lung cancer only occurred in female DNM carriers. Among TP53 hotspot mutations, R248Q is most enriched in DNMs.

Conclusions:

Our new statistical method Famdenovo provides the probability of a person carrying a de novo germline mutation in rare inherited syndromes when the mutation status of both parents is not available. Famdenovo is freely available as an R package from http://bioinformatics.mdanderson.org/main/Famdenovo. The LFS family cohorts not only served as a validation set for the accuracy of Famdenovo in predicting DNMs, but also benefited from using Famdenovo to discover additional individuals carrying DNMs in TP53. The computer-based identification of DNM carriers in TP53, who are otherwise hidden in a wide population, enabled epidemiological interpretation of the cancer outcomes for DNM carriers. Famdenovo is a general tool and can be applied to other cancer genes where there is a good understanding of the penetrance of the associated disease phenotype.

#2407

Stage shifting by modifying the determinants of cancer.

Gyanendra Pokharel,1 Paula J. Robson,2 Lorraine Shack,2 John J. Spinelli,3 Karen A. Kopciuk1. 1 _University of Calgary, Calgary, Alberta, Canada;_ 2 _Alberta Health Services, Calgary, Alberta, Canada;_ 3 _BC Cancer Agency, Vancouver, British Columbia, Canada_.

Early detection of cancer is a major challenge because most tumors are asymptomatic in the early stages. However, if the cancer is caught early, it can be treated more effectively thereby improving survival rates. Many environmental, lifestyle and genetic risk factors for developing cancer have been identified; however, they collectively do not fully explain its etiology. By using health and lifestyle information of people along with their cancer screening history before they develop cancer, we hope to identify factors that can be modified to avoid diagnosis at later stages of disease. These identified factors will be evaluated immediately using simulated data that mimics real data without waiting years for results from intervention studies. Hence, the aim of our study is to explore patient and health care system factors associated with stage of cancer diagnosis and to explore reduction in stages at diagnosis by modifying identified factors using simulation methods. The study is based on results obtained from the analysis of the data collected from the participants in Alberta Tomorrow Project (ATP), a prospective cohort of over 50,000 adults (aged ≥35 years). More than 3,500 participants have been diagnosed with cancer since their enrollment in the ATP. Close to 80% of the breast and colorectal cancers, and 70% of the lung cancer were diagnosed at early stage (stages I and II) and late stage (stages III and IV), respectively. Using these information as prior knowledge, we simulated the stages of breast, lung, and colorectal cancers at diagnosis along with risk factors and screening interventions. The outcome (stage at diagnosis) was modeled on the ordinal scale (I-IV) using a popular ordinal logistic regression model, so-called proportional odds and partial proportional odds models, that assesses the strengths of the associations between ordinal outcomes and measured risk factors. Preliminary findings from our simulation study for either ordinal response model found that a change in combination of risk factors could increase the probability of shifting the stage of diagnosis. We also plan to explore a shift towards earlier stage at diagnosis by modifying selected environmental and lifestyle risk factors - for example, reducing the amount of time spent in the midday sun (reduced risk exposure), and increase the amount of vitamin D (increase protective factor exposure). This will be done systematically to identify the impact of factors individually and collectively. The comprehensive modeling of factors associated with cancer stage at diagnosis provides information simply not attainable in empirical studies. Using a simple framework of stage at diagnosis, we expect to identify factors that can be used by screening and prevention programs to identify individuals who may benefit from individualized screening practices or from targeted prevention messages, thereby increasing the proportion of cases diagnosed at earlier stages.

#2408

**Analysis of the** CDKN2A **gene in FAMMM Syndrome families reveals early age of onset for additional syndromic cancers.**

Candace D. Middlebrooks. _NHGRI/NIH, Baltimore, MD_.

Familial atypical multiple mole melanoma (FAMMM) syndrome is a familial cancer syndrome that results from mutations in several genes including the CDKN2A gene. The syndrome is currently comprised of dysplastic nevi as well as melanoma and pancreatic cancer and is also associated with lung and breast cancer. The aforementioned set of cancers is referred to as "concordant" cancers. As families with known CDKN2A mutations have been studied longitudinally, clinicians observed an abundance of other cancers or "discordant cancers." However, it was unknown whether these cancers were related to the syndrome. We sought to determine whether these discordant cancers also occur at higher frequencies and at earlier age of onset in carriers than non-carriers.

We studied 10 FAMMM syndrome families (N = 1085 individuals) in which a causal mutation in the CDKN2A gene was identified. We performed survival analysis as well as a mixed effects cox regression with age at follow-up or cancer event as our time variable and presence or absence of a concordant or discordant cancer as our censoring variable. The survival curves showed a significant age effect with carriers having a younger age at cancer onset for concordant (as expected) as well as discordant cancers than that of non-carriers. The cox regression models were also highly significant (P = 1.24E-27 and P = 5.00E-13 for the concordant and discordant cancers, respectively). We then performed a sensitivity analysis where we removed non-melanoma skin cancers from the discordant cancer group (coding individuals with these cancers as unaffected with a discordant cancer) to ensure that these cancers were not driving the effect seen. The result was still highly significant. Furthermore, we observed a visible pattern where individuals with multiple primaries presented with melanoma first followed by various combinations of other cancer primaries later on. It is likely that melanoma is the initial cancer to occur in a series of cancer occurrences as the result of a CDKN2A mutation tumor-driving effect paired with almost unavoidable UV light exposure. The additional cancer primaries that occurred in these individuals are likely the result of additional germline or acquired somatic mutations and/or environmental exposures that have latent effects.

These analyses support the hypothesis that carriers of mutations in CDKN2A in FAMMM syndrome have increased risk for early onset of several additional cancer types that are not currently screened in these families. This suggests that early screening for these cancers would be beneficial to carriers. The implications from this study could impact clinical guidelines in cancer screening of CDKN2A mutation families as well as policies formulated in regards to insurance coverage. This in turn may reduce the morbidity of these additional cancers (due to earlier investigation and diagnosis and targeting of prevention strategies).

#2409

Predictive accuracy of Cox proportional hazards cure models with application in cancer studies.

Xiaoxia Han,1 Yilong Zhang,2 Yongzhao Shao3. 1 _Henry Ford Health System, Detroit, MI;_ 2 _Merck Research Laboratories, Rahway, NJ;_ 3 _New York University School of Medicine, New York, NY_.

With recent development in cancer screening, diagnosis and treatment, many early-stage cancer patients will never experience recurrence, metastasis or death due to their primary cancer, whom can be considered as clinically cured. In the era of precision medicine, it is of interest to develop predictive models for cure status as well as for survival among uncured patients. The area under the ROC curve (AUC) is commonly used for assessing discriminative accuracy for dichotomous outcome (cured/uncured). Yet the conventional AUC cannot be directly used here due to unobserved cured status. In this study, we extend the conventional AUC to Cox proportional hazards (PH) cure models for evaluating prognostic utility in the presence of latent cured patients. We develop consistent and asymptotically normal estimates with explicit formulas for sensitivity, specificity and AUC. Furthermore, we reviewed the prognostic accuracy estimators for survival time among uncured patients for Cox PH cure models. Numerical studies show that our proposed estimators perform well for finite sample size. A melanoma data is used to illustrate the utility of our proposed prognostic metrics. We also have developed an R package called evacure to efficiently compute these estimates and their confidence intervals.

#2410

Development of a custom genotyping platform and genetic prediction of prostate cancer risks in sub-Saharan Africa.

Joseph Lachance,1 Maxine Harlemon,1 Paidamoyo Kachambwa,2 Olabode Ajayi,2 Michelle Kim,1 Marcia Adams,3 Elizabeth Pugh,3 Lindsay Peterson,2 Timothy Rebbeck4. 1 _Georgia Institute of Technology, Atlanta, GA;_ 2 _Centre for Proteomic & Genomic Research, Cape Town, South Africa; _3 _CIDR at Johns Hopkins University, Baltimore, MD;_ 4 _Dana-Farber Cancer Institute, Boston, MA_.

Prostate cancer is a highly heritable disease that disproportionally affects African and African-American men. With this in mind, the MADCaP (Men of African Descent Carcinoma of the Prostate) Network has developed a custom genotyping platform. This array is optimized for detection of novel genetic associations in sub-Saharan African populations. The MADCaP Array contains approximately 1.6 million markers, many of which overlap the H3Africa Consortium Array and the OncoArray. It includes an imputation backbone that successfully tags 94% of common (MAF > 0.05) genetic variants in African populations (r2 threshold = 0.8). To aid in fine-mapping, the MADCaP Array has a high density of markers in genomic regions surrounding known cancer associations, including 8q24. Markers on the MADCaP Array also include over 27,000 prostate eQTLs. Using the MADCaP Array, we conducted a pilot study of 800 individuals with individual-level phenotype information. These samples include equal numbers of prostate cancer cases and controls, and they were collected from study sites in Ghana, Nigeria, Senegal, and South Africa. Here, we assess the extent to which polygenic risk scores are able to predict prostate cancer risks in African populations. We also identify non-African GWAS signals that replicate well in African populations. Additional analyses in this study include testing whether specific genetic ancestries are over-represented in cases, and quantifying the extent to which runs of homozygosity are found in the genomes of cases and controls.

#2411

A multidisciplinary approach for HCC risk prediction in patients with cirrhosis utilizing elastography, imaging, circulating tumor cells and genomics.

Emmanuel Thomas, Deukwoo Kwon, Sid Rawal, Ashutosh Agarwal, Beatrice Madrazo, Steven Chen, Eugene Schiff. _University of Miami Miller School of Medicine, Miami, FL_.

Background: HCV induced HCC is increasing in the United States. Therefore, it is imperative that we understand the genetic and cellular based mechanisms underpinning the linkages between HCV and HCC and the impact of race/ethnicity on the development of this deadly tumor. The aims of our study were to assess changes in transient elastography (TE) and Fibrosis-4 (FIB-4) score in a large cohort of patients and identify risk based on race/ethnicity. Patients were also stratified for HCC risk based on the Toronto HCC Risk Index (THRI), genomics, imaging data with inclusion of screening for circulating tumor cells.

Methods: Our cohort included 1,943 patients with liver disease, including HCV, HBV, NASH, etc., were assessed by Fibroscan and comparisons made with clinical parameters of liver disease. Approximately 500 patients had liver biopsies. Statistical analysis with Kruskal-Wallis and Chi-Square tests was carried out. Values reported are mean ± standard deviation. The estimated stage of liver fibrosis based on TE was categorized as F0-F2 (<9.4kpa), or F3 (9.5 - 12.4 Kpa), or F4/cirrhotics (TE >12.5 kpa).

Results: In our cohort, African Americans (AAs) had higher BMIs (27.8±5.2, p<0.01) and lower albumin levels (4.2±0.5 g/dl, p=0.01). Platelet (p=0.79) and AST values (p=0.17) were comparable between races; however, ALT was highest among non-Hispanic whites (67±68, p=0.02). TE measurement was highest in AAs and Hispanics (12.2±12 and 12.2±12 kPa, respectively) and lowest in non-Hispanic whites (12.2±12 kPa) (p<0.01), while FIB4 Index was not statistically different (p=0.23). Risk of developing HCC, as measured by THRI, was highest in AAs (234±65) and lowest in Hispanics (214±68, p<0.01). Stratifying by Hepatitis C (HCV) status, the majority of non-Hispanics had HCV, whereas most Hispanics had non-HCV liver disease (p<0.01). HCV positive patients were older (59±11 vs 54±14 years, p<0.01), had higher AST (60±71 vs 45±58, p<0.01), ALT (67±67 vs 55±75, p<0.01), THRI (238±64 vs 189±68, p<0.01), TE scores (12.4±11.6 vs 10.6±11.5 kPa, p<0.01), and FIB4 (3.0±3.2 vs 2.0±1.7, p<0.01), but lower BMI (26.4±4.5 vs 27.4±4.9, p<0.01), platelets (187.0±72 vs 204.1±74 109/L, p<0.01), and albumin (4.2±0.5 vs 4.4±2.3 g/dl, p<0.01).

Conclusions: Liver fibrosis stage, as determined by TE, increased with HCC risk as determined by THRI. THRI identified the subpopulation of African Americans as having generally greater risk of HCC, despite comparable platelet and FIB4 levels. Hispanics had similarly high TE scores as AAs, but lower risk of developing HCC. The patients who remain at risk for HCC will be further stratified for increased propensity to develop HCC utilizing genomics, advanced imaging and screening for circulating tumor cells. These results suggest the need for enhanced investigation of key drivers of HCC, with particular attention to racial/ethnic disparities.

#2412

Development of a gene signature to predict positive or negative sentinel nodes as a prognostic marker in melanoma.

Audrey J. Majeske, Timothy J. Geddes, Richard Keidan, George D. Wilson. _Beaumont Health, Royal Oak, MI_.

In recent years, sentinel lymph node (SLN) biopsy has been adopted as a less invasive technique to assess the prognostic outcome of melanoma, whereby draining the key lymph nodes for presence of a tumor decreases unnecessary lymph node dissections and reduces the risk of developing lymphedema. However, this technique only has therapeutic value in patients with SLN-positive tumor nodes, which only represents about half of the population diagnosed with melanoma. In a pilot study, we assessed the predictive efficacy of using a commercialized multi-biomarker next-generation sequencing (NGS) assay (Illumina® Comprehensive Cancer Panel) containing selected genes which have been shown to have a prognostic impact in melanoma progression, to determine whether these can identify primary tumors which do not require SLN biopsy. We used laser capture microdissection to capture pathologist-identified areas of tumor from four groups of samples 1) skin lesions from SLN-positive patients, 2) skin lesions from SLN-negative patients, 3) node samples from SLN-positive patients and node samples from SLN-negative patients; some cases of skin lesions and nodes were matched for the same patient. Sequencing identified 14 genes containing variants in >70% of the positive node samples, which were absent in samples with negative nodes. This included EGFR, PDGFR, ATM, PIK3CA, TP53 and ERBB4. Of note, variants in ERBB4 is of interest as it has been shown in the past to be a potential molecular target in the treatment of melanoma. We are currently validating these preliminary findings in a larger cohort of patients (n=31) and will report on the NGS findings of this study and the efficacy of using genetic biomarkers in skin biopsy samples as a potential prognostic marker for early stage melanoma patients who may not require SLN biopsy.

#2413

Genomewide expression profiling identifies a novel gene-expression signature for recurrence prediction in patients with early-onset colorectal cancer (EOCRC).

Kensuke Yamamura,1 Lina Zhu,2 Raju Kandimalla,1 Takatoshi Matsuyama,3 Yusuke Kinugasa,3 Francesc Balaguer,4 Hideo Baba,5 Xin Wang,2 Ajay Goel1. 1 _Baylor Scott & White Research Institute, Charles A. Sammons Cancer Center, Baylor University Medical Center, Dallas, TX; _2 _City University of Hong Kong, Hong Kong, Hong Kong;_ 3 _Tokyo Medical and Dental University, Tokyo, Japan;_ 4 _University of Barcelona, Barcelona, Spain;_ 5 _Graduate School of Medical Science, Kumamoto University, Kumamoto, Japan_.

Purpose: Although the incidence of CRC has declined over the last decade, recent evidence indicates that the incidence of early onset CRC (EOCRC; patients younger than age 50) is actually on the rise. The disease presentation in patients with EOCRC is quite distinct, with often the disease diagnosis at more advanced stages, poorer histological grade, and higher local recurrence rates, compared to late onset CRC (LOCRC). Hence, availability of biomarkers that can predict recurrence in high-risk EOCRC patients is much needed as it will lead to improved prognosis, and tailoring personalized treatment regimens in this population. Herein, we have conducted a genomewide biomarker discovery approach, followed by robust bioinformatic analysis to establish a novel gene-based recurrence prediction classifier in patients with EOCRC.

Methods: We undertook a comprehensive and systematic biomarker discovery effort by first analyzing six independent, genomewide expression datasets (GSE14333, GSE17538, GSE3311, GSE37892, GSE39084 and GSE39582) of EOCRC patients to identify (discovery dataset; n=81) and validate (validation dataset; n=75) a gene signature for predicting recurrence in EOCRC. Subsequently, the robustness of this signature was analytically evaluated in an in-house clinical training cohort (n=54). Using recursive feature elimination approach based on random forest classification, the signature was further refined and the gene signature was validated in another independent clinical cohort (n=54).

Results: We initially identified a 25-gene signature from a total of 3591 mRNAs, which showed significant potential for predicting recurrence free survival (RFS) in both the in-silico discovery (HR, 32.83, 95% confidential interval (CI), 10.73 - 100.51, P<0.001) and validation datasets in the discovery phase (HR, 4.11, 95% CI, 1.77 - 9.56, P <0.001). Subsequently, based on our clinical training cohort, we developed a reduced and optimized 5-gene signature, which demonstrated robust prognostic power in both the clinical training cohort (HR, 3.67; 95% CI, 1.48-9.52; P=0.003) and an independent validation cohort (HR, 7.53; 95% CI, 1.73-51.3; P=0.007). In univariate and multivariate cox regression analysis, our 5-gene signature emerged as an independent predictor of worse RFS. Furthermore, a combination signature comprising of lymph node metastasis and our 5-gene signature demonstrated an even superior predictive performance compared to individual factors in both cohorts (training cohort: HR 4.56; 95% CI, 0.65-0.88; validation cohort: HR 8.25, 0.86; 95% CI, 0.74-0.94).

Conclusion: Our novel 5-gene signature provides a robust prognostic tool for patients with EOCRC; which can be clinically transformative in guiding post-surgical decision-making to improve survival and management of young patients suffering from this malignancy.

#2414

Prognosis prediction of colorectal cancer using gene expression profiles.

Feixia Pan,1 Tianhui Chen,2 Xiaohui Sun,1 Kuanrong Li,3 Xiyi Jiang,2 Asta Försti,4 Yimin Zhu,1 Maode Lai1. 1 _Zhejiang University, Hangzhou, China;_ 2 _Zhejiang Academy of Medical Sciences (ZJAMS), Hangzhou, China;_ 3 _Guangzhou Medical University, Guangzhou, China;_ 4 _German Cancer Research Center (DKFZ), Heidelberg, Germany_.

Background: Investigation on prognostic markers for colorectal cancer (CRC) deserves efforts, but data from China are scarce. This study aimed to build a prognostic algorithm using differentially expressed gene (DEG) profiles and to compare it with the TNM staging system in their predictive accuracy for Chinese CRC prognosis.

Methods: DEGs in six-paired tumor and corresponding normal tissues were determined using RNA-Sequencing. Subsequently, matched tumor and normal tissues from 127 Chinese patients were assayed for further validation. Univariate and multivariate Cox regressions were used to identify informative DEGs. A predictive index (PI) was derived as a linear combination of the products of the DEGs and their Cox regression coefficients. The predictive performance of the PI was evaluated with receiver operating characteristics (ROC) curves and the area under curves (AUCs). A logistic regression model was built, including both the DEGs-based PI and tumors' TNM stages.

Results: We identified significant associations of 13 DEGs (out of 75 candidate DEGs) with CRC survival. A PI based on these 13 DEGs (PI-13) predicted CRC survival probability more accurately than the TNM staging system [AUCs for 3-year survival probability 0.76 (95% confidence interval: 0.67, 0.83) vs. 0.63 (0.53, 0.71)] but comparable to a simplified PI (PI-5) using five DEGs (LOC646627, SCARA5, CDKN2A, ATP6V1A, and DNMT3B). The predictive accuracy was improved further by combining PI-5 and the TNM staging system [AUC for 3-year survival probability: 0.81 (0.73, 0.88)].

Conclusion: Prognosis prediction based on informative DEGs might yield a higher accuracy than the TNM staging system.

#2415

Polygenic risk-tailored screening for prostate cancer: A cost-effectiveness analysis.

Thomas Callender,1 Mark Emberton,1 Steve Morris,1 Ros Eeles,2 Zsofia Kote-Jarai,2 Paul Pharoah,3 Nora Pashayan1. 1 _University College London, London, United Kingdom;_ 2 _Institute for Cancer Research, London, United Kingdom;_ 3 _University of Cambridge, Cambridge, United Kingdom_.

Background Screening for prostate cancer with prostate specific antigen (PSA) remains controversial. Risk-tailoring based on age and polygenic profile might conserve the benefits of PSA screening whilst reducing overdiagnosis. This study evaluates the cost-effectiveness and benefit-to-harm balance of polygenic risk-tailored screening for prostate cancer.

Methods A life-table approach was used. A hypothetical cohort of 4.48 million men in England between the ages of 55 and 70 was followed to the age of 90. Three scenarios were compared: no screening, age-based screening (PSA testing every four years from 55 to 70), and risk-tailored screening based on age and polygenic risk profile (men above the risk threshold receive PSA testing four-yearly from the age they reach the risk threshold to 70). The number of prostate cancer cases, deaths from prostate cancer, overdiagnosed cases, life-years and quality-adjusted life-years (QALYs), incremental cost per QALY, and net monetary benefit were calculated. Sensitivity analyses investigated the impact of parameter uncertainty, and the impact of varying the risk threshold, on outcomes. Costs were estimated from the perspective of the UK National Health Service; both costs and benefits were discounted at 3.5%.

Results The incremental cost per additional QALY for age-based screening compared to no screening was £58,254 ($75,899), with 27% simulations having an incremental cost-effectiveness ratio of less than £20,000 ($26,058), the threshold set by the UK National Institute for Health and Care Excellence for an intervention to be considered cost-effective. In risk-tailored screening, as the 10-year absolute risk-threshold was progressively increased from 2.6%, equivalent to the 10-year absolute risk of a 55-year old man, to 7.1%, the corresponding risk of a 69-year old man, the cost per additional QALY gained reduced from £36,065 ($46,989) to £26,210 ($34,149), when compared to no screening. By comparison with age-based screening, risk-tailored screening with a threshold between 2.6% and 7.1% led to 8 (2.5%) and 45 (13%) fewer deaths prevented from prostate cancer per 10,000 men, with a 0.1% rise and 10% reduction in costs, respectively. Raising the risk threshold from 2.6% to 7.1% led to fewer men being screened (cumulative total of 35% of men screened by comparison with 83%), 53% fewer overdiagnosed cases, 22% fewer biopsies and 10% lower costs, at the expense of 10% fewer prostate cancer deaths averted.

Conclusion Risk-tailored screening reduces the harms of screening for prostate cancer whilst preserving much of the mortality benefit. This study raises the prospect of risk-tailored population-based screening for prostate cancer; the precise risk-thresholds selected will depend on societal judgements regarding the appropriate trade-offs between harms and benefits.

#2416

Vitamin D concentration and the risks of Barrett's esophagus and esophageal adenocarcinoma: A Mendelian randomization study.

Jing Dong, Aaron Thrift. _Baylor College of Medicine, Houston, TX_.

Background Epidemiological studies on circulating concentrations of 25 hydroxy vitamin D (25(OH)D) and risks of esophageal adenocarcinoma (EAC) have shown conflicting results. Here we conducted a Mendelian randomization (MR) study to determine the associations between circulating concentrations of 25 hydroxy vitamin D (25(OH)D) and risks of EAC and its precursor, Barrett's esophagus (BE).

Methods We conducted a MR study using a two-sample (summary data) approach. Six single-nucleotide polymorphisms (SNPs) (rs3755967, rs10741657, rs12785878, rs10745742, rs8018720 and rs17216707) associated with circulating 25(OH)D concentrations were used as instrumental variables. Data from 6,167 BE patients, 4,112 EAC patients and 17,159 control participants were from studies participating in the Barrett's and Esophageal Adenocarcinoma Consortium (BEACON), as well as studies from Bonn, Germany, and Cambridge and Oxford, United Kingdom. Analyses were performed separately for BE and EAC.

Results Overall, we found no evidence for an association between genetically estimated 25(OH)D concentrations and the risk of BE or EAC. The odds ratio (OR) per standard deviation unit increase in genetically estimated 25(OH)D concentrations for BE risk estimated by combining the individual SNP association using inverse variance weighting was 1.21 (95% confidence interval, CI, 0.77-1.92; P = 0.41). The OR for EAC risk estimated by combining the individual SNP association using inverse variance weighting was 0.68 (95% CI, 0.39-1.19; P = 0.18).

Conclusions This Mendelian randomization study found that genetically estimated low 25(OH)D concentrations were not associated with increased risks of BE or EAC.

#2417

Unsupervised deep-learning to identify histopathological features among breast cancers in the Cancer Prevention Study-II Nutrition Cohort.

Samantha Puvanesarajah,1 James M. Hodge,1 Jacob L. Evans,2 William Seo,2 Michelle Yi,2 Michelle M. Fritz,2 Mary Macheski-Preston,2 Ted Gansler,1 Susan M. Gapstur,1 Mia M. Gaudet1. 1 _American Cancer Society, Atlanta, GA;_ 2 _Slalom Consulting, LLC, Seattle, WA_.

Background: Machine learning (ML) methods are becoming more feasible for use in clinical and epidemiologic research of breast cancer, particularly when characterizing histopathology. Compared to supervised ML methods, unsupervised approaches represent an opportunity to distinguish features heretofore unknown. The purpose of this study was to use unsupervised deep learning methods to identify histopathological features in diagnostic breast cancer hematoxylin and eosin (H&E) slides that are associated with clinical characteristics and patient outcomes.

Methods: One H&E slide was scanned (Leica Biosystems Aperio Versa scanner) at 20x magnification for each of 1,716 women diagnosed with breast cancer from the Cancer Prevention Study-II Nutrition Cohort. In the pre-processing phase, the scanned images underwent color normalization, artifact detection, and tiling. We then used an un-pretrained VGG16 autoencoder with data augmentation for feature learning and extraction from tiles. These features were two-tiered clustered using the K-means algorithm. Each tile was assigned the cluster with the highest probability. The tiles were reassembled into whole slide images. For each slide, the proportion of tiles in each cluster was calculated. We will associate clusters with clinical features and 5- and 10-year breast cancer-specific survival using multivariable logistic and Cox proportional hazards regression models, respectively.

Results: Mean age at baseline enrollment (1992-1993) and breast cancer diagnosis for the cases was 60.6 years (SD=6.0) and 71.5 years (SD=7.0), respectively. The majority of cancer diagnoses occurred after 1999 (79%) and 81% of women included were diagnosed invasive breast cancer. The final pipeline for the full set of images is currently being built. Preliminary runs at the 1x magnification level with 100 cases (N=21,472 tiles) have shown clustering based on macro-level features such as adipose, stromal and epithelial content. Second-tier clustering (clustering within clusters) shows further delineation of groups within clusters of interest (i.e. epithelial-cell rich regions). The final output with all 1,716 slides will be based on analysis at the 5x magnification level.

Discussion: We expect that some histopathological features identified by ML models will be associated with conventional pathology features, clinical features, and breast cancer-specific survival. Utilization of ML methods for analyzing histology slides provides additional data that can be integrated into epidemiological studies. Future directions include analyzing images at higher magnifications (10x or 20x) and assessing the association between ML histopathological characteristics and breast cancer risk factors and incorporating these characteristics into prognostic models.

#2418

Are we underestimating cancer mortality? A mediation model shows a larger impact from smoking.

Ronald C. Eldridge,1 W. Dana Flanders,1 Colin Adler,1 Deborah W. Bruner,1 Canhua Xiao2. 1 _Emory University, Atlanta, GA;_ 2 _Yale University, New Haven, CT_.

Purpose: Cancer mortality is estimated using underlying cause of death. For instance, to estimate the risk of head and neck cancer (HNC) mortality attributed to smoking, 'death by HNC' is the outcome (ie, smoking → HNC death). This conventional approach has notable flaws. It relies on accurate reporting on cause of death, a potential source of misclassification, and it excludes any mortality effects in cancer patients who die from other causes, a potential source of selection bias. Regarding HNC, 80% of all patients are excluded using this approach. These potential biases may result in underestimating cancer mortality from important public health factors such as smoking, obesity and physical activity. To overcome these biases, we are proposing a new approach to estimate cancer mortality. The method uses a causal mediation model and we demonstrate it in a smoking and HNC example.

Methods: The model estimates cancer mortality through a mediated pathway. In our example, smoking is the exposure, incident HNC is the mediator, and all-cause mortality is the outcome (smoking → HNC incidence → all deaths). The mediated effect is the risk of mortality attributed to HNC, which in turn, is attributed to smoking. We compare the results in the mediation model to the conventional approach using Aalen's additive hazards, which estimates the absolute increase in deaths in smokers compared to non-smokers; the mediation model also uses logistic regression for the HNC mediator. Both models are adjusted for age, sex, race, body mass index, diet, alcohol use, physical activity, marital status, education, and reported health. We used the NIH-AARP Diet and Health cohort, which has smoking information at baseline, and cancer incidence and mortality during follow-up on 518,100 U.S. subjects.

Results: In the study, 2,279 subjects developed HNC during follow-up, among whom, 508 (22%) died from HNC; 125,442 subjects died from any cause. Under the conventional method, 11.0 more deaths (per 100,000 person-years) are attributed to HNC from current smokers relative to never smokers (95% CI: 8.0, 13.9); 1.8 deaths in former smokers (95% CI: 0.4, 3.2). Under the mediation method, 13.4 more deaths are attributed to HNC from current smokers relative to never smokers (95% CI: 9.3, 16.8); 2.2 deaths in former smokers (95% CI: 1.2, 3.4). Comparing the two estimates, the conventional approach underestimates HNC mortality attributed to smoking by 18%. Noteworthy, the underestimate for current smokers manifests primarily in men, while the underestimate for former smokers manifests primarily in women.

Conclusions: The mediation method overcomes limitations in prior approaches of cancer mortality. In our example, the method estimated a stronger effect on HNC mortality by smoking. Further study is warranted as published estimates of cancer mortality by notable public health risk factors (eg, smoking, obesity, physical activity) may be underestimating their impact.

#2419

A polygenic risk score predicts breast cancer risk in Latinas.

Yiwey Shieh,1 Laura Fejerman,1 Sarah D. Sawyer,2 Donglei Hu,1 Scott Huntsman,1 Esther M. John,3 Lawrence H. Kushi,4 Gabriela Torres-Mejia,5 Jeffrey N. Weitzel,6 Christopher A. Haiman,7 Elad Ziv,1 Susan L. Neuhausen6. 1 _University of California, San Francisco, San Francisco, CA;_ 2 _Natera, San Carlos, CA;_ 3 _Stanford University, Palo Alto, CA;_ 4 _Kaiser Permanente, Oakland, CA;_ 5 _National Institute of Public Health, Cuernavaca, Mexico;_ 6 _City of Hope, Duarte, CA;_ 7 _University of Southern California, Los Angeles, CA_.

Background: Genome-wide association studies (GWAS) have identified over 180 single nucleotide polymorphisms (SNPs) associated with breast cancer risk. Polygenic risk scores (PRS) represent the combined effects of multiple SNPs and have been consistently shown to improve the performance of existing risk models. Most SNPs were discovered in women of European ancestry. Thus, there are limited data on how PRS perform in Latinas, whose ancestry is a mixture of European and Indigenous American. We therefore sought to validate the performance of a 184-SNP PRS in Latinas.

Methods: We conducted a pooled analysis of U.S. Latina and Mexican participants from 6 existing studies. Our analysis included 3,441 women with breast cancer and 7,773 women without breast cancer. We constructed a PRS containing 184 SNPs associated with breast cancer in prior GWAS, p < 5 x 10-8. We used multivariable logistic regression to evaluate the associations between the PRS and breast cancer, adjusting for genetic ancestry and study of origin. To assess discrimination, we calculated the area under the receiver operating characteristic curve (AUROC). To test the performance of the PRS by genetic ancestry, we stratified our analysis by quartiles of Indigenous American genetic ancestry.

Results: The PRS was associated with breast cancer, with an odds ratio (OR) per standard deviation increment of 1.51 (95% CI 1.44-1.59). The PRS showed intermediate discrimination for cases vs. controls, with an AUROC of 0.62 (95% CI 0.60-0.63). When we stratified the analysis by genetic ancestry, the PRS performed worse in women with higher Indigenous American/lower European ancestry. Specifically, the discrimination of the PRS was least in the top quartile of Indigenous American ancestry and greatest in the bottom quartile, with AUROC of 0.60 (95% CI 0.57-0.62) vs. 0.65 (95% CI 0.62-0.67), respectively (p = 0.01). The corresponding ORs per standard deviation of the PRS were 1.40 (95% CI 1.29-1.53) for the top quartile and 1.70 (95% CI 1.52-1.89) for the bottom quartile of Indigenous American ancestry (p = 0.006).

Conclusions: A 184-SNP PRS predicts breast cancer in Latinas, with the AUROC of 0.62 in our study being comparable to previous estimates in Europeans. However, the performance of the PRS varied according to genetic ancestry. While our results suggest the PRS may be used as-is in Latinas with higher European ancestry, future efforts should focus on refining the PRS in Latinas of higher Indigenous American ancestry through discovery and replication of novel predictive SNPs in this subgroup.

#2420

The development of novel detection system for circulating tumor DNA using DNA chip system.

Noriaki Nakamura,1 Hiroshi Okamura,1 Hirofumi Yamano,2 Hiromichi Ito,3 Hitoshi Zembutsu4. 1 _New Business Development Life Science Group, Toyo Kohan Co., Ltd., Tokyo, Japan;_ 2 _Life Science Department, Toyo Kohan Co., Ltd., Yamaguchi, Japan;_ 3 _Department of Gastroenterology Surgery, Cancer Institute Hospital, Japanese Foundation for Cancer Research, Tokyo, Japan;_ 4 _Cancer Precision Medicine Center, Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan_.

Gene panels covering hundreds of mutation hotspots for detection of cell-free DNA (cfDNA) have been developed for the establishment of clinically practical circulating tumor (ctDNA) detection system. Although amplicon-based or capture-based genomic profiling of genes commonly mutated in cancer using next-generation sequencing (NGS) system have been developed, they are not sufficiently sensitive nor accurate to be used for clinical diagnosis in hospital. As a result of preliminary study using 87 advanced pancreatic cancer (stage lll/lV), we observed that one or more KRAS mutation(s) could be detected in 60.9% of patients with advanced pancreatic cancer by using the amplicon-based deep sequencing system. To develop more sensitive and accurate ctDNA detection system, we optimized conditions of our DNA chip system. We firstly used 100 synthetic DNA samples, whose KRAS mutant allele frequencies were from 0.1% to 10.0%, and investigated the quantitativity of our DNA chip system. We amplified target regions by PCR to obtain 2,000 copies of synthesized DNA. The PCR product was hybridized with the probe on the DNA chip. The fluorescence ratio (mutant allele fluorescence intensity / total fluorescence intensity) of 100 synthetic DNA samples were clearly distinguished to that of negative control by using the DNA chip system. We defined an appropriate cut-off values of small quantities (0.1%) of mutations. Furthermore, to confirm the accuracy and reliability of this method, we verified our DNA chip system using 15 plasma cfDNA samples from patients with mutant KRAS (0.1~10.0%) and 15 samples from patients without mutant KRAS as negative control. 10 ng of ctDNA which extracted from blood sample is used in PCR. We observed that detected mutant status by DNA chip was completely concordant (100%) with those by amplicon-based deep sequencing system. Our data confirmed that mutant allele in cfDNA can be sensitively and accurately detected by DNA chip system. These results suggest that the ctDNA detection system developed in this study could be a novel diagnostic tool for tumor burden, and could realize the ultra-early detection of refractory tumors including pancreatic cancer.

#2421

Factors associated with abnormal cervical cancer screening results among screening compliant women in the United States.

Onyema Greg Chido-Amajuoyi, Sanjay Shete. _MD Anderson Cancer Center, houston, TX_.

Introduction:

Formal guidelines recommend routine cervical cancer screening using the Papanicolaou (Pap) test and / or screening for high risk Human Papillomavirus (HPV). This study aims to describe the factors associated with abnormal cervical cancer screening outcomes.

Methods:

We analyzed data from 7,716 women, aged 30 to 65 years, who were respondents of the 2015 Cancer Control Supplement (CCS) of the National Health Interview Survey (NHIS). Weighted multinomial logistic regression was used to examine the factors associated with i) abnormal Pap test, ii) positive high-risk HPV test, as well as iii) abnormal Pap and positive high-risk HPV tests.

Results:

Overall, 16.8% [95% CI, 15.6 to 18.0] of women who screened for cervical cancer in the last 3 years, had an abnormal result. Risk of abnormal cervical cancer screening results decreased as age increased. Compared to the younger age group (30-45 years), those aged 56-65 years had lower odds of an abnormal Pap test (aOR: 0.71, 95% CI: 0.55, 0.92), positive high-risk HPV test (aOR: 0.51, 95% CI: 0.27, 0.97) or abnormal Pap and positive high-risk HPV tests (aOR: 0.55, 95% CI: 0.35, 0.88). Similarly, women who were married or living with a partner were less likely to have an abnormal Pap test (aOR: 0.83, 95% CI: 0.69, 1.00), than unmarried women. On the contrary, geographic region, race and cigarette smoking were found increase the odds of an abnormal cervical cancer screening result. Blacks were more likely to have abnormal screening results compared to Whites. Current smokers had increased odds of abnormal Pap test (aOR: 1.33, 95% CI: 1.02, 1.73) and positive high-risk HPV test (aOR: 1.94, 95% CI: 1.13, 3.32), compared to never smokers.

Discussion:

Study findings call for continued surveillance of cancer screening outcomes. More so there is a need for further studies to examine factors promoting an increased likelihood of abnormal cervical cancer screening results in populations identified in this study.

#2422

Discordant opinions about cancer screening for the LGBTQ+ community.

Kevin Ko,1 Jerry Chen,2 Amy E. Leader,1 Marissa Ruggiero,1 Brittany A. Simone,1 Edith Mitchell,1 Nicole L. Simone1. 1 _Thomas Jefferson University, Philadelphia, PA;_ 2 _Wayne State Universtiy, Detroit, MI_.

Background: Recent studies have shown that these sexual and gender minorities often forgo care due to difficulties accessing health insurance coverage or fear of physician stigmatization. Therefore, using a nation-wide survey of providers and members of the LGBTQ+ community, we understand opinions on cancer screening since national guidelines have not been instated.

Method: An anonymous provider survey was created in Survey Monkey and sent to primary care providers and specialists such as oncologists, surgeons and radiologists. Questions (n=21) were designed to identify physicians understanding of issues faced by the LGBTQ+ community and also the provider and their clinics readiness to care for this population. In addition, a similar anonymous survey was designed in RedCap for members of the LGBTQ+ community (n=22) and posted on social media platforms such as Reddit, Facebook, and newsletters of national LGBTQ+ organizations. This survey focused each community member's understanding of their medical needs and asked specifically about existing barriers to their cancer screening and prevention. Both surveys end with free response questions to allow respondents a way to vocalize areas for providers to improve patient care and experience.

Results: Among the physicians, only 47% of physicians were confident regarding the health concerns unique to the LGBTQ+ community and only 28.1% of them had formal LGBTQ+ competency training. Over 70% of physicians believe that providers and support staff would benefit from more training regarding creating an environment conducive to increased comfort and care for LGBTQ+ and potential unique health issues. Among the members of the LGBTQ+ community, 65.6% of the group was not sure which cancer screenings are needed and 71.6% did not know when to start their screening. Close to 80% are aware that each sub-group under the umbrella term LGBTQ+ have unique health concerns. Notably, when visiting a provider, 83.6% reported feeling nervous, 67.3% overwhelmed, 56% excessively worried, and 45.8% depressed or hopeless.

Conclusions: Although some physicians feel comfortable addressing cancer screening needs of patients within the LGBTQ+ community, the majority of physicians would welcome additional training to learn about specific patient needs. Moreover, patients often do not understand their own medical needs, and often do not feel comfortable seeing a provider. To remedy both provider and patient confusion, we recommend the creation of 1) national guidelines for cancer screening for the LGBTQ+ population and 2) resources for the education of physicians, support staff, and patients population to improve the cancer health outcomes of this vulnerable population.

#2423

Longitudinal examination of breast cancer screening adherence among women in a prospective familial cohort.

Marcy L. Schaeffer,1 Betty J. May,1 Brenna C. Hogan,1 Mikiaila M. Orellana,1 Dana Petry,2 Katie Fiallos,2 Michelle S. McCullough,1 Deborah K. Armstrong,2 Kelly Myers,3 Kala Visvanathan1. 1 _Johns Hopkins Bloomberg School of Public Health, Baltimore, MD;_ 2 _Johns Hopkins Sidney Kimmel Cancer Center, Baltimore, MD;_ 3 _Johns Hopkins School of Medicine, Baltimore, MD_.

Background: Since 2007, U.S. national guidelines recommend cancer-free women at ≥20% lifetime risk of breast cancer (BC) undergo BC screening with mammogram and breast MRI. Little is known about long-term adherence to BC screening among young high-risk women. To address this knowledge gap, we examined predictors of screening adherence over time among women with varying BC risk.

Methods: Eligible women were ≥ 30 years old, had no history of BC/ovarian cancer, an intact breast, are enrolled in the Breast and Ovarian Surveillance Service (BOSS) Cohort, and visited the Johns Hopkins Cancer Genetics Clinic for risk assessment ≥ 2004 but prior to cohort enrollment (N = 374). Screening recommendations were determined from Tyrer-Cuzick BC lifetime risk estimates obtained at the clinic visit. Screening was self-reported at baseline, 4, and 8 years. A subset has been validated. An adherent screen before 2007 was mammography for women <20% risk at age ≥ 40 years or ≥20% risk/BRCA1/2 positive (high-risk). After 2007, an adherent screen for high-risk women was mammography and MRI. Cumulative adherence up to 2017 was also calculated. Associations between estimated risk, patient characteristics, and screening adherence were quantified using logistic regression.

Results: At baseline, the median age was 47 years, 31% had lifetime risk <20%, 60% had risk ≥20%, 8.8% were BRCA1/2 positive, and 69.5% were adherent to population BC screening recommendations. Over a median follow-up of 7.5 years, 9% of women underwent prophylactic bilateral mastectomy and 5% were diagnosed with BC. At each time point, proportion of mammography and MRI was ~80% and ~22.5%, respectively. The ≥20% group had better mammography adherence than the <20% group at 4 years [OR=2.0; 95%CI=1.1-3.7] and 8 years [OR=2.2; 95%CI=1.2-3.8], but was less adherent to risk-appropriate screening than the <20% group at 4 and 8 years [OR=0.2; 95%CI=0.1-0.4] [OR=0.4; 95%CI=0.2-0.6], respectively. Adherence was similar in the ≥20% and ≥30% high-risk groups. Risk-appropriate adherence at 4 years was predictive of adherence at 8 years [OR=11.1; 95%CI=5.5-22.5], adjusted for estimated risk, age, year of risk assessment, parity, other types of screening, comorbidities, and baseline adherence. In a multivariable model of cumulative risk-appropriate adherence, women with ≥20% risk (vs. <20%) or women with comorbidities (vs. no comorbidities) were less likely to consistently screen over 8 years [both OR=0.3; 95%CI=0.1-1.0], while women of older ages (vs. younger) were more likely [OR=1.2; 95%CI=1.1-1.3].

Conclusion: High-risk young women were not adherent to the more intensive screening regimen, and adherence did not improve over time. Other strong predictors of adherence included older age, prior adherence, and fewer comorbidities. New screening alternatives and interventions targeting reasons for non-adherence should be evaluated for high-risk women.

#2424

Smoking and mammography screening: An analysis of knowledge, barriers, self-efficacy, benefits, and perceived risk.

Andrew R. Marley,1 Lisa Carter-Harris,2 Wambui Gathirua-Mwangi,2 Victoria L. Champion2. 1 _Indiana University Richard M. Fairbanks School of Public Health, Indianapolis, IN;_ 2 _Indiana University School of Nursing, Indianapolis, IN_.

Introduction: Breast cancer is the second leading cause of cancer death among women in the United States. Although mammography screening is supported by the U.S. Preventive Services Task Force and has been found to effectively reduce mortality, only about 65% of women over 40 years of age are adherent to mammography screening guidelines, a rate far below the 81.1% goal proposed for Healthy People 2020. Screening rates are even lower among smokers compared to non-smokers; however, the reasons for this disparity remain unclear. Overall, knowledge, barriers, benefits, self-efficacy, and perceived risk play a role in decision making and may influence cancer screening behavior. Therefore, the purpose of this study was to examine the association between smoking status and mammography adherence, and to determine whether differences in knowledge, barriers, benefits, self-efficacy, and perceived risk could account for screening disparities between smokers and non-smokers.

Methods: A prospective, randomized screening intervention of women aged 50-75 (n=1,196) was conducted in Indiana from 2013-2015. A total of 846 women had complete smoking and mammography data for use in this analysis. Women were surveyed at baseline to ascertain current smoking status and mammography history, as well as knowledge, barriers, self-efficacy, benefits, and perceived risks for mammography. Binary logistic regression was used to estimate multivariate-adjusted odds ratios (OR) and 95% confidence intervals (CI) for the association between smoking and mammography adherence. T-tests were used to evaluate differences between current smokers and non-smokers with regard to knowledge, barriers, benefits, self-efficacy, and perceived risk. Regression analyses were adjusted for age, education, income, insurance coverage, ever receiving a physician recommendation for mammography, and family history of breast cancer.

Results: Smokers were significantly younger, less educated, and had lower income relative to non-smokers. Smokers were less likely to be adherent to mammography screening relative to non-smokers [OR = 0.54 (0.32 - 0.90), p=0.020)]. Smokers also demonstrated significantly less knowledge of breast cancer and mammography (p=0.0016), reported more barriers to screening (p=0.020), had less self-efficacy (p=0.0018), and had a lower perceived risk of breast cancer (p=<0.0001) compared with non-smokers. No differences were observed for benefits to screening (p=0.81).

Conclusions: Women who smoke are less likely to adhere to mammography screening guidelines than non-smokers, possibly due to less knowledge about breast cancer and mammography, more barriers to screening, less self-efficacy, and less perceived risk. Future interventions to increase mammography should consider smoking status disparities and tailor intervention content accordingly.

## PREVENTION RESEARCH

### Behavioral Science and Cancer Control

#2425

Moderate to vigorous physical activity reduces visceral adipose tissue in obese breast cancer survivors undergoing a weight loss intervention.

Carol J. Fabian,1 Jennifer R. Klemp,1 Jeffrey M. Burns,1 Eric D. Vidoni,1 Lauren Nye,1 Christie A. Befort,1 Jill M. Hamilton-Reeves,1 Debra K. Sullivan,1 Teresa A. Phillips,1 Stephen D. Hursting,2 Bruce F. Kimler1. 1 _University of Kansas Medical Center, Kansas City, KS;_ 2 _University of North Carolina, Chapel Hill, NC_.

Background: Body composition, adipose distribution, and fitness are likely superior to BMI alone in predicting outcomes in obese breast cancer survivors. Visceral adipose tissue (VAT) is thought to be disproportionately responsible for the metabolic and inflammatory changes linking obesity and breast cancer recurrence.

Purpose: Feasibility of achievement of high volume moderate-to-vigorous physical activity (MVPA) and its effect on body composition including VAT and other risk biomarkers in older sedentary, obese breast cancer survivors participating in a behavioral weight loss intervention.

Methods: Modest caloric restriction (350-500 kcal per day) was combined with a study provided YMCA membership and twice weekly personal trainer sessions. MVPA (40-80% of heart rate reserve in older women) was assessed by Garmin Vivoactive smart watches linked to GarminConnect. Total activity was escalated from 100 minutes week 1 to a total of 300 minutes per week by week 9 with a goal of > 200 min/wk as MVPA. Women were evaluated pre- and post-intervention for peak oxygen consumption (VO2peak), body composition by dual energy x-ray absorptiometry (GE Lunar iDXA), and serum levels of adipokines, cytokines, and hormones.

Results: 18 breast cancer survivors were enrolled and completed the 3 months of the behavioral weight loss intervention which included the YMCA membership and twice weekly personal trainer. Median age was 60, 11 had received prior chemotherapy, and 13 were currently taking aromatase inhibitors. Median (and range) baseline anthropomorphic values were BMI 37 (31-43) kg/m2, total mass 95 (76-125) kg, total fat 46 (34-66) kg, and VAT 1.7 (1.0-3.0) kg. All but one had a baseline VAT > 1.17 kg, consistent with elevated risk of metabolic syndrome. 17/18 achieved at least 150 min/wk of MVPA, with a median of 176 (range 55-291) min/wk for weeks 9-12. Fitness measured as VO2peak increased from 18.9 (13.7-25.3) to 21.1 (17.1-31.4) ml/kg/min (p=0.0003; Wilcoxon signed rank test). At 3 months, significant reductions were observed for total mass (median 7%), fat mass (median 13%), and VAT (median 20%, range 1-41%)(p<0.0003); as well as serum leptin, insulin, and leptin: adiponectin ratio (p<0.01). A strong correlation was observed between week 9-12 MVPA and leptin: adiponectin ratio (p=0.001) with more modest correlations between week 9-12 MVPA and VAT loss (p=0.043), and leptin: adiponectin ratio and VAT loss (p=0.013).

Conclusion: High volume moderate-to-vigorous physical activity can be achieved by older, sedentary, obese breast cancer survivors; and this translates to favorable modulation of body composition including VAT measures and serum risk biomarkers.

#2426

Familial reinforcement and family history interact to influence knowledge about pancreatic cancer.

Maria Stevens,1 Dalia Mikhail,1 Amanda Toledo Hernandez,2 Sarah Fagan,3 Kari Chaffee,1 Gloria M. Petersen4. 1 _Mayo Clinic, Rochester, MN;_ 2 _University of Puerto Rico, San Juan, PR;_ 3 _National Cancer Institute, Bethesda, MD;_ 4 _Mayo Clinic College of Medicine, Rochester, MN_.

Introduction: Family reinforcement (praise, reminders) may influence health behaviors and affect cancer knowledge, including risk factors. There has been insufficient research on the influence of familial factors in cancer prevention, especially for individuals with a family history of pancreatic cancer (PC). Objectives were to assess: 1) associations between family history of PC and familial reinforcement of cancer prevention with PC knowledge, and 2) interaction between family history of PC and familial reinforcement of cancer prevention on PC knowledge.

Methods: A sample of 778 unaffected relatives with ≥1 affected first (FDR) or second degree relatives (SDR) were ascertained from Mayo Clinic's Biospecimen Resource for Pancreas Research, a familial PC registry, along with 685 primary care controls. Participants with a personal history of cancer were excluded with the exception of non-melanoma skin cancer. Participants completed health/behavior questionnaires (June 2008 to June 2018). Items were summed to create factor scores for familial reinforcement of cancer prevention and PC knowledge. PC family history was categorized by number of relatives with PC: 1) none, 2) 1 FDR and/or ≥1 SDR, and 3) ≥2 FDRs. We employed linear models utilizing generalized estimating equations to assess the association between PC family history and familial reinforcement with PC knowledge, and the interaction based on PC family history, adjusting for age, sex, and education, and accounting for familial relatedness. Estimates of the linear coefficient β and 95% confidence intervals (CI) are reported, and p-values < 0.05 were considered significant. Analyses were conducted using SAS 9.4 (SAS Institute, Cary, NC).

Results: There was a significant association between PC family history and PC knowledge such that those with 1 FDR and/or ≥1 SDR (β=3.0; 95% CI: 2.5-3.4; p<0.0001) or ≥2 FDRs (β=4.4; 95% CI: 3.7-5.0; p<0.0001) with PC had increased PC knowledge after adjusting for age, sex, and education. There was a significant association between familial reinforcement and PC knowledge such that individuals with increased familial reinforcement (β=0.1; 95% CI: 0.03-0.12; p=0.0026) had higher PC knowledge after adjusting for pertinent covariates. The interaction between PC family history and familial reinforcement was significant such that increased familial reinforcement was associated with higher PC knowledge in individuals with ≥2 FDRs with PC compared to controls (β=0.2; 95% CI: 0.09-0.34; p=0.001); however, this relationship was not significant in individuals with 1 FDR and/or ≥1 SDR.

Conclusions: Individuals with a stronger family history of PC may benefit from increased familial reinforcement of cancer prevention to enhance knowledge about PC. Further efforts should be targeted toward incorporating the family in cancer care to raise familial awareness about PC.

#2427

Patterns of BRCA testing by provider type based on biological and non-biological factors among a diverse sample of young breast cancer survivors.

Sonya Reid,1 Deborah Cragun,2 Ann Tezak,1 Anne Weidner,1 Ingrid Mayer,1 Xiao-ou Shu,1 Susan Vadaparampil,3 Tuya Pal1. 1 _Vanderbilt University Medical Center, Nashville, TN;_ 2 _University of South Florida, Tampa, FL;_ 3 _Moffitt Cancer Center, Tampa, FL_.

Introduction: Per national practice guidelines, pre-test genetic counseling through a provider experienced and proficient in genetics (such as a genetics health professional (GHP)) is recommended prior to genetic testing for hereditary cancer. We sought to determine if there are differences in clinical and demographic variables between young breast cancer (BC) survivors who saw a GHP prior to genetic testing compared to those tested without involvement of a GHP.

Methods: A population-based sample of Black, Hispanic and non-Hispanic white (NHW) women diagnosed with invasive BC < age 50 in 2009-2012 was recruited through the Florida State Cancer Registry. Participants were asked to complete a questionnaire and medical records release to verify clinical information and genetic test results. Women tested through a GHP were compared to those tested through a non-GHP based on the following variables: 1) Socioeconomic status (SES) (income, college education and private insurance); 2) Racial group (Black, Hispanic and NHW); and 3) Clinical factors (age at diagnosis, family history (FH) of breast or ovarian cancer and triple negative breast cancer (TNBC) status) using Pearson chi-squared analyses.

Results: Of 1618 study participants, 1080 reported genetic counseling and/or testing, of which provider information was verified on 981 based on documentation in medical record and/or genetic test result report. GHP involvement (either through consultation and/or test ordering) was reported in 193 women (19.7%) whereas the remaining 788 women (80.3%) had no documentation of GHP involvement. There was a significantly higher rate of GHP involvement among women with TNBC compared to those without TNBC (26.1% vs. 19.1%; p=0.05); and non-significant increases in those with and without a FH of BC (21.1% vs. 17.6%; p=0.17); and ovarian cancer (22.8% vs. 19.2%; p=0.34). GHP involvement did not vary among: 1) women diagnosed < or > age 45 (19.6% vs. 19.8%; p=0.96); 2) Blacks (20.1% vs. 19.6%; p=0.88); 3) Hispanics (23.5% vs. 18.8%; p=0.15); and 4) NHW (18.5% vs. 22%; p=0.19). Regarding SES-related indicators, GHP involvement was lower among women with private insurance versus those without (18.3% vs. 29.3%; p=0.002), annual household income > $25,000 versus those < $25,000 (18.8% vs. 30.8%; p=0.004) and a college education (55.4% vs. 60.9%; p=0.17).

Conclusions: Our results demonstrate that TNBC status was associated with testing through a GHP, while other clinical and demographic variables were not associated with GHP involvement. This finding may be due to widespread recognition of a more aggressive disease phenotype and association of TNBC with hereditary disease. Furthermore, the inverse relationship between higher SES-related indicators and GHP involvement suggests that those with higher SES are less likely to receive care through a GHP, as recommended per national guidelines.

#2428

New school health care: Utilizing technology in the treatment and support of women with breast cancer in the ambulatory setting.

Tameka N. Jones,1 Andrew J. Paladino,2 Gregory A. Vidal,1 Rebecca Krukowski,2 J. Carolyn Graff,2 Ryan Blue,2 Ilana Graetz,2 Lee Schwartzberg,1 Mehmet Kocak,2 Janeane Anderson2. 1 _West Cancer Center, Germantown, TN;_ 2 _University of Tennessee Health Science Center, Memphis, TN_.

Background Adjuvant endocrine therapies (AETs) (e.g. tamoxifen and aromatase inhibitors (AIs) are almost always prescribed to early stage breast cancer (BrCa) patients as AETs have been shown to reduce risk of recurrence and mortality for women with hormone receptor-positive early stage breast cancer. Despite proven efficacy and patient education efforts , AET adherence is suboptimal. Most patients report symptom burden as the most common cause for non-adherence. We conducted focus groups among this patient population to aid in the refinement of a communication app for patients to report symptoms to their healthcare team and receive tailored feedback messages. The app aims to improve AET adherence, symptom management, and quality of life.

Method Four focus groups (N=28), stratified by race (Black and White) and patient length of time on AET ( < 6 months AET use or > 6 months AET use), were conducted in Memphis, Tennessee. The focus groups were audiotaped, transcribed, and analyzed to identify key themes.

Results Participants indicated they would be open to utilizing technology throughout their cancer treatment. The app was viewed as a conduit in the exchange of information between the patient and their healthcare team. Even patients who typically did not use or "like" technology thought the messages and symptom management information would be beneficial. Reporting symptoms through the app and then receiving a phone call from their healthcare team to respond to those symptoms was viewed as the most beneficial component to the patients. One participant stated how she could see the benefit of the symptom reporting feature when first initiating AET therapy: "There were times when I wanted to go tell my doctor, "STOP THIS MEDICINE RIGHT NOW, find me something else to do, find something else besides this because this stuff is not working for me." In regards to the tailored feedback messaging through the app, participant stated, "I like the one that says, 'Take meds regularly is an effective way to prevent cancer from coming back,' because for me that is the only reason why I take it SOME days just to make sure that it doesn't come back because I HATE the stuff."

Conclusions Early stage hormone receptor positive breast cancer patients may benefit from utilizing technology in the management of their care. The use of a communication app with tailored feedback messaging has the potential to increase medication adherence, improve symptom management and quality of life, improve the provider- patient relationship, and ultimately reduce mortality. As healthcare shifts toward focusing on patient-centered care and preparing patients to manage their care at home, apps like the one examined in the current study may play a vital role in helping these and other patient populations.

#2429

Social Support as a Moderator in the Relationship between Intrusive Thoughts and Psychological Symptoms among Spanish-speaking Latinas with Breast Cancer.

Cristian Escalera,1 Anna Maria Napoles,1 Jasmine Santoyo-Olsson,2 Carmen Ortiz3. 1 _National Institutes of Health, Bethesda, MD;_ 2 _University of California San Francisco, San Francisco, CA;_ 3 _Círculo de Vida Cancer Support and Resource Center, San Francisco, CA_.

Background: Intrusive thoughts are associated with higher levels of psychological symptoms and impaired quality of life in the year after breast cancer treatment. To our knowledge, no studies have examined social support as a potential moderator between intrusive thoughts about breast cancer and psychological symptoms among Latinas. This study aims to assess the moderating effects of different dimensions of social support on the association between intrusive thoughts and psychological symptoms among Spanish-speaking Latina breast cancer survivors. Methods: Analyses were performed using baseline data from a randomized control trial of a stress management intervention delivered to 151 Spanish-speaking Latinas with non-metastatic breast cancer. Survey measures on intrusive thoughts, four dimensions of social support (emotional/information, tangible, affectionate, and positive social interaction), and psychological symptoms (depression, anxiety, and somatization) were obtained at baseline via a structured survey. Information on age, time since diagnosis, breast cancer treatment, surgery type, breast cancer stage, history of depression and marital status was obtained via medical records review and served as covariates. Generalized linear models were used to investigate bivariate and multivariate associations and to explore moderation effects of the four dimensions of social support. Results: In the bivariate models, intrusive thoughts were associated with more depressive (β=0.024, p=0.0014), anxiety (β=0.047, p<.0001) and somatization symptoms (β=0.019, p = 0.0036). In multivariable models, intrusive thoughts remained strongly associated with anxiety symptoms (β=0.132, p=0.0001). In contrast, the effects of intrusive thoughts on depressive and somatization symptoms was attenuated in the multivariate models. Receiving affectionate social support was found to moderate the relationship between intrusive thoughts and anxiety symptoms (β=-0.022, p=0.0055). No dimension of social support was found to have a moderating effect in the relationship between intrusive thoughts and either depressive or somatization symptoms. Conclusions: Our findings indicate that social support, particularly affectionate support, had a protective effect on anxiety symptoms among Spanish-speaking Latinas with breast cancer experiencing intrusive thoughts. Affectionate social support is a possible protective factor that can be developed and encouraged in socio-behavioral interventions for this vulnerable population.

#2430

Assessing patient navigation and support services at FQHCs.

Lisa Aponte-Soto. _University of Illinois at Chicago, Chicago, IL_.

Cancer is the leading cause of morbidity and mortality for Latinx followed by African Americans (CDC, 2017). Screening can increase the rate of early detection and treatment success. However, the rates of compliance for screening are typically lower among Latinx, which may contribute to decreased diagnosis and increased risk of disease in comparison to their Euro-American counterparts (ACS, 2015). Access to screening services and ability to pay are among the commonly cited barriers for screening compliance. The IDPH recommends incorporating patient navigator (PN) models in community health clinics and communitywide events to increase health literacy, knowledge, and delivery, and quality of care including compliance with screening methods. The University of Illinois (UI) Cancer Center (UICC) works in partnership with the UI Health Mile Square Health Center (MSHC) to implement a bilingual, bicultural PN model for conducting comprehensive cancer prevention and screening services among Latinx, African Americans, and underserved communities in Chicago. A mixed methods approach was used to evaluate the patients' perceptions and experiences with patient navigator encounters and support services. The authors conducted a semi-structured interviews and abstracted data from patient records. Provider interviews were conducted to understand provider knowledge and attitudes of navigator services. Navigator interviews captured benefits and challenges of incorporating navigation services at the clinics. Data were entered in REDCap and analyzed using Dedoose v. 8.0.42. This paper validates the implementation of PN models at federally qualified health centers (FQHCs) as an asset to patients and providers in facilitating health education, increasing patient screening compliance, and improving access to health care services. Comprehensive cancer screening maximizes resources and improves the quality of life and health outcomes of underserved Latinx and African American patients who are navigated to services. PN-provider partnerships are introduced as a hallmark of the program's success in identifying at-risk patients and navigating them to appropriate and timely medical screening, diagnostic, and treatment services. Future directions for incorporating and evaluating culturally responsive, bilingual, bicultural PN models at FQHCs, community clinics, and school based clinics are shared to inform public policy for rapid cycle monitoring of protective health cancer interventions and early detection services for Latinx and African American patients in Chicago.

#2431

Working with stakeholders to adapt a cancer screening education program for Native American women with intellectual disabilities.

Julie S. Armin,1 Heather J. Williamson,2 Julie Baldwin,2 Janet Rothers,1 Ellen Cherup,2 Tara Chico-Jarillo,1 Jennifer Etcitty,2 Bailey Lockwood,1 Bonny Nasimi2. 1 _University of Arizona, Tucson, AZ;_ 2 _Northern Arizona University, Flagstaff, AZ_.

Background: Women with intellectual and developmental disabilities (IDD) are less likely than the general population of women to receive recommended breast and cervical cancer screenings. In addition, rates of breast and cervical cancer screenings are lower among Native American (NA) women than among White women. Using a community-informed approach, this project addresses the specific cancer screening needs of NA women with IDD and their networks of support in order to adapt an evidence-based cancer screening education program for women with IDD.

Methods: In this formative research project, the research team has engaged in a step-wise project that adheres to best practices for conducting ethical community-informed research with NA communities and ensures a feasible and acceptable cancer education program. 1) The university-based research team established a 6-person advisory board to guide partnership development, identify potential community needs, and contribute to study recruitment. 2) An ecosocial qualitative needs assessment examined facilitators and barriers to women's screening practices. In-depth interviews with Native women with IDD (N=24), family caregivers (N=24), health care providers (N=24), and leaders in partnering rural and urban Native and Native-serving communities (N=12) were completed. 3) Working with urban and rural partners to understand and interpret the team's qualitative data, the team will culturally adapt a cancer screening education program for diverse groups of NA women with IDD.

Results: During advisory board interactions, community meetings, and event participation, the research team identified individual, interpersonal, community, institutional and policy components influencing NA women with IDDs cancer screening. In a review of the cancer education curriculum, advisory board members identified several areas for adaptation, including: length of program, discussion of women's body parts, involvement of spiritual leaders and family members, involvement of health care providers, and integration with customary transition rituals for Native women. During community discussions, the team learned that NA women may not have seamless access to cancer treatment; we are working with the advisory board and community stakeholders to limit the gaps between cancer screening and treatment programs for women who are part of the project.

Conclusions/Next Steps: After working with community partners to analyze qualitative data and adapt the cancer education curriculum for Native women with IDD, the team will pilot test the adapted curriculum for feasibility, acceptability, and preliminary knowledge and self-efficacy outcomes.

#2432

Prevalence of depression and other chronic diseases among uninsured cancer survivors: A free clinic study.

Sayeef Mirza,1 Shams Rahman,2 Madeline Macdonald,1 Alexandra Towne,1 Katherine Robinson,1 Amber Todd,1 Wei Wang,2 Deanna Wathington2. 1 _University of South Florida, Tampa, FL;_ 2 _Bethune-Cookman University, Daytona Beach, FL_.

Introduction: Free clinics play an important role in providing health care services to the poor and uninsured. Limited data exist on chronic disease burden and patient characteristics of cancer survivors outside the network of insured health systems. In this study we describe characteristics of cancer patients utilizing free clinics for chronic disease management and mental health services in order to evaluate associations between chronic conditions.

Methods: Nine free clinics in the Tampa Bay area were included in this study. A retrospective review of patient charts from 01/01/2016 to 12/31/2017 was conducted. Data on patient characteristics and prevalence of chronic diseases were abstracted through an online questionnaire using Redcap software. Patients with a documented history of cancer were considered cancer survivors. Chi-square test was used to compare patient characteristics and p-values were reported. Logistic regressions were used to examine associations between diseases and socio-demographic factors. Odds ratios (OR) and 95% confidence intervals (CI) were reported.

Results: From 2016 to 2017, 208 cancer survivors were managed in nine free clinics. The average age was 54 years and 138 (66%) were women. The prevalence of depression was 11.14%. The prevalence of depression was higher among women (12.7%) compared to men (8.8%), p-value 0.001. The prevalence was significantly highest among patients who were 18-44 years, non-Hispanics, white race, unemployed and considered low-income. In univariate analyses, the prevalence of depression was significantly associated with cancer (OR 2.12; 95%CI: 1.23-3.67), diabetes (OR 1.59; 95%CI: 1.21-2.08), cerebrovascular accident (OR 3.25; 95%CI: 1.71-6.21), cardiovascular disease (OR 2.10; 95%CI: 1.26-3.51), hypertension (OR 1.96; 95%CI: 1.58-2.43), and hyperlipidemia (OR 2.30; 95%CI: 1.79-2.94). Unemployment, lower income level, and higher BMI were also factors positively associated with the prevalence of depression. Additional relationships and multivariate analyses are available.

Conclusion: Despite having access to limited resources, free clinics receive mainly poor, women, middle-aged, and uninsured patients. Comorbidities such as cancer and other chronic conditions are often directly associated with depression among patients managed in free clinics. Mental health support is critically needed in free clinics, especially for those who are cancer survivors.

#2433

The NCI Cancer Center Cessation Initiative (C3I): Characteristics and reach of tobacco treatment programs among NCI-designated cancer centers in the C3I.

Heather D'Angelo,1 Betsy Rolland,1 Rob Adsit,1 Michael Fiore,1 Marika Rosenblum,1 Glen Morgan2. 1 _University of Wisconsin Madison, Madison, WI;_ 2 _National Cancer Institute, Rockville, MD_.

Quitting smoking after a cancer diagnosis leads to better outcomes for cancer patients, including lower risk for a second primary cancer and increased survival. However, few cancer patients receive smoking cessation services during their oncology healthcare visits, and disparities in the receipt of such smoking cessation services exist. As part of the Cancer Moonshot, the National Cancer Institute (NCI) has dedicated funding to expand and enhance smoking cessation services at NCI-Designated Cancer Centers for all patients who smoke. We report on characteristics of tobacco treatment programs (TTPs) at the 22 Cancer Centers funded through the Cancer Center Cessation Initiative (C3I) for 6 months before funding was awarded ("pre-funding period"), and for 6 months during the first year of funding. We will also report on the second half of the first year of funding. Characteristics measured included referral methods to cessation services (e.g. referrals via electronic health records (EHR)), and types of cessation services offered. TTP reach (the percentage of smokers who engaged in any type of TTP) was calculated overall and by patient demographics for Centers providing aggregate patient data (n=13). Data were collected in 2018. Between the pre-funding period and first year of funding, the number of C3I funded Centers offering in-person smoking cessation counseling increased from 10 to 15 (45.5% to 68.2%). EHR referrals to tobacco treatment programs increased from 31.8% of Centers to 68.2%. Four Centers (18.2%) offered text and web based cessation programs in the first funding year compared with only 1 Center (4.5%) in the pre-funding period. During the first funding period, TTP reach on average was 20.2%, but varied by Center, ranging from 0.5% to 86.5%. During the first funding period, 16.4% of Black, 15.8% of White, 11.8% of Hispanic, 10.6% of Asian, and 6.2% of American Indian/Alaska Native smokers received smoking cessation services. About 13% of smokers aged 18-24 received cessation services, compared with those aged 25-44 (17.7%), 45-64 (22.9%) and 65+ (18.7%). The majority of C3I funded Cancer Centers offered some type of TTP; however, on average only 20% of smokers were reached by a TTP. Further, TTP reach varies by race, ethnicity, and age. The C3I provides an opportunity to improve the reach and effectiveness of smoking cessation services for cancer patients who smoke, and reduce disparities in the receipt of cessation services by providing financial and technical support for Centers to build and implement comprehensive evidence-based smoking cessation programs.

#2434

Characterizing age-of-onset of multiple primary cancers and specific cancer types in families with Li-Fraumeni syndrome.

Seung Jun Shin,1 Elissa Dodd,2 Gang Peng,3 Jasmina Bojadzieva,2 Jingxiao Chen,2 Jing Ning,2 Phuong L. Mai,4 Sharon A. Savage,5 Mandy L. Ballinger,6 David M. Thomas,6 Ying Yuan,2 Louise C. Strong,2 Wenyi Wang2. 1 _Korea University, Seoul, Republic of Korea;_ 2 _MD Anderson Cancer Center, Houston, TX;_ 3 _Yale University, New Haven, CT;_ 4 _Magee Women's Hospital, Pittsburg, PA;_ 5 _National Cancer Institute, Besthesda, MD;_ 6 _Garvan Institute, Darlinghurst, Australia_.

Purpose: Li-Fraumeni syndrome (LFS) is a rare autosomal dominant disorder associated with a germline TP53 mutation that increases an individual's risk for a spectrum of cancers as well as multiple primary diagnoses. Penetrance estimations of multiple primary cancer (MPC) risk and cancer specific (CS) risk associated with TP53 germline mutations remains a challenge because of limited data available for statistically meaningful estimation and the difficulty of accounting for variables like previous primary cancer effect or ascertainment bias inevitable in rare diseases. Accurate penetrance estimations are crucial to improve the clinical characterization and management of high-risk individuals of LFS. Modeling MPC and CS risks are also relevant questions for the general cancer population.

Methods: Through a novel non-homogenous Poisson process to model the non-recurrent event approach, we estimated the MPC penetrance from a large pediatric sarcoma cohort from MD Anderson Cancer Center (MDACC). We used data from all individuals, with or without TP53 genotype information, and specifically considered age as well as ages of first primary cancer diagnosis for model parameter estimation. Next, we developed a model under the competing risk framework that takes into account the pedigree structure into the penetrance estimation and corrects for ascertainment bias to address the TP53 penetrance for three cancer subtypes (breast, BR/sarcoma, SA/others, OT) on the same pediatric cohort.

Results: We then validated the risk prediction performance of the two sets of penetrance estimates via independent cohort datasets: a clinically ascertained dataset collected at MDACC (total number of individuals=774; SPC=264; MPC=252) for MPC penetrance validation and a LFS dataset collected at NCI combined with a dataset from the International Sarcoma Kindred Study (total number of individuals=1,083, BR=85, SA=540, OT=158) for CS outcome validation. Among carriers, the MPC penetrance estimates, which is dependent on TP53 genotype, gender, current age and age of first primary diagnosis are significantly different from those of a single primary cancer (SPC). We achieved an AUC=0.73 for predicting individual outcomes of multiple primaries vs. a single primary cancer. For the CS penetrance of TP53 mutation carriers for breast cancer, sarcoma, and other cancers related to LFS, we obtained AUCs of 0.92 (BR), 0.75 (SA), and 0.81 (OT).

Conclusion: We have obtained two new sets of penetrance to accurately characterize the age-of-onset of multiple primary cancers and specific cancer types like breast, sarcoma or others in families with LFS. Incorporating associated risk prediction models into LFS counseling can potentially facilitate clinical decision-making to both patients and their physicians. Future work will be focused on integrative analysis of multi-cohort datasets.

#2435

Knowledge and attitudes towards anal cancer and anal cancer screening among hispanic women at increased risk of anal cancer.

Ana P. Ortiz-Martinez,1 Sandra García Camacho,1 Jeslie M. Ramos Cartagena,1 Claudia Amaya Ardilla,2 Vivian Colón López,1 Josefina Romaguera2. 1 _University of Puerto Rico Comprehensive Cancer Center, San Juan, PR;_ 2 _University of Puerto Rico Medical Science Campus, San Juan, PR_.

Objective: Research shows that women with HIV infection, lower genital tract neoplasia (LGTN), organ transplants, Chron's disease and lupus have an increased risk of anal cancers and its precursor lesion, anal intraepithelial neoplasia (AIN). Thus, an expert review, including the American Society for Colposcopy and Cervical Pathology and the International Anal Neoplasia Society, recommends anal cytology screening for these women when referral to high-resolution anoscopy (HRA) and treatment is available. Although Puerto Rico has a specialized anal neoplasia clinic since 2014, little is known about anal cancer knowledge and attitudes in women at risk for this malignancy in this population. Thus, this study aims to describe knowledge and attitudes towards anal cancer and anal cancer screening among Hispanic women at increased risk of anal cancer and AIN in Puerto Rico.

Methods: Ongoing clinic-based cross-sectional study (May-November 2018) among women receiving services at the colposcopy, gynecology oncology and lower genital tract clinics of the University of Puerto Rico. All women with gynecological neoplasia within the clinic complete an interviewer administered questionnaire and the medical record is reviewed for LGTN diagnosis. From 235 study participants (response rate=67.5%), 83 (35.3%) had a diagnosis of LGTN, HIV, lupus, Chron's disease, or were transplant patients, and thus were considered at increased risk for anal cancer and AIN, and included in this analysis.

Results: 71.8% of women have cervical, 8.4% vulvar, and 9.6% vaginal LGTN. Meanwhile, 10.1% have HIV, 3.6%, are transplant patients, 8.4% have lupus and 2.8% Crohn's disease. Mean age is 54.9 years (SD=1.73) and 57.8% have an annual income <$15,000. Only 12.0% of study participants had ever had an anal pap, despite their increased risk. The most common reason reported for not having had an anal pap was being unaware that a test was available (56.6%). In terms of perceived risk, only 31.7% of women agree/strongly agree that they are at greater risk of anal cancer as compared to other persons of their age, although 74.7% agree/strongly agree that it concerns them a lot to develop anal cancer. Although 87.8% of women say to know nothing/little about anal cancer, 92.7% are interested in learning about this malignancy and 97.5% are willingly to undergo anal cytology if a doctor recommend it. Regarding risk factors, a moderate proportion of women knew than HIV infection (50.6%), tobacco consumption (45.1%), and anal sex (71.1%) are risk factors for anal cancer.

Conclusion: Despite low perceived risk and limited knowledge of anal cancer, there is high interest among this high-risk population to learn more about anal cancer and screening procedures. Results show the importance and the need for education in this topic for patients and physicians, to have an impact on anal cancer prevention. 

## BIOINFORMATICS AND SYSTEMS BIOLOGY

### Convergence Science for Tumor Biology, Molecular Biology, and Microenvironment

#2436

Systematic computational analysis of histologic-genomic associations in triple-negative infiltrating ductal carcinomas of the breast.

Mohamed Amgad,1 Uday Kurkure,2 Habiba Elfandy,3 Hagar H. Khallaf,4 David A. Gutman,5 Carlos S. Moreno,6 Michael Barnes,7 Lee A. Cooper1. 1 _Department of Biomedical Informatics, Emory University School of Medicine, Atlanta, GA;_ 2 _Roche Tissue Diagnostics, Digital Pathology, Mountain View, CA;_ 3 _Department of Pathology, National Cancer Institute, Cairo University, Cairo, Egypt;_ 4 _Faculty of Medicine, Cairo University, Cairo, Egypt;_ 5 _Department of Neurology, Emory University School of Medicine, Atlanta, GA;_ 6 _Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA;_ 7 _Roche Diagnostics Information Solutions, Belmont, CA_.

Background: Triple-negative breast cancer (TNBC) is characterized by rapid progression and lack of therapeutic targets. There is a pressing need for in-depth characterization of the biological correlates (and potential prognostic biomarkers) in TNBC. We performed a systematic analysis of genomic correlates of histologic markers of tumor aggressiveness and immune infiltration in 125 infiltrating ductal TNBC patients from The Cancer Genome Atlas.

Methods: Fully-convolutional networks of the VGG16-FCN8 architecture were trained to classify various tissue regions in H&E slides (0.956 AUC on unseen slides), and morphologic descriptors of tumor aggressiveness, invasion, and immune infiltration were extracted from predictions. Expression levels of 17,052 genes were entered into sample purity-adjusted linear regression models, and a Gene Set Enrichment Analysis was performed using the model coefficients to find gene set-level associations.

Results: Significant associations are summarized in Table 1. Expression of mTOR pathway genes (especially HIGD1C and PDE6H) is positively associated with large, dense tumor nests with a smooth tumor-stroma boundary. Boundary complexity is also positively associated with the oxidant stress response of NFE2L2. In contrast, some genes downregulated by the TGF-β pathway (including FGF6, PSG2 and CHRNG) are associated with a large tumor nest phenotype. The cell cycle regulator E2F1 (through PRM1, KRT72, and DBF4) is associated with dense immune infiltration, a known marker of good prognosis, and also small tumor nest size.

Conclusion: mTOR and NFE2L2-mediated mechanisms are significantly associated with features of tumor aggressiveness in TNBC, while E2F and some TGF-β targets are associated with morphological markers of less aggressive tumors. Further research is needed to elucidate the biological basis of these associations and their potential significance in therapeutic targeting of TNBC.

Table 1: Significant histologic-genomic associations in infiltrating-ductal TNBC.

---

Histologic phenotype (feature description) | Enriched gene set (MSigDB Oncogenic Signatures) | Gene set description | NES(P-value, FDR) | Leading-edge genes

Tumor nest size (Mean tumor nest area) | TGFB_UP.V1_DN | Genes down-regulated by TGFB1 | 1.55(p<0.001, FDR=0.036) | FGF6; PSG2; CHRNG

Tumor-stroma interface (non-)complexity (mean solidity of tumor nests) | NFE2L2.V2 | Genes upregulated with knockout of NFE2L2 gene (involved in oxidant stress response and inflammation) | -1.51(p<0.001, FDR=0.027) | DEFB119; CNTNAP5; SCGB1D1

MTOR_UP.V1_DN | Genes downregulated by everolimus (an mTOR inhibitor) | 1.43(p=0.0017, FDR= 0.094) | HIGD1C

Small, solitary tumor nests | E2F1_UP.V1_UP | Genes up-regulated when E2F1 is over-expressed (cell cycle regulation) | 1.48(p<0.001, FDR<0.001) | PRM1

MTOR_UP.V1_DN | Genes downregulated by everolimus (an mTOR inhibitor) | -1.50(p=0.0027, FDR= 0.079) | PDE6H; HIGD1C

Small tumor nests with abundant surrounding immune infiltration (a spatial descriptor meant to capture immune success) | E2F1_UP.V1_UP | Genes up-regulated when E2F1 is over-expressed.(cell cycle regulation) | 1.55(p<0.001, FDR= 0.0019) | PRM1; KRT72; DBF4

#2437

Microfluidic device integrated with lipid nanoprobe for extracellular vesicle isolation toward non-invasive cancer diagnosis.

Siyang Zheng,1 Yuan Wan,1 Mackenzie Maurer,1 Yi-Qiu Xia,1 Si-Jie Hao,1 Wen-Long Zhang,1 Nelson S. Yee2. 1 _The Pennsylvania State University, University Park, PA;_ 2 _The Pennsylvania State University, Hershey, PA_.

Extracellular vesicles (EVs) can mediate intercellular communication by transferring cargo proteins and nucleic acids between cells. Recently, they have been demonstrated for potential value in cancer diagnostics and treatment monitoring. However, relevant clinical translation is mainly limited by technical challenges in EV isolation. We previously reported lipid nanoprobes, which is a nanomaterial system of labeling probe and capture probe. The labeling probe is constructed with a lipid molecule as the active component to label EVs rapidly and spontaneously with high efficiency, while the capture probe can isolate labeled EVs magnetically. Herein, we report on the development of a microfluidic device with integrated lipid nanoprobe for on-chip EV isolation towards instrument development for clinical translation. The nanostructured substrate of the device is integrated with a micromixer for one-step isolation of EVs from 1-ml plasma. We validated the device with plasma collected from pancreatic cancer patients, followed by DNA mutation detection of EV cargo. Size-tunable nanostructures are fabricated by patterning high-resolution iron thin films on fused silica substrates. By judicious thermal annealing, homogeneously dispersed iron nanoparticles are formed by fission and fusion on the substrate, which are served as a mask for silica dry-etching. Subsequently, selected lipid nanoprobes, which can instantaneously insert into lipid bilayers of EVs, are grafted onto the nanostructured substrate in a micromixer. This platform is verified first to isolate cultured cancer cell-derived EVs spiked-in plasma samples that emulated clinical samples. This platform is further applied to isolate EVs from pancreatic cancer patients' plasma and identified KRASmutations in EV DNA with highly sensitive droplet digital PCR. Together, this platform enables rapid and efficient isolation of EVs from plasma specimens, and thus, holds great potential in clinical translation.

#2438

Rapid fluorescence based assessment of RNA integrity quality using machine learning.

Scott T. Clarke,1 Dylan Poulsen,2 Chris Vonnegut,1 Debra Gale,1 Kathy Free1. 1 _Thermo Fisher Scientific, Eugene, OR;_ 2 _Washington College, Chestertown, MD_.

Much attention has been focused on RNA transcript bias. The quality of isolated mRNA used for RNA-seq and other downstream analyses can contribute to sample specific bias of transcript abundance. If the library is prepared from polyA enrichment of degraded RNA or with reverse transcriptase using oligo dT priming then 3′ positional bias must be considered (Irizarry 2016). Isolated total RNA is routinely examined for its quality prior to committing to costly gene expression analysis. Since total RNA consists of ~85% rRNA, analysis primarily reflects rRNA quality and the mRNA quality is inferred. Typically, measurements consist of 260/280 nm ratio, 28S/18S rRNA ratio and capillary electrophoresis fragment analysis or gel-based separation and imaging methods. These methods which have become industry standards of quality generate an RNA score such as RNA integrity number (RIN) between 0-10 with >8 being acceptable for RNA-seq. Generating RIN score requires experienced technical ability, specialized equipment, as well as additional cost and time prior to committing to the next step of building the library for sequencing. In this presentation we examine the integrity of the RNA using a combination of nucleic acid specific fluorescent dyes which display different binding specificity depending on the primary and secondary structure of the RNA. Fluorescence signal is analyzed using machine learning based algorithm. For machine learning, a training data set of data was generated, followed by a test set of data used to determine accuracy. Data depicting the actual vs. predicted class are reported in a confusion matrix. Variable of importance analysis was used to determine the factors most important for predicting accurate RNA integrity quality (IQ). With machine learning the initial determination of accuracy using the test data, ranged between 92.7 - 95.7% over the entire range depending on the algorithm. With further refinement of the machine learning approach, RNA IQ had an accuracy of ±0.65 and ± 0.39 standard deviation (SD) for IQ range 0 - 10 using the test data set. The accurate and low SD generated using nucleic acid binding dyes makes for a quick, simple and easy to use assessment of isolated RNA. The dye binding and fluorescent measurement is rapid and requires only a few minutes to generate. Comparison of the dye/RNA based fluorescence based determination of RNA IQ was benchmarked against the industry standards using the BioAnalyzer system (Agilent) to generate a RIN score and other gel analysis methods to determine concordance and utility of this method. Technical ability, cost and time investment are minimal allowing for rapid sample characterization prior to further analysis and commitment to downstream sequencing steps.

#2439

A semi-supervised deconvolution method for quantifying the composition and activity of tumor-infiltrating cell types.

Wennan Chang,1 Changlin Wan,1 Yifan Sun,2 Yan Han,1 Siyuan Qi,1 Xiongbin Lu,2 Sha Cao,2 Chi Zhang2. 1 _Indiana University Purdue University Indianapolis, Indianapolis, IN;_ 2 _Indiana University School of Medicine, Indianapolis, IN_.

Tumor microenvironment (TME) plays a key role in tumorigenesis, disease progression and the acquirement of drug resistance. An accurate assessment of TME cellular compositions may not only shed light on how TME interacts with cancer cells, but also bring new insights for translational researchers in studying the non-responding mechanism of immunotherapy. Traditional deconvolution methods infer the relative proportions of predefined cell types based on a tissue omics data through either regression- or enrichment- based approaches. However, there are several challenges that remain unsolved in the current formulations, including (1) identifying the Immune/Stromal (I/S) cell types that truly exists in a TME, (2) identifying the marker genes for each cell type that are specifically expressed by one or a few I/S cell types in a TME, (3) co-linearity among to-be-assessed I/S proportions due to their co-infiltration. We have developed a novel semi-supervised deconvolution method namely ICTD (Inference of Cell types and Deconvolution), addressing the three challenges via (i) developing a Bi-Cross Validation (BCV) based matrix rank test to assess the significance level of the existence of cell types and signature genes, (ii) utilizing a constrained Non-negative Matrix Factorization (NMF) to eliminate the effect of co-linearity. We validated ICTD on bulk tumor datasets simulated using single-cell RNA-seq data. Our analysis suggested that ICTD has a largely improved prediction accuracy of TME compositions comparing to existing methods, and particularly, it is capable of identifying novel or sub-cell types. We applied our method to TCGA and other gene expression data of breast and prostate cancer. Subsets of CD8+ T cells with varied cytotoxicity levels and subtypes of fibroblast cells were identified. Moreover, integrated with an analysis of an independent single-cell and cell line gene expression dataset, we identified genes specifically expressed by cancer cells that are associated with decreased T cell cytotoxicity level.

#2440

**The microRNA-mRNA interactions in** ALK **-rearranged lung adenocarcinoma.**

Sophia Subat,1 Kentaro Inamura,1 Hironori Ninomiya,1 Hiroko Nagano,1 Sakae Okumura,2 Yuichi Ishikawa1. 1 _Japanese Fndn. for Cancer Res. Cancer Inst., Tokyo, Japan;_ 2 _Japanese Fndn. for Cancer Res., The Cancer Inst. Hospital, Tokyo, Japan_.

Background: ALK-rearranged lung adenocarcinoma, which is responsive to ALK inhibitors, exhibits distinct clinicopathological and molecular features. In ALK-rearranged lung cancer, miRNA/mRNA expression signatures or individual miRNA-mRNA interactions have been reported. However, no study has performed integrative analysis to identify comprehensive miRNA-mRNA interactive network in this type of tumor. Therefore, we conducted this study to reveal a landscape of miRNA-mRNA interactions present in ALK-rearranged lung adenocarcinoma.

Methods: To identify differentially expressed miRNAs and mRNAs according to ALK-rearranged status, microarray expression profiling was conducted using 77 surgically resected specimens of lung adenocarcinoma with a known ALK status (42 ALK-positive and 35 ALK-negative cases). We made a multistep bioinformatics approach to build an miRNA-mRNA regulatory network observed in ALK-rearranged lung tumors.

Results: Seventy-three miRNAs (comprising 56 downregulated and 17 upregulated miRNAs) were differentially expressed according to ALK-rearranged status. The 56 miRNAs included miR-19a-3p, miR-362-5p, and miR-340-5p, all of which have been reported to be downregulated in ALK-rearranged lung tumors. On the basis of miRNA expression data, we identified 80 distinct biological processes in ALK-positive tumors, which included "proteoglycans in cancer", "FoxO signaling pathway", "pathways in cancer", "mTOR signaling pathway", and "HIF-1 signaling pathway". The mRNA expression signature of ALK-rearranged tumors included ALK, ETV1, and CX3CL1, all of which have been

associated with lung tumorigenesis or tumor progression. Integrative analyses of miRNA and mRNA expression data revealed a refined list of putative miRNA-mRNA correlations. Of 136 putative miRNA-mRNA interactions, we identified 101 distinctive interactions with potential involvement in oncogenic machinery in ALK-positive tumors, such as miR-19a-3p/TLR2, miR-362-5p/SLC34A2, miR-1260b/ETV1, and miR-4481/CX3CL1 pairs. Network structural analysis provided a comprehensive view of complex miRNA-mRNA interactions in ALK-rearranged lung adenocarcinoma.

Conclusion: Overall, this observational study provides insight into the unique miRNA-mRNA regulatory network present in ALK-rearranged lung adenocarcinoma. Our findings, if validated, would inform future research examining the interplay of miRNAs and mRNAs in ALK-rearranged tumors.

#2441

Association between NRF1 regulatory gene networks and cancer and aging.

Kaumudi Bhawe, Quentin Felty, Changwon Yoo, Deodutta Roy. _Florida International University, Miami, FL_.

There is a growing convergence between molecular aspects of aging and cancer. Cellular senescence acts as a barrier through preventing the proliferation of cells under oncogenic stress. Therefore, we decided to examine the role of nuclear respiratory factor 1 (NRF1) transcription factor in human brain cancer and brain aging. In contrast to higher expression of NRF1 in human glioma tumors, lower NRF1 levels are observed in Alzheimer's disease (AD) brain tissue. Using the glioma and AD transcriptomic data of NRF1 target genes, we conducted Transcription Factor Target Enrichment Analysis that detects transcription factor (TF) activity based on quantification of the collective transcriptional activation of their target genes. NRF1 TF activity was significantly lower in AD brain tissues from four independent cohorts compared to non-AD brain tissues. In contrast, we observed higher NRF1 transcription factor activity in glioma. NRF1 activity as well as expression of several cell cycle pathway genes including CDK1,2,6,9 and NRF1 target genes such as NR2E1, APOE and MAPT are significantly altered in these two population cohorts. We also discovered that expression of NRF1, RB1 and E2F influenced survival in glioma. Specifically, the mean overall survival for patients with a high NRF1, E2F1,2,6,7,8 and RB1 gene signature was 20.91 months (95% CI = 9.37-32.46), compared to 101.99 months (95% CI = 74.28-129.70), in high NRF1 expressing patients that lacked the specific gene signature. NRF1 based gene signatures combined with IDH1 mutation status are necessary and sufficient for explaining the observed differences in survival curves. We examined the association of NRF1, the E2Fs and RB1 through Bayesian graphical modeling and observed gender differences in gene networks between NRF1 and E2F family members in the glioma patient sub-populations as compared to normal aging human brain samples. Furthermore, the differentially expressed NRF1 regulated genes are part of the KEGG pathways associated with spliceosome, ribosome and lysosome function as well as a diverse array of other functions are enriched, indicating ways in which NRF1 may be involved in glioma biology. In summary these findings support our postulate that NRF1 is an important transcription factor in the human brain, where it plays an active role in both aging and cancer.

#2442

Assessing how macrophages attain distinct iron handling phenotypes in a tumor via a mathematical model.

Luis Sordo Vieira,1 Joseph Masison,2 Jacqueline Adams,3 Suzy Torti,2 Reinhard Laubenbacher,1 Julia Chifman3. 1 _The Jackson Laboratory for Genomic Medicine, Farmington, CT;_ 2 _UConn Health, Farmington, CT;_ 3 _American University, Washington, DC_.

Tumor-associated macrophages are critical regulators of iron levels. It is well established that macrophages display distinct iron handling phenotypes based on the several signals they receive from other cells in the tumor microenvironment. In order to shed light on how macrophages respond to these different signals, we are building a knowledge-based mathematical model for how macrophages attain distinct, yet related, iron handling capabilities.

#2443

Predicting the impact of somatic mutations on chromatin conformation using deep learning.

Tuan Trieu, Ekta Khurana. _Weill Cornell Medical College, New York, NY_.

DNA in eukaryotes is packed into hierarchical layers of loops resulting in the 3D structure of the genome. 3D genome organization plays vital roles in many biological processes of the cell such as gene regulation, cell division and stem cell differentiation and its disruption can lead to gene dysregulation and diseases, including cancer. We developed a deep learning model to predict the impact of single nucleotide variants, indels and structural variants on 3D chromatin loop structures of the genome. We first built a deep convolutional neural network to learn DNA sequence patterns of boundaries of 3D chromatin loops. The learned patterns of boundaries are then used to train another neural network to learn if two boundaries are likely boundaries of a chromatin loop given their DNA sequences. The model produces a probability indicating the likelihood of the two boundaries to form a chromatin loop (loop probability). It is trained with DNA sequence only, and therefore, it can detect effects of DNA variants on 3D chromatin loops as changes in loop probabilities. We trained our model with 80% of 3D chromatin loop from the cell line GM12878. Our model to predict boundaries of loops can distinguish DNA sequences of boundaries from random sequences with AUC of 0.96 on a balanced test-set from the cell line GM12878 and also performs well on data from the K562 cells with AUC of 0.93. Our result shows that variants in lymphoblastoid cells GM12878 do not change loop probabilities as much as variants in leukemia cells K562 do, consistent with the greater disruptive role of tumorigenic variants in the cancer cells. We next applied our model on whole-genome sequences of 240 malignant lymphoma patients to identify loop-disrupting variants that may act as drivers of cancer. We find that genes in predicted altered loops show more differential expression in tumor samples compared to genes in unchanged loops. Our method can be applied on large cohorts of patients with whole-genome sequences to analyze the chromatin conformation changes caused by somatic variants.

#2444

The use of CT radiomics to predict immune infiltrate in pancreatic ductal adenocarcinoma.

Caitlin A. McIntyre, Jayasree Chakraborty, Maura Koszalka, Jared Bassmann, Yuting Chou, Mithat Gonen, Peter J. Allen, T. Peter Kingham, Michael I. D'Angelica, Jeffrey A. Drebin, William R. Jarnagin, Richard K. Do, Vinod P. Balachandran, Amber L. Simpson. _Memorial Sloan Kettering Cancer Center, New York, NY_.

Background: Prognostic and/or predictive biomarkers for patients with pancreatic adenocarcinoma are limited. Radiomics is a non-invasive method to quantitatively analyze tumors with imaging. Previous data have demonstrated that CT radiomics is predictive of overall survival in PDAC. Based on evidence that enhanced intratumoral adaptive immunity is associated with improved survival in PDAC, we sought to build an immune infiltration prediction model with CT radiomics.

Methods: A tissue microarray (TMA) was constructed with tumor arranged in triplicate from short-term and long-term survivors of PDAC who underwent resection between 2005 and 2010. Multiplexed immunohistochemistry (IHC) was performed for intratumoral mature dendritic cells (DC-LAMP+), regulatory T cells (CD3+FoxP3+), CD8\+ T cells, (CD3+CD8+), activated cytolytic CD8\+ T cells (CD3+CD8+GrB+), and macrophages (CD68+). The pancreatic tumor in the portal venous phase on preoperative CT was manually delineated, and 255 radiomic features were extracted and analyzed for significance using univariate linear regression. A multivariate linear regression analysis was then performed to correlate radiomic features with immune infiltrate for each of the immune phenotypes.

Results: There were 22 patients included in the analysis, 7 of whom were short-term survivors (median survival of 9 months) and 15 were long-term survivors (median survival of 74 months). Feature selection resulted in 17 significant radiomic features for mature dendritic cells, 7 for regulatory T cells, 11 for CD8\+ T cells, 16 for cytolytic CD8\+ T cells and 12 features for macrophages. Five regression models were then constructed using the significant radiomic features to predict intratumoral immune infiltrate. There was a strong association between radiomic features and mature dendritic cells (R2=0.83, 95% CI 0.761-0.906, p<0.001) and cytolytic CD8\+ T cells (R2=0.85, 95% CI 0.811-0.89, p=0.07) in PDAC, however the correlation was weaker (R2<0.7) for the other three cell types.

Conclusion: A strong association was found between imaging phenotypes and both dendritic cells and activated cytolytic T cells, demonstrating that non-invasive imaging techniques may help identify tumors with active adaptive immunity. Further analysis is underway in a larger cohort of patients to validate these findings.

#2445

Pan-cancer analysis of tumor microenvironment using deep learning-based cancer stroma and immune profiling in H&E images.

Kyunghyun Paeng,1 Geunyoung Jung,1 Sarah Lee,1 Soo Youn Cho,2 Eun Yoon Cho,2 Sang Yong Song2. 1 _Lunit Inc., Seoul, Republic of Korea;_ 2 _Department of Pathology and Translational Genomics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea_.

Introduction: Tumor purity and deconvolution of cell subsets in tumor environments has been developed based on DNA sequencing data or gene expression profiles. We have developed the artificial intelligence named Lunit SCOPE, identifying and quantifying various histologic components using deep learning from H&E-stained whole slide images. The Lunit SCOPE can define the proportions of cancer epithelium, cancer stroma and immune cells infiltration. We hypothesized that cell proportions analyzed by Lunit SCOPE can accurately predict cell subsets in tumor microenvironment with biologically-reliable results.

Method: Deep learning-based H&E image analyzer, Lunit SCOPE, has been trained by 1,191 H&E-stained breast cancer whole slide images with histological components such as cancer epithelium, cancer stroma, normal cells, fat, necrosis, lymphocyte, and fibroblast, annotated by over 10 pathologists. Lunit SCOPE trained by the annotated dataset can profile the cancer stroma ratio and tumor infiltrating lymphocyte (TIL) density in cancer epithelium and cancer stroma. We have selected 21 types of cancers excluding mesenchymal origin cancer in The Cancer Genome Atlas (TCGA). Finally, H&E slides from 7,837 cases have been analyzed by Lunit SCOPE.

Result: Pan-cancer analysis has shown that the cancer stroma ratio from Lunit SCOPE was the highest in pancreatic adenocarcinoma. Especially, Consensus Molecular Subtype 4 (CMS4) has shown the highest cancer stroma ratio among colorectal cancer (P < 2.2 x 10-16). The density of tumor infiltrating lymphocyte (TIL) was highest in renal clear cell carcinoma, melanoma and bladder cancer in that order, and lung cancer, esophageal cancer, and head and neck cancer have also presented high TIL density. Moreover, high mutational burden (> 10/Mb) was significantly related to higher TIL density across all cancer types (P = 4.06 x 10-5). In addition, the stromal TIL density was higher in basal type than other subtypes of breast cancer (P = 2.45 x 10-7). Pan-cancer survival analysis showed that higher cancer stroma ratio (HR 1.14, 95% CI 1.04-1.25, P = 0.00419) and higher stromal TIL density (HR 1.17, 95% CI 1.07-1.28, P = 0.000732) were associated with poor prognosis, but higher intratumoral TIL density had good prognosis (HR 0.89, 95% CI 0.82-0.98, P = 0.0126).

Conclusion: In this study, TCGA pan-cancer dataset have been analyzed by deep learning-based H&E image analyzer, Lunit SCOPE. As a result, it has been proven that the ratio of cancer stroma is high in pancreatic cancer and CMS4 colorectal cancer, and immunogenic tumors and high mutational burden is related to high TIL density. In summary, Lunit SCOPE has significantly correlated with previously well-defined biological features in pan-cancer analysis. Furthermore, Lunit SCOPE can be developed for the novel tissue-agnostic predictive biomarkers of cancer immunotherapy.

#2446

Stochastic calibration of an agent-based tumor growth model using time-resolved microscopy data.

Ernesto A. Lima, Danial Faghihi, Russel Philley, Jianchen Yang, Jack Virostko, Thomas E. Yankeelov. _University of Texas, Austin, TX_.

Like every biological process, tumor development is not purely deterministic, as it is subject to random perturbations from the environment. Moreover, mathematical and computational tumor growth models are subject to uncertainties in the parameters, which may arise from experimental variations or intratumor and intrapatient heterogeneities. Tumor growth models are typically deterministic representations of growth, neglecting the aforementioned uncertainties. However, carcinogenesis must be modeled as a stochastic process to generate more informative predictions of the tumor microenvironment, which is particularly important when describing the action of various therapies. The model developed here aims to reflect the stochastic proliferation and differentiation of tumor cells. We present, for the first time, a methodology for performing a stochastic calibration of an agent-based tumor growth model.

The agent-based model reproduces the interactions among different tumor cell phenotypes (e.g., proliferative and dead cells). The cell movement is driven by the balance of a variety of forces (e.g., cell-cell adhesion and repulsion) according to Newton's second law. Transitions among cell phenotypes are defined by rules that depend on glucose concentration and time spent in each phase of cell cycle. The model captures the phenomena at the cell scale, tracking the velocity, position, and confluence, as well as the glucose concentration in the microenvironment. The experiments were done using a HER2+ breast cancer line (BT-474). Cells were seeded at a density of 3.2×104, 4.0×104 and 4.8×104 cells/well on a 96-well tissue culture plate, with four glucose levels (0, 2, 5 and 10 mM), and four replicates for each combination of initial density and glucose levels. The cells were incubated in the IncuCyte live cell imaging system (Essen BioScience, USA). Multiple images were acquired and stitched together automatically by this system with a 4× objective to obtain whole well images for each well. IncuCyte Cytotox Red Reagents (Essen BioScience, USA), a highly sensitive cyanine nucleic acid dye was added into the medium to estimate cell death. The wells were imaged every 2 hours and cell segmentation was performed in Matlab (The Mathworks, Inc., USA). Segmented images from phase-contrast and fluorescent images were employed to determine the confluence of the live cells. The dead and live cells from our agent-based model were calibrated using a Bayesian framework. The likelihood used during the calibration process considers the stochasticity from our model and the variance in the in vitro experiments. Preliminary efforts indicate the average difference between the calibrated model and the experimental data is below 5.6±4.5%. The results of the proposed model suggest that the computational framework developed can correctly calibrate the agent-based model while considering the stochasticity of the model and data.

#2447

Accurate prediction of the signaling pathway activity elicited by variants of uncertain significance (VUS) and their drug sensitivity using deep convolutional neural networks on florescent microscopy images of cells.

Lior Zimmerman, Arie Aizenman, Yutti Daitsh, Zohar Barbash, Gabi Tarcic, Michael Vidne. _Novellusdx, Jerusalem, Israel_.

Sequencing based methods to tailor the treatment of cancer patients are at the cutting edge of personalized medicine and have remarkably advanced in the past few years. However, a large number of mutations that are found when sequencing tumors are poorly characterized in the literature and are thus labeled as VUS. Because of their uncertain significance, it is not possible to assign them a function and hence are overlooked when designing treatment regimens, and this can have deleterious effects. Here we report the development of a Deep Convolutional Neural Network (DCNN) platform trained using Deep Learning algorithms, that is able to accurately predict the activity elicited by specific mutations. The DCNN is based on a high-throughput platform to predict the activation of signaling pathways elicited by the specific mutations via changes to the intra-cellular localization of fluorescently labeled protein reporters. For our training set we used images of 6 billion cells expressing either WT or mutations with known activity of the analyzed genes. After training, the network was presented with images of cells transfected with VUS that the network has never seen before. We show that the network was able to correctly predict the level of activity of a large number of VUS in KRAS, BRAF and cKIT and on multiple signaling pathways, specifically the MAPK and JAK-STAT pathways. These findings were further confirmed by both biochemical assays as well as literature search. Furthermore, images of cells expressing known mutations of BRAF and cKIT and treated with increasing doses of the targeted treatments Vemurafenib and Imatinib, respectively, were analyzed by the network and we were able to generate high resolution dose response curves in specific signaling pathways. Lastly, we present the predicted dose response curves in specific signaling pathways of a variety of VUS which have never been characterized before and show that different VUS respond differently to the tested drugs. Overall, we provide evidence that this novel tool enables robust and highly accurate calculation of activity profiles of a wide range of genes and signaling pathways. Each mutation is highly specific in the magnitude of activity it elicits. The high degree of resolution allows the investigation of the efficacy of different targeted therapies on a wide range of mutations.

#2448

Elucidating the metabolic plasticity of cancer by coupling gene regulation with metabolic pathways.

Dongya Jia,1 Mingyang Lu,2 Kwang Hwa Jung,3 Jun Hyoung Park,3 José N. Onuchic,1 Benny Abraham Kaipparettu,3 Herbert Levine1. 1 _Rice University, Houston, TX;_ 2 _The Jackson Laboratory, Bar Harbor, ME;_ 3 _Baylor College of Medicine, Houston, TX_.

Metabolic plasticity allows cancer cells to adjust their metabolic phenotypes to grow and metastasize in hostile environments. Both glycolysis and oxidative phosphorylation (OXPHOS) are adapted by cancer cells to meet their bioenergetic and biosynthetic requirements in a context-dependent manner. Despite the advance in studies focusing only on glycolysis or OXPHOS in cancer, it remains largely unknown how cancer cells orchestrate different metabolic phenotypes for better survival. To address this question, there is an urgent need to develop systemic approaches to quantitatively study the interplay between glycolysis and OXPHOS in cancer. Mathematical modeling approaches have been employed to elucidate cancer metabolic reprogramming. Constraint-based models including flux balance analysis based on conservation of mass have been the most widely used approaches to simulate cancer metabolism. In addition, modeling efforts have also been developed to identify anomalous gene activity involved in cancer metabolism. These computational studies offer a quantitative and dynamical perspective of cancer metabolism mostly focusing on either metabolic pathways or gene activities. However, the alteration of the metabolic activity is often coupled with the change in gene activity, and vice versa. Thus, to comprehensively characterize cancer metabolic reprogramming, a mathematical modeling framework integrating gene regulation with metabolic pathways is urgently needed. Here, we establish a theoretical framework to elucidate cancer metabolic plasticity through systems biology analysis of the coupling of gene regulation and metabolic pathways. Our modeling results demonstrate a direct association between the activities of AMPK and HIF-1, master regulators of OXPHOS and glycolysis respectively, with the activities of three metabolic pathways: glucose oxidation, glycolysis and fatty acid oxidation (FAO). Guided by the model, we develop metabolic pathway signatures to quantify the activities of glycolysis, FAO and the citric acid cycle of tumor samples by evaluating the expression levels of enzymes involved in corresponding processes. The association of AMPK/HIF-1 activity with metabolic pathway activity, predicted by the model and verified by analyzing the well-annotated metabolomic and transcriptomic data from a breast cancer patients' cohort, is further validated by in vitro studies of aggressive triple negative breast cancer cell lines. We further investigate the existence of an aggressive hybrid metabolic phenotype that enables cancer cells metabolic plasticity for better survival and a metabolically inactive phenotype that may be employed by cancer cells under pressure. To the best of our knowledge, we are the first, or at least one of the first, to couple gene regulation with metabolic pathways to elucidate cancer metabolic plasticity.

#2449

Unsupervised machine learning methods reveal metabolomic based clusters in breast cancer patients.

Jocelyn Gal,1 Caroline Bailleux,1 David Chardin,1 Thierry Pourcher,2 Lun Jing,2 Jean-Marie Guignonis,2 Jean-Marc Ferrero,1 Renaud Schiappa,1 Emmanuel Chamorey,1 Olivier Humbert1. 1 _Antoine Lacassagne Center, Nice, France;_ 2 _University of Nice Cote d'Azur, Nice, France_.

Background Transcriptomic have led to the now widely used sub-type based classification of breast cancer first described by Perou in 2000. Yet there persists heterogeneity in biological behaviors within breast cancer subtypes, underlining the need to refine the taxonomy of breast cancer. Metabolomics is a rapidly expanding field dedicated to the study of metabolism which integrates the impact of the environment on cell biology. The aim of this study was to identify new biological breast cancer clusters using different unsupervised machine learning (ML) methods based on metabolomic features. Those methods, in which no a priori class label information is given to guide the algorithm, seem suitable to address this type of problem.

Methods 52 patients with breast cancer and an indication for adjuvant chemotherapy between 2013 and 2016, were retrospectively included. Tumor resection specimens were analyzed. 1300 metabolomic were extracted by combined liquid chromatography-mass spectroscopy and processed using MZmine software and the "Human Metabolome" database. 5 unsupervised ML methods were used: PCA-Kmeans, Sparcl, SIMLR, Spectral clustering and K-sparse. Clinical differences between clusters and variations for every metabolite of interest were analyzed for each clustering method. Cluster separability and homogeneity was evaluated using the silhouettes method and t-sne visual evaluation.

Results Among the 5 clustering methods, with a partitioning optimum parameter k=3, only K-sparse and SIMLR methods generated 3 clusters with significant clinical differences, unmatched to traditional subtypes. These differences concerned: tumor stage, axillary lymph node invasion, histological grade, ki-67 proliferation index, and tumor phenotype. With a silhouette average of 0.84 and 0.85 for K-sparse and SIMLR methods respectively, those 2 methods gave the best score in terms of silhouette average and they showed a better gradient for tumor aggressiveness compared to the 3 other methods. Among them, 42 and 55 metabolites were selected for the construction of tumor metabolome profiles for K-sparse and SIMLR, respectively. Among selected metabolites we found a significant increase of L-methionine, L-phenylalanine, L-isoleucine and L-proline along with a significant decrease in glutathione (also characteristic of oxidative stress) and glutamate in the cluster associated with poorer histopronostic factors. This high concentration of proteinogenic amino-acid and low concentration of amino-acid precursors could be correlated to poorer prognosis.

Conclusion Unsupervised ML methods generate heterogeneous results when applied to metabolomics data extracted from breast cancer patients. K-sparse and SIMLR were able to identify three different groups based on tumor metabolome. Tumors with the worst histopronostic factors seemed to present higher concentrations of protienogenic amino-acids.

#2450

Single-cell profiling reveals programs mediating oncogenic kinase-independence and minimal residual disease in BCR-ABL-rearranged acute lymphoblastic leukemia.

Mark A. Murakami,1 Peter S. Winter,2 Andrew W. Navia,2 Laura L. Bilal,2 Haley Strouf,2 Kay Shigemori,1 Alejandro Gupta,2 Mahnoor Mirza,2 Catharine S. Leahy,1 Nick Calistri,2 Kristen Jones,1 Alexandra Van Scoyk,1 Huiyun Liu,1 Foster Powers,1 Robert Kimmerling,2 Mark Stevens,2 Kristen E. Stevenson,1 Scott R. Manalis,2 David M. Weinstock,1 Alex K. Shalek2. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Massachusetts Institute of Technology, Cambridge, MA_.

Targeted inhibitors of oncogenic kinases have transformed cancer therapy by inducing high rates of clinical response across diverse tumor types, yet they cure few patients due to the persistence of minimal residual disease (MRD), which seeds relapse. Characterizing MRD biology would promote development of more effective therapies tailored to relapse-initiating cells. However, these efforts have been complicated by the technical challenges of isolating and profiling low-frequency tumor populations. We overcome these hurdles by measuring the mass and short-term growth rate of single cells ex vivo using a series of suspended microchannel resonators (SMR) followed by single-cell RNA-seq of the same cells. This platform enables measurement of low input samples to facilitate characterization of MRD subpopulations and functionally drug test MRD-specific vulnerabilities. PDX models of BCR-ABL-rearranged acute lymphoblastic leukemia (BCR-ABL ALL) treated with targeted kinase inhibitors provide a tractable experimental system in which to deploy this platform to interrogate in vivo tumor evolution, MRD, and acquired resistance. We conducted a statistically powered 4-arm randomized phase II-like trial combining ponatinib and the novel allosteric BCR-ABL inhibitor ABL001 in a panel of 13 PDX models of BCR-ABL ALL (total evaluable mice: n=10 vehicle, n=14 ABL001, n=30 ponatinib, n=33 combination). Six of 30 animals receiving ponatinib (20% [95% CI, 8-39%]) and a significantly higher proportion receiving combination (10 of 33, 30% [95% CI, 16-49%]) met the primary endpoint of progression-free survival at 120 days on treatment (p=0.030). No mice in the vehicle or ABL001 arms met this endpoint, and all mice eventually progressed or were found to harbor MRD when sacrificed after treatment courses of up to 248 days. These durable remissions prior to relapse allowed us to delineate in vivo tumor evolution, focusing on MRD, via serial bone marrow aspirates. Drug-persistent marrow-resident leukemia cells exhibit strong enrichment for cellular quiescence programs that extend through MRD and into relapse. These programs are driven by expression of genes known to govern quiescence in hematopoietic stem cells (e.g., EGR1, NR4A1), and their expression correlates with a rare subset of biophysically small cells (mass <25 pg) detected in pretreatment specimens. At MRD these small cells can be subdivided into at least two transcriptionally distinct populations, one poised to re-enter the cell cycle and likely to seed relapse, the other senescent (e.g. CDKN2A high) and therefore unlikely to contribute. Leukemia cells at relapse maintain low mass despite cell cycle re-entry. We are integrating these MRD quiescence programs with genetic and transcriptional mechanisms of tumor progression to develop rational therapies for overcoming MRD in BCR-ABL ALL.

#2451

Physical, genomic, and proteomic characterization of a cancer cell line panel in an integrated dataset.

Milen Nikolov,1 Brian White,1 Adrian Pegoraro,2 Debra Hope,3 Mariam Eljanne,3 James Eddy,1 Paul Janmey,4 Parag Mallick,5 Kristen Dang1. 1 _Sage Bionetworks, Seattle, WA;_ 2 _Harvard University, Cambridge, MA;_ 3 _National Institutes of Health, Bethesda, MD;_ 4 _University of Pennsylvania, Philadelphia, PA;_ 5 _Stanford University, CA_.

Introduction: Recent work has linked cancer phenotype (e.g., metastatic ability) to physical properties (e.g., nuclear deformability), as well as genetic alterations (e.g., mutations in lamin genes affecting nuclear stiffness). However, while the genomics of immortalized human cell lines - important models for studying cancer disease pathogenesis and progression - have been relatively well studied, an integrative analysis of physical cell phenotypes with genomic, transcriptomic, and proteomics assays, spanning tissues of origins and culture environments (CE), has been lacking. The Physical Sciences in Oncology Network (PS-ON), a trans-disciplinary project established by the NCI, has generated a curated open dataset that is accessible to researchers via various database interfaces to address this need.

Methods: A panel of 39 cancerous and non-malignant cell lines frequently used in cancer research were selected to cover a set of nine tissues: breast, lung, pancreas, brain, prostate, colon, ovary, blood, and skin. Thirty of these cell lines (from all tissues except blood) were assayed by traction and atomic force microscopy and imaging to measure their cell morphology, proliferation, motility, and nuclear volume. Measurements were performed in seven CE of varying physical properties (e.g., stiffness) to recapitulate a range of realistic tissue conditions and frequently used reagents. All 39 cell lines were assayed via whole exome sequencing, mRNA-seq, and miRNA-seq (in a single CE), while nine cell lines were subjected to proteomic analysis (across seven CEs).

Results: All raw measurements and summary metrics from the study were incorporated into a data model compatible with established data stores (e.g., GDC, miRBase) and integrated in an open-access relational database (RDB). We performed several integrative analyses using the RDB. A broad set of interactions emerged between cell line CE characteristics (e.g., integrin ligands), the phenotypes of cells (e.g., motility) and gene expression, with effect-size varying by cell-line tissue of origin and cancer diagnosis. For instance, motility was significantly (FDR < 5%) correlated with expression of a small set of genes across tissues (e.g., BNP1 and PFKP in both skin and prostate cancers).

Conclusion: This PS-ON cell line characterization allows correlative analyses across CE, cells' morphological and proliferation properties, and omics data. The integrated RDB facilitates these analyses via a unified interface and data model for the large collection of files comprising the resource. We demonstrated the utility of the RDB by using it to infer the relationship between differential gene expression, CE and resulting phenotypes, with potentially important downstream effects on hallmarks of cancer malignancy.

#2452

Patient segmentation using machine-learning based literature and genomic data synthesis uncovers novel cohorts of NSCLC and mesothelioma patients.

Enrique Garcia-Rivera,1 Aaron S. Mansfield,2 Karthik Murugadoss,1 Murali Aravamudan1. 1 _nference, Cambridge, MA;_ 2 _Mayo Clinic, Rochester, MN_.

Current unbiased approaches to mine the large amounts of patient-level data on mutations, structural variations and gene expression result in an unwieldy amount of interactions and correlations, which cannot be parsed to identify disease drivers. Here we present an approach to encode mutational and structural variant data at a patient level in a semantic association space. This approach transforms the presence of a mutation (or other feature) in each patient into the quantitative semantic association score of the corresponding gene and the phenotype of interest, which we have trained on all publicly available literature using word-embedding neural networks. Using data from The Cancer Genome Atlas (TCGA), we encoded the mutation or structural variant status (incl. copy number, fusion and chromothripsis) of all patients in the Lung Adenocarcinoma and Mesothelioma cohorts into our semantic space. For each cancer, we first defined the set of genes that are most associated to it according to the literature. To project each patient into this semantic space, we next determined if each patient had a mutation in the genes representing the disease semantic vector (e.g. NSCLC). For TCGA data we only counted non-Silent mutations and represented them as a binary number for each gene, i.e. 0 if the patient had no mutations in that gene and 1 if the patient had a non-Silent mutation in the gene. Each patient was then encoded in a binary vector with each member corresponding to a gene from the disease semantic vector. For example, lung adenocarcinoma was associated to 1,367 genes in our semantic space. A lung adenocarcinoma patient's vector would them be composed of 1,367 binary numbers dictating if the gene is mutated or not in that patient. We then multiply these binary vectors with the semantic disease vector to obtain the patient's projection in the disease space, which in effect replaces the binary number with the Semantic Association Score between the gene and the disease. Contrary to clustering patient samples by their mutation or structural variant data alone, our projected patient vectors clustered patients together into 22 groups with high patient-to-patient similarity. These clusters recapitulate canonical knowledge about the disease, e.g. Lung Adenocarcinoma patients form clusters that include EGFR-driven and KRAS-driven cohorts. We also see novel groups of patients driven by genes such as MET, STK11 and MALAT1. These clusters can be further stratified by their survival status and other clinical features. We validated our approach with a non-TCGA Mesothelioma cohort, revealing similarities in patient stratification regardless of the data source. This approach represents a dramatic shift in patient segmentation, delivering real-time grouping of patients and biomarker identification, which can accelerate clinical trial design and therapeutic development strategy.

#2453

A new deconvolution algorithm for accurate assessing immune and stromal cell populations in mouse transcriptomic data.

Xiaoyu Lu,1 Szu-wei Tu,1 Wennan Chang,2 Yan Huo,3 Pengcheng Wang,4 Yu Zhang,5 Chi Zhang,2 Sha Cao6. 1 _Department of BioHealth Informatics, Indiana University‐Purdue University Indianapolis, indianapolis, IN;_ 2 _Department of Medical and Molecular Genetics and Center for Computational Biology and Bioinformatics, Indiana University School of Medicine, indianapolis, IN;_ 3 _School of Fundamental Science, China Medical University, indianapolis, IN;_ 4 _Department of Computer & Information Science, Indiana University‐Purdue University Indianapolis, indianapolis, IN; _5 _College of Computer Science and Technology, Jilin University, Changchun, China;_ 6 _Department of Biostatistics and Center for Computational Biology and Bioinformatics, Indiana University School of Medicine, indianapolis, IN_.

Multiple deconvolution methods have been developed for investigating the heterogeneous immune and stromal (I/S) cell types in human cancer tissue by deciphering their relative abundances using transcriptomic data. However, there is a lack of a user-friendly software for mouse transcriptomic data deconvolution. Key challenges for developing such a tool include: (1) limited training data sets for available for deriving signature genes of I/S cell types in sporadic mouse data; (2) mouse models of diverse geno-/pheno-types may have varied expressions of I/S cell marker genes; (3) the transcriptomic data may be collected from mouse with certain levels of immuno-deficiency; and (4) the transcriptomic data may come from highly diverse experimental platforms. To solve these challenges, we (i) developed a novel non-parametric analysis method to derive potential I/S cell signature genes from a large collection of mouse data sets; (ii) implemented a low rank sub matrix identification method with a non-negative matrix factorization (NMF) based deconvolution method; and (iii) enabled the flexibility that certain I/S cell types may be absent if their respective cell markers do not form a significant low rank structure. The new method is applied to mouse prostate cancer data sets to infer the level of anti-cancer immune cell populations. Genes expressed by cancer cells that are negatively associated with the anti-cancer immune cells are further inferred, and compared with the results derived in human data. A user-friendly R package of the deconvolution method is released through GitHub.

#2454

Cancer RNA:DNA interactome analysis and prediction.

Vladimir A. Kuznetsov. _SUNY Upstate Medical University, Syracuse, NY_.

Transcriptional R-loops are three-stranded RNA:DNA hybrid structures essential for many normal and pathobiological processes. Previously, we have proposed and validated a quantitative R-loop forming structure (RLFS) model, QmRLFS, and predicted about 660,000 RLFSs in the human genome; most of them are localized in total within 75% of the coding and non-coding genes and gene-flanking regions, transcribed enhancer and pseudogene regions. RLFSs are also commonly co-localized as the singleton or clusters at regulatory sites and disease-associated genomic loci. Recently, it was computationally predicted and experimentally shown that RNA:DNA hybrid/R-loop interactome may play critical roles in mutagenesis, chromosome breaks, genome instability and cancer development. Herein, we conducted a comprehensive comparative analysis between the RLFSs, their clusters, publically available DRIP-seq and DRIPc-seq, and our own experimental datasets. QmRLFS demonstrated high accuracy (82-92%) and unbiased performance with resolution on the nucleotide, gene and genome scales. A preferential co-localization of the RLFSs with promoters, U1 splice sites, gene ends, enhancers and non-B DNA structures, such as G-quadruplexes, provides evidences for the mechanic links between RLFSs and dynamical DNA tertiary structures, transcription initiation and critical regulatory genome signals. We introduced and characterized a class of 25000 paired reverse-forward RLFS clusters highly enriched with non-B DNA structures, which co-localized in the transcribed promoters of sense-antisense gene pairs and enhancer's flanks. In the cancer genomes, we found the RLFSs that form RNA:DNA hybrids/R-loops and could preferentially link open promoters and transcriptionally active enhancer regions. This suggests new mechanisms for in trans RNA:DNA hybrid-mediated 3D-chromatin loop formation. Finally, we found the RLFS clusters supported by cancer cell RNA:DNA hybrids/R-loop data, which were overlapped with hyper-mutated clusters, kataegis expression signatures. Overall, our study provides a rationale for the prediction, discovery, and characterization of the RLFSs as novel functional structures for an emerging quantitative biology and pathobiology of RNA-DNA interactomes.

#2455

Single-color accurate and precise quantification of wild-type versus mutant allele using a novel integrated digital PCR platform.

Megan E. Dueck,1 Christina Wood-Bouwens,2 Andrew Zayac,1 Robert Lin,1 Steve Gallagher,1 Hanlee Ji,2 Paul Hung1. 1 _COMBiNATi, Burlingame, CA;_ 2 _Stanford University, Palo Alto, CA_.

In this study we present a single-step, one-color quantification of mutant versus wild-type allele using a novel integrated, clinic-optimized digital PCR (dPCR) platform. Allelic variants in DNA or RNA can be distinguished and quantified using Polymerase Chain Reaction (PCR) in concert with intelligent primer and probe design. Most commonly, a researcher or clinician will use unlabeled forward and reverse primers that bind to conserved regions outside of the allelic variation in addition to two differently labeled hydrolysis probes that bind at the region of allelic variation. Here we describe a novel method that leverages the precision of digital PCR (dPCR) technology and requires only one-color fluorescence utilizing probe-free, intercalating dye chemistry. The expense in developing or purchasing a complete PCR assay is normally embedded in the cost of the fluorescently labeled probe. The method described here eliminates the need for the fluorescently labeled probe, thereby reducing chemistry cost. Furthermore, technical complexity is reduced by requiring only one-color fluorescence. In our assay, we use a common unlabeled reverse primer that binds downstream of the allelic variation. Two unlabeled, allele-specific forward primers bind preferentially to either the wild-type or mutant allele at the site of allelic variation. The mutant forward primer contains a non-annealing tail (ie: stretch of the DNA primer that does not anneal to the initial DNA template) that will cause the ultimate mutant PCR product to be longer than the wild-type product. Digital PCR is characterized by dividing up a bulk PCR reaction volume into thousands of fluidically isolated partitions or droplets. In the presence of an intercalating dye, a longer amplicon will result in a higher endpoint fluorescence in that specific dPCR partition. As such, using our novel assay, dPCR partitions containing mutant versus wild-type amplicons can be distinguished by differing levels of end-point partition fluorescence. In this study we used the BRAF gene and the clinically relevant BRAF V600E mutation to benchmark this assay using a novel integrated one-step dPCR platform. This dPCR platform consists of a single, fully-integrated instrument along with a novel micro-injection molded plastic consumable providing a simple, single-step workflow. The instrument is dry and contamination-free, making it an attractive instrument for easy transition to the clinic. Parameters including thermal-cycling time and primer concentration were serially optimized for the novel integrated dPCR platform. This work highlights that we can accurately and precisely quantify samples containing varying ratios of wild-type versus V600E BRAF down to 0.1% mutant/wild-type ratio using one color of fluorescence and a novel integrated single-step walkaway dPCR workflow.

#2456

NetZen: A comprehensive network-based pathway and target discovery platform.

Son Le, Alberto Riva, Changwang Deng, David D. Tran. _University of Florida, GAINESVILLE, FL_.

BACKGROUND: Success in precision medicine depends on the ability to accurately and rapidly identify master regulators of critical gene subnetworks. To achieve this goal requires a seamless computational target discovery platform that integrates global network visualization with identification of master regulatory subnetworks (MRS) and their key influencers.

METHODS: We have created NetZen - a computer cluster-capable computational suite allowing for 1) robust network generation with million edges; 2) network-based MRS identification; 3) hierarchical analysis with automated annotations; and 4) whole-network 3D visualization of all 20,000 genes. To identify cancer MRS, we applied NetZen to expression datasets of tumors and normal tissues, tumor-initiating cells (TICs), non-TIC cancer cells, and normal stem and non-stem cells from TCGA, GEO and internal sources. For validation, we introduced these subnetworks in normal cells to recapitulate TIC phenotypes in vivo or disrupted them in patients-derived TICs and mouse xenografts (PDX). To identify MRS of mortality, we applied NetZen to survival data in TCGA.

RESULTS: Networks of 28 human cancers were generated, integrating transcriptomics, microRNAs, proteomics, and clinical data. For cancers with TIC datasets, we identified TIC-specific MRS with clear-cut hierarchy controlling immune surveillance, EMT and developmental functions. Interestingly, in some cancers, the immune subnetwork including immune checkpoints was entirely encompassed in the developmental subnetwork. We present representative validation on glioblastoma TIC (GIC) MRS with features common in other cancers' TICs. Exogenous introduction of this subnetwork in normal astrocytes forced them to become GICs and form tumors in mice. Disrupting this subnetwork in 10 independent PDXs profoundly blunted tumor growth and prolonged survival. Lastly, we present detailed MRS of mortality analysis of multiple cancer types and identify common pathways directly influencing outcomes.

DISCUSSION: NetZen provides a complete solution to big data analysis by effortlessly processing million-edge hierarchical networks with biological insights through automated annotations. The findings that the immune and developmental subnetworks were intimately linked suggest that immune escape by TICs is intrinsic of cellular transformation rather than a product of selection. Lastly, detailed network hierarchy provides a logical roadmap for cancer therapeutic development.

#2457

Prediction of selective genetic vulnerabilities from large-scale functional screens in cancer cells.

Benedikt Rauscher. _German Cancer Research Center (DKFZ), Heidelberg, Germany_.

Cancer genomes often harbor hundreds of molecular aberrations. Such genetic variants can be drivers or passengers of tumorigenesis and create new vulnerabilities for potential therapeutic exploitation. To identify genotype-dependent vulnerabilities, a large number of forward genetic screens in different genetic backgrounds have been conducted. To aggregate and mine functional data from published screens in a standardized format, we recently developed GenomeCRISPR, a curated database for high-throughput CRISPR-Cas9 experiments. We then devised MINGLE, a computational framework that integrates genetic screens in large panels of cancer cell lines to predict genetic interactions between variant and knockout alleles. We showed that our method identifies specific known and previously unknown genetic relationships. For example, we identified a synthetic lethal dependency between RNF43 and members of the Glucosidase II complex. Additional experiments confirmed that the Glucosidase II complex acts as a regulator of Wnt/beta-catenin signalling. In our first study we analyzed a dataset consisting of screens performed in sixty cell lines. Since then the amount of data available for this analysis has increased several fold. We are continuously integrating the new data that become available as we work towards the next iteration of a genetic interaction map in cancer cells. We are specifically interested in using the increased resolution of this new map to understand how genetic networks differ between individual cancer types.

#2458

Integrated proteogenomic characterization of clear cell renal cell carcinoma.

David J. Clark, The CPTAC Consortium. _The Johns Hopkins University, Baltimore, MD_.

Clear cell renal cell carcinoma (ccRCC) is the most predominant histology of renal cancer, representing 75% of all cases and accounting for the majority of associated deaths. Towards understanding the underlying molecular alterations that can drive renal oncogenesis, The Cancer Genome Atlas (TCGA) has performed extensive genomic and transcriptomic profiling, and comparative analyses. To gain insight into the impact of genomic alterations on the functional modules that drive ccRCC tumorigenesis, we leveraged comprehensive proteogenomic characterization of 110 ccRCC tumors and 84 corresponding normal adjacent tissue (NAT) samples. Pairing state-of-the-art mass spectrometry based proteomic and phosphoproteomic strategies with the comprehensive genomic analysis, we integrated global and phosphoproteomic measurements with genomic and transcriptomic data to elucidate the dysregulated cellular mechanism resulting from genomic alterations. Arm-level loss of 3p, involving genes VHL, SETD2, BAP1 and PBRM1 is a frequent event in ccRCC and investigation of chromosomal number variations (CNV), mutations, and methylation profiles of these genes, revealed a unique pattern of BAP1 regulated proteins relative to the other 3p loci genes. Comparative protein profiling of ccRCC tumors and NATs identified pathways associated with immune response and glycolysis to be regulated in ccRCC tumors, however correlating mRNA and protein revealed a non-linear relationship in cellular processes including Warburg Effect-related metabolism and protein translation. Employing unbiased consensus clustering identified three proteomic subtypes of ccRCC, discriminated by differential abundance of proteins involved in adaptive immune response, innate immunity, ribosome activity, and metabolic processes. Further evidence of tumor heterogeneity was observed in the phosphoproteome, with phosphopeptide co-expression modeling generating phospho-signaling modules that overlapped with global protein processes (i.e. adaptive and innate immunity), as well pathways only detected at the phosphoproteomic level (i.e. Notch signaling and DNA repair). The fact that ccRCC tumors are highly immunogenic prompted us to explore the degree of immune infiltration in our cohort. We deconvoluted immune, stromal, and microenvironment cell gene signatures, identifying 60 cell types in our cohort, and three immune-based subtypes of ccRCC: Inflamed, Immune-excluded, and Immune desert. These immune subtypes displayed unique patterns of PD-1, PD-L1, and CTLA4 expression, and could be discriminated by the up-regulation of distinct protein pathways including interferon-γ, HIF1, and EMT. Our results integrated multi-level "omics" analyses to provide greater depth and expand our understanding of ccRCC biology, while identifying novel proteomic, phosphoproteomic, and immune ccRCC subtypes for personalized, precision-based care.

#2459

Identifying genetic drivers of uterine serous carcinoma patient prognosis with regulatory network analysis.

Emily Myers,1 Lynn K. Tran,1 Paul M. Tran,1 David Mysona,2 Sharad Purohit,1 J. J. Wallbillich,1 Jin-Xiong She1. 1 _Medical College of Georgia at Augusta University, Augusta, GA;_ 2 _University of North Carolina Chapel Hill, Chapel Hill, NC_.

Uterine serous carcinoma (USC) is a subtype of endometrial cancer (EC), which is the most common gynecological malignancy in the developed world. Though USC is a Type II EC, which are only responsible for 20% of EC cases, Type II EC causes 74% of EC deaths. Understanding the molecular mechanisms driving USC tumor biology could reveal novel therapies and improve patient outcomes. We developed and validated gene expression signatures capable of predicting USC patient's prognosis. However, little was understood about why differential expression of these particular genes predicts patient prognosis. As such, we investigated the key transcription factors driving the phenotypic difference between the two groups by using the Passing Attributes between Networks for Data Assimilation (PANDA) algorithm. As described by Glass et al., PANDA uses message passing to integrate information from multiple data types to construct robust interaction networks between transcription factors and genes. Following regulatory network generation, we selected potential key regulators based on network analyses. First, we use linear regression analysis to select TFs involved in gene interactions with the greatest edge weight differential between the two groups. Second, we identify TFs involved in unique regulatory interactions to each group. Third, we analyze the TF-gene signature gene interaction networks for both patient populations to select hub transcription factors. TFs selected in all three analyses as potential key regulators are ranked using summed residual values to determine in which patient group the TFs were weighted more strongly. A literature review revealed that the TFs most heavily weighted in the poor survival group are implicated as suppressors of tumor growth. The TFs most heavily weighted in the good survival group are implicated in promoting tumor growth. This suggests that fast-growing tumors correlate with good survival and slow-growing tumors correlate with poor survival. We hypothesize that this effect is due to the impact of tumor growth rate on chemosensitivity, and we plan to test this hypothesis in future studies.

#2460

A comprehensive computational framework for interpretation of high-throughput cancer data using annotated pathways.

Pourya Naderi Yeganeh, M. Taghi Mostafavi. _UNIV. Of North Carolina Charlotte, Charlotte, NC_.

Introduction: Identification of pathways that are associated with experimental gene expression profiles is critical for understanding the biological mechanisms in cancer studies. Current standard pathway analysis methods such as Over-representation Analysis and Gene Set Enrichment Analysis often only use the gene symbols of the annotated pathways and typically disregard the biological interactions. This approach is not sensitive to the dysregulation of central genes in the pathway networks whose perturbations may critically affect the biological mechanisms.

Methods: In this study, we have developed a framework that leverages the networks of interactions from the KEGG database to infer the underlying pathways from high-throughput gene expression profiles of cancer data. This methodology uses novel network analysis models with optimized sensitivity towards genes with key positions in pathways, particularly cancer-genes. We have previously shown that our novel network models create a distinction between the topological position of cancer genes and non-cancer related genes in pathways. In this study, we have devised a statistical pipeline to incorporate the network evidences in refining standard pathway analysis models. We have also tested our model on a battery of multiple cancer datasets and incorporated synthetic data evaluation approaches to verify the performance of our model.

Results: The results show that our network-based model is capable of detecting known and well-studied pathway associations when other methods fail to capture them. For example, our model identifies the PI3K-Akt signaling pathway from 274 samples (benign and malignant) from GSE9899 ovarian cancer dataset, when only a few number genes (19) are perturbed (adjusted p-value = 0.003). In comparison, over-representation analysis only produces an adjusted p-value of 0.15 using the same data for the same pathway. This observation outlines an instance where our model makes informative interpretations, given that the dysfunction of PI3K-Akt pathway is studied in ovarian cancer. On the same dataset, our methodology identifies the association of Ras signaling pathway with the ovarian cancer from only 7 perturbations (adjusted p-value = 0.034). In comparison, over-representation analysis produces an adjusted p-value of 1.00.

Discussion: The network-based model of this study provides a new perspective for interpretation of high-throughput cancer data by accounting for the topological position of the genes with respect to their associated pathways. This methodology allows researchers to identify pathways that are associated with the perturbations. It also enables to evaluate the importance of the perturbation with regards to the organization of the pathways. This methodology is potentially beneficial to applications in biomarker discovery and drug target development.

### Databases and Computational Tools for Cancer Discovery

#2461

PDX Finder: A free and global catalog of patient tumor derived xenograft models.

Nathalie Conte,1 Jeremy C. Mason,1 Csaba Halmagyi,1 Abayomi Mosaku,1 Steven Neuhauser,2 Dale A. Begley,2 Debra M. Krupke,2 Helen Parkinson,1 Terrence F. Meehan,1 Carol J. Bult2. 1 _EMBL-EBI, Hinxton, United Kingdom;_ 2 _The Jackson Laboratory, Bar Harbor, ME_.

Patient-derived tumor xenograft (PDX) mouse models are an important oncology platform for cancer research, drug development and personalized medicine that are available from academic labs, large research consortia and contract research organizations (CROs). Because of the distributed and heterogeneous nature of repositories, finding models of interest is a challenge. To address this issue, The Jackson Laboratory and EMBL-EBI have co-developed the PDX Finder† (www.pdxfinder.org), a comprehensive, free and global catalogue of approximately 2000 PDX models and their associated data across repositories. To support the integration of data and make the discovery of relevant PDX models easier, we coordinated a community initiative to develop the PDX Minimal Information standard (PDX-MI). PDX-MI defines the metadata necessary for describing key elements of a PDX model including clinical attributes of a patient's tumor, xenograft methods of implantation, host strain and model quality assurance methodology††. Using PDX-MI, model attributes are harmonized within PDX Finder into a cohesive ontological data model that supports integration from different resources and allows for comprehensive search and filtering options. PDX Finder also provides access to patient treatment information, genomic and drug dosing study data when available. Advanced search and filtering options allow researchers to find PDX models based on multiple attributes such as diagnosis (e.g. invasive ductal carcinoma), various tumor attributes (e.g. metastasis or primary), availability of specific datasets (e.g. mutation, dosing studies), markers (e.g. KRAS V600E) or results from drug resistance/sensitivity studies (e.g. resistance to cetuximab). From PDX Finder, direct links to originating resources are provided to allow users to contact the relevant institution for model acquisition and collaboration. PDX Finder is formally collaborating and providing critical software components to support several worldwide consortia including NCI's PDXNet and EurOPDX. Individuals and organizations that generate or distribute PDX models are encouraged to register their models with PDX Finder. We also encourage researchers to explore the website to find PDX models and provide feedback as we continue to build this rich resource. Software components developed by the PDX Finder team are freely available under an Apache 2 license and source code is available at GitHub (github.com/pdxfinder). PDX Finder is supported by NCI U24 CA204781 01, R01 CA089713 and the European Molecular Biology Laboratory.

† Conte et al, 2019. PDX Finder: A Portal for Patient-Derived Tumor Xenograft Model Discovery. NAR, in press.

†† Meehan, Conte et al, 2017. PDX-MI: Minimal Information for Patient-Derived Tumor Xenograft Models. Cancer Res. 2017 Nov.

#2462

HemeBase: a comprehensive model-to-patient genomic map for AML and DLBCL.

Kathleen A. Burke,1 Tyler Faits,2 Adriana E. Tron,1 Jay Mettetal,1 Andrew Bloecher,1 Zhongwu Lai,1 Jonathan Dry,3 Bolan Linghu1. 1 _AstraZeneca, Waltham, MA;_ 2 _Boston University, Boston, MA;_ 3 _AstraZeneca, Boston, MA_.

Acute myeloid leukemia (AML) and Diffuse Large B-cell Lymphoma (DLBCL) are challenging hematological malignancies due to complex mutational patterns within and across patients and have unmet medical needs for therapies tailored for different disease subtypes. Translating AML and DLBCL preclinical findings into meaningful clinical outcomes can be inefficient and costly, as evidenced by failure of 85% of oncology drugs entering clinical testing to gain FDA approval and costs of bringing drugs to market exceeding $2 billion dollars. There is a pressing need for disease focused comprehensive knowledgebases to provide insight into whether preclinical models represent patient disease, assist in selecting accurate models to define proof of mechanism and advance therapeutic strategies.

We provided Hemebase as the first comprehensive functional-genomic map for a one-stop-shop solution to enable real-time assessment of the relationships between thousands of patients and preclinical models within DLBCL or AML. Hemebase is an interactive, web-based tool to browse, compare, and visualize models and patients from the largest functional-genomic knowledgebase to date, including over 600 AML patients, over 1000 DLBCL patients, over 60 AML/DLBCL cell lines and sources of PDX mouse models (Crown/Champion). The multimodal data incorporated into Hemebase include somatic mutations, copy number alterations, expression values, pathway activity scores, drug screening data, and clinical features (i.e. diagnosis, previous treatment, and clinical outcomes). Importantly, several unbiased methods of clustering cross preclinical models and patients are incorporated, including a non-negative matrix factorization (NMF) that enables interactive assessment of model-to-patient similarity on molecular basis. NMF integration allowed us to recreate the methods used in the Schipp lab's recent Nature Medicine paper to determine where cell lines lie within the the relevant 5 clusters of published patient data and perform real time analysis of how the clusters change based on varying the model inputs. To enable predictive biomarker discovery, clinically relevant filters can be simultaneously applied across models to analyze drug sensitivity - this was used to reveal the specific patient subpopulations that carry mutations that conferred drug sensitivity. These methods enabled the identification of an AML patient population that closely resembles previously used PDX models in terms of genomics and drug response, allowing for broader understanding of pharmacological response between these two models and a path for future investigation. Taken together, Hemebase provides unique opportunities to reveal the full landscapes of AML and DLBCL disease biology and therapeutics from enabling a comprehensive model-to-patient genomic map for AML and DLBCL.

#2463

Prototype open-access biomarker knowledgebase for genetic tests for breast cancer.

Dara Baker,1 Natalie Fedorova Abrams,2 Amanda Bell,1 Maureen Colbert,2 Hayley Dingerdissen,1 Evan Holmes,1 Samir Gupta,3 Robel Kahsay,1 Heather Kincaid,4 David Liu,4 A. S. M. Ashique Mahmood,3 Frédéric B. Bastian,5 Marc Robinson-Rechavi,5 Elena Schwartz,2 K. Vijay-Shanker,3 Daniel Crichton,4 Raja Mazumder1. 1 _George Washington University, Washington, DC;_ 2 _National Cancer Institute, Rockville, MD;_ 3 _University of Delaware, Newark, DE;_ 4 _NASA, Pasadena, CA;_ 5 _University of Lausanne, Lausanne, Switzerland_.

Cancer biomarkers have become integral components of many clinical protocols and research studies. Nonetheless there still is an urgent need for more accurate, less invasive, and cost-effective biomarker tests to advance precision oncology. Despite considerable success in the preclinical setting, most candidate biomarker tests have yet to receive regulatory clearance or approval and gain market acceptance. To better understand the challenges facing modern biomarker research and facilitate access to biomarker-related information, this study aims to delineate the paths to approval of genetic tests for breast cancer. Public databases were searched to identify approved tests and respective biomarkers. A prototype format for unified biomarker information was developed iteratively due to the highly variable presentation of this data. A knowledgebase of FDA-approved genetic biomarkers for breast cancer was built as a pilot implementation of this format. This model is extensible to a larger database of biomarkers to be hosted in OncoMX (https://oncomx.org), which integrates cancer mutation, differential expression, literature mining, and pathway information. Fields in the prototype database were populated based on findings from the FDA website, PubMed, and other open-source databases. The resulting dataset is structured with the corresponding readme following the BioCompute Object (BCO) model. This BCO-compliant approach enables provenance capture and transparency of data processing, both of which are critical to large-scale data integration efforts. The adherence to BCO standards is expected to enhance the usability of the resulting data and to streamline the subsequent integration of multiple biomarker datasets. In the rapidly evolving field of oncologic biomarker research, this open-source biomarker knowledgebase offers clinicians and researchers a streamlined access to genetic tests information including corresponding genes, guides to evidence, clinical trial data, links to archival databases, and context for the behavior of the implicated gene in other disease and normal processes.

#2464

Gabriella Miller Kids First Data Resource Center: Harmonizing clinical and genomic data to support childhood cancer and structural birth defect research.

Allison P. Heath,1 Deanne M. Taylor,1 Yuankun Zhu,1 Pichai Raman,1 Jena Lilly,1 Phillip Storm,1 Angela J. Waanders,1 Vincent Ferretti,2 Christina Yung,3 Michele Mattioni,4 Brandi Davis-Dusenbery,4 Zachary L. Flamig,5 Robert Grossman,5 Samuel L. Volchenboum,5 Sabine Mueller,6 Javad Nazarian,7 Nicole Vasilevsky,8 Melissa A. Haendel,9 Adam Resnick1. 1 _Children's Hospital of Philadelphia, Philadelphia, PA;_ 2 _CHU Sainte-Justine, Montréal, Quebec, Canada;_ 3 _Ontario Institute for Cancer Research, Toronto, Ontario, Canada;_ 4 _Seven Bridges Genomics, Cambridge, MA;_ 5 _University of Chicago, Chicago, IL;_ 6 _University of San Francisco, San Francisco, CA;_ 7 _Children's National Medical Center, Washington, DC;_ 8 _Oregon Health & Science University, Portland, OR; _9 _Oregon State University, Corvallis, OR_.

Childhood cancers and structural birth defects share a common context of altered developmental biology, but the potential role of shared, genetic alterations and/or pathways across pediatric cancers and birth defects is not well explored. It is increasingly critical that genomic data are paired with high-quality clinical data to drive translational research by elucidating the relationship between genomic alterations, treatments, outcomes, and other phenotypic characteristics. The NIH Common Fund Gabriella Miller Kids First Program represents a first-in-kind national, collaborative initiative focused on large-scale clinical and genomic data sharing for childhood cancers and structural birth defects. As part of this program, the Kids First Data Resource Center (DRC) is charged with empowering collaborative discovery across Kids First datasets. Through newly developed cloud-based platforms, researchers will be able to rapidly and interactively access standardized and harmonized clinical and genomic data. A better understanding of common developmental programs could spur advancements in prevention, detection, and therapeutics that will improve the outcomes of affected children and families.

Approximately 8,000 patient samples were available at the launch of the Kids First DRC portal, with an initial focus on whole genome sequencing (WGS) of trios and families. More than 25,000 WGS are expected to be processed by 2019, making the DRC one of the largest pediatric data resources of its kind across a diversity of diseases. The rise of cloud-based computing has greatly reduced the burden on the researcher of large-scale genomic harmonization. In normal operations, the DRC is capable of running two hundred workflows simultaneously with considerable scalability on demand. Additionally, there is a strong focus on harmonizing and structuring seemingly disparate clinical and phenotypic data types to make them more interoperable, discoverable and reusable by using ontologies. The data in the DRC is expertly curated and mapped to existing data standards, including NCI Thesaurus (NCIt), Human Phenotype Ontology (HPO), Monarch Disease Ontology (MONDO), and Uber-anatomy Ontology (Uberon). This allows for increased interoperability and semantic structure of the data. For example, MONDO integrates numerous disease terminologies into a single merged ontology, including the NCIt. The combination of harmonized genomic and clinical data across pediatric cancers and structural birth defect provides a key foundation for exploring and developing new methods to better understand the relationships between germline variants, cancer risk, and associated treatments and outcomes. Community standardization of this modeling is ongoing as part of GA4GH, and is critical for implementation of improved interpretation in EHR systems, for example via HL7 FHIR.

#2465

Genomic harmonization of the Data Resource Center for Gabriella Miller Kids First Pediatric Research Program.

Yuankun Zhu,1 Miguel Brown,1 Batsal Devkota,1 Bailey Farrow,1 Bogdan Gavrilovic,2 Allison Heath,1 Kyle Hernandez,3 Avi Kelman,1 Parimala Killada,1 Meen Chul Kim,1 Daniel Kolbman,1 Mateusz Koptyra,1 Milan Kovacevic,2 Maarten Leerkes,4 Alex Lubneuski,1 Michele Mattioni,2 Pichai Raman,1 Adam Resnick,1 Nikola Skundric,2 Deanne Taylor,1 Junjun Zhang,5 Bo Zhang,1 Phillip B. Storm1. 1 _The Children's Hospital Of Philadelphia, PA;_ 2 _Seven Bridges Genomics, PA;_ 3 _University of Chicago, Chicago, IL;_ 4 _National Institutes of Health, Bethesda, MD;_ 5 _Ontario Institute for Cancer Research, Ontario, QC_.

Gabriella Miller Kids First Pediatric Research Program (GMKF) is a nation-wide, multi-year initiative focused on the integration of large-scale clinically annotated genomic data for childhood cancers and structural birth defects supported by the NIH Common Fund. Awarded by GMKF, the Kids First Data Resource Center (DRC) is tasked to build infrastructure and workflows for data intaking, harmonization, integration and access authorization to empower collaborative discoveries across the GMKF and other integrated datasets. A key challenge for uniform analyses and empowered discovery of large-scale genomic data relates to the diverse genomic processing workflows and methods employed across the sequencing and bioinformatics community. The DRC genomic harmonization team aims to provide "analysis ready" datasets that are "functionally equivalent" across the Kids First datasets and other large-scale genomic data initiatives in order to accelerate the discovery process. Paired with the cloud-based workspace environments of the DRC, such harmonized dataset provide unprecedented opportunities for shared, reproducible discovery by a diverse, collaborative network of researchers. As such, DRC initial pipelines are developed with BWA-MEM alignment on genome build GRCh38 followed by the GATK best practices for germline variant calling and joint genotyping. Common Workflow Language (CWL) is used as the main workflow specification, while Docker technology has been applied to containerize all the tools used by the workflow. Our current workflows are tasked with data harmonization across a number of different experimental platforms including whole genome sequencing, exome sequencing, and RNA-seq. The data processing is done via CAVATICA, an Amazon Web Services (AWS) based cloud computing platform associated with the Kids First DRC Portal co-developed by Seven Bridges Genomics, where workflows feature scatter-gather parallelization and AWS resource optimization. By utilizing such a framework, the DRC team has harmonized over 10,000 WGS and 1,000 RNA-Seq samples across 12 study cohorts within 8 months. This dataset in its current release includes samples from 40 pediatric brain cancers as well as 8 childhood birth defects with the outcome of delivering 150TB harmonized CRAM and 60TB gVCF. With a highly optimized bioinformatics pipeline powered by an efficient cloud-based execution workflow, The DRC platform processes one genome in about 11 hours with an average compute cost of $15 for whole genome alignment and germline variant calling. Here we present our observed challenges and identified opportunities in the analysis and integration of multi-disease pediatric genomic data on a large scale.

#2466

A survey of molecular factors of DNA methylation and demethylation in a broad range of cancer cell lines.

Suleyman Vural, Julia Krushkal. _NCI/NIH, Rockville, MD_.

DNA methylation has a powerful influence on cellular processes. It plays important roles in cancer initiation and progression. Cancer cells exhibit frequent epigenomic alterations that affect expression of genes involved in apoptosis, cell proliferation, and DNA repair. We used expression information of known methylating and demethylating genes in cancer cell lines to analyze their association with methylation levels of individual probes and median epigenome-wide methylation levels. We utilized RNA-seq gene expression measures and Illumina Infinium HumanMethylation450 BeadChip DNA methylation data of 645 cancer cell lines from 23 cancer types from the Cancer Cell Line Encyclopedia and Genomics of Drug Sensitivity in Cancer resources. We analyzed 73 genes, products of which are involved in methylation and demethylation processes, and examined correlations between expression levels of these genes and methylation status of individual probes and epigenome-wide. In a combined analysis across all cancer types, we observed statistically significant correlations between expression of global DNA methyltransferase genes, DNMT1, DNMT3A, and DNMT3B, and probe methylation levels in 566, 909, and 230 genes, respectively. In particular, DNMT3A expression was associated with methylation status of growth factor and receptor genes FGF1, FGFR4, and EGFR. Expression of hydroxymethylating genes TET1, TET2, and TET3 was correlated with methylation status of probes in 363, 1, and 1055 genes, respectively, including associations of TET3 expression with methylation of probes in the FGF1, KIT, FHIT, IGF1R, IGF2BP2, and PARP3 genes, which may affect growth, proliferation, and DNA repair of tumor cells. APOBEC1, CBX2, UHRF1 and ZBTB38, products of which are involved in various molecular steps of DNA methylation or demethylation, had the largest numbers of associations in different cancer types. For example, in chronic lymphocytic leukemia cell lines we observed a strong negative correlation between ZBTB38 expression, the product of which binds methylated DNA, and methylation levels of probes in 204 genes. We also found a strong correlation between UHRF1 gene expression and methylation levels of probes in 150 genes in chronic lymphocytic leukemia cell lines, in agreement with reported roles of the product of this gene in binding DNA methyltransferases and affecting DNMT1 methylation activity. These results provide a better understanding of molecular mechanisms of epigenetic dysregulation in cancer cells and may provide strategies for future analysis of molecular targets of drug response that may be affected via epigenetic processes.

#2467

Heme-CMap: Generation and characterization of ~20K L1000 profiles across 11 hematologic malignant lines.

Chia-Ling Huang,1 Andrew Yang,2 Ted Natoli,2 Lev Litichevskiy,2 Frederic Vaillancourt,1 Alan Rolfe,1 Yonghong Xiao,1 Aravind Subramanian,2 Lihua Yu1. 1 _H3 Biomedicine, Cambridge, MA;_ 2 _Broad Institute, Cambridge, MA_.

The Next Generation Connectivity Map (CMap) provides a resource to explore the relationships between diseases, genetic perturbations and compound treatments at the transcriptome level. Current CMap focuses on 9 tumor cell lines of various cancer types, but none of hematological malignancies. Considering the distinctive features of hematologic malignancies at both transcriptome and genomics alteration level, we recognized the pressing need for a similar resource of hematologic malignancies to better understand their unique characteristics of compound treatment response. Here we generated the "Heme-CMap" which included 7 lymphoid and 4 myeloid lines. In order to better understand the compound effects on hematological malignant lines, especially lineage specificity of lymphoid versus myeloid lines, we selected 100 compounds targeting B-cell lymphoma and 38 compounds targeting cancer pathways and hallmarks in our reference compound list. Each compound was tested at 4 time points and 6 doses. To this end, the Heme-CMap encompasses 20,472 compound perturbations. The size of this dataset and compound collection allowed us not only to globally compare the classic CMap to the newly generated Heme-CMap, but also to explore cell lineage effects of lymphoid versus myeloid cell lines within Heme-CMap.

We found that several perturbation classes showed differential activity between classic CMap and Heme-CMap while some pathways show coherent activity. For example, EGFR inhibitors showed coherent activity in both classic CMap and Heme-CMap. On the other hand, BTK inhibitors showed coherent effects only in a subset of the lymphoid lines, but not the myeloid lines, consistent with the function in altering B cell signaling. We further investigated the compounds in Heme-CMap and observed samples clustered into two major groups by mechanism of action (MoA) of the compounds. One group contained samples treated with compounds targeting growth and survival, and B-cell signaling pathways, while the other group contained samples treated with compounds causing cell killing. Interestingly, we found both groups in the lymphoid lines but only the cell killing group in the myeloid lines. This may reflect our compound selection targeting signaling pathways implicated B cell lymphoma. These results suggest cell lineage plays an important role in compound response, and the experimental design of Heme-CMap allowed us to observe the differential compound effect in lymphoid and myeloid lines. We also found different types of compounds showed maximum effects at different timepoints and doses, suggesting various timepoints and doses can provide insights of compound MoA.

In summary, our results demonstrate that Heme-CMap with lineage specific cell lines can elucidate compound MoAs complementary to classic CMap. Additionally, this effort also serves as a roadmap for generating data collaboratively with CMap on a large scale.

#2468

Towards precise and cost-effective fusion discovery: A landscape of druggable gene fusions across TCGA cancers.

Roozbeh Dehghannasiri,1 Milos Jordanski,1 Donald E. Freeman,1 Gillian L. Hsieh,1 Jonathan M. Howard,1 Erik Lehnert,2 Julia Salzman1. 1 _Stanford University, Stanford, CA;_ 2 _Seven Bridges Genomics, Cambridge, MA_.

Gene fusions are one of the hallmarks of cancer and are among the most powerful biomarkers and drug targets in translational cancer genomics. We deploy sMACHETE (scalable MACHETE), a precise and sensitive fusion detection algorithm, particularly engineered for mining massive cancer sequencing databases, to provide a landscape of fusions across human primary cancers. sMACHETE consists of two main computational components: MACHETE-based component and Sequence Bloom Tree (SBT) checkpoint. MACHETE (Hsieh et al., 2017) is a precise fusion algorithm, which employs a statistical model to identify fusion junctions. The first component in sMACHETE is built on MACHETE and has undergone major algorithmic and computational improvements, such as the inclusion of well-known cancer fusions and a cloud-based implementation in Common Workflow Language, which makes the pipeline a good fit for large-scale studies. To control for false positives due to multiple testing in large datasets, the fusions called by the first component are then queried via SBT (Solomon and Kingsford, 2016), which is a kmer-based query algorithm. The fusions whose detection frequencies by MACHETE and SBT are statistically consistent could pass the checkpoint and are called by sMACHETE. sMACHETE achieved 100% positive predictive value, higher than any other top performing algorithm and comparable sensitivity on simulated benchmarking datasets. We have used sMACHETE to systematically analyze fusions in The Cancer Genome Atlas (TCGA) RNA-seq data datasets. sMACHETE calls 31,546 highly confident fusions in 9,946 TCGA tumor samples spanning 33 cancer types. Sarcoma (10 fusions per sample) and Esophageal Carcinoma (8 fusions per sample) have the highest abundance of fusions. We found 525 recurrent fusions, observed in at least 2 samples within a cancer type, in 12% of tumor samples. Our statistical analysis reveals a signature of selection for recurrent fusions and also for recurrent genes, which partner with more than one gene in fusions and are observed in 40% of samples, suggesting an evidence for their oncogenic role in tumorigenesis. Thyroid, Ovarian, Esophageal, and Lung Adenocarcinoma have rates of kinase fusions that exceed expectation by chance, strong evidence that they are unappreciated drivers of the disease. Having integrated our detected fusions with OncoKB database (Chakravarty et al., 2017), we detected druggable fusions in 3% of tumor samples. Our systematic and functional analysis highlights the substantial role of fusions as cancer drivers and their clinical implication in cancer treatment.

#2469

The COSMIC Cancer Gene Census - a comprehensive study of all mutated cancer-driving genes.

Zbyslaw Sondka, Helen E. Speedy, Sally Bamford, Charlotte G. Cole, Sari A. Ward, Simon A. Forbes. _Wellcome Sanger Institute, Cambridge, United Kingdom_.

The COSMIC Cancer Gene Census (https://cancer.sanger.ac.uk/census) is a catalogue of genes that drive all forms of human cancer. Since 2004, a consistent curation approach to the scientific literature has grown this resource into a comprehensive description of 723 genes, detailing how each gene contributes to disease causation The entire Cancer Gene Census has been fully re-evaluated, and each gene has been classified as Oncogene, Tumour Suppressor and/or Fusion gene - depending on their somatic mutation profile and functional role in carcinogenesis. Genes included in the CGC are characterised by the presence of somatic or germline mutations, which change the activity or expression of the protein product in a way that promotes one or multiple hallmarks of cancer. The minimum level of evidence required for CGC inclusion are at least 2 publications from independent research groups showing increased mutation frequency in at least one type of cancer; and 2 or more published experimental evidence of functional involvement of a gene in promoting the hallmarks of cancer. The functional description of how each mutated gene causes cancer is now underway, with approximately half the Census now described in both functional and mechanistic terms. These characteristics clearly show that many genes can participate in oncogenesis in multiple ways that are highly dependent on the type of genetic alteration and tissue in which the gene is expressed, as well as disease stage. Alongside this evidence-based characterisation of genes we know drive cancer, a "second tier" of genes is now encompassed in the Census, to describe genes implicated in oncology, but with less robust published evidence. These genes, often revealed by combining whole-tumour-genome sequencing studies are increasing rapidly in number and often underpin exciting new targets in oncology. Inclusion of these in the Census completes its ability to describe the contribution of inherited and acquired genetics to human oncology. With substantial research ongoing, the Census is updated with additional content every three months, included with every new release of the COSMIC database (https://cancer.sanger.ac.uk/).

#2470

A molecular oncology almanac for integrative clinical interpretation of molecular profiles to guide precision cancer medicine.

Brendan Reardon,1 Nicholas Moore,2 Nathanael Moore,3 Eric Kofman,1 Eliezer Van Allen1. 1 _Dana-Farber Cancer Inst., Boston, MA;_ 2 _Harvard Medical School, Boston, MA;_ 3 _Indianapolis University School of Medicine, Indianapolis, MA_.

Background: Tumor molecular profiling is increasingly used to detect first-order genomic alterations associated with therapeutic actions (e.g. BRAF V600E & RAF/MEK inhibition). Simultaneously, more complex molecular features are being discovered and applied to clinical scenarios (e.g. mutational signatures, somatic-germline interactions). As patients receive expanded profiling, such as clinical whole-exome and RNA sequencing, novel algorithms are needed to integrate interpretation of multiple data modalities. Furthermore, the clinical-preclinical gap continues to widen as data from high-throughput screens of cancer cell lines are generated without accessibility at the point of care. Here, we introduce a paired interpretation algorithm and knowledge system for cancer genomic data, the Molecular Oncology Almanac, to inform treatment decisions through rapid assessment of tumor actionability.

Methods: We implemented a cloud-based interpretation algorithm that annotates and evaluates variants from WES and RNA-seq (SNVs from WES and RNA-seq, InDels, CNAs, and fusions) and infers additional features such as mutational burden, mutational signatures, MSI, somatic-germline interactions, and aneuploidy. Predictive implication levels were assigned to reflect confidence in the database's catalogued relationships to therapeutic response and prognosis for each molecular feature. We also developed a patient-preclinical matchmaker function to expand the theoretical therapeutic modalities for any given patient. Towards timeliness of updates and knowledge system accessibility, we developed API endpoints, a browser extension for suggesting citations, and workflows in the FireCloud framework.

Results: A total of 260 patients with metastatic castration-resistant prostate cancer (n=150) and metastatic melanoma (n=110) were evaluated with 569 alteration-action relationships catalogued in the Molecular Oncology Almanac. Overall 80% of patients had at least one alteration suggesting therapeutic sensitivity based on FDA approval, clinical trials, or studies in humans; which increased to 95.8% by also considering preclinical and inferential associations. Per patient, the matchmaker function on average highlighted 1.56 additional therapies that would not have otherwise been nominated. At least one feature associated with resistance or prognosis was observed in 85% and 90% of patients, respectively.

Conclusion: Clinical actionability of sequence data was increased by including integrative molecular profiling of DNA and RNA, global molecular features, and preclinical alteration-action relationships. Increased accessibility of clinical interpretation through our cloud-based web portals and API endpoints may aid in sample contextualization.

Source code and a web portal for this project are available at moalmanac.org.

#2471

Pan-cancer catalog of Differentially Methylated Regions by Rocker-meth, a new computational method.

Matteo Benelli,1 Alberto Magi,2 Dario Romagnoli,1 Gian Marco Franceschini,3 Chiara Biagioni,1 Ilenia Migliaccio,1 Luca Malorni,1 Angelo Di Leo,1 Francesca Demichelis3. 1 _Azienda USL Toscana Centro, Hospital of Prato, Prato, Italy;_ 2 _University of Florence, Florence, Italy;_ 3 _University of Trento, Trento, Italy_.

DNA methylation (DNAm) is an essential player of gene regulation and therefore one of the most studied epigenetic mechanisms. Alterations of DNAm have been associated with a wide range of diseases, including cancer.

Most studies on DNAm focused on the characterization of methylation sites, despite the recognition that DNAm changes spanning entire genomic regions are common in cancer tissues with respect to benign cells, referred to as Differentially Methylated Regions (DMRs).

Here we report on a new computational method to identify DMRs both from array and Bisulfite Sequencing data. Rocker-meth consists of four main modules: 1) computation of Area Under the Curve (AUC) values from Receiver operating characteristic (ROC) Curve analysis of methylation levels (i.e., beta values) in tumor versus normal samples; 2) segmentation of AUC values by a tailored Heterogeneous Shifting Level Model (HSLM); 3) estimation of the statistical significance of AUC segments by Wilcoxon-Mann-Whitney test on beta values of tumor versus normal samples; 4) identification of sample specific DMRs by Z-score statistics. Tests on synthetic DNAm profiles demonstrated that Rocker-meth outperforms current state-of-the-art tools. Specifically, it showed markedly higher precision and recall in the presence of the lowest signal-to-noise ratios that mimic more than 40% of real human samples data. By exploiting well known features of DNAm in human cancers, we also demonstrated that Rocker-meth outperforms the most used array-based DMR detection tools in real data.

We then drafted the first pan-cancer catalog of DMRs based on the characterization of more than 6,000 human samples DNAm data of 14 tumor types from TCGA and other recent studies and studied the characteristics of hyper and hypo DMRs in terms of structure, genomic locations and functional role. Pan-cancer integrative analysis of DMRs and gene expression recapitulated well-known associations, validating the accuracy of our method. Finally, we demonstrated the reliability of Rocker-meth to perform sample-wise analysis. This was possible thanks to the intrinsic characteristic of DMRs to be extremely recurrent within a given tumor type, supporting our previous observation for methylation sites and in contrast to genomic alterations such as Single Nucleotide Variants (SNVs) and Somatic Copy Number Variants (SCNAs). Strikingly, DMRs detected by Rocker-meth were informative in segregating prostate and breast adenocarcinoma based on their molecular subtypes, confirming the results from DNAm site analyses. The pan-cancer catalog of DMRs with sample level characterization could be used to investigate across cancer mechanisms of tumorigenesis or to query synthetic lethal events.

#2472

LOCUS: Queryable database of cancer genomics and pharmacologic response enables rapid selection of in vitro and in vivo preclinical tumor models.

Armand Amin, Sandeep Sanga, Shanshan Gan, Yong Hu, Thomas Broudy. _BioDuro LLC, San Diego, CA_.

Preclincal drug testing in oncology research has benefited from the development of thousands of tumors models—both in vitro and in vivo. Within BioDuro laboratories alone, there is a collection of >500 models, including traditional 2D human cell lines grown in vitro, primary 3D lines, cell-derived xenograft (CDX), patient derived xenograft (PDX) and syngeneic murine models (SYN). Identifying the most relevant models to support cancer drug discovery is a challenge. In an era of precision medicine, the need to understand tumor model characteristics, such as functional drug response profiles or genomic status, has become a critical path.

In this report, we present LOCUS, a SQL backed database with graphical user interface, populated with functional pharmacologic response and genomic data for hundreds of tumor models including in vitro 2D, 3D, CDX, PDX and SYN. Models are readily searched by any number of conditions, including cancer type, model type, specific mutation, or target expression level.

LOCUS combines relational databases for pharmacology and genomics data with scientific statistical models through a spring integrator. A scientific data centered domain architecture allows for pharmacogenomic queries and analysis. Pharmacogenomic data is pushed to dynamic scientific statistical models running on a python environment, the results of which are visualized to a Java EE UI Framework.

All tumor models are genomically characterized, with searchable fields for single nucleotide variations, insertion deletions, and transcript expression levels. Extracted RNA was profiled using next generation sequencing performed on Illumina HiSeq using a paired end protocol.

For xenograft samples, reads were first aligned and filtered by the mouse reference genome using the Bowtie2 algorithm. The remaining reads were aligned to the GRCh38 human reference genome using the MapSplice2 algorithm.

Transcript- and Gene-level expression were determined by counting the number of sequence alignments per exon according to the RefSeq gene model, and computing Fragments Per Killobase Million (FPKM) and Transcripts Per Million (TPM) values using best practices. Opossum software was used to further prepare alignments produced by MapSplice2 algorithm for small variant calling. The Platypus variant caller was used to identify single nucleotide variant, small insertion, and small deletion events. The variation events were annotated using SnpEff and custom perl scripts to overlay known somatic mutations with oncogenic significance.

LOCUS provides users with rapid access to hundreds of pharmacology and genomic data sets that enable rational selection of tumor models for preclinical oncology drug testing. The database and structure are readily scaled, supporting future growth of thousands of more tumor models and characterization.

#2473

Scaling computational genomics to millions of individuals with GPUs.

Amaro N. Taylor-Weiner, François Aguet, Nicholas Haradhvala, Sager Gosai, Jaegil Kim, Kristin Ardlie, Eliezer M. Van Allen, Gad Getz. _Broad Institute, Boston, MA_.

Background: Current genomics methods and pipelines were designed to handle tens to thousands of samples, but will soon need to scale to millions to keep up with the pace of data and hypothesis generation in biomedical science. The computational costs associated with processing these growing datasets will become prohibitive without improving the computational efficiency and scalability of methods. For example, methods in population genetics, such as genome-wide association studies (GWAS) or mapping of quantitative trait loci (QTL), involve billions of regressions between genotypes and phenotypes. Currently, the state-of-the-art infrastructure for performing these tasks are large-scale clusters of central processing units (CPUs), often with thousands of cores that result in significant cost (960 cores on a standard Google Cloud machine costs $7,660.80 per day of compute). In contrast to CPUs, which feature relatively few cores (i.e. Intel's i9 has 6 cores), a single graphical processing unit (GPU) contains thousands of cores (i.e. Nvidia's P100 has 3,584 cores). Here, we show that implementation of genomics methods using recently developed machine-learning libraries for GPUs will significantly accelerate computations and enable scaling to hundreds of thousands of samples.

Results: To demonstrate this and benchmark the use of machine-learning libraries for large-scale genomic analyses, we re-implemented methods for two commonly performed computational genomics tasks: (i) QTL mapping (tensorQTL) and Bayesian non-negative matrix factorization (SignatureAnalyzer-GPU). To benchmark tensorQTL, we generated random data representing up to 50,000 people (with 107 variants) resulting in 500x109 all-against-all association tests. Our implementation enabled cis-QTL mapping >250x times faster than the current state-of-the-art implementation (FastQTL). Likewise, trans-QTL mapping (i.e., 500 billion regressions) took less than 10 minutes, a ~200x increase in speed compared to running without a GPU.

To benchmark SignatureAnalyzer-GPU (SA-GPU), we used the mutation counts matrix generated by the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which contains 2,624 tumors represented by 1697 mutational features of somatic single nucleotide variants and short insertions and deletions (defined based on their sequence contexts). Our GPU implementation ran approximately 200 times faster than the current implementation of SignatureAnalyzer (SA) in R with mean times for 10,000 iterations of 194.8 min using SA vs. 1.09 min using SA-GPU.

Conclusion: We anticipate that the accessibility of these libraries (e.g., TensorFlow, PyTorch), and the improvements in run-time will lead to a transition to GPU-based implementations for a wide range of computational genomics methods.

#2474

Automated somatic variant classifier to reduce false positives identified by tumor normal variant callers.

Alena S. Harley, Corine K. Lau, Eve Shinbrot. _Human Longevity, Inc., San Diego, CA_.

Accurate identification of somatic mutations is key to targeted cancer therapies. However, due to the complexity of next-generation sequencing, there are many variables that give rise to sequencing and alignment artifacts. Most sequencing workflows using tumor normal paired samples rely on the intersection or union of mutations by more than one somatic variant caller such as Mutect2, Strelka, VarScan2, Muse, and SomaticSniper. Yet, they still contain a number of false positive and false negative calls. Therefore, there is a need for a manual review of variants using Integrative Genomics Viewer (IGV) to incorporate information that is difficult to take into account in automated workflows.

To alleviate the burden of manual inspection, we present an automated classification method to pre classify detected somatic variants into true or false classes for further manual review.

Methods: We created a false positive classifier that utilizes automated IGV visualization of somatic variants. Our somatic workflow involves tumor normal DNA sequencing, BWAMEM alignment, deduplication of reads via Picard, somatic calling using two somatic callers, followed by the union of the detected variants, SnpEff annotation, and extracting nonsilent variants with variant allele fractions greater than 5 percent. We used the data for 1,413 nonsilent variants from a set of 10 tumor normal pairs of various cancer types. The variants were scored by cancer analysts using IGV. Initial false positive rate was 28 percent. We fine tuned Residual Deep Neural Network which utilizes weights from the network with the same architecture trained on ImageNet. Discriminative fine tuning was performed in two stages: first unfreezing the last fully connected layer only, and then unfreezing all the layers and training with differential learning rates.

Results and Conclusion: Our method achieves 93 percent overall accuracy. The classifier greatly reduces the false positive rate for the set of variant calls from 28 percent down to 4 percent at the expense of calling 3 percent of true variants false. Previously, deep neural networks have been shown to yield the state of the art performance in multiple domains including image processing, speech, and text processing. In this work, we describe a way to fine tune Residual Neural Network trained on a large image data set to perform false positive variant detection in tumor-normal variant alignment snapshots from IGV. Our general approach that significantly reduces false positive rate of putative variants can be extended to any subset of somatic callers.

#2475

**FLT3-Explorer: A bioinformatics pipeline for detecting low signal in** FLT3 **internal tandem duplications in acute myeloid leukemia.**

Christian Laing, Wenge Shi, Reinhold Pollner. _Navigate Biopharma, Carlsbad, CA_.

Introduction. Acute Myeloid Leukemia (AML) is the most common acute Leukemia among adults with a poor overall prognosis. With over 20,000 cases in the US alone, about a third of AML patients pertain to an important oncogenic driver composed of internal tandem duplication (ITD) somatic mutations in exons 14 and 15 of the FMS-like tyrosine kinase 3 (FLT3) gene. Detecting the presence of measurable residual disease in patients with AML who are in morphologic remission has been shown to be a powerful predictor of eventual relapse. Yet, despite its importance, the ability to detect FLT3-ITD mutations is hampered by the limited sensitivity of conventional PCR-based assays in general and in Next Generation Sequencing-based assays, in part due to the difficulty to align ITD-containing reads to the reference genome.

Methods. In order to facilitate the analysis of AML samples, we developed FLT3-Explorer, a comprehensive bioinformatics pipeline composed of open source software, combined with in-house algorithms to accurately detect low levels of FLT3-ITD mutations. The input of the pipeline is unmapped reads in fastq format. The pipeline then searches for either ITD sequences previously observed in our extensive database of historical data, or a de-novo detection procedure is used to determine mutation sequences of variable lengths within the FLT3 gene. To assess the performance of FLT3-Explorer, we considered a cohort of clinical samples from bone marrow and peripheral blood, as well as cell lines. For sample collection, genomic DNA was isolated, and polymerase chain reaction was performed using primers flanking exons 13 and 15 to amplify the FLT3 gene. DNA was then sequenced using NGS Illumina sequencing. Results were validated using fragment size analysis by Capillary Electrophoresis assays. Also, to determine the limits of detection, various dilutions were performed on cell lines. Furthermore, to assess the performance of our pipeline, we performed a comparison with other FLT3-ITD programs publicly available.

Summary. Results from our analysis showed that our method identifies FLT3-ITD mutations of variant allele fraction as low as 0.003%. Also, we found that our pipeline can detect as many FLT3-ITD mutants as other methods, or more, when the mutant signal is considerably low.

Conclusions. FLT3-Explorer constitutes a powerful solution for low FLT3-ITD detection signal of mutant sequences, of diverse lengths, that can be used on FLT3-ITD AML patients to track disease progression and relapse.

#2476

RECUR: Algorithm for directional allelic imbalance profiling and visualization from multi-sample data.

Yasminka A. Jakubek, F. Anthony San Lucas, Paul Scheet. _UT MD Anderson Cancer Ctr., Houston, TX_.

Somatic chromosomal alterations play an important role in the development and progression of cancer. Allelic imbalance (AI) resulting from such changes (gain, loss, or copy-neutral loss-of-heterozygosity; cnLOH) is defined as a deviation from the 1:1 ratio of inherited parental haplotypes. Observed "B allele" frequencies (BAFs) at germline heterozygous loci, from SNP array or next-generation sequencing data, are used to detect AI. In addition, log R ratio or read-depth data, can be analyzed alone or jointly with BAFs to detect gains and losses. When AI is detected in multiple intra-individual samples, spanning similar loci, it is natural to assess whether these signals reflect the same underlying mutation. One way to do this would be to note if the observed AI differs in mutation type (gain, loss), an approach that falls short for events of the same type and subtle AI events that cannot be classified as gains, losses, or cnLOH. In such a case, or with two mutations of the same type, samples may differ in their maternal/paternal haplotype balance and consequently their alleles will shift in opposite directions. This phenomenon is indicative of a recurrent or independent mutation, or an error in chromosome segregation that generates two "mirrored" clones, one with a gain and the other with a loss. Recent studies have used such a directional AI analysis to yield important insights into cancer initiation and progression (Jakubek et al. Cancer Research 2016, Jamal-Hanjani et al. New England Journal of Medicine 2017, Turajlic et al. Cell 2018). We developed REpeat Chromosomal changes Uncovered by Reflection (RECUR) to explicitly test directionality of AI from multiple samples with overlapping AI segments through the comparative analysis of AI profiles derived from SNP array and next-generation sequencing data. The algorithm accepts genotype calls and BAFs from at least 2 samples derived from the same individual. For a predefined set of genomic regions with AI, RECUR compares BAF values among samples. In the presence of AI, the expected value of a BAF can shift in two possible directions, reflecting an increased or decreased abundance of the maternal haplotype, relative to the paternal. RECUR detects such genomic segments of opposite haplotypes in imbalance and plots BAF values for all samples, using a two-color scheme for intuitive visualization. It can help identify genomic regions under selective pressure for example recurrent deletions/gains in the same genomic loci and/or regions with generalized genomic instability. Integration of directional AI, copy number, and somatic mutation data can help build more accurate phylogenetic trees and further illuminate the timing and distribution of somatic chromosomal aberrations to offer insights into cancer initiation and progression.

RECUR is available at scheet.org

#2477

CopyCat: estimating genomic copy number profiles from high-throughput single cell RNA seq data.

Ruli Gao, Nicholas Navin. _UT MD Anderson Cancer Ctr., Houston, TX_.

CopyCat is a computational approach for estimate genomic-copy number profiles from high-throughput single cell RNAseq data that is generated by technologies, such as Drop-Seq, 10X genomics or Nanowells. CopyCat involves first normalizing and transforming unique molecular index (UMI) count data matrices to calculate single cell copy number states from low-coverage datasets using gene-bin windows across the human genome, and does not require a reference normal cell control. The resulting binned data is normalized and used for segmentation to reduce noise from individual genes that can contribute to gene dosage effects along chromosomes. We show that this methods is highly effective at distinguishing diploid and aneuploid cells in human tumors or cancer cell lines, and can efficiently detect major (>10mb) chromosomal amplifications and deletions compared to copy number data generated by DNA sequencing of match samples. We further show that this method can resolve some clonal substructure within individual tumors, when subpopulations are distinguished by large chromosomal events. Our method outputs segmented copy number profiles and predicted classifications of normal (diploid) and tumor (aneuploid) cell states for individual single cells. It is implemented in R and is available on Github.

#2478

Cancer genome interpretation with CIViCpy.

Alex H. Wagner, Malachi Griffith, Obi L. Griffith. _Washington University in Saint Louis, Saint Louis, MO_.

The promise of precision medicine--in which a cancer patient's treatment is tailored to their specific cancer variants--requires concise, standardized, and searchable clinical interpretations. We recently developed the Clinical Interpretations for Variants in Cancer (CIViC; civicdb.org) crowd-sourced, open-access knowledgebase, providing a structured framework for evaluating genomic variants of various types (e.g., fusions, SNVs) for their therapeutic, prognostic, predisposing or diagnostic utility. CIViC is fully open-source, and has a documented API for accessing records of interpretation assertions, evidence, variants, and genes. However, this API was built around the framework for which the CIViC web tool was designed, with a focus on search and display of the interpretation of individual genomic variants in cancer. Third-party tools to analyze or access the contents of this database programmatically must therefore construct methods to leverage this API, often reimplementing similar functionality in the pursuit of common analysis tasks that are not within the scope of the CIViC web interface.

To address this shortfall, we have developed CIViCpy (civicpy.org), an analysis toolkit and Software Developer Kit (SDK) for extracting and exploring the content of the CIViC knowledgebase. CIViCpy lets you pull data from CIViC using the civic module and interact with records as dynamic objects in Python. With the aid of automatic caching, CIViCpy provides fully-documented data objects for CIViC records (e.g. assertions, genes, variants) without worrying about querying CIViC more than once for the same data. Thus, repeated requests for a given CIViC record in the same session will load quickly, enabling rapid identification of records matching desired characteristics. All CIViCpy objects are directly comparable for ease of data analysis. CIViCpy also performs sensible pre-caching, e.g. requests for variants pre-load related gene and evidence records. Like CIViC, CIViCpy is fully documented, open-source, and permissively (MIT) licensed. CIViCpy is also easily and freely installable from the python package index (PyPI).

In this study, we illustrate how the CIViCpy SDK is used to enable the analysis and export of CIViC entries. We highlight the use of the CIViCpy SDK to extract variants from CIViC and export them into the VCFv4.2 format through the built-in export tools, enabling their use in annotation pipelines or software that expects this common variant format. We also assess the viability of CIViCpy as a tool for advancing individualized patient care by examining how the CIViCpy interface can be used to systematically generate reports for the interpretation of observed variants in patient cancers. We evaluate this through the analysis of the 48,447 sequenced tumors of the AACR Project GENIE dataset, revealing clinical actionability at the patient level and addressing ongoing challenges in the programmatic analysis of genomic variants of cancers.

#2479

Designing an intuitive visualization of BRCAness scores for clinicians.

Hannah R. Allegakoen,1 Mary Goldman,2 Bennett Caughey,1 Martin Consunji,1 Christopher C. Benz,3 David Haussler,2 Jingchun Zhu,2 Eric A. Collisson1. 1 _University of California, San Francisco, San Francisco, CA;_ 2 _University of California, Santa Cruz, Santa Cruz, CA;_ 3 _Buck Institute for Research on Aging, Novato, CA_.

Objective: Genomic interrogation of tumors has changed the way we care for cancer patients. Monogenic, single mutation tests clearly and accurately define patient populations whose tumors will or will not likely respond to specific therapies. Next generation sequencing has enabled diagnostics derived from broader footprints, such as signatures of mutational processes. Currently, no universal guidelines exist on translating complex predictive signatures into the clinic, and their interpretability is a barrier to uptake by clinicians. Visually displaying genomic signatures may help clinicians interpret them. We designed a user-directed approach to developing understandable displays of an individual's BRCAness signature through testing at clinical cancer conferences.

Materials and Methods: We adopted a two-stage approach to optimizing user-testing methods. First, 44 attendees at the 2018 Gastrointestinal Cancer Symposium participated in a tablet-based test of a visualization of a patient's BRCAness signature. We refined our testing methods and continued tabled-based testing of 51 attendees at the 2018 American Society of Clinical Oncology. Participants viewed slides explaining the signature and visualization. They were asked to interpret a patient's BRCAness signature when provided information both as text and as a visualization. Participants received one of three similar versions of the visualization.

Results: Participants interpreted patients' BRCAness signatures faster when given a visual. When shown a visual indicating BRCAness scores of germline BRCA mutated tumors, participants interpreted the information fastest without sacrificing accuracy or confidence in their answer. Participants' interpretation of an individual's BRCAness signature was associated with their self-reported likelihood to recruit the individual to a clinical trial.

Conclusion: Visualizing distributions of BRCAness signatures with germline BRCA mutations displayed helped participants respond faster without sacrificing accuracy or confidence in their answer. Testing attendees at cancer conferences presents a potential platform to include clinicians and researchers in the development of genomics visualization tools.

#2480

MutSpliceDB: A database of splice sites mutations effects.

Alida Palmisano, Suleyman Vural, Yingdong Zhao, Dmitriy Sonkin. _NCI/NIH, Rockville, MD_.

Splice site mutations are one of the well-known classes of genetic alterations playing an important role in biology. Splice site mutations in cancer are most frequently observed as inactivating alterations in tumor suppressor genes (for example, TP53 or RB1) and to a lesser degree as activating alterations in oncogenes (for example MET). Splice site mutations may lead to alterations in mRNA transcripts, causing for example exon(s) inclusion/exclusion or, intron retention. Interpreting the consequences of a specific splice site mutation is not straightforward, especially if the mutation is located outside of the canonical splice sites. Accurate interpretation of the impact a splice site mutation has can further our understanding of biology, influence patient treatment, and in case of germline splice site mutations, may also have relevance to familial disease predisposition. To facilitate the interpretation of splice site mutation effects, we developed MutSpliceDB: https://brb.nci.nih.gov/splicing a public resource of splice sites mutations effects, documenting mutation effect(s) on splicing based on manually reviewed RNA-seq BAM files from sample(s) with particular splice site mutations.

#2481

Developing analysis platform for pan-cancer study of DNA methylation, mirna and lncrna expression based on tumor subtypes using TCGA data.

Darshan Shimoga Chandrashekar,1 Chad J. Creighton,2 Israel Ponce-Rodriguez,1 Sooryanarayana Varambally1. 1 _UAB, Birmingham, AL;_ 2 _Baylor College of Medicine, Houston, TX_.

Introduction: The Cancer Genome Atlas (TCGA) consortium performed high-throughput sequencing of thousands of tumor and normal tissues across 33 cancer types. This has led to molecular characterization of different cancers and many novel discoveries. Such diverse data offers an excellent opportunity to further address the questions associated with tumor heterogeneity. Systematic exploration of epigenetic features and non-coding gene expression could lead clinicians/cancer researchers towards unearthing new diagnostic biomarkers and therapeutic targets. Previously our group developed a cancer transcriptome web portal, UALCAN [http://ualcan.path.uab.edu, Google: UALCAN)] to study expression and survival profile of protein coding genes among TCGA cancers based on various subgroups and molecular subtypes of cancer. Although many data portals facilitating easy access and in-depth analysis of TCGA data exists, there is need for a user-friendly resource facilitating comprehensive DNA methylation profile and gene expression analysis of lncRNA and miRNA within tumor subgroups based on factors such as stage, tumor grade, age, sex, race and integration with the gene expression.

Methods: TCGA Level 3 RNA-seq, miRNA-seq and Illumina Infinium HumanMethylation450K data were downloaded using TCGA-Assembler 2 tool and Genomic Data Commons (GDC). The data were processed, organized based on tumor subtypes using custom R scripts. DNA methylation and gene expression analysis was performed using in-house PERL scripts, while statistical significance was estimated using "Statistics::TTest" module. The analyses results were stored as flat file database and hosted on a web portal developed using Apache2.0, PERL-CGI and HighChartJS JavaScript libraries.

Results: The user friendly web portal 1) allows users could obtain list of top over-/under-expressed miRNAs and lncRNAs in each of 33 TCGA cancer 2) facilitates analysis of promoter methylation level and gene expression for given set of miRNA/ lncRNAs in various tumor sub-groups based on individual cancer stages, tumor grade, race, body weight or other clinicopathologic features, 3) enables researchers to download high resolution graphics such as

heatmap, boxplots, dotplots as analyses outputs. The data can be downloaded in multiple output formats for use by researchers.

Conclusion: The current resource aids cancer researchers in identifying DNA methylation mediated epigenetic modification of gene expression in tumor subgroup specific manner. Furthermore, our platform will facilitate analyses of long non-coding RNAs and microRNAs, thereby help in discovery of novel biomarkers and better understanding of the tumor biology.

#2482

A portable bioinformatics pipeline for the FDA authorized IMPACT DNAseq assay.

Zuojian Tang, Nikhil Kumar, Allan Bolipata, Timothy Song, Cyriac Kandoth, Feng Liu, David Mcmanamon, Oliver Hampton, Nicholas Socci, David Solit. _Memorial Sloan Kettering Cancer Center, New York, NY_.

On November 2017, the US Food and Drug Administration (FDA) authorized Memorial Sloan Kettering Cancer Center's (MSKCC) IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets) tumor profiling assay and bioinformatics pipeline for clinical decisions in treating all solid tumors. The IMPACT test uses next-generation sequencing (NGS) to identify the presence of mutations in 468 unique genes, fusions at known sites, copy-number alterations, and microsatellite instability (MSI). A version freeze of the bioinformatics pipeline was made public in May 2017 in Zehir et al. "Mutational Landscape of Metastatic Cancer Revealed from Prospective Clinical Sequencing of 10,000 Patients", Nat Med. 23(6): 703-713. Active development of the pipeline has continued in its CLIA-compliant computing environment, and the resulting improvements have been incorporated into an open-source portable research-grade bioinformatics pipeline. Here, we present results from Roslin, our research bioinformatics pipeline built to be functionally equivalent to the IMPACT clinical reporting workflow that includes manual filtering/annotation and signout by molecular oncologists. Roslin can be installed and executed on laptops, workstations, local compute clusters, or cloud compute servers. As a research-grade pipeline it is flexible enough to handle new assays including exomes/genomes, and extensible enough to add or replace components like reference genomes, sequence aligners, variant callers, false-positive filters, and functional/clinical annotation. Tracking all components and parameters used in each pipeline run allows researchers to easily generate method sections for manuscripts that are sufficiently detailed for peer-review and reproducibility of published results. Roslin has been deployed and tested on multiple high-performance clusters and cloud computing resources. It offers end users GUI driven workflow logging, run reporting, and real-time tracking (https://github.com/mskcc/roslin). Evaluated on NGS data from 128 tumors with signed out IMPACT clinical reports, Roslin achieves a recall rate of 98% for both somatic substitutions and indels, and 60% for fusions. Precision was 96% for substitutions, and 86% for indels. We present our work in maximizing precision and recall rates while retaining the portability, modularity, and reproducibility of Roslin.

#2483

An integrated bioinformatics pipeline for profiling cancer-immune interaction from whole exome sequencing of pembrolizumab clinical samples.

Xiaoqiao Liu, Xinwei Sher, Hongchao Lu, Jun Zhuang, Weilong Zhao, Andrew Albright, Cinthia Umemoto, Christen Wudarski, Maureen Lane, Mark Ayers, Andrea L. Webber, Sandra C. Souza, Ping Qiu, Diane Levitan, Jennifer Cho, Deepti Aurora-Garg, Matthew Marton, Alexandra Snyder, Michael Morrissey, Andrey Loboda, Ronghua Chen, Razvan Cristescu. _Merck & Co., Inc., Kenilworth, NJ_.

Purpose We built an integrated analytic pipeline to robustly and comprehensively profile molecular features of pembrolizumab-treated tumors using whole exome sequencing (WES) data from clinical trials across different indications.

Experimental design We implemented a computational framework for analysis of WES data generated by different sequencing vendors for 1467 samples from 8 major tumor types. Reads were first processed by BWA-MEM, Picard, and GATK (v2) to generate analysis-ready BAM files. Quality controls (QC) before downstream analysis included Y/X reads ratio for matched normal sample as patient sex prediction and tumor/normal concordance and contamination estimation by Conpair. Key molecular features were evaluated, including tumor mutation burden (TMB) by MuTect (v1) and VEP; HLA-I typing by OptiType; neoantigen burden by NetMHC (v3.4); mutation signature by deconstructSigs; allele-specific copy number by VarScan2 and Sequenza; clonality by PyClone; presence of oncogenic viruses (eg, EBV, HBV, HPV); and LOH score indicating homologous recombination deficiency (HRD-LOH).

Results Concordance rate between predicted and clinical sex was 1446/1464 (98.8%), and 1420/1467 (96.8%) samples passed tumor-normal alignment QC. Highest TMB (median [range]) was detected in melanoma (245 [2-6246]) and urothelial carcinoma (UC) (124 [4-1579]), with lowest TMB in metastatic castration-resistant prostate cancer (52 [1-6143]) and ovarian cancer (OvCa) (49.5 [8-272]). The correlation of median TMB across 7 cancer types in our data and TCGA was Spearman R = 0.957. Within indications, there was no difference in TMB distribution for sequencing data originating from different sequencing vendors and TCGA data, which demonstrated concordance across data sets and robust TMB calling by our integrated pipeline. Neoantigen burden strongly correlated with TMB (N = 1420; Spearman R = 0.890). HPV was detected in 20/129 (15.5%) head and neck squamous cell carcinomas (HNSCC) and 4/6 (66.7%) anal cancers; EBV was detected in 8/129 (6.2%) HNSCC and 18/318 (5.7%) gastric cancers (GC). The dominant mutation signatures by disease included APOBEC for UC (135/236) and HNSCC (22/122), alcohol for HCC (19/35), HRD for OvCa (12/64) and triple-negative breast cancer (51/175), UV exposure for melanoma (145/176), and dMMR for GC (84/287) and CRPC (16/155). Samples with deleterious BRCA mutations showed significantly higher HRD-LOH score (N = 1420; AUROC = 0.61 [95% CI, 0.53-0.69]) and HRD mutational signature (N = 1316; DOR = 6.4 [95% CI, 3.7-11.1]).

Conclusion We assembled heterogeneous computational modules into an integrated pipeline to reliably profile diverse molecular features from WES data of nearly 1500 clinical samples across different tumor types. These data serve as a foundation for translational research efforts supporting pembrolizumab development.

#2484

An online tool to validate predictive biomarkers of therapy response using transcriptomic data of 3,651 ovarian cancer patients.

Balazs Györffy, Janos Fekete, Agnes Osz, Imre Pete, Marcell Szasz. _Semmelweis University, Budapest, Hungary_.

Systemic therapy of ovarian cancer can include chemotherapy and targeted therapy. Prognostic biomarkers are capable to predict survival and predictive biomarkers are capable to predict therapy response. To date, multiple online tools were established to identify prognostic biomarkers, but no platform is yet available for predictive biomarkers. In this study, we describe the first release of an online available tool capable to validate gene expression based predictive biomarkers using transcriptomic data of a large set of ovarian cancer patients.

Published gene expression data of 35 independent datasets was integrated with treatment data into a unified database. The classification is based on either author-reported pathological complete response (n=1,022) or relapse-free survival status at six months (n=1,347) or relapse-free survival status at twelve months (n=1,282). Treatment data includes chemotherapy (platin, taxol, docetaxel, paclitaxel, gemcitabine, topotecan) and targeted therapy (avastin). The transcriptomic database includes 54,675 probe sets corresponding to 20,089 distinctive genes. Finally, we performed a sample collection at the National Institute of Oncology (NIO cohort), and used these patient samples to validate the top six genes in ovarian cancer patients. Gene expression and therapy response were compared using receiver operating characteristics and Mann-Whitney tests.

In the validation of the tool we focused on paclitaxel-resistance associated genes. We selected the top genes after running the analysis across all samples and validated these by PCR in the NIO cohort of patients (n=80). The best performing paclitaxel-resistance biomarker candidates were ARAF (AUC=0.743, p=4.1E-09), PPCS (AUC=0.733, p=4.2E-07), GNL2 (AUC=0.718, p=2.7E-08), TFE3 (AUC=0.718, p=4.8E-06), PDXK (AUC=0.717, p=1.1E-05) and TOP1 (AUC=0.716, p=4.6E-05).

The analysis pipeline enables to validate and rank predictive biomarker nominees. By analyzing the candidate genes in a large set of independent patients, we can select the most reliable candidate and abolish those which are most likely to fail in a clinical setting. The registration-free interface of the online analysis platform is accessible at www.rocplot.org/ovar.

#2485

Somatic analysis services with best practice workflows in a cloud-based platform.

Junko Tsuji, Andrew Hollinger, Alyssa MacBeth, Brian R. Grander, Micah Rickles-Young, Tera Bowers, Carrie Cibulskis, Niall Lennon. _Broad Institute of MIT and Harvard, Cambridge, MA_.

With advances in high-throughput sequencing technologies and analytical tools, genomic analysis of tumors has led to the identification of various important somatic mutations that shed light on diagnosis, prevention, and treatment for cancer. However, detecting somatic variants is not a trivial task in terms of the technical aspects (e.g. filtering germline events and removing a variety of noises in tumor samples) and computational resources to handle large-scale cohort analysis. There is also a demand for maintaining stable software versions and the workflows for studies over extended periods of time, that need consistency and traceability, such as clinical trials. We introduce here, the Translational Analysis Group (TAG), a team which deploys, validates, and conducts scalable analytical workflows in a secure, cloud-based environment. We maintain 29 well-tested workflows with best practice methods and ample resources for both somatic and germline analysis. Our cloud platform, FireCloud, enables us to run workflows at any scale. Since May 2017, our team has performed nearly 10,000 analyses for mutation detection (SNV, InDel, CNV, and SV) and cohort analysis on tumor samples and cell-free DNA samples. TAG offers a range of options for somatic and germline variant detection, from legacy pipelines through recently validated contemporary pipelines to allow for continuity across long running projects.

#2486

Oncogene.iobio: A web app for real-time, integrative examination and functional prioritization of tumor mutations .

Stephanie Georges, Yi Qiao, Xiaomeng Huang, Gabor Marth. _University of Utah, Salt Lake City, UT_.

Genetic testing and clinical tumor sequencing are rapidly becoming part of the standard of care for a large subset of cancers. At present, a scattered framework of resources, often requiring expensive hardware and heavy computational skills, exists to aid clinicians and researchers in the bioinformatic analysis process. To assist in addressing the need for a comprehensive tool that operates intuitively, and at the pace of critical therapeutic timelines, we have developed oncogene.iobio. Utilizing our novel iobio analysis ecosystem (https://iobio.io), oncogene.iobio visualizes genomic, transcriptomic, and epigenetic data to aid in the discovery of variants with the highest potential for association with the patient's cancer.

Oncogene.iobio consumes primary sequencing data (in bam, vcf, fasta/fastq formats) sourced from local, or cloud-based repositories. In real-time, variants are identified and annotated on a gene-by-gene basis, and prioritized using functional annotations (sourced from SNPEFF, VEP, SIFT, PolyPhen) and cancer databases (COSMIC, cBioPortal). Uniquely, oncogene.iobio identifies compound heterozygotes between inherited cancer-implicated variants and somatically-acquired tumor mutations, dynamically filters mutations by allele frequency, and analyzes somatic variants within areas of chromosomal CNVs or LOH. Areas with large transcriptional change or allele-specific expression are highlighted when RNAseq data is present, along with hypo- or hyper-methylated sites given bisulfate-treated DNA sequencing reads. Ranked, candidate gene lists (sourced from ICGC, cBioPortal), as well as cancer-associated variants (sourced from GEMINI), are provided by oncogene.iobio to guide users in the analysis process, and to quickly elucidate suspect loci. Some analyses may benefit from the more advanced features of oncogene.iobio, such as the ability to recall variants in real-time, using our Freebayes algorithm attuned with tumor-specific parameters. This workflow assists in discovering false negatives, and large-scale deletions and duplications, normally missed in low-coverage areas analyzed using canonical methods. Additionally, oncogene.iobio provides the ability to corroborate true positives and reduce reference mapping biases using our graph-based adjudication tool GRAPHITE (https://github.com/dillonl/graphite). In conjunction with the rapidly-informative and visually-intuitive primary features, these options allow for critical biomedical findings sourced from high-confidence data.

With extensive integration and immediate feedback, oncogene.iobio aims to aid the discovery of impactful variants in oncological research and diagnostic care.

#2487

Application of RegTools to TCGA samples for the identification of tumor-specific splice variants.

Kelsy C. Cotto, Yang-Yang Feng, Zachary L. Skidmore, Obi L. Griffith, Malachi Griffith. _Washington University in St. Louis, Saint Louis, MO_.

The interpretation of variants in cancer is frequently focused on direct protein coding alterations. However, this analysis strategy excludes somatic mutations in non-coding regions of the genome and even exonic mutations may have unidentified non-coding consequences. To address this issue, we created RegTools (Feng et al. 2018), a free, open-source software package that integrates analysis of somatic variant calls from genomic data with splice junctions extracted from transcriptomic data in order to efficiently identify variants that may cause aberrant splicing in tumors. Initially, this software was developed using 188 cancer samples from 4 cancer types and enabled us to identify important non-coding variants which would otherwise be undervalued or perhaps even completely ignored by traditional methods and annotators. Here, we present the application of RegTools to over 10,000 TCGA samples in an effort to identify somatic variants that are associated with alternative splicing patterns within these tumors. In order to perform this analysis, we obtained all tumor samples that had somatic variant VCF files and RNA-seq bam files from the Genomic Data Commons (GDC). Included within these samples are more than 700 solid tissue normal samples, which will be used as a panel of normals in order to filter out variants and splicing events that are not tumor specific. We plan to present on the significance of our findings when analyzing these samples within their respective tumor type and across all tumor types. We also plan to present on whether these aberrant splicing events that result from somatic mutations might interfere with predicted neoantigens pVAC-Seq (Hundal et al. 2016). RegTools is freely available and open source (www.regtools.org).

#2488

CellMinerCDB and CellMiner web-applications for genomics and pharmacogenomics analyses of cancer cell lines.

William C. Reinhold, Sudir Varma, Yang-Hsin Wang, Fathi Elloumi, Yves Pommier. _National Cancer Institute, Rockville, MD_.

The CellMiner (http://discover.nci.nih.gov/cellminer)and CellMinerCDB (https://discover.nci.nih.gov/cellminercdb/) web-applications provide complimentary functionality and datatsets that may be used for comparisons of genomic, molecular and pharmacological data within the NCI-60 cancerous cell lines, Cancer Cell Line Encyclopedia (CCLE), Genomics of Drug Sensitivity in Cancer (GDSC), Cancer Therapeutics Response Portal (CTRP), NCI/DTP small cell lung cancer (SCLC), and NCI Almanac cell line sets. CellMiner contains data for the NCI-60, including the most extensive sets of molecular and drug activity data (generated by the NCI Developmental Therapeutics Program https://dtp.cancer.gov),found for any of the databases. CellMinerCDB contains all the above mentioned cell line sets, including the substantially increased cell line numbers and tissue of origin types found in the CCLE, GDSC, and CTRP. The two web-applications have separate but complimentary functionalities. Each cell line set has some variable number of data types, some of which measure the same parameters, and some that do not. The number and make up of cell lines also varies, from 60 for the NCI-60, 69 for the SCLC, and ~1000 for the CCLE, GDSC, and CTRP. As there are partial overlaps of cell lines between many of these cell line sets, one may fill in some data type gaps by merging data from two sources, as well as do quality control by comparisons of the same data from multiple institutions. This rich set of data and functions facilitates the exploration of the relationships between and among molecular alterations and pharmacological responses in cancer cell lines from the omic perspective.

#2489

Enabling data access, sharing, collaborative and reproducible research: The Frederick National Laboratory for Cancer Research (FNLCR) data coordinating center.

Paul Aiyetan, Paul Donovan, David Mott, Matthew Starr, Rajani Kuchipudi, Mahesh Yelisetti, Debra Hope, Corinne Zeitler, Uma Mudunuri, Andrew Quong. _Frederick National Laboratory for Cancer Research, Frederick, MD_.

The Frederick National Laboratory for Cancer Research hosts a Data Coordinating Center and Toolset (the Metadata Designer plus Validator) hereafter referred to as the DCC that embodies a scalable, next-generation biological and cancer research data repository that is flexible, intuitive, and adaptive. The DCC provides integrated management of datasets across all deposited projects making its data more accessible and easily reusable by the cancer research community. The DCC stores and manages access to data, enabling researchers or data depositors to grant controlled access only to specific collaborators while maintaining a user-specified embargo on deposited datasets. The DCC enables a data access and sharing capability aimed to facilitate the development of new biological insights. The DCC implements the ISA (Investigation-Study-Assay) paradigm. The ISA framework provides a rich description of experimental metadata that is agnostic and irrespective of sample characteristic, technology or measurement type. It provides clear and simple sample-to-data relationships that enables resulting data and discoveries to be reproducible and reusable. These data are in the standard Investigation-Study-Assay tab-delimited format (ISA-TAB) format, which describes a scientific investigation, its study or studies, and each study's assay(s). The DCC portal is a public repository of experiment-related information describing cancer and biomedical research investigations. The portal can be used to browse, search, and access data from uploaded datasets. Through its stand-alone Toolset (Metadata Designer plus Validator), the DCC offers data depositors and researchers a simple interface to create and validate ISA-Tab compatible metadata associated with data generated in their research. Deriving new insights from aggregates of datasets and the need for reproducible research has never been more apparent. However, a fundamental requirement for these is a comprehensive metadata annotation and documentation of the research processes - an art that is elusive to researchers. Beyond the resultant publication is the data and metadata. These two basic ingredients are building blocks for a successful reproducible research project. In addition, appropriately curated data and metadata are invaluable resources for meta-analyses of seemingly disparate research studies. Further emphasizing the importance of these basic elements, are the principles of Findability, Accessibility, Interoperability, and Reusability (FAIR) - to guide data producers and publishers around obstacles and help maximize added-value gained from published studies. The DCC provides a much-needed resource to the research community for simplifying the sharing of data sets that meet these principles. The DCC portal is located at https://cssi-dcc.nci.nih.gov/cssiportal/

#2490

R2: Genomics analysis and visualization platform.

Jan Koster, Richard Volckmann, Danny Zwijnenburg, Piet Molenaar, Rogier Versteeg. _Academic Medical Ctr. - Univ. of Amsterdam, Amsterdam, Netherlands_.

Given the complexity, omics data like RNA-seq are often analyzed by bioinformaticians, and not the wetlab researchers that performed the experiments. With the biomedical researcher (with limited or no bioinformatics skills) in mind as the end-user, we developed the user-friendly, publicly accessible R2 platform (http://r2.amc.nl), enabling biomedical scientists to work with their own data from anywhere at any time. R2 (cited in >850 peer-reviewed scientific publications) consists of an omics database, coupled to an extensive set of interactive tools to analyze/visualize the datasets. Interactive analyses within the software are highly connected, allowing quick navigation between various aspects of the datamining process. Besides password protected private data, R2 also contains an extensive body of publicly available omics data, enabling users to relate their findings to other diseases / tissues, or as validation in integrative analyses. Next to gene expression (bulk as well as single cell), R2 is also being employed for the integration, analysis and visualization of copynumber, SNP, methylation, miRNA, drug response, ChIP-seq, and NGS exome/WGS DNA sequencing data. Analyses include correlation, differential expression, gene sets, gene ontology, transcription factor binding sites, tSNE, PCA, k-means, Kaplan Meier survival scans, signature creation etc. Visualizations include various gene oriented plots, heatmaps, Circos plots, embedded genome browser, Venn diagrams, etc. The real power of the platform lies in the chaining of results of analyses; e.g. the results of a differential expression analysis can be further trimmed down using a Kaplan Meier analysis that in turn can be used for a Gene Ontology over-representation analysis. Furthermore, the webserver allows for overviews across different datasets (such as MegaSampler and 2D distribution), where a user can harness the power of thousands of measurements. Recently, integrated analyses for personalized medicine, where copy number, mRNA expression, methylation and mutation data are combined into a comprehensive overview, occasionally supplemented with PDX/organoid drug response data. These serve clinical decision making for a patient, but as growing cohorts also form a treasure trove for scientific discovery. R2 provides a central starting point from where data mining and analysis paths can be followed within one environment. Data/analyses can be shared between R2-users via our community options, making R2 an outstanding environment for discovery, hypothesis testing and scientific collaboration. 

## MOLECULAR AND CELLULAR BIOLOGY / GENETICS

### BCL-2 Family Proteins

#2491

Engineering and preclinical activity of MM-201, a best-in-class TRAIL receptor agonist.

Andrew J. Sawyer, Sara Ghassemifar, Christina Wong, Jennifer Richards, Stephanie Grabow, James Suchy, Alexander Koshkaryev, Maja Razlog, Eric Tam, Daryl C. Drummond. _Merrimack, Cambridge, MA_.

Early attempts at using TNF superfamily members for anticancer therapies, TNF and FAS, led to serious systemic toxicities. However, the discovery of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) introduced an agonist capable of killing tumor cells via apoptosis without the side effects observed with TNF and FAS agonists. First generation TRAIL agonists included a recombinant version of human TRAIL (dulanermin), as well as multiple DR4 and DR5 agonist antibodies. Despite some isolated responders, the initial clinical results were poor. The first-generation TRAIL agonists were limited by poor pharmacokinetics (the half-life for dulanermin was between 30 and 60 minutes) or by poor agonist activity and the need for Fc-mediated cross linking. Here we present the development and evaluation of two second generation TRAIL agonists, MM-201a and MM-201b. Both versions are composed of an IgG1 Fc fused to a single chain TRAIL trimer (Fc-scTRAIL). Mutations within the TRAIL domains, selected from a random mutagenesis library, were introduced to improve stability, expression, and DR5 binding. MM-201a has 5 mutations in each monomeric unit (R130G/N228S/I247V/Y213W/S215D) and MM-201b has 3 mutations in each monomer (R130G/N228S/I247V). In a panel of 27 colorectal carcinoma and sarcoma cell lines, both versions of MM-201 were observed to be significantly more active than all comparators, including the TRAIL cytokine and both DR4 and DR5 antibodies. MM-201a had a level of activity similar to ABBV-621, a single chain TRAIL fused to the N-terminus of an IgG1 Fc that is currently the subject of a Phase 1 trial. However, MM-201b was significantly more active than both MM-201a and ABBV-621, with up to 11-fold lower IC50 across a panel of 12 CRC cell lines. MM-201b treatment reduced cell viability to less than 20% in 10 out 12 colorectal cancer cell lines and in 8 of these cell lines, this was achieved at concentrations less than 1 nM. MM-201 also induced complete cell death at 1 nM or less in 3 of 8 synovial sarcoma and chondrosarcoma cell lines tested. For example, MM-201b reduced the viability of the SW-982 synovial sarcoma cell line to 17% at a dose of 1.5 pM, which is nearly twice the reduction in viability from the same dose of ABBV-621. We next evaluated both versions of MM-201 in multiple colorectal cancer and sarcoma patient-derived xenograft (PDX) models. In the Ewing's sarcoma PDX model TM01617, MM-201b treatment resulted in 90% tumor growth inhibition. In the same model, treatment with 10 mg/kg docetaxel resulted in 73% growth inhibition; however, in combination with MM-201a, the same dose resulted in a 100% complete response rate. Similar results were observed in the SK-UT1 uterine sarcoma xenograft. Based on this evidence, we believe that MM-201b is best in class and, when combined with an appropriate patient selection strategy, has significant potential for the treatment of sarcomas and colorectal cancer in patients.

#2492

**BRAF inhibitors synergize with BH3 mimetics to induce apoptosis in** BRAF **mutant colorectal cancer cells.**

Laura J. Jenkins,1 Fiona Chionh,1 Ian Y. Luk,1 Erinna F. Lee,2 Amardeep S. Dhillon,3 Niall Tebbutt,4 Walter D. Fairlie,1 John M. Mariadason1. 1 _Olivia Newton-John Cancer Research Institute, Melbourne, Australia;_ 2 _La Trobe Institute for Molecular Science, Melbourne, Australia;_ 3 _Deakin University, Geelong, Australia;_ 4 _Austin Health, Melbourne, Australia_.

Activating mutations in BRAF (BRAFV600E) occur in ~10% of colorectal cancers (CRCs) and drive tumorigenesis through constitutive activation of MAPK signaling. In metastatic CRC, BRAF mutations are associated with poorer prognosis and resistance to conventional therapies, necessitating an urgent need to develop new treatments for these patients. BRAF inhibitors such as vemurafenib and dabrafenib have significant clinical activity in BRAF-mutant melanoma, however BRAF-mutant CRCs are largely refractory to these agents, due at least in part to feedback-relief mediated reactivation of MAPK signaling or alternate signaling pathway activation. Strategies to enhance the activity of BRAF inhibitors in BRAF-mutant CRC are therefore needed. Consistent with clinical observations, treatment of a panel of BRAF-mutant melanoma and CRC cell lines with vemurafenib resulted in significantly increased apoptosis in melanoma cell lines compared to CRC cell lines, where effects were largely cytostatic. To determine the mechanisms for this differential response we interrogated vemurafenib-induced gene expression changes in the two tumor types, focusing on altered expression of components of the intrinsic apoptotic pathway. Vemurafenib induced a more pronounced increase in expression of the pro-apoptotic genes BIM, BMF and PUMA and suppression of pro-survival gene MCL1 in melanoma cells compared to CRC cells. These findings suggested that the extent to which expression of pro and anti-apoptotic genes are altered by vemurafenib in CRC cells may be insufficient to reach the threshold required for apoptosis initiation. We therefore postulated that BH3-mimetics may synergize with vemurafenib to induce apoptosis in BRAF-mutant CRC cells. Analysis of quantitative proteomic data of BRAF-mutant CRC cell lines revealed significantly higher basal expression of the pro-survival proteins Bcl-xL and MCL1 compared to BCL2 and BCLW, suggesting CRC cells may be particularly dependent on Bcl-xL and MCL1 for survival. Indeed, combination treatment of BRAF-mutant CRC cells with the Bcl-xL inhibitor A-1331852 significantly enhanced apoptosis in the majority of BRAF-mutant CRC lines. Comparatively, combination treatment of vemurafenib with the MCL1 inhibitor S63845 induced a modest increase in apoptosis, while combination treatment with the BCL2 inhibitor ABT-199 had no effect on apoptosis, consistent with the low levels of BCL2 expression in these lines. Finally, we investigated the effect of combination treatment of vemurafenib with inhibitors of both Bcl-xL and MCL1. The triple combination further enhanced apoptosis in 3/5 cell lines, suggesting these cell lines are likely dependent on both Bcl-xL and MCL1 for survival. Collectively, these findings demonstrate that combining BRAF-inhibitors with Bcl-xL and/or MCL1 inhibitors may represent a novel strategy for treating BRAF-mutant CRC.

#2493

Disulfide bond disproportionation agents activates the death receptor 5 pathway to kill highly resistant breast cancer.

Mengxiong Wang. _University of Florida, Gainesville, FL_.

There are significant unmet medical needs among patients who have HER2+ breast cancers that have acquired resistance to targeted agents, and patients with Triple-Negative Breast Cancers (TNBCs) that lack therapeutic targets. Novel treatment strategies are needed to target these cancers. Disulfide bond Disproportionation Agents (DDAs) represent a new chemical class of anticancer drugs and show prominent anti-cancer activities both in vitro and in animal model. Blockade of breast tumor growth by DDAs is associated with downregulation of EGFR/HER2/HER3 in parallel, induction of AKT dephosphorylation, and activation of ER stress. However, it is not known how DDAs trigger cancer cell death without affecting non-transformed cells. This study aims to investigate the death pathways involved in the anti-cancer activities of DDAs, and to develop strategies that target drug resistant breast cancer and cancer metastasis. This study demonstrates that DDAs induce breast cancer cell death by activating the Death Receptor 5 (DR5) pathway. DDAs are the first compounds identified that upregulate DR5 via both transcriptional and post-transcriptional mechanisms and uniquely cause DR5 accumulation and oligomerization by altering DR5 disulfide bond pattern. This study also shows that the combination of DDAs and Tumor Necrosis Family-Related Apoptosis-Inducing Ligand (TRAIL), a natural ligand of DR5, significantly overcomes TRAIL resistance that has been encountered in clinical trials with this ligand. Furthermore, DDAs and TRAIL synergistically kill Lapatinib-resistant breast cancer and cancer metastatic cells, and that this anti-cancer effect is amplified by the overexpression of EGFR/HER2/MYC. Together, this study highlights a promising novel pharmacological approach against drug resistant cancer and cancer metastasis.

#2494

Emodin inhibits colon cancer by altering BCL-2 family proteins and cell survival pathways.

Ian T. Saunders, Neeraj Kapur, Hina Mir, Shailesh Singh. _Morehouse School of Medicine, Atlanta, GA_.

The therapeutic outcome of colon cancer (CoCa) is often compromised due to the development of chemoresistance and toxicities associated with current therapeutics. Hence, identification of less toxic and efficacious agent is needed to treat CoCa. In this study, we tested the effect of natural agent, Emodin (6-methyl-1,3,8-trihydroxyanthraquinone) isolated from Asian plant rhubarb, on CoCa cell death and survival. Effects of Emodin on CoCa (DLD-1 and COLO-201) cell viability and death was determined by MTT and Annexin-V assays, respectively. Apoptosis-specific protein antibody array was used to define the Emodin induced molecular mechanisms involved in cell death vs. survival and results were confirmed by western blot analysis. Mitochondrial membrane potential and the expression/localization of Bcl-2 family protein were assessed via flow cytometry and WB, respectively. Our data show Emodin decreased the viability of CoCa cells and induced apoptotic cell death in a time and dose-dependent manner. Emodin-induced apoptosis resulted in increased death receptor expression, modulated caspase activation, and an imbalance in the Bax/Bcl-2 ratio. Changes in Bcl-2 family protein expression and localization correlated with loss in mitochondrial membrane potential. Emodin also negatively impacted MAPK/JNK, PI3K/AKT, NF-kB and STAT signaling pathways, which are associated with cell growth, differentiation, and Bcl-2 family expression or function. Modulation of cell survival and apoptotic pathways by Emodin and specifically the impact on Bcl-2 projects Emodin significance as promising therapeutic agent for CoCa treatment.

#2495

Glucosamine enhances TRAIL-induces apoptosis in DU145 and Hela cancer cell lines.

Chao Sun, Viktor Chesnokov, Keiichi Itakura. _Beckman Research Institute of City of Hope National Medical Center, Duarte, CA_.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has attracted wide attention and extensive research because of selective apoptosis of tumor cells but not of normal cells. However, the clinical trials using TRAIL alone were disappointed due to the resistance of cancer cells to the TRAIL-treatment. Several resistant mechanisms, such as low expression of death receptors, and overexpression of cFLIP, BCL-2 antiapoptotic family members and XIAP were proposed. Tunicamycin was reported to enhance the expression of death receptor 5 through the activation of CHOP. Similar to tunicamycin, glucosamine is known to increase the expression of CHOP by inducing ER stress. We thus investigated whether glucosamine could increase the expression of death receptor 5 to overcome TRAIL-resistance. We found that glucosamine treatment of DU145 cells did not increase the expression of death receptor 5 on the cell surface, even though the overall expression was increased. However, glucosamine significantly increased TRAIL-induced apoptosis in DU145 and Hela cells. Our investigation revealed that glucosamine sensitized cancer cells to TRAIL-induced apoptosis through both intrinsic and extrinsic apoptosis pathways, as evidenced by the cleavage of both caspase 9 and caspase 8. Our data further showed that the combination of TRAIL with glucosamine suppressed the expression of cFLIP, BCL-2 and XIAP, translocated BAK to the mitochondrial outer membrane, and released cytochrome C and SMAC from mitochondrial. Interestingly, in addition to the activation of apoptotic pathways, TRAIL-mediated inflammatory responses were attenuated by glucosamine treatment. Pretreatment of glucosamine reduced the TRAIL-induced nuclear NF-kB level and the expression of IL-6 and IL-8. Together, these results demonstrated that the combination treatment, TRAIL with glucosamine, may provide a promising novel strategy for treating different human cancers by overcoming TRAIL-resistance and inhibiting TRAIL-induced inflammation.

#2496

Dynamic BH3 profiling identifies active combinations with conventional chemotherapy in non-small cell lung cancer.

Danielle S. Potter, Anthony G. Letai. _Dana-Farber Cancer Institute, Boston, MA_.

Mitochondrial apoptotic priming (referred to as priming) determines a cell's 'readiness' for cell death and is regulated by the B-cell lymphoma 2 (BCL-2) family of proteins. It has been shown in multiple disease settings such as acute lymphoblastic leukemia, acute myeloid leukemia, multiple myeloma and ovarian cancer, that chemosensitivity correlates with priming. Patients with highly primed tumors exhibited superior clinical response to chemotherapy. In contrast, chemoresistant cancers and normal tissues were poorly primed. Priming is relative and can be measured using BH3 profiling. BH3 profiling is a functional assay which uses BH3 peptides derived from the BH3 domain of pro-apoptotic BH3-only BCL-2 family members to provoke a response from viable mitochondria. To identify drugs that enhance priming, tumor cells can be incubated with drugs prior to BH3 profiling, a method called dynamic BH3 profiling (DBP). DBP is a functional approach to precision medicine and measures early changes in death signaling after drug perturbation. An increase in priming after short term drug treatment (8-24 hours) has been shown to result in cell death days later. Moreover, it has been shown to predict in vivo response to therapy. Therefore, DBP can predict efficacious therapies within 24 hours.We hypothesized that drugs that enhance priming would render cancers more sensitive to conventional chemotherapy. To determine whether drugs that increase priming enhanced sensitivity to docetaxel and etoposide in non-small cell lung cancer (NSCLC), we first identified agents that enhanced priming via DBP. We found that targeted agents that increased priming of NSCLC tumor cells resulted in increased chemosensitivity in vitro. To assess whether targeted agents that increase priming might enhance the efficacy of cytotoxic agents in vivo, we carried out an efficacy study in PC9 xenograft mouse model. The BH3 mimetic navitoclax, which inhibits BCL-xL, BCL-w and BCL-2, consistently primed NSCLC tumors in vitro and in vivo. The BH3 mimetic venetoclax, which inhibits BCL-2, did not. In vivo navitoclax reduced tumor burden whilst mice were treated but as soon as therapy was stopped tumors recovered comparable to vehicle treated mice. Etoposide as a single agent had no effect on tumors. However, combining navitoclax with etoposide significantly reduced tumor burden after treatment was stopped, increasing mouse survival. Adding venetoclax to etoposide had no effect on tumor burden. These data suggest that targeted agents that increase priming, increased chemosensitivity resulting in reduction of tumor burden in vivo.

#2497

FCN-338, a novel and selective Bcl-2 inhibitor, exhibits potent anti-tumor activity in B-cell lymphoma.

Shu Lin,1 Xingdong Zhao,1 Hongbin Liu,2 Huajie Zhang,2 Zhifang Chen,2 Lihua Jiang,2 Yanxin Liu,2 Min Lin,2 Yue Rong,2 Rui Tan,2 Zuwen Zhou,2 Zongyao Zou,2 Yuwei Gao,2 Weibo Wang2. 1 _Fochon Pharma, Inc., San Leandro, CA;_ 2 _Fochon Pharmaceuticals, Ltd., Chongqing, China_.

B-cell lymphoma-2 (Bcl-2) family proteins are key regulators of apoptosis, which is vital for proper tissue development and cellular homeostasis. Apoptosis occurs via activation of two different pathways: the extrinsic pathway, triggered by a death ligand binding to a death receptor, such as TNF-α to TNFR1; and the intrinsic pathway, regulated by Bcl-2 family proteins and their complex protein-protein interactions. Bcl-2 family proteins are functionally classified as either anti-apoptotic, such as Bcl-2, Bcl-xL and Mcl-1, or pro-apoptotic, including Bid, Bim, Bad, Bak and Bax. Anti-apoptotic Bcl-2 family members are often found to be up-regulated in many types of human cancers, such as different subtypes of B-cell lymphoma and diverse solid tumors, and are frequently been correlated with decreased susceptibility to chemotherapeutics and to increased radio-resistance. Overexpression of Bcl-2 proteins is an independent indicator of poor prognosis in tumors including chronic lymphocytic leukemia (CLL), prostate cancer, and small cell lung cancer (SCLC). Therefore, targeting Bcl-2 and its pro-survival relatives that form the core anti-apoptotic machinery, has emerged as a promising therapeutic approach in cancer. To that end, we have developed a novel and selective Bcl-2 inhibitor, FCN-338, with nanomolar affinity to Bcl-2 and physicochemical properties suitable for oral administration. FCN-338 exhibits much greater affinity to Bcl-2 than Bcl-xL, suggesting its good potential to avoid thrombocytopenia caused by the inhibition of Bcl-xL. FCN-338 has shown remarkable anti-proliferation potency against a panel of Bcl-2-addicted human B-cell lymphoma cell lines, including DOHH2 follicular lymphoma (FL) cell line (IC50 2 nM), Mino diffuse large B-cell lymphoma (DLBCL) cell line (IC50 23 nM), and RS4;11 acute lymphoblastic leukemia (ALL) cell line (IC50 35 nM), but not in Bcl-xL-dependent H146 SCLC cell line (IC50 >4,500 nM). Correspondingly, in vivo efficacy of FCN-338 was demonstrated in a variety of xenograft models derived from FL, DLBCL and ALL tumors. FCN-338 dramatically caused tumor regression in a dose-dependent manner. In the non-clinical studies, FCN-338 exhibits much improved pharmacokinetic properties with higher T1/2, dose-normalized AUC and bioavailability in dogs compared with the FDA-approved Bcl-2 inhibitor ABT-199. Preferable safety profiles of FCN-338 were shown with no potential hERG inhibitory effect and less drug-drug interaction potential, as evidenced by no inhibitory effect (IC50 >50 μM) on CYP2C9 enzyme compared with ABT-199 (IC50 1.05 μM). Together, our findings highlight the therapeutic potential of FCN-338 to be used as effective therapy across a broad range of Bcl-2-addicted B-cell malignancies.

#2498

MCL1 binds and negatively regulates the transcriptional function of tumor suppressor p73.

Hayley Widden, Aneta Kaczmarczyk, Robert H. Whitaker, William J. Placzek. _Univ. of Alabama at Birmingham, Birmingham, AL_.

MCL1 is an anti-apoptotic member of the Bcl-2 family, which has become a popular therapeutic target in cancer research over the past decade due to its propensity to promote cancer cell survival and facilitate chemoresistance. Through a phage display screen, we identified a novel MCL1 binding motif that is the reverse of the canonical BH3-binding helix that mediates interactions amongst Bcl-2 family members. Upon a BLAST sequence analysis of native proteins, we identified a putative reverse BH3 (rBH3) motif in the tumor suppressor protein, p73. Here, we show that MCL1 and p73 bind through a direct protein-protein interaction. Using fluorescence polarization assay, we characterize the strength of this interaction and reduce binding down to the rBH3-containing alpha helix in the tetramerization domain of p73. To elucidate biological function, we employ electrophoretic mobility shift assay to show that excess MCL1 negatively impacts p73 binding to DNA. Finally, using two p53-/- cancer cell lines, we demonstrate that MCL1 inhibits the transcriptional function of p73 through analysis of p73 target gene expression. In summary, we characterize a novel protein-protein interaction between MCL1 and p73 and show that MCL1 negatively impacts p73 target gene activation. This work establishes a novel function of MCL1 outside of apoptotic regulation at the mitochondria and provides new evidence of cross talk between the Bcl-2 family and the DNA damage response pathway.

#2499

In vitro and in vivo efficacy of MCL1 inhibitor S63845 in small cell lung cancer.

Yuto Yasuda, Hiroaki Ozasa, Takahiro Tsuji, Takashi Nomizo, Tomoko Yamamoto, Hitomi Ajimizu, Hironori Yoshida, Yuichi Sakamori, Toyohiro Hirai, Young Hak Kim. _Kyoto University, Kyoto, Japan_.

MCL1 is a member of the BCL-2 family, which regulates apoptosis. Targeting MCL1 represents a potential breakthrough of cancer treatment. We tested S63845, a MCL1 inhibitor, in four SCLC cell lines (DMS114, DMS53, SW1271, and NCI-H69) and, in addition, one patient derived SCLC cell line (KTOR201). S63845 had greater efficacy in two of five SCLC cell lines (DMS114 and KTOR201). These two SCLC cell lines had higher expression of MCL1 and lower expression of BCL-XL, which is another member of the BCL-2 family. The other three SCLC cell lines (DMS53, SW1271 and NCI-H69) were resistant to S63845 and had a higher expression of BCL-XL or lower expression of MCL1. In vivo activity was evaluated in xenograft models. DMS114 was subcutaneously implanted into immunocompromised mice. When the tumor volume was reached 150-200 mm3, mice were administered with S63845 (25 mg/kg) or vehicle intravenously twice a week. S63845 showed significant antitumor efficacy in vivo. Finally, immunohistochemical evaluation of MCL1 and BCL-XL was assessed in SCLC obtained from 33 patients at Kyoto University Hospital. High MCL1 expression with low BCL-XL expression was observed in 16 specimens (48.5%).These data suggested that S63845 might be a powerful treatment of SCLC as a new therapeutic strategy. It is possible that the expressions of MCL1 and other members of the BCL-2 family predict the sensitivity of S63845.

#2500

Transient CDK9 inhibition with AZD4573 modulates Bfl-1 in preclinical lymphoma models.

Scott Boiko, Theresa Proia, Maryann San Martin, Lisa Drew, Wenlin Shao, Justin Cidado. _AstraZeneca, Waltham, MA_.

AZD4573 is a selective cyclin-dependent kinase 9 (CDK9) inhibitor under clinical development in patients with hematological malignancies. Transient CDK9 inhibition serves as an orthogonal approach for targeting Mcl-1, a labile anti-apoptotic protein essential for the survival of cancer cells. Across-broad hematological cancer models, anti-tumor responses with AZD4573 strongly correlate with selective Mcl-1 inhibitors, such as AZD5991 (R2=0.8). Despite compelling evidence for an Mcl-1 dependent mechanism of action, we also observed a subset of lymphoma models more sensitive to CDK9 inhibition compared to Mcl-1 inhibition, suggesting acute CDK9 inhibition could be targeting other labile proteins beyond Mcl-1 to induce apoptosis. We identified Bfl-1 as one such potential target and demonstrate lymphoma models expressing Bfl-1 are highly sensitive to CDK9 inhibition.

Bfl-1 belongs to the Bcl-2 family of anti-apoptotic proteins and was detected in over 20% of lymphoma cell lines evaluated (n=33). Cycloheximide experiments indicate Bfl-1 has a short protein half-life (<1h), similar to Mcl-1. Therefore, treatment with 100nM of AZD4573 in diffuse large B-cell lymphoma cell lines OCILY10 and TMD8 caused rapid down-regulation of both Mcl-1 and Bfl-1 by 4h, resulting in caspase cleavage by 6h. Evaluation of caspase activation following 6h treatment revealed an average maximum effect of 87% for AZD4573 compared to 45% with Mcl-1 inhibition, suggesting these cell lines are not exclusively Mcl-1-dependent. The hypothesis that survival of lymphoma cells may be co-dependent on both Mcl-1 and Bfl-1 was evaluated by siRNA knockdown. Following a dose-dependent suppression of Bfl-1 protein (>80%) in OCILY10 and TMD8 cells, viability loss was minimal (<30% reduction relative to control). However, when Bfl-1 knockdown cells were treated for 6h with an Mcl-1 inhibitor, the maximum caspase activation increased to over 90% in both cell lines, phenocopying a similar magnitude achieved with AZD4573-mediated CDK9 inhibition. In these models, depletion of both Bfl-1 and Mcl-1 was necessary to induce maximum apoptosis, with studies ongoing to evaluate single-gene Bfl-1 dependency in additional lymphoma models.

Consistent with the in vitro phenotype, intermittent dosing of the ABC-DLBCL xenografts OCILY10 and TMD8 with AZD4573 caused robust tumor regressions (198 and 184% TGI, respectively). AZD4573-mediated anti-tumor activity was associated with pharmacodynamic reductions of pSer2-RNAPII, Mcl-1 and Bfl-1, followed by caspase activation. Collectively, these findings support the ability to target Bfl-1 via CDK9 inhibition. Given the current absence of clinical small molecule Bfl-1 inhibitors and expanded monotherapy activity compared to selective Mcl-1 inhibition in a subset of preclinical models, CDK9 inhibitors have tremendous therapeutic potential in the treatment of patients with Bfl-1-expressing lymphoma.

#2501

Combining BH3-mimetics to target both BCL-2 and MCL1 has potent activity in diffuse large B-cell lymphoma (DLBCL) pre-clinical models.

Cory Mavis, Juan Gu, Matthew Barth, Sumera Khan, Pallawi Torka, Francisco Hernandez-Ilizaliturri. _Roswell Park Comprehensive Cancer Center, Buffalo, NY_.

Background: Upregulation of the anti-apoptotic protein Mcl-1 has been implicated in chemotherapy resistance and poor clinical outcomes in activated B-cell like (ABC)-DLBCL. We hypothesized that singular inhibition of Mcl-1 by AMG-176 and/or dual inhibition of MCL-1 and Bcl-2 by combining AMG-176 with venetoclax could be an effective strategy for inducing cell death in DLBCL pre-clinical models.

Materials and Methods: Using a panel of DLBCL cell lines, we conducted pre-clinical targeting of Mcl-1 with AMG-176. Cells were exposed to AMG-176 as a single agent (0-20uM) over 24, 48, 72 hrs and IC50 concentrations were calculated for each cell line. Subsequently, DOHH-2, OCI-LY1, DHL4, DHL6, TMD8, and VAL cells were exposed to AMG-176 (0-20 uM) in combination with Doxorubicin (0-1uM), Cisplatin (0-10uM), Cytarabine (0-10uM), Vincristine (0-10uM), Ixazomib (0-100nM), Carfilzomib (0-12.5nM), and Venetoclax (0-10uM) for 72 hours. Cell viability was determined by Cell Titerglo. Coefficient of synergy was calculated using CalcuSyn. In addition, induction of apoptosis was evaluated by flow cytometry (Annexin V/PI staining) as well as changes in cell cycle (PI staining). Bcl-2 family member protein expression was determined by Western blotting. Primary tumor cells isolated from a patient with a composite lymphoma: angioimmunoblastic T-cell lymphoma (AITL) and EBV-driven DLBCL were exposed ex vivo to AMG-176 +/- venetoclax. By immune-phenotype, the primary tumor cells isolated and expanded were ABC-DLBCL.

Results: In vitro, AMG-176 single agent exposure induced cell death in a dose- and time-dependent manner in all DLBCL cell lines tested. AMG-176 IC50 values varied from 0.03 to 11.42 uM for OCI-LY1 and ROS-50 respectively. Western blotting studies confirmed changes in Bcl-2 family members, specifically in Bok, Bcl-XL, Mcl-1 and Bcl-2 following in vitro exposure to AMG-176. The combination of AMG-176 with Doxorubicin, Cisplatin, Cytarabine, Vincristine, Ixazomib, Carfilzomib, or Venetoclax resulted in significant synergistic activity for each combination. The strongest coefficient of synergy was observed when AMG-176 was combined with venetoclax. Similar synergy results were noted in primary tumor cells isolated from the lymphoma patient. Annexin/PI staining with AMG-176 demonstrated caspase dependent apoptosis. No significant changes in cell cycle were observed with AMG-176 exposure.

Conclusion: Our data suggests that AMG-176 exhibits strong synergistic activity with various chemotherapy agents and small molecule inhibitors especially venetoclax. In vivo experiments are planned. Combination therapy targeting Mcl-1 and Bcl-2 may provide an alternative therapeutic approach in DLBCL.

#2502

Targeting anti-apoptotic Bcl-2 family for cancer therapy.

Elisabetta Valentini,1 Marta Di Martile,1 Marianna Desideri,1 Simona Carcasole,1 Alexandros Patsilinakos,2 Manuela Sabatino,3 Antonello Mai,4 Gianni Colotti,5 Rino Ragno,3 Daniela Trisciuoglio,5 Donalella Del Bufalo1. 1 _Preclinical Models and New Therapeutic Agents Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy;_ 2 _Alchemical Dynamics, Rome, Italy;_ 3 _Rome Center for Molecular Design, Department of Drug Chemistry and Technology, Sapienza University, Rome, Italy;_ 4 _Department of Chemistry and Technologies of Drugs, Sapienza University, Rome, Italy;_ 5 _Institute of Molecular Biology and Pathology, National Research Council, Rome, Italy_.

Introduction: Despite the recent advancement in treating melanoma, options are still limited for patients without BRAF mutations or in relapse from current treatments, and promising, immunotherapies are not without caveats. Thus, alternative options are still in need.

Bcl-2 protein is the main member of a family whose best-characterized function is to regulate apoptosis in several pathologies, including melanoma. To evade apoptotic checkpoints, melanoma cells often overexpress Bcl-2 and other antiapoptotic proteins. Consequently, along with the fact that antiapoptotic proteins play a critical role in the efficacy of most chemotherapeutics and play a relevant role in melanoma progression, they have emerged as attractive targets for therapeutic development. Structure-based efforts led to the development of ABT-199 (also known as venetoclax), recently approved by the Food and Drug Administration for the treatment of chronic lymphocytic leukaemia patients.

Experimental procedures: To identify new possible selective inhibitors towards antiapoptotic proteins belonging to the bcl-2 family, a computational medicinal chemistry protocol was set up. Both the PubMed/ChEMBL databases, containing known ligands for bcl-2, bcl-w and bcl-xL proteins and their associated affinities, and the RCSB Protein Data Bank listing 65 bcl-2 family protein structures, were used to build preliminary Quantitative Structure-Activity Relationships (QSAR) models. The QSAR models were used to screen the VITAS-M Laboratory database to select 60 chemical scaffolds as hypothetical novel Bcl-2 inhibitors. Surface Plasmon Experiments were carried out to assess affinity of compounds for Bcl-2. The sensitivity of human leukemia and melanoma cells to these compounds was evaluated in terms of cell survival, cell cycle distribution and apoptosis induction, by using different concentrations and time exposure.

Results: 9 out of 60 compounds (15%) were able to reduce cell viability at concentrations lower than 50µM, and to induce cell death in a dose-dependent manner, even though at different extend depending on the compound tested. Inclusion of the 60 selected compounds led to a new QSAR model to perform a second virtual screen on the NIH database from which 40 putative Bcl-2 target molecules were selected and whose efficacy to inhibit cell viability of both leukemia and melanoma cells was measured. 11 compounds are endowed with high affinity for Bcl-2, 2 out of 40 compounds were demonstrated to induce apoptosis in both tumor histotypes in a dose-dependent manner, while they did not affect cell viability of human normal fibroblasts.

Conclusion: Machine learning application coupled with experimental data have provided new insights into bcl-2 family's regulation of the apoptotic pathway both in leukemia and in melanoma, and suggest that inhibiting the major anti-apoptotic proteins is sufficient to induce cell death.

#2503

Predicting drug sensitivity to a novel BCL-2 and BCL-xL dual inhibitor in solid tumor PDX trials.

Jing Deng, Xiaojing Huang, Guoqin Zhai, Tingting Mao, Yan Yin, Douglas D. Fang, Dajun Yang, Yifan Zhai. _Ascentage Pharma (Suzhou) Co., Ltd, China_.

Background: BCL-2, an anti-apoptotic protein and oncogene, has been considered one of the top 10 targets for cancer drug development. The inhibition of BCL-2 and other anti-apoptotic proteins will help to restore the apoptotic pathway. Aiming to target much diverse BCL-2 family protein dependency in solid tumors, we have developed a potent BCL-2 and BCL-xL dual inhibitor APG-1252, which is currently in phase 1 trials for solid tumors in USA, China and Australia. Mechanistically, BCL-2 and BCL-xL protect cells from cell death by sequestering pro-death BH3-only proteins like BIM. Thus, BCL-2 and BCL-xL inhibitors are designed to disrupt the protein complexes and release pro-death proteins to trigger downstream apoptotic cascade. In this study, we intended to further understand the mechanism of action of APG-1252 in vivo and factors regulating its sensitivity. To this end, we conducted trials in SCID mice carrying patient-derived xenograft (PDX) tumors, and studied target engagement.

Methods and Experiments: We first selected eleven gastric and esophageal cancer PDX models that bore high levels of BCL-xL (BCL-xLhigh) but normal levels of MCL-1 (MCL-1nor) based on the gene expression profiles and the consideration of that BCL-xL may play a major role in tumorigenesis in solid tumors and MCL-1 a resistant factor for the dual inhibitor. Mice bearing PDX tumors were intravenously treated with 100 mg/kg of APG-1252 twice per week for three weeks. At the end of treatments, tumor samples were collected. An advanced ELISA system (MSD, Meco Scale Discovery) and Western blotting were performed to detect BCL-2 and BCL-xL protein complex and individual proteins.

Results: 1. The MSD assays revealed dominant BCL-xL:BIM complex signals in the solid tumor PDX samples in comparison with hematological malignancy cell lines, in which BCL-2:BIM was the major complex. 2. The dual inhibitor treatment disrupted BCL-xL:BIM complex, thus BCL-xL:BIM complex may serves as a pharmacodynamic (PD) marker for target engagement. 3. BCL-2 family protein profiles showed high BCL-xL and MCL-1 levels in the solid tumor PDXs. The treatment with APG-1252 led to a significant increase in BCL-2 protein levels, while there was no major change on pro-apoptotic BH3-only proteins BIM and PUMA. 4. The more efficacious responders to the treatment exhibited a more substantial decrease in BCL-xL:BIM complex signals and low MCL-1 protein levels, whereas a much less significant decrease in BCL-xL:BIM complex and higher MCL-1 levels were found in the poor or non-responders.

Conclusion: Both BCL-xL protein and BCL-xL:BIM complex are dominant in the solid tumor PDX tissues. The decrease of BCL-xL:BIM complex indicates the on-target activity after the treatment with APG-1252. Thus, the complex may be used to monitor the target engagement and, potentially, a predictive biomarker for APG-1252 efficacy. These assays may be applied to patient samples to guide the clinical trials.

#2504

Predictive biomarkers for Bcl2-inhibitor use in neuroblastoma.

Claudia Zapata,1 Jorida Coku,1 Kangning Liu,1 Annette Vu,1 Michael Goutnik,1 C. Patrick Reynolds,2 Kelly Goldsmith,3 Michael Hogarty1. 1 _Children's Hospital of Philadelphia/University of Pennsylvania, Philadelphia, PA;_ 2 _Texas Tech University Health Sciences Center, Lubbock, TX;_ 3 _Children's Healthcare of Atlanta/Emory University, Atlanta, GA_.

Apoptosis commitment is regulated by the interaction of Bcl2-family proteins at the mitochondria. Survival proteins such as Bcl2 and Mcl1 bind stress-activated BH3 proteins (like Bim, Puma and Bid), to prevent them from activating the obligate executioner proteins Bak and Bax and provides a survival advantage. Such apoptosis evasion is a cancer hallmark. Neuroblastoma (NB) is a highly lethal childhood tumor in which therapy resistance contributes to treatment failure. We used mitochondrial profiling and co-IP to define NB survival dependencies and develop biomarkers to predict response to Bcl2-inhibitors (BCL2i) such as Venetoclax as well as newer Mcl1 inhibitors (MCL1i). Mitochondrial profiling was used to interrogate isolated mitochondria with diverse BH3-only proteins to define their stress response "set-point" and infer their survival protein dependency. NBs can be classified as Bcl2- or Mcl1-dependent. The former have Bim sequestered by Bcl2 (confirmed by coIP) and a dominant mitochondrial response to BikBH3, the latter have Bim sequestered by Mcl1 (Bim:Mcl1 by coIP) and a dominant NoxaBH3 response. While Bcl2 and Mcl1 proteins are frequently co-expressed, a dominant survival protein is identified functionally. Bcl2-dependent NBs are highly sensitive to BCL2i's (Venetoclax and Navitoclax) with an IC50<<1 uM and displacement of Bim from Bcl2 by drug. Mcl1-dependent NBs are BCL2i resistant as anticipated (IC50>2 uM). Surprisingly, no NB was sensitive to the MCL1i, S63845, as monotherapy, including the Mcl1-dependent subset. However, S63845 markedly sensitizes Mcl1-dependent NBs to BCL2i's (shifting IC50>1 log in all tested). Ongoing mechanistic work tests a hierarchical model in which Bcl2 can sequester Bim displaced from Mcl1 (by an MCL1i), sensitizing a previously Mcl1-dependent NB to a BCL2i. In contrast, Bim displaced from Bcl2 by a BCL2i (in a Bcl2-dependent NB) does not bind Mcl1 but ostensibly interacts with Bak/Bax to induce death. To measure Bim:Bcl2 and Bim:Mcl1 as a predictive biomarker we are developing proximity ligation assays (PLAs) for use with FFPE-tumor slides. NB xenografts of defined survival dependency are being used to optimize these assays. Heterogeneous survival dependencies define NB sensitivity to Bcl2-family inhibitors. We propose a hierarchical binding model where BCL2i monotherapy sensitivity is defined by Bim:Bcl2 binding (Bcl2-dependency). Mcl1-dependent NBs are resistant to MCL1i's, yet by displacing Bim these agents restore sensitivity to BCL2i's. Lastly, development of clinically-relevant biomarker assays to define Bim binding will allow stratification of patients in clinical trials with Bcl2-family inhibitors.

### Cancer Genomics 3

#2505

Comprehensive molecular and genomic characterization of pancreatic tropism in metastatic renal cell carcinoma.

Nirmish Singla,1 Jacob Choi,2 Oreoluwa Onabolu,1 Layton Woolford,1 Christina Stevens,1 Vanina Tcheuyap,1 Tiffani McKenzie,1 Zhiqun Xie,1 Tao Wang,1 Renee McKay,1 Alana Christie,1 Payal Kapur,1 Brian Rini,2 James Brugarolas1. 1 _University of Texas Southwestern Medical Center, Dallas, TX;_ 2 _Cleveland Clinic, Cleveland, OH_.

Introduction & Objectives: Patients with metastatic renal cell carcinoma (mRCC) involving the pancreas have been shown to exhibit a relatively indolent course, yet the biologic explanation is unclear. We sought to characterize the genomic landscape of patients with mRCC harboring pancreatic metastases (PM) to identify molecular drivers of pancreatic tropism.

Materials & Methods: mRCC patients harboring PM from UTSW and Cleveland Clinic were identified. Clinicopathologic data and oncologic outcomes were analyzed. Samples were obtained from primary tumors, metastatic sites (including pancreatic or other distant metastases), and matched normal tissue. Whole exome (WES) and RNA sequencing of tumors was conducted. Patient-derived xenograft (PDX) models were generated from a subset of patients, and the engrafted tumors were analyzed.

Results: 31 mRCC patients with PM were included with 54 tumor samples derived from the primary tumor or thrombus (24), PM (21), or other metastatic sites (9). Median follow-up was 101 months. Clinicopathologic characteristics were similar between the two institutional cohorts, and all but one patient were favorable or intermediate IMDC risk. All patients had clear cell histology. 8 patients (26%) were metastatic at diagnosis, and median time to metastasis in the remaining patients was 74 months (IQR 32-120). Overall (OS) and cancer-specific (CSS) survival did not vary by IMDC risk group. OS was strikingly superior for mRCC patients with PM than a historic control of mRCC patients without PM (p<0.001), even after controlling for IMDC risk score. Morphologically, tumors largely displayed low-grade acinar patterns. WES with matched normal tissue and RNAseq were completed with adequate quality for 48 and 30 samples, respectively. 14 PDX lines were generated, of which 5 (36%) engrafted stably (≥2 passages). WES from 2 tumorgraft specimens revealed preservation of specific mutations in the corresponding human samples.

Conclusions: mRCC patients with PM exhibit remarkably favorable survival outcomes. The relatively indolent biology of these tumors is reflected histologically and genomically and can be recapitulated in PDX models. Understanding tumor heterogeneity may help refine prognostic models for mRCC and hold implications for improved personalization of therapy.

#2506

Exploring the complex etiology of oncogenic fusions in childhood cancer.

Nathaniel D. Anderson,1 Richard de Borja,1 Matthew D. Young,2 Fabio Fuligni,1 Andrej Rosic,1 Nicola D. Roberts,2 Nischalan Pillay,3 Jeffrey A. Toretsky,4 Yoshida Akihiko,5 Tatsuhiro Shibata,5 Markus Metzler,6 Gino Somers,1 Stephen W. Scherer,1 Adrienne M. Flanagan,3 Peter J. Campbell,2 Joshua D. Schiffman,7 Mary Shago,1 Ludmil B. Alexandrov,8 Jay S. Wunder,9 Irene L. Andrulis,9 David Malkin,1 Sam Behjati,2 Adam Shlien1. 1 _The Hospital for Sick Children, Toronto, Ontario, Canada;_ 2 _Wellcome Sanger Institute, United Kingdom;_ 3 _University College London, United Kingdom;_ 4 _Georgetown University, Washington, DC;_ 5 _National Cancer Center Hospital, Japan;_ 6 _University Hospital Erlangen, Germany;_ 7 _Huntsman Cancer Institute, Salt Lake City, UT;_ 8 _University of California and Moores Cancer Center, San Diego, CA;_ 9 _Lunenfeld-Tanenbaum Research Institute, Toronto, Ontario, Canada_.

Background, Rationale and Experimental Approach

Oncogenic fusions are generated via chromosomal rearrangements resulting in an exchange of coding or regulatory DNA sequences. These mutations play an important role in disease onset and subsequent cancer progression, however the exact timing and mechanisms by which they arise are unknown. Through the SickKids clinical sequencing program, KiCS, we explored how and when canonical fusions arise by studying the whole-genomes of childhood cancers with diagnostic or driver fusions. Our investigation began with the pediatric bone cancer, Ewing sarcoma, and later expanded to include other solid, blood, and brain cancers such as papillary thyroid carcinoma, myeloid leukemia, and ependymoma among others.

Results

The starting point of our investigation was ES, where we sequenced the whole-genomes of 124 cases. Ewing sarcoma (ES) represents the prototypical fusion-driven sarcoma as it is characterized and driven by the EWSR1-ETS fusion. In ~42% of cases, we found that the ES fusion gene arises by chromoplexy, a sudden burst of complex, loop-like rearrangements, rather than by simple reciprocal translocations as previously thought. We show that these rearrangements rapidly and dramatically altered the chromosomal landscape of ES tumors, producing the driver EWSR1-ETS fusion and disrupting numerous other genes in a short time. Remarkably, these complex rearrangements are enriched for genes, including those with a clear role in oncogenesis, and are associated with the earliest replicating portions of the genome. We then sequenced the genomes of 30 other childhood cancers with oncogenic fusions to study their timing and formation mechanisms. In doing so, we have identified several novel fusions in many cancer types, which have been validated by RNA sequencing and cytogenetics. In some cases, the presence of these chromoplectic fusions indicates these patients may benefit from targeted therapy due to the generation of druggable fusions. .

Conclusions

Our findings provide fundamental insights into the pathogenesis of gene fusions in human cancer. They reveal complex DNA rearrangements to be a mutational process underpinning gene fusions in cancer that influences tumorigenesis.

#2507

Genetic alterations affecting treatment response in locally advanced breast cancers.

Andreas Venizelos, Stian Knappskog, Inger Marie Loees, Hans Petter Eikesdal, Per Eystein Lønning. _University of Bergen, Bergen, Norway_.

Resistance to chemotherapy remains a main cause of death among cancer patients. The primary aim of the present study was to identify genetic alterations predicting resistance to chemotherapy by whole exome sequencing (WES) of tumors subject to neoadjuvant sequential treatment with epirubicin and docetaxel. A long term goal is to identify biomarkers that may be applicable in testing for drug sensitivity prior to commencement of therapy for individual patients, leading to early administration of optimal treatment and sparing patients from side-effects of inefficient treatment. From a total of 100 patients treated with sequential monotherapy in the neoadjuvant setting, we performed WES on 146 tumor samples and matching blood samples from 51 patients. Biopsies for research were taken before treatment start, at the time of treatment switch and at the time of surgery. In the present work, we have analysed WES data to assess the mutational- and the copy number landscape and compared these results between patients having a good or poor response to the two administered drugs. SNV analysis revealed imbalance in several well established breast cancer related genes, between the different response groups. Copy number assessments revealed a general increase in copy number gains in chromosome 16 and increase of copy number losses in chromosomes 1 and X after treatment with epirubicin, indicating that subclones harbouring different types of CNVs are selected for during treatment. Correspondingly, after treatment with docetaxel we found a general increase of copy number gains in chromosome 8 and an increase of copy number losses in chromosomes 1 and 8. Finally, we assessed overall mutational signatures and observed shifts in these profiles during the two treatments.

#2508

**Genomic alterations of** BRCA1/2 **genes in Chinese solid tumor patients.**

Junjian Wang,1 Lingxiang Liu,2 Bin Ni,3 Xiaoqian Chen,4 Ling Li,4 Junping Shi,4 Jierong Chen,4 Ming Yao4. 1 _Zhejiang Cancer Hospital, Hangzhou, China;_ 2 _Jiangsu Province Hospital, Nanjing, China;_ 3 _First Hospital Affiliated of Suzhou University, Suzhou, China;_ 4 _OrigiMed, Shanghai, China_.

Background: BRCA1 and BRCA2 are tumor suppressor genes that play an important role in DNA repair pathways. Germline mutations in BRCA1 and BRCA2 contribute to a significant number of familial and hereditary breast and/or ovarian cancers. Additionally, BRCA 1/2 alterations induce sensitivity to poly ADP ribose polymerase inhibitors (PARPi). Therefore, the detection of BRCA 1/2 mutations in solid tumor patients is essential to predict the sensitivity to PARPi therapy.

Methods: FFPE tumor and matched blood samples of 5269 Chinese patients with solid tumor were collected for next generation sequencing (NGS) based assay. Genomic alterations including single nucleotide variations (SNV), short and long insertions/deletions (Indel), copy number variations (CNV) and gene rearrangements and fusions in selected genes were assessed.

Results: Three hundred patients with BRCA1/2 mutations were identified in 5269 Chinese patients with solid tumor. The patients included 177 males and 123 females with a median age of 58. Highest yields of BRCA1/2 mutations were found in patients with ovarian (24.5%) and breast (12.3%) cancers. BRCA1/2 mutations were also identified in gastric cancer (8.9%), cholangiocarcinoma (8.8%), colorectal cancer (7.1%), esophageal cancer (6.1%), gallbladder cancer (5.1%), lung cancer (4.7%), soft tissue tumor (4.1%) and pancreatic cancer (4.0%). SNVs and short Indels (84.9%) were the most common variant types of BRCA1/2, while the percentages of gene rearrangements and fusions, CNV and long Indels were 12.0%, 1.6% and 1.5%, respectively. Among patients with BRCA1/2 mutations, 224 (74.7%) patients harbored somatic mutations, 73 (24.3%) patients harbored germline mutations, 3 (1.0%) patients harbored both somatic and germline mutations. Interestingly, all of the variant types of germline mutations were SNVs and short Indels. The mutation sites were distributed in the full length of BRCA1/2 genes. No hot spot mutation was observed.

Conclusions: Our data revealed that BRCA1/2 mutations occurred in 5.7% of Chinese patients with solid tumor. NGS targeted sequencing provides comprehensive and accurate information of BRCA1/2 mutations. Beyond ovarian and breast cancers, BRCA1/2 mutations could be detected in other solid tumors, which suggests potential clinical benefits from PARPi therapy.

#2509

Analysis of clonal hematopoiesis-associated mutations in the cell-free DNA of advanced cancer patients.

Jennifer Yen, Katie Quinn, Elena Helman, Andrey Chursov, Tracy Nance, Ariel Jaimovich, Kimberly Banks, Aleksandra Franovic, Kristin Gleitsman, John Latham, Arielle Yablonovitch, Marcin Sikora, Stephen Fairclough, Darya Chudova, Richard B. Lanman, AmirAli Talasaz. _Guardant Health, Redwood City, CA_.

Background: Clonal hematopoiesis (CH) is the acquisition of mutations in hematopoietic progenitor cells that can lead to clonal expansion. Recent studies suggest that CH-derived mutations can confound interpretation of cell-free DNA (cfDNA) sequencing results. To better understand the contribution of CH to cfDNA analysis in the metastatic cancer setting, we characterized CH-associated alterations observed in the cfDNA of late-stage cancer patients.

Methods: We analyzed somatic variants from cfDNA profiles of over 62,000 patients in four late-stage cohorts: lung (>35,000), gastro-intestinal (>14,000), urogenital (>4,700), breast (>8,600). Matched white blood cell (WBC) and cfDNA was obtained for a subset of patient samples. Plasma cfDNA was processed using a 73-gene (150Kb, Guardant360TM) or 500-gene (2Mb, GuardantOMNITM) panel and sequenced to average depth of ~5000 molecules, with a 95% limit of detection (LOD) of 0.3-0.4% and 0.15-0.6% variant allele fraction (VAF) for SNVs, and 0.2%-0.7% and 0.4-0.8% for indels (for G360 and OMNI, respectively).

Results: In samples processed on the 500-gene panel, DNMT3A, TET2, PPM1D, ASXL1 and SF3B1 were the most frequently mutated CH-associated genes (75% of samples). While the majority (73%) of these mutations were at low variant allele fractions (VAF) of <1%, CH VAFs ranged from 0.1% to 71.3%, and 20% of samples had ≥ 5 CH-associated variants. Tumor-associated VAFs across the same cohort ranged from 0.01% to 90.3%. In the 73-gene panel cohort of >60,000 samples, JAK2 V617F, a known CH mutation, was observed in 927 samples with VAFs between 0.29% and 98%. Across the combined cohort, we found that in samples with a known CH variant (n=7,717), half of samples had a max CH VAF less than 50% of max tumor VAF. Interestingly, in >25% of samples, the max CH VAF was higher than the max tumor VAF. Initial comparisons of matched plasma and WBC DNA show that 100% (16/16) of clinically relevant and 92% (84/91) of variants of unknown significance (VUSs) across the 73-gene panel were found exclusively in plasma DNA and not in WBC DNA testing.

Conclusions: We characterize the distribution of CH mutations across a large number of late-stage plasma samples and show that within highly pre-treated metastatic patients, the VAFs of CH mutations often differ from the tumor and can surpass the level of tumor shedding in circulation. Further investigation of these variants will enable improved understanding of CH in metastatic disease and its differentiation from the circulating tumor DNA.

#2510

Combining genetic and histopathologic features to predict response to anti-androgen therapy in aggressive prostate cancer.

Scott Wilkinson,1 Huihui Ye,2 Nicole Carrabba,1 Rayann Atway,1 Shana Y. Trostel,1 Thomas Hennigan,1 Ross Lake,1 Stephanie Harmon,1 Guinevere Chun,1 Baris Turkbey,1 Peter A. Pinto,1 Peter L. Choyke,1 Fatima Karzai,1 David J. VanderWeele,1 Kathleen Kelly,1 William L. Dahut,1 Adam G. Sowalsky1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Beth Israel Deaconess Medical Center, Boston, MA_.

Background: Genetic alterations in lethal, metastatic prostate cancer include the loss of PTEN, translocation of the TMPRSS2 and ERG genes, upregulation of the androgen receptor (AR), and disruption of the DNA homologous repair pathway driven by mutations to BRCA1, BRCA2, and ATM. Many of these mutations can be observed while the cancer is still localized in the prostate. Here, we seek to identify genetic features indicative of therapeutic response and novel drivers of cancer progression to inform clinical practice.

Methods: Using tissue from 33 patients in an intense neoadjuvant anti-androgen clinical trial at the NCI (NCT02430480), we examined genetic features that would predict exceptional or poor response to anti-androgen enzalutamide therapy. Each patient on trial presented with multiple tumor foci, allowing us to investigate the intratumoral heterogeneity across foci within individual patients, as well as investigating the tumor profiles across multiple patients. Several patients exhibited exceptional response with residual tumor burdens less than 0.5cc, while others had substantial treatment-resistant cancers. We developed a panel of 12 immunohistopathological stains and used this panel to guide laser capture microdissection on pre-treatment biopsies and spatially matched post-treatment radical prostatectomy specimens to isolate ultrapure tumor foci from each patient. DNA from these foci was used for whole exome sequencing, as somatic copy number alterations and mutations also confirm their evolutionary relationship. This enabled us to classify baseline specimens as responder or nonresponder, while examining variations in genetic features between the two cohorts.

Results: To date, focal PTEN loss was observed in all nonresponders, while focal ERG staining was absent in 100% of responders and present in 60% of nonresponders. Synaptophysin positivity was rare at baseline but predicted resistance to treatment with 100% sensitivity. Intriguingly, baseline copy number profiles highlight a 6q deletion in 100% of exceptional responders, but not in the non-responders. Together, these data suggest an immunostain panel to assess oncogene and tumor suppressor alterations can predict response to anti-androgen therapy, while also suggesting a novel role of 6q in resistance to anti-androgen hormone therapy. Current studies are examining the role of 6q in response to anti-androgen therapies, which remains an area of active interest in our laboratory.

Conclusions: These findings demonstrate the feasibility in identifying intratumoral heterogeneity based on prostate biomarker status both in pre-treatment and post-treatment specimens. Using these data, comprehensive molecular analysis of prostate cancer at diagnosis may better-enable physicians to predict response to anti-androgen therapy and provide tailored treatment based on gene expression status.

#2511

Analysis of circulating tumor DNA by targeted ultra-deep sequencing across various non-Hodgkin lymphoma subtypes.

Yeon Jeong Kim, Seung-Ho Shin, Danbi Lee, Woong-Yang Park, Seok Jin Kim, Won Seog Kim, Donghyun Park. _Samsung Medical Center, Seoul, Republic of Korea_.

Although targeted deep sequencing of cell-free DNA (cfDNA) was recently used to investigate tumor somatic mutations in particular subtypes of non-Hodgkin lymphomas (NHLs), the immense genetic heterogeneity across subtypes poses a hurdle to design a universal gene panel applicable for diverse subtypes of NHLs. We designed a panel targeting 66 genes associated with NHLs and performed targeted deep sequencing to analyze plasma cfDNA from patients with various subtypes of NHLs. Genetic profiling in plasma cfDNA using the method resulted in 88.0% sensitivity and >99% specificity in detecting mutations present at a frequency greater than 20% in the tumor biopsies. Furthermore, the level of ctDNA significantly decreased and increased depending on designated clinical responses to therapy and disease progression. These results demonstrated that ctDNA sensitively indicated the presence of cancer and reliably correlated with tumor burden, suggesting potential utility of the method for patients with various subtypes of NHLs.

#2512

A comprehensive genomics platform for precision immunotherapy: Simultaneously characterizing the tumor and tumor microenvironment from a single FFPE sample.

Richard O. Chen. _Personalis, Burlingame, CA_.

Immuno-genomic profiling of the tumor and the tumor microenvironment (TME) is critical for identifying new biomarkers of immunotherapy response, understanding resistance, and enabling personalized immunotherapies. However, running a comprehensive array of biomarker assays for each patient sample is often impractical given limited tumor sample, processing complexity, and prohibitive cost.

To address these challenges, we developed ImmunoID NeXT, a novel, augmented exome and transcriptome based diagnostic platform that simultaneously characterizes the tumor and TME from a single FFPE sample. We co-optimized the design of our sequencing assays and analytics to increase performance for detecting somatic mutations across ~20,000 genes, neoantigens, expression signatures, HLA-typing, T-cell receptor (TCR) and B-cell receptor repertoire, oncoviruses, tumor infiltrating lymphocytes (TILs), clinically actionable mutations, tumor mutational burden (TMB), and MSI status. We developed novel methods to sequence difficult regions of the exome and to extend coverage to key immuno-genomic biomarkers. Analytic pipelines were designed to utilize assay optimizations to achieve higher accuracy than achievable with other platforms. We then validated ImmunoID NeXT for diagnostic and therapeutic use.

With a minimum of 50ng of DNA per FFPE sample and co-extracted RNA, ImmunoID NeXT completely covers between 17% to 40% more genes compared to a non-augmented exome, thus increasing sensitivity to somatic mutations and putative neoantigens. For neoantigen performance, we generated immuno-peptidomic data from mono-allelic HLA transfected cell-lines and trained neural networks to predict neoepitope binding to MHC, demonstrating a higher precision (0.88) across alleles than publicly available tools (<0.7). For TCR clone abundance in tumor samples, we demonstrate strong correlation with a stand-alone TCR panel results (R2>0.94). For TILs, we developed signatures for CD4, CD8 T-cells, and other immune cells, demonstrating concordance with synthetic and CyTOF-derived validation sets. For HLA-typing, we achieve an accuracy of 99.1% for HLA Class I, and 95% for HLA Class II typing calls. We demonstrate sensitive detection of HPV, EBV, HCV, HTLV-1, and KSHV in known samples, and accurate MSI and TMB assessment. Finally, for diagnostic reporting, we achieve high sensitivity and specificity for clinically reportable mutations comparable to diagnostic cancer panels.

We have developed a novel immuno-genomics platform, ImmunoID NeXT, that can characterize both the tumor and TME from a single sample. By co-optimizing our assay and analytics for immuno-oncology, we enhance biomarker sensitivity compared to non-optimized genomics assays. Validation of the ImmunoID NeXT platform extends its use to diagnostics and personalized immunotherapies.

#2513

Mutational and transcriptional landscape of nodal marginal zone lymphoma.

Jiwon Koh,1 Seongmin Choi,2 Insoon Jang,2 Sehui Kim,1 Cheol Lee,1 Jin Ho Paik,3 Kwangsoo Kim,2 Yoon Kyung Jeon4. 1 _Seoul National University Hospital, Seoul, Republic of Korea;_ 2 _Biomedical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea;_ 3 _Seoul National University Bundang Hospital, Seongnam-si, Republic of Korea;_ 4 _Seoul National University College of Medicine, Seoul, Republic of Korea_.

Nodal marginal zone lymphoma (NMZL) is a rare B-cell neoplasm lacking disease-defining markers. In this study, we aimed to determine the mutational profile of Asian NMZLs and report novel genetic features of this disease. We performed WES on FFPE samples of eight Korean NMZLs and two matched non-neoplastic tissue samples. For unmatched cases, additional variant filtering was performed to minimize germline contamination. RNAseq was performed on six NMZL FFPE samples with acceptable samples quality. A total of 394 non-synonymous variants in 370 genes were found with the mean number of variants per case of 49.3 (range 14 – 98). We selected 93 candidate NMZL genes (CNGs) based on the following criteria: (i) genes with mutations in ≥ 2 patients; (ii) genes with mutations in either of two cases with matched control; (iii) genes with mutations in at least one case and reported to have biological roles in B-cell lymphomas. Mutations in genes involved in chromatin remodeling including KMT2D (25%), HIST1H1E (25%), and HIST1H1B (13%) were observed, which was consistent with previous studies. Genes encoding key components of B-cell receptor (BCR) signaling and related pathways were also affected, including CD79B (25%), CARD11 (13%) and BCL6 (13%). Of note, NFKBIE (c.759_762delTTAC) frameshift mutation was found in one patient; this mutation was proven to be loss-of-function in nature, resulting in deregulation of NF-κB signaling. Identical mutation was reported in Hodgkin lymphoma and primary mediastinal B-cell lymphoma, however, this is the first report of this mutation in NMZL. Genes involved in immune response including MYD88 (13%), PIM1 (13%), CD70 (13%) and TNFRSF14 (13%) were also mutated, suggesting the linkage between immune dysregulation and NMZL pathogenesis. At the transcriptome level, three samples were consistently clustered together. This group of patients was characterized by higher Ki-67 proliferation index compared to the others, and was associated with older age, higher serum LDH level and higher IPI score. Gene set analysis (GSA) based on lymphoma-specific gene sets from SignatureDB (https://lymphochip.nih.gov/signaturedb/) showed significant association between XBP1 target

genes and these high Ki-67 group (P = 0.024). In summary, this study showed high prevalence of mutations in chromatin remodeling and BCR signaling pathway, readdressing the importance of these pathways in NMZL. Several novel genetic alterations in NMZL were found including NFKBIE c.759_762delTTAC, warranting further validation to clarify the significance of these alterations. We also found that NMZLs with high Ki-67 index were associated with specific gene expression profile, providing clue to the explanation for the heterogeneity of NMZLs.

#2514

Inter-test variability in tumor mutational burden (TMB) quantification and identification of TMB thresholds.

Natalie Vokes,1 Elizabeth Jimenez Alguilar,2 Renato Umeton,1 Anika Adeni,1 Lynette Sholl,3 Matthew Hellmann,4 Hira Rizvi,4 Mark Awad,1 Eliezer Van Allen1. 1 _Dana Farber Cancer Institute, Boston, MA;_ 2 _3 University Hospital 12 de Octubre, Madrid, Spain;_ 3 _Brigham and Women's Hospital, Boston, MA;_ 4 _Memorial Sloan Kettering, New York, NY_.

Introduction:

Tumor mutational burden (TMB) is a possible predictive biomarker for response to immune checkpoint inhibitors (ICI). However, there is significant heterogeneity in the calculation of TMB and the thresholds that have been identified to associate with clinical benefit. We analyzed TMB and clinical outcomes across next generation sequencing (NGS) panels to address inter-panel heterogeneity and to identify consistent TMB thresholds.

Methods:

Non-small cell lung cancer sequencing data generated from three validated clinical platforms (OncoPanel n = 1155, MSK-IMPACT n = 1520, and Foundation Medicine n = 3377) and whole exome sequencing from The Cancer Genome Atlas (TCGA, n = 1144) were obtained. Tumor mutational burden (TMB) was calculated across cohorts as the number of nonsynonymous mutations per length of genome sequenced. The distribution of TMB was plotted for each cohort and compared to TCGA. For the subset of patients treated with ICI (OncoPanel n = 296; MSK-IMPACT,n = 227), durable clinical benefit (DCB) was defined as responsive/stable disease lasting ≥ 6 months and was associated with TMB.

Results:

There was high concordance across sequencing panels in the mean number of mutations per individual gene (OncoPanel vs TCGA R2 = 0.8914; Foundation vs TCGA R2 = 0.9025; MSK-IMPACT vs TCGA R2 = 0.8579). Absolute TMB values were higher in NGS panels compared to TCGA, likely due to enrichment for oncogenes and differences in germline filtration. Harmonization between NGS panels and WES was attempted by applying a linear transformation of panel TMB values to WES TMB values. Linear transformation constants were variable at the highest and lowest TMB values, and in low TMB subgroups such as EGFR mutants and never smokers. Consistent with prior studies, TMB was higher in patients with DCB compared to those with no durable benefit (NDB) (Oncopanel p-value=0.003; MSK-IMPACT p-value=0.006). Increasing TMB thresholds were associated with increase in DCB rate, likely due to enrichment of the highest TMB responders; DCB rates did not linearly associate with TMB at lower TMB values.

Conclusion:

There were significant differences in TMB quantification among NGS and TCGA panels, particularly at lower TMBs. Categorization, rather than fitting absolute values, may be an approach for harmonizing distinct panels. In these cohorts, the relationship between benefit of ICI and TMB was not consistent across the TMB spectrum but accentuated in the highest TMB patients. These findings may have implications for the application of TMB as a biomarker in clinical practice.

#2515

Comprehensive analysis of the histologically distinct components of an adenosquamous carcinoma of the lung.

Arthur Krause, Maria R. De Filippo, Thomas Lorber, Spasenija Savic, Salvatore Piscuoglio, Charlotte K. Ng, Lukas Bubendorf. _University Hospital of Basel, University of Basel, Basel, Switzerland_.

Background: Adenosquamous carcinoma (ASC) is a rare subtype of non-small-cell lung cancer, consisting of an adenocarcinoma (AC) and a squamous cell carcinoma (SCC) component. Little is known about the molecular makeup of the two components in ASC. In this study, we applied a comprehensive analysis including whole exome sequencing (WES) and RNA sequencing to understand the evolutionary relationship between the AC and SCC components of an ASC of the lung.

Methods:FFPE tumor tissue of three ASC patients was used in this study. Samples were stained with FastRed to visualize the nuclei. AC and SCC components were scratched separately from histological slides for DNA and RNA extraction. WES analysis was performed to assess the mutational profile and copy number alterations (CNAs). The transcriptome was investigated in each of the components using RNA sequencing.

Results:WES analysis of the AC and SCC components of one patient revealed a common trunk of 118 mutations, including driver mutations EGFR exon 19 deletion and TP53 p.192*, indicating a common clonal origin of the components and the presence of bona fide drivers in the common ancestor. AC shows more private mutations than SCC (234 vs 134). Potential drivers are private to AC included TP53 and AKT1 mutations, while TLX1 and TRBV5-1 mutations are restricted to SCC. AC and SCC have highly similar CNA profiles, suggesting that all CNAs are early evolutionary events of the ASC. Mutational signature of the truncal mutations is similar to that of the mutations private to AC, while mutations private to SCC showed a distinct pattern enriched in T>G, suggesting that SCC may have derived from an AC ancestor cell. Transcriptomic profiling shows that genes related to oxidative phosphorylation, fatty acid metabolism and peroxisome pathways are highly expressed in AC, in contrast to genes related to epithelial-mesenchymal-transition, hedgehog signaling and IL6-JAK-STAT3-signaling pathways in SCC.

Conclusion:Macroscopic dissection and separate analysis of the histologically distinct components of an ASC of the lung demonstrated the clonal relatedness of the AC and SCC. The diverse phenotype of the components is associated with distinct genetic profiles and mutational signatures. Analysis of additional ASCs of the lung may reveal genetic and/or transcriptomic underpinnings of the phenotypes.

#2516

A bioinformatics framework for high-sensitivity detection and monitoring of oncogenic gene fusions in plasma cfDNA.

Juber Patel, Maysun Hasan, Fanli Meng, Xiaohong Jing, Grittney Tam, Ian Johnson, Youyun Zheng, Chaitanya Bandlamudi, Caitlin Stewart, Helen Won, Oliver Hampton, Alison Schram, Ezra Rosen, Alexander Drilon, Anna Varghese, David Hyman, Dana Tsui, Brian Houck-Loomis, Michael Berger. _Memorial Sloan Kettering Cancer Center, New York, NY_.

Gene fusions represent key oncogenic driver alterations in many solid tumors that can be targeted therapeutically. Beyond FDA-approved drugs targeting ALK and ROS1 fusions in lung cancer, investigational agents targeting gene fusions involving RET, NTRK1/2/3, and FGFR2/3 have elicited significant and durable responses in a wide range of cancer types. Cell-free DNA (cfDNA) profiling provides a transformative opportunity to non-invasively and longitudinally monitor responses to these investigational therapies and identify acquired mutations that confer drug resistance. We have developed a pan-cancer, high-sensitivity NGS cancer assay (MSK-ACCESS, presented previously) to detect and monitor somatic mutations in plasma cfDNA. Here, we present an improved bioinformatics pipeline that detects gene fusions and achieves greater sensitivity and accuracy for detecting point mutations and indels.

MSK-ACCESS uses ultra-high depth sequencing (~20,000x) coupled with duplex unique molecular indexing (UMI) and dual sample barcodes to achieve high sensitivity through background error suppression. The panel was designed to capture key exons and domains of 129 cancer genes as well as introns harboring recurrent breakpoints in 10 commonly rearranged genes. We have now developed a bioinformatics module for sensitive detection of structural variation from cfDNA, leveraging the high-depth sequencing data and intronic coverage. We have also added a statistical polishing method that assigns confidence to detected somatic mutations by building frequency distributions for those mutations from a large set of normal samples that has been processed with the same laboratory protocol and bioinformatics pipeline as the patient samples. In parallel, we have further improved the wet lab protocols to address challenges of capturing sufficient numbers of cfDNA molecules and removing noise, both required for detection of somatic mutations with high sensitivity and specificity.

Using this method, we have successfully detected druggable fusions in plasma cfDNA from patients spanning a diverse set of cancer types. Through longitudinal cfDNA collection efforts embedded within early-phase clinical trials, we demonstrate that the levels of oncogenic driver fusions in cfDNA may scale with the burden of disease. Moreover, the ability to simultaneously call high-confidence mutations at low allele fractions has revealed novel acquired mutations indicative of polyclonal resistance to investigational targeted therapies.

#2517

Validation of a targeted sequencing workflow for sequence variants and focal copy number alterations (CNAs) in single circulating tumor cells (CTCs).

Paola Tononi, Valentina del Monaco, Alberto Ferrarini, Genny Buson, Marianna Garonzi, Claudio Forcato, Andrea Raspadori, Nicolò Manaresi. _Menarini Silicon Biosystems, Castel Maggiore, Italy_.

Background

Characterization of individual CTCs helps investigating intra-tumour heterogeneity, and provides longitudinal information about temporal evolution of genomic lesions following therapeutic evolutionary pressure, missed by one-time, bulk, single-biopsy analysis. Here we present the validation of a complete workflow to detect hotspot mutations and CNAs in single cells. It combines ligation-mediated PCR (LM-PCR) Whole Genome Amplification (Ampli1™ WGA kit), best-in-class in terms of low allelic drop-out (ADO) and reproducibility of amplification bias, with a tailored, WGA-aware, next generation sequencing (NGS) targeted cancer panel (Ampli1™ OncoSeek) and a fully-automated cloud-based platform for bioinformatic analysis (MSBiosuite).

Methods

Single-cells (n=24) of breast, prostate, lung and neuroblastoma cancer cell lines spiked in healthy-donor blood, alongside 15 single White Blood Cells (WBCs) from 5 healthy donors and CTCs from prostate, lung and breast patients were enriched with CellSearch® System, sorted with DEPArray™ NxT technology and WGA'ed with Ampli1™ WGA kit (Menarini Silicon Biosystems). NGS reference materials (Seraseq™ Breast CNV and Seraseq™ Lung & Brain CNV) with known CNAs were also WGA'ed. WGA products were used as input for the Ampli1™ OncoSeek Panel assay (Menarini Silicon Biosystems), a single-tube, Illumina®-compatible kit that covers 60 clinically relevant genes including more than 1500 mutation hotspots and CNAs for a subset of 19 genes. WGA-tailored primer pairs design and their concentrations were optimized so that targeted amplicons for sequence variants are sufficiently represented. Redundant amplicons were included for robustness of focal CNA detection. Bioinformatic analysis was performed with an assay-specific, cloud-based pipeline (MSBiosuite, Menarini Silicon Biosystems).

Results

Results on Seraseq NGS reference materials with known copy number gains (3, 6, 12) on 6 genes showed accurate detection of expected CNAs and high linearity of response (R2 = 0.97 ± 0.04). We observed low ADO rate (12.7% ± 4.2%). The Ampli1™ OncoSeek Panel assay detected known mutations and CNAs from cell lines at high sensitivity and the analysis of polymorphic variants in WBCs showed high agreement between biological replicates (overall agreement = 0.94 ± 0.06). Sequencing of CTCs from patients is ongoing and will be presented at the conference.

Conclusions

Here we presented a complete solution to detect hotspot mutations and focal CNAs that meets the need for accurate tumour characterization at single-cell level, suitable for individual CTC analysis.

#2518

APOBEC3-mediated C-to-U RNA editing in non-small cell lung cancer tumors is increased in patients with smoking history and squamous tumor histology.

Mariko Asaoka,1 Frank Zhang,1 Takashi Ishikawa,2 Kazuaki Takabe,1 Saikrishna Yendamuri,1 Santosh K. Patnaik1. 1 _Roswell Park Comprehensive Cancer Center, Buffalo, NY;_ 2 _Tokyo Medical University Hospital, Tokyo, Japan_.

Cytidine deaminase activity of APOBEC3 enzyme family is one of the causes of DNA mutations in many cancers. Members of this family are also RNA editors, capable of converting cytidine (C) bases at specific positions of RNAs to uracil (U). The prevalence and significance of APOBEC3-mediated site-specific C>U RNA editing in cancer is unknown. To study this in non-small cell lung cancer (NSCLC), we examined 1009 primary NSCLC tumors of The Cancer Genome Atlas project for RNA editing at 5,208 sites of 3,630 gene transcripts that are known to undergo APOBEC3-mediated RNA editing. A bioinformatics pipeline was developed to determine editing levels at these sites using whole exome (DNA) and RNA sequencing data. Cox regression, Spearman, and Welch's t tests were respectively used for analysis of survival, correlation, and group comparison. RNA editing could be determined for only some (mean 35, SD 64) of the examined sites because of poor sequencing coverage. For 333 sites (313 transcripts), editing was determinable for ≥10 tumors, with editing occurring at the sites in 2%-92% of tumors (mean 23%, SD 20%). Editing was most prevalent for SLC37A2 (92% of tumors), SERPIN1A (92%), and HPS1 (88%) transcripts. There were 366 tumors with editing determinable for ≥10 sites and occurring at ≥1 site. Among these tumors, editing occurred at 0.1%-100% of sites (mean 20%, SD 27%) at a level of 1%-18% (mean 6%, SD 3%). We summarized the site-specific editing determinations into an editing score, and used this measurement for further analyses. Editing score was on average 3.7x higher in tumors compared to adjacent normal tissues (P = 0.01). Tumors of squamous histology had 3.6x higher editing than adenocarcinoma (P <0.01). Patients with a history of smoking had 2x higher editing score than never-smokers (P = 0.02). There was no association of editing with pathologic tumor stage, but increased editing was associated with improved disease progression-free interval (HR = 0.4, P = 0.01). Correlation of tumor APOBEC3 gene expression levels with editing (r = 0.53 for APOBEC3A, 0.2-0.4 for others) was better than with APOBEC-mediated mutation burden (r = 0.29 for APOBEC3A, 0.07-0.27 for others). In conclusion, APOBEC3-mediated C>U RNA editing is prevalent in non-small cell lung cancer tumors, and reflects APOBEC3 gene expression better than APOBEC-mediated mutation burden. Additional research is needed to identify if editing occurs in cancer cells or in stromal cells of tumors and how it affects genesis and progression of cancer.

#2519

The landscape of FGFR alteration in Chinese solid tumor patients.

Kunpeng Bu,1 Zuping Lian,2 Xiangping Wang,3 Guoli Lv,4 Ruobing Ma,5 Qiang Cui,5 Ming Yao,5 Aodi Wang,5 Hui Chen,5 Youming Lei4. 1 _Affiliated Tumor Hospital of Guangxi Medical University. Nanning, China., Nanning, China;_ 2 _Ruikang Hospital Affiliated to Guangxi University of Chinese Medicine, Nanning, China;_ 3 _THE SENCOND NANNING PEOPLES HOSPITAL, Nanning, China;_ 4 _THE First Affiliated Hospital of Kunming Medical University, Kunming, China;_ 5 _OrigiMed, Hangzhou, China_.

Background: FGFR gene is a promising therapeutic target, and several inhibitors targeted FGFR have demonstrated clinical benefit in cancer patients, such as Erdafitinib in Metastatic Urothelial Cancer. However, the landscape of FGFR gene in Chinese patients with solid tumors has not been well understood, partially because the patients usually harbored different variations from other family gene members (FGFR1-4).

Methods: FFPE tumor samples were collected from 3736 Chinese patients with solid tumor for NGS-based targeted panel sequencing. Patients included lung cancer (39.3%, 1467/3736), hepatocellular cancer (8.6%, 322/3736), colorectal cancer (8.6%, 322/3736), pancreatic cancer (6.9%, 260/3736), gastric cancer (5.3%, 199/3736), intrahepatic cholangiocarcinoma (5.1%, 190/3736), and others. We measured the single base substitution, short and long insertions/deletions, copy number variations, gene fusions and rearrangements in tumor tissue against matched blood.

Results: In total, 6.6% of the 3736 patients with solid tumor had FGFR 1-4 variations. The incidences of genomic alterations on FGFR1, 2, 3, and 4 were 2.8%, 1.6%, 1.2% and 1%, respectively. Amplification was the most common type of FGFR variation , which accounted for 47% of all the genomic alterations, followed by mutations (41.5%) and fusions (11.4%). FGFR variations occurred in multiple cancers, among which FGFR variations were more likely to occur in uterine (4/17; 23.5%), squamous cell lung (29/167; 17.4%), breast (14/82; 17%), urothelial (2/12; 16.6%), intrahepatic cholangiocarcinoma (22/190; 11.6%), colorectal (31/322; 9.6%), gastric (16/199; 8%), small cell lung (7/93; 7.5%), pancreatic (11/260; 4.2%), and hepatocellular cancer (10/322; 3.1%). Most of these data was similar with previously reported published data or TCGA. However, compared with TCGA data, lower incidence was found in three types of cancers: Lung cancer 5.5% vs 19%; hepatocellular cancer 3.1% vs 11%; and pancreatic cancer 4.2% vs 10%. Interestingly, distribution of FGFR variations was also different between cancers. For example, in lung cancer, FGFR1 amplification was the most common variation of FGFR (2.8%), while few fusions were observed. In addition, FGFR2 fusion was mainly in intrahepatic cholangiocarcinoma (6.3%), while FGFR 1/3/4 fusions were rare.

Conclusions: This study, for the first time, revealed that FGFR genomic alterations were found in approximately 6.6% of Chinese patients across different solid tumors. FGFR1 amplifications and FGFR2 fusions were detected with higher frequencies in Lung cancer and intrahepatic cholangiocarcinoma. Our results indicated FGFR inhibitors would be a potential therapeutic strategy to be considered for Chinese patients with FGFR genomic alterations.

#2520

Analysis of the targeted sequencing with RNA Seq. results between primary breast cancer and metastatic sites: Matched paired analysis.

Kyung Hee Park,1 Eunjin Lee,1 Seri Park,1 Se Kyung Lee,1 Seok Won Kim,1 Jeong Eon Lee,1 Seok Jin Nam,1 Ji-Yeon Kim,1 Hee Kyung Ahn,2 Woong-Yang Park,1 Yeon Hee Park1. 1 _Samsung Medical Ctr., Seoul, Republic of Korea;_ 2 _Gachon University Gil Medical Center, Incheon, Republic of Korea_.

Background and Purpose In several breast cancer analyses of targeted gene panels, there was considerable concordance of mutations observed between primary tumors and matched metastases. We are to investigate to gain an understanding of the underlying genetics leading to BC metastasis to compare genomics data using paired biopsies between primary breast and several metastatic sites.

Methods We performed CancerSCANTM of forty-eight patients with paired primary and metastatic tumors, which is 380-gene targeted panel sequencing using HiSeq 2500 sequencing platform (Illumina). We made variant calls using Mutect for SNV, Pindel for INDEL, Contra for CNV, and in-house software for Fusion with default parameters. Tumor mutational burden (TMB) was measured by number of mutations per megabase (mb), and TMB was divided into two groups: high TMB (>=20mutations/mb) and low TMB (<20mutations/mb). Mutational signatures were estimated using R deconstructSigs package, and divided into high and low group by each signature's median value. Twelve patients among this cohort were performed whole-transcriptome sequencing with TruSeq RNA prep kit (Illumina) on primary and/or metastatic fresh-frozen tumors.

Results Seven among 48 patients (14.6%) showed discordant subtypes between primary and metastatic tumors, discordant patterns were diverse. Lung and pleural was the most frequent organ in metastatic tumors, and liver and lymph node were followed. There were 15 shared, 6 primary-specific, 4 metastatic-specific mutations per patient on average, and no significant difference in overall survival (OS) with respect to mutation-sharing patterns. TMB and number of mutations related to mismatch-repair (MMR) genes such as NF1 (P=0.0398) and PMS2 (P=0.1020) tended to be reduced in metastatic tumors compared to paired primary tumors. Mutational signature 3 (BRCA1/2) was increased in metastatic tumors, and signature 5 (General Cancer) was decreased (P>0.05). defect in MMR genes (P=0.0445) and germline BRCA mutation (P=0.0186) were significantly associated to OS, but TMB status, and mutational signature related to MMR respectively were not significant variables for OS according to the molecular profiles in primary tumors. Patients with PMS2 (P=0.156) and NF1 (P=0.0734) multi-hit mutations were better prognosis. Combining molecular profiles in primary tumors such as TMB and mutational signature 6 (MMR) and signature 13 (APOBEC) showed significant differences in OS: low TMB with high signature 6 plus 13 as worse, high TMB as intermediate, and low TMB with low signature 6 plus 13 as better prognosis (P=0.0228).

Conclusions With the combination of TMB and MMR-related molecular profiles, we could stratify patients into three groups according to OS.

#2521

Molecular profiling of T790M-negative NSCLC patients progressing on EGFR-TKI enrolled in the CL1-49076-003 trial with a MET/AXL/FGFR inhibitor in combination with gefitinib.

Monica Garzon Ibanez,1 Nuria Jordana Ariza,1 María González Cao,2 Ruth Román Lladó,1 Alejandro Martínez Bueno,2 Lidia Alonso Landeira,1 María de los Llanos Gil,2 Miguel Ángel Molina Vila,1 Niki Karachaliou,3 Veronika Smutna,4 Valerie Cattan,4 Rafael Rosell,2 Santiago Viteri2. 1 _Pangaea Oncology, Barcelona, Spain;_ 2 _Dr Rosell Oncology Institute (IOR), Barcelona, Spain;_ 3 _University Hospital Sagrat Cor, Barcelona, Spain;_ 4 _Servier, Orleans, France_.

Background Non-small cell lung cancer (NSCLC) tumors with mutations in the EGF receptor (EGFR) relapse to therapy with EGFR tyrosine kinase inhibitors (EGFR TKIs) due to a variety of mechanisms, such as emergence resistance mutations, dysregulation of AXL, MET, HER2 or FGFR1 receptors or histological transformation. The CL1-49076-003 trial of the MET/AXL/FGFR inhibitor S49076 in combination with gefitinib enrolled T790M-negative patients in progression to first-line EGF TKIs and showing dysregulation of MET and/or AXL. Molecular screening was performed in 47 patients, 23 of them met the molecular eligibility criteria and 14 were enrolled in the trial. Baseline biopsies of the 14 patients were submitted to molecular profiling.

Methods Ten patients had enough material available for Next Generation Sequencing (NGS), that was performed using the GeneRead QIAact Lung UMI Panel (Qiagen, Hilden, FRG). The panel included mutation detection in 16 genes, copy number variations in 5 and the MET exon 14 splicing variant. Amplifications detected by NGS were confirmed by FISH. Of the 4 patients with insufficient material for NGS, 3 were submitted to FISH for HER2 and MET and quantitative PCR for BRAF and PIK3CA hotspot mutations. Finally, of the remaining patient only FISH for MET could be performed.

Results Copy number gains were the most prevalent alterations in patients progressing to EGFR TKIs. Four of 14 evaluable patients (29%) showed MET amplification, 4/13 (31%) HER2 amplification and 3/10 (30%) EGFR copy number gains. FGFR1 amplifications were absent in the patient cohort. The baseline sensitizing mutation was confirmed in all cases. Two patients showed additional mutations that could be related to resistance, namely a p.G724S mutation in EGFR and a p.N784fs*2 mutation in MET. Remarkably, these two patients did not present gene amplifications. No other mutations were detected in the rest of genes analyzed, including BRAF, PIK3CA, KRAS, NRAS or ERBB2. Among the 4 patients with more than 6 months of progression free survival, 2 had MET amplifications. The patient with the p.G724S experienced rapid progression of target lesions.

Conclusion Next Generation Sequencing can be used to determine mechanisms of resistance to EGFR TKIs at progression, and can give useful clinical information in order to select therapies for second line treatment

#2522

RNA rescue somatic mutations and RNA editing in esophageal cancer.

Amie J. Radenbaugh,1 J Zachary Sanborn,1 Yulia Newton,1 Charlie Vaske,1 Katherine Van Loon,2 Eric Collisson2. 1 _NantOmics, Santa Cruz, CA;_ 2 _University of California San Francisco, San Francisco, CA_.

The detection of somatic single nucleotide variants is a crucial component to the characterization of the cancer genome. Traditional mutation calling algorithms have focused on comparing the normal and tumor DNA from the same individual. For projects like The Cancer Genome Atlas (TCGA), it became routine to also sequence the tumor RNA. Our computational method, RADIA (RNA and DNA Integrated Analysis), combines the patient-matched normal and tumor DNA with the tumor RNA to detect somatic mutations. The inclusion of the RNA increases the power to detect somatic mutations, especially at low DNA allelic frequencies or when tumor purity is low. By integrating an individual's DNA and RNA, we are able to detect mutations that would otherwise be missed by traditional algorithms that examine only the DNA. Mutations with high support in the RNA and low support in the DNA are termed RNA Rescue mutations. RNA editing is a post-transcriptional modification of pre-mRNA that has recently been identified as an additional epigenetic mechanism relevant to cancer development and progression. Using patient-matched normal and tumor DNA along with tumor RNA, we are able to identify RNA editing events across the entire transcriptome.

There exists a remarkable geographic variability observed in incidence rates for Esophageal Cancer where more than 80% of cases and deaths occur in developing countries. There is an urgent demand to address the unmet clinical needs for these regions. Mutation of the tumor suppressor gene TP53 is the most frequent genetic alteration in both Esophageal Squamous Cell Cancer (ESCC) and Esophageal Adenocarcinoma (EAC). In a previous study of 59 tumors in Malawi a high proportion of tumors without TP53 mutations was reported. Here, we apply RADIA to a cohort of 61 tumors from Tanzania, and identify RNA Rescue Mutations that were previously missed by DNA mutation callers in significantly mutated genes such as TP53, CDK6, NOTCH1, VEGFA, KMT2D. RNA Editing events in the 3'UTR regions of genes are very common, especially when Alu elements are present, and are known to deregulate microRNA mediated gene regulation. MDM2 is a key oncogene in the p53 pathway with elevated gene expression in many tumor types and is known to be transcriptionally repressed by microRNAs. Here we detect transcriptome-wide RNA Editing events, and identify RNA Editing events in the seed regions of microRNA target sites of genes such as MDM2. We will also show how RNA Editing of certain genes are significantly associated with an RNA-Seq clustering solution. These genes show significantly differential expression between RNA-Seq clusters, indicating that these editing events have a functional impact on the genes they affect. We will further investigate these functional effects and downstream implications on the molecular characterization of the RNA-Seq subtypes.

#2523

Targeted exome sequencing profiles genetic alterations in oral squamous cell carcinoma from Japanese patients.

Yasushi Sasaki, Takafumi Nakagaki, Kazuhiro Ogi, Masashi Idogawa, Takashi Tokino. _Sapporo Medical University, Sapporo, Japan_.

Somatic mutation analysis is a standard practice in the study of human cancers to identify mutations that cause therapeutic sensitization and resistance. We performed comprehensive genomic analyses that used PCR target enrichment and next-generation sequencing on Ion Torrent semiconductor sequencers. Forty-seven oral squamous cell cancer (OSCC) samples and their corresponding noncancerous tissues were used for multiplex PCR amplification to obtain targeted coverage of the entire coding regions of 409 cancer-related genes (covered regions: 95.4% of total, 1.69 megabases of target sequence). The number of somatic mutations in 47 patients with OSCC ranged from 1 to 20 with a mean of 7.60. The most frequent mutations were in TP53 (61.7%), NOTCH1 (25.5%), CDKN2A (19.1%), SYNE1 (14.9%), PIK3CA (10.6%), ROS1 (10.6%), and TAF1L (10.6%). The majority of TP53 mutations (83.9%) are localized in the DNA binding domain of the protein (residues 100-300). Importantly, TP53 mutations tended to be more frequent in HPV-negative OSCCs compared to HPV-positive cases (p = 0.00026). We also detected copy number variations (CNVs) in the segments of the genome that could be duplicated or deleted from deep sequencing data. The genes most frequently affected were EGFR (gain in 38.3%) and CCND1 (gain in 34.0%), followed by PIK3CA (gain in 31.9%), RB1 (loss in 27.7%), ERBB2 (gain in 23.4%), MYC (gain in 23.4%), CCND2 (gain in 17.0%), MYCN (gain in 14.9%), ATM (loss in 14.9%), MDM2 (gain in 14.9%), CDKN2A (loss in 14.9%), NRAS (gain in 12.8%), HRAS (gain in 12.8%), BCL2L1 (gain in 12.8%), and KRAS (gain in 8.5%). We validated 100%, 100%, 83.3%, 83.3% and 100% of the OSCC tissues that showed CNV gain for CCND1, CCND2, HRAS, NRAS and KRAS respectively in quantitative PCR copy number analysis. Pathway assessment showed that the somatic aberrations within OSCC genomes are mainly involved in several important pathways, including cell cycle regulation and RTK-MAPK-PI3K. Univariate survival analysis revealed that mutations in NOTCH1 and PIK3CA were found to be associated with worse overall survival in OSCC patients (P = 0.0054 and P = 0.0162, respectively). Interestingly, RTK-MAPK-PI3K pathway alteration correlated to poor survival, suggesting that RTK-MAPK-PI3K pathway is potentially a therapeutic target in this disease. We detected the tumor-specific TP53 mutation in the plasma cell-free DNA from two OSCC patients, and after surgery, the test for these mutations became negative. This study may enable better selection of therapies and deliver improved outcomes for OSCC patients when combined with clinical diagnostics.

#2524

Hallmarks of kinase fusions in Chinese tumors.

Lin Shi,1 Limuge Ce,1 Yanyan Wang,1 Zisong Zhou,1 Jing Ma,1 Hua Dong,1 Yizhou Ye,2 Shuyang Sun3. 1 _3DMed, Shanghai, China;_ 2 _3Dmed, Shanghai, China;_ 3 _Shanghai Ninth People's Hospital, Shanghai, China_.

Kinase fusions are essential drivers to tumor genesis and generally response well to kinase inhibitors. Kinase fusion protein often represent ideal targets and many fusion genes have been well established as therapeutic targets as well as biomarkers, such as BCR-ABL fusion in CML and ALK fusion in lung cancer. In a CAP certified lab 3DMed we describe here a pan-cancer analysis of 8599 patient samples from assayed with comprehensive genomic profiling. Tissue samples were sequencing with a lab validated Masterview gene panel with intronic baiting for 30 fusion genes. Plasma samples were sequencing with a lab validated CGP150 gene panel with intronic baiting for 16 fusion genes. The kinase fusions genes we analyzed are included in both gene panels. Fusions were detected by a sensitive, breakpoint based method and the results were confirmed by manual inspection of the soft-clipped reads. Tumor mutational burden (TMB) was determined by coding region SNV and indel variant counts per Mb for tumor tissue sample only. We collect evidence for all possible gene fusion alterations in all the qualified samples and then focused on the analysis of kinase fusions in lung tissue samples. We surveyed gene fusions across 20 solid tumor types. 196/8599 (2.3%) samples harbored kinase fusion. 31.7% samples are from lung cancer patients and 74% of the 196 kinase fusions are found in lung cancer samples. The average of TMB score in kinase fusion positive samples was 5.17 vs. 11.35 (Wilcox test, P < 0.001) for lung cancer cases. 13% of the kinase fusions positive lung cancer samples co-occur with a known driver mutation such as EGFR or ALK mutation. TMB score in subgroup with kinase fusion (with or without another oncogene driver mutation) is significantly lower than that of subgroup without fusion (Wilcox test, P < 0.01). TMB scores in the kinase fusion positive patients is significantly lower than the fusion negative patients, which suggests that the kinase fusion positive subgroup may not response well to the immune checkpoint inhibitor. There are unmet clinical needs for more fusion kinase inhibitors with the increasing detection sensitivity for kinase fusions by NGS. Our finding have immediate clinical implications for cancer diagnosis and expand the therapeutic options for cancer patients' care, as approved or exploratory drugs exist for many of these kinases.

#2525

Comprehensive analysis of thymic epithelial tumors using an integrated bioinformatics platform reveals novel biological characteristics.

Milan Radovich,1 Jeffrey P. Solzak,1 Wade Webster,2 Jesse Paquette2. 1 _Indiana Univ. School of Medicine, Indianapolis, IN;_ 2 _Tag.bio, San Francisco, CA_.

Introduction:Thymic epithelial tumors (TETs) are rare malignancies. Among TETs, thymoma is the most predominant, characterized by a unique association with myasthenia gravis, followed by thymic carcinoma which is less common but more clinically aggressive. The TCGA recently published a comprehensive analysis of 117 TETs using molecular profiling by exome-seq, RNA-seq, RPPA, and methylation. Using an integrated informatics platform we re-analyzed the TCGA TET data to identify novel biological characteristics of these rare tumors.

Methods: Molecular and clinical data from the TCGA TET project was downloaded from the cbioportal database and imported into the tag.bio informatics platform. Tag.bio is a commercial cloud-based analysis platform that combines data, algorithms and question-based analysis protocols. It is used to for the rapid and reproducible statistical comparison of cohorts.Statistical analysis for differential expression, methylation, RPPA and mutational enrichment was performed using the Mann-Whitney test with multiple correction testing. Pathway and upstream regulator analysis were performed using Ingenuity Pathway Analysis.

Results: Differential expression analysis of myasthenia gravis (MG) patients (N=32) compared to those with no history of MG (N=85), revealed the most significant gene as PPARGC1A (overexpressed, p<1e-8). This gene has not been previously associated with MG. PPARGC1A is highly expressed in skeletal muscle and mediates mitochondrial biogenesis. Supporting this observation, upstream regulator analysis was performed which identifies regulators that can explain the observed gene expression changes in a dataset. The results demonstrated that PPARGC1A as the most activated transcriptional regulator in MG+ tumors and may represent a novel autoantigen for MG. TETs are relatively indolent tumors, and only a small subset metastasizes. Differential expression in a subset of Stage IV tumors (N=6) identified overexpressed FGFR3. Of note, a previous study identified two patients with activating mutations in FGFR3. A genomic hallmark of Thymic Carcinoma (TC) is Chr16q loss. Several tumor suppressors reside on 16q, including:CYLD, CBFB, CDH1, CDH11, CTCF, ZFHX3, but the exact causative candidate is unknown. Analysis of the mutational data identified 2 of 10 patients with concurrent 16q loss and deleterious mutations in CYLD, congruent with previous reports of recurrent mutations in CYLD in TC. CYLD has been previously demonstrated to regulate STAT3. Differential expression followed by upstream regulator analysis of TC vs Thymoma demonstrated activated STAT3 in TC.

Conclusion:Re-analysis of the TCGA TET dataset using a novel integrated bioinformatics platform identified novel biology of TETs including: PPARGC1A association with MG; FGFR3 expression in TET metastasis; and CYLD/STAT3 axis in TC.

#2526

Measuring DNA quantity, FFPE degradation, and genomic DNA contamination of ccfDNA in a single assay.

Trista L. Schagat, Jennifer Mook, Leta Steffen, Doug Wieczorek, Curtis Knox. _Promega Corporation, Madison, WI_.

FFPE tumor biopsies are being increasingly used as a source of DNA for translational research in pursuit of personalized medicine. In addition, circulating cell-free DNA in liquid biopsy fractions shows promise as a means of tracking tumor burden, tumor regression, and/or cancer relapse. However, both sources come with significant DNA quality risks related to sample preparation that can result in downstream sequencing failures or false negatives. For FFPE samples, chemical fixation of the tissue crosslinks DNA, resulting in DNA fragmentation and damage that cannot be completely reversed. For liquid biopsy samples, contamination with genomic DNA can reduce sensitivity for detecting the tumor-specific fraction of ccfDNA. The ProNex® DNA QC Assay is a multiplex qPCR assay that provides both quantification and quality control for small or fragmented DNA samples. ProNex® DNA QC Assay includes a 75bp, 150bp, and 300bp target as well as an internal positive control. Unlike absorption, fluorescent DNA binding dyes, and electrophoresis-based methods, qPCR can be used to quantify specifically amplifiable DNA able to participate in downstream PCR or sequencing applications. The ratio of amplification at 75bp and 300bp sizes can be used to assess fragmentation of FFPE samples, which is correlated with improved coverage uniformity in NGS applications. With ccfDNA, the ratio can also be used to determine the degree of genomic DNA contamination and indicate when further clean-up is necessary prior to sequencing.

#2527

**Genomic aberrations of** ALK **in head and neck squamous cell carcinoma.**

Lan Wang,1 Yuchen Liu,1 Wenying Piao,1 Peony Hiu Yan Poon,1 Chun Kit Yeung,1 Amy Bik Wan Chan,1 Chin Wang Lau,2 Yuxiong Su,3 Jason Ying Kuen Chan,1 Vivian Wai Yan Lui1. 1 _The Chinese University of Hong Kong, Hong Kong, Hong Kong;_ 2 _Yan Chai Hospital, Hong Kong, Hong Kong;_ 3 _The University of Hong Kong, Hong Kong, Hong Kong_.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase encoded by the gene ALK, which belongs to a subfamily of the insulin receptor superfamily. ALK signaling has been shown to be involved in cell proliferation, differentiation, and development, etc. Thus far, ALK aberrations are known to be involved in the oncogenesis of non-small cell lung cancer and glioblastoma. Head and neck cancer squamous cell carcinoma (HNSCC) is an aggressive cancer with genomic heterogeneity as revealed by recent whole-exome studies.

Though ALK aberrations are found in a notable subset of HNSCC, their biological importance is largely unknown. Comprehensive pan-cancer analysis of 33 cancer types (TCGA, Pan-cancer, Provisional, N=11617, www.cbioportal.org) showed that ALK aberrations (mutations, amplification and gene copy gain) are commonly noted in 27 cancers, with HNSCC ranking 9th having ~22% cases of primary tumors harboring these ALK genomic aberrations. Within the US TCGA HNSCC dataset (Provisional; N=510), 3.5% of HNSCC patient tumors harbor somatic ALK mutations, 0.4% cases with ALK amplification, and 17.4% cases with ALK gain. Further, RNA-seq analysis revealed tumor specific ALK mRNA upregulation in 43 pairs of normal-tumor tissues (~2-fold, P=0.001***), and 457 HNSCC tumors (~1.5-fold, P=0.039*). There are currently 172261 single-nucleotide polymorphism (SNP) on ALK exons in human reported in the dbSNP database. We noted 18.9% of Asian HNSCC patients (N=53) bearing ALK germline variants (N=10) with unknown clinical significance.

Interestingly, ALK aberrations (mutations, amplification, gene copy gain) are noted to be significantly associated with TP53 mutations (P<0.0001), HPV-negativity (P=0.0094) and the male gender (P=0.0041; Fisher's Exact test), but not associated with HNSCC patient survival (P=0.734). Further, patients with ALK mutation and amplification are largely advanced cases (93.33%, Stage III and Stage IV). Gene Set Enrichment Analysis (GSEA) of mRNA expression shows that ALK-altered HNSCC tumors (vs unaltered tumors) appear to have significantly downregulation of immune response gene set [NES (normalized enrichment score)=-6.773, P=0.0026] and inflammatory response gene set (NES=-5.54, P= 0.0022), while nervous system development and cell development signaling gene sets are significantly upregulated (P=0.0015, 0.0016, respectively). Proteomic analysis (TCPA, N=209) showed that ALK-altered HNSCC (vs. -unaltered tumors) have increased PIK3CA and CHEK2 protein expression (P<0.001, 0.0001, respectively), as well as reduced PRAS40(pT246) and VEGFR2 protein expression (P<0.001, 0.001, respectively). In conclusion, ALK aberrations may contribute to HNSCC aggressiveness, potentially via reduction of immune response and activation of PI3K signaling pathway. Future investigations studying the biological effects of ALK aberrations on HNSCC oncogenesis are warranted.

#2528

Mapping tumoral and immune heterogeneity in PD-1 responsive glioblastoma.

Paula Restrepo, Raymund Yong, Ilaria Laface, Nadejda Tsankova, Sacha Gnjatic, Adilia Hormigo, Bojan Losic. _Icahn School of Medicine at Mount Sinai, New York, NY_.

Clonal evolution and the immune microenvironment in glioblastoma (GBM) ultimately determine the potential benefit of checkpoint inhibition. We present a case study of a 67- year-old patient who underwent a resection of a large right temporal GBM. The patient received standard-of-care treatment consisting of tumor resection, radiation therapy and concurrent temozolomide. Her tumor recurred as a gliosarcoma after 2 cycles of adjuvant Temozolomide. Subsequently, she received 26 cycles of a PD-1 checkpoint inhibitor and 20 cycles of bevacizumab, a VEGF-A inhibitor. She survived 25 months after diagnosis including six after immunotherapy and VEGF-A blockade was discontinued. We integrated RNA-seq and whole exome sequencing data from the primary tumor and several recurrent biopsies to characterize the genomic and molecular landscape within the context of clonal evolution. Specifically, we surveyed tumor-immune interactions by computing the mutational and neo-epitope tumoral burden, along with the magnitude and clonality of infiltrating lymphocytes using VDJ-sequencing. Furthermore, we intersected genomic instability measures with the regional gene expression profiles among the sectors. Finally, we integrated TCGA-GBM RNA-seq data to examine the expression of our patient sectors among the spectrum of known GBM variation. We found significant regional heterogeneity in the predicted neoantigen and infiltrating lymphocyte landscape, with a pronounced shift towards lower predicted immunogenicity and lower immune infiltration in all recurrent biopsies. We also observed profound large-scale immune variance among the regional sectors when examined in the context of TCGA. Overall, this case study quantifies intra-tumoral clonal and immune heterogeneity with multiregional glioblastoma omics data, providing reasonable priors for further assessing the efficacy of checkpoint inhibition for GBM.

#2529

Comprehensive genomic analyses of a case series of bilateral renal cell carcinoma.

Florian Büttner,1 Carolin Meyerhoff,1 Siarhei Kandabarau,1 Stefan Winter,1 Steffen Rausch,2 Arnulf Stenzl,2 Falko Fend,3 Christopher Schroeder,4 Peter Bauer,4 Per Hoffmann,5 Markus M. Nöthen,5 Jens Bedke,2 Matthias Schwab,1 Elke Schaeffeler1. 1 _Dr. Margarete Fischer-Bosch - Institute of Clinical Pharmacology and University of Tuebingen, Stuttgart, Germany;_ 2 _Department of Urology, University Hospital Tuebingen, Tuebingen, Germany;_ 3 _Institute of Pathology and Neuropathology, University Hospital Tuebingen, Tuebingen, Germany;_ 4 _Institute of Medical Genetics and Applied Genomics, University Hospital Tuebingen, Tuebingen, Germany;_ 5 _Institute of Human Genetics, University Hospital of Bonn, Bonn, Germany_.

Renal cell carcinoma (RCC) accounts for 5% in men and 3% in women of all oncological diagnoses worldwide (Capitanio et al., Eur Urol 2018). In 0.7-4.7% of patients both kidneys are affected by cancer. Although hereditary forms of RCC do exist (VHL disease, hereditary papillary RCC), the majority of bilateral RCC are sporadic. RCC comprises histologically distinct subtypes, and clear cell renal cell carcinoma (ccRCC) constitutes 65-70% of all RCC. Here, we present genomic analyses of three cases with bilateral ccRCC. In particular, we are interested in understanding the origin and evolution of these bilateral tumors. Our study includes bilateral tumors from three patients (P1 - P3). Tumors from patients P1 and P2 are metachronous. Patient P3 showed synchronous bilateral ccRCC, with two distinct tumors in one kidney. Two samples were retrieved per tumor to take account of intratumor heterogeneity. In addition one non-tumor sample was obtained per patient. Paired-end whole exome sequencing (WES) was performed using DNA isolated from all tissue samples. Single nucleotide and short indel variants were called using the Mutect2 software. Additionally, DNA samples were sequenced at ultra-deep coverage (median 1000) using a custom NGS panel comprising 33 genes frequently mutated in RCC to confirm results obtained from WES. The deepSNV method (Gerstung et al., Nat Commun 2012) was used to call somatic mutations from targeted sequencing data. Further, WES data as well as SNP data deriving from microarray analysis (Infinium Global Screening Array-24 v2.0) will be used to reconstruct somatic copy number aberrations for each tumor. We have analyzed relationships between the bilateral tumors by means of somatic mutations from WES data that were shared within and between tumors of a patient. In patient P1 Venn diagram analysis revealed that 1.9% (n=4) of 213 mutations detected in at least in two samples were shared between samples from different tumors. 32% (n=35) of 108 non-unique mutations were shared between tumors from patient P2; 16 mutations were even present in all four samples of P2. 3% (n=9) of 301 non-unique mutations occurred in samples from different tumors in patient P3. None of these mutations was present in all three tumors. Pairwise comparison between tumors of P3 revealed 1.6%, 0.6%, and 2.2% of non-unique mutations overlapping between tumor pairs, respectively. These first observations suggest a common origin of tumors from patient P2, whereas bilateral tumors from patients P1 and P3 seem to be independent events. Data from deep sequencing support these findings in all three cases, e.g. they confirm one of the ubiquitous mutations in P2 that is located in the von Hippel-Lindau gene. In summary, we present a comprehensive genomic analysis of a small case series of bilateral RCC indicating that even a metachronous bilateral RCC can share the common origin.

The work was supported by the Robert Bosch Stiftung, Stuttgart, Germany.

#2530

Understanding cancer heterogeneity in relation to minor sub-clones at single cell profiling level in renal cell carcinoma.

Enrique I. Velazquez Villarreal, John D. Carpten, David W. Craig. _USC Keck School of Medicine, Los Angeles, CA_.

Renal cell carcinoma (RCC) is one of the most lethal urological malignancies that would benefit from understanding cancer heterogeneity to identify molecular signatures providing potential innovative therapeutic targets. Renal cell carcinoma is characterized by extensive cancer heterogeneity. Single cell sequencing has the potential to improve our understanding of cancer heterogeneity by providing clonotype detection. We examined cancer heterogeneity using single-cell copy-number analysis and clonotype detection in four renal cancer tumors. The technology used for processing single cell was 10x ChromiumTM Technology that provides detection of 100 Kb CNV events, calling clonotypes down to 10 of 1000 cell inputs. By processing each of the four samples it resulted 469 total cells from sample one where 268 were tumor and 171 were normal; sample two resulted with 888 cells where 51 were tumor and 820 were normal; sample three resulted with 673 total cells where 167 were tumor and 485 normal; finally, sample four resulted with 1298 cells where 511 were tumor and 758 normal. Genome-wide ploidy analysis of the four tumor cells showed variable median ploidies. The regional copy number was estimated by processing our data in 20 Kb cases of reads. Multiple sub-clones were identified in sample number one where four clusters of sub-clones characterized cancer heterogeneity. Fast maximum-likelihood (ML) genetic clustering and Bayesian Information Criterion (BIC) were implemented on sample one to select the optimal clustering solution and provide a better insight of sub-clonal evolution. These sub-clones reported events such as copy number changes on entire chromosomes arms. The single cell resolution provided the ability of variant detection where we identified VHL gene mutations that are consistent with recent reports that describe its role as a molecular signature of disease outcome and drug efficacy in RCC. In conclusion, our findings describe sub-clones with large-scale events that provide new insights in understanding cancer heterogeneity in RCC.

#2531

Genomic landscape of breast cancer patients in Korea: Early data from K-MASTER project.

Jae Sook Sung,1 Hee-Joon Chung,2 Yoon Ji Choi,3 Won Jin Chang,3 Jung Yoon Choi,3 Ju Won Kim,3 Kyong Hwa Park,3 Yeul Hong Kim3. 1 _Korea University, Seoul, Republic of Korea;_ 2 _K-MASTER Cancer Precision Medicine Diagnosis and Treatment Enterprise, Seoul, Republic of Korea;_ 3 _Korea Univ. Hospital, Seoul, Republic of Korea_.

The K-MASTER cancer precision medicine diagnosis and treatment enterprise (K-MASTER project) is a precision medicine cancer treatment clinical trial platform, which uses NGS assay to screen the actionable mutations in 10,000 patients with refractory solid tumors and assigns the patients to matched treatment. K-MASTER project was initiated in Korea since June 2017. From October 2017, we have analyzed next-generation (NGS)-based cancer panel to detect cancer genomic variations in breast cancer. This study is ongoing with 36 sites. Tumor DNA was analyzed by CancerSCAN (Samsung Genomic Institute) which allows to detect variations of 375 genes. As of October 31 in 2018, total 164 breast cancer patients were submitted and 95 samples were analyzed. The failure rate of sequencing was 4.1% of patients with samples submitted. The mean depth of CancerSCAN was 701x. Average turn-around time (TAT) of CancerSCAN was 3 weeks.In Korean breast cancer patients, variations were mainly identified in TP53 (63.6%), PIK3CA (35.2%), BRCA1 (25.0%), BRCA2 (23.9%), ERBB2 (23.9%), PTCH1 (21.6%), NOTCH1 (19.3%), FGFR1 (12.5%), PTEN (11.4%) and MET (9.1%). Further analysis will confirm amplification and deletion in breast cancer. Until March 2019, we will finish the genomic profiling of 350 breast cancer patients and will be reported. In conclusion, this project is efficient to detect actionable variations in breast cancer. This novel knowledge provides an intriguing background to investigate new target approaches and to progress precision medicine. (This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &Welfare, Republic of Korea (Grant number: HI17C2206).)

#2532

High-throughput single-cell targeted DNA sequencing from frozen, fixed and preserved solid tumor samples reveals complex genomic variation and clonal propagation.

David Ruff, Pedro Mendez, Daniel Mendoza, Nianzhen Li, Adam Sciambi, Kaustubh Gokhale, Dalia Dhingra, Keith Jones, Dennis Eastburn. _Mission Bio, Inc., South San Francisco, CA_.

Biologically annotated specimens such as frozen, fixed and preserved tissues are key sources of cells for genomic analysis. Bulk NGS using archived solid tumor samples is inadequate to fully characterize somatic variation buried in the landscape of cellular populations. Single-cell targeted DNA sequencing provides an essential solution to elucidate and map genomic variation in such materials. Although the study of frozen, fixed and preserved tissues at the single-cell level is compromised by preservation processes, the isolation of nuclei allows the recovery of suitable gDNA templates. Common tissue disaggregation processes can be complicated by persistence of conglomerated cellular components, ruptured nuclei, and other insoluble extracellular matrices. For challenging samples, we developed a nuclei isolation protocol that demonstrates optimal performance for high-quality targeted DNA sequencing from archived human solid tumor samples. This process begins with physical maceration of ~10-100 mg preserved tissue or 20-100 uM sections, suspension followed by enzymatic treatment, filtration and centrifugal collection. After cell straining, nuclei are ready for counting, staining and sequencing. Fluorescent microscopy using membrane, cytoplasmic and nuclear stains reveal highly purified intact nuclei, recovering at least 500K nuclei from 30-50 mg of tissue containing greater than 70% nucleated cells by H&E-staining. Many human samples provide outstanding quality nuclei suspensions, including cryopreserved cell lines, PBMCs, bone marrow, liver, brain, breast, colon, lung, prostate and melanomas. Fixed and preserved specimens also yield nuclei readily interrogated by targeted DNA sequencing panels. Nuclei suspensions are readily processed with the Tapestri Platform for single-cell DNA analysis. Leveraging droplet microfluidics and barcoding, the workflow enables high uniformity of ~90%, low ADO of ~10%, and typical nuclei recovery rates exceed 10%. Up to 20,000 nuclei can be interrogated in each run with catalog or custom panels for any tumor type. Here, a 59-gene tumor hotspot panel was used to study five melanoma metastases. The single-cell data enabled the unique reconstruction of tumor sample clonal phylogeny unresolved by bulk analysis. Also, low prevalence metastatic subclones masked in bulk NGS data were detected in normal liver samples. In summary, we show that diverse types of archived tumor tissues are readily dissociated with this universal nuclei protocol. Researchers now have a highly sensitive, targeted, customizable solution for revealing genomic variation and clonal propagation in complex archived solid tumor samples.

#2533

The genomic and transcriptomic landscape of left versus right sided breast cancer in 410 cases.

Yulia Newton,1 Christopher Szeto,1 Charles Vaske,1 Leia Reddy,2 Sandeep Reddy2. 1 _Nantomics, LLC., Santa Cruz, CA;_ 2 _NantHealth, LLC., Culver City, CA_.

Background: Studies have shown that unilateral breast cancer is more common on the left (L) than right (R) and that the L breast is often larger than the R. No clear explanation for this finding has been described in the literature. Hypotheses range from complex genomic effects to environmental, hormonal, and anatomical causes.

Methods: We performed a retrospective analysis of data from the NantHealth database of 410 commercial cases where WGS (35x germline, 75x somatic) or WES (∼150x). Primary and metastatic breast tumors were included in the analysis. Of these 410 cases, 39.3% were R, 38.5% were L, and 22% were unspecified. Bases adjacent to variant sites were used to determine genomic context (i.e. mutation signatures) as previously reported. Additionally, paired deep whole transcriptomic sequencing (RNA-Seq; ∼200x106 reads per tumor) was performed on 376 of the 410 cases. Nant Breast Cancer Intrinsic Subtypes sorting based on RNAseq assay classified breast tumors into 4 subtypes (LumA/B, Basal-like, and HER2-enriched).

Results: Luminal A subtype was significantly overrepresented in R v L (OR=0.53, p=0.015). Similarly, R-side cases were significantly enriched for estrogen response gene expression by GSEA. However, only slight differential expression of ESR1 and MKI67 was observed specifically in individuals between 40 and 50 years old. Germline variants in the following genes were associated with sidedness: MEN1, KIT, JAK2, TET2, EP300, MET, FLCN, and KMT2D, and somatic variants within the following genes were associated with sidedness: CACNA1E, AGRN, RIMS2, TTYH3, KRTAP10-6, and DDX60L. However, significance of the variant frequency differences was lost after multiple-hypothesis correction. No significant differences between L and R were noted in tumor stage, HER2, ER, PR, somatic CNV, and mutation signatures, including signature 3 (BRCAness).

Conclusions: Despite performing WGS/WES a paucity of differentially mutated genes were seen between left and right sided breast tumors. RNAseq analysis reveals that right sided breast cancers may be more likely to involve ESR1-signaling and be Luminal A subtype. Right-sided ESR1-signaling may be especially relevant in perimenopausal cases, however the current cohort is underpowered to achieve significance in this analysis.

#2534

A comparative analysis of fraction genome altered vs tumor mutational count in prostate cancer.

Goutam Chakraborty,1 Mohammad Atiq,1 Subhiksha Nandakumar,1 Ying Z. Mazzu,1 Joshua Armenia,1 Yuki Yoshikawa,1 Nabeela Khan,1 Gwo-Shu M. Lee,2 Lorelei Mucci,3 Philip W. Kantoff1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Dana-Farber Cancer Institute, Boston, MA;_ 3 _Harvard T.H. Chan School of Public Health, Boston, MA_.

Prostate cancer is the second leading cause of cancer related death among men in the United States. Although 5 new life-prolonging agents have been FDA-approved for castration resistant prostate cancer in the past 8 years, drug resistance limits the durability of responses to these agents for most patients. We hypothesized that biomarkers of genomic alterations, defined as fraction genome altered (% of copy number altered chromosome regions out of measured regions) and mutational count (number of mutational events per cases), were prognostic biomarkers which might provide insights into resistance mechanisms. We used The Cancer Genome Atlas (TCGA; n= 490 primary prostate cancer) dataset to analyze fraction of genome altered and tumor mutational count in prostate tumors. Patients whose primary prostate cancer harbored a high frequency of fraction genome altered exhibited significant disease progression in TCGA cohort . In contrast, we did not observe a significant association between high tumor mutational count and disease progression. Fraction genome altered was more significantly associated with Gleason grade compare to tumor mutation count. Interestingly, our preliminary observation showed that primary prostate cancer patients who harbor high fraction genome altered (>6%), but low tumor mutational count (<30/cases) exhibited shorter disease-free survival, indicating that those primary prostate cancer patients who harbor high fraction genome altered but low mutational count may have more aggressive prostate cancer. We have also developed a transcriptional signature which is associated with high fraction genome altered. . A detailed understanding of these molecular features will help us to design appropriate therapeutic approaches for different subsets of the disease. We hypothesize that cases with a high fraction of genome altered are associated with aggressive disease and those with lower mutational count may be associated with a diminished immunoresponsiveness.

Tumor mutational count and fraction genome alteration in various stages TCGA prostate cancer

---

|

Disease Status

(Gleason unadjusted) | p value | Gleason | p value

(trend)

|

Disease

Free | Recurred/

Progressed | |

6 (n=45) | 7 (n=244) | >=8 (n=198)

|

Fraction

Genome Altered

(Mean + SEM) | 0.08248

± 0.0049 | 0.1254

± 0.0112 | 0.0003 | 0.02504

±0.0037 | 0.05985

± 0.0039 | 0.1425

± 0.0087 | < 2.2e-16

Mutational

Count

(Mean + SEM) | 35.05

± 1.354 | 39.88

± 2.655 | 0.3944 | 29.42

± 1.749, | 34.69

± 0.9089, | 38.58

± 1.272, | =9.971e-05

(7 vs >=8;

p value 0.0445)

### Cell Cycle Biology and Signaling

#2535

Enhanced gene expression of replication fork and other E2F targets genes is associated with sensitivity and, paradoxically, also with acquired drug resistance, to the Chk1 inhibitor prexasertib.

Wayne D. Blosser, Jack A. Dempsey, Ann M. McNulty, Wenjuan Wu, Philip J. Ebert, Jason C. Ting, Yue W. Webster, Trent R. Stewart, Xueqian Gong, Farhana F. Merzoug, Sean Buchanan, Ricardo Martinez. _Eli Lilly, Indianapolis, IN_.

Many cancers are under replicative stress (RS) arising from the combined influence of oncogenic drivers (i.e. Cyclin E, E2F, Myc), genomic instability and/or deficiencies in DNA Damage Repair (DDR). Tumors survive RS by upregulating checkpoints such as those driven by the ATR/CHK1 pathway. Dependence of tumors under high RS on the Chk1 pathway is currently being explored in the clinic with the Chk1 kinase inhibitor prexasertib. Preclinical and clinical studies have demonstrated evidence of efficacy in various cancers supporting the notion that this drug has the potential to improve the standard of care for patients with these malignancies. Two pivotal steps to ultimately fulfill the promise of a novel therapeutic in the clinic are, 1) to enable a patient selection strategy via predictive biomarkers of drug response or de novo resistance; 2) to gain insight into mechanisms underlying the development of acquired resistance, a common phenomenon of cancer therapeutics that contributes to non-durable response. Two experimental objectives to address these challenges for prexasertib were set, 1) to profile a large pan-cancer tumor cell line panel for prexasertib response and explore the association between drug response and baseline molecular profile via transcriptomic profiling, (2) to establish an in vitro tumor line with acquired resistance to prexasertib and characterize it via transcriptomic and targeted proteomic profiling to probe the mechanism of acquired resistance to prexasertib. Key observations from this study: A) pathways corresponding to E2F targets, G2M checkpoint and the Spindle Assembly Checkpoint (SAC), with a distinct enrichment for replication fork genes emerged across three different studies, (a) across a pan-cancer tumor line panel associated with prexasertib sensitivity, (b) associated with resistance in a tumor cell line with acquired resistance to prexasertib and (c) as top hits in an unbiased functional shRNA screen aimed at identifying genes that upon knockdown sensitize the resistant tumor cell line to prexasertib. B)knockdown of specific replication fork components in the drug-resistant tumor line was associated with marked sensitization to prexasertib and concomitant evidence of RS and DNA damage.To explain these paradoxical observations, we postulate that tumors may upregulate the expression of E2F target/G2M/SAC genes to resist oncogene-induced RS and that they may rely on similar genes to survive prexasertib-induced RS. Based on this data, transcriptomic profiling may prove of utility in the identification of patient tailoring biomarkers for prexasertib and as tool to unravel mechanisms of acquired resistance.

#2536

O-GlcNAcylation of Ewing sarcoma EWSR1/FLI1 protein impairs relocation of Aurora B to the midzone during mitosis.

Mizuki Azuma. _Univ. of Kansas, Lawrence, KS_.

Ewing sarcoma is a pediatric bone cancer expressing chimeric EWSR1/FLI1 protein derived from a t(22;11)(q24;12) translocation. Our previous study demonstrated that EWSR1/FLI1 impairs the relocation of Aurora B kinase from the centromere to the midzone during the metaphase-anaphase transition (referred to as "Aurora B-relocation"). Aurora B-relocation to the midzone is essential for faithful cell division, whereas compromising this process will lead to formation of a binucleated cell or chromosomal mis-segregation. This cellular activity might play a role in the pathogenesis of Ewing sarcoma; however, how EWSR1/FLI1 impairs Aurora B-relocation remains unclear. In the present study, we tested the hypothesis that O-GlcNAcylation of EWSR1/FLI1 impairs Aurora B-relocation. The results showed that the increase in cellular O-GlcNAc levels in Ewing sarcoma A673 cells induced by treatment with the O-GlcNAcase inhibitor Thiamet-G (TMG) increased the level of aberrant Aurora B-relocation, and the O-GlcNAcylation level of endogenously expressed EWSR1/FLI1. To map the amino acid(s) required for O-GlcNAcylation of EWSR1/FLI1, HeLa cells were used because they do not express EWSR1/FLI1 endogenously. Among the putative point mutant constructs of EWSR1/FLI1, the substitution of Thr79 of EWSR1/FLI1 by Asp (phosphor mimic mutant) significantly reduced the O-GlcNAcylation level of EWSR1/FLI1, suggesting that Thr79 is the site of O-GlcNAcylation. Finally, the EWSR1/FLI1-T79D and EWSR1/FLI1-T79A mutant constructs failed to induce aberrant Aurora B-relocation in HeLa and A673 cells. Therefore, we propose that O-GlcNAcylated EWSR1/FLI1-T79 is required for the induction of aberrant Aurora B-relocation. In addition, EWSR1/FLI1-dependent disruption of Aurora B-relocation leads to defects in chromosome instability (CIN). Here, we demonstrate a unique role of EWSR1/FLI1 that may be a part of the pathogenesis of Ewing sarcoma.

#2537

Targeting chromosomal passenger complex by disruption of INCENP function inhibits tumor progression in neuroblastoma.

Ming Sun, Veronica Veschi, Norris Lam, Sukriti Bagchi, Man Xu, Arnulfo Mendoza, Zhihui Liu, Carol J. Thiele. _National Cancer Institute, Bethesda, MD_.

Chromosomal passenger complex (CPC), which is composed of Aurora B and Survivin, INCENP and Borealin, regulates crucial mitotic events including chromosome alignment, segregation and cytokinesis during the mitosis. Disruption of CPC function by Aurora B and Survivin inhibition has been demonstrated to be effective at killing tumor cells and show promising results in many different cancer types including neuroblastoma (NB). However, chemical inhibition of either Aurora B or Survivin is unable to target CPC specifically due to off-target effects of Aurora B inhibitors on the other kinases and antiapoptotic activities of Survivin independent of this complex. In a chromatin-focused siRNA screen, we found that NB cells were particularly vulnerable to the silencing of INCENP, a gene encoding a key scaffolding and regulatory component of the CPC. In this study, we found that INCENP was highly expressed in NB cells and its expression levels were decreased upon Retinoic Acid (RA)-induced NB cell differentiation. Genetic silencing of INCENP reduced the growth of both MYCN single copy and MYCN amplified NB cell lines in vitro and led to significant decreases in NB xenograft growth and increases in murine survival in vivo. Elevated levels of INCENP were significantly associated with poor prognosis in primary NB tumors whereas low INCENP expression levels were predictive of better outcomes. Mechanistically, we found that INCENP depletion suppressed NB cell growth by inducing massive polyploidization, mitotic arrest, senescence and cell death (apoptosis). We also observed that in the majority of NB cell lines tested in vitro, cell death represented the primary cell fate after INCENP silencing due to a strong induction of DNA damage response and activation of the p53-p21 axis. Therefore, targeting INCENP phenocopies treatment with Aurora B inhibitor, providing a novel strategy to disrupt the activity of CPC and inhibit tumor progression in NB.

#2538

p53 activation induces cell cycle arrest by promoting DREAM and RB repression of cell cycle genes.

Amy E. Schade,1 Martin Fischer,2 James A. DeCaprio3. 1 _Harvard Medical School, Brookline, MA;_ 2 _Leibniz Institute on Aging - Fritz Lipmann Institute, Jena, Germany;_ 3 _Harvard Medical School, Boston, MA_.

p53 activation results cell cycle arrest at the G1/S checkpoint and the repression of early (G1/S) and late (G2/M) cell cycle gene expression in a p21 dependent manner. The mechanism how p53 and p21 repress cell cycle gene expression is unclear. p21 is known to repress CDK2 which is a key inhibitor of the pocket protein family members RB and p130. During a normal cell cycle, p130 as part of the DREAM complex (DP1, RB-like p130, E2F4, and MuvB) and RB cooperate to repress expression of early (G1/S) and late (G2/M) cell cycle gene expression. DREAM binds and represses late (G2/M) cell cycle promoters through MuvB binding and early (G1/S) cell cycle promoters though repressor E2F binding. RB is only able to bind and repress early (G1/S) cell cycle promoters through its interaction with activator E2Fs. The requirement for DREAM and RB for control of cell cycle gene expression after p53 activation was unknown. We hypothesized that DREAM and RB would cooperate to repress cell cycle gene expression after DNA damage. Using primary human foreskin fibroblast cells lacking RB and/or p130, we found repression of early and late cell cycle gene expression is differentially regulated after p53 activation. We found that while RB is required for repression of early (G1/S) cell cycle genes after p53 activation, DREAM is required for repression of expression of late (G2/M) cell cycle genes. Surprisingly, DREAM was unable to repress early cell cycle genes after p53 activation, suggesting that RB is the key regulator of G1/S gene expression during cellular stress like DNA damage. Further, the requirement for DREAM to repress late cell cycle gene expression underlies the importance of DREAM to prevent expression of mitosis inducing genes in a state like DNA damage when mitosis could result in propagation of mutated DNA. We found that p107, a protein reported be able to replace p130 in DREAM, does not potently repress early cell cycle genes in HFFs under contact arrest and serum starvation. However, we found that p107 strongly represses expression of late cell cycle genes during G0 in cells lacking p130 alone or in combination with RB. These data provide evidence for a specific role for p107-DREAM not previously appreciated. Together, our data show that control of cell cycle gene expression after p53 activation is split in two: RB represses early cell cycle genes while DREAM represses late cell cycle genes.

#2539

Discovery of degradation pathway for maternal embryonic leucine zipper kinase (MELK): Implications for breast cancer therapy.

Shyam Nyati,1 Benjamin Chandler,1 Anna Michmerhuizen,1 Andrea Pesch,1 Cassandra Ritter,1 Leah Moubadder,1 Meilan Liu,1 Meleah Cameron,1 Eric Olsson,1 Kari Wilder-Romans,1 Dipankar Ray,1 Theodore S. Lawrence,1 Felix Y. Feng,2 Lori J. Pierce,1 Corey Speers1. 1 _Univ. of Michigan Medical School, Ann Arbor, MI;_ 2 _University of California at San Francisco, San Francisco, CA_.

Background: Despite previous studies suggesting that MELK is not involved in proliferation in cancer, many groups have shown that MELK is a critical mediator of radioresistance and may be an ideal target for radiosensitization. Furthermore, additional studies suggest MELK functions to regulate the cell-cycle and that its mRNA and protein levels are regulated during cell-cycle, although the mechanisms of this regulation are currently unknown. We sought to identify mediators of MELK protein regulation, determine whether it was ubiquitin-ligase mediated, and if so, which E3-ubiquitin ligase machinery degrades MELK during cell-cycle progression.

Methods: Sequence alignments were carried out to identify degron motifs in MELK. Site directed mutagenesis were performed to mutate degrons in MELK. CDC20 and CDH1 were over-expressed to identify the co-activators of the E3-ubiquitin ligase complex. Cells were synchronized by double thymidine block or by nocodazole. CDC27/APC3 and CDC20 antibodies were used to pull-down the Anaphase Promoting Complex/Cyclosome (APC/C) and Mitotic Checkpoint Complex (MCC), respectively. Resulting pellets were probed with antibodies against BubR1/Mad3, Bub3, CDC20, CDC27, MELK, Cyclin B1, CDK1, BUB1, and Mad2. A role for MELK kinase activity on its half-life and formation of APC/C and MCC was evaluated using kinetically inactive (MELK-KD) mutant or OTSSP167. siRNA against MELK was used to confirm a requirement of MELK for formation of APC/C and MCC. The effect of MELK degron mutant on radiosensitization will be evaluated in MELK CRISPR TNBC cell lines.

Results: In this study we provide evidence that MELK has two D-boxes and a KEN box which are largely conserved from C. elegance to H. sapiens. We identified that MELK is degraded by the E3-ubiquitin ligase APC/C with the coactivator being CDC20. Rescue with MG132 confirms that MELK is degraded by a proteosomal-mediated pathway. CO-IP studies show that MELK is required for APC/C and MCC formation and that MELK interacts with CDC20, CDK1, BUB1, and BUBR1. Cycloheximide chase studies show D-Box1 and KEN box mutants of MELK are more stable while D-BOX2 mutants are least stable. The KEN and D-Box mutant MELK show impairment in APC/C and MCC formation, altered Cyclin B1-CDK1 and Cyclin B1-CDC20 interactions. MELK KD and OTSSP prove that kinase activity of MELK plays a role in its stability. We anticipate that the least stable MELK mutant will result in higher radiosensitivity compared to MELK-WT.

Conclusions: MELK is regulated by APC/CCDC20 through its D-Box1, KEN and D-Box2. Degron mutant and MELK KD show altered protein stability which leads to attenuated APC/C and MCC formation. These results provide proof that MELK is a cell-cycle checkpoint kinase and support the rationale for developing clinical strategies to degrade and inhibit MELK in combination with radiation therapy.

#2540

Mitotic Golgi vesiculation enables human papillomavirus to evade cGAS/STING.

Brittany L. Forte, Shauna M. Bratton, Samuel K. Campos. _University of Arizona, Tucson, AZ_.

Oncogenic human papillomaviruses (HPVs) infect and replicate in differentiating mucosal epithelium, causing 5% of cancers worldwide and essentially all cervical cancers. During infection, HPV must traffic viral genome (vDNA) to the nuclei of basal keratinocytes. Minor capsid protein L2 facilitates intracellular transport of vDNA to the Golgi; upon mitosis, the L2-vDNA complex penetrates limiting membranes and localizes to mitotic chromosomes, ensuring infection of both daughter cells.

Cytosolic DNA sensor cGAS recognizes cytosolic dsDNA and produces second messenger cGAMP; cGAMP causes activation and relocalization of ER-resident STING to the Golgi, where it recruits TBK1 to phosphorylate and activate IRF3, initiating a type-I IFN response. The cGAS/STING pathway is assumed, though never demonstrated, to be inactive during mitosis to avoid detecting self-DNA. Since the Golgi is a platform for STING/TBK1/IRF3 recruitment and activation, we hypothesize natural Golgi dispersal deactivates cGAS/STING during mitosis. Further, we hypothesize HPV has evolved to traffic to and translocate from the mitotic Golgi as an immunoevasive tactic to avoid detection by cGAS/STING during mitosis.

HaCaTs, a human keratinocyte line, were transfected with DNA or infected with HPV pseudovirions and analyzed for cGAS/STING activation. DNA transfection resulted in IRF3 phosphorylation and nuclear translocation, and STING translocation to the Golgi, indicating cGAS/STING activity. Strikingly, chemical disruption of the Golgi ribbon potently blocked IRF3 activation in response to foreign DNA, suggesting Golgi morphology might modulate cGAS/STING activity during mitosis. In accordance, DNA-dependent IRF3 phosphorylation was transiently reduced in synchronized mitotic cells, but chemical impairment of mitotic Golgi vesiculation enabled cGAS/STING activation, even without foreign DNA transfection. Further, HPV infection resulted in minimal IRF3 phosphorylation, indicating HPV can efficiently evade detection during initial infection.

To determine if HPV's unique trafficking enables evasion, we used cationic liposomes to permit premature virion translocation across limiting membranes. Such treatment renders a non-infectious, translocation-defective mutant HPV infectious, yet susceptible to cGAS/STING sensing. Overall, the cGAS/STING pathway may be inactivated by mitotic Golgi dispersal, allowing HPV to evade detection during mitosis.

#2541

Mastl regulates EGFR signaling to promote pancreatic cancer progression.

Iram Fatima,1 Shailender S. Chauhan,1 Srijayaprakash Babu Uppada,1 Geoffrey A. Talmon,1 Amar B. Singh,2 Surinder K. Batra,2 Punita Dhawan2. 1 _University of Nebraska Medical Center, Omaha, NE;_ 2 _University of Nebraska Medical Center, Buffet Cancer Center, Omaha, NE_.

Background: Anti-cancer treatments act primarily by damaging the DNA of cancer cells. Upon DNA damage, cells stop proliferation at cell cycle checkpoints, which provides them time for DNA repair. Hence, pharmacological inhibition of checkpoint kinases in combination with the DNA damaging anti-cancer therapies is now emerging as promising cancer treatment strategy. In this regard, pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies with a frightening resistance to chemotherapeutic and targeted approaches. Here, along with the KRAS-dependent signaling, receptor tyrosine kinases especially the EGF receptor (EGFR) signaling is strongly upregulated in PDAC. Importantly, Greatwall (Mastl; Microtubule-associated serine/threonine-protein kinase-like) promotes normal G2-mitosis transition and is highly upregulated in multiple cancer types, and have been demonstrated to associate with therapy resistance. However, potential role of Mastl in pancreatic cancer progression/therapy resistance remains unknown. Our central hypothesis is that MASTL expression is (a) involved in cancer progression and intrinsic drug resistance (b) constitutes a potential therapeutic target for PDAC (c) correlates with EGFR expression and regulation.

Methods: We used immunoblotting, immunohistochemistry and TCGA database analysis to examine Mastl expression, its association with EGFR in pancreatic cancer progression, and patient survival. Genetic and pharmacological manipulations for Mastl expression were performed and effects on proliferation, apoptosis and cell cycle were determined.

Results: A robust increase in Mastl expression characterized pancreatic cancer cells when compared with the non-transformed pancreatic cells. A similar upregulated expression of Mastl was found in samples originating from KPC mouse model of PDAC. Analysis of the human pancreatic cancer samples and the TCGA database strongly supported this outcome and suggested a positive association of the Mastl expression with cancer progression and patient mortality. Interestingly, genetic inhibition of Mastl expression in pancreatic cancer cells not only inhibited the ability of these cells to proliferate and invade through the matrix but also inhibited activation of the ErbB-family of tyrosine kinase receptors. Further studies revealed that overexpressing K-ras/mutant p53 pathway in HPNE cells (untransformed pancreatic cells) modulates Mastl expression, and thus suggested a causal correlation of Mastl with K-Ras/EGFR signaling in pancreatic cancer cells.

Conclusion: Our results suggest a cross talk of Mastl with K-ras/EGFR signaling pathway in pancreatic cancer progression. We hypothesize that combinatorial therapy targeting Mastl along with Gemcitabine would overcome the drug resistance in the PDAC. Thus, this study identifies a novel approach for the treatment of tumors resistant to traditional EGFR inhibitors.

#2542

Cell cycle-dependent fluctuated expression of long non-coding RNA RGMP regulates G2/M progression and can be therapeutically targeted by a currently used clinical drug.

Mengfeng Li, Shihua Liu, Hongyu Guan. _Sun Yat-sen University, Guangzhou, China_.

A comprehensive profiling of long non-coding RNA (lncRNA) expression in the course of cell cycle progression remains largely uninvestigated. In this study, RNA-seq was performed to detect lncRNA expression signature during cell cycle progression in non-small cell lung carcinoma (NSCLC). We found that the abundance of lncRNA RGMP (regulator of G2/M progression) fluctuated in different phases of cell cycle progression, peaking in M phase, followed by reduction in G1 phase. Furthermore, overexpression of RGMP robustly promoted cell proliferation and increased the G2/M population in NSCLC cells, whereas silencing RGMP inhibited cell proliferation and G2/M progression. The pro-proliferative role of RGMP was also demonstrated in a xenografted NSCLC mouse model. Subsequent RNA pull-down and RNA immunoprecipitation analyses demonstrated that RGMP directly interacted with the cyclin-dependent kinase 1 (CDK1)/cyclin B1/phosphorylase CDC25c complex, resulted in dephosphorylation and activation of CDK1. The activated CDK1/cyclin B1 consequently promoted G2/M progression. Moreover, we further sought to screen small molecules capable of interrupting the interaction between RGMP and CDK1 and thereby preventing cancer cell proliferation, and as a result, we identified that a currently used clinical drug could effectively block the above interaction and suppress tumor cell proliferation. Taken together, our findings reveal a previously unknown lncRNA-mediated mechanism involved in cell cycle regulation and highlight the importance of RGMP as a therapeutic target for NSCLC.

#2543

Cyclin-dependent kinase 1(CDK1)-mediated phosphorylation of SET in mitosis inhibits its oncogenic activity.

Ling Yin, Yongji Zeng, Jixin Dong. _University of Nebraska Medical College, omaha, NE_.

SET is a protein phosphatase 2A (PP2A) inhibitor and functions as an oncogene in multiple human cancer types. Here we revealed a novel phosphorylation site of SET in mitosis and its biological significance in tumorigenesis. We found that the mitotic kinase cyclin-dependent kinase 1 (CDK1) phosphorylates SET in vitro and in vivo at serine 7 during antimitotic drug-induced G2/M phase arrest and in normal mitosis. SET deletion resulted in massive mitotic defects including centrosome amplification, multipolar spindles, and chromosome missregregation. Moreover, we revealed that SET mitotic phosphorylation is required for proper mitotic progression and its oncogenic function in vitro and in xenograft animal models. Furthermore, we found STAT3 and p53 signaling are regulated by SET. Collectively, our findings suggest a novel layer of regulation for SET in mitosis and its role in tumorigenesis. (This is supported by Fred and Pamela Buffett Cancer Center Support Grant P30 CA036727)

#2544

Overexpression of the Polo-like kinase 4 (PLK4) contribute to tumor metastasis.

Yeung Sai Fung, Yick Pang Ching. _The University of Hong Kong, Hong Kong, Hong Kong_.

The Polo-like kinase 4 (PLK4) is an important mitotic kinase that lies at the head of centriole duplication pathway. Aberration of PLK4's level in cancer cell often results in supernumery centrosome. PLK4 transcripts was upregulated in a number of cancers including breast, colon gastric and lung cancer, and PLK4 overexpression had been linked to chemoresistance and cancer metastasis. In our preliminary data, knocking down PLK4 in 97L and HLE Hepatocellular Carcinoma (HCC) cell reduced their migration and growth rate. Overexpression of PLK4 in well-differentiated hepatoma HepG2 and Huh7 cell showed enhancement in migration. Centrinone B, a highly potent PLK4 inhibitor, inhibits HCC cells' colony-forming ability and wound healing rate. To gain the insight into the mechanism how PLK4 contribute to cell migration, microtubule regrowth assay was carried out. The results indicating that PLK4 overexpressing cell had a higher rate of microtubule nucleation, which could contribute to directional movement of the cell. Further supporting that PLK4 contribute to HCC progression, we look into the clinical samples. Using qRT-PCR, we found PLK4 transcript is upregulated in a local cohort of HCC samples (n=48), we also confirmed that PLK4 is upregulated in the TCGA and two GEO available datasets. Data from the cancer dependency map showed PLK4 is a common essential gene in a panel of HCC cell line. Suggesting targeted therapy against PLK4 might be useful in reducing tumor growth or metastasis.

#2545

Nek2 and other mitotic kinases are downstream of the E2F activators, and may modulate the epithelial-to-mesenchymal transition in breast cancer.

Yainyrette Rivera-Rivera,1 Mihaela Marina,2 Harold I. Saavedra1. 1 _Ponce Health Sciences University, Ponce, Puerto Rico;_ 2 _Meditech Media, Atlanta, GA_.

The E2F transcription factors have been extensively studied in cancer since they are known for their role in the regulation of cell cycle progression, centrosome duplication, cell survival, and other functions. We recently demonstrated that the E2F activators (E2F1, E2F2, and E2F3) regulate mitosis through the elevated expression of multiple mitotic proteins including Sgo1, Nek2, Hec1, BubR1, and Mps1. We have shown that Mps1 triggers EMT through multiple mechanisms. NIMA-related kinase 2 (Nek2) is a centrosome-associated protein kinase particularly abundant in cells during the G2-M phase, which primary function is to phosphorylate centrosome linker proteins to cause centrosome disjunction and the formation of the bipolar mitotic spindle. Previous results from our lab showed that overexpression of Nek2 in HCC1954 (Her2+ breast cancer cell line) with a knock down for E2F3 rescued a high centrosome amplification pattern and enhances the formation of invasive protrusions. cBioportal analysis using the TCGA database showed Nek2 mRNA alterations in 9.1% of breast cancer patients and is predominantly present in tumors that are ER-, PR-, and Her2- (25%). Similarly, analysis using the METABRIC database showed Nek2 mRNA alterations in 6.1% of breast cancer patients and is predominantly present in tumors from basal (24%), Her2+ (10%), and luminal B (13.9%) subtypes. Based on these results, we hypothesize that Nek2 overexpression has a role in the epithelial-to-mesenchymal (EMT) pathway leading to cell migration and invasion as prelude to metastasis in breast cancer. To address this, we used MCF10A non-tumorigenic mammary cells with or without the overexpression of Nek2 (MCF10A/GFP and MCF10A/GFP-Nek2). Our preliminary data indicates that overexpression of GFP-Nek2 in mammary epithelial cells enhances levels of vimentin, whereas decreased the levels of E-cadherin, suggesting a role of Nek2 in a mesenchymal state. Likewise, siRNA-mediated silencing of Nek2 in mesenchymal breast cancer cells modulates markers of EMT. Because inhibitors are available for Nek2, it would be feasible to target this kinase to combat metastatic HR- breast cancers. Our work will allow the identification of mechanistic molecular pathways influenced by Nek2 role in driving invasion and metastasis to highlight this mitotic kinase as a potential prognostic marker for high-risk breast tumors.

#2546

**Impact of CDK4/6 inhibition on tumor immune microenvironment and immunomodulatory effects PD-L1 blockade in a physiologically relevant 3D-tumoroid model of fresh patient tumor tissue** ex vivo **.**

Melba Marie Page, Melanie Mediavilla Varela, Vijayendra Agrawal, Jenny Kreahling, Soner Altiok. _Nilogen Oncosystems, Tampa, FL_.

Introduction: In mammalian cells, exposure to growth factors triggers de novo synthesis of D-type cyclins, which then associate with their catalytic partners, cyclin dependent kinase CDK4 or CDK6 to regulate the cell cycle. Recent studies implied the role of CK4/6 inhibitors such as Abemaciclib in modulating tumor immune microenvironment and response to PD1/PD-L1 inhibitors. However, the mechanisms by which CD4/6 inhibitors affect the tumor microenvironment is not well understood. Here we investigated the effect of CDK4/6 inhibition by Abemaciclib on the tumor immune microenvironment and response to PD1/PD-L1 inhibitors using Nilogen's 3D-EX ex vivo drug screening platform utilizing un-propagated 3D tumoroids of fresh patient tumor tissue with an intact tumor microenvironment.

Materials and Methods: All non-small cell lung carcinoma tumor samples were obtained with patient consent and relevant IRB approval. For the ex vivo assays 3D tumoroids measuring 100-150 micron in size were treated with Abemaciclib, pembrolizumab (Keytruda) alone or in combination. Treatment-mediated changes in tumor immune cell composition including CD4 and CD8 T-cells, Tregs, NK cells, macrophages and cell surface expression of checkpoint proteins as well as T-cell activation were evaluated by multiplex flow cytometry. Multiplex cytokine assays were performed to assess drug-induced changes in cytokine release. High-content confocal analysis was used to quantify tumor cell killing.

Results: We showed that drug treatments had significant impact on immune cell compositions, specifically on M1/M2 macrophage polarization as well as on CD4 and CD8 lymphocyte populations in selected tumors, which was accompanied by significant changes in pro-inflammatory cytokine release. We further characterized pharmacological interaction between Abemaciclib and pembrolizumab and correlated with tumor cell killing in quantitative imaging studies.

Conclusion: In this comprehensive study we documented the pharmacological effect of CDK4/6 inhibition on tumor immune microenvironment in a physiologically relevant 3D tumoroid model of ex vivo drug testing. We further characterized the pharmacological interaction between Abemaciclib and pembrolizumab to better understand the mechanisms by which CDK4/6 inhibition may enhance therapeutic response to PD1 blockade in patient tumors.

#2547

TERT promoter mutations in B viral humanhepatocarcinogenesis.

Young-Joo Kim,1 Jeong Eun Yoo,2 Youngsic Jeon,2 Jae Uk Chong,2 Gi Hong Choi,2 Dae-Geun Song,1 Sang Hoon Jung,1 Bong-Kyeong Oh,3 Young Nyun Park2. 1 _Korea Inst. of Science and Technology, Gangneung, Republic of Korea;_ 2 _Yonsei University College of Medicine, Seoul, Republic of Korea;_ 3 _Hanyang University College of Medicine, Seoul, Republic of Korea_.

Telomerase reverse transcriptase (TERT) promoter somatic mutations, related to telomerase activation, have been known to frequently occur at two hot spots located at -124 and -146 bp relative to the start codon in various cancers. In the present study, we investigated the occurrence and implications of the genetic alterations of the TERT promoter in B viral hepatocarcinogenesis. TERT promoter mutations, especially -124C>T mutation, obviously enhanced TERT promoter activity in hepatocellular carcinoma (HCC) cell lines. We identified prospero homeobox protein 1 (PROX1) as a novel transcriptional activator for TERT gene through binding to the promoter regions containing two hot spots. PROX1 binding affinity was strong to the mutant TERT promoter harboring a consensus E-twenty six/ ternary complex factor (ETS/TCF) binding sequence (CCGGAA). The incidence of TERT promoter mutations gradually increased according to the progression of human B viral multistep hepaticarcinogenesis, which was found in 9.0% of low grade dysplastic nodules (LGDNs), 13.5% of high grade dysplastic nodules (HGDNs), 27.3% of early HCCs (eHCCs) and 28.4% of progressed HCCs (pHCCs). The occurrence of TERT promoter mutations correlated with lower levels of alpha-fetoprotein (AFP) (p=0.046) and a poor overall survival (p=0.012) in B viral HCC patients. On the contrary to in vitro data, TERT mRNA expression was lower in B viral HCCs with the mutant TERT promoter compared to those without. In addition, mRNA level of PROX1 was not correlated with that of TERT in B viral HCCs, in contrast that such correlation was evident in non-B viral HCCs. Interestingly, induction of stable HBx expression inhibited PROX1-mediated TERT expression in vitro study. In conclusion, our findings suggest that TERT promoter somatic mutations are early events in B viral human multistep hepatocarcinogeneis and HBx can induce a loss of PROX1 function as transcriptional activator for TERT expression.

#2548

DNA damage response gene expression in CHK1 inhibitor responsive and resistant mouse models of MYC driven B-cell lymphoma.

Nicola L. Hannaway, Jill E. Hunter, Alastair Greystoke, Neil D. Perkins. _Newcastle Universtity, Newcastle upon Tyne, United Kingdom_.

Enhanced tumor replication stress and genomic instability can lead to disruption of the DNA damage response, leading to tumor survival and dependence on essential checkpoint controls. Drugs to target intact pathways, such as CHK1 inhibitors, are therefore desirable and currently in clinical trials as potential future anticancer therapies. The underlying mechanisms underpinning the response and resistance to CHK1 inhibitor treatment are still unclear. We have demonstrated that NF-κB can regulate the response to oncogene induced DNA replication stress in vivo through regulation of CLSPN (Claspin) gene expression. Claspin is an essential adaptor protein required for the phosphorylation and activation of CHK1 by ATR. Using the Eµ-MYC mouse model of B-cell lymphoma we have found that mutation of NF-κB subunits (knockout of c-Rel or a T505A transactivation domain mutation of RelA) leads to earlier lymphoma onset that correlates with loss of Clspn expression and inhibition of CHK1 activity. Moreover, while wild type Eµ-MYC lymphomas are highly responsive to CHK1 inhibitor treatment, by contrast Eµ-MYC NF-κB mutant cRel-/- or RelAT505A lymphomas are resistant. To understand more about how MYC driven lymphoma cells respond to CHK1 inhibitor treatment, we have investigated changes in the expression of genes encoding members of DNA damage response pathways. Wild type mice reimplanted with Eµ-MYC lymphoma cells were treated with the CHK1 inhibitor CCT244747 and harvested at multiple fixed timepoints. Quantitative PCR analysis of mRNAs encoding proteins in the ATR/CHK1 and ATM/CHK2 pathways was performed, with RPL13A used as reference gene. Interestingly, while dramatic changes were seen in wild type Eµ-MYC cells, these were not seen in CHK1 inhibitor resistant cRel-/- lymphomas. We observed an upregulation of the DNA damage and cell cycle responsive genes in the CHK1 inhibitor sensitive wild-type Eµ-MYC mice. This is consistent with with previously published in vitro studies and suggests an S phase cell cycle arrest in response to CHK1 inhibitor induced DNA damage. This response, however, is absent in the CHK1 inhibitor resistant Eµ-MYC NF-κB mutant lymphomas. It is hoped that by understanding these differences in gene expression that biomarkers of CHK1 inhibitor response can be identified to help stratify future clinical candidates.

#2549

Allostatic conditions in human glioblastoma stem cells are maintained with the contribution of CLIC1 membrane protein functional expression.

Ivan Verduci,1 Valentina Carlini,1 Federica Barbieri,2 Antonio Daga,2 Tullio Florio,2 Michele Mazzanti1. 1 _Univ. of Milano, Milano, Italy;_ 2 _Univ. of Genova, Genova, Italy_.

Allostasis is a cellular condition physiologically occurring during transient hyper-activated state of the cell. When stress is persistent, cells are unable to restore a basal homeostatic state and allostasis become chronic. Most of the cells are not able to cope with these prolonged conditions and die. Conversely, solid tumors reveal a persistent stress state strengthen by chronic overexpression of several elements and hyper-activation of intracellular pathways. In glioblastoma stem cells (GSCs), the most evident allostatic outcome is a high rate of cell division. Several proteins present in the allostatic GSC plasma membrane are deregulated in either expression or function. Altering any of these proteins disrupts the allostatic equilibrium, causing drastic functional changes and, in some cases, cell death. Several of these proteins, potentially used as valuable pharmacological targets, are also crucial for the physiological activity of the cells. Consequently, modifying their functions would affect the survival of both cancer and healthy cell population. On the contrary, chloride intracellular protein 1 (CLIC1) is active as a membrane charge carrier only during periods of chronic stress, while during homeostatic conditions it is essentially irrelevant. Inhibition of NADPH oxidase or the NHE1 proton pump, both overexpressed in CSCs, causes death of both GSCs and normal mesenchymal stem cells. Conversely, impairing CLIC1 activity delays GSC cycle progression but leaves healthy cells functions unaltered. Our work suggests that CLIC1 protein and its associated chloride permeability are a crucial element involved in the stabilization of GB CSC allostasis. (This work is supported by a GRANT from AIRC to MM)

#2550

Genetically determined telomere length impacts colorectal cancer recurrence and survival.

Brooke Druliner, Donna Felmlee-Devine, Kim Kossick, Miguel Munoz-Gomez, Jorge Loyo-Lopez, Lisa Boardman. _Mayo Clinic, Rochester, MN_.

Since colorectal cancer (CRC) is the second leading cause of cancer death in the United States, predicting survival for a CRC patient is crucial for individualizing care. Telomere length is a recognized biomarker in multiple cancers and is associated with CRC patient survival, but the comprehensive telomere profile of the individual has yet to be integrated into a clinically actionable survival prediction model for CRC patients. We measured telomere length and the telomere-related genotype (TERT, TERC and OFBC1) of peripheral blood leukocyte (PBL) DNA in 324 patients with Stage III colon cancer who were participants in our IRB approved Biobank for Gastrointestinal Health Research (BHGR) at the Mayo Clinic. We identified a significant association of PBL telomere length with both disease free survival and overall survival, and between telomere length and the telomere-shortening alleles (telomere-related genotype). We found that telomeres shortened with increasing number of telomere-shortening alleles; ptrend = 1.8*10-3. When we tested for an association between genetically and non-genetically determined PBL telomere length with the number of recurrences, we found that patients who had long PBL telomere length but were genetically predicted to have short telomeres and those with short PBL telomeres genetically predicted to have long telomeres were the two highest risk groups for CRC recurrence. Overall we have determined that Stage III CRC patients with genetically determined short telomeres have decreased cancer mortality.

#2551

Inhibition of mTOR abrogates mitosis progression.

Hsiao-Hui Kuo, Chieh-Ting Fang, Ling-Huei Yih. _Academia Sinica - Inst. of Cell. & Organismic Bio., Taipei, Taiwan_.

The mammalian target of rapamycin (mTOR) mediates the coordination of cell growth and cell cycle progression in response to nutrient supply. It is frequently activated in cancers and confers resistance to anti-cancer drugs. Accumulated studies have demonstrated that inhibition of mitotic kinases (PLK1 or aurora kinase A) enhances mTOR activities and the mTOR activation is associated with cell survival to anti-mitotic drugs. In this study, whether mTOR signaling is involved in mitosis progression is explored. Treatment of cells with mTOR inhibitors for 24 h resulted in the accumulation of G2 and mitotic cells and increased the percentage of mitotic cells with spindle abnormalities and chromosome missegregation. In addition, mTOR inhibitors sensitized cancer cells to taxol by enhancing apoptosis. Our results indicate that mTOR signaling pathway may be required for faithful mitosis progression and represent a potential resistance mechanism to the use of anti-mitotic drugs.

#2552

Senescence induced by CDK4/6 inhibition facilitates anti-tumor T cell responses.

Ashlyn Blevins,1 Stacey Mont,1 Hunter Lawrence,1 Nabil Saleh,1 Sheau-Chiann Chen,2 Ann Richmond,3 Anna Vilgelm1. 1 _Vanderbilt University, Nashville, TN;_ 2 _Vanderbilt University Medical Center, Nashville, TN;_ 3 _Tennessee Valley Healthcare System, Nashville, TN_.

Cancer immunotherapies have shown remarkable clinical activity across several tumor types but responses have been modest in breast cancers. This is attributed to low levels of neo-antigens in breast tumors and immunosuppressive nature of mammary tumor microenvironment. Here we investigated if we can counteract these obstacles to an effective immunotherapy by inducing senescence in breast cancer cells. Senescence is one of the physiological stress response programs characterized by loss of proliferative potential and secretion of a variety of pro-inflammatory markers which promotes identification and removal of senescent cells by the immune cells. Here we hypothesized that senescence will stimulate anti-tumor immune response by establishing immune-permissive microenvironment. We utilized immunocompetent and humanized PDX-bearing mice to investigate senescence-induced modulation of tumor secretome proteome, transcriptome and immune cell recruitment and activity. An FDA-approved small molecule CDK4/6 inhibitor was used to induce senescence. We detected induction of multiple markers of senescence after CDK4/6 inhibitor treatment of cells and tumors. Senescent cells and tumors had increased secretion of chemokines CCL4, CCL5, CXCL9, CXCL10 and CXCL11 which facilitated T cell homing into the tumor. This was a result of their transcriptional induction by NF-κB. Consequently, we observed the dynamic enrichment of tumor immune microenvironment with T cells in tumors treated with CDK4/6 inhibitor. Furthermore, the recruitment of adoptively transferred ex-vivo activated anti-tumor T cells was significantly enhanced in tumors that were treated with CDK4/6 inhibitor. Finally, CDK4/6 inhibitor-treated tumors became responsive to T cell-activating immunotherapy with antibody agonists of T cell co-stimulatory receptors OX40 and 4-1BB. In summary, our findings suggest that CDK4/6 inhibition can facilitate T cell recruitment into the mammary tumors which can lead to an improved immunotherapy response. This pre-clinical study provides rationale for clinical development of CDK4/6 inhibitors and T cell agonists and uncovers the potential of senescence-inducing approach for sensitizing tumors to immune therapy.

#2553

Integration of genomic studies and the patient-derived xenograft model in nasopharyngeal carcinoma precision medicine research.

Cheng-Lung Hsu,1 Hsin-Pai Li2. 1 _Chang Gung Memorial Hospital, Taipei, Taiwan;_ 2 _Chang Gung Memorial Hospital/University, Taipei, Taiwan_.

Patient-derived xenograft (PDX) tumor model has become a new approach to identify druggable tumor mutations, and to screen and evaluate personalized anti-cancer drugs. Here, we established five nasopharyngeal carcinoma (NPC) PDXs in mouse model and performed whole-exome sequencing (WES) and copy number variations (CNVs) analyses. Relatively high incident rate of CNV aberration in cell cycle pathway was detected. Among these five NPC-PDXs, three had cyclin D1 (CCND1) amplification while four had cyclin-dependent kinase inhibitor CDKN2A deletion. Selected inhibitors were tested for their anti-tumor effects in two NPC-PDX mouse models. Palbociclib, a cyclin-dependent kinase inhibitor, revealed efficient anti-tumor effects by inducing G1 arrest in the PDX tumor according to RNA sequencing. In this study, cyclin D1 overexpression was also observed in >90% clinical metastatic NPC tumors (87/91) and was associated with poor outcomes. One NPC patient with liver metastatic who was prescribed with Palbociclib had stable disease and a drop in Epstein Barr virus (EBV) DNA titer. CNV analysis disclosed that plasma CCND1/CDKN2A ratio is correlated with EBV DNA load in NPC patients' plasma and could serve as a screening test to select potential CDK4/6 inhibitors treatment candidates. Taken together, our findings indicate that integrated information from sequencing-based genomic studies and tumor transcriptomes before and after drug treatment in NPC-PDX models provide novel guidelines for personalized precision treatments.

#2554

Branched chain amino acid transaminase 1 in claudin-low breast cancer.

Lisa C. Reynen, Robert Jones, Roger Moorehead. _University of Guelph, Guelph, Ontario, Canada_.

Breast cancer, the most commonly diagnosed cancer in women, can be classified into five distinct subtypes. One subtype, claudin-low breast cancer, accounts for approximately 7% of the breast cancer cases and these tumors are aggressive and highly metastatic. RNA sequencing of human claudin-low breast cancers by other groups and RNA sequencing of a murine claudin-low mammary tumor cell line by our group has revealed that Bcat1 is significantly up-regulated in this breast cancer subtype. Bcat1 regulates the metabolism of branched chain amino acids and has been linked to numerous pathologies including heart disease, diabetes, and cancer. Based on this data we hypothesized that the expression of Bcat1 in claudin-low mammary tumors is driving the aggressive and metastatic nature of this cancer subtype and disrupting Bcat1 will deter these features. Elevated expression of Bcat1 in the murine claudin-low cell line RJ423, compared to the murine luminal mammary tumor cell line RJ345, has been confirmed at the mRNA and protein level. Bcat1 has been transiently down-regulated ~70% in RJ423 cells using siRNA and this suppression of Bcat1, contrary to the anticipated result, showed no effect on proliferation based on phospho-histone H3 immunofluorescence. However, when the cell population doubling time was investigated, a significant difference was seen between the RJ423 cell line and the cell lines with suppressed Bcat1 expression. To elucidate these contradicting results, cell cycle analysis using Bromodeoxyuridine and 7-AAD through flow cytometry is currently underway. RJ423 stably expressing Bcat1 shRNA and RJ345 stably expressing a Bcat1 expression vector are currently being selected. Further in vitro and in vivo studies will utilize these stably transfected cell lines. This study will determine whether further investigation into the effects of Bcat1 on claudin-low human breast cancer is prudent and if Bcat1 may be used as a therapeutic target.

#2555

Modulation of protein interaction states in the cell cycle and during cancer drug action.

Pär Nordlund. _Karolinska Institutet, Stockholm, Sweden_.

The cellular thermal shift assay (CETSA) is a biophysical technique allowing direct studies of ligand binding to proteins in cells and tissues. The proteome-wide implementation of CETSA with mass spectrometry detection (MS-CETSA) is an efficient method to discover targets for orphan clinical drugs and hits from phenotypic screens, as well as to identify off-targets, explain poly-pharmacology and toxicity. Recently implemented highly sensitive multidimensional MS-CETSA techniques can now also access binding of physiological ligands to proteins, such as metabolites, nucleic acids and other proteins. MS-CETSA can thereby provide comprehensive information on modulations of protein interaction states in cellular processes, including downstream effects of drugs and transitions between different physiological cell states (Dai, Cell 2018, Tan, Science 2018). Such horizontal information on ligand modulation in cells is largely orthogonal to vertical information on the levels of different proteins, and therefore opens novel opportunities to understand operational aspects of cellular proteomes in more detail. We will discuss recent application of MS-CETSA to understand the cell cycle as well as cancer drug action and resistance.

References:

\- Modulation of Protein-Interaction States through the Cell Cycle. Dai L, Zhao T, Bisteau X, Sun W, Prabhu N, Lim YT, Sobota RM, Kaldis P, Nordlund P. Cell. 2018 173(6):1481-1494.

\- Thermal proximity coaggregation for system-wide profiling of protein complex dynamics in cells. Tan CSH, Go KD, Bisteau X, Dai L, Yong CH, Prabhu N, Ozturk MB, Lim YT, Sreekumar L, Lengqvist J, Tergaonkar V, Kaldis P, Sobota RM, Nordlund P. Science. 2018 359(6380):1170-1177.

#2556

Enhanced expression of human cyclin G1 (CCNG1) gene in metastatic cancer, a novel biomarker in development for CCNG1 inhibitor therapy.

Erlinda M. Gordon,1 Christopher Szeto,2 Joshua R. Ravicz,1 Sandeep Reddy,3 Michael Morse,4 Sant Chawla,1 Frederick Hall5. 1 _Sarcoma Oncology Center/Cancer Center of Southern California, Santa Monica, CA;_ 2 _Nantomics, Culver City, CA;_ 3 _Nanthealth, Culver City, CA;_ 4 _Duke University Medical Center, Durham, NC;_ 5 _Delta Next-Gene, LLC, Santa Monica, CA_.

Background: Metastatic cancer is associated with, hitherto, an invariably fatal outcome. DeltaRex-G, a CCNG1 inhibitor, has induced long term (9-12 year-) survivorship in patients with metastatic melanoma, sarcoma, lymphoma and cancer of pancreas and breast.

Purpose: To evaluate CCNG1 expression in tumors as a novel biomarker for identification of patients who are likely to benefit from CCNG1 inhibitor therapy.

Methods: RNA sequence analysis was used to compare CCNG1 expression in tumor (TCGA, N=9161) versus normal tissues (TGCA, N=678 and GTEx, N=7187) from 20 organ sites, and in primary vs metastatic melanoma (N=106 & 367 respectively). Using IHC staining, the significance of differences between the number of CCNG1+ and Ki-67+ cells in normal tissues vs tumors were evaluated using paired Student's t test. Pearson's coefficient of correlation was used to evaluate the relationship between the number of Ki-67+ vs CCNG1+ cells.

Results: CCNG1 gene expression, by RNA sequence analysis, was significantly enhanced in melanoma, sarcoma, leukemia, and cancers of the lung, thyroid, brain, liver, prostate, testicle, head and neck, bladder, kidney, adrenal, cervix, and pancreas, compared to normal tissues (p = 0.006- 1.8E-58), and even greater, in metastatic vs primary melanoma. In a subset of melanoma tumors, there was overall positive correlation with progression/metastasis in both Ki67 and CCNG1 expression levels. Consistent with these findings, IHC showed significant differences when comparing CCNG1 nuclear staining percentages between analogous healthy and cancerous tissues (p = 0.002, df = 11). The nuclear staining percentages of CCNG1 and Ki-67 share a statistically significant, positive correlation (r = .936977, p < 0.00001). Further, in some patients, the level of CCNG1 expression by IHC fluctuated over time.

Conclusion: Taken together, these data indicate that (1) CCNG1 expression, is frequently enhanced in cancerous tumors compared to their normal analogous counterparts, and (2) the level of CCNG1 expression may change over time, necessitating real time analysis in order to determine the optimal timing for CCNG1 inhibitor therapy. A Phase 2 basket study of DeltaRex-G cell cycle checkpoint inhibitor therapy is planned to correlate treatment outcome parameters with CCNG1 expression in patients' tumors and circulating tumor cells. 

### DNA Damage and Repair 2

#2557

X-ray repair cross complementing 1 (XRCC1) is a critical factor in response to DNA damaging agents in triple negative breast cancer.

Kevin J. Lee, Joel Andrews, Natalie R. Gassman. _Univ. of South Alabama Mitchell Cancer Inst., Mobile, AL_.

Triple negative breast cancer (TNBC) is a breast cancer subtype that is characterized by the lack of expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). The lack of these targetable molecules makes cytotoxic chemotherapies a first-line choice, thus there is a pressing need to better understand how TNBC will respond to alternative or emerging treatments. Base excision repair (BER) proteins play an essential role in DNA damage response and repair, yet they have been overlooked in the analysis of chemotherapeutic treatment response. XRCC1 is a critical scaffold protein that mediates the protein-protein and protein-DNA interactions involved in BER and single-strand break repair. Several studies have linked single nucleotide polymorphisms (SNPs) within XRCC1 to increases in breast cancer incidence, yet XRCC1's role in modulating treatment response is unknown. Here we show XRCC1 expression in a panel of TNBC cell lines correlates to an increase in basal DNA damage levels. DNA damage was analyzed using the Repair Assisted Damage Detection (RADD) assay which measures the broadest spectrum of DNA damage in vitro. We show MDA-MB-157 with low levels of XRCC1 correlate to low levels of basal DNA damage, MDA-MB-231 and HCC1806 have moderate levels of XRCC1 and DNA damage, and MDA-MB-468 contain high expression levels of XRCC1 that correlate with high levels of DNA damage. XRCC1 localization is also variable across the panel of TNBC cell lines with XRCC1 being mislocalized to the cytoplasm in the HCC1806 cells and highly localized to the nuclear compartment in the MDA-MB-468. These expression and localization changes in XRCC1 alter the ability of cells to repair single strand break (SSB) lesions induced by 355 nM laser microirradiation and DNA damaging chemicals. Here we show that TNBC cell lines respond differently to alkylating damage (methyl methanesulfonate) and PARP inhibition (olaparib) based on XRCC1 expression and localization in combination with levels of basal DNA damage. Taken together it is important to consider XRCC1 in context with DNA damage levels and expression of other BER proteins in TNBC in order to effectively design experiments utilizing DNA damaging agents or chemotherapeutics targeting DNA repair pathways.

#2558

Regulation of the expression of the DNA repair endonuclease 8-like 3 gene in hepatocellular carcinoma.

Wenya Huang. _National Cheng Kung University, Tainan, Taiwan_.

Hepatocellular carcinoma (HCC) is the fifth commonest malignancy in the world, but the molecular mechanism of HCC development is still unclear. Previous studies had showed that the endonuclease 8-like 3 (Neil3) gene, a DNA glycosylase for oxidative DNA repair, overexpressed in most of cancers including HCC. In our previous cDNA microarray analysis in HCC tissues, it was found that the Neil3 gene was highly expressed in mouse and human tumors. The results of real-time RT-PCR analysis in 113 HCC patients showed that the Neil3 gene mRNA was increased in most human tumors. The Neil3 gene expression levels were also highly correlated with HCC recurrence in the patients receiving the hepatectomy surgeries, suggesting that it can serve as a prognostic marker for HCC. In this study we characterized the molecular mechanisms of the Neil3 gene expression regulation in HCC. We analyzed the methylation status in the Neil3 gene promoter regions by DNA methylation array (Agilent) and found that the Neil3 gene promoter was hypo-methylated in HCC tumors. Also, by the promoter reporter assays it was found that the main regulatory region for its transcription activity was from -123 to +94 upstream to the transcription start site. Furthermore, the DNA affinity precipitation assay (DAPA) was performed to screen for the transcription factors that bind to the Neil3 promoter and identified that cell cycle regulator E2F1, Yin Yang 1 (YY1) and histone deacetylase 1 (HDAC1) bind to the Neil3 gene promoter. It suggested that E2F1, YY1 and HDAC1 play important roles in regulating Neil3 gene expression. These transcription factors have been known to enhance cell proliferation and potentially the NEIL3-mediated oncogenic properties. And treatments of the HDAC inhibitor SAHA increased the Neil3 mRNA and protein levels. The functional effects of these pro-oncogenic transcriptional factors to the NEIL3-relevant HCC progression are currently in investigation. The results of these studies will clarify the regulatory mechanisms for the Neil3 gene induction in HCC progression.

#2559

Functional characterization of a RECQL4 mutation in Rothmund Thomson Syndrome.

Tianyi Wu,1 Robert L. Walker,1 Marbin Pineda,1 MiYoung Lee,2 Yuelin Zhu,1 Ashok R. Venkitaraman,2 Paul S. Meltzer1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _University of Cambridge, Cambridge, United Kingdom_.

Osteosarcoma (OS) is the most common malignancy of the bone, typically diagnosed during the adolescent years. Despite early advances in treatment, OS survival rates have not improved in recent times. Targeted treatment remains elusive because OS is a genetically complex and heterogeneous disease and the low incidence of sporadic OS presents a challenge in dissecting the causes and drivers of tumor development. Type II Rothmund-Thomson Syndrome (RTS), a cancer predisposition disorder, presents a potential model for studying OS pathogenesis as roughly 30% of Type II RTS patients are eventually diagnosed with OS. Type II RTS is associated with germline mutations of the DNA helicase RECQL4 that exclusively target the C-terminal helicase domain thought to be involved in various cellular mechanisms of DNA repair. The N-terminal SLD2-homology domain, required for the assembly of the CMG complex at origins of replication, is spared. We used CRISPR/Cas9 gene editing to generate cell lines containing a common RECQL4 mutation identified in Type II RTS patients to further study its effects. Unexpectedly, a mutation previously reported as a nonsense mutation instead upregulates the activation of a known cryptic splice site in exon 14. This splice variant results in the in-frame deletion of the final 66 amino acids of exon 14. Our cell lines exhibit a mildly increased sensitivity to the DNA double-strand break inducing agent neocarzinostatin consistent with RECQL4's reported functions in DNA damage repair. We will use next generation sequencing techniques to further characterize DNA damage repair signatures in RTS RECQL4 mutant cell lines.

#2560

Werner syndrome helicase is a selective vulnerability of microsatellite instability-high tumor cells.

Simone Lieb,1 Silvia Blaha-Ostermann,1 Elisabeth Kamper,1 Katharina Ehrenhöfer-Wölfer,1 Andreas Schlattl,1 Andreas Wernitznig,1 Jesse Lipp,1 Kota Nagasaka,2 Gerd Bader,1 Ralph Neumueller,1 Norbert Kraut,1 Mark Pearson,1 Simon Woehrle,1 Mark Petronczki1. 1 _Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria; _2 _Research Institute of Molecular Pathology (IMP), Vienna, Austria_.

Targeted cancer therapy is based on exploiting selective dependencies of tumor cells. By leveraging recent large-scale genomic profiling and functional screening of cancer cell lines we identified Werner syndrome helicase (WRN) as a novel specific vulnerability of microsatellite instability-high (MSI-H) cancer cells. MSI, caused by defective mismatch repair is frequently detected in human malignancies, in particular in colorectal, endometrial and gastric cancers. We demonstrate that WRN inactivation selectively impairs the viability of MSI-H but not microsatellite stable (MSS) colorectal and endometrial cancer cell lines. In MSI-H cells, WRN loss results in the emergence of chromosome breaks, chromatin bridges and micronuclei highlighting defective genome integrity. WRN variants harboring mutations abrogating the ATPase function of WRN helicase fail to rescue the viability phenotype of WRN-depleted MSI-H colorectal cells. Our study suggests that pharmacological inhibition of WRN helicase function might represent a novel opportunity to develop a targeted therapy for MSI-H cancers.

#2561

DNA-PKCS deacetylation by SIRT2 promotes DNA double-strand break repair by non-homologous end joining.

PamelaSara E. Head,1 Nagaraju P. Ganji,1 Shi-Ya Wang,2 Duc Duong,1 Hui Zhang,1 Waaqo Daddacha,1 Shuyi Li,1 Nicholas T. Seyfried,1 David M. Smalley,3 Ya Wang,1 Xingming Deng,1 William S. Dynan,1 Bassel El-Rayes,1 Anthony J. Davis,2 David S. Yu1. 1 _Emory Univ. School of Medicine, Atlanta, GA;_ 2 _UT Southwestern Medical School, Dallas, TX;_ 3 _Georgia Institute of Technology, Atlanta, GA_.

DNA-dependent protein kinase (DNA-PK) plays a critical role in non-homologous end joining (NHEJ), the predominant pathway that repairs DNA double-strand breaks (DSB) in response to ionizing radiation (IR) to govern genome integrity. How DNA-PK is activated in response to DSBs has remained elusive. Here, we show that the SIRT2 sirtuin deacetylase and tumor suppressor directs the activation of DNA-PK through deacetylation of its catalytic subunit (DNA-PKcs). SIRT2 deacetylase activity governs cellular resistance to DSB-inducing agents and promotes NHEJ. SIRT2 furthermore interacts with and deacetylates DNA-PKcs in response to IR, which facilitates its interaction with Ku and recruitment to DSBs, thereby leading to DNA-PKcs autophosphorylation and DNA-PK signaling to downstream NHEJ substrates. Moreover, SIRT2 inhibitor sensitizes resistant cancer cells and tumors to IR. Our findings define a mechanism for DNA-PK activation by SIRT2-mediated deacetylation, elucidating a critical upstream signaling event initiating the repair of DSBs by NHEJ to promote genome integrity and govern IR resistance, which can be exploited for improvements in cancer therapy.

#2562

Functional characterization of Polθ in esophageal squamous cell carcinoma.

Jian Li, Josephine Ko, Wei Dai, Lvwen Ning, Hoi Yan Ng, Valen Zhuoyou Yu, Maria Lung. _The University of Hong Kong, Hong Kong_.

Introduction: Wild-type (WT) Polθ, encoded by POLQ gene, is a specialized DNA polymerase which helps to protect cells against genomic instability by allowing DNA double-strand break (DSB) repairs through the alternative end-joining (AltEJ) pathway. In our previous targeted gene sequencing studies, we identified a high frequency of POLQ germline variants associated with esophageal squamous cell carcinoma (ESCC) in an endemic high-risk region of China. Here in this study, we aim to determine the functional impact of POLQ in ESCC, examine the synthetic lethality relationship of POLQ-mediated AltEJ DNA repair pathway and the canonical homologous recombination (HR) pathway, and eventually explore its potential roles as a biomarker or therapeutic target in ESCC.

Methods: To determine the functional roles of Polθ, POLQ knock out (KO), FANCD2 KO and POLQ/FANCD2 double KO ESCC cell lines were generated using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technique. MTT assay was used to assess cell viabilities with or without additional treatments that induced DNA damage or replication stress. DSB repair efficiency was examined by the ⲅH2AX foci recovery assay. The single cell gel electrophoresis/Comet assay was used to evaluate the genome stability. The expression level of POLQ was compared between ESCC patients' samples and paired non-tumoral tissues using Q-PCR.

Results: Among all ESCC cell lines we tested, 8 out of 13 (61.5%) exhibited higher levels of POLQ mRNA/protein than the normal immortalized esophageal epithelial cell line NE1. A 2-fold or more elevation of POLQ was found in 17 out of 28 Hong Kong ESCC patients' tumor samples when compared with their paired non-tumoral tissues. Depletion of POLQ in high-POLQ expressing ESCC cell lines (KYSE180TS and SLMTorT3) only slightly diminished the cell viability. Double KO POLQ and FANCD2, one of the HR genes, significantly sabotaged cell proliferation, as compared with single KO POLQ or FANCD2, especially after treated with Cisplatin or Hydroxyurea. The percentage of ⲅH2AX foci positive cells increased considerably upon POLQ KO at 8 hours post 4Gy ionizing radiation, hinting the compromised DSB repair efficiency in POLQ depleted cells. The POLQ-deficient cells also showed a higher degree of genomic instability as suggested by the Comet assay.

Conclusions: The results suggested that Polθ is overexpressed in ESCC tumors and cell lines. The Polθ mediated DSB repair pathway may work as a backup plan of HR to prevent the genomic instability in ESCC. Targeting Polθ might be a potential therapeutic approach for the better management of HR-deficient ESCC. Acknowledgments: Research Grants Council Collaborative Research Fund grant number 106150246 and Asian Cancer Research Fund to MLL. We thank Prof. Jean-Sébastien Hoffmann for providing the Polθ antibody.

#2563

A comparison of microsatellite instability analysis methods for detection of mismatch repair deficiency.

Samantha R. Lewis, Shaun Peterson, Kathryn Oostdik, Heather Tomlinson. _Promega Corporation, Madison, WI_.

Use of biomarkers for patient treatment stratification is an increasingly important topic with the recent FDA approval of Pembrolizumab and Nivolumab for microsatellite instability (MSI) high/mismatch repair (MMR) deficient cancers. This ground-breaking approval allows physicians to make patient treatment decisions based on molecular biomarker status. Thus, performance of detection methods for these biomarkers is of great importance. The gold standard for microsatellite instability (MSI) analysis is PCR followed by fragment analysis using capillary electrophoresis to resolve DNA fragment size. Recently, PCR followed by melt curve analysis and Next Generation Sequencing (NGS) have been presented as alternative approaches. In this set of experiments, these methods will be compared using a subset of human cancer samples.

A cohort of matched cancer and adjacent normal FFPE samples were used for this comparison. From this cohort, individuals were selected that contained subtle MSI shifts, low tumor volume, or heterozygosity at microsatellite loci. Samples from these individuals were tested for MSI by either fragment or melt curve analysis or by sequencing. Samples were then classified as MSI-High, MSI-Low or microsatellite stable (MSS) according to manufacturer specifications for melt curve analysis or by an interpretive software for fragment analysis and NGS. Preliminary results indicate that there may be differences in sensitivity when using challenging samples with melt curve analysis compared to the gold standard method, fragment analysis.

The standard method for determining MSI status utilizes PCR and fragment analysis. Recently, melt curve analysis and NGS have been proposed as alternative methods. We present preliminary findings of decreased sensitivity with melt curve compared to fragment analysis. Additional studies with a larger, more diverse sample set will be required to define this relationship further.

#2564

Tracing extrachromosomal DNA inheritance patterns in glioblastoma using CRISPR.

Eunhee Yi, Roel Verhaak. _Jackson Laboratory for Genomic Medicine, Farmington, CT_.

Glioblastoma (GBM) is the most lethal brain tumor; it is characterized by poor response to standard post-resection radiation and cytotoxic therapy, resulting in a dismal prognosis with a five-year survival rate of 10%. Recurrence after therapy for GBM is unavoidable. There are substantial differences among the cells of GBM tumors in the abundance and types of genetic material. This heterogeneity likely is the major cause of therapy failure, the development of treatment resistance, and ultimately recurrence. A recent study has suggested that the majority of GBM activate oncogenes through amplification extrachromosomal DNA (ecDNA). Despite the speculation that ecDNA is a key factor of tumor heterogeneity, how ecDNA is propagated and distributed among—and how it behaves within—cancer cells is completely unknown. To address this gap in knowledge, this study focused on developing a novel cytogenetic tool that enables visualization and tracking ecDNA behavior in live GBM cells. We found breakpoint sequences resulting from genome rearrangements during ecDNA formation by performing computational analysis whole genome sequencing data from a pair of primary/recurrent GBM neurospheres. Each breakpoint represents a unique target sequence for ecDNA-specific labeling. Breakpoint were validated by breakpoint-PCR, focusing on breakpoints shared by the primary and recurrent neurosphere. We determined location and quantity of all ecDNA breakpoints through breakpoint-FISH (BP-FISH) analysis, which showed that all signal was found outside of the conventional chromosomes in the metaphase spread, validating the extrachromosomal nature of the ecDNA breakpoints. The breakpoint quantities, reflecting the number of ecDNA elements per cell, varied between primary and recurrent neurosphere suggesting that ecDNAs quantitatively changed during GBM evolution. These results emphasize the necessity of tracking ecDNA dynamics to answer the question of how ecDNA is distributed into the daughter cells. Our findings will be strong evidence to make ecDNA-specific CRISPR system in further research. Tracing ecDNA dynamics will provide new insight into the impact of ecDNA on cancer evolution.

#2565

Type I interferons enhance chemosensitivity of breast cancer by inhibiting DNA repair.

Na-Lee Ka, Ga Young Lim, Mi-Ock Lee. _Seoul National University, Seoul, Republic of Korea_.

Type I interferons (IFNs) are known to have antineoplastic effects against several tumors by stimulating anticancer immune response. In this study, we identified a novel function of type I IFNs in DNA repair, which enhanced chemosensitivity in breast cancer cells. We investigated that IFNα and IFNβ inhibited both non-homologous recombination and homologous recombination double strand breaks repair. Moreover, IFNs accelerated the release of DNA into the cytoplasm in response to DNA damaging agent, doxorubicin. In agreement, IFNs synergistically activated DNA damage-induced STING pathway and increased the production of chemokines, CCL5 and CXCL11. In agreement, IFNβ greatly increased the sensitivity of cells to doxorubicin-induced suppression of cell survival. This sensitizing effect of IFNβ was also supported by in vivo xenograft experiments. Thus, combination therapy with chemotherapeutic agents and IFNs may be effective against breast cancer, particularly in patients with defective in DNA damage response pathways. The downstream effectors that modulate the effect of IFNs should be investigated as predictive biomarker in the treatment of breast cancer.

#2566

CYT01B suppresses RAD51 focus formation and induces replication catastrophe following AID induced DNA damage.

Melinda Day, Amber Cyr, Tyler Maclay, Kevin Mills. _Cyteir Therapeutics, Lexington, MA_.

Activation Induced Cytidine Deaminase (AID) is a DNA-directed cytidine deaminase that plays a critical role in somatic hypermutation and immunoglobulin class switching in activated B- lymphocytes. AID is often ectopically expressed and promotes genomic hypermutation in a range of lymphoid malignancies. Deamination of genomic cytidines results in point mutations, single strand DNA breaks (SSB), and double strand DNA breaks (DSB). The resulting DNA damage stress leads to an obligate dependency on homologous recombination for cellular survival. RAD51 is a critical component of the homologous recombination pathway, forming nucleoprotein filaments at sites of DNA damage or replication fork stalls. RAD51 mediates homologous DNA strand exchange to promote recombinational repair of breaks and damaged replication forks. We have previously shown that RAD51 depletion leads to accumulation of DNA breaks, and ultimately cell death, in AID expressing cells. We now describe the mechanism of action for a novel small molecule, CYT01B, that targets RAD51 mediated recombination. Treatment of AID-expressing cells with CYT01B, attenuates RAD51 focus formation, reduces the nuclear concentration of RAD51, and promotes RAD51 protein degradation. CYT01B reduces homologous recombination activity in cells, culminating in cytologically visible chromosome fragmentation following DNA damage. Finally, CYT01B treated cells undergo replication catastrophe associated with caspase 3/7 activation and persistent PCNA nuclear signal. We conclude that CYT01B is a novel homologous recombination inhibitor, and propose a new synthetic lethality mechanism, induced by CYT01B, mediated by DNA replication stress and replication catastrophe.

#2567

TRACERx: Intra-tumor subclonal driver mutation results in defective DNA damage response (DDR) and genome instability.

Wei-ting Lu,1 Lykourgos-Panagiotis Zalmas,1 Thomas Webber,1 Nnennaya Kanu,2 Charles Swanton1. 1 _The Francis Crick Institute, London, United Kingdom;_ 2 _UCL Cancer Institute, London, United Kingdom_.

TRACERx is a longitudinal clinical study, which explores the evolutionary trajectories of NSCLC by temporally dissecting genomic aberrations through multi-region sequencing. Analysis of the first 100 patients revealed extensive intra-tumor heterogeneity (ITH) and chromosomal instability (CIN), manifesting as subclonal somatic mutations and copy number aberrations. Although mutations in "trunk" clonal drivers such as Ras, C-Myc, p53 or MAPK pathway have been thoroughly characterized after the herculean efforts of TCGA consortium, the roles of sub-clonal drivers remain largely obscure as their identification is only made possible by multi-region sequencing.

Harnessing the power of the TracerX consortium, we investigate the molecular mechanisms that underpins the role of sub-clonal drivers in ITH. By means of high content imaging of DNA damage markers and tracking human artificial chromosome loss, we aim to elucidate the link between subclonal drivers and CIN events (Zalmas et al, in press). Importantly, we found the ablation of a large proportion of subclonal drivers result in genomic instability. This suggests that genome instability is not merely a standalone hallmarks of cancer, but also a major step that drivers sub-clonal expansion and functionally connect genome instability to tumor evolution. However, the molecular pathways directly linking genome maintenance, chromosomal instability events and cancer evolution is unclear. More intriguingly, only a fraction of these subclonal drivers has been previously reported to be involved in canonical DNA damage response (DDR). This suggests the error-prone DNA repair is favored during tumor clonal evolution.

Here, we present previously uncharacterized subclonal driver that are important for error-free DNA repair pathway. The ablation of these genes causes mis-localization of key homologous repair (HR) proteins, such as BRCA1 and Rad51, to DNA damage sites after IR irradiation. Strikingly, early signaling events such as ATM phosphorylation, phosphorylation and ubiquitylation of histone H2A.X, and 53BP1 localization remain unaffected. The exact molecular mechanism the leads to DNA repair pathway selection will be discussed.

#2568

Investigation of Fanconi Anemia pathway downstream genes.

Bianca Nguyen,1 Li Gao,1 Abeer Almiman,1 Shirley Tang,2 Kathleen Dotts,2 Miguel A. Villalona-Calero,3 Wenrui Duan1. 1 _Florida International University, Miami, FL;_ 2 _Comprehensive Cancer Center at Ohio State University College of Medicine and Public Health, Columbus, OH;_ 3 _Miami Cancer Institute, Miami, FL_.

Introduction: A key response mechanism to DNA damage is the Fanconi Anemia repair pathway (FA), which involves homologous recombination DNA repair and is activated through mono- ubiquitination of FANCD2. FA deficiency is considered to increase the sensitivity of tumors to particular DNA-targeted agents, and may prove to be a target of cancer treatment. We hypothesize that FA deficient tumors have a low growth rate and reduced ability for DNA repair compared to FA functioning tumors. Given that genetic modifications can interfere with FA functionality, we aim to explore the association between the FA pathways and downstream genes that influence tumor growth. To date, few studies have examined gene expression associated with FA deficiency in lung cancer cells. Identification of the FA downstream genes may provide insight on DNA repair networks that impact cancer treatment.

Methods: To generate FANCD2 knockdown cells, human lung cancer cell lines A549 and H1299 were transduced with FANCD2-specific short hairpin RNA expressing and puromycin-resistant lentiviral particles or control shRNA lentiviral particles. The cells were cultured in growth medium, and successful FANCD2 knockdown was confirmed by western immunoblot analysis. RNA deep sequencing was completed with Illumina RNA-Seq. We compared gene expression between knockdown FANCD2 and control samples across three cell lines and ranked significant gene expression changes, defined as a five-fold change in upregulation or downregulation. The fold change was calculated by dividing FANCD2 deficient expression by FANCD2 efficient expression.

Results and discussion: 13436 genes were evaluated across three cell lines and 17 genes demonstrated gene expression change by at least 5-fold with FANCD2 knockdown in three cell lines. FANCD2 knockdown resulted in 14 downregulated genes and 3 upregulated genes. The downregulated genes RP11-618G20.1, RP5-1021I20.4, RP11-219A15.1, XXbac-BPG32J3.20, and BMS1P17 demonstrated significant expression change across three cell lines. Of the 14 downregulated genes, 13 genes had literature supporting oncogenic function.

FA downstream genes confers oncogenic function. As FANCD2 is considered to promote cell proliferation, downregulation of oncogenic genes expression was expected with FANCD2 knockdown. However, the literature suggested that the 3 upregulated genes with FANCD2 knockdown also have oncogenic function. These genes may have functioning beyond the scope of carcinogenesis which may explain gene upregulation with FANCD2 knockdown. Pinpointing genes related to FA pathway deficiency may provide insight into genetic phenomena that drive cancer. Our results provide a starting point for developing targets to specific downstream genes associated with FA deficient tumors, which may prove to limit cancer progression. Further investigation is needed to determine how FANCD2 interacts with these genes to promote cell proliferation.

#2569

Synergism between RAD51AP1 and RAD54 during late stages of homologous recombination DNA repair.

Platon Selemenakis, David Maranon, Claudia Wiese. _Colorado State University, Fort Collins, CO_.

Genomic instability is one of the enabling hallmarks of cancer and can arise by exposure to several factors: endogenous challenges such as stress to DNA replication forks or exogenous compounds such as exposure to ionizing radiation (IR) and other environmental mutagens. These insults can lead to the induction and propagation of mutations through the generation of multiple DNA lesions, the most toxic one of which is a DNA double-strand break (DSB). DSBs can be repaired by several different repair pathways. One of these pathways is homologous recombination (HR), a relatively faithful DSB repair pathway that relies on the availability of a homologous template for DNA repair synthesis.

DNA strand exchange in HR is mediated by the RAD51 recombinase which forms a nucleoprotein filament on single-stranded DNA for strand invasion. In human cells, RAD51-mediated strand invasion is supported by the DNA motor protein RAD54 and by the RAD51-Associated Protein 1 (RAD51AP1). While RAD54 translocates on double-stranded DNA and promotes DNA strand separation, RAD51AP1 enhances the assembly of the synaptic complex. However, if RAD54 and RAD51AP1 work independently or together in the HR reaction is unclear. Since functional loss of either RAD54 or RAD51AP1 each leads to a moderate defect in HR, we speculate that RAD54 and RAD51AP1 may act in parallel in the promotion of RAD51 activity. We hypothesize that simultaneous inactivation of both RAD54 and RAD51AP1 may lead to a synthetic phenotype in HR impairment.

The main focus of this study is to investigate the potential synergism between RAD51AP1 and RAD54 during late stages of the HR reaction to improve targeted cancer therapy. We are investigating if loss of RAD54 in human cells exacerbates the phenotype of RAD51AP1-deficiency and renders cells more sensitive to DNA damaging agents. We have generated RAD51AP1 knockout HeLa cells using CRISPR/Cas9 technology. In these cells, we have also inactivated the RAD54 gene using a very similar approach. Moreover, we have generated RAD54 knockout (KO) cells in a wild type RAD51AP1 background. All cell lines are tested for their response to chemotherapeutic agents in cell survival and DNA replication assays. Our first results show that the double KO cells are more sensitive to mitomycin C exposure than single KO cells, suggesting that RAD51AP1 and RAD54 function largely independently of each other in the HR reaction.

The results from our investigation are expected to lead to the potential development of new cancer drugs targeted at ancillary factors of RAD51 in late stages of the HR reaction.

#2570

Colonic adenocarcinoma-associated DNA-binding site mutations in Rad52 perturbs its ssDNA binding polarity and function in homologous recombination.

Emma A. Tillison, Nilisha Pokhrel, Edwin Antony. _Marquette University, Waukesha, WI_.

Double strand DNA breaks (DSB) are repaired by the Homologous recombination (HR) pathway. Defective HR leads to accumulation of mutations that result in genomic instability and a plethora of associated cancers including, breast, ovarian, and skin cancers. In HR, the DSB is recognized and resected to yield single-stranded DNA (ssDNA) that are immediately coated by replication protein A (RPA). The Rad51 recombinase, which is the central engine for HR, needs to bind to ssDNA buried under RPA and form a nucleoprotein filament. Cells possess mediator proteins that either promote or inhibit the exchange of RPA for Rad51 on ssDNA. RAD52 is one such pro-recombinogenic mediator and functions analogous to the Breast Cancer type 2 Susceptibility Protein (BRCA2) to promote the formation of the Rad51 filament. However, the mechanistic details underlying how Rad52 mediates RPA-Rad51 exchange on ssDNA remains a mystery. Mutations in mediator proteins such as BRCA2 and RAD52 are prevalent in many cancers. For example, the COSMIC database shows there have been 139 sampled cases of mutations of Rad52 in solid tumors.

To understand how Rad52 fulfills its mediator functions, it is imperative to first understand how Rad52 associates with DNA. The recent crystal structure of human RAD52 (PDB ID) proposed two ssDNA binding site for RAD52: an outer and an inner binding site, where Arg55 is the interface amino acid between the two sites. To systematically characterize how Rad52 associates with ssDNA, we conducted stopped flow experiments with fluorescently labeled ssDNA. Our results show that Rad52 binds cooperatively to the 5′ end of DNA. Additionally, we created mutations in Rad52-ssDNA binding sites to investigate their role in polarity and cooperativity in depth. These residues and adjacent amino acids are hotspots for mutagenesis in cancers. Abolishing the outer binding site results in a loss of cooperative binding and higher order Rad52-ssDNA complex formation. Mutating the inner binding site abolishes ssDNA binding entirely. Additionally, abolishing the interface arginine residue results in non-selective, rapid binding. This exact amino acid has been mutated in two cases of colonic adenocarcinoma. Our result suggests that both sites are required for maintaining cooperativity and polarity of Rad52 binding to ssDNA. A detailed mechanistic insight into Rad52-ssDNA binding will aid in the development of small-molecule inhibitor drugs that can be used as effective chemotherapeutic agents against cancers in the future.

#2571

Role of XRCC4 downregulation in bioflavonoid-induced chromosomal translocations.

Anindita Ghosh, Christine A. Richardson. _Univ. of North Carolina at Charlotte, Charlotte, NC_.

The faithful repair of DNA double strand breaks (DSBs) is crucial for genome integrity, and aberrant repair can result in chromosomal translocations, including those associated with infant leukemia. Evidence from our lab and others suggests that environmental and dietary compounds such as bioflavonoids have the potential to promote chromosomal translocations in utero. DSBs can be repaired by either classical non-homologous end joining (C-NHEJ) that is mostly accurate or by microhomology-mediated alternative end joining (Alt-EJ) that is more error prone and can cause chromosomal translocations. Ku70/80, Ligase 4 (LIG4), X-Ray Repair Cross-Complementing factor 4 (XRCC4), and XRCC4 like factor (XLF) are central to C-NHEJ. Poly ADP Ribose 1 (PARP 1) and C terminal binding protein interacting protein (CtIP) participate in Alt-EJ. CtIP favors Alt-EJ pathway by promoting the resection of DNA ends to uncover microhomologies supporting the formation of large deletions and chromosomal translocations. Studies suggest that loss of efficient activity in the C-NHEJ repair pathway may promote the use of the other more error-prone pathway such as Alt-EJ. The purpose of the study is to understand the relationship between bioflavonoid exposure, induction of DNA repair proteins, and repair pathway choice to understand the underlying mechanisms of bioflavonoid-induced infant leukemias. Embryonic stem (ES) cells were exposed to genistein 1 hr at doses 0μM-100μM based on determined LD50 values. Following exposure, cells were recovered 4hr, 24hr and 48 hr, followed by protein extraction and Western blotting. Protein levels of γH2AX, Ku70, XRCC4, XLF, CtIP and PARP-1 were measured by densitometry and analyzed using one way ANOVA. CRISPR-Cas9 was used to downregulate C-NHEJ DNA repair protein XRCC4. XRCC4-/- cells will subsequently be exposed to genistein, recovered, followed by protein extraction and Western blotting. Protein levels of γH2AX, Ku70, XRCC4, XLF, CtIP and PARP-1 will measured by densitometry. Protein levels before and after gene editing of XRCC4 will be compared and analyzed using two way ANOVA. We have downregulated the XRCC4 gene in ES cells and anticipate that following exposure to genistein in the absence of XRCC4, we will detect elevated chromosomal translocation frequency in those stem cells by stimulating the Alt-EJ pathway. This research will be highly impactful in understanding how bioflavonoid-induced DNA damage is repaired by cells and whether these compounds promote genome instability analogous to that observed in infant leukemia.

#2572

Integrated viral sequences in esophageal cancer tissue.

Victoria A. Patten,1 Lamech Mwaphaga,1 Christopher G. Mathew,2 Denver T. Hendricks,1 M. Iqbal Parker1. 1 _University of Cape Town, Cape Town, South Africa;_ 2 _University of Witwatersrand, Johannesburg, South Africa_.

The human genome exhibits a significant degree of invasion by viral genomes. During the course of evolution, several RNA and DNA viruses have become integrated into the vertebrate genome and there is no reason to believe that the process has ended. Integrated viral DNA may account for as much as 10% of the human genome and it is very likely that they may act as carcinogens by altering normal cellular gene expression.

In this study we have compared the viral DNA sequences present in normal and tumor tissues from esophageal squamous cell carcinoma (ESCC) samples. DNA was extracted from paired normal and tumor biopsies and subjected to whole genome sequencing. Raw data run folders were converted to FASTQ files and progressed through a QC pipeline. Normal and tumor data sets were then blasted and aligned against an extensive viral sequence database using specialized DRAGEN software, so that all integrated viral DNA fragment sequences were identified. The identified viral sequences were then compared between the paired normal and tumor samples.

Using this approach, a large number of viral DNA sequences were identified; these included human papilloma viruses, Herpes simplex viruses, adenoviruses and Hepatitis C viruses. Furthermore, some unexpected viruses of interest were also identified. These include the Autographa Californica Nucleopolyhedrovirus, the Emiliana Huxleyi virus 86, the Gryllus bimaculatus nudivirus and the Trichodysplasia spinulosa-associated polyomavirus, present in different tumour DNA samples.

Amplification and rearrangements of endogenous retroviruses such as the Human Endogenous Retrovirus K113 were also observed. Endogenous retroviruses (ERV) can serve as long-lasting viral reservoirs and provide evidence for the coexistence between retroviruses and their hosts over many generations. Defective ERVs can also recombine to affect the expression of adjacent genes.

It has been proposed that integrated viral sequences may play a possible role in the etiology and promotion of diseases such as cancer, thus targeted studies of specific viral sequences may elucidate the mechanisms of gene disruption and may ultimately lead to the development of more effective therapeutic interventions.

#2573

DNA damaging drugs stimulate tRNA cleavage through SLFN11 to inhibit protein synthesis and induce cell death.

Manqing Li, Elaine Kao, Dane Malone, Xia Gao, Jean Y. Wang, Michael David. _University of California San Diego, La Jolla, CA_.

DNA damaging agents (DDAs) based chemotherapies are the first and still the most widely used anticancer treatments. However, quickly developed drug resistance of cancer cells often impedes their efficacies. Two independent transcriptome analyses in vast collections of cancer cell lines discovered that human SLFN11 is the only gene whose expression significantly sensitizes cancer cells to DNA damaging agents (DDAs) including Top1 and Top2 inhibitors, DNA alkylating agents, and DNA synthesis inhibitors. But the mechanism was unclear. We showed previously that SLFN11 inhibits HIV viral proteins syntheses by modulating tRNAs abundances and exploiting the biased codon usage of the virus. We hereby report that upon DDAs treatment, SLFN11 preferentially inhibits translation of genes involved in DNA damage response and repair, including ATR and ATM, based on their distinct codon usages, while the early DNA damage response signaling remains unaffected. Type II tRNAs, which include all serine and leucine tRNAs, are cleaved in a SLFN11-dependent manner in response to DDAs. The syntheses of proteins encoded by genes with high TTA (Leu) codon usage such as ATR display utmost susceptibility to SLFN11 expression due to the extreme low abundance of cognate tRNA-Leu-TAA. Specific inhibition of tRNA-Leu-TAA expression with special antisense oligonucleotides (Gapmers) suppressed ATR protein expression and restored DDA sensitivity of SLFN11-deficient cancer cells. Our study uncovered a novel mechanism of DNA damage response regulation mediated by SLFN11-dependent tRNA cleavage and codon-specific translational inhibition. Further investigation will elucidate the roles of SLFN11 in DNA damage-related tumorigenesis. More importantly, bioinformatics analysis revealed high frequency of codon TTA (Leu) usage in most genes involved in DNA damage response signaling and repair mechanisms including Homology Directed Repair (HDR), Nonhomologous End-Joining (NHEJ), and Mismatch Mediated Repair (MMR). Instead of targeting single gene product, novel therapies based on the modulation of tRNA-Leu-TAA expression would effect by inhibiting the protein synthesis of a wide spectrum of genes involved in DNA damage response and repair, thus preventing the development of drug resistance by cancer cells.

#2574

Werner syndrome helicase is required for the survival of cancer cells with microsatellite instability.

Lorn Kategaya,1 Senthil K. Perumal,1 Jeffrey H. Hager,2 Lisa D. Belmont1. 1 _IDEAYA Biosciences, South San Francisco, CA;_ 2 _IDEAYA Biosciences, San Diego, CA_.

Werner Syndrome protein (WRN) is a RecQ enzyme involved in the maintenance of genome integrity. Germline loss of function mutations in WRN lead to premature aging and pre-disposition to cancer. We evaluated synthetic lethality (SL) between not only of WRN, but also of another human RecQ helicase, BLM, with DNA damage response genes in cancer cell lines. We found that WRN exhibited SL with a DNA mismatch repair protein MutL homolog 1 (MLH1), loss of which is associated with high microsatellite instability (MSI-H). We then demonstrated that MSI-H cells exhibited increased double-stranded DNA breaks, altered cell cycles and decreased viability in response to WRN knockdown. Although WRN is the only human RecQ enzyme with a distinct exonuclease domain, only loss of helicase activity drives the MSI SL interaction. This SL interaction in MSI cancer cells positions WRN as a relevant therapeutic target in patients with MSI-H tumors.

#2575

EZH2 mediates resistance to cisplatin in small cell lung cancer through nucleotide excision repair.

Allyson E. Koyen. _Emory University, Atlanta, GA_.

Small cell lung cancer (SCLC) is the most aggressive form of lung cancer, with a five-year survival rate of 7%. Cisplatin-based chemotherapy is the first line treatment for SCLC; however, many patients develop treatment resistance and experience tumor recurrence. Targeting proteins critical to the repair of cisplatin DNA crosslinks is a strategy for overcoming acquired cisplatin resistance in SCLC, but many proteins that mediate crosslink repair have yet to be identified. To address this issue, we performed a synthetic lethal siRNA screen in cisplatin resistant SCLC cells, and identified EZH2 as one of the strongest mediators of cisplatin resistance. EZH2 localizes to sites of DNA damage which are induced by UVA-crosslinking laser microirradiation and interacts in a complex with DDB1, and DDB2, members of the nucleotide excision repair (NER) pathway. Loss of EZH2 sensitizes SCLC cells to UV damage, and further, loss of EZH2 and DDB1 together are epistatic in the sensitization of SCLC to cisplatin, confirming a role for EZH2 in NER. Specifically, EZH2 promotes the stability of DDB2, which is responsible for the detection NER lesions. This occurs independently of EZH2's methyltransferase activity on H3K27. Finally, we found EZH2 expression correlates with cisplatin resistance across SCLC cell lines. Together, this data suggests that EZH2 functions as a novel regulator of NER, and that EZH2 is a promising target for cisplatin resistant SCLC.

#2576

BMI1 and RING1A are involved in H2A ubiquitination at sites of platinum-induced damage.

Shruthi Sriramkumar, Heather M. O'Hagan. _Indiana University Bloomington, Bloomington, IN_.

Ovarian cancer (OC) is a lethal gynecological malignancy with a 5-year survival rate of 46%. Surgical debulking followed by platinum and taxane based chemotherapy is the standard of care for OC patients. However, the major obstacle in the use of these agents for treatment of OC patients is development of chemoresistance. An epigenetic mechanism commonly associated with development of platinum resistance is promoter DNA methylation and associated gene silencing. Transcriptional silencing of tumor suppressor genes like MutL homolog 1 (MLH1) by this mechanism has been well established in development of platinum resistance. However, the mechanism of initiation of this aberrant promoter DNA methylation is not known. Platinum agents are DNA damaging agents which crosslink guanines and form intra-strand or inter-strand adducts. We hypothesize that platinum induced DNA damage or repair of the damage results in recruitment of proteins involved in transcriptional repression to sites of damage. Recruitment of repressive proteins results in transient repression of transcription in the vicinity of damage to promote repair. However, similar to our findings with enzyme induced double strand breaks, repair of damage can occasionally result in retention of repressive proteins at sites of damage causing persistent transcriptional repression and gene silencing. Such persistent repression of key loci potentially contributes to the development of platinum resistance. We demonstrate that treatment of platinum sensitive OC cells with the IC50 dose of cisplatin for 8 hours resulted in ubiquitination of H2A/H2AX by western blot analysis. We hypothesize that this ubiquitination occurs on K119 of H2A/H2AX. H2A/H2AX ubiquitination at K119 mediated by Polycomb repressive complex 1 (PRC1) has been associated with transcriptional repression and gene silencing during development and differentiation and also during DNA repair. We observe a reduction in platinum induced H2AX ubiquitination on knockdown of PRC1 complex member RING1A using western blot analysis. Using immunofluorescence, we observe that BMI1, another PRC1 complex member, localizes to sites of platinum induced DNA damage. Platinum induced lesions are repaired by different repair pathways including – Nucleotide excision repair pathway (NER), Fanconi Anemia repair pathway (FA) and Homologous recombination repair (HRR). We demonstrate that knockdown of proteins in global genome NER and HRR pathways results in a decrease in platinum induced ubiquitination. Through on-going studies we seek to further elucidate the mechanism of recruitment of BMI1, RING1A to sites of platinum induced DNA damage and their role in long-term gene silencing. Understanding this mechanism will enable us to design inhibitors to prevent the occurrence of platinum resistant tumors in OC patients.

### miRNA-based Diagnostics and Therapeutics

#2577

Circulating micro RNA/isomiRs as novel biomarker of esophageal squamous cell carcinoma.

Yuta Ibuki, Yasuhiro Tsutani, Daisuke Ueda, Yumiko Koi, Hidetoshi Tahara, Morihito Okada. _Hiroshima University, Hiroshima, Japan_.

Background: Micro RNAs(miRNAs) are small noncoding RNA molecules comprising 19 - 25 nucleotides that regulate the expression of messenger RNA. Micro RNAs are promising diagnostic biomarkers they differ between patients with cancer and healthy individuals. Recent deep sequencing studies have found that highly stable miRNA isoforms(isomiRs) circulate in bloodstream. Although circulating isomiRs could potentially be powerful biomarker like mature miRNAs, their application as cancer biomarkers not been investigated in detail. The present study aimed to determine whether circulating mature miRNAs and isomiRs detected by deep sequencing could serve as biomarkers of esophageal cancer.

Methods: We enrolled serum samples from 62 patients who were histologically diagnosed esophageal squamous cell carcinoma (ESCC) and 60 age and sex matched healthy control (HC) individuals without a history of cancer. The expression of miRNA/isomiRs was investigated using Ion S5 XL sequencer (Thermo Fisher Scientific). Samples were classified into primary (18 ESCC and 12 HC), secondary (30 ESCC and 30 HC), and tertiary (14 ESCC and 18 HC) groups. The definition of a diagnostic biomarker is detected in > 90% of the ESCC and HC samples, mean read numbers significantly differed over twice between them (p < 0.05, t-test). Candidates which met the criteria in the primary group were validated in secondary group. Diagnostic panel was generated using miRNA/isomiRs that consistently confirmed in primary and secondary groups by stepwise multiple linear regression model. Thereafter, accuracy of the diagnostic panel was tested in the tertiary group.

Results: We detected 5452 miRNA/isomiRs at least one sample. Among them, 88 miRNA/isomiRs met the criteria for a diagnostic biomarker in primary group, and were validated in secondary group. As a result, 28 candidates (8 mature miRNAs and 20 isomiRs) met the criteria in primary and secondary group. Multiple linear regression model selected four mature miRNAs and three isomiRs. The index calculated from the panel was significantly higher in patients with ESCC than in the control (87.7 ±11.7 vs. 9.3±1.8, p < 0.001). The area under the receiver operating characteristics (ROC) curves (AUC) of the panel index to predict ESCC patients was 0.993 (95% CI, 0.98 - 1.0, p < 0.001) in the primary and secondary groups, and 0.91 (95% CI, 0.78 - 1.0, p < 0.001) in the tertiary group. The panel index of patients with esophageal adenocarcinoma was significantly lower than that with ESCC (28.5±8.93 vs. 87.7±11.7, p < 0.001). The postoperative panel index tended to be lower than the preoperative index (36.3±5.73 vs. 50.1±37.6, p = 0.13).

Conclusion: To the best of our knowledge, this is the first study to show the usefulness of isomiR to diagnose esophageal cancer. Our diagnostic panel including isomiR had high accuracy and specificity for diagnosing ESCC. IsomiRs detected by deep sequencing could serve as a novel biomarker of ESCC.

#2578

Serum analysis of obese and diabetic African American colon adenoma patients reveals novel potential miRNA markers of carcinogenesis.

Hassan Brim,1 Mohammad Daremipouran,2 Gail Nunlee-Bland,1 Adeyinka Laiyemo,1 Hassan Ashktorab1. 1 _Howard University Hospital, Washington, DC;_ 2 _Howard University Hospital, DC_.

We have previously demonstrated that Adiponectin, leptin, TNF-alpha, and IGF-1 were highly prevalent in patients with colon adenomas when compared to healthy individuals. In the present study, we aimed to establish specific miRNA signatures in obese and diabetic African American patients developing colorectal adenomas.

Patients & Methods: Serum samples from 72 individuals (18 normal, 18 adenoma-obese, 18 adenoma-diabetic, and 18 adenoma-obese-diabetic) seen at Howard University Hospital were collected. All selected patients were age and gender-matched across the 4 groups. Serum RNA extracts were used for library preparation and sequencing on an Illumina NextSeq 500 using SR75 High Output kit. Sequencing data were processed through the miasma-Seq pipeline. CutAdapt was used to trim Illumina adapters and remove reads that were less than 15 bp long. Reads were aligned to the UCSC hg38 human genome reference sequence using Bowtie2. Annotation data in the form of a GFF file from mirBase was used to count the number of reads falling in each hairpin locus. To estimate differential expression between different groups of samples, the count data were used in the DESeq2 R package and the 4 patient groups comparisons were performed. MiRNAs with statistically significant (p < 0.05) expression difference from normal and log 2 FC > 2 and/or < -2 were selected for functional analysis using Ingenuity Pathway Analysis (IPA).

Results: Comparisons to normals revealed that 11 miRNAs [hsa-mir-34a, hsa-mir-133a-1, hsa-mir-127, hsa-mir-99b, hsa-mir-485, hsa-mir-378d-2, hsa-mir-3168, hsa-mir-3679, hsa-mir-378e, hsa-mir-4635, hsa-mir-6509] were down-regulated in Adenoma-Obese; 3 miRNAs were down-regulated [hsa-mir-1228, hsa-mir-3912, hsa-mir-4665] and 3 up-regulated [hsa-mir-455, hsa-mir-219b, hsa-mir-8072 ] in Adenoma-Diabetic patients; and 2 down-regulated [hsa-mir-487b, hsa-mir-6777] and 1 up-regulated miRNA [hsa-mir-4525] in Adenoma-Obese-Diabetic patients. The IPA analysis revealed that many of the altered miRNA have known roles in carcinogenic and metabolic syndrome pathways.

Conclusion: From the pairwise comparison, diabetes seems to be associated with more alterations of the miRNA profile in the analyzed samples when compared to patients developing colon adenomas on an obesity background. MiRNA-4525 can be used as a potential serum marker in obese patients while miRNA-455, miRNA-219b, and miRNA-8072 can serve as such in diabetic patients. Except for miRNA-455 that was previously described as a promotor of migration and invasion in breast cancer, the other mi-RNAs are novel and their mechanisms of action need further study.

#2579

Genomewide transcriptomic profiling identifies a novel miRNA signature for predicting lymph node metastasis in patients with pancreatic ductal adenocarcinoma.

Satoshi Nishiwada,1 Kensuke Yamamura,1 Raju Kandimalla,1 Takahiro Akahori,2 Kota Nakamura,2 Hideo Baba,3 Masayuki Sho,2 Ajay Goel1. 1 _Baylor Scott & White Research Institute, Dallas, TX; _2 _Nara Medical University, Japan;_ 3 _Kumamoto University, Japan_.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is an extremely lethal cancer, and is projected to become the second leading cause of cancer-related deaths in the US by 2030. The lymph node (LN) status is an important predictor of survival outcomes in PDAC patients undergoing curative resection. The National Comprehensive Cancer Network guidelines recommend neoadjuvant therapy in patients with high-risk features such as large regional LNs, elevated CA-19-9 levels, large primary tumors, excessive weight loss, and extreme pain. Therefore, a pre-treatment diagnosis of LN metastasis (LNM) is critical in developing a more personalized treatment strategy in PDAC patients. Herein, for the first time, we performed a genomewide analysis to identify and develop a miRNA signature for the detection of LNM in PDAC patients.

Methods: We analyzed a total of 518 PDAC patients, which included 269 patients from three genomewide miRNA-expression datasets (TCGA, GSE24279 and GSE35688) and 249 from two, independent clinical patient cohorts. During the biomarker discovery phase, we used rigorous computational and bioinformatic approaches to identify candidate miRNA biomarkers, which later were subjected to in-silico validations. The robustness and performance of this miRNA-signature was subsequently interrogated in two independent clinical cohorts (training cohort: n=150, validation cohort: n=99) using qRT-PCR assays. Furthermore, the clinical potential value of the miRNA signature was evaluated and compared to several clinicopathological factors using logistic regression analysis.

Results: Through a comprehensive miRNA expression profiling analysis of LNM-positive and negative patients, we identified a panel of 7 miRNAs that significantly predicted LNM [the area under curve (AUC)=0.76, 0.75 and 0.92, respectively]. Using logistic regression analysis, we then optimized and trained a 6-miRNA risk-prediction model in the training cohort of patients, which robustly distinguished PDAC patients with LNM (AUC: 0.84, 95%CI: 0.76-0.89). The performance of this trained model was subsequently confirmed in an independent, large clinical validation cohort (AUC: 0.73, 95%CI: 0.64-0.82). Interestingly, multivariate analysis which incorporated several pre-operative clinical factors, revealed that our 6-miRNA signature was as an independent predictor of LNM in both patient cohorts (training cohort: OR=34.97; P<0.01, validation cohort: OR=10.11; P<0.01, respectively).

Conclusion: We identified and developed a novel 6-miRNA signature that is highly robust in predicting presence of LNM in PDAC patients; highlighting the clinical impact of these findings in more appropriate patient selection and development of improved individualized treatment strategies for PDAC patients.

#2580

Investigation of plasma exosomal miRNA as a biomarker and its potential function in prostate cancer castration resistant development.

Tianyu Guo,1 Yang Wang,1 Xueying Mao,1 Lei Xu,1 Jacek Marzec,1 Edwina Burke,1 Glenda Scandura,1 Elzbieta Stankiewicz,1 Caitlin R. Davies,1 John Hines,2 Greg Shaw,3 Jonathan Shamash,4 Daniel Berney,1 Prabhakar Rajan,1 Karen Tipples,5 Alistair Grey,6 Yong-jie Lu1. 1 _Bart Cancer Instistute, QMUL, London, United Kingdom;_ 2 _Department of Urology, Barts Health NHS, London, United Kingdom;_ 3 _University Collage London Hospitals NHS Foundation Trust, London, United Kingdom;_ 4 _Department of Medical Oncology, Barts Health NHS, London, United Kingdom;_ 5 _Bart Health NHS, London, United Kingdom;_ 6 _University Collage London, London, United Kingdom_.

Prostate cancer (PCa) ranks as the second most frequent cancer and the fifth leading cause of cancer death in men worldwide. Androgen-deprivation therapy is the principal treatment for locally advanced and metastatic disease. Although a majority of patients initially respond well to ADT, most will progress to castration-resistant prostate cancer (CRPC), which contributes to the majority of PCa deaths. Exosomes are small vesicles that contain numerous molecular constituents, including lipids, proteins, RNAs and DNAs, and can mediate cell-cell communications. The aim of this study is to identify plasma exosomal miRNAs correlated with CRPC development, which may serve as a biomarker to monitor disease status and may reveal new role of exosome in CRPC development. Next-generation sequencing (NGS) of plasma exosomal miRNAs was performed in 24 treatment-naive PCa patients and 24 CPRC patients. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to validate candidate miRNAs in 108 treatment-naive PCa patients and 42 CRPC patients and a group of 38 non-CRPC patients under hormone therapy (treated non-CRPC). To study the miRNA function, hormone-dependent PCa cell line LNCaP was cultured in hormone-depleted media. MiRNA was overexpressed by transient transfection of miRNA mimic. Post transfection, transwell assays and cell viability assays were performed to determine the effect of miRNA on cell migration and proliferation. RNA NGS generated an average of approximately 5-million reads per sample and identified differentially expressed miRNAs. QRT-PCR validation showed six miRNAs were significantly differentially expressed between treatment-naïve PCa and CRPC (p-value<0.05). It also showed that five of these miRNAs were differently expressed in CRPC compared treated non-CRPC patients (p-value<0.001). When receiver operating characteristic curve was applied, one of the exosomal miRNAs, miR-423-3p, achieved area under the curve (AUC) = 0.784 when predicting CRPC from treatment-naïve PCa and AUC = 0.883 when predicting CRPC from treated non-CRPC. Overexpression of miR-423-3p had no effect on LNCaP proliferation, but significantly increased cell migration. This study demonstrated that plasma exosomal miRNAs play important roles in CRPC development and may serve as a biomarker for CRPC occurrence prediction.

#2581

Identification and establishment of a novel, circulating miRNA signature, for pre-operative diagnosis of occult distant metastasis in patients with pancreatic ductal adenocarcinoma.

Kensuke Yamamura,1 Susan Tsai,2 Jasjit K. Banwait,1 Fuminori Sonohara,3 Douglas B. Evans,2 Yasuhiro Kodera,3 Hideo Baba,4 Ajay Goel1. 1 _Baylor Scott & White Research Institute and Charles A. Sammons Cancer Center, Baylor University Medical Center, Dallas, TX; _2 _Medical College of Wisconsin, Milwaukee, WI;_ 3 _Nagoya University, Nagoya, Japan;_ 4 _Kumamoto University, Kumamoto, Japan_.

Purpose: In spite of recent advances in radiographic imaging technologies, not all patients with resectable pancreatic ductal adenocarcinoma (PDAC) with occult distant metastasis (ODM; including peritoneal or liver metastasis) can be robustly diagnosed with such approaches. A more accurate pre-operative diagnosis for ODM is of clinical significance, as it could facilitate selection of patients that are most appropriate candidates for such complex surgeries. Currently there is lack of availability of biomarkers, which can help diagnose presence of ODM in PDAC patients. Herein, using a systematic and comprehensive biomarker discovery and validation approaches, we aimed to develop a blood-based miRNA signature for the diagnosis of ODM in PDAC patients.

Methods: During a biomarker discovery step, we analyzed serum miRNA expression profiles in stage I-III vs. stage IV PDAC patients (GSE59856: n=100) to identify a circulating miRNA-signature for ODM. This miRNA signature was subsequently validated in sera from a clinical training cohort (n=129), which included 44 stage-IV and 85 stage I-III PDAC patients. Using a multivariate logistic regression model, this circulating miRNA signature was further refined, and its performance was evaluated in an independent patient cohort of PDAC patients who were clinically diagnosed stage I to III by radiographic imaging, for the detection of ODM pre-operatively.

Results: The expression profiling of 2556 miRNAs during the biomarker step led to the identification of a panel of 10 differentially expressed miRNAs in stage-IV PDAC patients (miR-99b,1304-3p, 3609, 4728-5p, 4788, 6802-5p, 6820-3p, 6823-5p, 6858-5p, 6867-5p); and a combination signature of these 10-miRNAs yielded an impressive diagnostic accuracy for distinguishing stage-IV vs. stage I-III PADC patients (AUC=0.85, 95% Confidence interval (CI) 0.76 - 0.91). We next examined the expression of this 10-miRNA panel by qRT-PCR assays in a serum training cohort, which resulted in a reduced and optimized 4-miRNA signature, with a demonstrated robust diagnostic accuracy (AUC=0.84, 95% CI, 0.76 - 0.90) in distinguishing stage-IV PDAC patients. We subsequently validated the diagnostic performance of this 4-miRNA signature for the presence of ODM in an independent patient cohort (AUC=0.82, 95% CI, 0.72 - 0.90). Finally, using a multivariate logistic regression analysis for ODM, we developed a diagnostic risk-assessment model by combining the 4-miRNA signature, tumor location and tumor size, which exhibited an even superior diagnostic performance vis-à-vis individual factors (AUC= 0.94, 95% CI, 0.86 - 0.98).

Conclusions: We for the first time have identified and established a novel, noninvasive, serum-based miRNA signature that can pre-operatively diagnose PDAC patients with high risk occult distant metastasis.

#2582

Investigating the origin and function of circulating miRNA in lung cancer.

Tommaso Colangelo,1 Francesco Mazzarelli,1 Roberto Cuttano,1 Elisa Dama,1 Valentina Melocchi,1 Paolo Graziano,2 Fabiola Fiordelisi,2 Fabrizio Bianchi1. 1 _IRCCS-Casa Sollievo della Sofferenza, Unit of Cancer Biomarkers, San Giovanni Rotondo, Italy;_ 2 _IRCCS-Casa Sollievo della Sofferenza, Unit of Pathology, San Giovanni Rotondo, Italy_.

MicroRNAs (miRNAs) are small, highly conserved noncoding RNA molecules involved in the regulation of gene expression. MicroRNAs are resistant to harsh conditions and stably exist in body fluids (e.g. saliva, urine, breast milk, blood). Circulating cell-free miRNAs (cf-miRNA) were shown to be effective biomarkers for the early diagnosis of cancer. Furthermore, cf-miRNA were also found in exosomes that are nano-sized extracellular vesicles which exchange molecular information among cells. However, the understanding of origin of cf-miRNAs and of biological function still remains elusive. We recently identified a signature of cf-miRNAs diagnostic for lung cancer which we now hypothesize being released by lung cancer cells (EP-cf-miRNA) and by tumor microenvironment (TME-cf-miRNA). In particular, two cf-miRNAs, miR-29a and 223-3p, were selected as prototypes of EP- and TME-cf-miRNAs for further investigations.We found that the expression of miR-223-3p was higher in TME than in cancer cells of a cohort of 19 lung adenocarcinoma. In contrast, miR-29a-3p was frequently overexpressed (≥2 fold) in cancer cells. Interestingly, the serum concentration of cf-miR-29a was lower than miR-223-3p and this also in exosomes.We next investigated the expression profile of miR-29a/223-3p in a panel of lung normal and adenocarcinoma cell lines (N=14). miR-29a/223-3p were both expressed intracellularly and in exosomes. Of note, the miR-29a expression level resembles that observed in lung cancer cells from FFPE samples. Conversely, miR-223-3p expression was lower in the adenocarcinoma cell lines which further confirm a more TME origin. We are now expanding our analysis by screening the whole set of cf-miRNAs by high-throughput qPCR profile (OpenArray technology) and by applying laser capture microdissection to investigate single cancer epithelial cells and TME. Our work provides proof of principle demonstration of an effective approach to investigate cf-miRNA origin to unravel the role of cf-miRNA in lung adenocarcinoma.

#2583

DNA-linked gold nanoprobe assay: An efficient technology for direct and high-sensitive quantification of serum microRNAs in prostate cancer.

Prakash Kshirsagar, Satyanarayana Rachagani, Parthasarathy Seshacharyulu, Sakthivel Muniyan, Sunandini Sharma, Ram Mahato, Surinder K. Batra, Maneesh Jain. _Univ. of Nebraska Medical Ctr., Omaha, NE_.

Background: Serum-based biomarkers in conjunction with advanced biosensing techniques for early detection or screening of indolent vs. aggressive prostate cancer (PCa) can improve patient care and outcmomes. Recent discoveries have identified miRNAs (miRs) as promising biomarkers and opened potential avenues for earliest diagnosis and examining aggressiveness of PCa. However, quantitation of ultra-low levels of miRs in sera or tissue of patient remains challenging with traditional techniques. The present study describes the development and application of the DNA-linked gold nanoprobe (DNA-AuNPr)-based fluorescence assay for quantification of target miRs in serum with high sensitivity.

Methodology: Thorough meta-analysis followed by global in-silico analyses were performed to evaluate the differential expression of miRs among PCa studies. Further, the identified miRs were validated using TaqMan assay in human and mouse PCa serum. Thereafter, we synthesized DNA-AuNPr specific to target miRs, by conjugating FAM-tagged DNA-probes to the PEG-coated gold nanoparticle (AuNP) via cross-linker chemistry and fully characterized with UV, DLS and TEM. Sensitivity and target specificity of nanoprobe-based assay were determined using the Synergy-H1 multimode reader. As a proof of principle, a real-time evolution of fluorescence signal upon enzymatic digestion of DNA-probe in DNA-RNA heteroduplexes was tested. Finally, we utilized human (PCa n=44, control n=43) and Pten conditional knockout mouse (PCa n=8, control n=8) sera for our training and blank validation sets to determine the diagnostic significance of miRs in PCa.

Results: TaqMan assays detected a significantly higher level of both miR-21 and miR-375 in the PCa compared to normal control. Analyses of DNA-AuNPr shown uniform-sized, highly stable AuNP core tagged with multiple target-specific DNA-probes (~150±21 per NP). Additional assay validation indicated an excellent sequence specificity of DNA-AuNPr in discriminating the single-base mutation (mismatch) in miRs sequence. The uniqueness of proposed assay was its capability to quantitate miR targets in the range 50 pM to 10 nM with a lower detection limit of ~5 pM (0.3 fmol) in just ~4 h of total assay time and using ~10 μL of total RNA isolated from serum. Finally, the assay performance was validated in serum for both miR-21 and miR-375. The two miRs efficiently differentiated PCa from the healthy control in training sets (AUC=0.94) as well as in independent validation sets (AUC=0.90), which reconfirmed their diagnostic potential.

Conclusion: We developed a direct (without converting target miR into cDNA), highly sensitive and technologically efficient miR-quantitation strategy for diagnosis of PCa.

#2584

Exosome molecular reference standards (Exo-Ref) generated from CRISPR/Cas9 engineered cell Lines for oncology diagnostic applications.

Vigneshwaran Mani, Suhani Thakker, Debapriya Sengupta, Sravanthi Kondapalli, Gianluca Roma, Qi Zheng, Sonika Saddar. _Accuref Diagnostics, milpitas, CA_.

Introduction: Exosomes (commonly known as extracellular vesicles) have generated tremendous interest for the therapeutic and diagnostic applications for oncology and other diseases. Specifically, exosome molecular cargo (proteins, nucleic acid, and small molecules) profiling have been subject of intense research for potential biomarkers for cancer. Currently, there exist only few exosomal nucleic acid (ExoRNA) biomarkers that have been realized for cancer diagnostics and treatment monitoring. This is due to the lack of exosome reference standards for assay development, assay performance validation, and interpretation of results. Exo-Ref products are ideal reference standards, since they mimic physical properties and genomic composition of exosomes isolated from patient biofluids. Further, Exo-Ref are biologically relevant, reproducible, and uses engineered cell lines that have previously demonstrated a source of reference materials (ctDNA, FFPE, and cells). We have developed Exo-Ref featuring RNA SNPs including point mutations in EGFR (c.2369C>T, c.2573T>G), NRAS (c.181C>A), and PIK3A(c.1633G>A). Herein we present the isolation of exosomes from engineered cell lines and characterization of exo-RNA SNPs transcripts using RT-digital PCR and Next Generation Sequencing (NGS). The validation of variants using the two methods show a great promise for Exo-Ref products for oncology exosome-based diagnostic applications. Methods: CRISPR/Cas9 targeting reagents were transfected into either HCT116 or RKO cell line. Exosomes are produced by culturing the cells in exosome free culture media. Exosomes were then isolated from ExoRNeasy kit (Qiagen). For genetic analysis, exo-RNA was isolated from using trizol/membrane filter using the ExoRNeasy kit and allelic rare mutations in RNA were verified by RT dPCR and validated by NGS. Results: Engineered, homozygous clones were conclusively identified by Sanger Sequencing. We were able to reproducibly achieve fragmentation profile of exo-RNA centered at approximately 25bp. Digital RT-PCR validated cellular and exosome mutant transcripts. Targeted variants including EGFR-T790M, EGFR-L858R, PIK3CA-E45K, NRAS-Q61K showed measurable copies of EV-RNA and cell-RNA from engineered cells. Digital PCR verified variants in exo-RNA was also detected by NGS and were consistent with dPCR. NGS sequence validation of exo-RNA transcript were verified at 100% mutation frequency. Conclusion CRISPR/Cas9 engineered cell lines are biologically relevant materials and allow for a reproducible and renewable source of Exo-RNA reference standards for assay development and quality control for use in clinical exosome-based diagnostic workflows.

#2585

The development of a novel microRNA replacement therapy for ovarian cancer - a potential of patient derived-exosomes as a carrier.

Masaki Kobayashi, Kenjiro Sawada, Aasa Shimizu, Mayuko Miyamoto, Erika Nakatsuka, Mahiru Kawano, Yuri Matsumoto, Michiko Kodama, Kae Hashimoto, Seiji Mabuchi, Tadashi Kimura. _Osaka University Hospital, Osaka, Japan_.

Objective: microRNAs (miRNAs) affect key features of cancer cells and some are identified as tumor suppressor (TS) miRNAs. In spite of many preclinical studies with promising results, all translational clinical trials with TS miRNAs have failed due to the difficulties of miRNA systemic delivery. Therefore, the development of a novel carrier is urgently needed. Exosomes, non-sized vesicles secreted from cells, are stable in body fluid and therefore can serve as a promising candidate for miRNA carrier. In ovarian cancer treatment, omentum should be taken at surgery and normal fibroblasts can be collected from omentum. The aim of this study is to pursue the possibility of fibroblast derived-exosomes as a carrier for miRNA replacement therapy to treat ovarian cancer.

Methods: At surgery, omentum was collected and fibroblasts were primary cultured. Thereafter, exosomes were collected from culture media. miR-199a-3p was selected as a TS miRNA. Synthesized miR-199a-3p was corporated into exosomes by electroporation. miR-199a-3p-loaded exosomes (M199-exosomes) were treated with ovarian cancer cells and the expression levels of miR-199a-3p and target genes were assessed. The treatment effect of M199-exosomes was analyzed focusing on peritoneal dissemination.

Results: The treatment of M199-exosomes drastically increased miR-199a-3p expression in ovarian cancer cells (CaOV3; 8592-fold, SKOV3; 67188-fold, OVCAR3; 2280-fold). Treatment with M199-exosomes suppressed c-Met expression and significantly inhibited the proliferation and invasion of ovarian cancer cells in vitro (P<0.01). In a xenograft study, M199-exosomes significantly inhibited peritoneal dissemination and extended the survival of mice.

#2586

**Therapeutic potential of the topical treatment of** miR-634 **ointment for cutaneous squamous cell carcinoma.**

Jun Inoue,1 Kentaro Gokita,1 Masahiro Kishikawa,1 Hidetoshi Hamamoto,2 Katsunori Kobayashi,2 Kyoko Fujiwara,3 Takahiro Asakage,1 Kazuyuki Kojima,1 Johji Inazawa1. 1 _Tokyo Medical & Dental Univ., Tokyo, Japan; _2 _MEDRx. Co. Ltd., Kagawa, Japan;_ 3 _Nihon University School of Medicine, Tokyo, Japan_.

Cutaneous squamous cell carcinoma (CSCC) is the second most common nonmelanoma skin cancers. Surgery represents the gold standard for CSCC treatment, however it is needed to develop the noninvasive and effective treatments as the nonsurgical options for CSCC patients whose conditions are not suitable for or who refuse operation. On the other hand, microRNAs (miRs) are endogenous small non-coding RNAs negatively regulate gene expression by interfering with the translation or stability of target transcripts. Some tumor-suppressive miRs can target multiple cancer-promoting genes concurrently and may be useful as a therapeutic agent for cancer therapy. We have previously found that overexpression of miR-634 activated the mitochondrial apoptotic pathway by directly targeting genes associated with cytoprotective processes, including mitochondrial homeostasis, anti-apoptosis, antioxidant ability, and autophagy, in various cancer cells. The development of drug delivery system (DDS) is critical for the implementation of miR-based therapeutics. While topical pharmacotherapy represents a nonsurgical option to consider in CSCC, we validated the therapeutic potential of topical treatment of miR-634 ointment in mice model of CSCC. In xenograft tumors of A431 cells, a human epidermoid carcinoma cell line, in mice the topical treatment of ointment incorporated double-strand miR-634 mimics significantly reduced tumor growth. Furthermore, the topical treatment of miR-634-ointment also inhibited tumor growth in carcinogen-induced mouse skin papilloma model. Taken together, these findings suggest that topical treatment of miR-634-ointment may be useful as a noninvasive and effective treatments for CSCC.

#2587

MicroRNAs targeting versican, a proteoglycan in bone marrow microenvironment inhibit multiple myeloma progression.

Nidhi Gupta, Raman Kumar, Tulika Seth, Bhavuk Garg, Alpana Sharma. _All India Institute of Medical Sciences, New Delhi, India, Delhi, India_.

Background Multiple Myeloma (MM) is second most common hematological malignancy characterized by uncontrolled proliferation of abnormal plasma cells in bone marrow (BM). These myeloma cells require BM niche consisting of proteoglycans, cytokines and growth factors, etc. for their growth. One of the chondroitin sulfate proteoglycan, Versican (VCAN) has gained consideration in milieu of solid tumors where it has shown to promote tumor progression but there is dearth of literature in hematological malignancies including MM. Hence, involvement of VCAN in MM and its regulation by microRNAs has been studied.

Materials and Methods 30 MM patients & 20 controls were recruited and BM Mononuclear Cells (BMMNCs) were isolated. BM Stromal Cells (BMSCs) were harvested from BMMNCs by primary culture. Relative mRNA expression of VCAN and relative microRNA expression of miR-144, miR-199 & miR-203 were examined in BMMNCs, BMSCs and MM cell lines (RPMI8226 & U266). VCAN levels were correlated with microRNAs by spearman correlation analysis. Conditioned medium (CM) of BMSCs was examined for presence of VCAN by ELISA and its effect was studied on pathogenesis of MM cells in presence or absence of VCAN antibody. Further, signaling pathways altered by VCAN were identified. VCAN regulation by microRNAs was then assessed by transfecting miR mimics (mir-144 & mir-199) in primary BMSCs and CM thus obtained was supplemented to MM cells to investigate alteration in effects caused by VCAN with or without miR mimics. Significance was determined using Mann-Whitney U test and student's t-test.

Results Relative mRNA expression of VCAN was found significantly higher in MM patients in both BMMNCs and BMSCs with higher expression in BMSCs while relative microRNA expression was significantly lower in patients. VCAN levels showed negative correlation with microRNAs. VCAN being produced in stroma found at lower levels in MM cell lines. Moreover, BMSCs CM showed presence of VCAN which upon supplementing to MM cells alter the parameters in favour of myeloma progression, however, this effect was neutralized by VCAN antibody. The downstream signaling of VCAN was found to activate FAK and STAT3 which subsides by using VCAN antibody. The transfection of mir-144 and mir-199 mimics in BMSCs leads to significant reduction in levels of VCAN. The effect caused by VCAN on MM cell lines has also been found to be neutralized by using miR mimics.

Conclusion Augmented levels of VCAN in BM of patients imply its involvement in BM niche of MM. The neutralization of oncogenic effect of BMSCs CM by VCAN antibody affirms plausible role of VCAN in progression of MM. Moreover, myeloma promoting effect of VCAN has also been reversed by miR mimics of mir-144 and mir-199. These findings open up new avenues for exploring VCAN as a novel therapeutic target and microRNAs as a mean to target VCAN. This approach has great potential for treatment of MM after validation in future.

#2588

Restoring downregulated microRNAs miR-195-5p and miR-497-5p increases sensitivity to chemotherapy in colorectal cancer cells.

Lenka N. Boyd, Dennis Poel, Tim Schelfhorst, Thang V. Pham, Sander R. Piersma, Jaco C. Knol, Connie R. Jimenez, Henk M. Verheul, Tineke E. Buffart. _Amsterdam UMC, Amsterdam, Netherlands_.

Background: Patients with advanced colorectal cancer (CRC) are commonly treated with systemic combination therapy, however the success of treatment is hampered by drug resistance. MicroRNAs (miRNAs) have recently emerged as important players in therapy resistance in CRC. The aim of this study was to improve therapy efficacy by sensitizing CRC cells to the chemotherapeutics currently used in clinical practice for patients with advanced CRC by restoring downregulated miR-195-5p and miR-497-5p.

Methods: Sensitivity to 5FU, oxaliplatin (OHP) and irinotecan before and after transfection with miR-195-5p and miR-497-5p mimics was analyzed using tetrazolium reduction and clonogenic assays in CRC cell lines HCT116, RKO, DLD1 and SW480. Transfection with cel-miR-39-3p and untransfected cells were used as controls. Proteomic analyses of transfected and untransfected cells were implemented to identify possible targets involved in sensitivity to chemotherapy. Group comparisons by beta-binomial statistics were performed to analyze significantly altered peptides after restoring miRNA expression.

Results: After transfection with miR-195-5p and miR-497-5p mimics, RKO showed an increased sensitivity to OHP (IC50 decreased from 1100 nM to 900 nM and 800 nM, respectively) and HCT116 showed increased sensitivity to OHP (IC50 decreased from 600 nM to 200 nM and 180 nM, respectively) and 5FU (IC50 decreased from 3500 nM to 2900 nM and 2800 nM, respectively). No increased sensitivity to irinotecan was observed. Clonogenic assay revealed that HCT116 formed 20% and 50% less colonies when treated with 5FU after transfection with miR-195-5p and miR-497-5p mimics, respectively, and 60% less colonies when treated with OHP after transfection with either mimic. RKO showed a 20% decrease in colonies when treated with OHP after transfection with miR-497-5p mimic. No differences in sensitivity to OHP, 5FU or irinotecan were found in transfected SW480 and DLD1. Unsupervised clustering of the proteome showed that HCT116 and RKO cells transfected with miR-195-5p and miR-497-5p mimics could be distinguished from the cells transfected with a control mimic and the untransfected cells. Transfected and control SW480 and DLD1 could not be distinguished based on the proteome profiles. In the combined analyses of HCT116 and RKO 12 peptides showed significantly altered expression (FDR<0.1) after transfection. No significantly altered peptides were detected in transfected SW480 and DLD1 cell lines.

Conclusion: Restoring miR-195-5p and miR-497-5p expression enhanced sensitivity to OHP in HCT116 and RKO cells and to 5FU in HCT116 cells. Proteomics revealed 12 peptides with significantly altered expression after restoring expression of these miRNAs. Treatment with miRNA mimics to sensitize tumor cells to chemotherapy could be an alternative systemic treatment strategy for patients with advanced CRC.

#2589

A systematic study on the anti-tumor effect of combinatorial miRNA expression in hepatocellular carcinoma.

Feng Xu, Man Tong, Stephanie Ma, Alan SL Wong. _The University of Hong Kong, Hong Kong, China_.

Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third leading cause of cancer mortality worldwide. Treatment options for HCC patients are limited, especially for those with advanced HCC. Deregulation of microRNA (miRNA) expression is frequently observed in HCC, and many cellular targets of miRNAs are genetic regulators of tumorigenesis. In addition, miRNAs interact with each other to coordinate targeted gene expressions. The potential of devising combinatorial miRNA-based therapies for HCC, however, remains largely unexplored. Here, we performed high-throughput CombiGEM screens to characterize the anti-tumorigenic effect of combinatorial miRNA expression. We assembled a library of 1,849 pairwise combinations for 43 human miRNAs that are downregulated in HCC patients/cell lines, and identified four miRNA combinations that exert anti-proliferative effect in HCC cells both in vitro and in vivo. Further experiments are underway to validate and characterize the anti-tumor effect of these newly identified miRNA combinations in HCC.

#2590

Nano-exosomes with miRNA: A new therapeutic approach.

Sudeepti Kuppa,1 Balazs Rada,2 Arthur Roberts,1 Mandi M. Murph1. 1 _University of Georgia College of Pharmacy, Athens, GA;_ 2 _University of Georgia, Athens, GA_.

Access to reliable and effective therapeutics for patients suffering from aggressive triple-negative breast cancer (TNBC) has been clinically challenging to achieve due to lack of targetable mutations and high heterogeneity in the tumor cells. Therapeutic approaches to reduce mortality in the TNBC patient subset requires an innovative and targeted approach to drug delivery, which is presented here in the form of labeled-exosomes. In this study, we have developed a novel method to bioengineer exosomes, where an antibody is covalently bonded to a fatty acid, which is then anchored into the exosomal membrane to create labeled-exosomes. Our data demonstrate that labeled-exosomes can contain up to 700,000-fold higher miRNA concentrations and can deliver miRNA cargo with increased efficiency into MDA-MB-231 TNBC cells. Results further reveal that insertion of a specific antibody-label to the exosomal membrane significantly increases microRNA uptake into MDA-MB-231 cells, and is further enhanced in cells overexpressing that same antibody marker on their surface. The customizable bioengineering of the labeled-exosomes makes it a highly versatile tool to target cells based on surface marker expression and increase miRNA or other therapeutic cargo delivery. Most importantly, the relevancy of this model reaches far beyond its success in just TNBC cells and can also be translated to several different disease models that require specific targeting with an encapsulated miRNA or therapeutic. We believe this is a novel and exciting utilization of exosomes with a high degree of customization based on surface markers on the target cell, as well as microRNA cargo loaded into the exosome to inhibit a specific oncogene.

#2591

miR-193b is a novel regulator of Inhibition of Notch signaling by for targeting cancer stem cell and tumor microenvironment.

Ummu Guven,1 Hamada Ahmed Mokhlis,1 Nermin Kahraman,1 Cristina Ivan,1 Fahriye Duzagac,1 Gabriel Lopez-Berestein,1 Gulperi Oktem,2 Bulent Ozpolat1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Ege University Faculty of Medicine, Izmir, Turkey_.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive human cancers with extremely poor survival rates with 6 months of median patient survival, representing an unmet therapeutic challenge. The exact mechanisms by which PDAC progresses and still remain poorly understood. Strategies targeting only tumor cells have proven to be unsuccessful in eliminating PDAC and prevent metastasis, drug resistance and patient deaths. Accumulating evidence suggests that cancer stem cells (CSC) are also responsible for relapses and resistance to therapies as well as promoting invasive and metastatic growth of the tumor. Notch signaling has been implicated in the survival of CSC, suggesting that NOTCH pathway can be targeted for treatment of PDAC. In four known transmembrane Notch receptors, antibodies targeting Notch 2/3 have shown efficacy in solid tumors and PDAC in phase I clinical trials, suggesting that these are viable targets. Targeting Notch 2 expression has been shown to induce apoptosis in PDAC but its role and regulation of in PDAC CSC is not well understood. We found that Notch2 significantly upregulated in pancreatic cancer stem cells and its expression is associated with shorter overall patient survival in pancreatic cancer. Recent discoveries revealed that small non-coding RNAs such as microRNAs (miRNAs) play significant role in the pathogenesis of human cancers. Therefore, microRNAs have attracted great interest as a tool for cancer treatment and entered clinical trials in the US. Using in silico prediction algorithms we found that miR-193b-3p has binding sites on the 3'-untranslated region (3'-UTR) of NOTCH2 gene and demonstrated that transfection of PDAC and CD133+/CD44+/EpCam+ pancreatic CSC cells with miR193b-3p mimic or NOTCH 2 siRNA suppressed NOTCH2 expression by Western blot and qPCR analysis and proliferation, migration and metastasis of PDAC (Panc-1 and MiaPaCa-2) and the tumorosphere forming ability of CSC cells in Mammocult Human Tumorosphere Culture. CD133+/CD44+/EpCam+ pancreatic cancer stem cells transfected with miR-193b inhibited Notch 2 and colony forming capabilities in soft agar. Currently we are performing a gene reporter assay to show miR193b directly binds to 3'-UTR of NOTCH2 mRNA and inhibits its expression. We demonstrated that injection of nanoliposome incorporated miR193b-3p into mice from tail vein (0.15 mg/kg once a week) significantly inhibited PDAC tumor growth. However, animal studies transplanted with CD133+/CD44+/EpCam+ pancreatic cancer stem cells after miR193b-3p transfection are underway to detect in vivo effects of targeting miR193 in CSC and tumor growth. Overall, our findings provide new insights into the tumor suppressor role of miR-193b by targeting of Notch 2 signaling and suggest that miR-193b-based therapy may be a potential therapeutic strategy to targeted pancreatic cancer stem cells.

#2592

Development of a novel modified miR-15a mimic with enhanced therapeutic potential for treatment of pancreatic ductal adenocarcinoma.

Shixiang Guo,1 Andrew Fesler,1 Wenjie Huang,2 Yunchao Wang,2 Jiali Yang,2 Xianxing Wang,2 Ga-Ram Hwang,1 Matthew Godwin,1 Huaizhi Wang,2 Jingfang Ju1. 1 _Stony Brook Univ. Medical Ctr., Stony Brook, NY;_ 2 _Institute of Hepatopancreatobiliary Surgery, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China_.

Treatment of pancreatic ductal adenocarcinoma (PDAC) remains a clinical challenge associated with resistance and early recurrence. There is an urgent need to discover and develop new strategies to enhance treatment efficacy and improve prognosis for pancreatic cancer patients. microRNAs (miRNAs) play critical roles as oncogenes or tumor suppressors in the regulation of cancer development and progression. Our previous study identified miR-15a as a tumor suppressor miRNA and potential therapeutic candidate in PDAC. The expression of miR-15a is significantly down-regulated in PDAC compared to normal pancreas and is associated with proliferation and epithelial mesenchymal transition (EMT) through targeting genes such as BMI-1. In addition to BMI-1, we have identified Wee1, Chk1, and Yap-1 as direct targets of miR-15a in PDAC. We also analyzed the correlation between expression of miR-15a and its downstream target Cdc2 in PDAC clinical samples by real-time PCR and immunochemistry (IHC). Our results show that miR-15a expression is inversely correlated with Cdc2 expression in PDAC tissues and miR-15a expression is significantly associated with prognosis. To further explore the therapeutic potential of miR-15a in PDAC, we have designed a modified version of miR-15a, by replacing uracil residues in the guide strand of miR-15a with 5-Fluorouracil (5-FU). This modified miR-15a mimic combines the therapeutic powers of 5-FU and miR-15a. The miR-15a mimic has improved stability and efficacy compared to the native miR-15a. miR-15a mimic also retains target specificity for Wee1, Chk1, BMI-1 and Yap-1, and regulates the Wee1/Chk1-Cdc25c-Cdc2 signaling pathway. Another unique feature of this miR-15a mimic is that it can be delivered to cancer cells without the aid of a delivery vehicle. The miR-15a mimic has enhanced ability to inhibit the proliferation of a panel of pancreatic cancer cell lines compared to native miR-15a, with IC50 values of 18.713 nM and 37.541 nM respectively. miR-15a induces significant cell cycle arrest at the G1/S checkpoint and miR-15a mimic induces S phase arrest. In addition, we demonstrated that there is a synergistic effect by combining miR-15a mimic and gemcitabine in reducing pancreatic cancer cell proliferation, with reduced IC50 for both drugs. The therapeutic potential of miR-15a mimic was also demonstrated in vivo using a metastatic mouse pancreatic cancer model. miR-15a mimic (3.2 mg/kg) inhibits metastatic tumor growth at a dose that is 15-fold less than that of gemcitabine (50 mg/kg) and the inhibitory effect is enhanced in combination with gemcitabine. While mice treated with gemcitabine alone developed resistance, we did not observe any resistance in mice treated with miR-15a mimic. This study demonstrates that miR-15a mimic has great potential to be further developed as a novel therapeutic drug for treatment of PDAC.

#2593

A double dose of DNA damage: Overcoming drug resistance using targeted oncolytic viruses.

Taylor R. Jamieson, Carolina S. Ilkow. _Ottawa Hospital Research Institute, Ottawa, Ontario, Canada_.

Background: Hereditary breast and ovarian cancers make up 5-10% of breast cancer and 10-15% of ovarian cancer cases. These cancers tend to be difficult to treat and progress in an aggressive manner. Poly(ADP-ribose) polymerase inhibitors (PARPi) are a type of drug that targets the DNA repair pathway; many of these drugs are undergoing clinical trials and some are presently in use for cancers harbouring mutations in their DNA repair machinery, such as hereditary breast and ovarian cancers. Unfortunately, numerous patients become resistant to PARPi, leaving limited options for further treatment. Oncolytic or "cancer-killing" viruses are an innovative approach for treating even the most complex cancers. Oncolytic viruses target and destroy cancer cells, leaving normal cells unharmed, all while activating a patient's own immune system to fight the cancer. Our group has demonstrated that oncolytic rhabdoviruses may be used to deliver therapeutic payloads by encoding targeting sequences to act on genes via RNA interference (such as shRNA).

Objective: The aim of this project is to overcome PARPi resistance by engineering cancer specific viruses that will target genes relevant to the DNA repair pathway, sensitizing them to PARPi treatment in a synthetically lethal manner.

Methods: shRNA sequences targeting components of the DNA repair pathway have been cloned into the genome of an oncolytic rhabdovirus (a non-targeting control sequence has been inserted into the same virus as well). Prior to encoding shRNA sequences into the virus, validation of these targets was performed using siRNA transfection. Downregulation of targeted genes are being assessed via qPCR and Western Blot analysis following transfection and infection. Cell viability following treatment with PARPi +/- viruses will be assessed by Alamar Blue and Crystal Violet assays.

Results: Downregulation of siRNA targeted genes has been validated at the mRNA level via qPCR analysis (Western Blot analysis is currently ongoing). Validation of the newly engineered viruses to show downregulation of their specific gene targets is currently underway. Changes in cell viability for seven unique human/mouse cell lines following treatment with varying concentrations of two different PARPi to establish a dosing scheme for combination therapy with viral infection has been completed.

Conclusions and project impact: This project is currently in the early stages of development; however, preliminary experiments testing the combination of siRNA knockdown with PARPi have revealed a significant decrease in cell viability compared to siRNA knockdown or PARPi alone in MCF-7 breast cancer cells. By engineering OVs that specifically replicate in and kill cancer cells while delivering PARPi sensitizing sequences, this combination approach may enhance and expand the utility of oral PARPi therapeutics in numerous cancers with alterations in DNA repair genes.

#2594

Exosomes co-expressing aquaporin-5-targeting miRNAs and IL-4 receptor-binding peptide inhibit the migration of human breast cancer cells.

Eui-Jung Park,1 Hyun Jun Jung,2 Hyo-Jung Choi,1 Hyo-Ju Jang,1 Hye-Jeong Park,1 Tae-Hwan Kwon1. 1 _School of Medicine, Kyungpook National University, Daegu, Republic of Korea;_ 2 _School of Medicine, University of Maryland, Baltimore, MD_.

Water channel aquaporins (AQPs) play a critical role in transcellular water transport. Recent studies revealed that dysregulation of AQPs is highly correlated with the migration of cancer cells and metastasis of tumor in vitro and in vivo models. In particular, we have demonstrated that increased expression of aquaporin-5 (AQP5) is associated with breast cancer cell migration, metastasis, and poor prognosis in human patients. We aimed to identify novel AQP5-targeting miRNAs and to examine the strategy for the post-transcriptional regulation of AQP5 using exosome-mediated microRNA (miRNA) delivery. Multiple tools of bioinformatics were used to identify AQP5-targeting miRNAs and miRNA target enrichment analysis revealed the cellular signaling pathways associated with the identified miRNAs. To examine the exosome-mediated miRNA delivery systems, exosomes containing both AQP5-targeting miRNAs and a peptide (CRKRLDRNC: IL-4RPep1) targeting the interleukin-4 receptor (IL-4R), which is highly expressed in breast cancer cells, were applied to triple-negative breast cancer cell line MDA-MB-231. Putative AQP5-targeting miRNAs were identified using DIANA Tool. Thereafter, three additional bioinformatic tools (TargetScan, miRDB and MiRanda Tools) were applied to select the miRNAs that were commonly identified from at least 3 different programs. Three miRNAs, miR-1226-3p, miR-19a-3p and miR-19b-3p, were identified as putative regulators of AQP5. Protein expression of AQP5 was decreased when mimic of each miRNA was transfected into MDA-MB-231 cells. Quantitative real-time PCR demonstrated a decrease of AQP5 mRNA expression by miR-1226-3p mimic and luciferase reporter assay revealed a reduction of AQP5 translation by miR-19b mimic in MDA-MB-231 cells. Consistently, cell migration was inhibited by mimic of each miRNA. miRNA target enrichment analysis showed that target genes of three miRNAs were predominantly enriched in the gap junction regulatory pathway. This suggested that these miRNAs are associated with breast cancer cell migration and, also, they might be involved in cell-cell adhesion. For the delivery of AQP5-targeting miRNAs to MDA-MB-231 cells, exosomes containing both AQP5-targeting miRNAs and a peptide (IL-4RPep1) were constructed in HEK293T cells. Both AQP5 protein expression and cell migration were significantly decreased in MDA-MB-231 cells when they were incubated with the exosome-containing culture media obtained from the culture of HEK293T cells expressing both AQP5-targeting miRNAs and IL-4RPep1. In conclusion, AQP5-regulating miRNAs are identified and their target genes are found to be mainly involved in the gap junction regulatory pathway. The exosome-mediated delivery of AQP5-targeting miRNAs could be exploited for the inhibition of breast cancer cell migration associated with AQP5.

### Oncogenic Transcription Factor Function

#2595

**HMGA1 induces the** HOXB13 **developmental gene to drive tumor progression in androgen receptor negative, castrate-resistant prostate cancer.**

Lionel Chia, Guangjing Zhu, Mikhail Gorbounov, Lingling Xian, Briyana Chisholm, Mohammad Heydarian, Dorhyun Johng, William B. Isaacs, Karen Reddy, Linda S. Resar. _Johns Hopkins School of Medicine, Baltimore, MD_.

While High Mobility Group A1 (HMGA1) chromatin remodeling proteins are frequently dysregulated in diverse cancers, their molecular underpinnings in carcinogenesis remain poorly understood. HMGA1 are small, non-histone proteins that bind to AT-rich regions in DNA, bend chromatin, and recruit transcriptional complexes to modulate gene expression. Prior studies in prostate cancer (PCa) suggest that HMGA1 overexpression associates with higher pathologic grades and increases in HMGA1 protein levels correlate with metastatic potential in rat PCa cells. Overexpression of HMGA1 in human PCa cell lines induces unbalanced chromosomal rearrangements in vitro. Thus, we hypothesized that HMGA1 drives PCa progression through epigenetic reprogramming of transcriptional networks involved in development and chromosomal instability. To test this, we focused on androgen receptor (AR)-negative, castrate-resistant PCa (CRPC) cell lines as tumors with these features are resistant to therapy and associated with metastatic progression and early death. Here, we uncover a novel role for HMGA1 in regulating HOXB13 to drive tumor progression and cancer stem cell properties. We found that silencing HMGA1 in patient-derived metastatic cell lines (PC3-Epi, PC3-EMT, DU145) halts proliferation. Cell morphology changed most dramatically in PC3-EMT cells, transforming spindle-shaped, mesenchymal cells to more cuboidal, epithelial-like cells. Both migration and invasion were disrupted, but primarily in more invasive, mesenchymal cell lines (PC-EMT, DU145). Colony formation and 3D sphere formation were also blocked in all CRPC lines. Immunohistologic analysis in primary tumors revealed that HMGA1 nuclear staining associates with more advanced Gleason scores in PCa. To elucidate transcriptional networks downstream of HMGA1, RNA-seq was performed in PC3-EMT and PC3-Epi cell lines + HMGA1 silencing. Intriguingly, HMGA1 regulates pathways involved in inflammation in the more epithelial PC3-Epi cells, while pathways involved in mitosis, cell cycle progression, DNA damage, and checkpoint regulation predominated in the mesenchymal PC3-EMT cells. Pathways involved in proliferation and development were regulated by HMGA1 in both settings. We focused on HOXB13, a developmental gene linked to cell fate and prostate carcinogenesis. Both HOXB13 and HMGA1 are co-regulated in CRPC cell lines at the gene expression and protein level. HMGA1 occupies at least 1 site within the HOXB13 promoter region by chromatin immunoprecipitation. Strikingly, silencing HOXB13 recapitulates HMGA1 phenotypes, impairing proliferation, colony formation, and 3D sphere formation. These findings reveal a novel role for HMGA1 in CRPC progression by dysregulating developmental networks. Together, these results also suggest that targeting the HMGA1-HOXB13 pathway could be effective therapy in CRPC.

#2596

SOX11 is a key epigenetic regulatorin the adrenergic MYCN amplified neuroblastomas.

Amber Louwagie. _Universiteit Gent, Ghent, Belgium_.

Neuroblastoma (NB) is a pediatric cancer of the developing sympatho-adrenergic nervous system, responsible for 15% of childhood cancer deaths. Understanding the fundamental underlying mechanisms of NB is key to early detection and appropriate treatment. High risk NBs are predominantly driven by DNA copy number alterations, including MYCN amplification, rare recurrent amplifications (affecting critical oncogenes including LIN28B, ALK and MDM2) and large 2p and 17q gains. Here we report on the role of rare recurrent focal gains and amplifications of the SRY-related HMG-box transcription factor SOX11. In a series of 842 NB samples, we observed five tumors with high-level gain or amplification of SOX11. Copy number versus mRNA expression analysis for SOX11 suggests dosage sensitivity (p=1.82e-09). Kaplan-Meier analysis in two large independent cohorts of NB primary tumors showed decreased overall survival for patients with higher SOX11 levels (p=4.19e-07) suggesting SOX11 contribution to tumor aggressiveness. SOX11 is upregulated by a super-enhancer in adrenergic neuroblastoma cells while absent in mesenchymal cells, indicating a putative role in cell identity. SOX11 knockdown in NB cell lines showed reduced colony formation capacity and G1-S cell cycle arrest indicating cellular dependency on high SOX11 levels. Integrated ChIP-, ATAC- and RNA-seq analysis revealed binding of SOX11 to promotors and enhancers and regulation of genes involved in cell cycle, DNA replication, DNA repair as well as chromatin remodeling. IP-MS analysis identified MYCN as interacting protein and SOX11 and MYCN expression levels show a positive correlation (R= 0.576, p=1.9e-26). A putative cooperativity is further suggested by a significant number (in average 26%) of common DNA binding regions including PRC2 components and accessory proteins and specific components of the SWI/SNF complex. Moreover, one of the most robustly regulated SOX11 targets is MYB, a known oncogene, presumed pioneer factor and master regulator. SOX11 was previously shown to preferentially bind to enhancer regions. In view of recent data that show that highly increased MYCN levels cause the protein to invade enhancers, we propose that SOX11 plays a well-defined developmental role in controlling the epigenetic enhancer landscape which is subsequently high-jacked through the MYCN enhancers invasion process. We will present enhancer landscaping on NB cells upon MYCN only versus MYCN/SOX11 combined knockdown as recently reported for MYCN/TBX2 (Decaesteker et al, Nat Commun, 2018). Initial tests on a panel of NB cell lines of compounds known to block MYB functionality showed effects on viability and proliferation and will be further tested in organoids and in vivo for patient-derived xenografts. In conclusion, we suggest an important role for SOX11 in cooperation with MYCN in NB development and propose the SOX11 regulated gene MYB as a novel therapeutic target in NB.

#2597

The Cyclin D1 carboxyl terminus encodes an epigenetic reader domain.

Gabriele Di sante,1 Agnes Tantos,2 Mathew Craig Casimiro,1 Peter Tompa,2 Richard G. Pestell1. 1 _Baruch S. Blumberg Institute, Wynnewood, PA;_ 2 _Institute of Enzymology, Budapest, Hungary_.

Purpose of the study: The cyclin D1 gene has been shown to promote chromosomal instability CIN) in cultured cells and in vivo using transgenic mice, through a kinase-independent mechanism. Correlative studies suggest the transcriptional function of cyclin D1 governs the induction of CIN. The cyclin D1 gene encodes a regulatory subunit of a holoenzyme complex that phosphorylates and inactivates the retinoblastoma protein (Rb) and the nuclear respiratory factor 1 (NRF1) proteins to thereby regulate DNA synthesis and mitochondrial metabolism. In addition, cyclin D1 regulates gene expression. Cyclin D1 promotes oncogene-dependent growth, and genetic ablation of the cyclin D1 gene in mouse models produces mice resistant to Ras or ErbB2-induced mammary tumorigenesis and APC-induced gastrointestinal tumorigenesis. The abundance of cyclin D1 is increased early in the malignant process and cyclin D1 abundance is regulated at different molecular levels, (transcription, translation and post-translational). In our previous studies, we identified, via a genome wide ChIP-Seq analysis, cyclin D1 binding regions in genes governing CIN including Top2A, AurkB, Cenpp, Mlf1ip, Zw10, Ckap2.

Experimental procedures: Cyclin D1 promotes CIN via accessing gene regulatory regions at the local chromatin level. We deployed a histone peptide array and a peptide pull-down assays. Subsequent, we analyzed the biochemistry of the specific interactions between cyclin D1 and histone modifications via Surface Plasmon Resonance (SPR).

Results: Cyclin D1 selectively recognized histone residues of H2B and H3 that were acetylated, methylated, phosphorylated or citrullinated (H2B-S14pho, H2B-K15ac), (H3R26Cit H3K273me). Mutational and deletion analysis showed that the cyclin D1 C terminus was sufficient to augment transcription of CIN genes and cell proliferation and that the E-box region (aa272-aa280) was necessary for cyclin D1 binding to modified histones. Cyclin D1 ΔE mutant (lacking the E-box motif) abolished the interaction with modified histones and abolished the transcription response and the augmentation of cell proliferation. In the peptide pull-down assay, we confirmed the specific physical interaction between cyclin D1 and H2B-S14pho. We further investigated this strength of the interaction using SPR. This approach showed a strong binding between cyclin D1 and H2B-S14pho and a decreased affinity for the unmodified histone H2B. The interaction was proven to be abolished between H2B-S14pho and cyclin D1 when the cyclin D1 ΔE mutant was introduced.

Conclusion: Collectively these findings are consistent with a model in which cyclin D1 is recruited into chromatin through binding directly to an epigenetic histone code. Such binding may tether cyclin D1 to participate in transcriptional activity at specific target genes.

#2598

Developing small molecule inhibitors that target androgen receptor signaling in castration-resistant prostate cancer.

Balaji Chandrasekaran,1 Ashish Tyagi,1 Venkatesh Kolluru,1 Aakash M. Vadhanam,1 Becca von Baby,1 Suresh Gorle,2 Suman Sirimulla,2 Srinivasa R. Ramisetti,3 Arun K. Sharma,3 Murali K Ankem,1 Chendil damodaran1. 1 _University of Louisville, Louisville, KY;_ 2 _University of Texas, El Paso, TX;_ 3 _Pennsylvania State University, Hershey, PA_.

The transcription factor, androgen receptor (AR) is a key driver and plays an important role in prostate cancer. Androgen ablation therapy and brachytherapy remains the standard treatment of advanced prostate cancer, but unfortunately, it is not curative, and eventually the disease returns as lethal castration-resistant prostate cancer (CRPC). Previously we have reported that Urolithin A (Uro A), a natural compound, inhibits AR signaling and suppresses the growth of CRPC. Now, we have developed a series of pro-drug conjugates of Uro A and our initial structure-activity relationship (SAR) studies led to the identification of three small molecules as lead compounds. Hence, we investigated the effects of all three compounds and dissected their molecular mechanism in cell culture and mouse models of CRPC. All the three compounds more effectively inhibited the growth CRPC cell lines than the parent compound Uro A. Based on their IC50 concentration; we identified a novel compound (ASR-600) that demonstrated better efficacy by inhibiting AR signaling in CRPC cell lines than the parental compound Uro A. The model system and molecular dynamics (MD) stimulation studies suggest that ASR600 bind to the ligand-binding domains of AR and blocks the conformation changes, that allows AR to phosphorylate and degrade in the cytosol. Further, our thermal shift assay confirmed ASR 600 binds to AR in presence and absence of Dihydrotestostreone. Our ongoing in vivo studies, may suggest whether oral administration of ASR-600 effectively inhibits the tumor growth raised from CRPC cell lines (C4-2B, 22RV1 and enzalutamide resistance C4-2B) in xenografted mice. These observations provide a rationale for devising novel therapeutic agent based on ASR 600 for the treatment of CRPC

#2599

Pleiotropic effects of PPARD accelerate colorectal tumorigenesis progression and invasion.

Yi Liu,1 Yasunori Deguchi,1 Rui Tian,2 Daoyan Wei,1 Ling Wu,1 Weidong Chen,3 Weiguo Xu,4 Min Xu,5 Fuyao Liu,1 Shen Gao,1 Jonathan C. Jaoude,1 Sarah P. Chrieki,1 Micheline J. Moussalli,1 Mihai Gagea,1 Jeffrey S. Morris,1 Russell Broaddus,1 Xiangsheng Zuo,1 Imad Shureiqi1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Affiliated Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China;_ 3 _Affiliated the Second Xiangya Hospital, Xiangya Medical College, Central South University, China;_ 4 _Affiliated Hospital of North China University of Science and Technology, China;_ 5 _Affiliated Hospital of Jiangsu University, Zhenjiang, China_.

APC mutations activate aberrant β-catenin signaling to drive colorectal cancer (CRC) initiation. CRC progression however requires additional molecular mechanisms. PPAR-delta (PPARD) is a downstream target of β-catenin and upregulated in CRC. PPARD germline genetic deletion has however been reported to promote intestinal tumorigenesis in Apcmin mice, which has questioned PPARD effects on β-catenin aberrant activation and CRC. Addressing this knowledge gap is important because PPARD is a druggable protein. Mice with targeted PPARD overexpression/deletion into intestinal epithelial cells (IECs) were bred with mice with intestinally targeted ApcΔ580 mutation via CDX2-cre (ApcΔ580) or CDX2-cre-ERT2 (ApcΔ580-TMX). APCΔ580 mice treated with PPARD agonist (GW501516) or antagonist (GSK3787), and human CRC organoid cells were also tested. PPARD expression was examined in human CRC invasive fronts and their paired colorectal adenomas and cancer centers. PPARD's mechanisms to promote CRC were screened by Functional Proteomics Reverse Phase Protein Assay (RPPA) and subsequently validated in in-vitro and in-vivo studies. PPARD overexpression/deletion in IECs in mice augmented/suppressed β-catenin activation via upregulation/downregulation of BMP7/TAK1 signaling and strongly promoted/suppressed CRC .PPARD downregulation by siRNA in human CRC organoid cells inhibited BMP7/β-catenin signaling and suppressed organoid self-renewals. GW501516 enhanced while GSK3787 suppressed CRC tumorigenesis in ApcΔ580 mice. PPARD expression was significantly higher in human CRC invasive fronts versus their paired invasive tumor centers and adenomas. RPPA and validation studies identified PPARD upregulation of multiple other pro-invasive pathways: PDGFRβ, connexin 43, AKT1, EIF4G1 and CDK1, Our data demonstrate that PPARD strongly potentiates multiple pro-tumorigenic pathways to promote CRC progression and invasiveness.

#2600

Interaction of host cell factor-1 with the oncoprotein transcription factor MYC.

Tessa M. Popay,1 Lance R. Thomas,1 Jing Wang,2 Qi Liu,2 William P. Tansey1. 1 _Vanderbilt University, Nashville, TN;_ 2 _Vanderbilt University Medical Center, Nashville, TN_.

Host cell factor (HCF)-1 is a highly-conserved epigenetic scaffolding protein that is essential for development, cell cycle progression, and cellular proliferation. Through integration into several chromatin modifying complexes, HCF-1 plays a critical role in regulating the activity of key transcription factors, such as the E2F family of proteins. HCF-1 is also linked to leukemogenesis, through its regulation by the BAP1 tumor suppressor and its incorporation into the oncogenic MLL complex. Despite its role in the cell cycle, presence at chromatin, and well-defined interactions, the function of HCF-1 in cancer remains under-studied and undetermined. Here we describe the role of HCF-1 in the context of its interaction with the transcription factor and oncoprotein MYC. MYC is overexpressed in most cancers and drives tumorigenesis by regulating the expression of genes involved in ribosome biogenesis, cell growth, and metabolism. The region of MYC responsible for interaction with HCF-1 is evolutionarily conserved in all vertebrate MYC family members, and we have previously demonstrated that mutations in MYC that selectively disable interaction with HCF-1 attenuate the tumorigenicity of MYC in vivo. This demonstrates that HCF-1 is an important co-factor for MYC-driven cancer. Using a battery of contemporary genomic approaches, including ChIP-seq, SLAM-seq, RNA-seq, and PRO-seq, coupled to a decisive CRISPR/Cas9 editing approach, we reveal the relationship of MYC and HCF-1 at chromatin and how this modulates the function of MYC as a transcription factor and oncoprotein.

#2601

HMGA1 **is induced by procarcinogenic bacteria within the microbiome where it drives expansion in the colon stem cell pool and tumorigenesis.**

Linda M S Resar, Lingling Xian, Jawara Allen, Shaoguang Wu, Lionel Chia, Cynthia Sears. _Johns Hopkins Univ. School of Medicine, Baltimore, MD_.

Cancer cells undergo chromatin remodeling and epigenetic reprogramming to hijack stem cell transcriptional networks and drive tumorigenesis, although the molecular underpinnings remain unclear. High Mobility Group A (HMGA1) chromatin remodeling proteins are architectural transcription factors that bind AT-rich sequences where they "open" chromatin and recruit transcriptional complexes to modulate gene expression. The HMGA1 gene is highly expressed during embryogenesis, but silenced postnatally in differentiated tissues. HMGA1 is re-expressed in aggressive cancers where high levels portend adverse clinical outcomes. In colorectal cancer, HMGA1 is among the genes most overexpressed compared to normal colon epithelium. We previously found that HMGA1 induces genes involved in EMT and drives tumor progression in colon cancer models. Hmga1 transgenic mice develop aberrant proliferation and polyposis in the small intestine and colon. In intestinal stem cells, Hmga1 amplifies Wnt signals to enhance self-renewal. Surprisingly, Hmga1 also induces Sox-9 to drive Paneth cell differentiation and "build" a stem cell niche. To determine how HMGA1 functions in the colon, we examined mouse models with varied levels of Hmga1. Hmga1 overexpression in colon stem cells leads to expansion of the colon stem cell pool. Both goblet cells and colonic mucous are increased. In DSS models of inflammatory bowel disease, Hmga1 enhances repair and tissue regeneration, while Hmga1 overexpression promotes aberrant proliferation and polyposis with advancing age. To determine how HMGA1 is induced during carcinogenesis, we examined mice harboring a heterozygous Apc mutation (Min+/-) following colonization with the human symbiotic bacteria, enterotoxigenic Bacterioides fragilis (ETBF). ETBF triggers increased Wnt signaling by inducing E-cadherin cleavage with release of membrane bound β-catenin, leading to enhanced distal colon tumorigenesis in mice. Further, ETBF colonization in humans is linked to colon cancer. ETBF colonization in mice induces Hmga1 and increases stem cell self-renewal in preliminary studies. Inflammatory cytokine genes are also induced. Bacteroides fragilis toxin (BFT) induces Hmga1 in wildtype and Min+/- mouse colon organoids. HMGA1 also increases modestly in human HT29/C1 cells after exposure to BFT. Studies are underway to identify transcriptional networks and epigenetic alterations governed by HMGA1 in this setting. This work not only provides new insights into the role of HMGA1 in colon epithelial homeostasis by maintaining both the stem cell pool and promoting regeneration following injury, but also suggests that HMGA1 can be aberrantly induced by signals from the microbiome to promote tumorigenesis. Our results also highlight the interactions between the microbiome and HMGA1 as a potential therapeutic opportunity in colon carcinogenesis.

#2602

AKT1-mediated activation of HSF1 by phosphorylation and an association with metastasis-free survival.

Wen-Cheng Lu,1 Haimanti Ray,1 Ramsey Omari,1 Sha Cao,2 Richard Carpenter1. 1 _Indiana University, Bloomington, IN;_ 2 _Indiana University, Indianapolis, IN_.

Breast cancer is the most common cancer affecting women worldwide. The overwhelming majority of deaths from breast cancer are caused by metastasis. The transcription factor heat shock factor (HSF1), which classically regulates the heat shock response, has been linked to tumor progression and metastasis. Our previous data indicate HSF1 is activated in approximately 50% of patient breast tumors, independent of subtype and is associated with promoting epithelial-to-mesenchymal transition (EMT). Our laboratory has shown that AKT1 phosphorylates Ser326 of HSF1, leading to HSF1 activation in breast cancer cells and EMT. However, it remains unclear what phosphorylation sites are most critical for HSF1 transcriptional activity and whether AKT1 also regulates other HSF1 sites. To answer this question, HSF1 was subjected to an in vitro kinase assay. We observed Ser326 phosphorylation via immunoblotting. These phosphorylated HSF1 proteins were further subjected to mass spectrometry wherein we identified three novel sites of AKT1-mediated phosphorylation in Thr142, Ser230, and Thr527. All of these sites, along with Ser326, have previously been shown to promote HSF1 transcriptional activity. Interestingly, incubation with a pan-AKT inhibitor also suppressed the heat shock response by HSF1. However, when HSF1 protein was incubated with AKT2 or AKT3, there was no phosphorylation at Ser326. Furthermore, transcriptional activity of HSF1 and expression of Hsp70, a known HSF1 target gene, were increased by AKT1 but not by AKT2 or AKT3. Thus, we have further identified that AKT1 phosphorylates several activating residues on the HSF1 protein whereas AKT2 and AKT3 do not phosphorylate HSF1 to promote its activation. The importance of promoting HSF1 transcriptional activity by AKT1 cannot be understated as the PI3K pathway has been found to be genetically activated in ~77% of breast cancer. Additionally, it has recently been reported that this pathway is activated in almost 40% of all human tumors. However, whether HSF1 activity has any relation to EMT or metastasis in these other tumor types is unknown. To address this, we developed a gene expression signature for HSF1. We found this gene signature was strongly associated with metastasis-free survival in a broad range of solid tumors including breast cancer, lung cancer, ovarian cancer, melanoma, pancreatic cancer, and prostate cancer. In summary, we established a more definitive mechanism by which AKT1 phosphorylates and activates the HSF1 protein by phosphorylation of several activating sites. Furthermore, due to the seemingly ubiquitous nature of PI3K/AKT signaling across human tumors, we found that HSF1 is also potentially activated in these tumor types and has a strong association with patient outcomes, in particular metastasis.

#2603

Herbal extract SH003 suppresses tumor growth and metastasis of MDA-MB-231 breast cancer cells by inhibiting STAT3-IL-6 signaling.

Yu-Jeong Choi,1 Myeong-Sun Kim,1 SooYeon Kang,1 Seo Yeon Lee,1 Ji Hye Kim,1 Youn Kyung Choi,2 Sukjoong Oh,3 Seong-Gyu Ko1. 1 _Kyung Hee University, Seoul, Republic of Korea;_ 2 _Korea Institute of Ocean Science and Technology (KIOST), Jeju, Republic of Korea;_ 3 _Kangbuk Samsung Hospital, Seoul, Republic of Korea_.

Cancer inflammation promotes cancer progression, resulting in a high risk of cancer. Here, we demonstrate that our new herbal extract, SH003, suppresses both tumor growth and metastasis of MDA-MB-231 breast cancer cells via inhibiting STAT3-IL-6 signaling path. Our new herbal formula, SH003, mixed extract from Astragalus membranaceus, Angelica gigas, and Trichosanthes kirilowii Maximowicz, suppressed MDA-MB-231 tumor growth and lung metastasis in vivo and reduced the viability and metastatic abilities of MDA-MB-231 cells in vitro. Furthermore, SH003 inhibited STAT3 activation, which resulted in a reduction of IL-6 production. Therefore, we conclude that SH003 suppresses highly metastatic breast cancer growth and metastasis by inhibiting STAT3-IL-6 signaling path.

#2604

Constitutive activation of the human aryl hydrocarbon receptor in mice promotes hepatocarcinogenesis independent of its coactivator Gadd45b.

Peipei Lu, Xinran Cai, Zihui Fang, Wen Xie. _University of Pittsburgh, Pittsburgh, PA_.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), or dioxin, is a potent liver cancer promoter through its sustained activation of the aryl hydrocarbon receptor (Ahr) in rodents. However, the carcinogenic effect of TCDD and AHR in humans has been controversial. It has been suggested that the inter-species difference in the carcinogenic activity of AhR is largely due to different ligand affinity in that TCDD has a 10-fold lower affinity for the human AHR compared to the mouse Ahr. It remains unclear whether the activation of human AHR is sufficient to promote hepatocellular carcinogenesis. The goal of this study is to clarify whether activation of human AHR can promote hepatocarcinogenesis. Here we reported the oncogenic activity of human AHR in promoting hepatocellular carcinogenesis. Constitutive activation of the human AHR in transgenic mice was as efficient as its mouse counterpart in promoting diethylnitrosamine (DEN)-initiated hepatocellular carcinogenesis. The growth arrest and DNA damage-inducible gene 45 β (Gadd45b), a signaling molecule inducible by external stress and UV irradiation, is highly induced upon AHR activation. Further analysis revealed that Gadd45b is a novel AHR target gene and a transcriptional coactivator of AHR. Interestingly, ablation of Gadd45b in mice did not abolish the tumor promoting effects of the human AHR. Collectively, our findings suggested that constitutive activation of human AHR was sufficient to promote hepatocarcinogenesis.

#2605

Mithramycin enhances the efficacy of etoposide in Ewing sarcoma cells and a mouse xenograft tumor model.

Anish Ray,1 Umesh T. Sankpal,2 Lina Albeer,2 Abigail Hunter,2 Holly Lout,2 Kathryn Dunlap,2 W. Paul Bowman,2 Don Eslin,3 Riyaz M. Basha2. 1 _Cook Children's Medical Center, Fort Worth, TX;_ 2 _Univ. of North Texas Health Science Ctr., Fort Worth, TX;_ 3 _Arnold Palmer Hospital for Children, Fort Worth, FL_.

Ewing sarcoma (ES) tumors arise in bone or soft tissue of children, adolescents, and young adults. The fusion protein and transcription factor, EWS-FLI1 is over-expressed in >85% of ES tumors. High throughput screening with approximately 50,000 compounds identified Mithramycin (MIT) as an inhibitor of EWS-FLI1. A previous clinical trial to test the efficacy of MIT was not successful, presumably due to toxicity from the high dose required to inhibit EWS-FLI1. We hypothesize that the efficacy and safety of treatment can be enhanced if MIT is used along with standard chemotherapeutic agents. Accordingly, preclinical experiments were conducted to test combination treatment with chemotherapeutic agents used for the treatment (standard care) of ES. After screening several chemotherapeutic agents used in the standard treatment of ES, Etoposide (VP-16) was tested for further experiments. ES cells, CHLA10 and TC205 were treated in the presence of vehicle (Dimethyl sulfoxide), or optimized doses of MIT, VP-16 or MIT+VP-16. The effect of these treatments on cell growth inhibition was studied at 48-hour post-treatment using a luminescent (CellTiter-Glo®) cell viability assay kit. The effect of these treatments on programmed cell death was measured by evaluating the apoptotic cell population using Flow cytometry with Annexin V Apoptosis Detection Kit and the expression of a protein marker, Cleaved-Poly (ADP-ribose) polymerase (c-PARP), by Western blot analysis. Nude mice were injected subcutaneously with TC205 cells, treated with MIT, ETO or MIT+VP-16 and the tumor growth inhibition was assessed. As expected, all treatments showed a dose and time-dependent effect on ES cell growth inhibition; however, the combination treatment of MIT+VP-16 was significantly (p<0.01) more effective than individual agents. The cell growth inhibition in combination treatment was accompanied by an increase in apoptotic cells and c-PARP expression in both ES cell lines. The combination treatment significantly (p<0.01) decreased the tumor weight and volume when compared to control or the groups treated by either MIT or VP-16. The combination of MIT and VP-16 is effective when compared to individual treatments in the TC205 and CHLA10 cell lines and inhibited tumor growth in a mouse model. Among the two cell lines tested, CHLA10 cell line expresses EWS-FLI1 while mTC205 cell line does not express EWS-FLI1. These results demonstrate that the combination of MIT and VP-16 is inhibiting both cell lines, suggesting a novel mechanism of targeting the ES cells even in the absence of EWS-FLI1 expression. These results demonstrate that MIT in combination with the chemotherapeutic agent VP-16 increases therapeutic efficacy in a preclinical model for ES and demonstrates strong potential for translational application.

#2606

Genetic contribution of MYC to the development of primary prostate cancer.

Nichelle C. Whitlock,1 Shana Trostel,1 Rayann Atway,1 Elizabeth Walton,1 Brian J. Capaldo,1 Huihui Ye,2 Adam G. Sowalsky1. 1 _NIH, Bethesda, MD;_ 2 _Beth Israel Deaconess Medical Center, Boston, MA_.

Despite the predictive value of Gleason score (Gs), the molecular characteristics and associated biology that underpin specific Gleason patterns (Gp) remain understudied. Based on previous data showing increased MYC activity in higher-grade Gp4 versus lower-grade Gp3 tumor foci and addressing the paradox that prostate cancer is not hyper-proliferative, we sought to examine the genetic contribution of MYC to locally-advanced prostate cancer development. We screened a series of Gs7 cancers in which the Gp3 and Gp4 components were adjacent and represented contributing elements of the index lesion. Tumor foci were collected by laser capture microdissection based on MYC staining and purified for RNA; transcriptome profiling was performed. To further guide our bioinformatics analyses, we generated a LNCaP prostate cancer cell line harboring MYC knockdown. RNA-seq and ChIP-seq was performed to identify differentially bound regions of the cistrome that correlate with altered gene expression and elucidate potential mechanism(s) of action for MYC in primary prostate cancer. In contrast to other reports, MYC knockdown had the effect of increasing global transcription. Moreover, the publicly available Cancer Genome Atlas (TCGA) prostate cancer dataset was curated according to MYC status based on extreme case design using reverse phase protein assay data. Preliminary results show: (1) increased MYC protein levels, but not proliferation, in Gp4 tumor foci as compared to adjacent Gp3, (2) distinct histology and biology based on MYC tumor status, and (3) found recurrent enrichment for gene sets cross-referencing differentially expressed genes between TCGA, tissue, and our engineered cell line. Of note, we observed recurrent enrichment for the Acosta Proliferation Independent MYC Targets Up gene set, which correlated with MYC activity. When this gene set was cross-referenced between all three datasets, the gene NOLC1 was identified. Nucleolar and coiled-body phosphoprotein 1 (NOLC1) plays an important role in rRNA synthesis and ribosome biosynthesis; its role in tumorigenesis appears context dependent. NOLC1 was found to be positively correlated with MYC expression and may thus contribute to MYC-driven prostate cancer. Although further cases are needed to determine the frequency at which NOLC1 drives MYC-driven prostate cancer, our results suggest a novel role for MYC in mediating development of locally-advanced prostate cancer.

#2607

BPTF role in melanoma progression and BRAF resistance therapy.

Altaf A. Dar,1 Shahana Majid,2 David de Semir,1 Vladimir Bezrookove,1 Mehdi Nosrati,1 Mohammed Kashani-Sabet1. 1 _California Pacific Medical Ctr. Research Inst., San Francisco, CA;_ 2 _UCSF, San Francisco, CA_.

Bromodomain PHD finger transcription factor (BPTF) plays an important role in chromatin remodeling, but its functional role in tumor progression is incompletely understood. Here we reveal new pro-oncogenic roles for BPTF in promoting tumor cell proliferation and resistance to targeted therapies. shRNA-mediated BPTF silencing suppressed the proliferative capacity (by 65.5%) and metastatic potential (by 66.4%) of melanoma cells. Elevated BPTF mean copy number greater than 3 was observed in 28 of 77 (36.4%) melanomas. BPTF overexpression predicted poor survival in a cohort of 311 melanoma patients (distant metastasis-free survival, p=0.03, and disease-specific survival p=0.008), and promoted resistance to BRAF inhibitors in melanoma cell lines. Metastatic melanoma tumors progressing on BRAF inhibitors contained low BPTF-expressing, apoptotic tumor cell sub-clones, indicating the continued presence of drug-responsive sub-clones within tumors demonstrating overall resistance to anti-BRAF agents. These studies demonstrate multiple pro-tumorigenic functions for BPTF, and identify it as a novel target for anti-cancer therapy. They also suggest the combination of BPTF targeting with BRAF inhibitors as a novel therapeutic strategy for melanomas with mutant BRAF.

#2608

EGF signaling activates a TAZ-driven oncogenic program in glioblastoma.

Minling Gao, Yi Fu, Laterra John, Mingyao Ying. _Kennedy Krieger Research Inst., Baltimore, MD_.

Glioblastoma multiforme (GBM) is the most aggressive and lethal primary brain tumor in humans. Hyperactivation of the signaling pathway driven by the epidermal growth factor (EGF) and its receptor (EGFR) are commonly occurring in GBM. The oncogenic transcriptional program driven by EGF signaling is still incompletely understood in GBM. Here, we found that EGF induces the upregulation and nuclear translocation of TAZ, an oncogenic transcriptional co-activator. TAZ and its paralog gene YAP are commonly known as downstream mediators of the Hippo signaling pathway. TAZ has been found as a driver of mesenchymal transition in GBM cells and promotes GBM radioresistance. Our results support that EGF induces TAZ transcription and the activation of TAZ gene targets, through the signaling axis including EGFR, STAT3 and ERK. EGFRvIII is the most frequently occurring EGFR mutation in GBM causing constitutively active EGFR. We found that EGFRvIII causes EGF-independent activation of TAZ and its downstream oncogenic targets. Next, we used next-generation sequencing to establish a genome-wide map of TAZ gene targets in GBM cells after EGF stimulation. The results suggest that EGF signaling activates a TAZ-driven oncogenic program including autocrine and paracrine factors that promote GBM growth, angiogenesis, stemness and immunoescape. Moreover, we found that TAZ targeting by shRNAs or the FDA-approved drug Verteporfin effectively inhibits the TAZ-driven oncogenic program activated by EGF stimulation or the EGFRvIII mutant. TAZ targeting also inhibits GBM cell growth and xenograft formation. In contrast, enforced TAZ induces the TAZ-driven oncogenic program and promotes GBM cell growth and xenograft formation. In conclusion, our results reveal a novel TAZ-driven oncogenic program that is activated by EGF signaling in GBM. Our discoveries strongly support TAZ as a therapeutic target for GBM and likely other human cancer with hyperactivation of EGF signaling. Our preclinical studies also provide a clinically translatable strategy for GBM therapy based on using the FDA-approved drug Verteporfin.

#2609

A novel MYB-AR cross-talk promoting castration-resistance in prostate cancer.

Girijesh K. Patel. _Univ. of South Alabama Mitchell Cancer Inst., Mobile, AL_.

Progression of prostate cancer from castration-sensitive to castration-resistant disease is a complex process, which likely involves multiple genetic and epigenetic alterations. Here we report a novel mechanism where MYB acts as a novel binding partner of AR enabling its ligand-independent activation to support castration resistance in prostate cancer. MYB and AR interact and co-localize with each other predominantly in the nuclei. Androgen-depletion or enzalutamide treatment does not interfere with MYB-AR interaction, and MYB-overexpressing prostate cancer cells retain AR in the nucleus even when cultured under androgen-deprived condition. Transcriptional activity of KLK3 (an androgen-responsive gene encoding PSA) promoter is increased in MYB-overexpressing cells, while sustained under androgen-depleted condition. In silico analysis identifies a MYB-binding region in KLK3 promoter in close proximity to the AR binding site, and MYB is shown to cooperatively promote AR binding to the KLK3 promoter. MYB-overexpressing prostate cancer cells exhibit greater tumorigenicity when implanted orthotopically and quickly regain growth following castration leading to the poorer survival of mice, compared to those carrying low MYB-expressing prostate tumors. Together, these findings establish a novel and significant role of MYB-AR cross-talk in prostate cancer, which could be exploited for its therapeutic management.

#2610

**The FOXC2 transcription factor promotes growth of melanoma cells in both** in vitro **and** in vivo s **ettings.**

Corey J. Williams, Coleman E. Johnson, David Z. Bushhouse, Kristian M. Hargadon. _Hampden-Sydney College, Hampden-Sydney, VA_.

Melanoma, a cancer derived from pigment-producing melanocytes of the skin, is a highly aggressive cancer characterized by rapid growth and metastasis. Recent evidence has emerged demonstrating that overexpression of the FOXC2 transcription factor contributes to tumor progression in many cancer types, but the role of FOXC2 in melanoma progression has not previously been studied. Using CRISPR-Cas9 gene editing technology, we have generated and characterized a novel FOXC2 knockout B16-F1 melanoma cell line (B16-F1ΔFOXC2). In comparative in vitro and in vivo studies with wild-type B16-F1 and B16-F1ΔFOXC2 melanomas, we show for the first time that the FOXC2 transcription factor is a critical regulator of melanoma cell growth. The B16-F1ΔFOXC2 melanoma cell line described herein will serve as a useful tool for investigating the function and target genes of FOXC2 in melanoma.

#2611

Activated pentose phosphate pathway mediated by FBP1 upregulation supports progression of acute myeloid leukemia with high EVI1 expression.

Hideaki Mizuno,1 Yuki Kagoya,1 Junji Koya,2 Yosuke Masamoto,1 Mineo Kurokawa1. 1 _The University of Tokyo, Tokyo, Japan;_ 2 _National Cancer Center Research Institute, Tokyo, Japan_.

EVI1 (ecotropic viral integration site 1) is a transcription factor essential for the development and progression of acute myeloid leukemia (AML). Since high EVI1 expression, seen in approximately 10% of AML patients (EVI1high AML), is associated with poor clinical outcome, development of a novel therapeutic strategy is required. In this study, we investigated key molecules that are induced by aberrant EVI1 expression and modulate the metabolomics of leukemia cells. We conducted whole transcriptome analysis by RNA-sequencing in murine leukemia cells induced by retroviral transduction with EVI1 followed by transplantation into irradiated mice. We identified 45 genes with more than 10-fold expression in EVI1-transduced bone marrow cells compared to those with the mock vector at both pre-leukemic phase and leukemia phase. Intriguingly, the fructose bisphosphatase 1 gene (Fbp1), which encodes a key enzyme of gluconeogenesis was highly upregulated in EVI1-induced AML cells. We confirmed that FBP1 mRNA expression is quickly upregulated by ectopic expression of EVI1 in murine LSK (Linneg, c-Kitpos, Sca-1pos) cells. Moreover, we observed an enrichment of EVI1 in the promoter and enhancer region of Fbp1 by chromatin immunoprecipitation followed by qPCR analysis in murine hematopoietic cells, suggesting that Fbp1 expression is directly regulated by EVI1. Although high FBP1 expression suppresses glycolytic metabolism and negatively affects cellular proliferation in various types of solid tumor, its role in AML has not been investigated. In contrast to the results previously reported in other types of cancers, pharmacologic inhibition or short hairpin RNA (shRNA)-mediated knockdown of FBP1 significantly decreased colony-forming cell (CFC) capacity in EVI1-transduced murine LSK cells and significantly delayed progression of leukemia in the mice secondarily transplanted with EVI1-induced AML cells in vivo. These results suggest that aberrant expression of FBP1 is important for progression of EVI1high AML. While high FBP1 enzymatic activity negatively affects the glycolytic pathway, it in turn enhances the pentose phosphate pathway (PPP) through elevating the fructose-6-phosphate level. Activated PPP results in enhanced nucleotide synthesis essential for rapidly dividing cells. Importantly, individual knockdown of main PPP enzymes (G6pd, Pgd, Rpia) by shRNA transduction significantly decreased the CFC capacity of EVI1 transduced murine LSK cells, further confirming an important role of PPP in driving proliferation of EVI1high leukemia cells. Collectively, these results indicate that the activated PPP through transcriptional upregulation of FBP1 is crucial for progression of EVI1-driven leukemia. Since inhibition of FBP-1 did not compromise normal hematopoiesis, targeting the enzyme can be a promising therapeutic approach for the EVI1high AML.

#2612

Roles of USF1 in breast tumorigenesis and disease progression.

Jessica Lewis, Iqbal Mahmud, Guimei Tian, Daiqing Liao. _University of Florida College of Medicine, Gainesville, FL_.

Breast cancer accounts for nearly one-quarter of all cancer diagnoses and is the principal cause of cancer-related mortality in women worldwide. Triple negative breast cancer (TNBC) is a clinically aggressive subtype of breast cancer commonly resistant to therapeutics that have been successful in increasing survival in patients with ER+, PR+ and HER2+ breast cancer subtypes. As such, identifying factors that contribute to poor patient outcomes and mediate the growth and survival of TNBC cells remain important areas of investigation. USF1 (upstream stimulatory factor 1), a gene linked to drive lipogenesis and cellular proliferation, is overexpressed in human malignancies, yet its contribution to cancer remains unclear. In analyzing large number of genomic datasets including The Cancer Genome Atlas (TCGA), we found that USF1 expression is significantly higher in TNBC patient samples. Also, USF1 gene expression positively correlates with key lipogenic enzymes. Significantly, we found that high expression of USF1 in breast cancer correlates with decreased patient survival. We therefore hypothesize that USF1 promotes breast tumorigenesis and progression by activating lipogenic gene expression. We conducted pilot in vitro studies to determine the influence of USF1 expression and cell proliferation. It was demonstrated that knockdown of USF1 decreased cellular proliferation in 2D cell culture of the TNBC cell line MDA-MB-231. We also assessed the effects on USF1 expression levels on in vivo tumor growth. We found that USF1 overexpression and knockdown enhanced and reduced tumor growth in vivo, respectively. Further studies are underway to determine the mechanisms by which USF1 promotes TNBC tumorigenesis and metastatic progression. Our studies will shed lights on roles of USF1 in breast cancer tumor biology. (Supported by McKnight Foundation, Bankhead-Coley Cancer Research Program and James and Esther King Biomedical Research Program, Florida Department of Health.)

#2613

**MYC controls DNA methylation on a global scale through** DNMT3b **upregulation in T-ALL and Burkitts Lymphoma.**

Candace J. Poole, Atul Lodh, Jan van Riggelen. _Augusta University, Augusta, GA_.

Background: Oncogenic MYC is implicated in 60-70% of all human cancers, including T-cell acute lymphoblastic leukemia (T-ALL) and Burkitts lymphoma (BL). Genetic inactivation of MYC can cause tumor regression through cellular senescence in mouse models (exhibiting oncogene addiction), making it a promising therapeutic target. However, a pharmacological MYC inhibitor remains elusive, thus these findings cannot be translated into clinic. We are the first to demonstrate that MYC can control DNA methyl-transferase (DNMT) expression and thus, genome-wide DNA methylation; as an alternative strategy to disrupt the MYC network indirectly, we have chosen to target MYC-dependent DNMTs as a tumor-cell specific strategy.

Experimental Procedures and Results: Utilizing expression profiling in patient-derived cell lines and clinical specimens, we found DNMT 1, 3a, and 3b to be overexpressed in MYC-driven T-ALL and BL compared to non-malignant tissue. Using transgenic inducible "tet-off" MYC-models in mouse T-ALL (EµSRα-tTA;tet-o-MYC) and human Burkitt's lymphoma (P493-6), we have shown that DNMT1 and DNMT3b expression depend on high MYC expression levels in T-ALL and BL. Using patient-derived cell lines, we've shown that depletion of endogenous MYC caused a reduction of both DNMT1 and DNMT3b expression. Chromatin Immunoprecipitation (ChIP) profiling for MYC binding, further establishes the DNMT1 and DNMT3b promoters as bona fide targets of the MYC oncogene. Furthermore, shRNA-mediated knockdown of DNMT3b or pharmacological inhibition using NanaomycinA, indicates a tumor promoter function in the context of MYC-driven tumor maintenance in T-ALL. To further elucidate the consequence on DNA methylation and gene expression upon DNMT3b loss of function, Reduced Representation Bisulfite Sequencing and RNA-sequencing was performed to analyze alterations in DNA methylation and gene expression, respectively. We found that DNMT3b maintains CpG island DNA methylation near promoters, responsible for gene silencing during tumor maintenance. Accompanying changes in DNA methylation, we identified approximately one thousand differentially expressed genes upon DNMT3b knockdown, with upregulated genes responsible for controlling differentiation, transport, and development; while downregulated genes controlled processes including signal transduction, apoptosis, and regulation of cellular proliferation.

Conclusion: Together, we provide novel evidence that MYC directly controls global DNA methylation through deregulation of DNMT1 and DNMT3b expression to maintain gene programs for T-ALL tumor maintenance. Furthermore, we provide evidence that targeted inhibition of individual DNMTs may reveal alternative tumor-specific therapies for T-ALL, BL, and other MYC-driven cancers.

#2614

CCAR2 acetylation establishes a BET/BRD9 acetyl switch in response to combined deacetylase and bromodomain inhibition.

Gavin S. Johnson,1 Praveen Rajendran,1 Li Li,1 Ying-Shiuan Chen,1 W. Mohaiza Dashwood,1 Nhung Nguyen,1 Ahmet M. Ulusan,1 Furkan Ertem,1 Mutian Zhang,1 Deqiang Sun,1 Yun Huang,1 Shan Wang,1 Hon-Chiu E. Leung,2 David Lieberman,3 Laura M. Beaver,4 Emily Ho,4 Mark T. Bedford,5 Kyle Chang,5 Eduardo Vilar,5 Roderick H. Dashwood1. 1 _Texas AM Univ. Health Science Ctr., Houston, TX;_ 2 _Baylor College of Medicine, Houston, TX;_ 3 _Oregon Health & Science University, Portland, OR; _4 _Oregon State University, Corvallis, OR;_ 5 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

There continues to be interest in targeting epigenetic 'readers, writers and erasers' for the treatment of cancer and other pathologies. A mechanistic understanding is frequently lacking, however, for the synergy observed when combining deacetylase and bromodomain inhibitors. We identified cell cycle and apoptosis regulator 2 (CCAR2) as an early target for acetylation in colon cancer cells treated with sulforaphane (SFN). N-terminal acetylation of CCAR2 diminished its interactions with histone deacetylase 3 (HDAC3) and β-catenin, interfering with Wnt coactivator functions of CCAR2, including in cells harboring genetically encoded CCAR2 acetylation. Protein domain arrays and pull-down assays identified acetyl 'reader' proteins that recognized CCAR2 acetylation sites, including BRD9 and members of the bromodomain and extraterminal domain (BET) family. The BET inhibitor JQ1 synergized with SFN in colon cancer cells, and suppressed tumor development effectively in a preclinical model of colorectal cancer. Studies with SFN+JQ1 in combination implicated a BET/BRD9 acetyl switch, and a shift in the pool of acetyl 'reader' proteins in favor of BRD9 regulated target genes. These results highlight the competition that exists among the 'readers' of acetylated histone and non-histone proteins, and provide a mechanistic basis for potential new therapeutic avenues involving epigenetic combination treatments.

#2615

Systematic approaches to predict oncogenic transcriptional regulatory circuitries identify important nodes in high-grade serous ovarian cancer.

Brian J. Abraham,1 Jessica Reddy,2 Marcos A. Fonseca,2 Isaac A. Klein,1 Lena K. Afeyan,1 Rosario I. Corona,2 Paloma Cejas,3 Felipe Segato,4 Beth Y. Karlan,2 Simon A. Gayther,2 Matthew L. Freedman,3 Houtan Noushmehr,5 Myles Brown,3 Ursula A. Matulonis,3 Kate Lawrenson,2 Richard A. Young1. 1 _MIT Whitehead Inst. for Biomed. Resea, Cambridge, MA;_ 2 _Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA;_ 3 _Dana-Farber Cancer Institute, Boston, MA;_ 4 _Ribeirão Preto Medical School, University of São Paulo, Sao Paolo, Brazil;_ 5 _Henry Ford Hospital, Detroit, MI_.

Previous work indicates that identifying core transcription regulatory circuitries (CRCs) predicts tumor-specific genetic dependencies, in the form of transcription factors responsible for establishing this circuitry (Durbin, Zimmerman, Dharia, et al., 2018), which suggest targetable, onco-requisite factors. However, a systematic effort to identify CRCs across tumors has been challenging, since current approaches rely on data difficult to acquire in heterogeneous or low cell number samples. Here, we combine -omics approaches, including a newly developed algorithm to predict super-enhancer—driven CRCs in a range of tumor types. Our work leverages two characteristics CRC members: association with super-enhancers, defined using ChIP-Seq data, and highly cell type-specific expression, analyzed using the Cancer Cell-Type-Specificity (CaCTS) algorithm. CRC members uncovered with the CaCTS algorithm recover known drivers of specific tumor types and known misregulated pathways. We deeply characterized the CRC of high-grade serous ovarian cancer (HGSOC), a tumor type with poor overall prognosis for which few known driver mutations or targetable oncogenes are known. Our CRC models contain onco-requisite transcription factors that dominate the HGSOC gene expression program, including ESR1, MECOM (EVI1), and NOTCH2, each of which play important roles in other malignancies. Many of these CRC members represent HGSOC-selective dependencies in CRISPR/Cas9 screens. Profiling CRCs in HGSOC cells and presumed cells-of-origin (fallopian tube and ovarian tissue) highlights a set of CRC genes whose expression appears to have been evolved by HGSOC cells, including an isoform of the estrogen receptor. The dependence of HGSOC cells on transcription factors suggests a state of "transcriptional addiction," which has been targeted with molecules against transcription apparatus. HGSOC lines are correspondingly susceptible to treatment with small molecules against transcriptional CDKs (CDK7 and CDK12). Also, some HGSOC-specific SEs are associated with genes druggable with existing small molecules. Thus, identifying the CRC governing HGSOC predicts dominant factors and suggests therapeutic avenues in this underserved disease. Similar predictions of CRCs in other poorly-studied tumors may also enhance understanding of those tumor cell identities and targetable nodes.

#2616

A novel iron-chelating agent reduces MYC transcription via E2F gene family regulation.

Emanuele Valli,1 Chengyuan Xue,1 Leanna Cheung,1 Laura Gamble,1 Ruby Pandher ,1 Simone Di Giacomo,2 Catherine Burkhart,3 Natalia Fedtsova,4 Francesca Ferrucci,5 Sergei Makarov,6 Thomas Telfer,7 Rachel Codd,7 David Scott,8 Giovanni Perini,2 Andrei Osterman,8 Andrei Gudkov,4 Murray Norris,1 Michelle Haber1. 1 _Children's Cancer Institute, Randwick, NSW, Australia;_ 2 _University of Bologna, FaBiT, Bologna, Italy;_ 3 _Cleveland BioLabs, Inc., Buffalo, NY;_ 4 _Roswell Park Cancer Institute, Buffalo, NY;_ 5 _Oslo University Hospital Rikshospitalet, Oslo, Norway;_ 6 _Attagene Inc., Morrisville, NC;_ 7 _The Univeristy of Sydney, Camperdown, NSW, Australia;_ 8 _Sanford-Burnham Medical Research Institute, La Jolla, CA_.

Childhood neuroblastoma, a disease of the sympathetic nervous system, is the most common solid tumour of infancy. Amplification of the MYCN oncogene, which occurs in approximately 25% of patients with neuroblastoma, is one of the most powerful independent prognostic markers for this disease. MYCN belongs to the Myc gene family that codes for transcription factors often aberrantly expressed in both solid and liquid malignancies. However, despite their importance in adult and childhood malignancies, Myc oncoproteins have to date proven to be 'undruggable'. Using a chemical library of 34,000 compounds we aimed to identify novel MYCN inhibitors, and among a number of molecules identified, M606 was found to markedly reduce MYCN protein levels as well as its downstream targets in MYCN-amplified SK-N-BE(2)-C neuroblastoma cells. Bioinformatic analysis of signalling pathways affected by M606 using FACTORIAL™ technology (Attagene Inc) demonstrated that this compound inhibits Myc-mediated transcription and simultaneously activates the HIF1α pathway. MYCN/MYC protein and mRNA stability were unaltered whereas their mRNA levels were significantly decreased. Metabolomics analysis suggested that the mechanism by which M606 inhibits MYCN transcription involves its capacity to chelate iron and, in this regard, M606-mediated inhibition of MYCN was reversed by the addition of iron. Progressive deletions of the MYCN promoter in luciferase reporter assays identified a minimal promoter region responsive to M606 that contains two E2F sites. Microarray assays performed on SK-N-BE(2)-C cells after M606 treatment showed significant changes in the expression level of the E2F gene family. Chromatin immune precipitation (ChIP) and gel shift (EMSA) assays revealed a reduction in E2F binding at the MYCN promoter region after M606 treatment. Moreover, western blot analysis showed that the RB protein, which is involved in E2F binding and transcriptional activation, becomes inactive due to hypophosphorylation following M606 treatment. On the other hand, M606 lost its inhibitory activity on a pRB-deficient osteosarcoma cell line SAOS-2, underlying the importance of the RB/E2F axis for M606 function. In conclusion, M606 is a novel Myc inhibitor and iron chelator that directly downregulates MYCN/MYC transcription via the E2F signalling pathway, providing a potentially valuable therapeutic approach in the treatment of cancers overexpressing Myc oncoproteins.

#2617

FOXC2-associated regulation of melanoma cell adhesion to the extracellular matrix and lymphatic endothelial cells.

Coleman E. Johnson, Corey J. Williams, Kristian M. Hargadon. _Hampden-Sydney College, Hampden-Sydney, VA_.

The FOXC2 transcription factor has emerged as a tumor-promoting oncogene in many cancer types. We have recently investigated the impact of Foxc2 gene disruption and FOXC2 overexpression in melanoma on tumor cell adhesion to extracellular matrix (ECM) proteins and lymphatic endothelial cells. We show that melanoma-associated FOXC2 is a critical regulator of tumor cell adhesion to the ECM proteins fibrinogen and fibronectin as well as to lymphatic endothelial cells. These effects on tumor cell adhesion correlate with FOXC2-mediated regulation of the integrins ITGA5 and ITGA9. As changes in cellular adhesion frequently accompany tumor cell transformation and progression, our data suggest that FOXC2 and genes regulated by this transcription factor may play key roles in promoting melanoma invasion and metastasis.

#2618

SOX9 controls TNBC growth and metastasis via regulating the expression of apoptosis and EMT genes.

Yanxia Ma, Lakshmi Bollu, Jamal Hill, Yun Zhang, William M. Tahaney, Abhijit Mazumdar, Powel H. Brown. _MD Anderson Cancer Center, Houston, TX_.

Background: There are few effective targeted therapies available for triple-negative breast cancers (TNBCs), the most aggressive form of breast cancer. A better understanding of the critical molecular regulators is necessary to develop effective targeted therapies for TNBCs. Our previous study focused on SOX9 transcription factor and determined that SOX9 is highly expressed in TNBCs which is required for TNBC cell survival and metastasis.

Hypothesis: SOX9 controls TNBC cell growth and metastasis via directly regulating cell apoptosis and epithelial-mesenchymal transition (EMT) genes.

Material and Methods: The breast cancer datasets were used to analyze the association between overall survival and SOX9 expression. Cell growth was measured using an automated cell counting assay. Cell migration and invasion were detected by transwell migration and invasion assays. A doxycycline-inducible Cas9-CRISPR knockout or siRNA knockdown system was used to inhibit SOX9 in cells. Xenograft mice model was established to evaluate the effect of SOX9 on TNBC cell growth in vivo. Protein and mRNA levels were examined by western blotting and qRT-PCR assays. An EMT PCR array and the CHIP-seq enrichment analysis based on published SOX9-CHIP-seq datasets were performed to identify effect of SOX9 on downstream pathways. SOX9 binding peaks in gene promoter region were determined and confirmed by SOX9-CHIP-seq datasets and CHIP assay. Data are presented as mean values ± SD. Statistical significance (p-values) was calculated using the Student's t-test unless otherwise indicated.

Results: Using available gene profiling datasets, we found that high expression of SOX9 was correlated with worse overall survival in Boersma breast dataset, but not in other datasets. Late stage breast cancers showed higher SOX9 expression compared to early stage breast cancers. Using SOX9 inhibited cells, we demonstrated that SOX9 is critical for TNBC cell survival and invasion in vitro and in vivo. To identify the molecular mechanisms of these functions, we used CHIP-seq data and CHIP assays to demonstrate that SOX9 directly binds to the promoter of several extrinsic apoptosis-inducing factors (such as fadd and tnfrsf10b) and represses their expressions. Loss of SOX9 caused up-regulation of the expression of these genes, consistent with the induction of apoptosis. We then investigated the effect of SOX9 on EMT-regulating genes. Loss of SOX9 induced down-regulation of most EMT genes. Using CHIP assays, we demonstrated that SOX9 directly binds the promoters of several EMT genes (such as vim and ctnnb1) and increases the expression of these genes.

Conclusion: Our results demonstrate that by directly binding and regulating cell apoptosis and EMT gene expression, SOX9 controls TNBC survival and induces EMT resulting in aggressive and potentially lethal tumors.

Grant Support: These studies were supported by a Breast Cancer Research Fund (BCRF) grant (PB).

#2619

DNA damage agents promote TFEB function and induce pro-survival autophagy signals.

Benoit Marchand, Marie-Josée Boucher. _Univ. of Sherbrooke, Sherbrooke, Quebec, Canada_.

The transcription factor EB (TFEB) was identified as a master regulator of autophagy and lysosomal biogenesis. We recently demonstrated that TFEB is aberrantly regulated in pancreatic cancer (PDAC) cells. Interference with TFEB impairs PDAC cell growth supporting an important role for TFEB in maintaining PDAC cell phenotype. Given the limited impact of chemotherapeutic drugs in the context of PDAC, we aimed at testing whether the aberrantly regulated TFEB in PDAC cells could confer resistance to DNA damage agents, commonly used in PDAC therapeutic regimens.

Methods: Experiments were performed in PDAC cells (MIA PaCa2, PANC1). The DNA damage agents gemcitabine, 5-FU, cisplatin and doxorubicin were used. Autophagic flux was measured upon 4-hours treatment with bafilomycin A1.

Results: Treatment with DNA damage agents 1) increased the autophagic flux in PDAC cells. This autophagic signal was associated with 2) the dephosphorylation and nuclear accumulation of TFEB. The mTORC1 pathway is well-known to regulate TFEB phosphorylation and nuclear localization of TFEB. 3) However, the impact of the DNA damage agents on TFEB appeared independent of the mTORC1 pathway since no modulation in the phosphorylation of the mTORC1 target S6K1 was observed. To delineate whether TFEB influenced the response to DNA damage agents, we generated stable populations of PDAC cells expressing a non-targeting or shRNA targeting TFEB. Interfering with TFEB function 4) prevented the autophagic response upon treatment with DNA damage agents. This correlated with 5) increased accumulation of DNA damage and 6) increased sensitivity to the DNA damage agents.

Conclusion: Our results suggest that DNA damage agents can induce a pro-survival autophagic response involving TFEB. Interfering with TFEB function limits the autophagic response and leads to increased sensitivity to DNA damage agents. Altogether, our results suggest that the aberrantly expressed TFEB in PDAC cells could confer resistance to DNA damage agents by, among others, promoting autophagy and limiting DNA damage accumulation.

#2620

Global epigenetic regulation of gene expression and tumor suppression in T-cell leukemia by Ikaros.

Yali Ding,1 Bo Zhang,1 Jonathon L. Payne,1 Kimberly J. Payne,2 Chunhua Song,1 Chandrika Gowda,1 Soumya Iyer,1 Pavan K. Dhanyamraju,1 Dhimant Desai,1 Suming Huang,1 Feng Yue,1 Sinisa Dovat1. 1 _Hershey Medical Center, Hershey, PA;_ 2 _Loma Linda University, Loma Linda, CA_.

Ikaros encodes a DNA-binding zinc finger protein that functions as a tumor suppressor. Loss of Ikaros function results in the development of high-risk B-cell and T-cell acute lymphoblastic leukemia. Previous studies suggested that Ikaros controls expression of its target genes via chromatin remodeling, and by forming complexes with the histone deacetylase, HDAC1. However, the mechanisms through which Ikaros regulates the global epigenetic signature in leukemia is unknown. We studied the role of Ikaros in regulating tumor suppression in Ikaros-defficient T-cell acute lymphoblastic leukemia (T-ALL) cells. The re-introduction of Ikaros at physiological levels via retroviral transduction into T-ALL cells, results in a cessation of cellular proliferation and cell cycle arrest within 4 days. To determine the dynamics of Ikaros-mediated epigenetic regulation of gene expression in T-ALL, we performed ChIP-seq of Ikaros, HDAC1, and histone modifications, along with microarray and ATAC-seq in wild-type T-ALL (that lacks Ikaros), and during each of 3 days following the re-introduction of Ikaros. Our results demonstrated that Ikaros directly regulates the global epigenomic landscape including genome wide de novo formation of enhancers in T-ALL. Further analysis showed that Ikaros induces the re-distribution of HDAC1 via direct recruitment of HDAC1 to promoter and enhancer regions of different target genes resulting in their transcriptional repression. Expression analysis identified a large number of novel signaling pathways that are directly regulated by Ikaros and HDAC1, and are responsible for the cessation of proliferation and the cell cycle arrest of T-ALL cells. Our results identified novel functions of Ikaros in the epigenetic regulation of gene expression and tumor suppression in T-ALL.

#2621

OLIG2 regulates stem cell maintenance and cell cycle in glioma stem cells.

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

The Cancer Genome Atlas project described a robust gene expression-based molecular classification of glioblastoma with the functional and biological significance of the subclasses yet to be determined. Here, we show that a comprehensive analysis of a panel of glioma initiating cell (GIC) lines can identify a group of stem cells with high OLIGg2 expression as in Proneural-like GBM subtype. In vitro differentiation studies showed that proneural GIC lines possess the potential to differentiate into astrocytic, neuronal, and oligodendrocytic lineages, whereas mesenchymal GICs exhibited limited potential for neural lineage differentiation following retinoic acid induction. A considerable decline of OLIG2 in proneural GIC lines was observed following retinoic acid treatment. We also showed that OLIG2 is a functional marker associated with cell proliferation in Olig2-high GIC lines. In addition, OLIG2 inhibiton disrupted cell-cycle control mechanism by decreasing CDK2 and CDK4 and elevating apoptosis-related molecules. Mechanistic investigations revealed molecular interactions between CDK2/CDK4 and OLIG2. Inhibition of CDK2/CDK4 activity disrupted OLIG2-CDK2/CDK4 interactions and attenuated OLIG2 protein stability. Further investigation on these mechanisms may lead to novel targeted therapy on GBMs with high OLIG2 expression. 

### Receptors and Growth Factors

#2622

Plasma growth diefferentiation factor-15 is and independen marker for aggressive disease in endometrial cancer.

Hilde Engerud. _University of Bergen, Bergen, Norway_.

Objective: Better biomarkers are needed in order to identify patients with endometrial carcinoma at risk of recurrence and who may profit from a more aggressive treatment regimen. Our objective was to explore the applicability of plasma growth differentiation factor 15 (GDF-15) as a marker for recurrent disease, as well as a marker for poor prognosis and lymph node metastases.

Methods: EDTA-blood samples were obtained from 235 patients with endometrial cancer before primary surgery. For 36 of these patients, matching blood samples were collected at time of recurrence. Blood samples were also collected from 78 patients with endometrial hyperplasia. Plasma GDF-15 was measured by an enzyme-linked immunosorbent assay (ELISA). Preoperative pelvic MRI scans for 141 patients were investigated in parallel for imaging variables.

Results: Preoperative plasma level of GDF-15 was significantly higher for patients who experienced recurrence (1780 ng/L; 95% CI; 518-9475 ng/L) than for patients who did not develop recurrent disease (1236 ng/L; 95% CI; 307-7030 ng/L) (p<0.001). Plasma levels of GDF-15 at recurrence (2818 ng/L, 95% CI 2088-3548 ng/L) were significantly higher than plasma levels of GDF-15 measured at time of primary diagnosis (1857 ng/L, 95% CI; 1317-2398 ng/L) (p=0.001). High plasma level GDF-15 independently predicts recurrent disease (OR=3.14; 95% CI 2.10-4.76) and lymph node metastases (OR=2.64; 95% CI 1.52-4.61). Patients with high plasma level of GDF-15 had significantly larger tumor volume (p=0.008).

Conclusion: Elevated plasma level of GDF-15 is associated with aggressive disease and lymph node metastasis in endometrial carcinoma. GDF-15 may be helpful in indicating recurrent disease.

#2623

**Modulating native GABA** A **receptors in medulloblastoma with positive allosteric benzodiazepine-derivatives induces cell death.**

Laura Kallay,1 Havva Keskin,1 Alexandra Ross,1 Manali Rupji,2 Olivia A. Moody,3 Xin Wang,4 Guanguan Li,5 Taukir Ahmed,5 Farjana Rashid,5 Michael Rajesh Stephen,5 Kirsten A. Cottrill,3 Austin Nuckois,3 Maxwell Xu,6 Deborah E. Martinson,2 Frank Tranghese,7 Yanxin Pei,8 James M. Cook,5 Jeanne Kowalski,1 Michael D. Taylor,4 Andrew Jenkins,2 Daniel Pomeranz Krummel,1 Soma Sengupta1. 1 _Emory University, Winship Cancer Institute, Atlanta, GA;_ 2 _Emory University - School of Medicine, Atlanta, GA;_ 3 _Emory University, Atlanta, GA;_ 4 _The Hospital for Sick Children, Toronto, Ontario, Canada;_ 5 _University of Wisconsin-Milwaukee, Milwaukee, WI;_ 6 _Johns Hopkins University, Baltimore, MD;_ 7 _Boston University, Boston, MA;_ 8 _Center for Cancer and Immunology Research, Brain Tumor Institute, Washington, DC_.

Pediatric brain cancer medulloblastoma standard-of-care results in numerous comorbidities. Medulloblastoma is comprised of four molecular subgroups. Group 3 subgroup patients have the highest relapse rates and 20% survival after standard-of-care. Group 3 tumors have high expression of GABRA5, which codes for the α5 subunit of the γ-aminobutyric acid type A receptor (GABAAR). We are advancing a therapeutic approach for group 3 based on GABAAR modulation using benzodiazepine-derivatives. We performed analysis of GABR and MYC expression in medulloblastoma tumors and used molecular, cell biological, and whole-cell electrophysiology approaches to establish presence of a functional 'druggable' GABAAR in group 3 cells. Analysis of expression of 763 medulloblastoma tumors reveals that group 3 tumors share high subgroup-specific and correlative expression of GABR genes, which code for GABAAR subunits α5, β3 and γ2 and 3. There are ~1000 functional α5-GABAARs per group 3 patient-derived cell that mediate a basal chloride-anion efflux of 2x109 ions/sec. Benzodiazepines, designed to prefer α5-GABAAR, impair group 3 cell viability by enhancing chloride-anion efflux with subtle changes in their structure having significant impact on potency. A potent, non-toxic benzodiazepine ('KRM-II-08') binds to the α5-GABAAR (0.8 μM EC50) enhancing a chloride-anion efflux that induces mitochondrial membrane depolarization and in response, TP53 upregulation and p53, constitutively phosphorylated at S392, cytoplasmic localization. This correlates with pro-apoptotic Bcl-2-associated death promoter (BAD) protein localization. GABR expression can serve as a diagnostic biomarker for group 3 tumors, while α5-GABAAR is a therapeutic target for benzodiazepine binding, enhancing an ion imbalance that induces apoptosis.

#2624

NRP2b promotes migration, drug resistance and AKT activation in lung cancers by recruiting GSK3â to phosphorylate and destabilize PTEN.

Anastasios Dimou,1 Cecile Nasarre,2 Monika Gooz,2 Rose Pagano,3 Yuri Peterson,2 Kent Armeson,2 Harry A. Drabkin,4 Patrick Nasarre,2 Chadrick E. Denlinger,2 Robert M. Gemmill2. 1 _Univ of Colorado Medical School, Aurora, CO;_ 2 _Medical Univ. of South Carolina, Charleston, SC;_ 3 _Carnegie Mellon Univ., Pittsburgh, PA;_ 4 _Univ. of Denver, Denver, CO_.

Background: Neuropilins (NRPs) are cell surface co-receptors known to enhance signaling from multiple ligands, including growth factors and their cognate receptors, such as HGF/MET. We have previously shown that the novel "b" isoform of NRP2 (NRP2b) is specifically associated with aggressive NSCLC and is required for TGFβ-induced EMT. We also found that NRP2b promotes AKT activation, drug resistance and metastatic spread, while the canonical NRP2a isoform inhibits these processes. However, the mechanisms supporting these differential functions remain obscure.

Results: Live cell imaging revealed that NRP2b expression led to slow internalization/rapid surface recycling of HGF-bound MET compared to NRP2a, which promoted rapid internalization and little/no recycling. Co-immunoprecipitation experiments showed that GSK3β interacted robustly with NRP2b, but only weakly with NRP2a. Our previous work demonstrated analogous differential recruitment of PTEN, but with reversed preferences; i.e., robust binding to NRP2a, but weak binding to NRP2b. Importantly, GSK3β inhibition blocked HGF-dependent migration of lung cancer cells expressing NRP2b, but not in cells expressing NRP2a. Given that PTEN is an established target of GSK3β, and that modification by GSK3β leads to its proteasome-mediated degradation, PTEN levels were examined in cells expressing individual NRP2 isoforms. PTEN was significantly reduced by expression of NRP2b, but not by NRP2a. The reduction of PTEN in the presence of NRP2b was blocked by inhibition of GSK3β, and by proteasome inhibition with MG132. Despite lower PTEN levels, phosphorylation of the GSK3β target site on Thr366 was specifically increased by NRP2b. Co-immunoprecipitation of GSK3β with NRP2b deletion mutants identified a C-terminal 15 amino acid peptide as the site of interaction. Mutants without this sequence failed to recruit GSK3β and failed to reduce PTEN levels. In silico modeling of this peptide suggested an amphipathic alpha-helical structure with a high affinity docking site (ΔG = -45 kcal) on the atomic structure of GSK3β.

Conclusion: Our results suggest that NRP2b promotes receptor signaling to AKT by differential endosomal trafficking and by recruitment of GSK3β. This kinase will phosphorylate and destabilize active PTEN recruited into the complex by NRP2a, which heterodimerizes with NRP2b. Increased AKT signaling following PTEN loss is likely responsible for the pro-tumorigenic properties of NRP2b, including resistance to targeted agents and metastatic spread. This novel mechanism for NRP2b function suggests that targeting its interaction with GSK3β might be a fruitful therapeutic strategy to reduce drug resistance and/or metastatic spread in lung cancer.

#2625

Identification of CTX-30916 as a novel antagonist of progesterone receptor signaling pathways.

Deepak Lala, Tasir Haque. _Context Therapeutics, Philadelphia, PA_.

Breast cancer is the most commonly diagnosed cancer in women and the second leading cause of cancer related death in women. Progesterone is a major mitogen in the adult human mammary epithelium that, in addition to estrogen, is a key driver of breast cancer cell proliferation. Activated PR signaling through MAPK-driven phosphorylation of the progesterone receptor (PR) is also an important driver of breast cancer stem cells. PR expression is associated with the presence of estrogen receptor-alpha (ESR1) mutations that are constitutively active and linked to resistance in the clinic in response to aromatase inhibitors, antiestrogens and CDK4/6 inhibitors. Thus, PR may play a role in tumor relapse and resistance in the clinic and blocking both estrogen and progesterone receptors would likely be an effective approach in 2L treatment for patients with advanced breast cancer. There is great interest in evaluating antiprogestins in the clinic in combination with antiestrogens or CDK4/6 inhibitors. Apristor®, an extended release formulation of onapristone, a full antagonist of PR, that has efficacy in multiple in vivo and in vitro models in combination with antiestrogestins and CDK4/6 inhibitors, is currently being evaluated in several human clinical trials.

In this study we have examined the in vitro properties of CTX-30916, a novel mono-N-desmethyl analog of onapristone. CTX-30916 binds potently to PR and acts as an antagonist in cell-based transfection assays. CTX-30916 displays selectivity against other steroid receptors in binding assays and is also a potent inhibitor of T47D breast cancer cell proliferation. Interestingly, CTX-30916 leads to a unique co-regulator binding pattern in the Microarray Assay for Real-time Co-regulator-Nuclear receptor interaction (MARCoNI) assay compared with onapristone and mifepristone indicating that the co-regulator peptide-PR interaction landscape is different between the various antiprogestins. These data suggest that CTX-30916 binding to PR leads to a distinct receptor conformation and antagonizes PR signaling pathways in cancer through a unique mechanism of action compared to other antiprogestins.

#2626

**Exquisite specificity of** PMEPA1 **isoforms in regulation of androgen signaling through AR protein degradation in prostate cancer.**

Shashwat Sharad,1 Natashia Benjamin,2 Zsófia Sztupinszki,3 Zoltan Szallasi,4 Shiv Srivastava,1 Inger L. Rosner,5 Jennifer Cullen,1 Hua Li,1 Albert Dobi1. 1 _Ctr. for Prostate Disease Research, Rockville, MD;_ 2 _University of the District of Columbia, Washington, DC;_ 3 _Technical University of Denmark, Lyngby, Denmark;_ 4 _Harvard Medical School, Boston, MA;_ 5 _Walter Reed National Military Medical Center, Bethesda, MD_.

Introduction: Prostate Transmembrene Protein, androgen Induced 1 (PMEPA1) gene was defined as an androgen and TGF-β responsive gene to inhibit androgen receptor (AR) and TGF-β signaling via negative feedback loops. Our previous studies identified two functionally distinct PMEPA1 isoforms: androgen responsive PMEPA1-b and TGF-β responsive PMEPA1-a. These two isoforms share high homology within the intracellular domains including PY1 and PY2 motifs essential for binding to E3 ubiqintin ligase, NEDD4. It is still unclear which PMEPA1 isoform with distinct N-terminus regulates AR protein stability in CaP cells.

Methods: An immunoprecipitation (IP) assay was used to study the protein-protein interactions between AR, PMEPA1 isoforms and NEDD4. HEK293 cells were used for co-transfection of the expression vectors harboring PMEPA1 isoforms, AR and NEDD4. RNA Seq data from TCGA datasets were utilized to analyze the correlation between transcript levels of both PMEPA1 isoforms and clinical outcomes including Gleason scores, overall survival and progression free survival of cancers of prostate, breast, lung and colon.

Results: Only PMEPA1-b isoform bound to wild-type and T877A mutated AR, directly. In contrast, PMEPA1 isoforms a, b and c bound to E3 ubiquintin ligase NEDD4. As expected, only PMEPA1-b bound to AR and NEDD4 for tri-complex interactome. Interestingly, the direct binding between AR and NEDD4 was not detected, which highlighted the adaptor function of PMEPA1-b in mediating proteasome-mediated AR degradation. Furthermore, the deletion of the transmembrane domain compromised binding of PMEPA1-b with AR. The TGF-β responsive PMEPA1-a was not found to bind to AR or form AR/NEDD/PMEPA1 complex. Consistently, ectopic PMEPA1-b down-regulated AR protein, inhibited AR signaling, and suppressed cell growth of LNCaP cells. But PMEPA1-a was not detected to interfere with AR signaling in CaP cells. Clinically, a higher ratio of PMEPA1-a/b was correlated with higher Gleason scores in CaP. And higher expression of PMEPA1-a and b was associated with more aggressive lung cancer and colon cancer. All of these findings highly suggest that the biological functions of PMEPA1 isoforms on TGF-β or AR signaling are tissue specific.

Conclusions: Our study highlighted PMEPA1 isoform b as an essential docking platform by interacting with AR at N-terminus and NEDD4 at C-terminus for mediating the protein degradation of AR by NEDD4. Additionally, the transmembrane domain at N-terminus of PMEPA1-b protein was essential for NEDD4 dependent AR degradation. These findings might help guide a novel anti-AR strategy in development of anti-CaP therapy. RNA seq data analysis further underscored the tissue-specificity of division of labor among PMEPA1 isoforms in tum

#2627

Inhibition of TGFβ in colorectal cancer cells is associated with a compensatory activation of AXL and p38 MAPK signaling pathways.

Davide Ciardiello,1 Pietro Paolo Vitiello,1 Nunzia Matrone,1 Valentina Belli,1 Claudia Cardone,1 Luca Poliero,1 Carola Borrelli,1 Gianluca Arrichiello,1 Giulia Martini,2 Vincenza Ciaramella,1 Giusi Barra,1 Floriana Morgillo,1 Teresa Troiani,1 Davide Melisi,3 Fortunato Ciardiello,1 Erika Martinelli1. 1 _Università degli studi della Campania "Luigi Vanvitelli", Naples, Italy;_ 2 _Vall D'Hebron institute of oncology, Barcelona, Spain;_ 3 _Università degli studi di Verona, Verona, Italy_.

According to the consensus molecular subtypes (CMS), almost 23% of human colorectal cancer (CRC) are classified in the CMS4 group, characterized by a mesenchymal signature and the activation of transforming growth factor β (TGF-β) signaling. AXL is a tyrosine kinase receptor involved in epithelial to mesenchymal transition, angiogenesis and immune modulation in CRC. We have previously demonstrated that AXL gene is amplified in approximately 5% of human CRC, and that AXL inhibition causes a significant blockade of CRC cell proliferation and migration. Here we have evaluated the role of TGF-β signaling and the potential interaction between TGF-β and AXL in human CRC cell lines. We assessed the expression and activation of TGF-β and AXL in a panel of six human CRC cell lines by western blot (WB) and real time PCR. We tested the sensitivity of HCT116 and LOVO cells to treatment with galunisertib ( LY21209761), a selective TGF-β R1 inhibitor, by MTT, cell invasion, wound healing and soft-agar colony forming assays. Further, intracellular CRC cell signaling was evaluated following galunisertib treatment by WB. TGF-β receptors 1 and 2 were expressed in all human CRC cell lines (HCT116, SW480, LOVO, LIM 1215, SW48 and CaCo2), whereas AXL expression was restricted to HCT116, SW480, LOVO cells. Treatment with galunisertib had little or no effect on cancer cell growth, whereas it partially reduced TGF-β induced cell migration, invasion and colony formation in HCT116 and LOVO cells (that co-expressed both TGF-β receptors and AXL). Interestingly, TGF-β inhibition resulted in a concomitant significant activation of AXL and p38 MAPK in both cell lines, that could represent a cancer cell adaptive mechanism of resistance to galunisertib treatment. These results suggest a potential functional cross talk between TGF-β, AXL and p38 MAPK signals in human CRC cells. In this respect, further experiments are ongoing to better understand this interaction with the aim of identifying potential novel anti-AXL and anti-TGF-β therapeutic strategies.

#2628

Non-canonical Wnt signaling in late-stage PDAC.

Payton D. Stevens,1 Adam Racette,2 Rochelle L. Tiedemann,1 Bart O. Williams1. 1 _Van Andel Research Institute, Grand Rapids, MI;_ 2 _University of Michigan, Ann Arbor, MI_.

Pancreatic cancer has a disproportionate death-to-incidence rate largely resultant from late stage diagnoses, leading to advanced cancers which have metastasized and have increased resistance to common chemotherapeutics. This malignant progression is characterized by the increase of many molecular markers, and developing novel therapeutics targeting these late stage markers is essential for improving patient prognoses. We have identified several proteins within the non-canonical Wnt pathway that are upregulated in advanced pancreatic ductal adenocarcinomas (PDAC), and we are exploring the mechanisms driving upregulation and the resulting alterations to cell signaling. Analysis of microarray data from human derived organoids (from 6 normal pancreas samples and 38 tumor samples) revealed that two receptors in the Wnt pathway, Frizzled 2 (FZD2) and Frizzled 6 (FZD6), have increased expression levels in late-stage PDAC. The temporal increase of these non-canonical, planar cell polarity altering receptors suggests a previously underappreciated role for Wnt signaling in the progression of pancreatic cancer. The increase of these two receptors in tumor tissue was confirmed through analysis of RNA-sequencing of patient samples, including 179 tumor and 171 normal samples. Interestingly, after analyzing patient overall survival and progression free survival we found that, out of the ten members of the Frizzled receptor family, higher levels of FZD2 and FZD6 result in the worst patient prognoses. Additionally, retrospective analysis of gene expression data from Moffitt et al. suggests that FZD2 and FZD6 are higher in the aggressive "basal-like" sub-category of pancreatic cancer. To begin to understand the mechanism for these changes we have used a panel of PDAC cell lines, grown in both traditional and 3-dimensional culture. We have found that decreasing levels of FZD2 and FZD6, through stable sh-RNA knockdown, results in cells which are less motile (observed via Transwell and scratch assays) and are also less capable of anchorage independent growth. Also, cell lines with increased levels of FZD2 result in cells migrating through 3D-matrix as small cell clusters rather than as single cells, which has also been observed in partial-epithelial to mesenchymal transition cell lines. Taken together, non-canonical Wnt signaling in PDAC, mediated by FZD2 and/or FZD6 receptors, results in phenotypic alterations which are consistent with those often observed during cancer progression. These changes may help explain why patients with increased expression of FZD2 and FZD6 have worse prognoses and may also warrant more exploration into specific Frizzled inhibitors.

#2629

Constitutive AhR signaling enhances androgen receptor signaling and growth in prostate cancer cells.

Maryam Ghotbaddini, Sakura McLaughlin, Kailen Turner, Devyn Pirtle, Joann B. Powell. _Clark Atlanta Univ., Atlanta, GA_.

The aryl hydrocarbon receptor (AhR) has been reported to interact with multiple signaling pathways during prostate cancer progression to castration resistant prostate cancer (CRPC) including the androgen receptor. Previously published data from our lab shows that AhR protein is overexpressed and constitutively active in castration resistant (DU145, PC3 and PC3M) prostate cancer cells. Pharmacological inhibition of AhR in these advanced prostate cancer cell lines reduced the growth rate under androgen depleted conditions. Furthermore, shRNA mediated depletion of AhR expression in castration resistant C4-2 cells, which were derived from hormone sensitive LNCaP cells, resulted in decreased expression of androgen responsive genes as well as a decreased growth rate which was comparable to LNCaP cell growth. In the present study, we evaluate cloneA (moderate increase in AhR) and CloneB (high increase in AhR) for alterations in AhR and AR signaling. AhR was overexpressed in LNCaP using PLNCX2 retrovirus vector containing AhR cDNA to determine if ectopic overexpression alone induces a castrate resistant phenotype. The highly overexpressed AhR clone (CloneB) illustrated further increase in transcriptional and promotor activity for AhR and AR compared to the moderately overexpressed AhR clone (Clone A) and control. Western blot analysis showed more AhR, AR, cSrc, and pSrc protein expression in clones compared to empty vector control. AhR overexpression was found to induce several biological properties such as migration, invasion, proliferation, and promotion of G1 to S phase during the cell cycle. Bicalutamide treatment had no effect on AR transcriptional activity in either clone, proving resistance to anti-androgen therapy. Our results confirm that overexpression of AhR induces constitutive AhR activity and stimulates androgen receptor signaling. This suggests a role for AhR in the development of CRPC.

#2630

Inhibition of bone morphogenetic protein receptor 2 increases mitochondrial permeability and the downregulation of XIAP in lung cancer cells.

Rachel E. NeMoyer, Elaine Langenfeld, John Langenfeld. _Rutgers Robert Wood Johnson, New Brunswick, NJ_.

Bone morphogenetic protein receptor (BMP) inhibitors have been shown to induce death of lung cancer cells, which involves the downregulation of x-linked inhibitor of apoptosis protein (XIAP). XIAP is a potent inhibitor of executioner caspases that promotes resistance to many chemotherapeutic agents. The mechanism by which inhibiting BMP signaling downregulates XIAP has not been elucidated. We find that the BMP inhibitors JL5 and DMH2 downregulate XIAP and enhances cell death of TRAIL, Taxol, and AEG a Smac mimetic. JL5, which inhibits both type I and type II BMP receptors, enhances cell death of cancer therapeutics by decreasing the expression of XIAP, which leads to an increase in caspase activity. The downregulation of XIAP is mediated by the inhibition of the BMP type II receptor and not the BMP type I receptors. Knockdown of BMPR2 but not the BMP type I receptors increases mitochondrial permeability resulting in the release of cytochrome c and Smac/Diablo into the cytosol. These studies suggest that the inhibition of BMPR2 increases cytosolic Smac/Diablo that inhibits XIAP enhancing apoptotic cell death of cancer therapeutics.

#2631

Effective solubilization and stabilization of functional G protein-coupled receptors.

Joanna Geddes, Christopher Wojewodzki, Barbara Kaboord, Kay Opperman. _Thermo Fisher Scientific, Rockford, IL_.

G protein-coupled receptors (GPCRs), spanning 7-transmembrane domains, are a large protein family of receptors that play a key role in transmitting signals across the cell membrane. These receptors are effective drug targets for genetic and immune system disorders and serve as potential biomarkers and therapeutic targets for cancer treatment. The challenges of stabilizing GPCRs in their native and functional form, post extraction, make studies of these receptors outside of the cell membrane extremely difficult. Most functional GPCR assays must be performed immediately following extraction of the receptor, with activity rapidly degrading over time. Here we report the development of a GPCR extraction reagent that not only efficiently solubilizes GPCRs, but also stabilizes the receptor, enabling researchers to look at structure and function of receptors outside of the cell. GPCRs solubilized with this extraction buffer can be stored for 7 days at 4⁰C and 1 month at -20⁰C with minimal loss of function. GPCR stability was verified using both overexpressed and endogenous receptors and assayed by receptor-ligand binding assays. The improved extraction efficiency, solubility and preserved activity obtained with this new reagent will give researchers greater breadth and flexibility in their GPCR research and targeted therapeutic studies.

#2632

Increased recurrence and clinical grade in renal cell carcinomas associated with novel EGFR splice variants.

George A. Coba, Saif Zaman, Jamie K. Teer, Timothy Robinson, Roger Li, Jingsong Zhang, Todd C. Knepper, Philippe E. Spiess, Wade Sexton, Matthew A. Smith, Mayer N. Fishman, Julio M. Pow-Sang, Michael A. Poch, Scott M. Gilbert, Anthony M. Magliocco, Theresa A. Boyle, Brandon J. Manley. _H. Lee Moffitt Cancer Center & Research Institute, Temple Terrace, FL_.

Background: It is well established that alterations of epidermal growth factor receptor (EGFR) are associated with the development and progression of epithelial tumors across several cancer types. Alternative splicing and alterations of EGFR splice sites can cause translational changes and EGFR alterations have historically demonstrated associations with clinical and therapeutic outcomes in lung cancer.

Objectives: To evaluate the prevalence and clinical significance of recently identified novel ΔEGFR-TKF splice variant in 102 renal cell carcinoma (RCC) tumors

Methods: The majority of the RCC samples were RNA sequenced through the Orien AVATAR™ pipeline, and 7 additional cases were analyzed through Moffitt STAR™. Moffitt STAR™ and AVATAR™ are next generation targeted sequencing assay that include both DNA and RNA analyses. Frequency tables were generated using the data collected. The Grehan-Breslow-Wilcoxon test was used to compare recurrence free survival (RFS) due to the higher weight given to survival difference at earlier time points. The rationale being that the majority of recurrence events occurred earlier than 25 months in EGFR ≥5% cases and 50 months in the EGFR <5% cases compared to the follow-up times of 75 months and 125 months in the study cohort.

Results: We identified a EGFR gene splice variant, predominantly c.2470-188_c.2470-2, between exons 20 and 21 in RCCs subjected to the sequencing assay. Of 1075 solid tumors analyzed, the presence of the EGFR gene splice variant was observed, in any amount, within 74 of 102 RCC cases (72.5%), with the only exceptions being one multiple myeloma and sarcoma case. Using an EGFR slice variant cutoff of ≥5%, 33 of 102 (32.4%) RCC were chosen to be further investigated. Upon further analysis, 32 EGFR slice variant tumors were clear cell RCC, while 1 was papillary type II RCC. Thirty cases involving ≥5% slice variants were clinically staged revealed 6 T1, 8 T2, and 16 T3/4 cases compared to tumors with <5% of the splice variants which were staged 28 T1, 10 T2, 21 T3/4, and 8 Tx (p=.011). The RFS curve trended to worse outcomes for patients with ≥5% EGFR splice variants compared to <5% EGFR splice variants (p=.0315). All variants were identified at the RNA level without obvious corresponding DNA alterations. There was an average of 106 unique reads (average of 7.7% of all reads) for these cases supporting this variant.

Conclusion: EGFR splice variants in RCC are a relatively frequent and specific molecular alteration. This novel splice variant may prove to be significant as a druggable target or as part of a kidney cancer screening protocol.

#2633

Deciphering the role of protein glycosylation in oral cancer: insights into tumor biology and treatment.

Kevin B. Chandler,1 Vanessa L. Stahl,1 Khalid Alamoud,2 Bach-Cuc Nguyen,2 Vinay Kartha,1 Khikmet Sadykov,2 Stefano Monti,1 Maria A. Kukuruzinska,2 Catherine E. Costello1. 1 _Boston University School of Medicine, Boston, MA;_ 2 _Boston University Henry M. Goldman School of Dental Medicine, Boston, MA_.

Head and neck cancer is a debilitating malignancy, with the majority of cases arising in the oral cavity as oral squamous cell carcinoma (OSCC). A major driver of OSCC is the epidermal growth factor receptor (EGFR), whose activity is aberrantly upregulated in >80% of tumors. EGFR is highly modified with N-linked glycans; fucosylation of N-glycans interferes with EGFR dimerization and activation. Thus, post-transcriptional changes may govern EGFR activity. In OSCC, EGFR signaling converges on Wnt/β-catenin activity, known to play pivotal roles in the pathobiology of this malignancy through the interaction of nuclear β-catenin with the histone acetyltransferase CREB-binding protein (CBP). We have shown that a small molecule inhibitor of β-catenin-CBP interaction, ICG-001, interferes with OSCC proliferation and aggressive features in cellular, zebrafish and murine models. Also, OSCC-cell line derived mouse tumor xenografts exhibit reduced EGFR abundance, and genomic analyses show a positive correlation between ICG-001 and EGFR inhibition. Given that modification of EGFR with N-glycans impacts its cell-surface localization and signaling, we hypothesized that ICG-001 affects EGFR N-glycosylation. To determine the effect of inhibition of β-catenin/CBP activity on cellular N-glycosylation programs, N-glycans from CAL27 and HSC3 cells treated with ICG-001 or vehicle control were released, permethylated, and analyzed via MALDI-TOF MS. Next, EGFR glycopeptides from CAL27 and HSC3 cells treated with ICG-001 or vehicle control, were analyzed with an Orbitrap Fusion™ Lumos™ Tribrid™ mass spectrometer (Thermo Scientific) using EThcD. In CAL27 cells, we observed higher levels of high mannose (less processed) N-glycans and complex fucosylated N-glycans, whereas in HSC3 cells we observed complex, afucosylated N-glycans. After ICG-001 treatment, HSC3 cells displayed higher levels of fucosylated N-glycans, suggesting that ICG-001, via inhibition of β-catenin/CBP signaling, promotes a more indolent-like glycan profile. Similarly, EGFR from CAL27 cells had highly fucosylated N-glycans, while EGFR from HSC3 cells displayed N-glycans with a paucity of fucose. Treatment of HSC3 cells with ICG-001 led to higher fucosylation, potentially inhibiting EGFR signaling. Parallel analyses of gene expression signatures in response to ICG-001 treatment in HSC-3 cells showed increased transcriptional expression of fucosyltransferases, FUT2 and FUT3. Our studies suggest that the β-catenin/CBP axis promotes EGFR signaling through downregulation of FUT2 and FUT3 expression and activity. Thus, inhibition of β-catenin/CBP signaling with ICG-001 may serve as a therapeutic approach to downregulate EGFR pro-tumorigenic activity in OSCC. Supported by NIH grants P41 GM104603 (CEC), F32 CA196157 (KBC), and by the Evans Center for Interdisciplinary Biomedical Research ARC #9950000118 (MAK).

#2634

The role of Eph A receptor 3 tyrosine kinase signaling in prostate cancer progression.

Marwah M. Al-Mathkour, Bekir Cinar. _Clark Atlanta University, Atlanta, GA_.

Dysregulation of the receptor tyrosine kinases (RTKs) by means of mutation, amplification or overexpression plays a crucial role in cell growth, cell survival, cell motility during cancer progression and metastasis. EPHA3 (erythropoietin-producing hepatocellular carcinoma cell surface type A receptor 3) is a member of the RTKs. Evidence indicates that the upregulation of the EPHA3 activity is implicated in the pathobiology of various cancers, including prostate cancer, and thus, it is a prime therapeutic target in cancer. However, the role of EPHA3 signaling in prostate cancer progression remains obscure. Currently, the development of castration-resistant prostate cancer (CRPC) poses a clinical challenge because it is lethal. The molecular mechanisms that contribute to lethal prostate cancer are largely unknown. The objective of this study is to investigate whether EPHA3 signaling plays a critical role in prostate cancer progression and therapeutic relapse. Our analysis of the prostate cancer public datasets revealed that the EPHA3 gene was amplified up to 19% of metastatic CRPC cases with the neuroendocrine phenotype. Our immunological assay confirmed the positive staining of EPHA3 protein in human prostate cancer specimens. Our semi-quantitative and quantitative PCR assays demonstrated that the levels of EPHA3 varies among established prostate cancer cell lines. Nevertheless, we consistently found that the levels of EPHA3 mRNA in CRPC cell line, C4-2, were 3-fold higher than its castration-sensitive parental LNCaP cells. Furthermore, we demonstrated that increase in expression of EPHA3 mRNA in C4-2 compared with LNCaP cells coincided with the upregulation of the EPHA3 protein, as independently confirmed by western blotting and immunofluorescence imaging. These findings indicate that EPHA3 may confer an aggressive prostate cancer cell phenotype. Because androgen receptor (AR) signaling is a potent mediator of CRPC cell growth and survival, the targeting of EPHA3 signaling alone or together with AR may improve the efficacy of current therapies for patients with advanced prostate cancer.

#2635

ROR1-CAVIN3 interaction required for caveolae-dependent endocytosis and pro-survival signaling in lung adenocarcinoma.

Tomoya Yamaguchi,1 Miyu Hayashi,2 Lisa Ida,2 Masatoshi Yamamoto,1 Can Lu,2 Taisuke Kajino,2 Jinglei Cheng,2 Masahiro Nakatochi,3 Hisanori Isomura,2 Motoshi Suzuki,2 Toyoshi Fujimoto,2 Takashi Takahashi2. 1 _Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan;_ 2 _Nagoya University Graduate School of Medicine, Nagoya, Japan;_ 3 _Nagoya University Hospital, Nagoya, Japan_.

TTF-1/NKX2-1, a homeobox-containing transcription factor indispensable for lung morphogenesis, was previously identified by four independent groups including us, as a "lineage-survival" oncogene involved in the pathogenesis of lung adenocarcinoma. We have further shown that TTF-1 induces expression of the receptor tyrosine kinase-like orphan receptor 1 (ROR1), which in turn sustains a favorable balance between pro-survival PI3K-AKT and pro-apoptotic ASK1-p38 signaling in both ROR1 kinase activity-dependent and -independent manner. It is interesting to note that while alterations in EGFR signaling are involved as a "driver" in lung adenocarcinoma pathogenesis, ROR1 appears to play a "sustainer role" for the EGFR-mediated signaling. Previously, we have also found an unanticipated function of this ROR1 receptor tyrosine kinase (RTK) as a scaffold of CAVIN1 and caveolin-1 (CAV1), two essential structural components of caveolae. This kinase-independent function of ROR1 facilitates the interactions of CAVIN1 and CAV1 at the plasma membrane, thereby preventing the lysosomal degradation of CAV1. Caveolae structures and pro-survival signaling towards AKT through multiple RTKs, including EGFR, MET, and IGF-IR are consequently sustained. Therefore, ROR1 is an attractive target for overcoming EGFT-TKI resistance due to various mechanisms such as EGFR T790M double mutation and bypass signaling from other RTKs. Caveolae function in vesicular and cholesterol trafficking, and have roles in internalization of cholera toxins and the SV40 virus, as well as glycosylphosphatidylinositol (GPI)-anchored proteins and various RTKs. Endocytosis supports various cellular functions, such as nutrient uptake, intracellular signaling, morphogenesis, and defense against pathogens, and is tightly regulated, while it also ensures an appropriate amplitude of growth factor signaling by recycling RTKs back to the cell surface or sorting them to lysosomes for degradation. Here we report that ROR1 possesses a novel scaffold function indispensable for efficient caveolae-dependent endocytosis. CAVIN3 was found to bind with ROR1 at a site distinct from sites for CAV1 and CAVIN1, a novel function required for proper CAVIN3 subcellular localization and caveolae-dependent endocytosis, but not caveolae formation itself. Furthermore, evidence of a mechanistic link between ROR1-CAVIN3 interaction and consequential caveolae trafficking, which was found to utilize a binding site distinct from those for ROR1 interactions with CAV1 and CAVIN1, with RTK-mediated pro-survival signaling towards AKT in early endosomes in lung adenocarcinoma cells was also obtained. The present findings warrant future study to enable development of novel therapeutic strategies for inhibiting the multifaceted scaffold functions of ROR1 in order to reduce the intolerable death toll from this devastating cancer.

#2636

Tumor associated MUC1 mediates TGF-β in pancreatic cancer.

Priyanka Grover,1 Ru Zhou,1 Mahboubeh Yazdanifar,1 Mohammad Ahmad,1 Angat Puri,1 Kajal Grover,2 Xinghua Shi,1 Pinku Mukherjee1. 1 _Univ. of North Carolina - Charlotte, NC;_ 2 _Univ. of North Carolina - Chapel Hill, NC_.

Pancreatic Cancer (PC) is the third leading cause of cancer-related deaths in the United States, with a five-year survival rate at 9%. Over 90% of PC is categorized as Pancreatic Ductal Adenocarcinoma (PDA). 85% of PDA overexpress tumor associated Mucin-1 (tMUC1), a membrane bound glycoprotein that is aberrantly glycosylated. Overexpression of tMUC1 in PDA plays a critical role in tumor progression and metastasis by promoting oncogenic signaling through its cytoplasmic tail (tMUC1-CT).

Transforming growth factor-β (TGF-β) plays pleiotropic roles during cancer development and progression. Within normal cells and early carcinogenesis, TGF-β functions as a tumor suppressor and induces apoptosis. This effect is mediated by activation of the canonical SMAD pathway via engagement of TGF-β Receptor 1 (TGF-βRI). However, during later stages of cancer, TGF-β becomes a tumor promoter and stimulates epithelial to mesenchymal transition (EMT), migration, and invasion thus enhancing metastasis through the non-canonical Erk1/2 pathway.

We have shown that high tMUC1 expressing PDA cells respond to TGF-β via activation of the non-canonical pathway and undergoes EMT whereas, low tMUC1 expressing PDA cells respond to TGF-β via activation of the canonical SMAD pathway and undergoes apoptosis.

tMUC1 plays a key role in regulating TGF-β's dual function in PDA. We hypothesize that signaling via tMUC1-CT enables TGF-β function to switch from a tumor suppressor to a tumor promoter.

The effects of TGF-β1 to induce apoptosis versus proliferation and invasiveness was determined in a panel of tMUC1 high and low PDA cell lines. Analysis of apoptosis and cell cycle was conducted using flow cytometry, while invasiveness was determined using the Boyden chamber assay. Proteins levels were determined using western blot. TGF-β pathway genes were studied in patient data collected from GTEx (healthy, n=171) and TCGA (PDA, n=178). It was compared using Pearson's correlation coefficient. Finally, we determined the anti-tumor efficacy of neutralizing TGF-β1 in vivo in high and low tMUC1-expressing PDA tumors.

In response to TGF-β, tMUC1 high PDA cells resisted apoptosis, enhanced invasiveness, and activated the Erk pathway, while tMUC1 low cells became apoptotic and activated the SMAD pathway. Cell cycle analysis confirms a significant increase in apoptosis in tMUC1 low PDA cells, however tMUC1 high PDA cells resist TGF-β induced apoptosis. Signal transduction was dependent upon tyrosine phosphorylation of tMUC1-CT domain by Src. RNA-Seq analysis in TCGA PDA tissue reveal eight genes that show significant correlation (low p-value OR p-value < 0.05) while it was insignificant in the GTEx healthy tissue. Finally, in mice, tMUC1-high PDA tumors responded to the neutralization of TGF-β1 by significantly reducing tumor growth, while having no effect on tMUC1-low PDA tumors. tMUC1 expression levels in PDA may serve as a marker for eligibility for potential anti-TGF-β therapies.

#2637

**Novel targeted therapy-resistant** FGFR3 **splice variant increases oncogenic phenotype in African American prostate cancer.**

Jacqueline Moy, Bi-Dar Wang, Norman H. Lee. _George Washington Univ., Washington, DC_.

Introduction: In this study, we investigate the role of alternative splicing in the receptor tyrosine kinase family on prostate cancer health disparities in African American men.

Background: Prostate cancer (PCa) is the most diagnosed cancer in men and the second leading cause of male-cancer related deaths in the U.S. Dramatic ethnic disparities have been observed in PCa patients, as African American (AA) men are 60% more likely to be diagnosed with PCa and have a 2.4 fold higher mortality rate compared to European American (EA) men. Increasing evidence suggests that, after accounting for epidemiological factors, a remaining component of this disparity is due to intrinsic genetic and biological factors. Interestingly, exon array data from our lab suggest that differential alternative splicing (dAS) may be occurring in AA PCa. We hypothesize that dAS involving exon 14 of FGFR3 is generating a shorter, more oncogenic variant in AA PCa, which is absent or weakly expressed in EA PCa. Differential splicing of FGFR3 in AA patients may be mechanisms contributing to AA PCa health disparities.

Results: Cloning confirmed the presence of the FGFR3-L variant (containing exon 14) and the FGFR3-S variant (without exon 14) from EA and AA PCa cell lines, respectively. RNA-seq data analysis suggests decreased survival of PCa patients with high FGFR3-S/-L expression ratios. EA cell lines overexpressing FGFR3-S show increased small molecule inhibitor (SMI) TKI258-mediated resistance to proliferation, invasion, and apoptosis in vitro. Additionally, mice injected with cells overexpressing FGFR3-S have increased tumor growth, metastasis, and resistance to TKI258 treatment.

Conclusions: We have identified an oncogene of interest, FGFR3, which undergoes exon skipping specific to AA PCa. We have successfully cloned this novel FGFR3 variant in cell lines and created stable cell lines overexpressing both short and long variants. In vitro and in vivo studies suggest FGFR3-S is more resistant to SMIs. Thus, dAS in FGFR3 may be one mechanism contributing to the increased aggressiveness of PCa in AA patients.

#2638

Galectin-3 activates the receptor tyrosine kinase Tyro3 and stimulates ERK signaling, cell survival and migration in human head and neck cancer cells.

Sassan Hafizi, Nour Al Kafri. _Univ. of Portsmouth, Portsmouth, United Kingdom_.

The beta-galactoside-binding protein galectin-3 (Gal-3) has been shown to influence the tumorigenic behaviour of cancer cells, and elevated serum levels of Gal-3 have been linked to tumor metastasis in several different cancers. Previously, Gal-3 was shown to be a ligand MerTK, a member of the TAM (Tyro3, Axl, MerTK) subfamily of receptor tyrosine kinases (RTKs) - these RTKs are implicated in tumorigenesis and/or enabling tumor chemoresistance in many different cancers. However, it is not known whether Gal-3 is also a ligand for the other two TAM RTKs, nor what signaling pathways it activates to regulate cancer cell functions. This study is aimed at determining the ligand properties of Gal-3 against Tyro3 and Axl RTKs in human cancer cells and the oncogenic signal pathways linked to them. The cultured human head and neck squamous cell carcinoma (HNSCC) cell line SCC-25 was determined by western blot and qPCR to express both Axl and Tyro3, but not MerTK. Recombinant Gal-3 and the TAM ligands Gas6 and ProS1 were added to serum-starved cells over a time-period of minutes and cells lysed to determine activation/phosphorylation of RTKs and intracellular signal mediators by western blotting. Longer incubations were performed to measure cell viability (MTS assay) and cell migration rate (scratch assay). All experiments were carried out a minimum three times. The known TAM ligands Gas6 and ProS1 stimulated TAM RTKs differentially in SCC-25 cells: Gas6 activated Axl strongly within minutes, with ProS1 having a weak effect. In contrast, ProS1 was the strongest ligand for Tyro3, activating it by fourfold, followed by Gal-3 at threefold and Gas6 at twofold. All three exogenous proteins also rapidly stimulated ERK1/2 phosphorylation by two-fold, whereas only Gas6 and Gal-3 stimulated Akt phosphorylation. All three proteins significantly maintained cell survival during acute induction of apoptosis by staurosporine. However, only Gas6 conferred a survival effect over long-term serum starvation-induced cell death. In addition, Gal-3 significantly stimulated cell migration during Axl blockade with the small molecule Axl inhibitor BGB324, further suggesting a role for Tyro3 in this effect. These results have shown that Gal-3 is a ligand for Tyro3 RTK, and that Gal-3 promotes cell survival and migration in HNSCC cells, coupled with activation of both the related signal pathways ERK and Akt. The data therefore reveal a new mechanism of Tyro3 activation; however, it remains to be determined whether Gal-3 interacts with the glycosyl chain on the RTK or else via other interfaces. Finally, cancer therapeutic strategies targeting Gal-3 action and/or cell surface glycosylation may be viable approaches in further research.

#2639

Insulin receptor alternative splicing during development and cancer.

Safiya Khurshid. _Nationwide Children's Hospital, Columbus, OH_.

It has been shown that alternative splicing of some genes changes during development from embryogenesis to adulthood. Intriguingly, transformation of cells is also often accompanied by a reversion to the embryonic spliced isoforms. Insulin receptor (IN-R) undergoes alternative splicing to produce two isoforms: the full-length IN-RB isoform and the exon 11-skipped IN-RA isoform. The IN-RB isoform encodes a receptor with high affinity for insulin, while the IN-RA isoform has high affinity for both insulin & IGF2 growth hormones. IN-RAexploits the IGF pathway to accelerate growth, proliferation and angiogenesis. It is known that during embryogenesis the IN-R gets spliced to primarily express the INR-A form. Recently, the A isoform has has also been shown to be expressed in certain cancer types. Because the insulin receptor signaling converges on the mTOR/AKTpathway that is one of the most critical pathways for organismal development as well in cancer, it is important to understand the expression of these isoforms and their role in signaling and cellular fate. However, a tissue and a temporal description of the expression of these two isoforms is lacking. Towards this end we have shown that the mouse insulin receptor gene is spliced similarly to the human gene and have used the mouse model to study IR splicing during development. We harvested tissues from mice at different developmental stages (E10, E18, P1, P7, 3 months and 6 months) and performed RT-PCR for the insulin receptor gene. Our data confirmed that the whole embryos at embryonic day 10 (E10) express mostly IN-RA (90%). However, as the embryo develops and we can obtain discrete tissues we are able to detect tissue-specific profiles of IN-Rsplicing. For example, in bone at E18 as well as postnatal day P1 and 7, IN-RBis highly expressed (65%) and decreases to (40%) as the mouse reaches 6 months of age. Likewise, the liver and pancreas, which are primarily involved with metabolic functions, express mostly IN-RB(90%) from postnatal day P1 and into adult hood (6 months). In contrast, when we analyzed IR-splicing in human cancers, we find that in both liver cancer and cancers of the bone (osteosarcoma and Ewing's sarcoma) there is a shift toward increased levels of the pro-proliferative isoform, IN-RA. We believe that understanding the molecular underpinnings of the splicing regulation during development will provide us with knowledge that will help us better understand the transformation process and how to control it, possibly through splicing corrective therapy.

#2640

DKK1 overexpression might induce crizotinib resistance.

Ha Young Seo. _Seoul National University, Seoul, Republic of Korea_.

NSCLC patients with pleural effusions tend to show progressive status and malignant features. Newly established lung cancer cell lines from pleural effusions, SNU-2550 and SNU-2563 were harboring ALK rearrangement and they were originally sensitive to crizotinib. After treated with regular crizotinib treatment and recovery at least 1 year, crizotinib-resistant cell lines (SNU-2550CR and SNU-2563CR) that became resistant to crizotinib more than 5 times were established. And then, whole exome sequencing (WES) and RNA-seq were operated in order to understand way to acquire resistance. According to WES result, there were differences in mutations between parental cell lines and resistant cell lines. For example, mutations in MKNK2, IGFN1, PRSS1 and FOXA2 genes were only present in SNU-2550CR not SNU-2550. Variations of ESX1, PRSS1, PCDHA4 and AGAP3 genes were observed in only SNU-2563CR. When the differences were studied and visualized via SPIA two-way evidence plot and heatmap, genes related with cell cycle, p53 and Wnt pathway were commonly changed in RNA expression level. In fact, there were expressional differences in genes related those pathways such as MAPK2, DKK1, APC and GSK3b genes. Among them, Dkk1 expression level was more than doubled in two set. Through those analysis, we examined differences between parental cell lines that have sensitive reactivity and resistant cell lines that acquired resistance. When crizotinib resistance was induced, Dkk1 expression was elevated. Augmentation of Dkk1 expression level induces the level of overall β-catenin expression, and cytoplasmic β-catenin tends to be concentrated adjacent to membrane and into nucleus region. Therefore, Dkk1 can be considered as a factor that is related to crizotinib resistance by up-regulating Wnt/β-catenin pathway in lung cancer. Acquired resistance to crizotinib is major predicament especially when there is ALK rearrangement. Studying the mechanism of crizotinib resistance therefore improves the prognosis of ALK rearrangement lung cancer. We found that Dkk1 overexpression might be correlated to crizotinib resistance.

#2641

**Transactivation of EGFR and mTOR pathway by H** 2 **Sin cancer cells.**

Jillian L. Higgins,1 Aileen K. Kraus,1 Dominic T. Arruda,1 Hao Guo,2 Mattew Lautato,1 Christopher Lautato,1 Eliana DaCunha,1 Ashley Chen,3 Ricky Yao,4 Dongqin Yang,5 Yinsheng Wan1. 1 _Providence College, Providence, RI;_ 2 _China Medical University, Shenyang, China;_ 3 _University High School, Tucson, AZ;_ 4 _University of Virginia, Charlottesville, VA;_ 5 _Brown University Medical School, Providence, RI_.

Hydrogen sulfide regulates a variety of physiological and pathological processes. Previous studies have suggested that H2S plays a significant role as a secondary messenger in cellular signaling. Interestingly, it has been found that this unique gaseous molecule, or gasotransmitter, is capable of producing both pro- and anti- apoptotic activity in cultured cells. It has been speculated that these gasotransmitters are endogenously generated in mammalian systems through enzymatic catalysis. Our pursuits focus on comparing the effects of H2S on various lines of cancer cells (CaOV3-ovarian cancer cells, A375-melanoma cells, HeLa-cervical cancer cells, and WM 266-4-melanoma cells). Using a water-soluble, slow-releasing H2S donor, GYY4137 (200 μM), we studied how both concentration and time affect cell viability and investigated further whether H2S causes pro- or anti- apoptotic activity. Existing data have indicated that GYY4137 may activate molecular targets, including EGFR, ERK and JNK. We hypothesize that GYY4137 activates mTOR (mammalian target of rapamycin) with result of phosphorylation of S6, leading to either cell death or cell proliferation. By monitoring various proteins through Western blot analysis and confocal microscopy we studied the activation of EGFR/mTOR pathway in response to GYY4137 treatment. Our data showed that the longer the treatment the more prevalent phosphor S6 and phosphor-EGFR are. Noticeably, we observed a significant difference between 15 minutes and 30 minutes, suggesting that GYY4137 has the largest effect after 30 minutes of treatment. We further studied cell growth and viability using MTT assay to investigate the effects of various concentrations of GYY4137 on the cells. Our data indicated that the stronger the concentration is, the lower the cell viability. Similarly, we used Mitotracker ® Red dye to stain for mitochondrial activity in CaOV3 and WM 266-4 cells treated at various time points. Overall, mitochondrial activity increased when cells were exposed to GYY4137, suggesting that H2S may cause anti-apoptotic activity, and instead favor cell growth at the concentration we used. Collectively, our data suggests that the H2S donor, GYY4137 has a significant effect on cell proliferation and survival in several cancerous cells likely mediated by transactivation of EGFR and mTOR pathways.

#2642

Antisense oligonucleotide-based induction of dominant-negative variants of STAT3 and EGFR in breast and lung cancer cells.

Prasad Subramaniam, Luca Cartegni. _Rutgers, The State Univ. of New Jersey, Piscataway, NJ_.

The signal transducer and activator of transcription 3 (STAT3) is linked to multiple cancers, including breast and lung adenocarcinomas, where it is known to activate multiple oncogenic pathways and serve as a bypass mechanism in drug resistance. For example, STAT3 is known to be a driver in some basal-like Triple-negative breast cancers (TNBC) and a resistance mechanism to EGFR inhibitors in non-small cell lung cancer (NSCLC). Thus STAT3 has become an attractive target in cancer therapeutics. A naturally occurring alternative splicing variant, STAT3-beta, is truncated and lacks the C-terminal trans-activation domain. Stat3-beta can act in a dominant negative fashion. We recently demonstrated that splice-switching oligonucleotides (SSOs) can be used to induce a shift in endogenous expression from the abundant, oncogenic STAT3-alpha to the truncated dominant-negative STAT3-beta isoform. This leads to decreased cell viability in triple-negative breast cancer cells and to tumor regression in mice, as compared to full STAT3 knock-down or controls. Our aim was to test a panel of next-generation SSOs in order to evaluate their efficacy in basal-like breast cancer cells, which are typically refractory to tyrosine kinase inhibitors. Based on our previous studies, we generated a panel of optimized SSOs targeting STAT3, and assayed the extent of the STAT3 alpha-to-beta switch, at the RNA and protein level, and at their efficacy at eliciting cell apoptosis in MDA-MB-468 TNBC cells, and identified one which show a 5-10 fold improvement of activity in vitro, compared to our previous leading published compound. The optimized STAT3 SSO sequence was also tested in RTK inhibitor-resistant H1975 non-small cell lung cancer cells and was found to be effective in killing the cancer cells as compared to Stat3 knockdown. We also used a similar antisense approach to induce the expression of a dominant-negative variant of the EGFR and tested in combination with our STAT3 SSO. While both the SSO-induced dominant negative variants of EGFR or STAT3 were able to effectively kill lung cancer cells individually, we observed a marked synergistic increase in inhibition when the two compounds where used in combination.

#2643

TNFAIP2 interacts with EGFR to modulate EGF-induced EGFR auto-phosphorylation and internalization and sequential ERK1/2 activation in nasopharyngeal carcinoma cells.

Hao-Ping Liu,1 Chih-Ching Wu2. 1 _National Chung Hsing University, Taichung, Taiwan;_ 2 _Chang Gung University, Taoyuan, Taiwan_.

TNFAIP2 (TNF-α-inducible protein 2) is identified as an angiogenic and proinflammatory factor and is essential for formation of tunneling nanotubes connecting between remote cells. Cancer-associated TNFAIP2 overexpression is first demonstrated in nasopharyngeal carcinoma (NPC) and is significantly correlated with poor survival of patients. We previously revealed that TNFAIP2 is associated with actin filaments, inducing actin remodeling and membrane protrusion and promotes cell motility of NPC cells. To elucidate functional roles of TNFAIP2 in cancer progression, we exploited co-immunoprecipitation (co-IP) coupled with mass spectrometry (MS)-based proteomic analysis to identify interacting proteins of TNFAIP2 in NPC cells. Epidermal growth factor (EGF) receptor (EGFR) is one of the identified candidates and is frequently overexpressed in various cancer types. We validated the interaction between TNFAIP2 and EGFR in two types of NPC cell lines; nevertheless, the interaction of TNFAIP2 with EGFR was reduced along with EGF stimulation. Reciprocal domain mapping analyses and immunofluorescence staining further demonstrated that the C-terminal amino acid residues 461-520 of TNFAIP2, which are dispensable for its association with the plasma membrane, are required for TNFAIP2 interaction with EGFR. On the other hand, the intracellular juxtamembrane domain (amino acids 645-689) and kinase domain (amino acids 690-945) of EGFR are needed for its interaction with TNFAIP2. Furthermore, EGF-triggered internalization of EGFR was significantly interfered in TNFAIP2-knockout NPC cells generated using the CRISPR-Cas9 system, accompanied by alleviated auto-phosphorylation of EGFR on the 1068 and 1173 tyrosine residues and subsequent activation of downstream ERK1/2. This impairment of liganded EGFR-transduced signaling in TNFAIP2-knockout NPC cells was correlated with a decrease in cell migration as revealed by transwell migration assays. These data collectively reveal a novel role of TNFAIP2 in modulation of EGF-triggered EGFR activation and sequential signaling transduction through protein-protein interactions. Efforts are made to pursue if the aforementioned events involve modulation of EGFR oligomerization or recruitment of internalized activated EGFR into endosomes. The results could shed light on how TNFAIP2 promotes cancer progression via coordinating with EGFR and the impact of TNFAIP2 on EGFR-targeted cancer therapy.

#2644

Histone deacetylase 6 plays a critical role in sulfiredoxin-mediated activation of the epidermal growth factor receptor signaling in human colorectal cancer.

Qi Ying, Hong Jiang, B. Mark Evers, Qiou Wei. _University of Kentucky College of Medicine, Lexington, KY_.

Mammalian cells develop various transmembrane receptors including the family of receptor tyrosine kinases (RTKs) to respond to extracellular stimuli. Epidermal growth factor receptor (EGFR) is the first identified member of the RTKs and it plays a pivotal role in colorectal cancer by stimulating cell proliferation and survival. Binding of extracellular ligands, mainly EGF, leads to the dimerization of EGFR that triggers the activation of downstream mitogen activated protein kinases. In addition to the amount of ligand binding, the strength and duration of EGFR signaling are also affected by the posttranslational modifications of the receptor itself, such as phosphorylation, ubiquitination, acetylation, etc. In the previous study, we demonstrated that Sulfiredoxin (Srx) enhances the EGFR signaling in colorectal cancer cells through the inhibition of receptor acetylation at lysine residue 1061. However, the molecular mechanism by which Srx regulates the level of EGFR acetylation is still unclear. In this study, we investigated (1) the mechanism of EGFR acetylation and its regulation by Srx; (2) the functional consequence of EGFR acetylation on EGF-induced receptor dimerization, recycling, trafficking and degradation. Human colon cancer cell lines were cultured and maintained in standard conditions. Western blotting, immunoprecipitation and Mass spectrometry were used to identify cysteine oxidation of HDAC6 and characterize enzymes that are involved in the acetylation of EGFR. Stable cells that overexpress or loss of target genes, such as Srx, HDAC6, EGFR and/or mutants, were established using the 3rd generation lentiviral ShRNA infection or the CRISPR-Cas9 system. The activity of histone deacetylases was determined using commercial kit in the presence/absence of pan- or isoform-specific chemical inhibitors of HDACs. Upon EGF stimulation, the dimerization, recycling, trafficking and degradation of EGFR were demonstrated through a combination of immunoblotting, flow-cytometry and immunofluorescent imaging. We demonstrated that HDAC6 plays a central role in the regulation of EGFR acetylation through its interaction with EGFR. Depletion of Srx in human colon cancer cells increases the levels of intracellular reactive oxygen species, leading to inhibition of HDAC6 activity through regulating the redox state of cysteines 407/417. Depletion of HDAC6 not only leads to increased level of EGFR acetylation but also suppresses the binding between EGFR and its adaptor proteins. EGF-induced receptor dimerization and recycling are altered in HDAC6 knockout cells, result in inhibition of EGFR downstream signaling. Taken together, this study indicates that Srx enhances the strength and duration of EGF signaling through the ROS-HDAC6-EGFR axis in human colon cancer cells.

#2645

Quantitative measurement of multiple signal transduction pathway activities in cell and tissue culture, including cancer, fibroblast, and immune cell types.

Anja Van De Stolpe, Marcia Alves de Inda, Eveline den Biezen-Timmermans, Laurent Holtzer, Henk van Ooijen, Wim Verhaegh. _Philips Research, Eindhoven, Netherlands_.

To improve pathophysiology research, biomarker discovery and drug development, cell culture models should adequately mimic human (patho)physiology and provide reproducible results. This requires comparison between cultured cells/tissue and actual histopathology in the patient, as well as standardization of culture experiments to ensure experimental reproducibility, preferably in a quantitative manner1. 10-15 signal transduction pathways govern major cellular processes, e.g. cell division, differentiation and migration. The past decade we developed tests to quantitatively measure functional activity of signal transduction pathways in individual cell/tissue samples, based on Bayesian computational model inference of pathway activity from measurements of mRNA levels of target genes of the transcription factor associated with the respective signalling pathway. Tests provide quantitative pathway activity scores and are intended to be used for diagnostics and life sciences research2-4.

Method: Tests have been developed for androgen (AR) and estrogen receptor (ER), Hedgehog (HH), Wnt, TGFβ, Notch, NFκB, PI3K, JAK-STAT 1/2 and 3, and MAPK pathways. After calibration and freezing of the models, extensive biological test validation was performed on healthy/diseased cell and tissue types, including multiple cancer, fibroblast and immune cell types. Using Affymetrix expression microarray data (GEO database) >900 cell lines of most cell and cancer types were analyzed, as well as primary cultures of most immune cell types. LnCaP (prostate), MCF7, BT474 (breast), HCC827 (lung) and A2780 (ovarian) cancer cell lines were compared across laboratories.

Results: Typical expected single or combined pathway activities were confirmed, e.g. ER activity in breast, AR activity in prostate, and Wnt activity in colon cancer; HH activity in soft tissue tumor, NFκB activity in lymphoma, frequently combined with PI3K and/or MAPK and/or JAK-STAT growth factor pathways. Quantitative pathway activities were reproducible within studies, but highly variable between labs, and dependent on culture conditions.

Conclusion: Our pathway tests measure signaling pathway activity in many cell and tissue types, and can be used as quantitative readout for cell/tissue culture. Applications are: standardization of cell/tissue culture to ensure reproducibility; comparison between culture-based disease model and patient histopathology; quantitative assessment of drug efficacy on disease models; assessment of toxicity on healthy cell/tissue models. A number of tests have been adapted to qPCR, enabling use on FFPE tissue and small samples. 1 Ben-David U, et al. Nature, 2018;560(7718):325; 2 Verhaegh W, et al. Cancer Res 2014;74(11):2936-45; 3 Verhaegh W, Stolpe A van de. Oncotarget, 2014:5(14):5196-7; 4 Ooijen H. van, et al. Am J Pathol 2018;188(9):1956-1972

#2646

**Proteomic approaches to study cetuximab resistance in** RAS/BRAF **wild type colorectal cancer.**

Alexandros Georgiou,1 Adam Stewart,2 George Vlachogiannis,2 Lisa Pickard,2 Nicola Valeri,1 David Cunningham,3 Steven R. Whittaker,2 Udai Banerji1. 1 _The Institute of Cancer Research/ The Royal Marsden NHS Foundation Trust, Sutton, United Kingdom;_ 2 _The Institute of Cancer Research, Sutton, United Kingdom;_ 3 _The Royal Marsden NHS Foundation Trust, Sutton, United Kingdom_.

Background: Cetuximab is a licensed anti-EGFR antibody treatment for RAS/BRAF wild type (WT) metastatic colorectal cancer (CRC) but is only effective in a proportion of these patients. Established mechanisms of resistance include primary and secondary activating RAS mutations. However, these cannot explain a proportion of resistant cases.

We investigated the differences in early adaptive signaling in cetuximab sensitive and resistant cells using phospho(p)-proteomics in order to elucidate p-proteomic signatures of response, understand resistance mechanisms and suggest future therapeutic strategies.

Methods: We used a panel of 12 cell lines (6 cetuximab sensitive and 6 resistant cell lines), cells isolated from CRC patients' ascites (n=4) and patient derived organoids (n=2). All CRC samples used were RAS/BRAF WT. Following 1 and 4h exposure to cetuximab, a multiplex antibody-based platform was used to simultaneously quantify changes in 60 p-proteins. Key results were validated with Western blotting. Proliferation assays and 2-week colony assays were used to test potential cetuximab combinations. Statistical analysis included logistic regression, T tests and Spearman's correlation

Results: Following 1 and 4h exposure in sensitive cell lines there was downregulation of pHER2 and other intracellular effector p-proteins that function within MAPK, PI3K and JAK-STAT pathways.

In contrast, in resistant cell lines at 1h there was a significantly lower degree of pHER2 downregulation and upregulation of pHER3 (p<0.05). Following 4h exposure, despite comparable pEGFR inhibition, in resistant cells there was statistically significant lack of downregulation/upregulation of known effector p-proteins of the PI3K pathway and also upregulation of other phosphorylated receptor tyrosine kinase (RTKs). When compared to sensitive cell lines, in resistant cells there were also statistically significant increase of pPDGFRα, pTIE2 and pIRS1. Other tyrosine kinases: pVEGFR, pFGFR1 and pSRC were also upregulated in ≥50% of resistant cell lines.

The upregulation/lack of downregulation of the PI3K pathway and upregulation of other pRTKs were confirmed in cetuximab resistant patient derived cells and organoid cultures.

We then investigated the combination of cetuximab with: PI3K inhibitor (i), AKTi, mTORi and IGF1Ri. The combination of cetuximab with PI3Ki pictilisib was able to sensitize to cetuximab in the resistant cell lines and organoid cultures with an effect that ranged from additive to synergistic.

Conclusion: RAS/BRAF WT sensitive and resistant CRC cells have distinct p-proteomic signatures following 1 and 4h cetuximab exposure. In resistant cells, early adaptive signaling involves upregulation of RTKs and the PI3K pathway. The combination of cetuximab and pictilisib showed a modest benefit, however, overlapping toxicity may render this difficult to deliver clinically.

### Targeting Hypoxia and Endoplasmic Reticulum Stress

#2647

MUC13 interacts with SOX2 leading to cell stemness and Anoikis resistance in colorectal cancer.

Kyle Doxtater, Chidi Zacheaus, Fatemeh Keramatnia, Mehdi Chaib, Manish Triparthi, Subhash Chauhan. _University of Tennessee Health Science Center, Memphis, TN_.

Colorectal Cancer(CRC) is one of the leading cause of cancer related death in the United States today in both men and women. With an expected 50,260 deaths in 2017 it is the second leading cause of cancer related deaths among men and the third among women in the United states. Often in colorectal cancer it is not the primary tumor that is the killer, but it is the metastatic sites that develop in the later stages of the disease that is the true killer. About 90% of all cancer related deaths are due to the development of metastatic sites in the body. What is metastasis? Metastasis is the transfer of disease from the primary tumor site to another unconnected site within the body through a process called the metastatic cascade. Metastasis develops slowly over time, due to it being an inefficient process having to overcome several natural defenses within the body. One of these defenses is Anoikis, cell death due to lack of attachment to the extracellular matrix. The development of Anoikis resistant is key in the process of metastasis, because of the need of the cells to detach and circulate the body before settling at a new site within the body. The inhibition of Anoikis resistance development in CRC could lead to improve patient outcome and the failure of metastasis to develop in the lifetime of the disease in the patient. Thus, characterizing and developing methods of inhibition of Anoikis resistance could lead to the decrease in cancer related deaths in the United States as well as finding effect therapeutic options. One of the key characteristics of Anoikis resistance in cells is the develop of stem cell like characteristics, and expression of key stemness factors of Sox2, Oct4, and NANOG. The expression of these factors allows for the cells to overcome their need to be attached to the ECM and travel through the blood stream. We have found through analysis that the membrane bound muicin MUC13 interacts with Sox2 increasing cell stemness leading to the development of anoikis resistance in Colorectal Cancer. Cell cycle analysis of Sw620 cells with high endogenous MUC13 expression showed an 80% less cell death when compare to isogenic Sw480 cells after 48 hours. Western blot anaylsis of the time points 0,24,36, and 48 hrs showed an increase in Sox2 expression and MUC13 expression in Sw620 cells during 24 and 36hrs. To further understand this relationship MUC13 knockdown and overexpressing cells lines will be analyzed to see if any there is a change in the Anoikis phenotype of Sw620 and Sw480 cells. This will also be done with Sox2 knockdown and overexpression. We will also test different therapeutic options, specifically a novel non-viral delivery of MUC13 Crispr Cas9 plasmid to see it effect on Anoikis resistances.

#2648

Hypoxia-induced NADPH oxidase 5 (NOX5) promotes tumor growth of head and neck squamous cell carcinoma through HIF-2á signaling.

Siqi Chen, Wei Gao, Jimmy Yu-Wai Chan, Thian-Sze Wong. _The University of Hong Kong, Hong Kong, Hong Kong_.

Background: Head and neck squamous cell carcinoma (HNSCC) has an increased content of reactive oxygen species (ROS). However, clinical trials of antioxidant resulted in no benefit towards HNSCC anti-cancer treatment. NADPH oxidase (NOX) family is found to be the major generator of ROS which might contribute to the failure of antioxidant therapy. For the past 10 years, NOX family have been found with a range of functions in different cancers. Of which, NOX5 is the least well-understood one with limited details of signaling roles.

Purpose of the study: To investigate the role of NOX5 in HNSCC.

Experimental procedures: Online databases were used to analyze the expression of NOX5 in HNSCC patients. Immunohistochemistry was performed to detect the NOX5 expression level and its co-expression with hypoxia-inducible factors (HIFs) in paraffin-embedded tumor tissues surgically resected from HNSCC patients in Hong Kong. Subcutaneous xenograft model was established using NOX5-silenced HNSCC cells to evaluate the tumor growth potential in vivo. Sulforhodamine B assay and colony formation assay were used to measure the proliferation potential of NOX5-silenced HNSCC cells in vitro. Hypoxia chamber was applied to mimic the hypoxic condition of tumor tissue in vitro. Flow cytometry with Dihydroethidium staining was performed to measure the production of superoxide when silencing or overexpressing NOX5. Superoxide scavenger TEMPOL was applied to test the signaling role of superoxide generated by NOX5. Quantitative PCR and western blot were used to measure the mRNA and protein expression level of NOX5 and HIFs in HNSCC cell lines.

Results: Relative mRNA expression of NOX5 was found significantly increased in HNSCC tumor tissues comparing to normal tissues both in Oncomine and TCGA datasets. Interestingly, NOX5 expression was found higher in central areas of tumor nests with the co-expression of HIF-1α and HIF-2α in our HNSCC patients' samples. Further, NOX5 was upregulated in HNSCC cell lines under hypoxic condition (1% oxygen). Knocking-down of NOX5 impaired HNSCC cell growth both in vivo and in vitro, and this reduction was more significant under hypoxic condition in vitro. Loss of NOX5 resulted in superoxide reduction and HIF-2α impairment but not HIF-1α. Overexpressed NOX5 induced superoxide production and HIF-2α expression, and this induction was attenuated by TEMPOL.

Conclusions: Hypoxia-induced NOX5 promotes HNSCC tumor growth by regulating HIF-2α via generating superoxide. NOX5 might serve as a novel therapeutic target for treatment of HNSCC.

#2649

A novel approach to fate-map hypoxic cells during tumor progression uncovers metastatic potency of post-hypoxic cells.

Inês Godet, Yu Jung Shin, Julia A. Ju, Soumitra Bhoyar, I Chae Ye, Guannan Wang, Saraswati Sukumar, Daniele M. Gilkes. _Johns Hopkins University, Baltimore, MD_.

Hypoxia occurs in 90% of solid tumors due to inefficient vasculature and has been demonstrated to promote a more aggressive phenotype, correlating with worse patient prognosis. Current methods to measure hypoxia in human tumors only identify cells that are hypoxic at the time the tissue is collected and do not delineate cells that were transiently exposed to hypoxia but survived and adapted. To overcome this challenge, we generated a Lox-Cre dual-vector oxygen-dependent reporter system. Under hypoxia, Cre expression leads to the cleavage of a DsRed reporter and permanent expression of a GFP reporter. We stably transduced breast cancer cell lines and demonstrated an irreversible reporter-switch that occurs when cells are exposed to 0.5% O2, the typical O2 level in human breast tumors, but does not occur under physiological conditions (8% O2). To explore the utility of our system, we embedded spheroids derived from MDA-MB-231 reporter cells in collagen and cultured them under 20% O2 conditions. A defined hypoxic core (GFP+) was visible after 15 days in culture and it correlated with hypoxic O2 measurements. We then generated orthotopic breast tumors by transplanting MDA-MB-231 reporter cells into the mammary fat pad of mice. Hypoxic cells (GFP+) were localized in the peri-necrotic region of the tumor and co-localized with hypoxyprobe and HIF1α labeling. RNA sequencing of GFP+ compared to DsRed+ tumor cells demonstrated that GFP+ cells were enriched for the hallmark signature of hypoxia. Additionally, we developed a transgenic mouse expressing the hypoxia-driven construct that was bred to a tdTomato-floxed GFP mouse. A female double fluorescent/hypoxia-reporter mouse was then bred to a male FVB/N-Tg(MMTV-PyMT) mouse in order to generate triple-transgenic mice that form breast tumors that spontaneously metastasize. We systematically studied hypoxia in spontaneous breast tumors from the development of ductal hyperplasia to late stage invasive carcinoma. Tumor hypoxia was first detected in early stage ductal carcinoma in situ (DCIS) lesions. Similarly to the spheroid model, mammary organoids derived from triple-transgenic mice tumors cultured under 20% O2 conditions also exhibited a defined hypoxic core after 15 days in culture. Both orthotopic and transgenic mouse models revealed an increased potency of post-hypoxic cells at arriving to distant sites in early stage metastasis. We utilized RNA sequencing to identify potential mechanisms of enhanced metastatic capacity. Ultimately, our system allowed the fate-map of hypoxic cells during tumor progression to metastasis. We show for the first time that 3D in vitro organoids derived from PyMT tumors develop regions of hypoxia under normal tissue culture conditions. In vivo, we show that hypoxic cells are more potent at arriving at distant sites and that they retain a different gene expression that potentially confers a metastatic advantage.

#2650

Characterizing CHCHD2 subcellular localization in response to hypoxia and redox perturbations in glioblastoma cells.

Jan C. Lumibao, H. Rex Gaskins. _University of Illinois at Urbana-Champaign, Urbana, IL_.

BACKGROUND: Glioblastoma (GBM) is the most common and malignant form of primary brain tumor in adults and remains incurable. Inter-organelle communication between mitochondria and nuclei is a potential compensatory signaling pathway that may contribute to therapeutic resistance and cellular plasticity in the face of a hypoxic tumor microenvironment. Coiled-coil-helix-coiled-coil-helix domain-containing protein 2 (CHCHD2) is a protein that colocalizes to the mitochondrial inter-membrane space and the nucleus, where it exhibits hypoxia-sensitive transcription factor capabilities. We demonstrated previously that CHCHD2 knockout in hypoxic U87 GBM cells harboring epidermal growth factor receptor (EGFR) variant III (U87vIII) abrogated the increase in proliferation and invasion exhibited by U87vIII WT cells expressing CHCHD2. The C-terminus of CHCHD2 contains redox-sensitive cysteines amenable to thiol/disulfide formation, which determine protein folding. The objective of this study was to characterize the effect of hypoxia and cellular redox status on CHCHD2 subcellular localization. We hypothesized that reducing the C-terminal CX9C motif would induce CHCHD2 mitochondrial export and nuclear accumulation.

METHODS: U87 and U87vIII cells were incubated in standard culture oxygen conditions (~21% O2), 7% O2, 4% O2, and pathophysiological hypoxia (1% O2). Confocal immunofluorescence microscopy was used to observe CHCHD2 subcellular localization, and nuclear fluorescence intensity was quantified using Axiovision software. Western blot of whole cell lysates was used to quantify CHCHD2 protein levels in response to decreasing oxygen tensions. Chemical reduction of cells was achieved using dithiothreitol (DTT) at designated concentrations and time courses.

RESULTS: The amount of nuclear CHCHD2 did not differ between 21% and 7% O2. Incubation in 1% O2 increased the amount of nuclear CHCHD2 in both U87 and U87vIII cells, with the latter consistently exhibiting greater amounts of nuclear CHCHD2 at all oxygen tensions. Total CHCHD2 protein levels in U87 cells were maximal at 4% O2, but reduced at 1% O2, a trend also observed in U87vIII cells. Chemical reduction with 1 mM DTT for either 15 min or 24 h induced significantly greater CHCHD2 nuclear accumulation in U87vIII cells compared to U87. Increasing DTT dose 10-fold resulted in rapid accumulation of CHCHD2 in nuclei after 15 min, and complete disappearance from mitochondria after 30 min.

CONCLUSIONS: Hypoxia and chemical reduction are capable of driving CHCHD2 translocation from mitochondria to the nucleus in U87 GBM cells, which is enhanced in the presence of the EGFRvIII mutant. Ongoing work to expand our understanding of how CHCHD2 protein folding governs protein localization and function may inspire novel therapeutic strategies to undermine mitonuclear signaling in GBM.

#2651

Deep hypoxia and the genomic background cooperate to shape the metabolic profile of acute myeloid leukemia cells.

Samantha Bruno,1 Martina Pazzaglia,1 Claudio Cerchione,2 Simona Soverini,1 Michele Cavo,1 Lorenzo Montanaro,1 Giorgia Simonetti,2 Giovanni Martinelli2. 1 _Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy;_ 2 _Istituto Scientifico Romagnolo Per Lo Studio e la Cura dei Tumori(IRST)-IRCCS, Meldola, Italy_.

Hematopoietic stem cells (HSC) and leukemic stem cells (LSC) develop in the hypoxic bone marrow (BM) niche. Hypoxia contributes to the development and maintenance of LSC and might influence chemosensitivity in acute myeloid leukemia (AML). In this study we analyzed the transcriptomic and metabolomic profile of 2 AML cell lines with diverse genomic background, in order to better understand the impact of hypoxia in AML. Gene Expression Profiling (GEP) and Liquid Chromatography-Tandem Mass Spectroscopy were performed on OCI-AML3 (FAB M4,NPM1 and DNMT3A mutations) and KASUMI-1 (FAB M2, t(8;21) and KIT mutation) cell lines, after 20 hours of incubation under normoxia or hypoxia (1% O2). GEP analysis was performed by Transcriptome Analysis Console (Affymetrix), DAVID tool and Gene Set Enrichment Analysis (GSEA). Hypoxia alters the transcriptional level of 1301 and 276 genes in OCI-AML3 and KASUMI-1, respectively. Hypoxia induces MYC down-regulation in both lines, combined with increased expression of several HIF-1α-related genes (e.g. ARNT, CXCR4, S100A4). Moreover, hypoxia upregulates HIF-1α target genes associated with the glycolytic-pathway, as HK2, GPI, PFKP, PKM, LDHA, ALDOA and ENO1 (p<0.01). Differentially expressed transcripts were significantly enriched for genes involved in metabolic pathways including regulation of cellular amino acid metabolic process, canonical glycolysis, gluconeogenesis, pyruvate metabolism and glycolytic process (p<0.01). Conversely, gene sets related to tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OxPhos), cytochrome complex assembly and mitochondrial respiratory chain were enriched in normoxia. Hypoxia forced metabolic adaptation that was dependent on the cell type. In OCI-AML3 cells, deep hypoxia induced metabolic changes associated to the Warburg effect, with increased glycolysis and low efficiency of OxPhos. The metabolic hypoxia-related profile was characterized by an increased conversion of pyruvate to lactate and alanine with high levels of fumarate and succinate, which are intermediates of the TCA cycle, elevated 2-hydroxiglutarate and glycerol 3-phospate, suggesting a reduction of energy production by OxPhos. The observed increase of glutamine levels and the reduction of glutamate, that is catabolized by TCA cycle, was associated with down-regulation of MYC expression, induced by hypoxia. Kasumi-1 showed an increase of lysolipid and fatty acid metabolism, with low impact on TCA intermediates and increased levels of alanine, glutamine and glutamate. The observed cell-line specific metabolic response to hypoxia suggests that a deep characterization of stem cells residing in the hypoxic BM niche is required in each patient. Moreover, the analysis of the metabolic profile may help define specific vulnerabilities and guide personalized therapeutic approaches.Supported by: ELN, AIL, AIRC, FP7 NGS-PTL

#2652

Defining the role of AP1 in molecular adaptation to hypoxia in colorectal cancer.

Eric Vancauwenberghe,1 Hannah Bolland,1 Christopher Carroll,1 Leonardo Da Motta,1 Anna Grabowska,2 Francesca Buffa,3 Adrian Harris,4 Alan McIntyre1. 1 _Hypoxia and Tumour Microenvironment Group, Cancer Biology, Division of Cancer and Stem Cells, University of Nottingham, Medical School, Queen's Medical Centre, Nottingham, United Kingdom;_ 2 _Cancer Biology, Division of Cancer and Stem Cells, University of Nottingham, Medical School, Queen's Medical Centre, Nottingham, United Kingdom;_ 3 _Computational Biology and Integrative Genomics, Department of Oncology, University of Oxford, Oxford, United Kingdom;_ 4 _Molecular Oncology Laboratories, Department of Oncology, The Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom_.

Background:

The aim of this project was to investigate the role of Activator Protein 1 (AP1) in molecular adaptation to hypoxia in colorectal cancer. Colorectal cancer is the 3rd most common cancer worldwide. Low oxygen (hypoxia) found in 30-50% of colorectal tumors is associated with resistance to chemotherapy, radiotherapy and poor patient prognosis. Hypoxia stabilizes the transcription factors HIF1α and HIF2α which enable molecular adaptation to the hypoxic insult at a transcriptional level, in cooperation with additional transcription factors. To identify genes that regulate hypoxic survival in colorectal cancer, we carried out a lentiviral shRNA unbiased screen targeting 6142 genes, in HCT116 cells. We identified AP1 transcription factor subunits as key mediators of hypoxic cell viability that were required in hypoxia and in 3D spheroid culture in colorectal cancer

Methods: Our experiments were conducted in a panel of cancer and normal colorectal cell lines (HCT116, LS174T, SW620, HT29 and CCD 841 CoN) in normoxia and hypoxia (1%O2) and in 3D spheroid cultures. The association of the different AP1 subunits and the impact of AP1 subunits on hypoxia-regulated expression and cell phenotypes was investigated using a variety of cell and molecular biology approaches.

Results: Our results identify that AP1 subunits cJUN, JUNB, JUND, FOSL1 and FOSL2 are upregulated by hypoxia in colorectal cancer in a HIF independent manner. We observed that individual AP1 subunit knockdown in particular FOSL2 and JUND knockdown significantly decreased cancer cell survival in hypoxia. FOSL2 and JUND decreased the cell survival respectively by 40% and 60% in HCT116 and by 45% and 60% in LS174T (n=3, p<0.001). This effect was not observed when the cells were treated with T-5224 (10 µM), an AP-1 inhibitor suggesting a complex modulation of the transcriptional response by AP1 in hypoxia. In hypoxic conditions, by Co-IP studies, we established in HCT116 and LS174T that a selected pattern of AP1 heterodimers in particular FOSL2/JUNB and FOSL2/cJUN heterodimers were induced to mediate the transcriptional response to hypoxia. siRNA knockdown demonstrated that AP1 subunits modulate the expression of a specific set of hypoxia-regulated genes such as CA9 (Carbonic Anhydrase 9) and ANGPTL4 (n=3, p<0.05), important modulators of the hypoxic response.

Conclusions:

These data identify AP1 as a key mediator of the molecular adaptation to the hypoxic insult in the colorectal tumour microenvironment. Targeting AP1 subunits is a likely therapeutic approach for the treatment of the therapy resistant hypoxic regions of colorectal cancers.

#2653

Usefulness of plasma exosomes to characterize hypoxic phenotype in primary prostate tumors.

Gatikrushna Panigrahi,1 Yixin Su,1 Leslimar Rios-Colon,1 Taylor Peak,1 Kiran Sai,1 Prakash Praharaj,1 Jingyun Lee,1 Cristina Furdui,1 Ashok Hemal,1 Deepak Kumar,2 Gagan Deep1. 1 _Wake Forest Baptist Medical Center, NC;_ 2 _North Carolina Central University, NC_.

Hypoxia in primary prostate cancer (PCa) selects aggressive clones and causes treatment failure. However, there are several clinical challenges in measuring hypoxia in PCa. For example, MRI needs sophisticated instruments and highly trained personnel; needle electrodes could cause bleeding; and pimonidazole and immuno-histochemistry (IHC) for hypoxia biomarkers require surgical removal of tumor. Hence, novel non-invasive measures are needed to assess hypoxia. Exosomes are nano-vesicles (40-150 nm in size) secreted by cells for intercellular communication and are present in all biofluids loaded with unique cargo that could predict the cell of their origin and cellular physiologic state. Our primary goal here was to use exosomes to non-invasively assess hypoxia in PCa. We employed ultracentrifugation, mass spectrometry (MS), immunogold labeling, electron microscopy, PET, IHC, xenograft and immune-pull down methods. First, we isolated exosomes from the conditioned media of African American PCa MDA PCa 2b and E006AA-hT cells cultured under normoxia (21% O2) and hypoxia (1% O2) by ultracentrifugation. Next, through a novel method involving exosomal surface shaving and LC-MS/MS, we identified 13 unique proteins present only on the surface of exosomes secreted by PCa cells under hypoxia. Among the 13 unique proteins, Annexin A6 expression was highly correlated with 44 hypoxia signature genes in TCGA-PRAD. Next, Annexin A6 expression was confirmed on the surface of exosomes isolated from the blood of nude mice implanted with human PCa 22Rv1 xenografts. Hypoxia in xenografts was confirmed by F18MISO-PET imaging, and pimonidazole staining in xenograft tissues. Next, we pull-down Annexin A6+ exosomes using biotin-tagged human Annexin A6 antibody and streptavidin tagged agarose resin, and analyzed proteins loading by MS. Interestingly, Annexin A6+ exosomes showed a hypoxia signature as 35/172 loaded proteins were hypoxia and HIF1α regulated. We next isolated Annexin A6+ exosomes from the blood of African American and Caucasian PCa patients (n=5 each) with high grade disease (Gleason score >7) and confirmed Annexin A6 expression on their surface. Importantly, for similar Gleason score disease, Annexin A6+ exosomes concentration was 2.6 times higher in African American patients, and could be associated with disease aggressiveness. Together, these results suggest that exosomes could be useful to non-invasively assess hypoxia in PCa.

#2654

Synergistic inhibition of cancer cell viability and suppression of HIF1α by CDK4 inhibitors and HSP90 inhibitors across cancer types.

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

Hypoxia is common and hypoxia-inducible factor 1α (HIF1) promotes various aspects of cancer, such as angiogenesis, or metastasis, and confers therapy resistance. Targeting HIF's is a promising cancer treatment strategy. We previously showed that cyclin-dependent kinase 1 and 4 (CDK1, CDK4) stabilize HIF1α protein in cancer cells (Warfel et al., Cell Cycle, 2013). We also found that CDK activity facilitates HSP90 stabilization of HIF1α and that targeting CDK1 or CDK4/6 and HSP90 simultaneously reduces HIF1α levels and inhibits colorectal cancer (CRC) cell viability. The CDK4/6i palbociclib plus HSP90i ganetespib synergistically inhibit cell viability in HCT116 CRC cells under normoxia and hypoxia, show anti-tumor efficacy in vivo, and reduce both HIF1 and HIF2 levels. To investigate the translational potential of the combination strategy, we explored use of a HSP90i in clinical trials as no HSP90i has been FDA-approved for cancer treatment. Onalespib is a HSP90i undergoing phase I trials for solid tumors and lymphoma. and XL888 is being tested in GI cancer and melanoma. A third HSP90i, TAS116, is under investigation in solid tumors. We found that all these HSP90 inhibitors, in combination with palbociclib, reduce HIF1α levels in CRC cells. Cell viability was inhibited by the combination treatment in a synergistic manner. Similar effects were observed in pancreatic cancer, breast cancer and other tumor types upon treatment with TAS116 and palbociclib. As palbociclib is not the only FDA-approved CDK4/6 inhibitor, we examined the anti-tumor effects of HIF targeting effect using an alternative FDA-approved CDK4/6 inhibitor, abemaciclib. Abemaciclib, in combination with TAS116, decreased HIF1α levels and inhibited cancer cell viability. Our findings demonstrate a class effect towards HIF1α inhibition and cancer cell viability across multiple tumor types from the combination of a CDK4/6i and HSP90i. Ongoing experiments are examining the role of HIF in the sensitivity to the drug combination and the impact of the combined inhibitor therapy on cancer cell migration and invasion. Further clinical translation of this concept is warranted for cancer therapy.

#2655

Oxygen tension regulates lysosomal activation and receptor tyrosine kinase degradation.

Jaewoo Hong, P. Charles Lin, Todd Wuest, Yongfen Min. _National Cancer Institute, Frederick, MD_.

Oxygen sensing is crucial for adaptation to variable habitats and physiological conditions. Low oxygen tension, or hypoxia, is a common feature of solid tumors and hypoxic tumors are often more aggressive and resistant to therapy. Here we show that, in mammalian tissue culture cells, hypoxia suppressed lysosomal acidification/activation and receptor tyrosine kinase (RTK) degradation. Hypoxia down-regulated mTORc1, reducing its ability to activate transcription factor EB (TFEB), a master regulator of v-ATPase, the lysosomal proton pump. Hypoxia prevented epidermal growth factor receptor (EGFR) degradation in tumor tissues, whereas activation of lysosomes enhanced tumor cell response to anti-EGFR treatment. Our results linkoxygen tension and lysosomal activity, provide a molecular explanation of the malignant phenotype associated with hypoxic tumors, and suggest activation of lysosomes may provide therapeutic benefit in RTK-targeted cancer therapy.

#2656

Regulation of HIF-1a by MDM2 in prostate cancer cells.

Saad Ebrahim Alobid,1 Appu Rathinavelu2. 1 _Nova Southeastern Univ. College of Pharmacy, Fort Lauderdale, FL;_ 2 _Rumbaugh Goodwin Institute for Cancer Research, Nova Southeastern Univ. College of Pharmacy, Fort Lauderdale, FL_.

MDM2 is an oncogene amplified in approximately 40% of all human prostate carcinomas, and such amplification is correlated very well with aggressive tumor phenotype, distant metastasis, poor treatment outcomes and overall increase in mortality. Interestingly, in cancer cells, hypoxia inducible factor-1alpha (HIF-1α) is activated to facilitate adaptation to the non-conducive tumor microenvironment, through the regulation of downstream genes to ensure tumor survival and progression through modification of protective biological processes. Our previous investigations have outlined a significant role for MDM2 overexpression in regulating HIF-1α under both normoxic and hypoxic conditions in MDM2 expressing LNCaP and MDM2 transfected LNCaP-MST cells, leading to angiogenesis and other detrimental effects. Therefore, our study was aimed to identify how HIF-1α level is regulated in these prostate cancer cells, as a result of MDM2 overexpression, under both normoxic and hypoxic conditions. Analysis of mRNA levels exhibited a significant transcriptional upregulation of HIF-1α in LNCaP-MST compared to LNCaP cells under normoxic condition. Interestingly, targeting the transcriptional machinery with Actinomycin D resulted in a significant downregulation of HIF-1α protein levels also under normoxic conditions. On the other hand, inhibition of proteasomal degradation with MG-132 resulted in further elevation of HIF-1α protein levels in normoxic condition. However, under hypoxic condition, the levels of HIF-1α mRNA seem to be unchanged in both cell lines. In support of these findings, LNCaP-MST cells exhibited a significant downregulation of VHL (Von Hippel-Lindau) levels compared to LNCaP cells under hypoxic condition. The VHL protein typically facilitates polyubiquitination of HIF1α that are marked for proteasomal degradation subsequent to prolyl hydroxylation. So far, our results have confirmed a positive correlation between MDM2 expression and HIF-1α levels under normoxic conditions, while protein degradation may play a significant role during hypoxic conditions. In conclusion, our study provides new evidences that are linking MDM2 overexpression to HIF-1α protein levels in prostate cancer cells under both normoxic and hypoxic conditions, which may be important while considering targeted therapies. (The financial support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida is gratefully acknowledged).

#2657

GSTP mediated S-glutathionylation of GRP78 attributes to multiple myeloma resistance.

Zhiwei Ye, Jie Zhang, Kenneth D. Tew, Danyelle M. Townsend. _Medical University of South Carolina, Charleston, SC_.

The proteasome inhibitor bortezomib (Btz) is used in the treatment of multiple myeloma (MM), but its efficacy is restricted by the wide-spread occurrence of resistance. MM cells have the highest rate of secretion of proteins with disulfide bonds and are exquisitely dependent upon redox balance and accurate protein folding. Our results showed that some important redox enzymes, including glutathione transferase P (GSTP) are upregulated in Btz-resistant cells. While GSTP was originally described as a phase II detoxification enzyme, our laboratory has ascribed a thiolase activity catalyzing the forward S-glutathionylation reaction, a post-translational modification (PTM) through reversible addition of glutathione to thiolate anions of cysteines in target proteins. Although primarily cytosolic, GSTP has been reported in the nuclear and mitochondrial compartments. We recently showed that GSTP is localized at high concentrations in the endoplasmic reticulum (ER) and regulates a number of redox active proteins through S-glutathionylation; these include glucose-regulated protein (GRP78). Proteomic analysis revealed that both cys41 and cys 420 of GRP78 were subject to S-glutathionylation and this PTM altered the activity of GRP78, enhancing its holdase activity, while concomitantly decreasing its foldase activity. This alters protein folding and contributes to the Btz-resistant phenotype of MM. Our data also support the principle that drugs that target the ER protein folding machinery significantly alter local redox conditions. Thapsigargin (Thg) is an inhibitor of SERCA, interfering with the functional activities of calcium-dependent chaperones, while tunicamycin (TuM) inhibits N-linked glycosylation. Btz resistant cells showed marked levels of cross-resistance (2-10 fold) to both. Linking such results with GSTP/redox, the absence of GSTP (from GSTP knockout mice) makes both bone marrow derived dendritic and mouse embryonic fibroblast cells more sensitive to Btz, ThG and TuM. The nature of the cross-resistance patterns and the fact that the presence of GSTP in drug naïve cells also confers resistance provides a rational platform for studying redox-based mechanisms as causative in resistance. Moreover, inhibition of GSTP with Telintra restored Btz sensitivity, decreased S-glutathionylation and increased expression of markers of UPR induced apoptosis. We conclude that altered GSTP expression and S-glutathionylation of key ER-localized proteins are significant contributors to drug response and resistance in MM treated with Btz.

#2658

PFKFB3 inhibition reprograms malignant pleural mesothelioma to glycolytic stress-induced macropinocytosis and ER stress as independent binary adaptive responses.

Sayantani Sarkar Bhattacharya, Ling Jin, Debarshi Roy, Deokbeom Jung, Prabhu Thirusangu, Yinan Xiao, Julie Staub, Julian Molina, Viji Shridhar. _Mayo Clinic, Rochester, MN_.

The metabolic signatures of cancer cells are often associated with elevated glycolysis which promotes alteration in cellular bioenergetics. 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), a critical control point in the glycolytic pathway has emerged as a potential anti-cancer target associated with many aspects of neoplastic progression. Pharmacological (PFK158 treatment) and genetic inhibition of PFKFB3 activity, decreases glucose uptake, ATP production, and lactate dehydrogenase activity and inhibits cell proliferation in vitro by arresting the cells in the G0/G1 phase induced cell death in malignant pleural mesothelioma (MPM). It induces an early response of increased macropinocytosis and drastic endosomal vacuolization to overcome the nutrient stress both in sarcomatoid (H28) and epithelioid (EMMeso) MPM cells. Inhibition of PFKFB3 leads to the activation of the endocytic Rac1-Rab5-Rab7 pathway resulting in a unique form of cell death called "methuosis". Transmission Electron Microscopy analysis showed that pharmacological and genetic inhibition of PFKFB3 activity leads to the formation of nascent macropinocytotic vesicles, which rapidly coalesced to form large vacuoles with compromised lysosomal function. Confocal microscopy revealed F-actin and α-tubulin disassembly leading to the disruption of cell structure integrity. In parallel, PFKFB3 expression inversely modulates endoplasmic reticulum (ER) stress in MPM. Pharmacological and genetic inhibition of PFKFB3 led to an escalation in ER activity and aggravated ER stress mostly by upregulating BiP and GADD153 expression levels. However, EHT1864, a Rac1 inhibitor, could not rescue MPM cells from ER stress, suggesting that the activation of macropinocytosis and ER stress are a result of two independent PFKFB3 modulated adaptive responses. Both immunofluorescence microscopy and co-immunoprecipitation analyses revealed that the two crucial biomolecules of each pathway, Rac1 and Calnexin, interact with each other only at the later stages of PFKFB3 inhibition. Finally, PFK158, in a combination of with CBPt, demonstrated a significant reduction in tumor weight in mesothelin overexpressed EMMeso xenograft model. Since most cancer cells exhibit an increased glycolytic rate, these results provide evidence for PFK158, in combination with standard chemotherapy, may have a potential in the treatment of MPM.

#2659

IRE1á-dependent increase of Gá12, a gep oncogene, augments ER stress-induced catastrophic cell death.

Jihoon Tak, Tae Hyun Kim, Sang Geon Kim. _College of Pharmacy, Seoul National University, Republic of Korea_.

Hepatocellular carcinoma (HCC) belongs to the most deadly types of cancer since it has a poor prognosis due to its aggressive metastatic phenotype. Endoplasmic reticulum (ER) stress is closely associated with the incidences of a variety of liver diseases including liver injury, regeneration, fibrosis, cirrhosis and often HCC. G protein-coupled receptor (GPCR) signals lead to activation of their downstream pathways through G proteins. Previously, we reported that Gα12 overexpression enhances HCC malignancy, promoting cancer migration and microvascular invasion. However, the role of Gα12 in ER stress-associated pathophysiology in the liver was unclear. Given the link between ER stress and HCC malignancy, this study investigated the effect of ER stress on Gα12, the role of Gα12 in ER stress-mediated hepatocyte death, and the underlying pathway. In the analyses of RNA-sequencing data from mouse liver or primary mouse embryonic fibroblasts challenged with ER stress, Gα12 and C/EBP homologous protein (CHOP) levels correlated with each other. Increases in Gα12 level by unfolded protein response (UPR) under ER stress condition was verified using primary hepatocytes or hepatocyte-derived cell line, confirmative of the close association between Gα12 and UPR. Of note, enforced Gα12 expression under ER stress condition augmented IRE1α level in a RhoA/Rock-dependent pathway. Among the canonical pathways, IRE1α gene silencing distinctly inhibited Gα12 induction elicited by ER stress in hepatocytes. Consistently, overexpression of IRE1α enhanced Gα12 level upon tunicamycin treatment. Similar outcomes were obtained in cells or mice treated with acetaminophen, a representative liver toxicant inducing ER stress response. Overall, our results provide evidence that IRE1α-dependent increase of Gα12 potentiates ER stress response in the liver as mediated by the RhoA-ROCK pathway, enhancing catastrophic death of hepatocytes.

#2660

Selective inhibition of HSP90 by its C-terminal inhibitors is more effective to suppress bladder cancer cell growth.

Jun Ling,1 Connor Magura,1 Vaibhav Sharma,1 Maisara Rahimi,1 Heinric Williams2. 1 _Geisinger Commonwealth School of Medicine, Scranton, PA;_ 2 _Geisinger Clinic, Danville, PA_.

HSP90 (heat shock protein 90) is a central chaperone to regulate protein folding for a large array of client proteins as important drivers for cancer development. Its overexpression in bladder cancer, especially the most aggressive muscle-invasive subtype (MIBC), attracts a lot of attention for drug development. Although many HSP90 inhibitors have been tested in clinical trials for many cancers, none of them has been approved by FDA for cancer therapy. One of critical problems is the co-activation of heat shock response (HSR), an integrated stress response pathway with strong pro-survival function. Our previous studies also observed the robust activation of HSF1 (an effector of HSR) and HSP70 (a target gene of HSF1) associated with the inhibition of HSP90 by its N-terminal (NT) inhibitors, thereby impairing the effect of HSP90 NT inhibitor. Intriguingly, HSP90 inhibitors tested in the clinical trials so far are all NT inhibitors, further promoting us to explore the HSP90 C-terminal (CT) inhibitors as a new strategy for MIBC therapy. In this study, a number of MIBC cell lines representing different cancer stages were utilized to generate more physiologically relevant data. When UMUC3, T24, SW-780, and J82 cells were treated by novobiocin (Novo, a HSP90 CT inhibitor) at a series of concentrations, it was found that Novo inhibited the proliferation of all cells. However, their sensitivities to Novo were different in the following order: T24> SW-780> UMUC3> J82. This effect was partially correlated with the aggressiveness of cancer stage, wherein higher grade MIBC cells are less sensitive to Novo treatment. Western blotting (WB) analysis indicated that HSF1 and HSP70 were not activated. More importantly, client kinase AKT1 was highly activated in SW-780 but not in all other three cells; ERK1/2 was highly activated in UMUC3 but not in all other three cells, suggesting that the activation of kinases is differential and cell type-dependent. Confocal fluorescence microscopic analysis revealed that HSP90 cellular localization was also altered by Novo from more diffused cytoplasmic distribution to more perinuclear localization, whereas the predominant nuclear localization of HSF1 was not changed by Novo treatment, suggesting the localization of HSP90, different modified forms, or the interaction with other proteins may also contribute to HSP90's functions in cell growth. These aspects are under investigation by various biochemical and cell biology techniques to validate the advantage of HSP90 CT inhibitors for MIBC therapy. (* corresponding authors)

#2661

Targeting endoplasmic reticulum-resident proteins for the treatment of B cell cancer.

Chih-Hang Anthony Tang,1 Juan R. Del Valle,2 Chih-Chi Andrew Hu1. 1 _The Wistar Inst., Philadelphia, PA;_ 2 _The University of South Florida, Tampa, FL_.

IRE-1 splices XBP-1 mRNAs, leading to activation of the functional XBP-1 transcription factor. We showed that genetic deletion of XBP-1 in chronic lymphocytic leukemia (CLL) cells decelerated malignant progression of CLL in mice. We synthesized and characterized a specific inhibitor, B-I09, which could block the RNase activity of IRE-1 with high potency and efficacy. B-I09 clearly suppressed activation of the IRE-1/XBP-1 pathway, as evidenced by the decreased mRNA and protein levels of XBP-1 in intact cells. B-I09 specifically targeted mouse CLL cells in vivo by inducing apoptosis. Because XBP-1 deficiency could compromise the BCR signaling, a crucial survival signal for CLL, we tested whether pharmacological inhibition of XBP-1 could enhance the effect of inhibitors of the BCR signaling by combining B-I09 with ibrutinib (a Bruton's tyrosine kinase inhibitor) to treat human CLL cells. A strong pharmacological synergism was determined using the Chou-Talalay combination index method, suggesting that B-I09 could help ibrutinib to achieve higher cytotoxicity at a lower dose, addressing ibrutinib's toxicity issue. Our studies also led us to discover that IRE-1 interacted with STING, an ER-resident protein critical for cytoplasmic DNA sensing and interferon production. We showed that the IRE-1/XBP-1 pathway was required for the interferon-producing function of STING, and that agonists of STING selectively triggered mitochondria-mediated apoptosis in malignant B cells. Upon stimulation, STING was degraded inefficiently in malignant B cells, implying that prolonged activation of STING could lead to apoptosis. In CLL-bearing mice, injection of the STING agonist, 3'3'-cGAMP, induced apoptosis and regression of leukemia. Similarly efficacious effects were elicited by 3'3'-cGAMP injection in syngeneic or immunodeficient NSG mice grafted with malignant B cells. These data suggested that STING agonists could directly eradicate CLL and other B cell malignancies in vivo. IRE-1 and STING are thus useful ER-resident protein targets for the treatment of B cell cancer.

#2662

Redaporfin induced immunogenic cell death through destruction of the endoplasmatic reticulum and the golgi apparatus.

Lígia Catarina Gomes-da-Silva,1 Luís G. Arnaut,1 Oliver Kepp,2 Guido Kroemer2. 1 _University of Coimbra, Coimbra, Portugal;_ 2 _Gustave Roussy Cancer Campus, Villejuif, Paris, Portugal_.

Redaporfin is a halogenated bacteriochlorin with high antineoplastic effects as well as indirect immune-dependent destruction of malignant lesions, which is currently being evaluated in clinical trials for head and neck cancer (NCT02070432). In this work, the mechanisms through which photodynamic therapy (PDT) with redaporfin kills cancer cells were deeply investigated. When activated, redaporfin caused rapid reactive oxygen species-dependent perturbation of endoplasmic reticulum - golgi apparatus (ER/GA) compartments as observed by electron microscopy, which was correlated with ER stress and a general inhibition of the GA-dependent secretory pathway. Redaporfin-based PDT also induced signs of ER stress namely phosphorylation of eIF2α, which was identified to be mediated by the heme-regulated eIF2α Kinase (HRI), also known as EIF2AK1. Phosphorylation of eIF2α was recently described as a hallmark of immunogenic cell death (ICD). Indeed, redaporfin-based PDT induced other hallmarks of ICD such as plasma membrane calreticulin exposure, as well as the release of ATP and HMGB1 from cancer cells in vitro. Accordingly, redaporfin/PDT-killed cancer cells injected subcutaneously into syngeneic mice were able to fully protect a fraction of the animals against rechallenge with live cancer cells of the same type and to reduce tumor growth in the remaining mice. In this work it is demonstrated that ER/GA play a role upstream of mitochondria in the lethal signaling pathway triggered by redaporfin-based PDT. Indeed, pharmacological perturbation of GA function or homeostasis, with Brefeldin-A or Golgicide-A, reduces mitochondrial permeabilization and subsequent cell death. In contrast, removal of the pro-apoptotic multidomain proteins BAX and BAK or pre-treatment with proteases inhibitors, reduced cell killing, yet left the GA perturbation unaffected. Altogether, these results point to the capacity of redaporfin to kill tumor cells in an immunogenic way through ER/GA destruction.

#2663

**Regulation of ER stress-mediated cell death by calcium mobilization: A potential mitochondrial pathway for 15-deoxy-Δ** 12,14 **prostamide J** 2 **cytotoxicity in melanoma.**

Daniel Alexander Ladin, Estefani Cota, Rukiyah Van Dross. _East Carolina Univ., Greenville, NC_.

Melanoma is the most aggressive and deadly form of cutaneous cancer in the United States, representing a major clinical challenge. Our lab previously demonstrated that the endocannabinoid metabolite, 15-deoxy, Δ12,14 prostamide J2 (15d-PMJ2), was an effective and selective inducer of apoptotic cell death in B16F10 murine melanoma cells. Furthermore, we found that 15d-PMJ2 mediated apoptosis was regulated by endoplasmic reticulum (ER) stress. Numerous studies have determined that ER stress causes Ca+2 flux, and that this action initiates cellular apoptosis. As such, we investigated Ca+2 mobilization in the context of 15d-PMJ2-mediated ER stress. In B16F10 melanoma cells, 15d-PMJ2 significantly increased cytosolic Ca+2 levels. Blockade of Ca+2 channels using ruthenium red (a pan Ca+2 channel inhibitor) or 2-APB (an IP3 selective inhibitor) decreased the cytotoxicity of 15d-PMJ2. To investigate the role of ER stress in Ca+2 mobilization, cells were pretreated with the ER stress inhibitors, phenylbutyric acid (PBA) and GSK2602 (a PERK-selective inhibitor). Both inhibitors significantly lowered cytosolic Ca+2 levels suggesting Ca+2 flux is mediated by ER stress. Moreover, BAP31, an important regulator of endoplasmic Ca+2 release and ER stress was activated following exposure to 15d-PMJ2. To understand the impact of elevated Ca+2 levels, we investigated cysteine proteases (calpains) and mitochondrial Ca+2, both of which induce apoptosis in a Ca+2-dependent manner. Our results show that mitochondrial Ca+2 levels were significantly increased by 15d-PMJ2 and that this effect was prevented by blocking ER stress. In contrast, 15d-PMJ2 increased calpain-2 activity however, the inhibition of calpain had no effect on the cytotoxicity of 15d-PMJ2. These findings indicate that mitochondrial Ca2+ flux, but not calpains regulate ER stress apoptosis induced by 15d-PMJ2. It has been reported that the activity of J-series prostaglandins is conferred by an electrophilic double bond contained within its cyclopentenone ring. To evaluate the significance of this moiety in 15d-PMJ2, a neutral analog (neutral-15d-PMJ2) lacking the double bond was synthesized. We determined that that the accumulation of intracellular and mitochondrial Ca2+ was dependent on the electrophilic double bond, suggesting it is a critical structural component required for activity. Taken together, these data indicate that the mobilization of calcium to the cytoplasm and mitochondria may critically regulate ER stress apoptosis that is mediated by 15d-PMJ2.

#2664

Differential activation of the integrated stress response correlates with anti-tumor activity of imipridones ONC201 and ONC206 in pediatric sarcomas.

David V. Allegakoen,1 Rohinton S. Tarapore,2 Joshua E. Allen,2 Amit J. Sabnis,1 Trever G. Bivona1. 1 _UCSF, San Francisco, CA;_ 2 _Oncoceutics, Inc., Philadelphia, PA_.

Cure rates for pediatric sarcomas have minimally improved with intensification of genotoxic chemotherapy. We have previously shown that activation of the Integrated Stress Response (ISR), a cellular stress response that can lead to apoptosis, is a novel, effective means of selectively inducing the death of rhabdomyosarcoma (RMS) cells. However, context-specific wiring of homeostatic components such as protein chaperones dictates unique barriers to ISR activation. We hypothesized that just as HSP70 inhibition can preferentially activate the ISR in RMS, but not Ewing sarcoma, alternative small molecules might highlight dependencies unique to other sarcoma subtypes.

ONC201 is a selective dopamine receptor (DRD2/3) antagonist that has previously shown antitumor activity through ISR activation in solid tumor models. ONC206 is a more potent analogue of ONC201 that was shown to be effective in Ewing sarcoma cell lines with sub-micromolar IC50. We used these molecules to test the hypothesis that DRD2/3 antagonism can activate the ISR in pediatric sarcomas, identifying a new therapeutic strategy.

We measured the growth inhibitory effects of ONC201 and ONC206 across a panel of four rhabdomyosarcoma (RMS) and three Ewing sarcoma (ES) patient-derived cell lines. Across all sarcoma cell lines tested, ONC201 and ONC206 inhibited growth with low micromolar and sub-micromolar IC50 values respectively. ES cell lines were more sensitive than RMS lines, with mean ONC201 IC50 of 7.7 micromolar vs 3.2 micromolar (p = 0.0016).

To explore this difference, we measured ISR activation by immunoblot. The ISR initiates with phosphorylation of eIF2α, leading to a halt in protein translation. RMS cells showed a strong increase in phosphorylated eIF2α one hour after treatment with either drug that was sustained through 24 hours, but attenuated by 48 hours of treatment. ES cells, however, did not show increased eIF2α phosphorylation until 48 hours of treatment, at which point levels of phosphorylation were much higher than in RMS cells. Correspondingly, PARP cleavage was relatively modest in RMS cells, but robust in ES cells at 48 hours of treatment. We conclude that two phenotypes of ISR activation – early and attenuated, versus late and sustained – correlate with the therapeutic efficacy of these agents.

Our data suggest that regulation of the ISR downstream of DRD2/3 antagonism underlies differential response to two clinically relevant agents. The development of biomarkers of the strength and duration of ISR activity may thus guide the clinical application of ONC201, ONC206, or other ISR-activating therapies in pediatric sarcomas. Ongoing work will test the expression and activity of eIF2α phosphatases and the necessity and sufficiency of terminal ISR effectors in controlling this differential response, as well as the efficacy of these agents in relevant in vivo models of disease.

#2665

Zinc dysregulation as an emerging molecular target in epithelial cancers.

Jessica Perer, Rebecca Justiniano, Anh Hua, Georg T. Wondrak. _University of Arizona, Tucson, AZ_.

Zinc plays an established role in structure and function of enzymes and transcription factors, and zinc signaling has now been recognized as a molecular determinant of epithelial differentiation and cell fate. Intracellular zinc homeostasis is regulated by zinc importers (ZIPs encoded by the SLC39A gene family). Cumulative evidence suggests that dysruption of zinc homeostasis is an emerging hallmark of various tumor types, characterized by downregulation of ZIP expression upstream of intracellular zinc depletion. In nonmelanoma skin cancer (NMSC) examined in TMA format we have observed downregulation of ZIP2 (involved in epidermal differentiation) that occurs with zinc depletion and impaired differentiation. Likewise, in pancreatic ductal adenocarcinoma (PDAC) tumor tissue, downregulation of ZIP3 and zinc depletion are detectable. We therefore tested feasibility of pharmacological induction of intracellular zinc overload for chemotherapeutic intervention. Using the small molecule ionophore ZnPT (zinc pyrithione) as an experimental chemotherapeutic, rapid induction of zinc overload followed by metal stress response signaling and cell death were observable in a panel of malignant epithelial cell lines (NMSC: SCC-25; HaCaT-ras II-4; PDAC: BxPC-3, MIA PaCa-2, PANC-1). Remarkably, PDAC cell lines displayed nanomolar sensitivity to ZnPT-induced cytotoxicity. In BxPC-3 cells, pronounced upregulation of stress response gene expression (MT2A, HSPA6, HMOX1) and signaling (p-p38, p-eIF2α, HO-1) was detectable within one hour of ZnPT exposure. Accumulation of protein-ubiquitin conjugates further substantiated the rapid occurrence of ZnPT-induced proteotoxic stress and UPR, consistent with a role of ZnPT in the inhibition of proteasome-associated deubiquitinating enzymes (DUBs). Next, we confirmed NMSC-directed anti-tumor activity of ZnPT in a photocarcinogenesis SKH1 mouse model, followed by exploratory chemotherapeutic intervention targeting BxPC-3 PDAC xenografts. Taken together, our data suggest that ZIP-based dysruption of intracellular zinc homeostasis may be a valid molecular target in specific epithelial cancers. [This research was funded in part by NCI: P30 CA023074 & R03 CA230949.]

#2666

ARD1-mediated Hsp70 acetylation protects cancer cells against the cellular stress.

Ji Hae Seo, So-Jin Shin, Jin Young Kim, Seungmee Lee, Hyewon Chung, Chi-Heum Cho. _Keimyung University School of Medicine, Daegu, Republic of Korea_.

The 70 kDa heat shock proteins (Hsp70s) are a family of ubiquitously expressed intracellular proteins that are required for the maintenance of protein homeostasis. Hsp70 is highly induced in response to cellular stressors, including oxidative stress, hyperthermia, hypoxia and changes in pH, contributing to cellular resistance to stress-induced cell death. Most tumor cells, which live under continuous stress conditions, express elevated levels of Hsp70 to combat these harsh conditions and suppress apoptosis. During cellular stress, Hsp70 maintains protein homeostasis through two opposing mechanisms: protein refolding and degradation. However, the mechanisms by which Hsp70 balances these opposing functions under stress conditions remain unknown. Here, we demonstrate that Hsp70 preferentially facilitates protein refolding after stress, gradually switching to protein degradation via a mechanism dependent on ARD1-mediated Hsp70 acetylation. During the early stress response, Hsp70 is immediately acetylated by ARD1 at K77, and the acetylated Hsp70 binds to the co-chaperone Hop to allow protein refolding. Thereafter, Hsp70 is deacetylated and binds to the ubiquitin ligase protein CHIP to complete protein degradation during later stages. In addition, this switch is essential for the maintenance of protein homeostasis and ultimately rescues cancer cells from stress-induced cell death. Therefore, ARD1-mediated Hsp70 acetylation is a key regulatory mechanism that enables Hsp70-expressing cancer cells to be more resistant to proteotoxic stress, and regulation of Hsp70 K77 acetylation might be helpful to cancer treatment.

#2667

Genetic removal of metazoan SpoT homolog I (MESH1) inhibits proliferation through the repression of HIPPO effector TAZ.

Tianai Sun, Chien-Kuang Ding, Jen-Tsan A. Chi. _Duke University, Durham, NC_.

The capacity of tumor cells to survive various stresses is an essential property that endows them invulnerability. One of the bacterial key adaptive strategies against nutrient deprivation is stringent response. In bacteria, nutrient deprivation leads to an elevation of a signaling alarmone, guanosine tetraphosphate ((p)ppGpp) to repress the protein synthesis and proliferation activity in order to maintain cell viability. The accumulation of (p)ppGpp is partially caused by the repression of its hydrolase, SpoT. However, this strategy still remains elusive in humans, but the human ortholog of SpoT-MESH1 has been discovered. MESH1 also has the hydrolase activity on (p)ppGpp in vitro, but human cells lack the required alarmone (p)ppGpp and its synthetase, which restricts the discovery of stringent response in humans. Unexpectedly, our recent data suggests that MESH1 depletion allows cell survival under cystine deprivation, an extremely oxidative stress, yet with a slow proliferation rate. These correspond to bacterial stringent response phenotypes. Moreover, our gene expression profiling shows that MESH1 silencing triggers a global transcriptional reprogramming with a similarity to bacterial transcriptional responses of stringent response. Altogether, these evidences inspire us to speculate on the existence of human stringent response with similar cell protective functions, albeit in the absence of (p)ppGpp. In this study, I aim to identify the molecular pathways and regulatory mechanisms that MESH1 adopts to explain the MESH1-related stringent response phenotypes in human cancer cell lines. One interesting phenomenon we observed from our genome-wide RNA screen is that MESH1 silencing significantly represses TAZ RNA level. TAZ serves as a key co-activator involved in the HIPPO pathway that has been well studied to regulate cell proliferation, cell stemness, and etc, which are perfectly relevant to the MESH1-related stringent response phenotypes. Therefore, I will specifically determine the functional roles of TAZ repression upon MESH1 removal in the expression of stringent response features by genetic tools. Furthermore, I will characterize the transcriptional control of TAZ and other gene signatures of human stringent response. We observed that MESH1 depletion significantly represses the acetyl-CoA:CoA ratio and further inhibits the histone acetylation activity that presumably turns off/on certain gene transcription. Based on these facts, I will determine the importance of histone modification on the transcriptional reprogramming upon MESH1 silencing. Successful completion of this study will equip us with more thorough understandings of stress response mechanisms in humans so that we can improve the treatment for stress-related diseases, such as cancer, by providing a novel therapeutic target, MESH1.

#2668

Stress granule as mechanism of tumor cell fitness and drug resistance.

Elda Grabocka. _Thomas Jefferson University, Philadelphia, PA_.

Acquisition of a gain-of-function mutation in the KRAS gene is a driver of tumorigenesis in a wide number of tissues including adenocarcinomas of the pancreas, colon, and lung. Mutant KRAS-driven cancers are extremely refractory to standard chemotherapeutic treatments. Studies over the recent years have established that stress-coping strategies play a crucial role in disease progression and therapy response. Consequently, mechanisms that determine the stress-coping capabilities of cancer cells may constitute an attractive target for therapy. We have recently reported that stress granules (SGs), non-membranous organelles consisting of protein and mRNA that form when cells are exposed to stress, constitute a novel stress-adaptive mechanism that is engaged by mutant KRAS cancer cells to enhance their fitness. We stratified mutant KRAS cancers based on their stress granule levels and found that high SG levels correlated with poor prognosis. We have observed that the growth of mutant KRAS tumors was significantly impaired when SG formation was compromised. Furthermore, SG inhibition sensitized mutant KRAS tumors to chemotherapy and led to tumor regression. The pro-tumorigenic and drug resistant function of SGs was mediated by their regulation of cell plasticity and stress resistance pathways. Together these data identify a dependency of mutant KRAS cancers on SGs for tumor progression and therapy response, and underscore the potential significance of SGs as a therapeutic target.

#2669

**A role for** HOXA5 **in the transcriptional response of ovarian and fallopian tube surface epithelial cells to norepinephrine.**

Sweta Dash,1 Anxhela Gjyshi,1 Ling Cen,1 Chia-Ho Cheng,1 Chaomei Zhang,1 Sean J. Yoder,1 Jamie K. Teer,1 Guillermo N. Armaiz-Pena,2 Alvaro N. Monteiro1. 1 _Moffitt Cancer Center, Tampa, FL;_ 2 _Ponce Health Sciences University/Ponce Research Institute, Ponce, PR_.

Chronic stress has been suggested to play a role in tumor initiation, progression and patient survival. Studies have shown that activation of the sympathetic nervous system and increased levels of stress-associated hormones such as norepinephrine can enhance migration, invasion and growth of ovarian cancer cells. However, the extent to which stress contributes to tumor initiation remains unknown. The purpose of this study was to explore the effects of norepinephrine on the transcriptional program of immortalized normal cell lines presumed to be the origin of epithelial ovarian cancers. We treated normal immortalized ovarian (iOSE) and fallopian tube (iFTE) surface epithelial cells with 10uM norepinephrine for 15 min, 1h and 4h and generated global transcriptomic profile of treated vs untreated cells by RNA-Seq. The most significant gene expression changes (P(adj) < 0.05) were observed at the 1h time point; with an overlap of 45 differentially expressed genes in both iOSE and iFTE cells. Genes in immune-related and developmental pathways were found to be highly enriched in the norepinephrine treated samples set. By the 4h time point, the number of differentially expressed genes had increased to 313 genes from 234 at 1h in iFTE cells, while it reduced to 34 genes from 53 in iOSE cells. Additionally, the number of differentially expressed genes common to iOSE and iFTE cells had reduced to 14. Gene ontology analysis of the differentially expressed genes revealed 'transcription factor' to be the most significantly overrepresented class (based on FDR value) at both 1h and 4h time-points. Promoter enrichment analysis identified HOXA5, GFI1 and FOXI1 transcription factor binding sites to be highly enriched (based on Z-score and Fisher-Score) at both time points. HOXA5 ranked consistently at the top in both cell lines and at both 1h and 4h time points. These observations suggest that HOXA5 may play a central role in regulating the early response to norepinephrine in normal ovarian and fallopian tube cells. We will further assess the function of HOXA5 through ChIP-Seq and shRNA- mediated silencing.

#2670

Spatial potassium buffering among clustered glioblastoma cells is proposed to enable survival scavenging of nutrients released by interior necrotic cells.

Marie Beckner. _Kent State University, Willoughby Hills, OH_.

Glioblastoma cells are known for surviving harsh environments and for forming secondary structures of Scherer (cell clusters around neurons/axons, blood vessels, regions of coagulative necrosis, & in the subpia). The astrocytic lineage of glioblastomas suggests recapitulation of astrocytic-type syncytia that buffer potassium released from neurons/axons. Here, multiple glioblastoma cell lines were stressed by nutrient withdrawal to observe cell behavior in view of their astrocytic lineage & known genome sequence of 1 cell line. Methods: Single cell suspensions of 4 glioblastoma cell lines (U87, LN229, T98G & A172) were added to a settling chamber (Neuro Probe, Gaithersburg, MD). After 0.5 hr of gradual nutrient withdrawal, slides (Diff Quik stain) were analyzed, 2 replicate wells per cell line. U87 cells with cytoplasmic vacuoles were quantified to indicate the degeneration in cell clusters. When vacuoles collectively had a diameter at least 1/3 of an ave. nuclear diameter in the same 100X microscopic field, the vacuolated cell was deemed +. Potassium ion channel genes potentially used by glioblastoma cells per the Online Mendelian Inheritance in Man (OMIM) database were checked in genome sequence results for U87 cells (Clark MJ, et al. PLoS Genet 2010 6(1): e10000832). Results: All glioblastoma cell lines displayed cell adherence & wrapping around single swollen, often heavily vacuolated degenerating cells of the same lineage after nutrient withdrawal. As clustered U87 cells (putative syncytia) increased in cell number, the number of constituent cells with vacuoles decreased (Pearson regressions, p=.04, p=.006) in 2 replicate wells. Whereas single cells were +, 72% and 51%, vacuolated cells fell to 18% and 11% in putative syncytia of over 20 cells in 100X microscopic fields across the width (7 mm) of each well. Thus, within increasing-sized putative syncytia, which offer increasing potassium buffering capacity, significantly fewer constituent cells displayed vacuoles. Presumably this was due to available nutrients from one to few necrotic cells in their midst during nutrient withdrawal. Potassium ion channeling within astrocytic-type syncytia, termed "spatial buffering" explains how viable glioblastoma cells adhere to degenerating cells despite high potassium levels. Genome sequencing of U87 cells revealed that only KCNMB2 & KCNJ16 of 47 potassium channeling genes pertinent to brain, were negatively affected by genetic defects. Functional losses of KCNMB2 as an enhancer for KCNMA1 (intact), and KCNJ16 as a co-assembler with other family members (intact), are unlikely to prevent potassium buffering. Preservation of nearly all potassium channeling genes in U87 suggests that maintenance of spatial buffering in astrocytic-type syncytia permits viable cells to surround necrotic cells and salvage released nutrients as a survival strategy.

#2671

Omics unveils a specific signature of tumor dormancy in two murine models of leukemia and melanoma.

Yasmine Touil,1 Loïc Lemonnier,2 Pascaline Segard,3 Martin Figeac,4 Frédéric Leprêtre,4 Audrey Vincent,3 Lucile Noyer,2 Maxence Wisztorski,5 Jean-Pascal Gimeno,5 Jean-Pascal Meneboo,4 Guillemette Marot,6 Isabelle Fournier,5 Thierry Idziorek,3 Bruno Quesnel7. 1 _Inserm UMRS- 1172 University of Lille, Lille, France;_ 2 _Inserm U1003, University of Lille, Lille, France;_ 3 _Inserm, Lille, France;_ 4 _University of Lille, Platform of Functional Genomics, Lille, France;_ 5 _Inserm U1192, PRISM, University of Lille, Lille, France;_ 6 _University of Lille, BiLille Bioinformatics Platform, Lille, France;_ 7 _Inserm UMR-S1172 University of Lille, Lille, France_.

The goal of the project was to integrate "omics" data, including genomic, epigenetic, transcriptomic and proteomic profiles, to identify a specific signature of tumor dormancy.

Materials and Methods: Two murine models of tumor dormancy, B16F1 malignant melanoma and DA1-3b myeloid leukemia, were generated and exome sequencing of dormant cells and parental cells was performed.Mutations were identified. Epigenetic alterations were detected by ChIP sequencing. Briefly, repressed and activated genes were identified by sequencing DNA fragments obtained after immunoprecipitation with antibodies against H3K9 and H3K27 and against histone H3K4me3, respectively. DNA Methylation analysis was assessed by Methylation Specific PCR (MSP). Transcriptomic analysis was conducted using Agilent arrays. Proteomes were compared by mass spectrometry. For the calcium homeostasis analysis, time courses of cytosolic Ca2+ concentration were measured using the ratiometric dye Fura-2.

Results: Exome analysis identified a specific gene signature of tumor dormancy in each model. Previously characterized mutations in human melanoma and myeloid leukemia were found in the murine models of B16F1 melanoma (e.g., Pten, Brca2, and CKit) and myeloid leukemia (e.g., Flt3 and Dnmt3b), thus reinforcing the relevance of these models for translational research on human pathology. ChIP sequencing revealed a specific epigenetic signature of dormant cells compared to parental cells. Global proteomic analysis showed a deregulation of proteins involved in many metabolic pathways in both models. Despite the absence of common gene mutations in the two models tested, several genes involved in calcium homeostasis were found to be mutated (Transient Receptor Potential, Store Operated calcium channels ). Regarding their epigenetic regulation assessed by MSP, no difference was observed in methylation level. Nevertheless, functional assays showed profound alterations in calcium homeostasis in the two dormant cell lines compared to the parental cell lines, suggesting a functional role of one or several receptors or ion channels in calcium signaling in tumor dormancy.

Conclusions: Taken together, "omics" analyses indicate that the regulation of tumor cell dormancy is extremely complex. Therefore, an integrated approach is crucial to fully understand tumor dormancy. Bioinformatics and integrative analyses will provide a better understanding of mechanisms controlling dormancy. Finally, functional analysis of calcium homeostasis revealed a common calcium signature in dormant tumor cells compared to parental cells. The genes involved in this altered intracellular calcium flux are currently being elucidated.

#2672

Effect of conjugating gemcitabine with novel temperature responsive polymer in improving drug targeting, retention, and release in the KPC mouse model of pancreatic cancer.

Jarwei Fang, Porakrit Leophairatana, Jeffrey Koberstein, Tamas Gonda, Chathuranga De Silva. _Columbia University, New York, NY_.

Introduction: Pancreatic cancer remains a devastating disease where most chemotherapies are modestly effective in part because of limited drug penetration into the stroma rich hypovascular tumors. Our goal was to develop a drug delivery mechanism that allows cancer specific release of cytotoxic agents and minimizes off-target effects. We have developed a gemcitabine-polyacetal drug conjugate that has a highly selective drug release profile via temperature response, pH-degradation, and ability for tuning reverse temperature transitions. The gemcitabine-polyacetal conjugates release drug selectively in acidic environments but remain inert in others.

Objective: Here, we present in vitro and in vivo data on the efficacy of gemcitabine-polyacetal drug conjugates in pancreatic cancer cell lines and KPC (KrasG12D/+;Trp53R172H/+;Pdx1-Cre) derived allografts.

Methods: The drug release of polyacetal-drug conjugates were previously characterized using Gel Permeation Chromatography and Nuclear Magnetic Resonance. In the current study, drug release is characterized using viability of KPC-derived cell lines and BxPC3 cells. We assessed the drug efficacy of solo-gemcitabine and gemcitabine-polyacetal drug conjugates at 24, 48, and 72-hour time points at two pHs: physiological pH using HEPES media, and PDAC tumor pH by phosphate buffer set at pH 6.8. Drug free polyacetal was used as a negative control.

Results: In the two KPC and one BxPC3 cell lines tested, statistically significant differences in cell viability were observed. Gemcitabine-polyacetal conjugate at pH 6.8 showed 3-6% cell viability at drug concentrations less than 5μmol within 24 hours, while the conjugate in HEPES media (at pH ~8.0) showed more than 90% cell viability up to 72 hours. In contrast, solo-gemcitabine showed 5-8% cell viability in both pH's. Percutaneous injection of 25μM dose of gemcitabine-polyacetal drug conjugate into allograft tumors showed a significant decrease in % of Ki67+ cells (p=0.0.009) compared to controls injected with PBS.

Conclusions: The gemcitabine-polyacetal conjugate serves as a drug deactivator in its conjugated form. In acidic pH, the drug is activated and released with high selectivity. This site-specific drug release could significantly reduce off-target effects that are reported with gemcitabine. Here, we showed significant effect on direct tumor injection of the conjugated gemcitabine. As previously shown, polyacetal-drug conjugates can be synthesized using dozens of chemotherapeutic drugs without chemical modification, making polyacetal conjugation a versatile approach to minimize off-target effects and ultimately systemic cytotoxicity in a variety of chemotherapeutic treatments. These results support further development of this delivery strategy in treatment of pancreatic cancer.

## CLINICAL RESEARCH

### Present and Future of Biomarkers for Anticancer Immunotherapy

#2673

Association of PD-L1 Combined Positive Score and Immune Gene Signatures With Efficacy of Nivolumab (NIVO) ± Ipilimumab (IPI) in Patients With Metastatic Gastroesophageal Cancer (mGEC).

Ming Lei,1 Nathan Siemers,1 Dimple Pandya,1 Han Chang,1 Teresa Sanchez,1 Cecile Dorange,1 Christopher Harbison,1 Peter M. Szabo,1 Yelena Janjigian,2 Patrick A. Ott,3 Padmanee Sharma,4 Johanna Bendell,5 Jeffry Evans,6 Filippo de Braud,7 Ian Chau,8 Zachary Boyd1. 1 _Bristol-Myers Squibb, Princeton, NJ;_ 2 _Memorial Sloan Kettering Cancer Center, NY;_ 3 _Dana-Farber Cancer Institute, Boston, MA;_ 4 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 5 _Sarah Cannon Research Institute at Tennessee Oncology, Nashville, TN;_ 6 _Beatson West of Scotland Cancer Centre, University of Glasgow, Glasgow, United Kingdom;_ 7 _Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy;_ 8 _The Royal Marsden NHS Foundation Trust, Sutton, Surrey, United Kingdom_.

Background: In the CheckMate 032 phase 1/2 study, NIVO ± IPI demonstrated clinically meaningful antitumor activity in patients with chemotherapy-refractory mGEC. Archival or fresh tumor biopsies were analyzed to determine whether expression of PD-L1 and selected immune gene signatures were predictive of response to NIVO ± IPI. Methods: Pooled analyses included all treatment regimens (NIVO 3 mg/kg, NIVO 1 mg/kg + IPI 3 mg/kg, NIVO 3 mg/kg + IPI 1 mg/kg, and patients treated with NIVO 1 mg/kg + IPI 1 mg/kg in the dose-escalation phase). PD-L1 immunohistochemistry (IHC; Dako PD-L1 IHC 28-8 pharmDx assay) was used to evaluate tumor PD-L1 expression, referred to as tumor proportion score (TPS). Combined positive score (CPS) was determined by evaluating PD-L1 expression on previously stained IHC slides using the CPS algorithm. Gene expression profiling (GEP) by RNA sequencing was used to evaluate immune cell activation and infiltration signatures, a Bristol-Myers Squibb (BMS) inflammatory signature, and PD-L1 gene expression. Results: The pooled CPS (N = 104), TPS (N = 130), and GEP (N = 40) cohorts were mostly representative of the overall CheckMate 032 mGEC cohort (N =163). At a median (range) follow-up of 23.4 (17.0-35.4) months, CPS at higher cutoffs correlated better with efficacy and had higher prevalence than TPS in all analyses (Table). For all immune gene signatures examined, responders had higher signature scores in aggregate. For the BMS inflammatory signature, the association between signature score and response was significant (P = 0.004; false discovery rate = 0.037). Conclusions: CPS demonstrated stronger association with efficacy of NIVO ± IPI than TPS in mGEC. Prevalence of CPS was higher than that of TPS. Among immune gene signatures examined, the BMS inflammatory signature achieved the best association with efficacy and warrants further investigation.

All treatments combined

---

Method | N | Cutoff | Response rate, % | Prevalence, %

All patients | 163 | NA | 9.8 | 100

CPS | 104 | 10 | 26.5 | 33

5 | 19.2 | 50

1 | 14.1 | 68

TPS | 130 | 10 | 9.1 | 8

5 | 7.7 | 10

1 | 17.5 | 31

NIVO 1 mg/kg + IPI 3 mg/kg

Method | N | Cutoff | Response rate, % | Prevalence, %

All patients | 49 | NA | 20.4 | 100

CPS | 33 | 10 | 54.5 | 33

5 | 41.2 | 52

1 | 28.0 | 76

TPS | 42 | 10 | 0 | 2

5 | 0 | 2

1 | 40.0 | 24

CPS = combined positive score (number of PD-L1-staining tumor cells, lymphocytes, and macrophages relative to all viable tumor cells x 100); IPI = ipilimumab; NA, not applicable; NIVO = nivolumab; TPS = tumor proportion score

#2674

Immune profiling of in NSCLC shows expression of PD-L1 in macrophages is better associated with outcomes than PD-L1 in tumor cells in PD-1 axis immunotherapy treated patients.

Yuting Liu, Jon Zugazagoitia, Brian Henick, Kurt A. Schalper, David L. Rimm. _Yale University, New Haven, CT_.

Background: The immune checkpoint target and companion diagnostic, PD-L1 is expressed in both tumor cell and immune cells and both are read by the pathologist and associated with response to therapy in different organ systems. Here, we examine the expression of PD-L1 using molecular assessment of cellular localization to determine which cell type carries the predictive value of the companion diagnostic test.

Methods: We used the quantitative immunofluorescence (QIF) methods (AQUA analysis of images from the PM2000 and inForm analysis of images from the PerkinElmer Vectra and Polaris Automated Quantitative Pathology Imaging System) and measured the expression of PD-L1 in multiple immune cell types by phenotyping each cell. We also acquired high resolution images from the multiplex for precise identification of co-localization using confocal microscopy. The PD-L1 localization was measured using two multiplex QIF panels including CD56 for natural killer (NK) cells, CD68 for macrophages and CD8 for cytotoxic T cells. This multiplex was performed on three retrospective Yale NSCLC cohorts (457 non-immunotherapy-treated cases and 62 PD-1 axis immunotherapy treated cases) represented in tissue microarrays.

Results: PD-L1 localization was significantly higher in macrophages compared to NK cells and CD8 T cells in both tumor and stroma compartment. In stroma, 80% of PD-L1 is expressed by macrophages, while 5% and 3% by NK cells and CD8 T cells, respectively. High CD8 level (top tertile) was associated with better overall survival (P <0.05) predominantly driven by the stroma CD8 (P <0.05), independent of sex, stage, smoking status, and age. Elevated PD-L1 in macrophages was associated with high PD-L1 level in tumor as well as CD8 level and CD68 level (P <0.05). High PD-L1 expression in CD68+ macrophages was correlated better overall survival (OS; P = .0448) while high PD-L1 expression in tumor cells was not.

Conclusion: In over 500 lung cancer cases the predominant immune cell type that expresses PD-L1 CD68 positive macrophages, as compared to NK cells and CD8 T cells. The level of PD-L1 in macrophages is significantly associated with the level of PD-L1 in tumor and with high levels of CD8+ T cells, suggesting a connection between high PD-L1 and "hot" tumors. Similar to our previous findings in melanoma, we find that high levels of PD-L1 in macrophages is a positive predictive biomarker for PD-1 pathway blockade therapy. Due to the small size of the treated cohort in the study, further work is required to confirm this observation.

#2675

Assessment of inflammation biomarkers in relation to clinical outcomes in nivolumab-treated patients with advanced hepatocellular carcinoma in CheckMate 040.

Ignacio Melero,1 Jaclyn Neely,2 Bruno Sangro,3 Richard Finn,4 Ghassan K. Abou-Alfa,5 Ann-Lii Cheng,6 Thomas Yau,7 Junji Furuse,8 Joong-Won Park,9 Samir Wadhawan,2 Hao Tang,2 Christine Delacruz,2 Carlos Baccan,2 Zach Boyd,2 Anthony El-Khoueiry10. 1 _Universidad de Navarra, Pamplona, Spain;_ 2 _Bristol-Myers Squibb, Princeton, NJ;_ 3 _Clinica Universidad de Navarra and CIBEREHD, Pamplona, Spain;_ 4 _University of California Los Angeles, Los Angeles, CA;_ 5 _Memorial Sloan Kettering Cancer Center, NY;_ 6 _National Taiwan University Hospital, Taipei, Taiwan;_ 7 _University of Hong Kong, Hong Kong, China;_ 8 _Kyorin University Faculty of Medicine, Tokyo, Japan;_ 9 _National Cancer Center, Goyang, Democratic People's Republic of Korea;_ 10 _USC Norris Comprehensive Cancer Center, Los Angeles, CA_.

Introduction: Nivolumab (NIVO), a programmed death-1 (PD-1) inhibitor, demonstrated durable responses, manageable safety, and promising long-term survival in patients (pts) with advanced hepatocellular carcinoma (HCC), regardless of etiology, with/without prior sorafenib treatment in CheckMate 040 (El-Khoueiry, Lancet 2017). NIVO is approved in several countries for the treatment of sorafenib-experienced pts with advanced HCC. We report findings on additional exploratory biomarker analyses from NIVO-treated pts with advanced HCC from CheckMate 040.

Methods: In CheckMate 040, pts with advanced HCC received NIVO monotherapy in the phase 1/2 dose-escalation (ESC; 0.1–10 mg/kg) and dose-expansion (EXP; 3 mg/kg) phases every two weeks, regardless of programmed death ligand 1 (PD-L1) status and HCC etiology. For pts in the ESC and EXP phases receiving NIVO 3 mg/kg, baseline tumor biopsy samples were analyzed using immunohistochemistry for the following markers: expression of PD-L1, PD-1, T-cell markers (CD3, CD4, CD8, FOXP3), and macrophages (CD68, CD163). Baseline tumor biopsy samples from a subset of pts in the ESC and EXP phases were evaluated using RNA sequencing to assess inflammatory signatures. Results were correlated with clinical outcomes: response (complete response [CR] + partial response [PR] vs stable disease [SD] or progressive disease [PD]) and overall survival (OS). Associations with characteristics such as etiology and geographical region (non-Asia vs Asia) were also assessed. The data cutoff date was June 2018.

Results: In pts with evaluable data (n = 184), increased tumor cell PD-L1 expression was significantly associated with response (CR+PR vs SD [P = 0.00009]; CR+PR vs PD [P = 0.0007]) and OS (P = 0.03). Increased PD-1 expression was also significantly associated with response (CR+PR vs SD [P = 0.05]; CR+PR vs PD [P = 0.009]) and OS (P = 0.05). Of the T-cell markers assessed, CD3 expression was significantly associated with response (n = 182, CR+PR vs SD; P = 0.05). In pts positive for CD3 or CD8, there was a trend towards improved survival (P = 0.08). No association between CD68- and CD163-expression and clinical outcomes was observed. For the subset of pts for whom RNA sequencing data was available (n = 37), the median Bristol-Myers Squibb (BMS) inflammatory signature score was significantly higher in pts with a PR vs SD (P = 0.05) and was correlated with improved OS (P = 0.01). For all of the inflammation markers assessed, there was no association with HCC etiology or geographical region.

Conclusions: In pts with advanced HCC, improved survival and response to nivolumab may be associated with higher PD-L1, PD-1 and CD3 expression, and higher BMS inflammatory signature scores. These data support the role of PD-1 inhibition in the treatment of HCC. Further investigation of these biomarkers is required.

#2676

Associations of peripheral biomarkers to outcomes to anti-PD-L1 immune checkpoint blockade in metastatic urothelial cancer.

Kobe C. Yuen, Vinita Gupta, Congfen Li, Deepali Rishipathak, Edward E. Kadel, Ying-Jiun J. Chen, Leonard D. Goldstein, Zora Modrusan, Sanjeev Mariathasan. _Genentech, SOUTH SAN FRANCISCO, CA_.

Introduction Immune checkpoint blockade such as inhibition of programmed death-1 (PD-1) or programmed death-ligand 1 (PD-L1) has been shown to be effective in metastatic urothelial cancer (mUC) and many other types of cancers. Despite its robust efficacy, a large proportion of patients still fail to show durable responses; therefore, it is important to understand the mechanisms that drive clinical responses, as well as the pathways associated with lack of response. Circulating peripheral biomarkers are particularly useful as readily accessible and minimally invasive markers to enable monitoring both tumor growth and responses to treatment.

Methods Baseline plasma from 337 patients treated with an anti-PD-L1 (atezolizumab) in the mUC Ph2 trial IMvigor210 (NCT02108652) were analyzed using a Myriad-RBM panel. To further characterize the findings, single-cell RNA sequencing (scRNAseq) was performed with PBMC isolated from responders and non-responders (n=5 each) to atezolizumab in the same trial. NanoString gene expression levels of the PBMC isolated from mUC patients in IMvigor210 was used to validate the identified signatures.

Results High levels of CXCL9 (HR: 0.692, CI: 0.529-0.903, p=0.006) and leptin (HR: 0.633, CI: 0.485-0.825, p=0.001) were associated with better overall survival (OS), whereas high levels of pro-inflammatory cytokines IL6 (HR: 2.744, CI: 2.084-3.612, p<0.0001) and CXCL8/IL8 (HR: 2.639, CI: 1.997-3.489, p=0.006) were associated with poor OS. In addition, high levels of angiogenesis and cell adhesion proteins such as TIMP1 (HR: 2.317, CI: 1.773-3.029, p<0.0001), VWF (HR: 1.571, CI: 1.207-2.045, p=0.001), and ICAM1 (HR: 1.349, CI: 1.034-1.76, p=0.026) correlated with poor OS. High plasma IL6 levels and high tumor IL6 expression are significantly enriched in non-responders in the T cell excluded phenotype, further suggesting association of IL6 with stroma/angiogenesis. Results from scRNAseq show response (CR/PR) to checkpoint blockade was associated with adaptive immune signature (GZMM, PSMB8, PSMB9) (FDR<0.0001) in lymphoid PBMC. Lack of response (SD/PD) was associated with complement coagulation (PF4, THBS, TIMP1) (FDR<0.0001) and innate inflammation (IL8, IL1B, IL18) (FDR=<0.0001) signatures in the myeloid cells. We confirmed these outcome-associated signatures with OS modeling with gene expression levels in PBMC.

Conclusion Peripheral biomarker analysis revealed that adaptive immune signatures to be associated better clinical outcome, whereas innate inflammation and angiogenesis signatures to be correlated with worse clinical outcome to anti-PD-L1 immunotherapy. The link between IL6 and angiogenesis in the tumor microenvironment will be further characterized. Understanding the genes and pathways associated with outcomes will shed light on peripheral biomarker development.

#2677

MSIClass - **Identification and classification of microsatellite unstable using cell-free DNA from ultra low pass sequencing.**

Yosef E. Maruvka,1 Ruslana Frazer,1 Jonna Grimsby,1 Carrie Cibulskis,1 Viktor Adalsteinsson,1 Ryan Corcoran,2 Gad Getz1. 1 _Broad institute, Cambridge, MA;_ 2 _Massachusetts General Hospital, Boston, MA_.

The acquisition of mismatch repair deficiency is common in many tumor types. A unique subtype of mismatch repair deficient tumors, called tumors with microsatellite instability (MSI), has been recently approved for treatment with PD-1\PD-L1 blockade immunotherapy. Therefore, developing a quick, inexpensive, and easy-to-implement technique for classifying tumors as MSI is of utmost importance. Here, we describe such a method using next-generation sequencing data, and show that it can detect MSI from cell-free DNA (cfDNA), even when there is low fraction of tumor DNA in the cfDNA.

We first analyzed the unique mutational features of MSI vs. microsatellite stable (MSS) tumors. Using our SignatureAnalyzer tool, we analyzed the signatures of the different insertions and deletions within microsatellite loci (MS-indels), and found that MSI tumors harbor elevated MS-indels of certain types, which we further validated using whole genome sequencing data from The Cancer Genome Atlas.

We next used this information to develop a method (MSIClass) that analyzes the WGS data of a given sample by capturing the features unique to MSI tumors. This method assigns each sample a score based on aggregating information from all MS loci simultaneously; since MSIClass is a sample-based tool, it can call the MSI status of tumor samples that have no patient-matched normal sample. We further demonstrate that MSIClass requires minimal genomic information; indeed, sequencing coverage as low as 0.05x (current sequencing cost of ~$2/sample) is sufficient for accurate classification by MSIClass.

Using in silico and in vitro DNA mixing we show that MSIClass can accurately identify MSI cases in very low purity cases, at 0.1% tumor DNA (using 0.5x sequencing coverage). We show that MSIClass, applied on cfDNA, could accurately classify 29 colon cases -- 22 MSS and 7 MSI cases, 3 of which are early stage disease and with low tumor content.

These results demonstrate that MSIClass is an inexpensive MSI test that can be applied in various cases: bulk sequencing where a matched normal is not available, cases of very low purity tumors, and even using cfDNA from blood in cases a biopsy is not available or possible. Finally, this methodology can be the basis for monitoring minimal residual disease, and potential be used for early detection of MSI tumors.

#2678

A clonal expression biomarker improves prognostic accuracy: TRACERx lung.

Dhruva Biswas,1 Nicolai J. Birkbak,2 Rachel Rosenthal,1 Crispin T. Hiley,1 Emilia L. Lim,3 Krisztian Papp,4 Marcin Krzystanek,5 Dijana Djureinovic,6 Yin Wu,1 David A. Moore,1 Marcin Skrypski,1 Christopher Abbosh,1 Maise Al Bakir,3 Thomas B. Watkins,3 Selvaraju Veeriah,1 Gareth A. Wilson,3 Mariam Jamal-Hanjani,1 Arul M. Chinnaiyan,7 Patrick Micke,6 Jiri Bartek,5 Istvan Csabai,4 Zoltan Szallasi,5 Javier Herrero,1 Nicholas McGranahan,1 Charles Swanton3. 1 _UCL, London, United Kingdom;_ 2 _Aarhus University, Aarhus, Denmark;_ 3 _Francis Crick Institute, London, United Kingdom;_ 4 _ELTE Eötvös Loránd University, Budapest, Hungary;_ 5 _Danish Cancer Society Research Center, Copenhagen, Denmark;_ 6 _Uppsala University, Uppsala, Sweden;_ 7 _University of Michigan, Michigan, MI_.

At the point of cancer diagnosis, molecular biomarkers aim to stratify patients into precise disease subtypes predictive of outcome independent of standard clinical parameters such as tumour stage. Although prognostic gene expression signatures have been derived for many cancer types, seldom have they been shown to improve therapeutic decision making, limiting their clinical use. While intra-tumour transcriptomic heterogeneity (RNA-ITH) has been shown to bias existing biomarkers, efforts to control for this biological parameter have not been considered in biomarker development. Here, we analyse multi-region RNA-seq and whole-exome data for 156 tumour regions from 48 TRACERx patients to explore RNA-ITH in NSCLC. We show that chromosomal instability is a major driver of RNA-ITH, through the generation of heterogeneous copy number events within tumours, and that existing prognostic gene expression signatures are vulnerable to sampling bias. To address this issue, we develop the Outcome Risk Associated Clonal Lung Expression (ORACLE) assay, comprised of genes expressed homogeneously within individual tumours but heterogeneously between patients. These genes are enriched in modules associated with cell proliferation, such as mitosis and nucleosome assembly, that are often selected for through copy number gain events occurring early in tumour evolution. Our approach to identify "clonal" transcriptomic biomarkers in NSCLC overcomes tumour sampling bias, improves survival risk forecasting over current clinicopathological risk factors, and may be generalised to other cancer types, whilst revealing the early evolutionary selection of high risk DNA copy number events driving poor clinical outcome.

#2679

Integration of DNA repair deficiency and immune biomarkers to predict which early stage triple negative breast cancer patients are likely to respond to platinum containing regimens vs. immunotherapy: The neoadjuvant I-SPY 2 trial.

Denise M. Wolf,1 Christina Yau,1 Julia Wulfkuhle,2 Emanuel Petricoin,2 Lamorna Brown-Swigart,1 Gillian Hirst,1 Smita Asare,3 I-SPY 2 Consortium, Douglas Yee,4 Angela DeMichele,5 Hope Rugo,1 Olufunmilayo Olopade,6 Rita Nanda,6 Minetta Liu,7 Laura Esserman,1 Laura van t' Veer1. 1 _University of California, San Francisco, CA;_ 2 _George Mason University, Fairfax, VA;_ 3 _QuantumLeap Healthcare Collaborative, San Francisco, CA;_ 4 _University of Minnesota, Minneapolis, MN;_ 5 _University of Pennsylvania, Philadelphia, PA;_ 6 _University of Chicago, Chicago, IL;_ 7 _Mayo Clinic, Rochester, MN_.

Background: Triple negative breast cancer (TNBC), generally considered aggressive with few options beyond standard chemotherapy, is currently experiencing a period of optimism, with multiple trials showing increased efficacy of platinum containing regimens (with and without PARP-inhibition), and more recently, immunotherapy. In I-SPY 2, veliparib/carboplatin (VC) and the immune checkpoint inhibitor pembrolizumab (Pembro) graduated in the TN subtype. As part of the I-SPY 2 biomarker program, we previously identified the signature PARPi7, a measure of DNA repair deficiency (DRD), as predicting response to VC. As well, immune signatures, including one representing dendritic cells, predicted response to Pembro. Here we investigate the overlap between these biomarkers to identify patient subgroups more likely to respond to immunotherapy or platinum-based therapy and estimate response rates to VC and Pembro in patients positive for one, both, or neither biomarker.

Methods: 153 TNBC patients (Control: 85; VC: 39; Pembro: 29) were considered in this analysis. Continuous DNA repair deficiency and immune signatures assayed at the pre-treatment time point were evaluated as published (Wolf, 2017; Danaher, 2017). To identify optimal dichotomizing thresholds, 2-fold cross-validation was repeated 500 times. Bayesian logistic regression was used to estimate pCR rates by arm for patient subsets defined by biomarker combinations. Our study is exploratory with no claims for generalizability; and this analysis does not adjust for multiplicities.

Results: Using the optimal dichotomizing cutpoints, 54% of TNBC patients are classified as DRD+, and 67% Immune+. As expected, DRD+ patients have a high estimated pCR rate to VC (75%); and Immune+ patients have a high estimated pCR rate to Pembro (82%). Combining these biomarkers, 40% of TNBC are positive for both biomarkers, 40% for only one biomarker (26% Immune+/DRD-, 14% Immune-/DRD+), and 20% for neither. The Immune+/DRD+ TN subset predicted sensitive to both VC and Pembro has a higher estimated pCR rate to both VC and Pembro relative to control (estimated pCR rates: VC: 75%, Pembro: 73% vs. control: 18%). In contrast, the Immune+/DRD- group, predicted to be sensitive to Pembro, has the highest pCR rate to Pembro (VC: 48%, Pembro: 83% vs. control 21%), whereas the Immune-/DRD+ group is most responsive to VC (VC: 63%, Pembro: 41% vs. control 29%). For the 20% of Immune-/DRD- TNBC patients, their estimated response rates are lower than the biomarker-positive patients (VC: 27%, Pembro: 43% vs. control: 19%).

Conclusion: Molecular phenotypes capturing immune activation and DNA repair deficiency may predict response of TNBC and help prioritize selection of platinum or immunotherapy containing regimens.

## EPIDEMIOLOGY

### Exposures and Genetics in Cancer Risk

#2680

Intake of whole grain and dietary fiber and risk of hepatocellular carcinoma among U.S. adults.

Wanshui Yang,1 Yanan Ma,1 Yue Liu,1 Smith-Warner Stephanie,2 Tracey G. Simon,3 Dawn Chong,4 Jeffrey A. Meyerhardt,5 Edward L. Giovannucci,6 Andrew T. Chan,6 Xuehong Zhang6. 1 _Harvard Medical School, Boston, MA;_ 2 _Harvard T.H. Chan School of Public Health, Boston, MA;_ 3 _Massachusetts General Hospital, Boston, MA;_ 4 _National Cancer Centre Singapore, Singapore, Singapore;_ 5 _Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA;_ 6 _Brigham and Women's Hospital and Harvard Medical School, Boston, MA_.

Background Higher intake of whole grain and dietary fiber has been associated with lower risk of insulin resistance, hyperinsulinemia, and inflammation, which are known predisposing factors for hepatocellular carcinoma (HCC). To our knowledge, no epidemiological study has yet examined the association between whole grain intake and HCC risk. Therefore, we hypothesized that long-term intake of whole grain and dietary fiber may be associated with lower risk of developing HCC.

Methods We tested this hypothesis by utilizing data from two large U.S. prospective cohorts, the Nurses' Health Study and Health Professionals Follow-up Study. Intake of whole grain and its subcomponents (bran and germ), and dietary fiber (cereal, fruit, and vegetable) were collected and updated almost every 4 years using validated food frequency questionnaires. Time-varying Cox proportional hazards regression model was utilized to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) of of HCC by tertiles of energy-adjusted whole grain and fiber, after adjusting for sex, race, age, body mass index, alcohol drinking, smoking, physical activity, regular aspirin use, and type 2 diabetes.

Results During up to 32 years of follow-up, we identified a total of 141 incident HCC cases. Higher whole grain intake was significantly associated with lower risk of HCC (the highest versus lowest tertile intake, HR=0.63, 95% CI: 0.41-0.96, Ptrend=0.04). A suggestive inverse HCC association was found for total bran (HR=0.70, 95% CI: 0.46-1.07, Ptrend=0.11), but not for germ. After mutual adjustment for bran and germ, the inverse association between total bran and HCC persisted (HR=0.66, 95% CI: 0.41-1.07, Ptrend=0.13). Increasing intake of cereal fiber (HR=0.68, 95% CI: 0.45-1.03, Ptrend=0.07), but not fruit or vegetable fiber, was also suggestively associated with reduced risk of HCC. In sensitivity analyses, the results did not materially change after excluding HCC cases with hepatitis B or C virus infections. In addition, we observed similar inverse associations for whole grain, bran and germ, or dietary fiber with risk of HCC subtypes by history of cirrhosis (i.e., cirrhotic versus non-cirrhotic HCC).

Conclusions Intake of whole grains, cereal fiber, and bran could be associated with reduced risk of HCC among U.S. adults. Future studies that carefully consider hepatitis B and C virus infections are needed to replicate our findings, to examine these associations in other racial or high-risk populations, and to elucidate the underlying mechanisms.

#2681

**Prediagnostic levels of urinary 8-** epi **-prostaglandin F** 2α **and prostaglandin E** 2 **metabolite, biomarkers of oxidative damage and inflammation, and risk of hepatocellular carcinoma.**

Jian-Min Yuan,1 Menno Grouls,2 Steven G. Carmella,3 Renwei Wang,1 Alicia Heskin,3 Yang Jiang,4 Yuting Tan,5 Jennifer Adams-Haduch,1 Yu-Tang Gao,5 Stephen S. Hecht3. 1 _Univ. of Pittsburgh, Pittsburgh, PA;_ 2 _Waginengen University and Research, Droevendaalsesteeg 4, Netherlands;_ 3 _Univ. of Minnesota, Minneapolis, MN;_ 4 _ETH Zurich, Zurich, Switzerland;_ 5 _Shanghai Cancer Institute, Shanghai, China_.

Background: Chronic inflammation causes persistent liver injury and consecutive regeneration, potentially leading to fibrosis and cirrhosis, and consequently, to the development of hepatocellular carcinoma (HCC). Prostaglandin E2 (PGE2) is one of the major end-products of the cyclooxygenase-2 (COX-2) pathway, an enzyme that is an important mediator of inflammation. Oxidative stress, which results from the generation of reactive oxygen species (ROS) by environmental risk factors or cellular mitochondrial dysfunction, may be involved in the progression of chronic liver disease to the development of HCC. 8-epi-prostaglandin F2α (8-epi-PGF2α) is a product of lipid peroxidation, which has been recognized as a specific, chemically stable, quantitative marker of systemic and integrated measure of oxidative stress.

Method: We evaluated the associations for urinary levels of 8-epi-PGF2α and PGE-M, a metabolite of PGE2, with HCC risk in a prospective cohort of 18,244 men in Shanghai, China. After 25 years of follow-up, 347 participants developed HCC. For each case, two control subjects were chosen and matched on age (±2 years), date of sample collection (±1 months), and neighborhood of residence. Urinary 8-epi-PGF2α and PGE-M were quantified using validated liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) assays. The logistic regression method was used to calculate the odds ratio (OR) and its 95% confidence interval (CI) associated with higher levels of both urinary 8-epi-PGF2α and PGE-M.

Results: 8-epi-PGF2α levels were significantly higher in HCC cases than control subjects (geometric means 0.92 vs 0.80 pmol/mg creatinine, P <0.001). The OR of HCC for the highest relative to the lowest quartile of 8-epi-PGF2α were 2.51 (95% CI 1.60 - 3.93, P trend < 0.001) after adjustment for age, cigarette smoking, alcohol consumption, liver cirrhosis, and hepatitis

1 B surface antigen. This association remained significant after excluding all HCC cases diagnosed within the first 10 years of follow-up. The present study did not find any significant association between urinary PEG-M and HCC risk.

Discussion: 8-Isoprostanes have been found to be significantly elevated in patients with non-alcoholic steatohepatitis (NASH). Our findings support a significant role of oxidative stress in the development of HCC, especially in those without traditional risk factors. The null finding on PGE-M and HCC risk is consistent with results of epidemiological studies that did not show a statistically significant association for HCC risk with use of COX-2 inhibitors.

#2682

Analysis of polygenic risk score interaction with coal use and risk of lung adenocarcinoma among never-smoking women in Asia.

Batel Blechter,1 Chao Agnes Hsiung,2 Zhihua Yin,3 Xiao-Ou Shu,4 H. Dean Hosgood,5 Jason Y.Y Wong,6 Jianxin Shi,6 Wei Hu,6 Bryan Bassig,6 Wei Jie Seow,7 Yu -ang Gao,8 Qiuyin Cai,4 Yong-Bing Xiang,9 I-Shou Chang,10 Baosen Zhou,3 Wei Zheng,4 Kyoung-Mu Lee,11 Stephen Chanock,6 Nilanjan Chatterjee,1 Nathaniel Rothman,6 Qing Lan6. 1 _Johns Hopkins Bloomberg School of Public Health, Baltimore, MD;_ 2 _National Health Research Institutes, Zhunan, Taiwan;_ 3 _China Medical University School of Public Health, Shenyang, China;_ 4 _Department of Medicine, Vanderbilt University Medical Center and Vanderbilt-Ingram Cancer Center, Nashville, TN;_ 5 _Albert Einstein College of Medicine, Bronx, NY;_ 6 _National Cancer Institute, Rockville, MD;_ 7 _Saw Swee Hock School of Public Health, National University of Singapore and National University Health System, Singapore;_ 8 _Shanghai Cancer Institute, Shanghai, China;_ 9 _Shanghai Cancer Institute & Shanghai Jiaotong University School of Medicine, Shanghai, China; _10 _National Institute of Cancer Research, National Health Research Institutes, Zhunan, Taiwan;_ 11 _Korea National Open University, Seoul, Republic of Korea_.

Background: Accounting for approximately 1.76 million annual deaths worldwide, lung cancer is a significant global health burden. While smoking is the most common cause of lung cancer, up to 25% of all lung cancer patients worldwide are never smokers. Lung cancer is the leading cause of cancer mortality in China, where most women do not smoke, making women in Asia an ideal population to study. Previously conducted genome-wide association studies (GWAS) of lung cancer risk among never-smoking women in Asia identified 10 lung cancer susceptibility loci. Indoor air pollution from coal burned for home cooking and heating is known to contain lung carcinogens and has been found to be causally associated with lung cancer. In the current analysis, we evaluated gene-environment interaction between a polygenic risk score (PRS) and coal use in relation to lung adenocarcinoma.

Methods: Three studies (Taiwan, Shanghai, Shenyang) from the Female Lung Cancer Consortium in Asia (FLCCA) were used for the primary analysis (1,419 cases; 1,446 controls). A replication study was conducted using samples from Xuanwei, China (159 cases; 572 controls), where lung cancer rates for never-smokers are among the highest in the world and attributed to widespread coal use. We calculated a PRS as the weighted sum of the risk allele counts across the 10 loci, and modeled PRS as a continuous variable scaled by the standard deviation in controls. Logistic regression was used to estimate the main effects of the PRS and coal use, and a likelihood ratio test was used to evaluate the interaction. Models were adjusted for age (<40, 40-49, 50-59, 60-69, ≥70 years), study, and significant eigenvectors.

Results: Coal use was associated with an increased risk of lung adenocarcinoma (OR=1.31, 95% CI: 1.01-1.68). We observed an exposure-response relationship between PRS and lung adenocarcinoma (p-trend= 2x10-16) and found a significant multiplicative interaction between PRS and coal use (p-interaction= 0.005). The association between PRS and lung adenocarcinoma was significantly higher among the never coal users (OR=1.68, 95% CI: 1.52-1.86) compared to ever coal users in the three studies (OR=1.24, 95% CI: 1.03-1.50) (p-interaction=0.005), as well as between never coal users in the three studies and ever coal users in Xuanwei (OR=1.25, 95% CI: 1.04-1.49) (p-interaction=0.004).

Conclusion: We observed an antagonistic interaction between PRS and coal use with lung adenocarcinoma, where the genetic effect was attenuated among those exposed to coal combustion in the home. We replicated the finding in Xuanwei. These results suggest that the pathogenesis of lung cancer among never-smoking women in Asia differs by exposure to coal combustion emissions and provides one of the few examples of sub-multiplicative gene-environment interactions in the cancer literature.

#2683

Biomarkers of glucose homeostasis and inflammation and risk of prostate cancer: A case-cohort study.

Ying Wang,1 Peter T. Campbell,1 Victoria L. Stevens,1 Christina C. Newton,1 Eric J. Jacobs,1 Michael Pollak,2 Susan M. Gapstur1. 1 _American Cancer Society, Atlanta, GA;_ 2 _McGill University, Montreal, Quebec, Canada_.

Background: Type 2 diabetes (T2DM) is associated with a lower risk of prostate cancer especially low-grade and localized prostate cancer. The association with aggressive prostate cancer is inconclusive. Few studies have directly examined the associations of diabetes biomarkers with prostate cancer risk. Methods: Among a cohort of 13,994 men who were on average 70.2 years (SD=5.5) and cancer free at blood draw between 1998 and 2001, 1,206 developed non-aggressive prostate cancer and 313 developed aggressive prostate cancer (AJCC stage 3-4 or Gleason score 8-10) during follow-up through June 2013. We conducted a case-cohort study including a random sub-cohort of 1,303 men, 391 non-aggressive cases and 313 aggressive cases to examine circulating hemoglobin A1c (HbA1c), c-peptide, and C-reactive protein (CRP) in relation to prostate cancer risk.

Results: HbA1c, a biomarker of hyperglycemia, was inversely associated with non-aggressive prostate cancer after adjusting for age, race, family history of prostate cancer, prostate-specific antigen screening, smoking, alcohol intake, physical activity, and total energy intake (relative risk (RR) per unit increase: 0.89, 95% confidence interval (CI): 0.79-1.00, P=0.04). Further adjustment for body mass index (BMI) attenuated the association. In contrast, HbA1c was associated with a higher risk of aggressive prostate cancer after adjusting for the covariates including BMI (RR per unit increase: 1.11, 95% CI: 0.98-1.25, P=0.11). In an analysis combining self-reported T2DM and HbA1c, men with T2DM and poor blood glucose control (HbA1c ≥6.5%) had a lower risk of developing non-aggressive prostate cancer (RR: 0.53, 95% CI: 0.30-0.95) than men without T2DM and good blood glucose control (HbA1c <6.5%). After excluding men with self-reported T2DM, HbA1c was statistically significantly positively associated with risk of aggressive prostate cancer (RR per unit increase: 1.25, 95% CI: 1.04-1.51, P=0.02) but was not associated with non-aggressive prostate cancer. C-peptide and CRP were not associated with risk of either aggressive or non-aggressive prostate cancer.Conclusions: The present study suggests that men with hyperglycemia may have a lower risk of developing non-aggressive but a higher risk of developing aggressive prostate cancer. Further studies are needed to examine the role of hyperglycemia in the etiology of aggressive prostate cancer and in prostate cancer progression.

#2684

Identification of 14 novel genetic loci for testicular germ cell tumor susceptibility.

Louise C. Pyle,1 John Pluta,1 Kevin T. Nead,1 Nandita Mitra,1 Javier Benitez,2 D. Timothy Bishop,3 Victoria Cortessis,4 Alberto Ferlin,5 Jourik Gietema,6 Mark Greene,7 Tom Grotmol,8 Ramneek Gupta,9 Rob Hamilton,10 Michelle A. Hildebrandt,11 Trine B. Haugen,12 Lambertus Kiemeney,13 Christian Kubisch,14 Davor Lessel,14 Paloma Martin,15 Thorunn Rafnar,16 Lorenzo Richiardi,17 Rolf Skotheim,18 Clare Turnbull,19 Fredrik Wiklund,20 Tongzhang Zheng,21 Ewa Rajpert-De Meyts,22 Stephen M. Schwartz,23 Katherine A. McGlynn,7 Peter A. Kanetsky,24 Katherine L. Nathanson,1 Testicular Cancer Consortium (TECAC). 1 _University of Pennsylvania, Philadelphia, PA;_ 2 _Spanish National Cancer Research Centre (CNIO), Madrid, Spain;_ 3 _University of Leeds, Leeds, United Kingdom;_ 4 _University of Southern California, Los Angeles, CA;_ 5 _University of Padova, Padova, Italy;_ 6 _University Medical Center Groningen, Groningen, Netherlands;_ 7 _National Cancer Institute, Bethesda, MD;_ 8 _Cancer Registry of Norway, Oslo, Norway;_ 9 _Technical University of Denmark, Lyngby, Denmark;_ 10 _University of Toronto, Toronto, Ontario, Canada;_ 11 _MD Anderson Cancer Center, Houston, TX;_ 12 _Oslo Metropolitan University, Oslo, Norway;_ 13 _Radboud University Medical Centre, Nijmegen, Netherlands;_ 14 _University Medical Center Hamburg-Eppendorf, Hamburg, Germany;_ 15 _Spanish National Cancer Centre (CNIO), Madrid, Spain;_ 16 _deCode Genetics, Reykjavik, Iceland;_ 17 _CPO Piemonte and University of Turin, Turin, Italy;_ 18 _Oslo University Hospital, Oslo, Norway;_ 19 _Institute of Cancer Research; Queen Mary University, London, United Kingdom;_ 20 _Karolinska Institutet, Solna, Sweden;_ 21 _Brown University, Providence, RI;_ 22 _Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark;_ 23 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 24 _H. Lee Moffitt Cancer Center, Tampa, FL_.

Testicular germ cell tumors (TGCT) are ideally suited to agnostic genome-wide association methods because of their known high heritability and homogeneous cell of origin. Here we report the most updated results from the international Testicular Cancer Consortium (TECAC) that extends published findings from our recent meta-analysis, which included 3,558 cases and 13,970 controls from five centers. To these existing samples, we added summary statistics from 300 cases and 151,991 controls provided by deCODE combined with imputed genotypes from an additional 5,602 cases and 5,006 controls assembled from 15 TECAC centers, almost all (97%) of which were centrally genotyped using the Illumina Human Core array. A total of 9,458 TGCT cases were included in our current meta-analysis, representing a 66% increase since our last publication. We identified 14 new and confirmed 41 previously reported loci that surpassed genome-wide significance (5 × 10-8). Fifteen previously reported loci did not reach this threshold, although one did so in subgroup analysis. In addition, 57 new loci attained nominal significance (5 × 10-8 ≤ p ≤ 1 × 10-5). As expected, a substantial proportion of top hits continue to map to genetic regions belonging to biological pathways essential to male germ cell development, sex determination, and chromosomal segregation. For example, newly identified genes include anaphase promoting complex subunit 2 (ANAPC2), BCL2 like 11 (BCL211), anti-Mullerian hormone receptor type 2 (AMHR2), and DEP domain containing mTOR interacting protein (DEPTOR). Our findings also further implicate genes located on the X chromosome, specifically the androgen receptor (AR). In analyses stratified by tumor subtype, we identified GATA-binding protein 4 (GATA4) as a susceptibility locus among men diagnosed with seminoma. Despite a similar effect size observed among men diagnosed solely with non-seminoma (i.e. excluding cases with a mixture of both seminoma and non-seminoma tumors), this marker did not surpass genome-wide significance in this subtype likely because of the smaller case sample size. GATA4 previously has been associated with overall risk of TGCT (Litchfield et al., Nat Genet. 2017). Biological mechanisms for all genome-wide significant loci are currently being explored using publicly available data sets, ATAC-seq of four TGCT cell lines, and SPATIaL-Seq (a novel high-resolution chromatin conformation capture (3C) assay) of the NTERA2 pluripotent human embryonal carcinoma cell line. Additional genotyping of top hits in an independent sample set of 1,103 TGCT cases and 1,210 controls is currently ongoing.

#2685

Identification of circulating protein biomarkers for colorectal cancer risk: A genetic instrument analysis.

Xiang Shu,1 Xiao-ou Shu,1 Jirong Long,1 Qiuyin Cai,1 Conghui Qu,2 Stephanie L. Schmit,3 Chenxu Qu,4 Sonja I. Berndt,5 Peter T. Campbell,6 Andrew T. Chan,7 Graham G. Giles,8 Andrea Gsur,9 Michael Hoffmeister,10 Mark A. Jenkins,11 Sanford D. Markowitz,12 Li Li,13 Gad Rennert,14 Kenneth Offit,15 David Conti,16 Annika Lindblom,17 Graham Casey,18 Stephen B. Gruber,16 Ulrike Peters,2 Wei Zheng1. 1 _Vanderbilt University Medical Center, Nashville, TN;_ 2 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 3 _H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL;_ 4 _USC Norris Comprehensive Cancer Center, Los Angeles, CA;_ 5 _National Cancer Institute, National Institutes of Health, Bethesda, MD;_ 6 _American Cancer Society, Atlanta, GA;_ 7 _Massachusetts General Hospital and Harvard Medical School, Boston, MA;_ 8 _Cancer Council Victoria, Melbourne, Australia;_ 9 _Institute of Cancer Research, Medical University Vienna, Vienna, Austria;_ 10 _German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 11 _Melbourne School of Population and Global Health, The University of Melbourne, Melbourne, Australia;_ 12 _Case Comprehensive Cancer Center, Case Western Reserve University, and University Hospitals of Cleveland, Cleveland, OH;_ 13 _Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH;_ 14 _Lady Davis Carmel Medical Center, Haifa, Israel;_ 15 _Memorial Sloan-Kettering Cancer Center, New York, NY;_ 16 _USC Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA;_ 17 _Karolinska University Hospital, Stockholm, Sweden;_ 18 _University of Virginia, Charlottesville, VA_.

Colorectal cancer (CRC) is one of the most common cancers in United States and many countries in the world. There is an urgent need to better understand its etiology and identify novel biomarkers to facilitate the early detection of CRC. Previous studies either lacked sufficient power to identify novel biomarkers or provided inconsistent results. In the current study, we attempted to uncover novel protein biomarkers for CRC through an integrated analysis of genomics and proteomics data. We first constructed study instruments using genetic variants identified from a large-scale protein quantitative trait loci (pQTL) analysis for over 1,400 circulating proteins. We used beta coefficients and standard errors for these pQTL variants from two large consortia of European-ancestry populations, the Colorectal Transdisciplinary (CORECT) Study (11,895 cases and 14,659 controls) and the Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO) (22,974 cases and 14,392 controls) for association analyses for genetically predicted protein levels with CRC risk using an inverse-variance weighted method. The genetically predicted levels of six proteins were associated with CRC risk after accounting for multiple comparisons (Benjamini-Hochberg FDR < 0.05). Among them, genetically predicted levels of VCAM-1, MIP-3b and LPH were inversely associated with CRC risk (VCAM-1: odds ratio [OR] per unit of increase = 0.65, Pmeta = 6.2 ×10-11; MIP-3b: OR = 0.68, Pmeta = 5.5×10-7; LPH: OR = 0.93, Pmeta = 8.0×10-5), while BMP-6, CRDL2 and laminin were positively associated (BMP-6: OR = 1.57, Pmeta = 3.0 ×10-9; CRDL2: OR = 1.36, Pmeta = 3.0 ×10-9; laminin: OR = 1.13, Pmeta = 6.7 ×10-5). Except for VCAM-1 (rs3184504, 12q24.12-SH2B3), other associations are not accounted for by any known CRC susceptibility variants. We observed a possible biological link connecting the genetic variants of LAMC1, LAMC1 expression, and laminin concentrations to CRC risk. Our study identifies potential novel biomarkers for CRC risk and provides novel insight into the disease etiology.

#2686

Associations between a polygenic risk score and risk of multiple myeloma and its precursor.

Alyssa I. Clay-Gilmour,1 Michelle A. Hildebrandt,2 Nicola J. Camp,3 Elad Ziv,4 Elizabeth E. Brown,5 Jonathan N. Hofmann,6 John J. Spinelli,7 Graham G. Giles,8 Parveen Bhatti,9 Wendy Cozen,10 Xifeng Wu,2 Dennis P. Robinson,1 Aaron D. Norman,1 Jason P. Sinnwell,1 Shaji K. Kumar,1 S Vincent Rajkumar,1 Susan L. Slager,1 Celine M. Vachon1. 1 _Mayo Clinic, Rochester, MN;_ 2 _MD Anderson Cancer Center, Houston, TX;_ 3 _Huntsman Cancer Institute & University of Utah, Salt Lake City, UT; _4 _University of California San Francisco, San Francisco, CA;_ 5 _University of Alabama at Birmingham, Birmingham, AL;_ 6 _National Cancer Institute, Bethesda, MD;_ 7 _BC Cancer Agency-Cancer Control Research (BCCRC), British Columbia, Canada;_ 8 _Cancer Council Victoria, Australia;_ 9 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 10 _Keck School of Medicine-University of Southern California, CA_.

Genome-wide association studies (GWAS) conducted among populations of European ancestry (EA) have identified 23 common single nucleotide polymorphisms (SNPs) associated with multiple myeloma (MM) risk. We hypothesize that the combination of these SNPs in a polygenic risk score (PRS) is likely to be a strong risk factor for MM. However, it is unclear whether the genetic variation associated with MM susceptibility also predisposes to monoclonal gammopathy of undetermined significance (MGUS). Thus, we calculated a PRS and evaluated the association with risk of MM and its precursor, MGUS.

We pooled genotype data for 2434 MM and 3446 controls from ten MM GWAS of individuals of EA within the Interlymph Consortium, for 23 MM risk SNPs identified by prior GWAS. An additional 754 MGUS cases were ascertained from Mayo Clinic and MD Anderson clinical practices. To calculate the PRS, we used the risk estimates corresponding to the 23 SNP associations from the largest published MM GWAS. The log of the odds ratio (OR) for each SNP was multiplied by the respective number of risk alleles and summed to generate a PRS for each individual. The PRS was examined continuously, per one standard deviation (SD), and as quintiles, based on the PRS distribution in the controls. Associations of PRS with MM and MGUS risk were examined separately, using multivariable logistic regression assuming an additive model to assess ORs and 95% confidence intervals adjusted for age, sex, and site. We also evaluated age and sex stratified models.

The distribution of sex within MM cases, MGUS cases and controls were each ~60% male and ~40% female. The median age was 61, 66, and 66 years for MM cases, MGUS cases and controls, respectively. PRS ranged from 1.52-4.91, with a median PRS of 3.21 for MM cases, 3.19 for MGUS cases, and 3.05 for controls. PRS was significantly associated with MM risk when assessed continuously (OR=1.19 per SD, p=2.2x10-16) and categorically; compared with the middle quintile (Q3), individuals in the highest quintile (Q5) had a 66% increased MM risk (OR=1.66, p=2.3x10-9) and those in the lowest quintile (Q1) had a 38% decreased MM risk (OR=0.62, p=1.3x10-6). PRS was also significantly associated with MGUS risk (OR=1.19 per SD, p=1.7x10-11); individuals with the highest PRS (Q5) had a 77% increased risk (OR=1.77, p=4.0x10-4) and those with lowest PRS (Q1) had 30% decreased risk (OR=0.70, p=0.04), compared with Q3. When stratified by age and sex, similar associations and trends were found.

Using an independent sample of MM / MGUS cases and controls, we showed that a PRS constructed from 23 common genetic variants for MM risk is associated with risk of both MM and MGUS, regardless of age or sex. A future direction of this work is testing associations with PRS and clinical characteristics of the MM cases, as well as differences between MGUS cases that progress and those that do not. Our results suggest that common genetic variation may predispose to MGUS as the precursor to MM.

## EXPERIMENTAL AND MOLECULAR THERAPEUTICS

### Novel Therapeutic Agents and Screening Approaches

#2687

Dolasynthen - A novel, homogeneous Auristatin F hydroxypropyl amide antibody-drug conjugate platform.

Dorin Toader, Marc Damelin, Anouk Dirksen, Shawn P. Fesler, Scott D. Collins, Barrett J. Nehilla, Jian Xu, Ling Xu, Kalli C. Cattcott, Alex Uttard, Winnie Lee, Susan Clardy, Cheri A. Stevenson, LiuLiang Qin, Patrick R. Conlon, Mariya V. Kozytska, Chen-Ni Chin, David H. Lee, Timothy B. Lowinger. _Mersana Therapeutics, Inc., Cambridge, MA_.

Dolasynthen is a novel, fully synthetic, structurally homogeneous platform that enables the construction of ADCs with tunable drug-to-antibody ratios (DAR), from a low of 2 to a high of 24. The resulting ADCs exhibit excellent physicochemical properties and fully homogeneous conjugates can be created through a variety of bioconjugation chemistries. Analogous to our first platform, Dolaflexin®, Dolasynthen is loaded with the proprietary payload Auristatin F hydroxypropylamide (AF-HPA) with precisely defined numbers of the cytotoxin per Dolasynthen scaffold.Studies that evaluate the tolerability and efficacy of Dolasynthen in preclinical models are described herein. ADCs containing a range of DAR values were generated following conjugation of Dolasynthen to two different monoclonal antibodies. The DAR of the ADCs was achieved by controlled reduction of native disulfide bonds in IgG1 antibodies, chromatographic fractionation, or through use of site-specific conjugation technologies. ADCs with both DAR6 and DAR12 were evaluated in vitro and also in vivo in the mouse, rat and monkey, for efficacy, tolerability and PK. Dolasynthen conjugates had excellent physicochemical properties and displayed the expected cell binding and in vitro cytotoxicities. In vivo pharmacology of Dolasynthen ADCs in in vivo xenograft models showed dose-dependent tumor growth inhibition at low mg/kg mAb doses. Tolerability in the rat at multiple doses was determined, including histopathological evaluation. Dolasynthen ADCs showed excellent PK characteristics in mouse, rat and NHP. Overall, the Dolasynthen platform appears to offer significant potential for clinical application.

#2688

SGN-CD228A: A novel humanized anti-CD228 antibody-drug conjugate for the treatment of solid tumors.

Sharsti L. Sandall, Marsha Mason, Devra Olson, Rebecca Mazahreh, Disha Sahetya, Lori Westendorf, Chris Leiske, Brian Schimpf, Liem Nguyen, Madhu Katepalli, Esther Trueblood, Christopher Hale, Albina Nesterova, Jason Wall, Timothy S. Lewis. _Seattle Genetics, Bothell, WA_.

Melanotransferrin (CD228/MFI2/MELTF) is a cell-surfaced glycosylphosphatidylinoitol (GPI)-anchored glycoprotein that belongs to the transferrin family of iron-binding proteins. CD228 was first described as an oncofetal protein highly expressed on malignant melanoma cells. Data from The Cancer Genome Atlas (TCGA) suggests that CD228 has broad expression across many types of carcinomas and it was recently described as a potential biomarker of invasive colorectal carcinoma. In this study, we characterize protein expression of CD228 using an immunohistochemical (IHC) assay and describe pre-clinical antitumor activity of SGN-CD228A, a potent CD228-targeting antibody-drug conjugate (ADC). We found that in addition to melanoma, CD228 is highly expressed in mesothelioma, non-small cell lung (NSCL), breast, colorectal, and pancreatic carcinomas. Monoclonal antibodies (mAbs) specific for human CD228 were evaluated and a lead antibody was selected based on binding characteristics, internalization properties, and cytotoxic activity as an ADC. SGN-CD228A is a humanized anti-CD228 mAb to which eight molecules of MMAE, a potent microtubule disrupting cytotoxic drug, have been conjugated via a β-glucuronidase-cleavable linker, which incorporates a PEG side chain and self-stabilizing maleimide to achieve homogenous conjugation with decreased plasma clearance and increased preclinical antitumor activity. Interestingly, when evaluating drug linkers, we found that changing the linker from a di-peptide to β-glucuronidase resulted in a striking improvement in the cytotoxicity of MMAE, likely due to unique trafficking and recycling of CD228. We examined 50 carcinoma cell lines and found 41 had >10,000 CD228 receptors per cell of which 60% had EC50 values <10ng/ml, and 30% had EC50 values between 10-100ng/ml when treated with SGN-CD228A in vitro. We also evaluated antitumor activity of SGN-CD228A in melanoma and NSCLC xenograft and PDX models. In the Colo-853 and Sk-Mel-5 melanoma models, a single dose of 0.33 mg/kg caused tumor delay whereas 1.0 mg/kg resulted in 5/8 and 4/8 durable complete responses (CRs), respectively. Similarly, in the squamous NSCLC model, Calu-1, a single dose of 1.0 mg/kg produced 6/6 durable responses (DRs). Similar results were obtained in NSCLC PDX models with 3 out of 4 models achieving tumor delay or DRs when dosed with 1.0 mg/kg and durable (CRs) at 3.0 mg/kg. Additionally, in a mouse TNBC PDX clinical trial (n=22), we found that SGN-CD228A achieved durable CRs even in low expressing PDX models. In summary, CD228 is a highly expressed carcinoma target and the novel glucuronide-MMAE ADC, SGN-CD228A, shows potent antitumor activity in vitro and in vivo.

#2689

High-content imaging and single-cell analysis of drug response ex vivo is predictive of clinical outcome for hematological cancer patients.

Gregory I. Vladimer,1 Berendd Snijder,2 Nikolaus Krall,1 Christoph Kornauth,3 Stefan Kubicek,4 Ulrich Jäger,3 Philipp B. Staber,3 Giulio Superti-Furga4. 1 _Allcyte, Vienna, Austria;_ 2 _ETH Zurich, Zurich, Switzerland;_ 3 _Medical University of Vienna, Vienna, Austria;_ 4 _CeMM, Center for Molecular Medicine, Vienna, Austria_.

The ability to predict clinical drug response is a fundamental goal of precision cancer medicine. Genetics has performed well in identifying the presence of targets and stratifying patients into clinical trials. However, particularly for late-stage and relapsed cancer patients, many genetic driven trials have yet to report improvements in clinical response. In parallel, directly measuring cytotoxicity of drugs ex vivo in patient samples with functional technologies has been gaining traction during the past five years, with some programs showing translational success.

In our approach, we use high throughput and high content microscopy to quantify single-cell phenotypic biomarkers of cancer cells, and healthy cells, that are indicative of cell death in order to determine a differential drug response. Cancer cells and healthy cells are distinguished using fluorescently-labeled extracellular-marker targeted diagnostic antibodies, and no long term ex vivo culture is necessary as the assay lasts only 24 hours. The cytotoxicity of cancer cells under drug treatment can be scored and compared to any cell death occurring in healthy cells within the same patient sample. As all is assessed by imaging of each individual cell within the same sample and set-up, the healthy cells of the patients can be used as internal controls, statistical analysis covers millions of events and images can be re-mined and re-analysed in future.

In a prospective basket trial testing 140 drugs in triplicate at two concentrations (768 tests, yielding 15.000 images per patient), we ranked drugs prospectively for late-stage patients diagnosed with hematological cancers. In this interim analysis, 15 (88%) of 17 patients receiving guided treatment had an overall response compared with four (24%) of 17 patients with their most recent regimen. 12 (71%) of 17 patients had a progression-free survival ratio of 1·3 or higher, and median progression-free survival increased by four times, from 5.7 weeks to 22.6 weeks. While continued follow-up of the study is warranted, this clinical use case foreshadows the success of functional drug screening using single-cell imaging, and further, big data medicine.

A unique byproduct of this big data medicine approach, is large pan-indication off-labeling drug-to-patient maps. These maps describe both on- and off-target drug responses, but also indication areas that lack ex vivo drug response - potentially enabling clinical re-use of drugs, and elucidating mechanism of action of drugs where it is unknown. The functional screening data over many patients, indications, and drugs, combined with clinical response data, can eventually be used for larger predictive studies.

#2690

Massively parallel multiplexed methods to screen hundreds of barcoded cancer cell line models with small molecules or genetic perturbations using next-generation sequencing.

Samantha Bender,1 Cong Zhu,1 Li Wang,1 Michael Rothberg,1 Joshua Dempster,1 Brenton Paolella,1 Mustafa Kocak,1 Massami Laird,1 Jordan Rossen,1 Karolina Stumbraite,1 Jordan Bryan,2 Vickie Wang,1 John Doench,1 Francisca Vazquez,1 Aviad Tsherniak,1 Todd Golub,1 Jennifer Roth1. 1 _Broad Institute, Cambridge, MA;_ 2 _Duke University, Durham, NC_.

Phenotypic screening is a valuable tool to identify compounds to treat cancer, but is limited as it is time and resource intensive to screen hundreds of cancer cell lines. In order to increase the throughput of phenotypic screening, we set out to expand and further develop the PRISM method (Profiling Relative Inhibition Simultaneously in Mixtures) [Yu et al., 2016]. PRISM is a method to barcode cell lines with a unique 24 nucleotide barcode and mix them together to screen simultaneously in a pool, decreasing the time and cost of screening. The previously described PRISM method was limited in that it used only 100 adherent cell lines of one cancer model context, it required the use of a highly specialized Luminex bead-based detection system, and it was only applicable to small molecule screening.

Here we report on the expansion of our barcoded cell line collection to 800 cancer cell lines of over 45 lineages, an improved method called PRISMseq using next-generation sequencing, and a novel method to perform genetic perturbations in 500 cancer cell lines simultaneously called PRISPR (PRISM/CRISPR). PRISMseq allows for assaying compound cell line sensitivity profiles in a large pool of hundreds of cell lines and detecting the DNA barcodes using next-generation sequencing (NGS). We recapitulate the known biology for established oncology drugs like the BRAF, EGFR, BCR-ABL and MDM2 inhibitor classes using this method with our cell line panel. We also further extended this method to be applicable to genetic perturbation using CRISPR/Cas9 knockout of individual genes concurrently in hundreds of cancer cell lines. We are able to recover expected biomarkers or genetic dependencies for our CRISPR/Cas9 validation reagents.

This expanded PRISM profiling approach increases statistical power due to the addition of cancer contexts in our cell line collection, and improves versatility by enabling the screening of both small molecules and genetic perturbations. We believe that this method has overcome the limitations of the original PRISM method. It has also overcome the limitations of phenotypic screening, as the resources required to screen hundreds of cell lines has been decreased by orders of magnitude.

#2691

4D printing of programmable smart material for drug screening in patient derived organoids.

Chen Yang,1 Michelle Chadwick,2 Howon Lee,1 Hatem E. Sabaawy2. 1 _Rutgers-The State University of New Jersey, Piscataway, NJ;_ 2 _Rutgers-The Cancer Inst. of New Jersey, New Brunswick, NJ_.

Numerous studies have contributed to a paradigm shift in cancer modeling, moving from traditional two-dimensional (2D) culture to three-dimensional (3D) sphere and organoid cultures, to recapitulate key aspects of in vivo multicellular, anatomical and functional organ complexity. In parallel, biomaterial 3D printing advances allowed generating devices, implants, and scaffolds for tissue engineering and drug delivery systems. Despite these advances, platforms that integrate genomic, proteomic, and bioprinting innovations for high-throughput screening (HTS) or selection of effective personalized cancer treatments are scarce. Here, we utilized cell-based 3D organoids with four-dimensional (4D) printing of smart biomaterial to generate devices and platforms for personalized and HTS of drugs. Because genomic assays alone are insufficient to identify therapeutic options for most patients, we integrated genomic sequencing, patient derived organoids (PDOs), histological and immunohistochemical (IHC) assessments with evaluating tumor cell responses to therapies. First, we developed a novel 4D printed temperature-sensitive smart material consisting of interconnected basket arrays that fit into 96-well plates. The temperature-responsive 4D-printed basket arrays were fabricated using projection micro-stereolithography (PμSL), with a spatial light modulator used as a reconfigurable photomask, and projection through reduction lenses into photo curable shape polymer resin. These 4D-printed baskets are 3.4 mm-deep and have a 1.8 mm-opening, into which cells, media and matrices could be plated (manually or automated) to generate 3D spheres and organoids, to be treated with different drugs. We have utilized these 4D-printed basket arrays to generate PDOs from glioblastoma (GBM) tissues that were subjected to exome sequencing. Single cell derived GBM were allowed to form PDOs for 2 weeks, followed by 72-hour treatments with various compounds, such as temozolomide, rapamycin, PI3K and BMI-1 inhibitors, among others. Following treatments, the entire 4D printed basket arrays were evolved, with a 10-min heating step at 50ºC, to their programmable cassette size to directly perform histological and IHC validation on the same day. PDOs were examined for cell proliferation by quantifying intracellular adenosine triphosphate (ATP), tumor invasiveness and pathway-specific markers to assess drug efficacy. This novel platform allowed the entire patient tissue and drug response assessment to be completed in ≤20 days. When including exome and/or single cell sequencing, histological, IHC and targeted therapeutic assays, our basket array platform could offer dynamic, automated, and quantitative drug analyses, thus allow the discovery of novel preclinical therapeutic approaches that can be assessed in clinical trials, and provides personalized therapies for individual patients in precision medicine.

#2692

Preclinical development of a novel, highly selective PI3Kδ inhibitor, IOA-244, for the treatment of solid malignancies.

Katherine Ewings,1 Amy MacQueen,1 Pritom Shah,1 Anna Tsapara,2 Evangelia Papakonstanti,2 Lars van der Veen,3 Michael Lahn,3 Zoe Johnson3. 1 _CRUK, Cambridge, United Kingdom;_ 2 _Medical School, University of Crete, Heraklion, Greece;_ 3 _iOnctura SA, Geneva, Switzerland_.

The Class I Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) family is an attractive target class for the treatment of hematological and solid malignancies. In addition to the direct effects of PI3Kδ inhibition on B cell malignancies, inhibiting PI3Kδ preferentially targets regulatory T cells and myeloid derived suppressor cells, thus breaking tumor-induced immune tolerance and restoring anti-tumor immunity. To date, the development of selective inhibitors of the Class I PI3K family has been hampered by the lack of isoform specificity as well as safety issues. IOA-244 is a novel, highly selective, orally bioavailable PI3Kδ inhibitor, with the unique property of being ATP non-competitive; these characteristics make IOA-244 the ideal drug to explore the hypothesis that PI3Kδ inhibition can modulate anti-tumor immunity as a monotherapy and in combination in clinical trials.

In mouse models, IOA-244 inhibited tumor growth when combined with either anti-PD-1 or anti-PD-L1. In the CT26 model the composition of the tumour infiltrating lymphocytes showed a marked decrease in the suppressor cell populations, i.e. Tregs and MDSCs, as well as tumor associated macrophages (TAMs). Conversely a concomitant increase in natural killer cells as well as the ratio of cytotoxic CD8\+ T cells: Treg cells was observed. Furthermore, in vitro studies using primary human T cells demonstrated a selective and concentration-dependent suppression of Treg cells by IOA-244 whilst leaving CD8\+ T cell proliferation intact.

In addition to effects on the immune system, we are exploring the potential of IOA-244 to mediate direct anti-cancer effects on solid tumors with a high expression of PIK3CD. IOA-244 induced a concentration-dependent inhibition of p-AKT in MDA-MB-231 cells. Furthermore, we demonstrate strong anti-proliferative activity by IOA-244 on hepatocellular tumor lines in vitro. Activity in these and other cell and patient derived lines is being studied, both in xenograft in vivo studies and in vitro models.

Based on GLP toxicology studies, IOA-244 has the potential to be a best-in-class PI3Kδ inhibitor with a safety and pharmacokinetic profile that is amenable for use alone and in combination with immunotherapies for the treatment of solid malignancies. IOA-244 is poised to enter phase I clinical testing in selected cancer indications in the first half of 2019.

#2693

Targeting the Hippo-YAP pathway with novel small molecule inhibitors of the YAP-TEAD transcription activity.

Tracy T. Tang, Andrei W. Konradi, Ying Feng, Xiao Peng, Sofie Qiao, Leonard Post. _Vivace Therapeutics, San Mateo, CA_.

We have discovered and are developing multiple novel small molecule medicines targeting the Hippo-YAP pathway. Consisting of upstream regulatory components, the Hippo core kinase components (MST1/2, MAP4K1-7, and LATS1/2), and the downstream transcriptional machinery (YAP, TAZ, and TEAD), the mammalian Hippo-YAP pathway is involved in the regulation of cell proliferation, survival, and cell migration. It functions normally to control tissue regeneration and homeostasis and limit organ size/shape. Genetic alterations of pathway components have been reported in a variety of human malignancies. YAP activity is also linked to resistance to targeted therapies and cancer relapse. Through high-throughput phenotypic screens, we have identified novel classes of small molecule compounds that inhibit the YAP-TEAD transcription activity. Medicinal chemistry efforts on lead optimization resulted in highly potent inhibitors that block YAP-TEAD transcription activity at single digit nM. The classes of inhibitors that were demonstrated to interact directly with the TEAD transcription factors by thermal shift assay, co-crystal structures, and inhibition of autopalmitoylation of purified recombinant TEAD proteins will be presented. In cell-based assays, these inhibitors block TEAD palmitoylation in cells and selectively inhibit proliferation of NF2-deficient mesothelioma. In vivo, complete growth inhibition of NF2 mutant xenograft tumors can be achieved at doses that are well tolerated in mice without adverse effect on body weight. With the discovery and development of potent TEAD inhibitors, we bring forth a new generation of cancer therapeutics targeting a thus far undruggable pathway that is dysregulated in many cancers and critically associated with targeted therapy resistance.

### Preclinical Drug Development

#2694

A first-in-class small molecule modulator of the p300/CBP interactome exhibits broad efficacy in xenograft models of solid tumors and hematological malignancies.

Bernd Hentsch,1 Valentino Cattori,1 Marc Labelle,2 Ulrich Kessler1. 1 _Inthera Bioscience AG, Waedenswil, Switzerland;_ 2 _ML Pharma Consulting LLC, Bedford, NH_.

Background: Dysregulation of the cellular transcription machinery is a fundamental feature of cancer. E1A binding protein (p300) and CREB binding protein (CBP) are two closely related paralog transcription co-activators involved in the expression of oncogenic drivers in cancer cells.

Methods: Small molecule ligands of the CH1/TAZ1 domain of p300/CBP were rationally designed and optimized for their anti-proliferative activity in vitro. Subsequent characterization focused on their ability to modulate downstream biological processes controlled by p300/CBP, including hormone-dependent androgen receptor signalling, the HIF-1 alpha/VEGF pathway and reactivation of p53 in HPV-positive carcinomas.

The in vivo efficacy of this novel compound class was assessed in a variety of patient and cell line derived xenograft models, and pharmacodynamic effects were investigated.

Results: Several modulators of p300/CBP exhibited anti-proliferative effects at low nanomolar concentrations in more than 60 cancer cell lines inducing both senescence and apoptosis in a highly context dependent manner. Transcriptome analyses revealed the downregulation of various drivers of cell cycle progression.

Once daily oral administration triggered strong growth-inhibitory effects in several xenograft models up to complete tumor eradication at well tolerated doses. p300/CBP dependent biomarkers like VEGF (colon cancer) and PSA (prostate cancer) were significantly downregulated in the tumors of treated animals. In an orthotopic model of acute myeloid leukemia, the growth of primary tumors and metastasis was almost completely inhibited.

Conclusions: Reprogramming the transcriptional profile of cancer cells by modulation of p300/CBP activity by targeting the CH1/TAZ1 domain represents a novel and broadly applicable approach for the treatment of cancer. The presented data illustrate the clinical potential of this new class of therapeutics against a broad variety of tumor types, including prostate and colon cancer as well as hematological malignancies.

#2695

Direct targeting of NOTCH transcription complex by a novel small molecule CB-103 circumvents dose limiting toxicities associated with pan-NOTCH inhibitors.

Rajwinder Lehal,1 Jelena Zaric,1 Michele Vigolo,1 Charlotte Urech,2 Maximilien Murone,2 Freddy Radtke1. 1 _EPFL, Lausanne, Switzerland;_ 2 _Cellestia Biotech AG, Basel, Switzerland_.

NOTCH signalling is a key development pathway whose aberrant activation plays an onco-driver role in human cancers. In human tumors the NOTCH pathway can be activated by various genetic lesions such as over expression of ligands/receptors, GOF mutations in NOTCH receptors, chromosomal translocations, or loss-of-function mutations in the negative regulators of the pathway. Activation of NOTCH due to above mentioned mechanisms can be addressed in part using blocking antibodies against NOTCH ligands/receptors or small molecule inhibitors of the gamma secretase enzyme (GSIs). In addition to their limitations in targeting tumors harbouring chromosomal translocations in NOTCH receptors, the use of specific blocking antibodies against NOTCH receptors/ligands allows targeting of only a narrow spectrum of NOTCH positive tumors. On the other hand, GSIs act as pan-NOTCH inhibitors and thereby are able to target human tumors positive for NOTCH1-NOTCH4 receptors. However, clinical development of these pan-NOTCH inhibitors is hindered due to dose limiting toxicities (DLTs) associated with GSIs. The main DLTs associated with GSIs have been diarrhoea and vomiting due induction of goblet cell metaplasia in intestine, where NOTCH is known to regulate proliferation and differentiation of intestinal stem cells. A sustained blockage of NOTCH signalling leads to an inhibition of stem cell proliferation and differentiation of stem cells into mucous producing goblet cells. The effect on stem cell proliferation and differentiation is mediated via a subset of specific NOTCH target genes. Effect on proliferation is mostly driven by downstream target genes such as HES1, 3, and 5, cMYC and BMI1. The effect on stem cell differentiation is predominantly mediated by repression of MATH1. Therefore, MATH1 upregulation upon NOTCH inhibition is a pre-requisite to induce goblet cell metaplasia. Given the role of NOTCH signalling in human cancers and DLTs associated with 1st and 2nd generation NOTCH targeting agents (GSIs and blocking Abs), there is a need to develop novel pan-NOTCH inhibitors able to circumvent these DLTs. We have previously reported discovery and development of a novel class of pan-NOTCH inhibitors that blocks signalling by directly targeting the NOTCH transcription complex.

Here we present further in vivo characterization of the development candidate CB-103. A comprehensive in vitro study demonstrated that CB-103 acts as a pan-NOTCH inhibitor. Furthermore, in vitro and in vivo pharmacological studies show that CB-103 circumvents DLTs, namely goblet cell metaplasia, associated with GSIs, due to the novel mode of action. We present molecular evidence that due to differential regulation of downstream target genes, CB-103 fully engages the NOTCH pathway in intestine and spares MATH1 gene repression and thereby does not cause goblet cell metaplasia.

#2696

Genetic and pharmacological evaluation of the ubiquitin ligase CBL-B as a small molecule, tumor immunotherapy target.

Jennifa Gosling,1 Ryan Rountree,1 Asad Taherbhoy,1 Chenbo Wang,1 Thomas Cummins,1 Frederick Cohen,1 Hiroko Tanaka,1 Dahlia Weiss,1 Mario Cardozo,1 Christopher Karim,1 May Tan,1 Joseph Juan,1 Austin Tenn-McClellan,1 Szerenke Kiss von Soly,1 Julie Sheung,1 Kathleen Boyle,1 Ketki Dhamnaskar,1 Katherine Kurylo,1 Jilliane Bruffey,1 Jennifer McKinnell,1 Dane Karr,1 Andria Christianson,2 Anne-Renee Van Der Vuurst de Vries,2 Pallavur Sivakumar,2 Mark Gallop,1 Paul A. Barsanti,1 Anjanabha Saha,1 Neil F. Bence,1 Christoph W. Zapf1. 1 _Nurix Therapeutics, San Francisco, CA;_ 2 _Celgene Corporation, Seattle, WA_.

E3 ubiquitin ligases play critical roles in directing cellular protein fate by controlling the specificity of ubiquitin conjugation to substrate proteins and targeting them for cellular relocalization or degradation by the ubiquitin proteasome system. The E3 ubiquitin ligase CBL-B is expressed in immune cell lineages and negatively regulates activity of the T-cell receptor (TCR) by imposing a requirement for a costimulatory signal to mount a productive immune response upon TCR engagement. Mice deficient in Cbl-b, and more specifically in the RING Zn-finger ligase domain of Cbl-b, demonstrate a tumor rejection phenotype mediated by CD8+ T cells (Paolino et al., JI, 2011). We have reproduced these results and demonstrate that Cbl-b deficient mice show enhanced anti-tumor activity. In addition, we show that CD4+ and CD8+ T cells from mice deficient in Cbl-b have 5 to 10-fold enhanced secretion of IL-2 and IFN γ when stimulated ex vivo. These data provide a genetic rationale for the development of a small molecule inhibitor of CBL-B ligase activity for use in patients with tumor-mediated immune suppression of effector T cells.

We have identified a series of small molecule inhibitors of CBL-B activity with biochemical potency at low nanomolar concentrations. CBL-B inhibitors increased cytokine secretion in vitro at low nanomolar concentrations, as measured by IL-2 and IFN γ secretion, in primary human and mouse T cells stimulated with CD3/CD28 or CD3 alone. The compounds also stimulated proliferation and elevated levels of the T cell surface activation markers CD25 and CD69. CBL-B inhibitors enhanced an antigen recall response in human PBMCs ex vivo, as measured by approximately 5-fold higher secretion of GM-CSF, TNF-α and RANTES, and demonstrated effects in an ex vivo model of exhausted T cell function.

Oral dosing of an optimized CBL-B inhibitor enhanced anti-CD3 stimulated T cell activation in mouse CD4+ and CD8+ T cells, demonstrating a dose proportional pharmacodynamic effect. Oral administration over 28 days in the syngeneic CT-26 tumor model was well tolerated and resulted in single agent tumor growth inhibition.

These data support the continued advancement of small molecule oral CBL-B inhibitors for future development in immuno-oncology.

#2697

YPN005, an oral CDK7 inhibitor, exhibits a significant antitumor activity in Myc-driven cancers.

Kwang-Ok Lee, Jakyung Yoo, Mi Jung Lee, Kang Woo Lee, Ji Eun Min, Jinhwan Kim, Ki-Nam Min, Tae Chul Roh, Kang-Sik Seo, Hae In Rhee, Jun Hee Lee, Da-Hye Jeon, Dae Seong Lim. _Yungjin Pharm, Suwon-si, Gyeonggi-do, Republic of Korea_.

Background: CDK7 plays an important role in regulating cell cycle progression and gene via activation of cell cycle kinases (CDK1, CDK2, CDK4 and CDK6) and RNA polymerase II (PolII). Recent studies indicate that the inhibition of CDK7 is an attractive strategy for the treatment of cancer by down-regulation of c-Myc expression. (Wang et al 2018) We have investigated the therapeutic efficacy of YPN005, a novel oral CDK7 inhibitor, in triple negative breast cancer (TNBC) and hepatocellular carcinoma (HCC).

Methods: We evaluated antiproliferative activity of YPN005 on TNBC and HCC cells and the expression of RNA PolII and c-Myc was studied by Western Blot analyses to investigate the mechanism of action. To identify a biomarker, we examined the correlation between the level of c-Myc expression and anticancer activity of YPN005 in vitro using HCC cells. The therapeutic efficacy of YPN005 was evaluated in TNBC xenograft mouse model.

Results: YPN005 significantly inhibited the proliferation of TNBC and HCC cells and IC50s was determined as 10-20 nM and 5-30 nM range, respectively. Inhibition of cell proliferation was accompanied by a decrease in the levels of RNA PolII phosphorylation and c-Myc expression. Western Blot analyses revealed that the sensitivity of HCC cells to YPN005 was correlated with the level of c-Myc expression. In vivo xenograft model of TNBC showed that oral daily administration of YPN005 for 21 days (once, and 10mg/kg) efficiently inhibited the growth of tumor. All mice survived during the dosing period without significant changes of the hematologic profiles.

Conclusion: We propose that oral administration of YPN005, an orally available CDK7 inhibitor, could be a potent and attractive approach to treat the Myc overexpressing cancers.

#2698

eFT226, a potent and selective inhibitor of eIF4A, is efficacious in preclinical models of lymphoma.

Peggy A. Thompson, Boreth Eam, Nathan P. Young, Sarah Fish, Joan Chen, Maria Barrera, Haleigh Howard, Eric Sung, Ana Parra, Jocelyn Staunton, Gary G. Chiang, Christopher J. Wegerski, Andres Nevarez, Jeff Clarine, Samuel Sperry, Alan Xiang, Chinh Tran, Christian Nilewski, Garrick K. Packard, Theodore Michels, Paul A. Sprengeler, Justin T. Ernst, Siegfried H. Reich, Kevin R. Webster. _eFFECTOR Therapeutics, San Diego, CA_.

Dysregulated messenger RNA (mRNA) translation drives the pathogenesis of multiple hematological malignancies. In lymphoma this includes the upregulation of key driver oncogenes and anti-apoptotic proteins (e.g., MYC, CCND1/3, BCL2 and MCL1) that contain a highly structured 5'-untranslated region (UTR) in their mRNA requiring enhanced eIF4A helicase activity for translation. eIF4A is a component of the eIF4F translation initiation complex and catalyzes the ATP-dependent unwinding of RNA duplexes and facilitates 43S ribosome scanning within the 5'-UTR. The activation of oncogenic signaling pathways, including RAS and PI3K, enhance eIF4A activity through phosphorylation of eIF4B, eIF4G and PDCD4 which facilitates formation of eIF4F and full activation of eIF4A. The PI3K/AKT/mTOR pathway is frequently activated in lymphoma, promoting the translation of oncogenes with complex 5'-UTRs that are required for tumor cell proliferation, survival and metastasis.

eFT226 is a potent and sequence selective eIF4A1 inhibitor that promotes eIF4A1 binding to specific 5'-UTR polypurine and/or G-quadraplex recognition motifs leading to a selective block in ribosome mRNA scanning. The sequence dependency of eFT226 translational inhibition was evaluated in cell-based reporter assays demonstrating >100-fold greater sensitivity for reporter constructs containing a polypurine motif in the 5'-UTR (IC50 ~2 nM). Direct binding studies also confirmed the formation of a stable ternary complex with increased drug residence time between eFT226, eIF4A1 and RNA oligonucleotides containing polypurine motifs. The ability of eFT226 to inhibit MYC or MCL1 expression was found to be dependent on the presence of their respective 5'-UTR supporting a translational regulation mechanism dependent on recognition elements within the 5'-UTR.

eFT226 shows potent anti-proliferative activity (GI50 < 15 nM) against a panel of B-cell lymphoma cell lines. Treatment with eFT226 leads to coordinated inhibition of MYC, CCND1/3, BCL2 or MCL1 protein expression resulting in significant anti-tumor activity. eFT226 has good pharmacokinetic properties and exhibits significant in vivo activity across a panel of diffuse large B cell lymphoma (DLBCL), and Burkitt lymphoma tumor models with ≤1 mg/kg/week IV administration. Further evaluation of predictive markers of sensitivity or resistance has shown that tumors with mTOR mediated activation of eIF4A are most sensitive to eFT226. In addition, tumors with PTEN mutations do not exhibit activated eIF4A and are generally resistant to induction of apoptosis by eFT226, resulting in reduced in vivo efficacy. The association of eFT226 activity with PI3K/mTOR pathway activation and mutational status provides a means to identify patient subsets during clinical development. Clinical trials in patients with lymphoma and other malignancies are planned.

#2699

Targeting an ubiquitin-activating enzyme in small cell lung cancer (SCLC).

Safa Majeed,1 Mansi K. Aparnathi,2 Lifang Song,2 Aaron D. Schimmer,2 Ming S. Tsao,2 Geoffrey Liu,2 Benjamin H. Lok2. 1 _University of Toronto, Toronto, Ontario, Canada;_ 2 _Princess Margaret Cancer Centre/University Health Network, Toronto, Ontario, Canada_.

Background: Despite valiant efforts to improve patient outcomes, small cell lung cancer (SCLC) (~15-17% of lung cancer) remains an aggressive lung cancer, with an overall five-year survival of 7%. Current first-line therapy (chemotherapy+/-radiation) has not changed in 30+ years. TAK-243, an inhibitor of the ubiquitin activating enzyme (UAE), E1 (encoded by UBA1), poses a novel approach for cancer therapy.

TAK-243 limits the formation of ubiquitin conjugates that ultimately mediate many cellular processes, including DNA repair signalling and protein degradation. Consequently, TAK-243 may disrupt cancer-specific dependencies of UAE to induce malignant cell death, and potentiate radiation-induced DNA damage. The discovery publication revealed that TAK-243 is especially potent in 2 SCLC cell-lines compared to healthy breast and fibroblastic tissues. With these limited data, the potential benefit of this novel agent as monotherapy and in combination with chemotherapy for SCLC has yet to be elucidated.

Methods: We assessed the anti-proliferative effect of TAK-243 monotherapy in a large panel (n = 10) of variant and classical SCLC cell-lines, including assorted MYC family member amplifications and different UBA1 mRNA expression levels. TAK-243 EC50 single agent doses were determined by treating cell-lines with incremental concentrations of TAK-243 (0-1μM). For combinatorial analysis, TAK-243 (0-1μM) was administered to cell-lines alongside cisplatin/etoposide (CE) at cell-line specific optimized doses yielding 30-35% kill by CE. Short-term efficacy was determined by a cell viability resazurin assay, 48 hours after treatment initiation. ∆EC50 was measured as the single agent EC50 subtracted by the EC50 of TAK-243+CE. Positive ∆EC50 values show potential synergy between TAK-243 and CE.

Results:

Single-agent therapy. SCLC cell-lines demonstrated a range of sensitives to TAK-243 monotherapy after 48 hours. The cell-lines tested are ranked from highest to lowest sensitivity: NCI-H82, NCI-H69, NCI-H1618, LX22 (ex-vivo cultured PDX), NCI-H1092, NCI-H446, SHP77 and NCI-H196; with EC50 values ranging from 18nM to 80nM. However, NCI-H526, NCI-H889, and SBC-5 were non-responsive to therapy at 1μM.

Combination therapy. TAK-243 upon combination treatment with CE revealed ∆EC50 values ranging from 5.23-14.54nM. NCI-H82, NCI-H69, SHP77 and NCI-H1092 demonstrated potential chemosensitivity to combination treatment.

Conclusions: These data begin to hint at synergy between TAK-243 and CE, where lower doses of TAK-243 may be required for certain SCLC cell-lines when used in combination. These preliminary results suggest that TAK-243 may have the potential to improve the current SCLC standard therapies in molecular defined subgroups. These results are being confirmed in additional cell-lines and through in vivo experiments using patient-derived xenograft models of SCLC.

*S.M and M.K.A contributed equally to this work

## IMMUNOLOGY

### Immune Cell Subsets in the Suppressive Tumor Microenvironment

#2700

CD8 T-cell infiltration into renal tumors requires a supportive antigen presenting niche.

Caroline S. Jansen, Nataliya Prokhnevska, Viraj A. Master, Jennifer W. Carlisle, Mehmet A. Bilen, Adriana M. Reyes, Haydn T. Kissick. _Emory University, Decatur, GA_.

Background: Tumor infiltrating immune cells have a clear prognostic benefit in many tumor types. Immune variables have independently improved prognostication in various cancer types, with tumor-infiltrating lymphocytes (TILs) more accurately predicting patient survival than currently employed methods. This has been shown using the Immuno-score, which predicts disease progression in colorectal cancer based on CD8 T cell infiltration. Many recent studies have also highlighted similar observations in other cancers, including breast cancer, lung cancer, and melanoma. These observations raise the question of whether the level of CD8 T cell infiltration into renal cell tumors may also predict patient survival, and more fundamentally, why some patients may mount a strong immune response to their tumors and others do not.

Methods: Tumor tissue was collected from 68 renal cell carcinoma (RCC) patients undergoing surgery at Emory University Hospital. Patients had a minimum follow up time of 24 months. Intraoperative tumor samples were processed and analyzed by flow cytometry and immunofluorescence.

Results: The proportion of CD8 TILs, measured by flow cytometry, was found to vary widely in RCC patients. This CD8 T cell response is independent of standard risk assessment tools, tumor size, pathology, and patient demographics. Significantly, an increasing percent of tumor CD8 T cells is associated with improved cancer-specific survival in these patients, and this association is particularly strong in a small cohort of stage III patients.

The phenotype and functional capacity of TILs were examined, and presence of a stem-like CD8 T cell—that can proliferate and differentiate—was required to generate a strong anti-tumor Tcell response. When this stem-like T cell is lost, there is a poor anti-tumor immune response and patients experience progressive disease. Flow cytometry analysis revealed that the number of dendritic cells in the tumor correlates with T cell infiltration, and immunofluorescence image analysis showed that stem-like T cells reside in areas of high antigen-presenting cell density. Tumors with poor T cell infiltration lack APC density, suggesting that an antigen presenting niche is required for a strong T cell response.

Conclusions: Measuring CD8 T cell infiltration in RCC predicts cancer-specific survival, particularly in patients with advanced disease. As this patient population is one for whom improved prognostication is a critical clinical goal, this study represents an opportunity to inform future prognostic measures and to direct reduction or intensification of therapy.

The T cell response was found to be maintained by a population of cells, which harbor both proliferative and differentiation capacity. These stem-like cells require a supportive niche inside the tumor in order to persist, and without this support, the T cell response collapses, resulting in disease progression.

#2701

Functionally specialized subsets of exhausted CD8+ T cells mediate tumor control and differentially respond to checkpoint blockade.

Brian C. Miller,1 Debattama R. Sen,1 Rose Al Abosy,1 Kevin Bi,1 Yamini V. Virkud,2 Martin W. LaFleur,1 Kathleen B. Yates,1 Ana Lako,1 Kristen Felt,1 Girish S. Naik,1 Michael Manos,1 Evisa Gjini,1 F. Stephen Hodi,1 Scott J. Rodig,1 Arlene H. Sharpe,3 W. Nicholas Haining1. 1 _Dana-Farber Cancer Inst., Brookline, MA;_ 2 _Massachusetts General Hospital, Boston, MA;_ 3 _Harvard Medical School, Boston, MA_.

T cell dysfunction in the tumor microenvironment (TME) is a hallmark of many cancers. Reinvigoration of T cell function by PD-1 checkpoint blockade can result in striking clinical responses, but is only effective in a minority of patients. The basis for T cell dysfunction in the TME, as well as the mechanisms by which anti-PD-1 therapy acts on dysfunctional T cells are not fully understood. Here we show that anti-PD-1 therapy acts on a specific subpopulation of CD8+ tumor-infiltrating lymphocytes (TILs) in melanoma mouse models, which can also be found in patients with melanoma. We find that dysfunctional CD8+ TILs possess canonical epigenetic and transcriptional features of T cell exhaustion, mirroring those seen in chronic viral infection. Similar to chronic viral infection, exhausted CD8+ TILs contain a subpopulation of "progenitor exhausted" T cells that have a distinct regulatory state. Progenitor exhausted TILs also have critical functional attributes that are not shared by the majority "terminally exhausted" TILs: they retain more polyfunctionality, persist following transfer into tumor-bearing mice, and differentiate to repopulate terminally exhausted TILs in the TME. As a result, progenitor exhausted CD8+ TILs are better able to control tumor growth than terminally exhausted cells. Progenitor exhausted, but not terminally exhausted, CD8+ TILs can respond to anti-PD-1 therapy but this occurs without reversion of their exhausted epigenetic state. Human melanomas contain CD8+ T cells with a progenitor exhausted phenotype and patients with a higher fraction of this subpopulation in their tumors have a significantly longer duration of response to combination checkpoint blockade therapy. Therefore, approaches to expand progenitor exhausted CD8+ T cells in the tumor microenvironment may be an important component of improving checkpoint blockade response.

#2702

Contribution of effector regulatory T cells to the regulation of antitumor immunity.

Michael L. Dixon, Lin Luo, Jianmei W. Leavenworth. _University of Alabama at Birmingham, Birmingham, AL_.

Regulatory T cells (Treg) belonging to the FoxP3 lineage stringently control immune responses and self-tolerance. Follicular regulatory T (TFR) cells, an effector Treg (eTreg) subset, not only suppress cellular responses but also regulate the magnitude and quality of the germinal center (GC)-antibody (Ab) response through interactions with activated follicular helper T (TFH) cells and GC B cells. Although the Blimp1 transcription factor has been used as a phenotypic marker for eTreg, its contribution to the lineage stability and suppressive activity of eTreg, particularly TFR, has been clouded in uncertainty. In contrast to the current view that Blimp1 may have a negative impact on the TFR response, our recent analysis of mice that harbor a FoxP3-specific deletion of Blimp1 has shown that expression of Blimp1 in eTreg (including TFR) is essential for the maintenance of FoxP3 expression and stable eTreg suppressive phenotype. We then generated B16 melanoma model in these mice and investigated the impact of Blimp1 deficient eTreg on the tumorigenesis. These mice had delayed tumor growth compared to WT controls. The enhanced antitumor immunity in mice with Blimp1 deficient eTreg reflected reprogramming of unstable eTreg into T-cells with effector activity (Teff) specifically within the tumor and loss of suppression on TFH-Ab responses, leading to intratumoral TFH expansion, Ab deposition and enhanced CD8+ T-cell and NK cell effector function. Further analysis of mice expressing a conditional and tamoxifen-inducible Blimp1 deletion within the FoxP3+ lineage revealed the specific contribution of Blimp1 deficient TFR cells to antitumor immune response. These results suggest that induction of eTreg lineage instability provokes intratumoral eTreg conversion into Teff −a new source of effector activity by T-cells within the tumor and enhances robust Ab responses. The increased cellular and humoral responses cooperatively promote antitumor immunity. Insights from our studies may suggest new approaches to manipulate eTreg for better vaccines or combined immunotherapeutic approaches to melanoma or other types of cancer.

#2703

Lymph node colonization promotes distant tumor metastasis through the induction of systemic immune tolerance.

Nathan E. Reticker-Flynn,1 Maria M. Martins,1 Pamela A. Basto,1 Weiruo Zhang,2 Alborz Bejnood,2 Andrew J. Gentles,2 John B. Sunwoo,2 Sylvia K. Plevritis,2 Edgar G. Engleman1. 1 _Stanford, Palo Alto, CA;_ 2 _Stanford, Stanford, CA_.

The majority of cancer-associated deaths are the result of distant organ metastasis, an event that is typically preceded by metastasis to regional or distant lymph nodes (LNs). LNs are education hubs of the adaptive immune system wherein antigens derived from pathogens or malignancies are presented to lymphocytes in a manner that facilitates elimination of the threat. Nonetheless, LN metastasis, which is typically attributed to passive drainage of tumor cells through lymphatics, frequently does not lead to the generation of an anti-tumor immune response, but instead correlates with poor prognosis and further disease progression. Here, we find that LN metastasis represents a critical step in tumor progression through the capacity of such metastases to induce systemic immune tolerance in a manner that promotes further dissemination of tumors to distant organs. Through serial in vivo passaging of a syngeneic melanoma in mice, we generate nearly 300 unique cell lines that exhibit an enhanced capacity to metastasize to LNs. Transcriptional profiling of these lines reveals increased expression of immune-related programs. We show that the presence of these LN metastases enables distant organ seeding of metastases in a manner that the parental tumor cannot, and this differential seeding is eliminated in mice that lack an adaptive immune response. To query the effects of the LN metastases on the systemic immune response, we perform organism-wide immune profiling by mass cytometry and identify a number of cellular mediators of tolerance. In particular, we find that LN metastases have the capacity to both resist NK cell cytotoxicity and induce regulatory T cells (Tregs) in vitro. Furthermore, depletion of NK cells in vivo enables non-metastatic tumors to disseminate to LNs, and ablation of Tregs using FoxP3-DTR mice eliminates the occurrence of lymphatic metastases. We further identify an interferon signaling axis that is constitutively activated within the LN metastases in the absence of exogenous interferon signaling. Through the use of ATAC-seq, we find that this program is conferred through epigenetic regulation of chromatin accessibility. Knockout of key interferon-induced genes using CRISPR/Cas9 in the LN-metastatic cells reveals that this program is required for enhanced LN metastatic seeding in vivo, and their overexpression increases LN metastasis of the non-metastatic cells. Using additional mouse models of pancreatic ductal adenocarcinoma and head and neck squamous cell carcinoma (HNSCC), we show that these findings are conserved across multiple malignancies. Additionally, we perform RNA-seq on sorted malignant populations from node-positive and node-negative HNSCC patients and confirm that these differences in transcriptional profiles extend to the human disease. Together, these findings demonstrate a critical role for LN metastasis in promoting tumor immune tolerance.

#2704

Immune cell subset composition in ductal carcinoma in situ of the breast.

Marie Colombe Agahozo,1 Mieke R. van Bockstal,1 Floris H. Groenendijk,1 Thierry P. van den Bosch,1 Pieter J. Westenend,2 Carolien H. van Deurzen1. 1 _Erasmus MC Cancer Institute, Rotterdam, Netherlands;_ 2 _Laboratory of Pathology Dordrecht, Dordrecht, Netherlands_.

Introduction: Ductal carcinoma in situ (DCIS) of the breast includes several subtypes with a divergent biological behavior. HER2+ DCIS is often detected at the in situ stage, while ER- HER2- DCIS is relatively rare. Besides, HER2+ DCIS has a more extensive growth pattern compared to other subtypes. Data regarding the composition of DCIS-associated immune cells and their potential role in DCIS progression is limited. We studied DCIS-associated immune response by characterizing immune cell subsets according to DCIS subtypes.

Methods: We evaluated DCIS-associated tumor infiltrating lymphocyte (TIL) density and distribution based on H&E stained sections of excision specimens from 473 patients with DCIS. These cases were subtyped based on ER, PR and HER2. Patients were categorized as TIL-high or TIL-low, whereby TIL-high was defined as high TILs density (>50% of the DCIS-associated stroma occupied by TILs) with a patchy or diffuse distribution. The DCIS-associated immune cells of TIL-high cases were immunostained on consecutive whole slides with CD4 (T-helper cells), CD8 (cytotoxic T-cells), CD20 (B-cells), CD68 (macrophages), FOXP3 (regulatory T-cells), PD-L1 (immune checkpoint ligand, clones SP142 and SP263). The percentage of CD4+, CD8+, CD20+ and CD68+ immune subsets was assessed relatively to one another, with a combined score of 100%. The percentage of FOXP3+ and PD-L1+ immune cells was determined as a proportion of all immune cells. PD-L1+ DCIS cells were scored using the histo-score (H-score). The immune cell composition according to DCIS subtypes was analyzed using a Kruskal Wallis or Mann-Whitney U test.

Results: DCIS was subtyped as ER+PR+/-HER2- (n=225), ER+PR+/-HER2+ (n=80), ER-PR-HER2+ (n=85), triple negative (TN; n=22) or missing (n=31). In total, 131/473 patients (27.7%) were considered as TIL-high and the percentage of TIL-high cases was significantly associated with DCIS subtype (11.4% of ER+PR+/-HER2-, 38.8% of ER+PR+/-HER2+, 61.2% of ER-PR-HER2+ and 63.6% of the TN subtype, P<0.0001). There was no statistical difference in the immune cell composition according to DCIS subtypes. However, individual DCIS subtype comparison showed that the ER+PR+/-HER2+ subtype was associated with a significantly higher proportion of CD8+ T-cells compared to the TN subtype (P=0.047). The ER-PR-HER2+ subtype was associated with a higher proportion of CD4+ T cells compared to the TN subtype, though significance was not reached (P=0.061). PD-L1 expression by both clones was low (range: 0-10% of immune cells, H-score 0-54 of DCIS cells). However, the mean value of PD-L1 SP263 was higher compared to PD-L1 SP142, for both TILs and tumor cells (P<0.0001).

Conclusion: High numbers of TILs are mainly observed in HER+ and TN DCIS subtypes. The ER+ HER2+ DCIS subtype attracts more CD8+ T-cells compared to the TN subtype. This suggests a more pronounced anti-tumor immunity in HER2+ DCIS, which could play a role in its biological behavior.

#2705

Differential stromal macrophagic profiles precede ER+ and ER- breast carcinogenesis.

Jodi M. Carter,1 Ethan P. Heinzen,1 Tanya L. Hoskin,1 Stacey J. Winham,1 Derek C. Radisky,2 Daniel W. Visscher,1 Amy C. Degnim1. 1 _Mayo Clinic, Rochester, MN;_ 2 _Mayo Clinic, Jacksonville, FL_.

Background: Macrophages are well-established components of the inflammatory repertoire in breast cancer (BC) progression. The role of macrophages in early breast carcinogenesis is less understood. Macrophages adopt microenvironment-driven pro- or anti-inflammatory phenotypes. We evaluated macrophagic subsets in benign breast stroma to evaluate their association with future or concurrent BC.

Methods: With institutional approval, FFPE tissues were identified from age-matched subjects, [with index benign breast biopsies and (a subset) interval-matched second benign tissues taken at the time of one of three events: ER+ BC, ER- BC or no cancer event (Controls) (index N=32, 26 and 30 respectively)]. Tissues were immunostained (CD68 or CD163) and stromal CD68+ and CD163+ macrophagic cell densities (MCD) (cells/mm2 stromal fibroadipose area) were digitally quantified. Statistical analysis was performed using Wilcoxon signed-rank tests for univariate analysis and conditional logistic regression models to adjust for BMI.

Results: In the index age-matched biopsies (median age 54y), within subjects in all groups, the median CD163+ MCD (54.5; range: 13.9-371.3) was significantly higher than CD68+ MCD (19.6, range: 3.5-78.5) but the two were strongly correlated (r = 0.76); neither correlated with BMI (r< 0.1 each). Across the groups, index benign biopsies of ER- BC had significantly lower CD68+ MCD and CD163+ MCD compared to ER+ BC (CD163: median 47.2 vs. 57.8, p=0.004; and median CD68+ MCD: 15.8 vs. 29.7, p=0.03); and ER- BC had significantly lower CD163+ MCD density than Controls (p=0.02), (all by univariate analysis and after adjusting for BMI). Comparing the index biopsy and second biopsy/excision within subjects, all showed significant increases in CD68+ MCD (median increase 27.9 for ER- BC, 16.1 for ER+ BC, and 17.8 for controls, p<0.01), and only ER-BC showed an increase in CD163+ MCD (median increase 26.6, p=0.01). Thus, in the second biopsies/excisions, the CD163+ MCD and CD68+ MCD were similar across groups (median CD163+ MCD: 69.3, 75.4, and 59.3 and median CD68+ MCD: 47.4, 45.2, and 36.0 for ER+ BC, ER- BC, and controls); however, all groups had decreased CD163+:CD68+ MCD ratios compared to the index biopsies.

Conclusion: Benign breast tissues have higher stromal CD163+ MCD than CD68+ MCD irrespective of future BC events and independent of BMI. However, between index and second biopsies/excisions, the stromal CD68+ MCD differentially increased in all groups, lowering CD163+:CD68+ MCD ratios, and likely shifting pro- and anti-inflammatory stromal macrophage functionality. Across groups, ER- BC index biopsies had significantly lower stromal MCD of both types compared to ER+ BC, and were the only group to increase stromal CD163+ MCD between index biopsy and cancer event. These data suggest that ER+ and ER- BC may have different pre-carcinogenic stromal inflammatory microenvironments.

#2706

Comprehensive immune profiling of primary tumor-infiltrating lymphocytes isolated from primary non-small cell lung cancer specimens.

Yoshinobu Koguchi,1 William Miller,1 Brian D. Piening,1 Venkatesh Rajamanickam,1 Brady Bernard,1 Zhaoyu Sun,1 Yaping Wu,1 Johanna K. Kaufmann,2 William L. Redmond1. 1 _Earle A Chiles Research Institute, Providence Portland Medical Center, Portland, OR;_ 2 _TESARO Inc, Waltham, MA_.

In the past years, multiple inhibitors of the immune checkpoint receptor (ICR) PD-1 or its ligand PD-L1 have been approved in the U.S. in a selected, yet growing number of indications including non-small cell lung cancer (NSCLC). In patients with advanced NSCLC, checkpoint inhibition significantly prolongs overall survival and, due to its overall responsiveness, additional novel immunotherapeutic combinations are being investigated. Rational design of future combination treatment with immunomodulatory agents like PD-1 inhibitors will greatly benefit from a detailed understanding of the immune contexture and the functional state of tumor infiltrating lymphocytes (TILs) within NSCLC. To this end, we characterized the immune infiltrate in 13 primary NSCLC resections using multiple orthogonal techniques: (1) surface expression of multiple ICRs on various T cell subsets as well as the functional markers granzyme B and Ki-67 were assessed by flow cytometry; (2) the spatial localization of immune infiltrates was analyzed by 7-color multiplex immunohistochemistry (mIHC); (3) gene expression profiling enabled in depth molecular characterization of TILs and indicated their functional status. Flow cytometry revealed differential contributions of ICR expression in various T cell compartments. Of note, around 30% (median=29.1%; 95% CI, 20.3-38.5%) of CD8+ T cells co-expressed CD39 and CD103, which represents a recently described population of tumor-reactive CD8+ T cells. Furthermore, approximately 90% of these double-positive CD8+ T cells expressed PD-1 (median=91.3%; 95% CI, 78.3-96.4%) and about 50% were granzyme B-positive (median=51.8%; 95% CI, 32.5-63.95%), indicative of functionally active effector status. Interestingly, more than 90% of PD-1+ CD8+ T cells co-expressed at least one more ICR (TIM-3 and/or LAG-3) (median=90.6%; 95% CI, 70.3-95%), suggesting ongoing development of T cell exhaustion in response to chronic T cell activation. In contrast, the CD4+ T cell compartment was dominated by PD-1 single-positive cells and contained a median of 15.4% (95% CI, 11.7-20%) regulatory T cells.The overall composition of the immune infiltrates in these patients were corroborated by mIHC, revealing strong immune infiltration within the tumor stroma. Furthermore, immune cell deconvolution of bulk RNA-seq data from FFPE tissues revealed a high concordance with mIHC. In conclusion, we established a pipeline for high content immune profiling of human TIL enabling correlative analyses of proteomic and transcriptomic data. The high proportion of markers indicative of TIL with anti-tumor effector potential was in line with the responsiveness of NSCLC to immunotherapy. Integration of additional gene expression data, mutational burden, and T cell clonality will deepen our understanding of the NSCLC tumor microenvironment.

### Rational Combinations of Immunotherapy

#2707

**A novel RAS inhibitor, MCI-062, inhibits colon tumor growth** in vivo **and activates antitumor immunity.**

Adam B. Keeton,1 Antonio Ward,1 Xi Chen,1 Jacob Valiyaveettil,1 Bing Zhu,1 Veronica Ramirez-Alcantara,1 Yulia Maxuitenko,1 Kristy Berry,1 Tyler E. Mattox,1 Michael R. Boyd,2 Gary A. Piazza1. 1 _Univ. of South Alabama Mitchell Cancer Inst., Mobile, AL;_ 2 _ADT Pharmaceuticals, LLC, Orange Beach, AL_.

Approximately 45% of colorectal cancers harbor mutations in the KRAS gene, resulting in constitutive activation of RAS signaling through effector proteins, primarily RAF and PI3K. Members of the RAS family are GTPases that function as a molecular switch, cycling between inactive (GDP-bound) and active (GTP-bound) states in cells to regulate proliferation and survival. Constitutive signaling from mutant RAS drives tumorigenesis, in part, by deregulation of the cell cycle, resulting in increased proliferation (mitosis) and decreased apoptosis of tumor cells. From an extensive medicinal chemistry/screening campaign, we identified a novel compound class that potently and selectively inhibits the growth of tumor cell lines harboring constitutively activated RAS by a mechanism involving the disruption of RAS nucleotide loading, blockage of effector activation, and consequent inhibition of signaling. A prototype of the class, MCI-062, inhibits the growth of KRAS-driven (HCT-116) colon tumor cells with low nanomolar IC50 values, while RAS-independent (HT-29) colon tumor cells harboring the BRAF V600E mutation, are essentially insensitive. A strong correlation was measured among a larger panel of tumor cell lines between potency to inhibit tumor cell growth and levels of activated RAS. The growth inhibitory effects of MCI-062 were sustained and irreversible as demonstrated by colony formation and apoptosis assays. MCI-062 treatment of HCT-116 colon tumor cells reduced levels of activated RAS and RAS-mediated signaling as measured by GST-RBD pulldown assays and phospho-specific immunoblotting. Within the same concentration range, MCI-062 induced mitotic arrest as measured by cell cycle analysis of DNA content and phospho-histone H3B immunofluorescence. Further analysis revealed that MCI-062 interfered with localization of the mitosis-inducing protein, PLK1 to kinetochores and decreased nuclear localization of its substrate, Cdc25C, a downstream target of RAS-RAF signaling involved in both mitotic entry and exit checkpoints. In vivo testing of MCI-062 in a syngeneic mouse model of KRAS-driven colon cancer (CT-26) demonstrated that MCI-062 engages its molecular target, depleting GTP-RAS and suppressing activation of the MAPK signal transduction pathway, and inhibiting tumor growth. MCI-062 also suppresses PD-L1 expression and activates anti-tumor immunity, which may contribute to its antitumor activity and suggests potential benefits of combining with immunotherapy. From these studies, we have identified a novel class of RAS inhibitors that potently and selectively inhibits RAS-driven tumor growth by disrupting downstream signaling, leading to cell cycle arrest and apoptosis. These findings support further development of MCI-062 for treatment of RAS-driven colorectal and other cancers. Funding provided by NCI grants R01CA131378, R01CA148817, R01CA197147, and R01CA155638.

#2708

Potent CAB CTLA4 antibody to reduce immune side effects and toxicities associated with single agent and combination cancer immuno therapies.

Leslie Sharp, Cathy Chang, Gerhard Frey, Haizhen Liu, Charles Xing, Jing Wang, Marlena Walls, Christina Wheeler, Yong Ben, William J. Boyle, Jay M. Short. _BioAtla LLC, San Diego, CA_.

CTLA4 is a receptor on T cells that acts as a brake on their activation during immune responses, and when blocked can stimulate cancer immune-surveillance. The use of anti-CTLA4 antibodies to unleash T cells has demonstrable anti-tumor efficacy in animal models and in certain patients in the clinic. However, blockade of CTLA4 as a single agent and in combination with chemotherapy, targeted, and other immunomodulatory therapies has been limited clinically in part due to systemic immune-response related toxicities. In addition, there is increasing concern that systemic immune activation may lead to sustained autoimmune-related disease in survivors. The Conditionally Active Biologics (CAB) technology generates antibodies that reversibly bind to target antigen in the context of diseased tissues, but not normal tissues, by taking advantage of the unique cancer microenvironment that is produced largely because of glycolytic tumor metabolism including the Warburg effect. Using our CAB technology, we have identified anti-CTLA4 Abs that reversibly bind human CTLA4 and enhance T cell response to bacterial super-antigen under in vitro tumor microenvironment, but not in normal tissue conditions. CAB-CTLA4 Abs have anti-tumor efficacy equal to that of ipilimumab analog, including tumor regression, against MC38 colorectal tumor in a syngeneic human CTLA4 knock-in mouse model. Interestingly, CAB CTLA4 antibodies modulate the activity and numbers of infiltrating T cell subsets within the tumor microenvironment, but unlike ipilimumab analog, the CAB CTLA antibodies do not increase CD4 effector T Cells in periphery, suggesting less systemic immune toxicities impacting normal tissues. Repeated co-administration of either CAB CTLA4 or ipilimumab analog in combination with an anti-PD-1 (nivolumab analog) into monkeys for four weeks was performed to access the peripheral systemic and normal tissue effects of combination treatments. Combination treatment with ipilimumab and nivolumab analogs (I+N) resulted in increases in T cell proliferation markers in peripheral blood cells, while the CAB CTLA4 plus nivolumab analog had normal immunophenotypic patterns. All animals in the I+N treated groups had significant gastrointestinal symptoms (diarrhea, loose stools) that presented early, were sustained throughout the treatment period and were associated with substantial mononuclear infiltration within the intestinal wall. In sharp contrast, the CAB-CTLA4 plus nivolumab treated groups showed no significant GI symptomology nor histopathology. These data indicate that our CAB CTLA4 molecule may have a superior safety profile when used in combination with PD-1 inhibitors and allow increased dosing levels to achieve superior efficacy to current anti CTLA4 therapy as a single agent or in combination with other anti-cancer therapies including immuno-oncology agents.

#2709

**Modulation of the tumor infiltrating lymphocyte population by PARP inhibitor Talazoparib in combination with anti-PD1 treatment significantly enhances overall survival in a murine BRCA1** -/- **breast cancer model.**

Anya Avrutskaya,1 Cordula Tschuch,2 Astrid Jensen,3 William Durham,1 Maycee Robinson,1 Charles Krause,1 Emily O'Koren,1 Gerhard Kelter,2 Anne-Lise Peille,2 Armin Maier,2 Anne-Marie Zuurmond,3 Julia Schüler2. 1 _Discovery Services Charles River, Morrisville, NC;_ 2 _Charles River Research Services Germany GmbH, Freiburg, Germany;_ 3 _Charles River, Leiden, Netherlands_.

Targeted therapy of BRCA-deficient cancers has been achieved using poly(ADP-ribose) polymerase (PARP) inhibitors, which block BRCA-independent DNA repair. With first approval in 2014 of Olaparib the concept of tumor-specific synthetic lethality was added to the treatment portfolio of cancer patients. Although the effects of PARPi have shown promising results in multiple cancer types, how and whether patients might benefit from combination with compounds modulating the immune landscape of a tumor is largely unknown. In the current study, we investigate the cross-talk between PARPi and immune checkpoint inhibition, in particular, anti PD-1 and anti CTLA-4, as the most advanced targets in the field. PARP inhibitors Niraparib, Rucaparib, Talazoparib and Olaparib were investigated in vivo using the murine EMT6/BRCA1-/- model in monotherapy as well as in combination with anti-CTLA-4 or anti-PD1 treatment. The four PARPi showed distinct activity profiles in the two breast cancer models. Talazoparib was the most active compound in the BRCA1-/- model (optimal T/C (test/control) of 60%), followed by Niraparib and Rucaparib (65% and 67%, respectively). Olaparib was considered inactive with a T/C value of 80% in monotherapy. The EMT6/BRCA1-/- turned out to be sensitive towards anti CTLA-4 treatment (optimal T/C of 35% - 38% in three independent experiments). Anti PD-1 treatment in monotherapy induced no significant reduction in tumor growth (optimal T/C of 80% - 82%, in two independent experiments). The combination of PARPi and anti CTLA-4 induced a transient but significant reduction of tumor load early in the treatment phase (p< 0.002, one-way ANOVA, day 8). However, anti PD-1 treatment significantly prolonged overall survival in combination with Talazoparib (p< 0.011 log-rank test). The analysis of tumor infiltrating lymphocytes (TILs) by flow cytometry revealed that Talazoparib as monotherapy and in combination with checkpoint (CP) inhibitors enhanced the number of gMDSC as well as the number of CD3/CD11b double positive T cells. Weekly cytokine analysis in the serum of tumor bearing mice will elucidate possible interactions between PARP and CP inhibitors and give guidance to optimal combination and schedules. First results indicate that PARPi as well as CPi induce unique cytokine profiles correlating well with the modified TIL composition of the respective treatment groups. Further mechanistic studies as well as comparative studies with the EMT6/BRCAwt model will elucidate the tumor biology behind these observations and might lead to beneficial combination strategies in patients suffering from triple negative and BRCA1-/- breast cancer.

#2710

Depletion of CXCR2-dependent myeloid-derived suppressor cells (MDSCs) overcomes anti-PD-1 resistance in a murine model of LKB1-deficient non-small cell lung cancer (NSCLC) with high mutational load.

Rui Li,1 Ramin Salehi-Rad,1 Stephanie Ong,1 Milica Momcilovic,1 Bin Liu,1 Raymond Lim,1 Linh Tran,1 Ziling Huang,1 Zhe Jing,1 Manash Paul,1 Kostyantyn Krysan,1 Stacy Park,2 John Minna,3 Michael Teitell,1 David Shackelford,1 Steven Dubinett1. 1 _UCLA, Los Angeles, CA;_ 2 _Kaiser Permanente, Los Angeles, CA;_ 3 _UT Southwestern, Dallas, TX_.

Checkpoint inhibitors such as PD-L1/PD-1 blockade have rapidly integrated into the paradigm of NSCLC treatment. However, a majority of patients do not benefit from monotherapy with checkpoint inhibitors. High tumor mutational burden (TMB), along with pre-existing intratumoral T cell infiltration and baseline high PD-L1 expression, predicts response to checkpoint blockade. Furthermore, a recent retrospective study identified LKB1 alterations as the most prevalent genomic driver of resistance to PD-1 axis inhibitors in KRAS-mutant lung adenocarcinoma. In this study, we investigate the mechanisms underlying LKB1-mediated immunosuppression in NSCLC. We show that loss of LKB1 in human bronchial epithelial cells (HBECs) and NSCLC cells leads to increased secretion of CXCR2 ligands, including CXCL1, CXCL2, CXCL3, CXCL5 and CXCL8. These CXCR2 ligands are also elevated in LKB1-deficient tumors from patient-derived xenografts and genetically-engineered murine models. We find abundant tumor infiltrating MDSCs in murine Lkb1-deficient NSCLC, consistent with the capacity for CXCR2 ligands to recruit MDSCs. MDSCs mediate potent immune suppressive activities at multiple levels including release of immunosuppressive cytokines, recruitment of regulatory T cells (Tregs), inhibition of CD8 T cell tumor infiltration and upregulation of PD-L1 expression. Although MDSC depletion activates interferon gamma signaling and decreases systemic Tregs in murine KrasK12D;Tp53-/-;Lkb1-/- (KPL) tumors, it does not sensitize KPL tumors to anti-PD-1 therapy. One of the major challenges in the preclinical assessment of lung cancer immunotherapy is that the commonly utilized murine models lack the mutational burden of human NSCLC. To assess this combination therapy in the context of a mutational burden that more accurately reflects the clinical disease, we generated tumors with high TMB by exposing KPL cells in vitro to the tobacco carcinogen N-methyl-N-nitrosourea. In the context of high TMB, MDSC depletion demonstrates remarkable anti-tumor effects in combination with anti-PD-1 therapy. Finally, we delineate the regulation of CXCR2 ligands by LKB1 which is dependent on the MARK-mediated NF-κB pathway. In conclusion, we find that LKB1 deficiency leads to increased CXCR2 ligand production and tumor infiltrating MDSCs. MDSC depletion enhances the efficacy of anti-PD-1 blockade in LKB1-deficient tumors bearing high TMB.

#2711

Rational combination of GITR agonism with PD-1 blockade.

Roberta Zappasodi,1 Cynthia Sirard,2 Yanyun Li,1 Sadna Budhu,1 Moshen Abu-Akeel,1 Cailian Liu,1 Xia Yang,1 Hong Zhong,1 Walter Newman,2 Jingjing Qi,1 Philip Wong,1 David Schaer,1 Henry Koon,3 Vamsidhar Velcheti,4 Margaret K. Callahan,1 Jedd Wolchok,1 Taha Merghoub1. 1 _Mem. Sloan Kettering Cancer Ctr., New York, NY;_ 2 _Leap Therapeutics, Boston, MA;_ 3 _Case Western Reserve University, Cleveland, OH;_ 4 _Cleveland Clinic, Cleveland, OH_.

The clinical successes of checkpoint blockade have demonstrated that proper modulation of T cell co-inhibitory/co-stimulatory pathways can achieve substantial anti-tumor activity. However, many patients are or become refractory to checkpoint blockade. Additional forms of immunotherapy targeting alternative immune pathways are thus needed. Antibodies (Abs) engaging the TNF receptor GITR can enhance T cell functions and counteract regulatory T cell (Treg) suppression and have shown potent anti-tumor activity in animal models. Based on this evidence, we initiated the first in-human phase-I trial with the humanized aglycosylated anti-GITR Ab TRX518 (NCT01239134). Here, we report the immune effects of a single ascending dose of TRX518 monotherapy in 37 advanced cancer patients in this phase-I trial and provide mechanistic preclinical evidence to rationally combine GITR agonism with checkpoint blockade. We found that TRX518 frequently reduces circulating Tregs. In 8 patients for whom pre- and post-treatment tumor biopsies were available, reductions in intra-tumor and circulating Tregs after TRX518 were positively correlated. Yet, these patients did not experience substantial clinical responses. To explain this outcome, we modeled sensitivity and refractoriness to anti-GITR by treating B16F10-melanoma-bearing mice with the Ab DTA-1 on day 4 (curative regimen) or day 7 (refractory regimen) after tumor implantation respectively. We found that Tregs were significantly reduced and CD8+:Treg and Teff:Treg ratios increased in both responding and refractory tumors. Interestingly, CD8+ T cells from refractory tumors overexpressed T cell exhaustion markers and did not up-regulate memory and functional markers. We thus tested whether counteracting exhaustion could overcome resistance of advanced tumors to anti-GITR. PD-1 blockade in combination with anti-GITR starting on day 7 after tumor implantation controlled tumor growth similar to the curative anti-GITR monotherapy regimen (day 4 treatment) and achieved 50% complete response rate associated with long-lasting anti-tumor immunological memory. This was associated with more activated and less exhausted profiles of intra-tumor CD8+ T cells, which displayed enhanced tumor-lytic capacity compared to CD8+ T cells from non-responding tumors treated with each agent alone. These results indicate that Treg reduction can serve as a pharmacodynamic biomarker of anti-GITR in patients. However, Treg elimination from advanced tumors may not be sufficient to activate cytotoxic CD8+ T cell responses unless the T cell exhaustion process is concurrently blocked. This provides the rationale to combine immunotherapies targeting Tregs with strategies able to counteract exhaustion, such as anti-PD-1, to regress advanced tumors. Based on these observations, we have started to investigate TRX518 in combination with PD-1 pathway blockade in patients with advanced solid tumors (NCT02628574).

#2712

Effect of combined PD-1 and IL-6 blockade on K-ras mutantlLung cancer.

Marco A. Ramos-Castaneda, Neha Daga, Stephen Peng, Shanshan Deng, Walter Velazco, Mauricio Da Silva Caetano, Seyed Moghaddam. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Lung cancer is the leading cause of cancer death and the second most common cancer diagnosed worldwide in both males and females. K-ras gene mutations are among the most common mutations encountered in non-small cell lung cancer (NSCLC), encompassing up to 30% of them, and are unfortunately associated with chemoresistance and poor prognosis. Recently, immunotherapy treatment of several cancers including NSCLC, particularly by targeting the co-inhibitory molecules program cell death protein 1 (PD-1) and program death-ligand 1 (PD-L1), have shown promising results given either in combination with standard chemotherapy or as neoadjuvant therapy. We have previously shown an important role for IL-6 in K-ras mutant lung tumorigenesis by reprogramming myeloid contexture in the lung tumor microenvironment (TME) and promoting tumor cell proliferation and angiogenesis through the activation of STAT3 pathway. Accordingly, we hypothesized that targeting both immunosuppressive TME and IL-6 driven pro-tumor immune response, through the use of immune checkpoint blockade (ICB), and inhibition of IL-6 would give us a synergistic/additive response and increases the tumor inhibitory effect of ICB. Briefly, starting at the age of 6 weeks, 4 cohorts of K-ras mutant mice (LSL-K-rasG12D/ CCSPCre) (CC-LR) were injected intraperitoneally with vehicle alone (PBS1x), anti-PD-1 (CD279) antibody alone (Clone: 29F.1A12- Bioxcell - BE0273-CUST) (200µg), anti-IL-6 antibody alone (Clone: MP5-20F3 - Bioxcell - BE0046-CUST) (20 mg/kg), or a combination of both. After 8 weeks of treatment, tumor burden and inflammation were studied and quantified. We found a significant (2-fold) decrease in tumor burden in the anti-PD1 alone treated group which was further reduced in the combined anti-PD-1 and anti-IL-6 treated group compared to age and sex matched vehicle treated group. There was also a significant difference in the lung macrophage infiltration in combined anti-PD1/anti-IL-6 treated group similar to what we previously observed in anti-IL-6 alone treated group. Our results suggest that immunomodulatory approaches by targeting cytokine networks like IL-6 blockade could be used to increase the efficacy of immunotherapy, i.e. PD-1 blockade, as a preventive and/or therapeutic strategy for K-ras mutant lung cancer.

#2713

Development and characterization of AZD8701, a high affinity antisense oligonucleotide targeting FOXP3 to relieve immunosuppression in cancer.

Charles Sinclair,1 Alexey S. Revenko,2 R B. Johnson,2 Alison Peter,1 Molly A. Taylor,1 Lisa A. Hettrick,2 Stephanie Klein,2 Anisha Solanki,1 Melissa Chapman,1 James Yates,1 Helen K. Angell,1 Andrew Watt,2 Danielle Gattis,2 Brett P. Monia,2 Simon T. Barry,1 Paul Lyne,3 Mark Edbrooke,1 Frederick Goldberg,1 A R. Macleod2. 1 _AstraZeneca, Cambridge, United Kingdom;_ 2 _Ionis Pharmaceuticals, San Diego, CA;_ 3 _AstraZeneca, Waltham, MA_.

Regulatory T cells (Treg) critically maintain immuno-suppression in the tumor microenvironment, representing an attractive immuno-oncology target. The Treg lineage is defined by expression of the FOXP3 transcription factor, which controls immune-suppressive functions. We have developed the clinical candidate AZD8701, a next-generation antisense oligonucleotide inhibitor of FOXP3 (utilizing the Ionis Gen 2.5 cEt-modified ASO platform). AZD8701 treatment knocked down FOXP3 in primary human Tregs via free uptake (IC50 65nM), which was also associated with modulation of known FOXP3 target genes including 25-50% reduction in CTLA4, ICOS, CCR8 and GITR. Tregs treated with AZD8701 further exhibited reduced suppressive functions in in vitro suppression assays, which confirmed the functional effects of FOXP3 modulation. Finally, AZD8701 promoted dose-dependent knockdown of FOXP3 in humanized mice, including >50% FOXP3 knockdown at doses that can be feasibly achieved with the Gen 2.5 ASO platform in the clinic. To support the importance of FOXP3 in immuno-oncological settings, we characterized murine surrogate FOXP3 ASOs in the context of syngeneic tumour bearing mice. Murine FOXP3 ASOs similarly promoted >50% FOXP3 knockdown in mice and were well tolerated with no overt toxicological findings at high doses, over a maximum of 12 weeks of treatment. Murine FOXP3 ASOs significantly attenuated tumour growth in A20 and ID8-VEGF syngeneic models, which was associated with some complete tumour regressions. Moreover, we found that mouse surrogate FOXP3 ASOs promoted additive/enhanced therapeutic effects when combined with immune checkpoint blockade. Collectively, FOXP3 ASOs represent a first-in-class strategy to target Tregs in cancer in a highly selective manner. The clinical application of AZD8701 may provide therapeutic benefit to patients either as a monotherapy or in combination with immune checkpoint blocking agents.

## MOLECULAR AND CELLULAR BIOLOGY / GENETICS

### Exploiting Metabolic Vulnerabilities of Cancer

#2714

Targeting MAT2A in CDKN2A/MTAP-deleted cancers.

Katya Marjon, Peter Kalev, Marc Hyer, Mark Fletcher, Peili Zhang, Elia Aguado-Fraile, Everton Mandley, Zenon Konteatis, Jeremy Travins, Kevin Marks. _Agios Pharmaceuticals, Cambridge, MA_.

While deletions of the p16/CDKN2A tumor suppressor were first discovered more than 30 years ago, therapeutics that selectively target such tumors have proven elusive. Recent work utilizing functional genomics has identified a synthetic lethal vulnerability that arises due to co-deletion of the adjacent metabolic gene, methylthioadenosine phosphorylase (MTAP). Loss of MTAP in these tumors leads to an accumulation of MTAP substrate 5'-methylthioadenosine (MTA), which partially inhibits the arginine methyltransferase PRMT5 and sensitizes tumors to shRNA-mediated depletion of PRMT5 and the upstream metabolic enzyme, methionine adenosyltransferase 2 alpha (MAT2A). To investigate the therapeutic potential of this finding, we utilized a biophysical binding screen followed by iterative structure-guided design to make the first highly potent, selective, and orally bioavailable inhibitors of MAT2A. MAT2A inhibitor treatment leads to potent inhibition of the growth of HCT116 MTAP-/- cells while sparing isogenic HCT116 MTAP+/+ cells. Tumor xenograft studies similarly demonstrated MTAP-selective growth inhibition in HCT116 MTAP-/- tumors compared to isogenic HCT116 MTAP+/+ tumors. Further, MTAP-deletion correlated with MAT2A inhibitor efficacy across a panel of >300 cell lines in vitro, and MAT2A inhibitor treatment was efficacious in a variety of MTAP-deleted patient-derived xenografts in vivo. Having demonstrated that potent MAT2A inhibitors selectively block the proliferation of MTAP-deleted cells and tumors, we sought to investigate the mechanism by which these effects arise. Using methylation proteomics we noted that MAT2A inhibitor treatment leads to selective inhibition of PRMT5 methylation activity in MTAP-deleted cancers in vitro and in vivo. RNA-seq analyses revealed that MAT2A inhibition leads to substantial defects in RNA splicing in MTAP-deleted cancers, consistent with published findings that PRMT5-mediated methylation of splicing complex proteins is critical for their function. MAT2A inhibitor treatment led to a substantial increase in detained introns, which were enriched in genes involved in cell cycle regulation and DNA damage response, thus implicating dysregulated splicing in the antiproliferative effects of MAT2A inhibition in MTAP-deleted cancer cells. Furthermore, we demonstrated substantial drug-drug synergy between MAT2A inhibitors and select agents inhibiting cell cycle progression or DNA repair. Importantly we validated key combination findings in vivo, including demonstration of synergy with the MAT2A inhibitor AG-270 and anti-mitotic taxanes. AG-270 is the first MAT2A inhibitor to enter clinical development and is under investigation in a Phase I trial that is currently enrolling patients with MTAP-deleted solid tumors (NCT03435250). Our findings suggest clinically-applicable combination strategies which may further enhance the efficacy of AG-270 in malignancies with this genetic lesion.

#2715

Deciphering a metabolic basis for single agent venetoclax efficacy in t(11;14) multiple myeloma.

Richa Bajpai,1 Abhinav Achreja,2 Changyong Wei,1 Arusha Siddiqa,1 Shannon M. Matulis,1 Vikas Gupta,1 Samuel K. McBrayer,3 Anjali Mittal,2 Manali Rupji,1 Hsiao-Rong Chen,1 Jeanne Kowalski,1 Sagar Lonial,1 Ajay K. Nooka,1 Lawrence H. Boise,1 Deepak Nagrath,2 Mala Shanmugam1. 1 _Winship Cancer Institute of Emory University, Atlanta, GA;_ 2 _University of Michigan, Ann Arbor, MI;_ 3 _Dana-Farber Cancer Institute, Boston, MA_.

Multiple myeloma (MM) is the second most common hematologic malignancy. In 2017, MM accounted for approximately 30,770 new diagnoses and 12770 deaths in the US. Although, advancements in treatment options have increased survival rates and life expectancy, MM remains incurable due to development of resistance. Venetoclax is a highly selective, potent BCL-2 antagonist, currently in phase I/II trials for MM and FDA approved for the treatment of CLL patients exhibiting 17p deletion. Venetoclax is effective in eliciting cell death as a single agent in a subset of MM with the (11;14) translocation (which we henceforth designate "sensitive") in contrast to the majority of MM that is resistant (which we henceforth designate "resistant"). We previously reported that glutamine deprivation increases BIM binding to BCL-2 thereby sensitizing MM to venetoclax, while alpha-ketoglutarate supplementation reversed this sensitivity. We were therefore interested to explore if there was a metabolic basis for t(11;14)-myeloma sensitivity to single agent venetoclax to aid in 1) identifying venetoclax sensitive MM and; 2) inform us of metabolic targets that could be inhibited to sensitize resistant MM to venetoclax. We first queried the CoMMpass MM patient RNAseq trial data and cell lines for electron transport chain (ETC) and TCA cycle gene expression differences in t(11;14) vs non-t(11;14) patients and cell lines. All sensitive cells exhibited varied suppression of TCA/ETC genes, and reduced TCA cycle metabolites and oxygen consumption rates (OCR) compared to resistant cells. Examination of TCA cycle activities connected to the ETC and OCR showed significant decrease in Complex II Succinate ubiquinone reductase (SQR) activity in sensitive cell lines and purified CD38+ve MM patient cells and elevated SQR activity in resistant cells. Furthermore, inhibition of SQR with thenoyltrifluoroacetone (TTFA) sensitized resistant cells to venetoclax. Consistent with SQR inhibition leading to ABT-199 sensitivity, overexpression of an SQR mutant (SDHCR72C) in SDHC-knockout resistant MM, increased venetoclax sensitivity, identifying a unique role for SQR in regulating BCL-2 dependence. In interrogating the mechanistic underpinnings of SQR inhibition-induced sensitivity to venetoclax, we identified increased expression of ATF4 and BIM upon SQR inhibition. ATF4KD or BIMKO restored viability in TTFA and venetoclax co-treated MM further confirming the role of SQR inhibition-induced ATF4 and BIM in venetoclax sensitivity. In testing translationally relevant compounds, we determined that the histone deacetylase inhibitor, panobinostat reduced SQR activity in a dose dependent manner and sensitized MM cells to venetoclax. Our study thus identifies SQR as a novel target and predictive marker to aid in identifying ABT-199-responsive MM patients in a functional biomarker informed manner.

#2716

Autophagy modulates lipid metabolism in liver kinase B1 (LKB1) - deficient Kras-driven lung tumorigenesis.

Vrushank D. Bhatt, Khoosheh Khayati, Zhixian Hu, Xiaoyang Su, Jessie Yanxiang Guo. _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ_.

Tumor suppressor liver kinase B1 (LKB1) activates 5'-adenosine monophosphate protein kinase (AMPK) to maintain energy homeostasis. LKB1 mutations are the third most frequent mutation detected in non-small cell lung cancer (NSCLC). Compared to p53 mutation, mutations in LKB1 causes more aggressive tumor growth and resistance to chemotherapy as well as immunotherapy. Thus, identifying a novel target to improve LKB1-deficient NSCLC treatment is urgently needed. Autophagy degrades and recycles macromolecules for cells to survive starvation. Using genetically engineered mouse models for NSCLC with Kras activation and LKB1 loss (KL), we found that autophagy (Atg7) deficiency prevents tumor initiation, reduced tumor growth and extended the mouse lifespan compared to mice bearing Atg7 wild-type (WT) tumors. Most importantly, tumor growth inhibition by Atg7 deficiency was much more dramatic in KL than Kras/p53 (KP) lung tumors. Furthermore, we generated mouse tumor-derived cell lines (TDCLs) and found that Atg7 null TDCLs were more sensitive to starvation-induced cell death than Atg7 WT TDCLs, which can be rescued by palmitate supplementation, indicating that autophagy is required to maintain free fatty acid (FA) level for cells to survive starvation. We further performed 13C glucose tracing study and found that Atg7 deletion increased de novo FA synthesis and reduced FAs elongation. Fasnall, a selective FA synthase inhibitor, inhibited Atg7 null cell proliferation. Treatment with Orlistat, a lipase inhibitor, did not impact cell proliferation in nutrient-rich conditions but accelerated starvation-induced Atg7 null cell death, indicating that lipase-medicated lipid degradation compensates for autophagy loss to provide free FAs in starvation. A carnitine palmitoyltransferase I inhibitor, Etomoxir treatment, accelerated the starvation-induced Atg7 null cell death and reduced oxygen consumption rate (OCR) of Atg7 null cells in starvation, compared with Atg7 WT cells, indicating that autophagy-deficient cells rely more on FA oxidation for energy production in nutrient deprivation. To probe the significance of autophagy in human lung cancer cell lines with LKB1 mutation, we knocked down ATG7 using siRNA. ATG7 knockdown (KD) inhibited clonogenic survival of LKB1 mutant cells in starvation, but had no effect in LKB1 WT cells. Supplementation of glutamine or palmitate rescued the survival of LKB1 mutant cells with ATG7 KD in starvation and maintained OCR, demonstrating that KRAS-driven human lung cancer cells with mutant LKB1 rely on autophagy to maintain mitochondrial metabolism to survive energy crises. Taken together, our results demonstrate that autophagy modulates lipid metabolism for KL cells to survive metabolic stress. Thus, a combination of autophagy inhibition with interruption of lipid metabolism could be a novel therapeutic strategy to specifically treat LKB1-deficient NSCLC.

#2717

**Role of NAD** + **level by NQO1 enzymatic action in regulation of hair regrowth that prevents chemotherapy induced alopecia.**

Dipendra Khadka,1 Gi-Su Oh,2 Hyung-Jin Kim,2 SeungHoon Lee,2 Su-bin Lee,2 Subham Sharma,2 Seon Young Kim,2 Tae Hwan Kwak,2 Sei-Hoon Yang,2 Hyuk Sim,2 Hong-Seob So2. 1 _Wonwang University, School of Medicine, Iksan, Jeonbuk, Republic of Korea;_ 2 _Wonwang University, School of Medicine, Iksan, Republic of Korea_.

Various types of chemotherapeutic agents are widely used to treat a variety of human neoplasms. Nonetheless, its clinical use is hampered because of severe adverse side effects such as alopecia, neutropenia, pain, cachexia, conception impairment etc. Chemotherapy-induced alopecia (CIA) is one of the fundamental unsolved problems of clinical oncology that often encountered in cancer treatment. CIA has been reported to be caused by skin inflammation through oxidative stress, DNA damage, and inflammatory responses. However, the best remedy for CIA is still not established. In a new strategy, we show that WK0202 compound as a strong NQO1 substrate that modulates the intracellular NAD+ levels by NAD(P)H quinone oxidoreductase 1 (NQO1) enzymatic action and plays important role in hair regrowth response to single (cyclophosphamide-CYP) or combined (taxol, adriamycin and cyclophosphamide-TAC) cancer therapy. Particularly, we demonstrated that cellular NAD+ levels as well as SIRT1 activity and its expression was decreased in skin with hair follicles after CYP and TAC treatment, and PARP-1 hyperactivation was associated with CIA through increased nuclear factor (NF)-κB p65 and p53 acetylation in wild type, NQO1-/- and K14-/- C57BL/6 mice. However, an increase in NAD+ levels by WK0202 completely restored SIRT1 activity and its expression, and subsequently deacetylated NF-κB p65 and p53 in wild type, however not in NQO1-/- and K14-/- mice thereby attenuating CIA. Therefore, modulation of NAD+ levels by NQO1 may be a novel therapeutic approach to prevent chemotherapy-associated hair loss, including CIA.

#2718

Insulin-like growth factor-1 signalling promotes mitochondrial turnover and protection in cancer cells.

Sarah Riis,1 Michael Coleman,2 Stephen D. Hursting,2 Rosemary O'Connor1. 1 _Univ. College Cork, Cork, Ireland;_ 2 _Univ North Carolina, Chapel Hill, NC_.

Mitochondrial activity and cellular metabolic reprogramming may influence cancer phenotype and contribute to therapy resistance. Our previous work showed that Insulin-like Growth Factor 1 (IGF-1) induces expression of a mitochondrial carrier protein for nucleotides (PNC1/SLC25A33). PNC1 has an essential function in maintenance of mitochondrial RNA, DNA and in cell growth 1, 2. This suggested an underlying role for IGF-1 signalling in mitochondrial homeostasis. To explore this further we investigated (1) the signalling pathways by which IGF-1 promotes mitochondrial maintenance, and (2) investigated whether PNC1 expression levels influence the phenotype of cancer cells.

We found that activation of the IGF-1 pathway promotes mitochondrial biogenesis through induction of the transcriptional co-activators PGC-1β and PRC in a range of cell lines3. MCF-7 breast cancer cells with acquired resistance to an IGF-1R tyrosine kinase inhibitor and consequently reduced IGF-1R activity (MCF-7R cells) exhibit lower expression of PGC-1β and PRC and impaired mitochondrial biogenesis compared to controls3. Interestingly, MCF-7R cells also exhibit mitochondrial dysfunction and a strong dependence on glycolysis for survival. IGF-1 also induces expression of the Nrf2/NFE2L2 gene, which is implicated in mitochondrial biogenesis, anti-oxidant responses, and regulating expression of the mitophagy mediator BNIP3. Interestingly, suppression of Nrf2 impaired the induction of BNIP3 in response to IGF-1. This suggests that Nrf2 integrates IGF-1-stimulated mitochondrial biogenesis with induction of mitophagy to regulate mitochondrial turnover and promote the survival of cancer cells.

To investigate the role of IGF-1-induced PNC1 in cancer cell metabolism we manipulated its expression in different cell lines. PNC1 suppression was associated with loss of cell growth, acquisition of an invasive phenotype, and impaired mitochondrial function indicated by reduced respiration. Ectopic expression of PNC1 was sufficient to restore mitochondrial function, and to suppress clonogenic growth and invasive characteristics.

Overall we conclude that IGF-1 signalling protects mitochondrial function in cancer cells by stimulating biogenesis and turnover. This mitochondria protective signal is likely to strongly influence responses to therapy and the phenotypic evolution of cancer.

References:

1. Floyd, S. et al (2007) The Insulin-like Growth Factor-I mTOR Signaling Pathway Induces the Mitochondrial Pyrimidine Nucleotide Carrier to Promote Cell Growth Mol. Biol Cell. 18:3545-3555.

2. Favre, C. et al (2010) Pyrimidine Nucleotide Carrier (PNC1) regulates mitochondrial biogenesis and the invasive phenotype of cancer cells. Oncogene 29:3964-3976.

3. Lyons, A. et al (2017) IGF-1- signalling is essential for mitochondrial biogenesis and mitophagy in cancer cells. J. Biol. Chem. 292:16983-16998.

#2719

Metabolic product of inositol-trisphosphate 3-kinase B confers chemotherapy resistance by managing redox homeostasis.

Chaoyun Pan,1 Sumin Kang,1 Jaemoo Chun,1 Austin Boese,1 Jihoon Kang,1 Jie Li,1 Lingtao Jin,2 Dan Li3. 1 _Emory University, Decatur, GA;_ 2 _University of Florida, Decatur, GA;_ 3 _National University of Singapore, Decatur, GA_.

How altered metabolism contributes to chemotherapy resistance in cancer cells remains unclear. Through a metabolism-related kinome RNAi screen, we identified inositol-trisphosphate 3-kinase B (ITPKB) as a critical enzyme that contributes to cisplatin-resistant tumor growth. We demonstrate that inositol (1,3,4,5) tetrakisphosphate (IP4), the product of ITPKB, plays a critical role in redox homeostasis upon cisplatin exposure by reducing cisplatin-induced ROS through inhibition of a ROS-generating enzyme NADPH oxidase 4 (NOX4), which promotes cisplatin- resistant tumor growth. Mechanistically, IP4 competes with the NOX4 cofactor NADPH for binding and consequently inhibits NOX4. Targeting ITPKB with shRNA or its small molecule inhibitor resulted in attenuation of NOX4 activity, imbalanced redox status, and sensitized cancer cells to cisplatin treatment in patient-derived xenografts. Our findings provide insight into the crosstalk between kinase-mediated metabolic regulation and platinum-based chemotherapy resistance in human cancers. Our study also suggests a distinctive signaling function of IP4 that regulates NOX4. Furthermore, pharmaceutical inhibition of ITPKB displayed synergistic attenuation of tumor growth with cisplatin suggesting ITPKB as a promising synthetic lethal target for cancer therapeutic intervention to overcome cisplatin resistance.

#2720

Mitochondrial ClpP-mediated proteolysis induces selective cancer cell lethality.

Jo Ishizawa,1 Sarah F. Zarabi,2 R Eric Davis,1 Ondrej Halgas,2 Takenobu Nii,1 Yulia Jitkova,2 Ran Zhao,1 Jonathan St-Germain,2 Lauren E. Heese,1 Grace Egan,2 Vivian R. Ruvolo,1 Samir H. Barghout,2 Yuki Nishida,1 Rose Hurren,2 Wencai Ma,1 Marcela Gronda,2 Todd Link,1 Keith Wong,2 Mark Mabanglo,2 Kensuke Kojima,3 Gautam Borthakur,1 Neil MacLean,2 John Man Chun Ma,1 Andrew B. Leber,2 Mark D. Minden,2 Walid Houry,2 Hagop Kantarjian,1 Martin Stogniew,4 Brian Raught,2 Emil F. Pai,2 Aaron D. Schimmer,2 Michael Andreeff1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada;_ 3 _Saga University, Saga, Japan;_ 4 _Oncoceutics, Inc., Philadelphia, PA_.

ClpP is a mitochondrial protease and a major protein quality control mediator that primarily interacts with metabolic enzymes in mitochondria. Here, we demonstrate that activation of this protease results in prominent anti-cancer activity, and propose ClpP activation as a novel therapeutic strategy for cancer and hematologic malignancies. We used genetic and chemical tools to activate ClpP. In a genetic approach, we tested the anti-cancer effects of ClpP activation by expressing a constitutively active ClpP mutant. Indeed, induction of the active ClpP mutant induced apoptosis in vitro and inhibited tumor progression in vivo. To further explore the antineoplastic effects of ClpP activation, we then performed a chemical screen of an in-house library of on-patent and off-patent drugs and identified imipridones (ONC201 and ONC212) as potent ClpP agonists. Imipridones are first-in-class antineoplastic agents and have shown preclinical efficacy in various malignancies in vitro and in vivo and are currently being evaluated in clinical trials in a diverse spectrum of cancers. Importantly, we and others have shown that their activity is agnostic to TP53 mutational status. Of note, molecular targets of imipridones that bind the drugs and are functionally important for their cytotoxicity have never been identified. Through extensive chemical investigations, including analysis of binding mechanism of the compounds to ClpP in cell free (ITC) and cell based assays (CETSA) as well as molecular analysis of the crystal structure, we demonstrate that these molecules bind ClpP non-covalently, and activate the protease by stabilizing the ClpP 14-mer, enlarging the axial pores of the complex, and inducing structural changes in the residues surrounding and including the catalytic triad. In leukemia, lymphoma and colon cancer cells including primary acute myeloid leukemia (AML) cells, both compounds displayed potent ClpP-dependent cytotoxicity with IC50s in low micro- or nanomolar ranges. Importantly, in primary AML samples, pretreatment ClpP levels correlated with response to imipridones. In lymphoma and AML xenograft models, both genetic and chemical activation of ClpP resulted in antitumor effects, while expression of inactive D190A ClpP mutant induced resistance. Mechanistically, ClpP activation leads to increased degradation of substrates of the enzyme including respiratory chain complex subunits and mitochondrial translation system. The resultant impaired mitochondrial structure and reduction in oxygen consumption is selectively cytotoxic to malignant cells that rely highly on mitochondrial energy production for their survival, whereas normal cells are not affected. In conclusion, ClpP activation is an entirely novel therapeutic strategy for malignant tumors. Our findings also suggest a general concept of inducing TP53-independent cancer cell lethality through activation of mitochondrial proteolysis.

### Structural and Functional Cancer Genomics

#2721

Establishment of a catalog of somatic genetic alterations of Japanese cancer patients across multiple tumor types at Shizuoka Cancer Center.

Hirotsugu Kenmotsu,1 Masakuni Serizawa,2 Takeshi Nagashima,3 Keiichi Ohshima,2 Keiichi Hatakeyama,2 Yuji Shimoda,3 Shumpei Ohnami,2 Koji Maruyama,2 Tohru Mochizuki,2 Yasuto Akiyama,2 Takashi Sugino,1 Kenichi Urakami,2 Masatoshi Kusuhara,2 Ken Yamaguchi1. 1 _Shizuoka Cancer Center, Shizuoka, Japan;_ 2 _Shizuoka Cancer Center Research Institute, Shizuoka, Japan;_ 3 _SRL Inc., Japan_.

Background: Recent progression of systematic tumor-molecular profiling study centered on genome sequencing have elucidated molecular signature underlying each tumor type. Tumor molecular profiling enables efficient identification of somatic genetic alterations as potential therapeutic targets. In January 2014, the Shizuoka Cancer Center of Japan launched Project HOPE, which is the first prospective molecular profiling study centered on genome sequencing across multiple tumor types in Japan. Herein, we describe the distributions of the tumor mutation burden (TMB; number of mutations/Mb), genetic alterations frequency, mutational signature across tumor types, and their associations with tissue of origin, histological type, carcinogenic factors, and ethnicity.

Methods: Between January 2014 and January 2017, 3,163 tumor samples from 3,011 patients who underwent surgery at the Shizuoka Cancer Center were collected with informed consent and subjected to whole-exome sequencing (WES) with an Ion Proton system. Corresponding peripheral blood samples were also subjected to WES for identification of tumor-specific genetic alterations. Oncogenic fusions were detected by targeted RNA sequencing. Among them, data of 2,126 primary tumor samples obtained from 2,091 patients without neoadjuvant chemotherapy and/or radiation therapy were selected for this study.

Results: The principal tumor types of ≥40 primary tumors were as follows (N, TMB): colon adenocarcinoma (COAD; 459, 3.6), rectal adenocarcinoma (READ; 366, 3.1), lung adenocarcinoma (LUAD; 290, 1.7), stomach adenocarcinoma (STAD; 221, 3.0), hepatocellular carcinoma (HCC; 112, 3.6), head and neck squamous cell carcinoma (HNSC; 112, 2.8), breast invasive ductal carcinoma (IDC; 82, 1.1), lung squamous cell carcinoma (LUSC; 81, 5.9). Approximately 8.9% of the samples (154 / 1,723) showed a high mutation burden above the threshold defined by the TMB distribution in each principal tumor type, and the mutation signature was related to a defect of DNA repair and exposure to environmental mutagens. In comparison of distribution of TMB in each tumor type between TCGA and this study, some differences in TMB distribution were observed, including LUAD, LUSC, and IDC. In gene mutations based on OncoKB site (October 2018), level 1 or 2 of somatic gene mutation was frequently observed in glioma (84.6%), gastrointestinal stromal tumor (77.4%), melanoma (40.0%), and LUAD (40.0%).

Conclusions: This established catalog of somatic genetic alterations across multiple tumor types in Japanese cancer patients can contribute as benchmark to clinical-trial design with the aim of expanding the number of patients suitable for molecular-targeted therapies in Japan. Based on these data, we are planning clinical trials of molecular-targeted therapies for patients with solid tumor harboring gene alterations.

#2722

The genomic landscape of high-grade serous ovarian cancer in long-term survivors.

Dale W. Garsed,1 Ahwan Pandey,1 Sian Fereday,1 Kathryn Alsop,1 Maartje C. Wouters,2 Flurina Saner,1 Jessica A. Beach,1 Katy Milne,2 Catherine J. Kennedy,3 Joy Hendley,1 Nadia Traficante,1 Celeste L. Pearce,4 Malcolm C. Pike,5 Multidisciplinary Ovarian Cancer Outcomes Group, Susan J. Ramus,6 Martin Köbel,7 Brad H. Nelson,2 Ellen L. Goode,8 Anna deFazio,3 David D. Bowtell1. 1 _Peter MacCallum Cancer Centre, Melbourne, Australia;_ 2 _British Columbia Cancer Agency, Victoria, British Columbia, Canada;_ 3 _The Westmead Institute for Medical Research, Sydney, Australia;_ 4 _University of Michigan School of Public Health, Ann Arbor, MI;_ 5 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 6 _University of New South Wales, Melbourne, Australia;_ 7 _University of Calgary Foothill Medical Center, Calgary, Alberta, Canada;_ 8 _Mayo Clinic, Rochester, MN_.

Purpose: The majority of patients with high-grade serous ovarian cancer (HGSC) develop progressive disease following primary treatment, with a five-year survival rate of ~30%. However, a subset of patients have an extraordinary response to treatment and ~15% survive more than ten years (long-term survivors). The Multidisciplinary Ovarian Cancer Outcomes Group (MOCOG) aims to uncover factors that influence long-term survival of HGSC patients. Here, we investigated the genomic and immunologic determinants of exceptional survival of this deadly disease.

Experimental Design: Patient characteristics and clinical histories were evaluated to identify patients diagnosed with advanced stage (Stage IIIC/IV) and histopathologically confirmed HGSC with greater than 10-year overall survival. Whole-genome sequencing (WGS) was performed on primary tumors (median 78x coverage) and germline samples (median 39x coverage) of 55 long-term survivors. Primary tumor samples were also characterised by RNA sequencing, DNA methylation profiling and immunohistochemistry.

Results: A total 38 (69%) of long-term surviving patients had residual disease following surgery, suggestive of highly chemo-sensitive disease. Most patients (41, 75%) were alive at last follow-up and 26 (47%) were progression-free. Somatic mutation burden was higher in primary tumors of long-term survivors relative to controls (316 unselected HGSC patients in The Cancer Genome Atlas). Genome-wide mutational signatures were predominantly Signature 3 (associated with homologous recombination deficiency), Signature 1 (age related) and Signatures 5, 8 and 16 (unknown etiology). Inactivation of the tumor suppressor RB1 by structural rearrangements or homozygous deletion was frequent in long-term survivors, with 33% of tumors showing associated loss of RB1 protein expression by immunohistochemistry compared to 13% of unselected HGSC controls (n=207; P = 0.001). Staining of adjacent tumor tissue revealed that RB1 loss was associated with increased numbers of PD-1+ tumor-infiltrating lymphocytes (P = 0.015) and MHC class I on tumor cells (P = 0.002). In an independent HGSC cohort (n=847), RB1 protein loss was associated with prolonged survival (HR: 0.75, P < 0.001) compared to patients with RB1 positive tumors. Furthermore, co-occurrence of germline mutations in BRCA1 or BRCA2 and RB1 loss was associated with a significantly longer overall survival compared to patients with retained RB1 protein expression and no germline BRCA mutation (HR: 0.44, P < 0.001).

Conclusions: This study delineates the full landscape of genomic alterations in HGSC of long-term survivors. Our findings indicate that specific mutations might be associated with enhanced host immune responses and long-term survival.

#2723

Defective homologous recombination DNA repair as therapeutic target in advanced chordoma.

Stefan Gröschel,1 Daniel Hübschmann,1 Francesco Raimondi,2 Peter Horak,3 Gregor Warsow,1 Martina Fröhlich,1 Barbara Klink,4 Laura Gieldon,4 Barbara Hutter,1 Kortine Kleinhenz,1 David Bonekamp,1 Oliver Marschal,5 Priya Chudasama,1 Jagoda Mika,1 Marie Groth,1 Sebastian Uhrig,1 Stephen Krämer,1 Christoph Heining,6 Christoph Heilig,1 Daniela Richter,6 Eva Reisinger,1 Katrin Pfütze,1 Roland Eils,1 Stephan Wolf,1 Christof von Kalle,1 Christian Brandts,7 Claudia Scholl,1 Wilko Weichert,8 Stephan Richter,9 Sebastian Bauer,10 Roland Penzel,11 Evelin Schröck,12 Albrecht Stenzinger,11 Richard Schlenk,1 Benedikt Brors,1 Robert Russell,2 Hanno Glimm,6 Matthias Schlesner,1 Stefan Fröhling1. 1 _DKFZ Heidelberg, Heidelberg, Germany;_ 2 _Bioquant Heidelberg, Heidelberg, Germany;_ 3 _NCT Heidelberg, Heidelberg, Germany;_ 4 _Technische Universität Dresden, Germany;_ 5 _Onkologische Schwerpunktpraxis, Braunschweig, Germany;_ 6 _NCT Dresden, Germany;_ 7 _Uniklinikum Frankfurt, Germany;_ 8 _Technische Universität München, Germany;_ 9 _Uniklinikum Dresden, Germany;_ 10 _WTZ Essen, Germany;_ 11 _Uniklinikum Heidelberg, Heidelberg, Germany;_ 12 _Technische Universität Dresden, Heidelberg, Germany_.

Chordomas are rare tumors of the axial skeleton and skull base with few therapeutic options and no clinically validated molecular drug targets. The value of comprehensive genomic analyses for guiding medical therapy of patients with advanced-stage chordoma is unknown. We performed whole-exome and genome sequencing of tumor and matched germline control samples from 11 patients with locally advanced or metastatic chordoma within the MASTER program, a prospective clinical sequencing program of the German Cancer Consortium. All patients were pretreated and had progressive disease prior to molecular analysis. Genomic profiling showed that advanced chordomas are frequently characterized by genomic patterns indicative of defective homologous recombination (HR) DNA repair. First, DNA copy number profiles showed high numbers of structural variants greater than 10 million base pairs in size in the majority of cases. Second, all patients harbored somatic aberrations of at least 2 genes known to be involved in HR, and 10/11 cases harbored somatic alterations in 3 or more HR pathway genes. For example, 8 patients showed heterozygous BRCA2 deletions, which were associated with heterozygous deletions of ERCC6 in 6 patients and RAD54L in 7 patients, as well as PTEN alterations (heterozygous deletion, heterozygous mutation and deletion of the wildtype allele or loss of heterozygosity). Other recurrently altered HR genes included ATR, CHEK2, FANCC, FANCD2, FANCG, RAD18, RAD51B, and XRCC3. Third, pathogenic germline alterations were detected in 3 patients. A heterozygous BRCA2 frameshift mutation (p.T3085fs*26; ACMG Class 5), a heterozygous NBN frameshift mutation (p.K219Nfs*16; ACMG Class 5), and a heterozygous CHEK2 missense mutation (p.R145W; ACMG Class 4) were accompanied by somatic deletion of the respective wildtype alleles. Fourth, a mutational signature associated with HR deficiency was significantly enriched in 72.7% of samples and coincided with genomic instability. The high prevalence of an HR deficiency "footprint" in chordoma patients prompted us to explore the clinical efficacy of the poly(ADP-ribose) polymerase(PARP) inhibitor olaparib, which is preferentially toxic to HR-incompetent cells. Olaparib treatment of a patient whose tumor showed a prominent exposure to an HR deficiency-associated mutational signature, a high degree of genomic instability, and 13 heterozygous HR gene alterations halted tumor growth for 10 months. Whole-genome analysis at progression revealed a PARP1 p.T910A mutation predicted to disrupt the autoinhibitory PARP1 helical domain, providing novel insight into the mechanisms of PARP inhibitor resistance. In summary, our study has uncovered a key biological feature of advanced chordoma that represents an immediately actionable therapeutic target and provides a rationale for genomics-guided clinical trials of PARP inhibition in this intractable tumor entity.

#2724

A combination of short-read and long-read RNA sequencing reveals NOVA1's role in telomere biology.

Andrew T. Ludlow,1 Mohammed E. Sayed,1 Aaron L. Slusher,1 Mark Ribick,1 Anisha Pancholi,1 Brian Sereni,2 Yu Qui,2 Elizabeth Tseng,3 Meredith Ashby,3 David C. Corney2. 1 _University of Michigan, Ann Arbor, MI;_ 2 _GENEWIZ, South Plainfield, NJ;_ 3 _Pacific Biosciences, Menlo, CA_.

Dysregulated alternative splicing underlies many aspects of cancer cell biology including tumor cell telomere maintenance. We recently described a role for the neuronal splicing factor, NOVA1, in non-small cell lung cancer in the regulation of full-length hTERT splicing, telomerase activity, maintenance of telomere length, and general tumor growth characteristics. We wanted to further define the role of NOVA1 in telomere biology. Cells that maintain telomeres with different mechanisms were studied to be able to decipher telomerase specific versus alternative lengthening of telomeres (ALT) specific perturbations by NOVA1. Both short-read (Illumina) and long-read (Pacific Biosciences) sequencing of control and NOVA1 siRNA knockdown cells in H920 non-small cell lung cancer cells (telomerase positive) and U2OS cells (ALT positive cells) were performed to detail the transcriptomes of these cells. A combination of rMATS analysis of the short-read sequencing, SQANTI analysis of the long-read sequencing, and pathway analysis was performed to identify genes related to telomere biology and alternative splicing related to telomerase. Skipped exon events were the primary focus of this study as this is NOVA1's main role in alternative splicing. rMATS analysis identified 4,530 significant events mapping to 2,950 genes in H920 cells and 2,534 significant events in 1,863 genes in U2OS cells. SQANTI analyzed 15,443 full-length transcripts (7,530 genes) in control and 14,463 transcripts (7,240 genes) in NOVA1 siRNA treated samples of the H920 cells and 12,133 transcripts (6,618 genes) in control and 10,126 transcripts (5,959 genes) in NOVA1 siRNA treated U2OS cells. There were 2,762 and 2,470 novel transcripts in control and NOVA1 siRNA treated H920 cells, respectively. In U2OS cells there were 2,178 novel transcripts in control and 1,688 novel transcripts in NOVA1 siRNA treated cells. The pathway analysis of rMATS cross-referenced with SQANTI revealed five top candidates that were specifically altered in NOVA1 knockdown cells and related telomere length maintenance by telomerase (POLD1, SUN2, ERCC1, SRSF2, and PCBP2). Interestingly, SRSF2 and PCBP2 were identified in our recent hTERT minigene loss of function screen for splicing regulatory genes of hTERT and have binding sites in hTERT near NOVA1s motif in intron 8. Long-read sequencing determined the full-length nature of these splicing events pointed out by the rMATS analysis. These data indicate that telomerase positive cancer cells with dysregulated expression of NOVA1 utilize different isoforms compared to ALT cells with NOVA1 to maintain telomeres. These new observations reveal that alternative splicing regulated in part by NOVA1 plays a larger and unexpected role in telomere biology. Manipulation of alternative splicing to shorten/deprotect telomeres should be pursued as a means to inhibit survival and growth of cancer cells.

#2725

The single-cell atlas of driver mutations in acute myeloid leukemia.

Kiyomi Morita,1 Feng Wang,1 Katharina Jahn,2 Yuanqing Yan,3 Robert Durruthy-Durruthy,4 Anup Parikh,4 Jairo Matthews,1 Latasha Little,1 Curtis Gumbs,1 Jianhua Zhang,1 Xingzhi Song,1 Erika Thompson,1 Keyur Patel,1 Carlos Bueso-Ramos,1 Courtney DiNardo,1 Farhad Ravandi,1 Elias Jabbour,1 Michael Andreeff,1 Jorge Cortes,1 Marina Konopleva,1 Guillermo Garcia-Manero,1 Hagop Kantarjian,1 Dennis J. Eastburn,4 P Andrew Futreal,1 Niko Beerenwinkel,2 Koichi Takahashi1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Swiss Federal Institute of Technology in Zurich, Zurich, Switzerland;_ 3 _The University of Texas Health Science Center at Houston, Houston, TX;_ 4 _Mission Bio, Inc., South San Francisco, CA_.

Assessment of intratumor genetic heterogeneity by next-generation sequencing (NGS) is confounded by tumor purity and zygosity of mutations. Furthermore, bulk NGS cannot visualize the cell-level co-occurrence or exclusivity among multiple mutations, causing the inaccurate inference of subclonal architecture. Here, we performed single cell DNA sequencing (scDNA seq) in 82 bone marrow samples from 70 patients with acute myeloid leukemia (AML) using a novel microfluidics-based platform covering 40 amplicons in 19 AML genes (Tapestri, Mission Bio). All samples were concurrently sequenced by the bulk NGS using 295 gene exome capture sequencing. Selected cases were also analyzed by SNP array (Illuminia Omni 2.5 array) to obtain allele-specific copy number data. In total, 319,406 cells (median 3,755 cells/sample) were genotyped with median allele drop-out rate of 8.7% (population frequency inferred from commonly heterozygous SNP loci). Each amplicon was covered at a median 24x/cell. The scDNA seq detected 230 of 238 (97%) bulk NGS-confirmed mutations. RUNX1 mutations were frequently detected as homozygous mutations and concurrent SNP array analysis detected copy number neutral loss-of-heterozygosity of RUNX1 in these cases. Additionally, in cases with homozygous FLT3-ITD mutations, SNP array detected allele-specific copy number gain of mutant loci, which likely resulted in homozygous calls. scDNA seq data unambiguously resolved the single-cell level co-occurrence of driver mutations in AML such as DNMT3A/FLT3-ITD/NPM1, SRSF2/IDH2, and ASXL1/RUNX1, confirming the cooperative function of these driver mutations. On the other hand, the data also revealed mutually exclusive relationships at cellular level between IDH1/IDH2, FLT3-ITD/TKD, NRAS/KRAS, NRAS/PTPN11, and SRSF2/EZH2 mutations, which supports the functional redundancy of these genes in leukemogenesis. Inference of phylogenetic trees using SCITE algorithm (Jahn et al. Genome Biology 2016) uncovered distinct patterns of clonal evolution in AML. The majority of the analyzed cases had a linear evolution pattern where the founder mutations linearly acquired sub-clonal mutations in a step-wise manner. We also detected convergent evolution in some cases where functionally similar driver mutations were acquired in parallel. DNMT3A, IDH1, IDH2 and U2AF1 mutations were frequently detected as trunk mutations, whereas FLT3, NRAS, and NPM1 mutations were usually detected as branch mutations. Analysis of longitudinal samples from 11 patients revealed the remodeling of clonal architecture at relapse. Patients who had 2 or more major subclones at baseline had significantly worse overall survival than those with one subclone (2-year OS 13 vs. 70 months, p = 0.0493). For the first time our data provides a comprehensive landscape of driver mutations and detailed clonal evolution history in AML at the single-cell level using high-throughput scDNA seq genotype information.

#2726

**Functional multiomic profiling identifies Aurora kinase inhibition as a therapeutic strategy in** RIT1 **-mutant lung adenocarcinoma.**

Athea Vichas, Naomi T. Nkinsi, Alice Berger. _Fred Hutchinson Cancer Research Center, Seattle, WA_.

Treatment of lung cancer is evolving from standard cytotoxic to personalized treatment based on the molecular alterations unique to each patient's tumor. In our recent work with TCGA and others, we identified somatic mutations in the gene encoding the RAS-related small GTPase, RIT1, in lung adenocarcinomas. In addition, somatic RIT1 mutations have also been identified in myeloid malignancies and germline RIT1 mutations are found in the developmental "RAS-opathy," Noonan syndrome. In all these diseases, RIT1 mutations are mutually-exclusive with other RAS/MAPK pathway mutations, including mutations in EGFR, KRAS, and ALK. While our previous work demonstrated that gain-of-function RIT1 mutations induce activation of PI3K and MEK signaling, the specific effectors used by RIT1 to promote tumorigenesis are unknown. To genetically dissect the signaling pathways downstream of RIT1, KRAS, EGFR, and PIK3CA, we performed genome-wide CRISPR/Cas9 screens in four isogenic lung adenocarcinoma cell lines in which cell survival is dependent on the expression of the oncogene. Through integrative analysis of essential genes across the four cell lines, we identified synthetic lethal relationships unique and shared in each isogenic background. Among the dependencies identified was the requirement of aurora kinase pathway genes for survival of RIT1-mutant cells, raising the possibility of therapeutic aurora kinase inhibition in RIT1-positive tumors. To evaluate whether the dependence on Aurora kinases extended to small molecule inhibition, we performed a screen of 160 small molecules in clinical development or approved by the FDA. Consistent with our CRISPR/Cas9 results, we find that RIT1-mutant cells depend on Aurora kinase activity for survival. Interestingly, global phosphoproteomic profiling identified RIT1 regulation of an Aurora kinase-Haspin axis. Taken together these results identify a new arm of RIT1 signaling and nominate Aurora kinase inhibition as a potential therapeutic strategy in RIT1-mutated tumors.

#2727

The pre-malignant state captured in the landscape of somatic mutations can reveal the cancer cell-of-origin.

Kirsten Kubler,1 Rosa Karlic,2 Nicholas J. Haradhvala,1 Kyungsik Ha,3 Jaegil Kim,1 Maja Kuzman,2 Wei Jiao,4 Sitanshu Gakkhar,5 Kent W. Mouw,6 Lior Z. Braunstein,7 Olivier Elemento,8 Andrew V. Biankin,9 Ilse Rooman,10 Mendy Miller,1 Christopher D. Nogiec,11 Edward Curry,12 Mari Mino-Kenudson,13 Leif W. Ellisen,13 Robert Brown,12 Alexander Gusev,14 Cristian Tomasetti,15 Hong-Gee Kim,3 Hwajin Lee,3 Kristian Vlahovicek,2 Charles Sawyers,7 Katherine A. Hoadley,16 Edwin Cuppen,17 Amnon Koren,18 Peter F. Arndt,19 David N. Louis,13 Lincoln Stein,4 William D. Foulkes,20 Paz Polak,11 Gad Getz1. 1 _The Broad Institute of MIT and Harvard, Cambridge, MA;_ 2 _University of Zagreb, Croatia;_ 3 _Seoul National University, Republic of Korea;_ 4 _Ontario Institute for Cancer Research, Ontario, Canada;_ 5 _Canada's Michael Smith Genome Sciences Centre, British Columbia, Canada;_ 6 _Brigham & Women's Hospital and Dana Farber Cancer Institute, MA; _7 _Memorial Sloan Kettering Cancer Center, NY;_ 8 _Weill Cornell Medical College, NY;_ 9 _University of Glasgow, United Kingdom;_ 10 _Vrije Universiteit Brussel, Belgium;_ 11 _Mount Sinai Health System, NY;_ 12 _Imperial College London, United Kingdom;_ 13 _Massachusetts General Hospital, MA;_ 14 _Brigham and Women's Hospital & Dana Farber Cancer Institute, MA; _15 _The Johns Hopkins Medical Institutions, MD;_ 16 _The University of North Carolina at Chapel Hill, NC;_ 17 _Hartwig Medical Foundation, Netherlands;_ 18 _Cornell University, NY;_ 19 _Max Planck Institute for Molecular Genetics, Germany;_ 20 _McGill University, Quebec, Canada_.

Despite increasing knowledge of tumorigenesis, the identity of the cancer cell-of-origin, i.e. the normal cell type that acquired the cancer-initiating event, remains largely unknown. Our approach of identifying the cell-of-origin is based on two observations: (1) the chromatin structure is cell-specific; and (2) the density of somatic mutations along the genome is associated with the regional profile of chromatin modifications.

We have previously developed a method that quantifies the ability to predict the mutational distribution along the cancer genome from the profile of epigenetic modifications in different normal cell types. Here we present the largest application of our method using 2,550 whole genomes representing 32 distinct cancer types. To identify the cell-of-origin, we determined the correlation between the observed density of mutations along the genome and the predicted values based on chromatin modifications from 104 different normal tissue types. The normal cell type that showed the strongest correlation with a specific cancer mutational landscape was the candidate cell-of-origin.

We found that in almost all cancer types the cell-of-origin can be characterized solely from DNA sequences. Interestingly, we found that the fallopian tube was the best match for high-grade serous ovarian cancer, providing independent evidence that this is the cancer's site of origin. For breast cancer we found that the four distinct subtypes best-matched cells from the luminal cell lineage: basal-like breast cancer likely originates from luminal progenitors, whereas all other subtypes from luminal mature cells. This association holds true even when accounting for different alterations in the homologous recombination repair pathway, suggesting that subtypes are more determined by the cell-of-origin than the specific DNA repair defect. In addition, we found that we could identify the cell-of-origin using metastatic samples -- a finding that may help in difficult clinical diagnoses. Moreover, we demonstrate that cancer drivers, both germline risk alleles and somatically mutated drivers, reside in active chromatin regions in the respective cell-of-origin.

Taken together, our findings indicate that many of the somatic mutations accumulated while the cells maintained a chromatin structure similar to the cell-of-origin (likely occurring prior to transformation). Therefore, this historical record, captured in the DNA, can be used to identify, the often elusive, cancer cell-of-origin. Our approach can ultimately help better understand the potential of particular normal cell types to transform and initiate cancer, as well as the association of the cell-of-origin with tumor subtypes and sensitivity to treatment.

## PREVENTION RESEARCH

### Molecular and Cellular Mechanisms in Cancer Prevention Research

#2728

Cholecystokinin receptor antagonist proglumide reverses nonalcoholic steatohepatitis and prevents hepatocellular carcinoma.

Martha D. Gay,1 Victor Ciofoaia,2 Sandeep Nadella,1 Hong Cao,1 Matthew Huber,1 Robin D. Tucker,1 Narayan Shivapurkar,1 Bhaskar Kallakury,1 Alexander H. Kroemer,1 Jill P. Smith1. 1 _Georgetown University, Washington, DC;_ 2 _Mayo Clinic Jacksonville, Jacksonville, FL_.

Background: The fastest growing cause of cancer related death is hepatocellular carcinoma (HCC), in part due to the rising prevalence of nonalcoholic steatohepatitis (NASH). HCC cases are expected to increase during 2015-2030. Cholecystokinin (CCK), blood levels are increased in those consuming high fat diets and CCK receptors have been identified in normal liver, fibroblasts, and immune cells. Hypothesis: We hypothesize that CCK receptor blockade could decrease NASH and prevent HCC in a mouse model.Methods: We examined whether CCK receptor blockade with proglumide could decrease NASH in the choline deficient ethionine supplemented (CDE)-diet mouse model. Two cohorts of Female C57BL/6 mice (N=65) mice were examined: (1) a preventive study in which mice were treated for 12 and 18 weeks to determine if proglumide could prevent NASH and HCC and (2) a 'reversal study' in which histologic and biochemical NASH was induced on a 75% CDE high fat diet for 12 weeks followed by treatment with proglumide for 6 weeks (until week 18) while remaining on the CDE diet to determine if CCK-receptor blockade could reverse NASH once established. Control mice received the same high fat diet with choline and half received proglumide therapy. Results: Alanine aminotransferase, aspartate aminotransferases, and bilirubin were increased in mice on the CDE/Regular (untreated water) diet and these values returned to near normal with proglumide therapy in the prevention study and also reversed after NASH was established. Histologic scoring showed significant decreased inflammation, fibrosis and steatosis in mice on the high fat CDE diet when treated with proglumide. 35% of the mice (N=23) at week 18 developed HCC or dysplastic nodules on the CDE diet while none of the mice in the CDE/proglumide prevention (N=15) or reversal therapy groups (N=15) had HCC. Mice on the lard-control diet had fatty livers at week 18 but no evidence of NASH histologically or biochemically and no HCC. Fibrosis was decreased in mice treated with proglumide as evidence by Masson's trichrome stain and Western blot for FAP. Gene expression of the liver's collagen-4, TGFβR11, PDGH-Rβ, and collagen-1α1 as determined by qRT-PCR decreased with proglumide treatment. Control diet fed mice had normal serum tests and fibrosis biomarkers. Conclusion: This data supports our novel hypothesis that CCK receptors play a role in NASH-associated HCC. CCK receptor blockade decreased histologic and biochemical evidence of NASH and prevented HCC. Since proglumide has already been tested in human subjects for peptic ulcer disease decades ago and is known to be orally bioavailable, results of our study could easily be translated into a clinical trial in human subjects to improve NASH to prevent HCC.

Supported by AGA Research Foundation, Ruesch Foundation, and a grant from NIH, TL1TR001431

#2729

Development of a targeted liquid biopsy for early gynecologic cancer detection leads to discovery of a highly prevalent genomic landscape of cancer driver gene mutations in uterine tissue from women without cancer.

Deep S. Pandya,1 Shannon Tomita,2 Olga Camacho,2 Sabina Swierczek,1 Catalina Camacho,2 Kelsey Engelman,2 Stephanie Polukort,1 Maria Mercedes Padron,2 Jordan RoseFigura,3 Jon Irish,3 Linus Chuang,1 Vaagn Andikyan,1 Samantha Cohen,2 Paul Fiedler,1 Steven Sieber,1 Ie-Ming Shih,4 Jean-Noel Billaud,5 Boris Reva,2 Robert Sebra,2 Peter Dottino,2 John A. Martignetti2. 1 _Danbury Hospital, Danbury, CT;_ 2 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 3 _Swift Biosciences, Ann Arbor, MI;_ 4 _Johns Hopkins University School of Medicine, Baltimore, MD;_ 5 _Qiagen, Redwood City, CA_.

Introduction: Ovarian and endometrial cancers are, respectively, the most lethal and most frequent female reproductive tract malignancies. Currently, there are no effective screening methodologies or protocols for these cancers. We hypothesized that the use of ultra-deep, targeted gene sequencing on a highly specific and gynecologic cancer-focused analyte source, namely, uterine lavage fluid, could detect somatic mutations associated with these cancers. Using these highly sensitive NGS-based assays, we demonstrated both (1) the ability to detect tumor-specific mutations in uterine lavage from women even with microscopic stage IA ovarian and endometrial cancers and (2) the existence of highly prevalent populations of cancer driver gene mutated cells in uterine tissue from women without cancer.

Methods: In total, and following written informed consent, samples were prospectively collected and analyzed from two institutions from more than 250 women at the time of their surgery. Sequencing was performed using both Ion Torrent and Illumina-based sequencing and additional orthogonal mutation validation was performed using dPCR and Sanger sequencing.

Results: Targeted sequencing of cellular and cell free DNA isolated from uterine lavage samples was performed on all samples in a blinded fashion. Histopathologic diagnosis and sample decoding, revealed that lavage mutations were present in nearly all women with ovarian and endometrial cancer, even those with microscopic stage IA disease. These mutations were demonstrated to be tumor-specific by sequencing their respective tumors. Notably, we also identified mutations in approximately half of all women without any evidence of cancer. To define the possible origin of these mutations in normal tissue, we analyzed uterine tissue from women without cancer. Two hundred biopsy samples were obtained from 24 uterine samples following hysterectomy for non-cancerous conditions. Remarkably, sequencing revealed relatively high allele fraction cancer-associated mutations in more than half of these samples. The overwhelming majority of samples harbored low frequency mutations.

Conclusions: Our studies demonstrate the ability to detect with very high sensitivity, tumor-specific somatic mutations in women with ovarian and endometrial cancers; suggesting the future possibility of earlier diagnosis and better outcomes. At the same time, our studies again highlight caveats regarding NGS-based results for cancer screening. Finally, the identification of a previously unknown, highly prevalent, cancer-driver mutation landscape in apparently normal uterine tissue opens the door onto new areas of biologic exploration for understanding tumorigenesis and the protective mechanisms which inhibit cancer development.

#2730

Exceptional activity of ARN-509 (apalutamide) in prostate cancer prevention in rats.

Genoveva Murillo,1 Xinjian Peng,1 Miguel Muzzio,1 Maarten C. Bosland,2 Elizabeth R. Glaze,3 Chen Suen,3 David L. McCormick1. 1 _IIT Research Institute, Chicago, IL;_ 2 _University of Illinois at Chicago, Chicago, IL;_ 3 _National Cancer Institute, Bethesda, MD_.

ARN-509 (apalutamide) is a "next-generation" non-steroidal androgen receptor (AR) antagonist that is approved by the FDA for treatment of non-metastatic castration-resistant prostate cancer. Studies were performed in a well-studied rat model to evaluate the efficacy of ARN-509 as an inhibitor of androgen-dependent prostate carcinogenesis. To support dose selection for the prostate cancer chemoprevention bioassay, a six-week pilot study was performed to evaluate the safety of ARN-509 in Wistar rats, and to characterize its PK parameters and dose-response relationships for pharmacodynamic activity (modulation of AR-dependent signaling pathways in the prostate). Several androgen-dependent signaling pathways were modulated by ARN-509; the most reliable biomarker of ARN-509 action in the prostate was down-regulation of kallikrein 1-related peptidase c9 (Klk1c9). Efficacy in prostate cancer prevention was evaluated using a rat model in which invasive prostate cancers are induced by sequential administration of anti-androgen, androgen, and chemical carcinogen (N-methyl-N-nitrosourea; MNU), followed by chronic androgen stimulation. Beginning one week after a single dose of MNU (30 mg/kg), male Wistar rats received daily oral (gavage) administration of ARN-509 at doses of 0 (vehicle control), 15, or 30 mg/kg body weight/day for 60 weeks; a fourth group received daily gavage administration of ARN-509 at 30 mg/kg/day for 60 weeks using an intermittent (one week on; one week off) schedule. Chronic oral administration of ARN-509 was well-tolerated by male Wistar rats; the compound is an extraordinarily potent inhibitor of prostate carcinogenesis. In comparison to a 53% incidence of accessory sex gland cancers (20/38 rats) and a 47% incidence of cancers that were clearly confined to the dorsolateral/anterior prostate (18/38 rats), cancer incidence in the accessory sex glands in both groups of rats receiving continuous daily administration of ARN-509 was 0% (30 mg/kg/day: 0/39 rats; 15 mg/kg/day: 0/37 rats). Intermittent (one week on; one week off) administration of the high dose of ARN-509 (30 mg/kg/day) was also highly effective in cancer prevention, but did not confer the complete protection seen with continuous daily administration: the group receiving intermittent exposure to ARN-509 demonstrated a 10% incidence of accessory sex gland cancer (4/39 rats), with 3% of animals (1/39 rats) demonstrating cancers that were clearly confined to the dorsolateral/anterior prostate. ARN-509 is the most potent inhibitor of prostate carcinogenesis identified in more than two dozen in vivo prostate cancer prevention bioassays performed in our laboratory, and is substantially more active in prostate cancer prevention in this model than is flutamide. These data suggest that ARN-509 merits consideration for evaluation in clinical trials for prostate cancer prevention. [Supported by HHSN261201500042I from NCI]

#2731

A novel PDE10/beta-catenin inhibitor, MCI-048, suppresses lung tumorigenesis to block metastasis.

Bing Zhu,1 Veronica Ramirez-Alcantara,1 Antonio Ward,1 Kristy Berry,1 Adam B. Keeton,1 Michael R. Boyd,2 Yulia Maxuitenko,1 Xi Chen,1 Gary A. Piazza1. 1 _University of South Alabama Mitchell Cancer Institute, Mobile, AL;_ 2 _ADT Pharmaceuticals, Orange Beach, AL_.

We previously reported that phosphodiesterase 10A (PDE10) is overexpressed during early stages of lung cancer and is essential for lung tumor cell growth. Here we characterize a novel PDE10 inhibitor, MCI-048, that was identified by screening a library of indene compounds. MCI-048 potently inhibited the growth and induced apoptosis of multiple human lung cancer cell lines expressing PDE10, while normal human airway epithelial cells lacking PDE10 expression were appreciably less sensitive. The mechanism of action of MCI-048 involves PDE10 inhibition, cGMP elevation, PKG activation, and phosphorylation of β-catenin at key residues that induce ubiquitination and proteosomal degradation of the oncogenic pool of β-catenin in cytoplasm to suppress the translocation of active β-catenin to the nucleus and Lef/Tcf-mediated transcription of genes encoding for proteins such as c-myc, cyclin D, and survivin, which are essential for tumor cell proliferation and survival. Pharmacokinetic and tissue distribution studies revealed a unique feature of MCI-048 to accumulate at high concentrations in lungs relative to plasma and other tissues. To assess the potential of MCI-048 for the treatment of lung cancer and blocking metastasis, we tested the drug in two mouse models of lung cancer involving either orthotopic implantation of KRAS mutant A549 lung tumor cells or the chemical carcinogen, urethane. Oral administration of MCI-048 significantly inhibited tumor growth and extended survival in the orthotopic model using two different protocols to either treat the primary tumor or metastasis. Similarly, MCI-048 significantly reduced tumor burden as measured by both surface counting and histopathological analysis in the urethane-induced model of lung tumorigenesis. Biochemical analysis showed that MCI-048 reduced levels of urethane-induced active Ras-GTP and PDE10 levels, as well as other oncogenic markers, including pEGFR and c-Myc. Both mouse models revealed that MCI-048 was well tolerated with no discernable toxicity, thus supporting preclinical development for the treatment of lung cancer. Funding provided by NCI grants R21CA182941, R01CA131378, R01CA148817, R01CA197147, and R01CA155638.

#2732

Frameshift neoantigen vaccination prevent Lynch syndrome mouse model intestinal cancer.

Ozkan Gelincik,1 Hamza Ibrahim,1 Mine Ozkan,2 Aysel Ahadova,2 Shizuko Sei,3 Robert Shoemaker,3 Mattias Kloor,2 Magnus Von Knebel Doeberitz,2 Steven M. Lipkin1. 1 _Weill Cornell Medical College in New York City, New York, NY;_ 2 _DKFZ, Heidelberg, Germany;_ 3 _NCI Division of Cancer Prevention, Rockville, MD_.

Microsatellite-unstable (MSI) cancers occurring in the context of Lynch syndrome elicit pronounced tumor-specific immune responses directed against frameshift peptide (FSP) neoantigens, which result from mismatch repair (MMR) deficiency-induced insertion/deletion mutations in coding microsatellites (cMS). We have recently completed a clinical phase I/IIa trial that successfully demonstrated safety and immunogenicity of an FSP neoantigen-based vaccine in MSI colorectal cancer patients (Clinical trial number: NCT01461148). The vaccine was safe and induced robust cellular and humoral immune responses in all vaccinated patients. To further develop a cancer preventive vaccine against MSI cancers in Lynch syndrome, we aimed to establish a preclinical mouse model. A systematic database search was performed to identify cMS sequences in the murine genome. Subsequently, intestinal tumors obtained from Lynch syndrome mice (Msh2flox/flox VpC+/+) were evaluated for mutations affecting these candidate microsatellites. Thirteen candidate cMS were detected that presented with a mutation frequency of 15% or higher. Epitope prediction using the netMHC4.0 algorithm was performed, and ten most promising FSP neoantigens were synthesized. Immunogenicity was evaluated after vaccination of C57BL/6 mice using IFN-gamma ELISpot. Four FSP neoantigens derived from cMS mutations in the genes Nacad, Maz, Xirp1, and Senp6 elicited strong antigen-specific cellular immune responses. CD4-specific T cell responses were detected for Maz, Nacad, and Senp6 and CD8-positive T cells were detected for Xirp1 and Nacad. Vaccination with peptides encoding these four intestinal cancer FSP neoantigens promoted anti-neoantigen immunity, reduced intestinal tumorigenicity and prolonged overall survival (P<0.01). Additionally, NSAIDs, which have chemopreventive efficacy for Lynch syndrome, increase T cell immunity against neoantigens. Mechanistic tumor mutation burden and adaptive immune response studies will be shown. In summary, these data support the further development of vaccination strategies for preventing cancers associated with Lynch syndrome.

#2733

**Novel mouse model carrying** APC **and** PIK3CA **mutations in colorectal cancer.**

Naghma Khan, Farah Jajeh, Emily L. Eberhardt, Devon D. Miller, Dawn M. Albrecht, Rachel Van Doorn, Melissa D. Hruby, Morgan E. Maresh, Linda Clipson, Hasan Mukhtar, Richard B. Halberg. _Univ. of Wisconsin-Madison, Madison, WI_.

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in the United States, despite substantial development in its early diagnosis and treatment. The animal models of CRC have been immensely useful for understanding CRC pathogenesis, investigating the effects of genetic modifications on CRC, and for the development of new chemopreventive/chemotherapeutic drugs. Most (>80%) CRC carry mutations in the APC gene and many (15-20%) carry mutations in the PIK3CA gene, encoding the p110 catalytic subunit of the PI3K kinase. We sought to better understand the interaction between APC and PIK3CA mutations in the mammalian intestine. To examine the effect of mutations in APC and PIK3CA on tumorigenesis, Min mice were crossed with the FC13K1 (FC13K1ApcMin/+). This cross resulted in a murine model with the loss of one allele of Apc throughout the intestine and the expression of a dominant active PI3K (3K1) in the distal small intestine and colon due to the expression of Cre under the control of the rat fatty acid binding protein-1 promoter (FC1). There was increased tumor multiplicity, size and a more aggressive and poorly differentiated phenotype as a consequence of synergy between APC and PIK3CA mutations. Tumors form as adenomas, but quickly progress to invasive adenocarcinomas that eventually metastasize to regional lymph nodes. Using this mouse model, we have recently shown that fisetin, a dietary flavonoid could be used as a preventive agent and an adjuvant with 5-fluorouracil (FU) for the treatment of PIK3CA-mutant CRC. Tumor incidence was markedly lower in fisetin-treated FC13K1ApcMin/+ mice in distal small intestine and colon, as compared to control animals, indicating that fisetin is a strong preventive agent. In addition, the combination of fisetin and 5-FU also reduced the total number of intestinal tumors. We extended our work by demonstrating that the effect of drugs on tumorigenesis was impacted by the mutation profile of the tumor and intratumoral heterogeneity. We developed the mouse model in which intestinal tumors were composed entirely of PIK3CA

wild-type cells, entirely of PIK3CA-mutant cells, or a mixture of both. We demonstrated that low dose aspirin blocked the development of heterogeneous tumors composed of PIK3CA wild-type and PIK3CA-mutant cells but not the development of homogenous tumors composed entirely of PIK3CA wild-type cells. Thus, this new model of CRC recapitulates the effect of aspirin that was observed in humans. Sustained exposure to low dose aspirin reduced the recurrence the PIK3CA-mutant CRC cancers in humans. This aggressive murine model is an exciting model of human CRC that has the potential to be instrumental in the development of targeted chemoprevention and therapeutics.

#2734

Poly-ligand profiling of plasma samples as a potential predictor of prostate biopsy results.

Xixi Wei, Xiaowei Liu, Radhika Santhanam, Daniel Magee, Anthony Helmstetter, Mark Miglarese, David Spetzler. _Caris Life Sciences, Phoenix, AZ_.

Prostate cancer is the most common cancer in men, excluding skin cancer, and is also one of the leading causes of cancer death. Currently the prostate-specific antigen (PSA) blood test is employed as an initial screening method in men without symptoms, and patients with blood PSA level higher than 4 ng/mL are commonly recommended for prostate biopsy for definitive diagnosis. However, other non-cancerous conditions may also cause elevation in PSA, such as prostatitis, benign prostatic hyperplasia and prostate intraepithelial neoplasia. Among men with PSA levels of 4-10 ng/mL, only about 1 in 4 will show positive (cancer) biopsy, and for those with PSA levels higher than 10 ng/mL, the chance of having prostate cancer increases to over 50%. Moreover, approximately 15% of men with a PSA levels below 4 ng/mL have a positive prostate biopsy. Because of these scenarios, as well as significant risks associated with prostate biopsies, there is a need for diagnostic adjuncts that provide additional data to inform decisions on whether or not to perform biopsies.

Here we describe a method using single stranded oligonucleotide (ssODN) libraries to perform Poly-Ligand Profiling on plasma exosomes from men scheduled for biopsy to classify those with or without a subsequent diagnosis of prostate cancer. Through alternating rounds of positive and negative selection in binding assays, we isolated ssODNs that selectively bound to exosomes from prostate cancer patients (PSA level of at least 4 ng/mL; Gleason Score range 7-8, and at least 3 positive cores from at least 10 biopsy cores) and not to benign cases (all negative cores from at least 10 biopsy cores). An independent test set of 29 prostate cancer cases and 28 benign cases was then evaluated in binding assays. Based on 100 runs of 10-fold cross validation with multiple statistic models, an average successful classification rate of 82% was achieved with an area under the ROC curve of 0.89. While further refinement and testing on larger cohorts are needed, these results suggest that Poly-Ligand Profiling may provide an additional approach to stratify men with suspicious PSA levels for prostate biopsies.

## TUMOR BIOLOGY

### Invasion and Metastasis 1: Cell-Intrinsic

#2735

Targeting HDAC prevents smoking-induced pancreatic cancer metastasis.

Jiyong Yang,1 Chintan Chheda,2 Dina Hauptschein,3 Latifa Zayou,2 Josiah Tang,3 Qiang Wang,3 Stephen J. Pandol,3 Mouad Edderkaoui3. 1 _Shanghai University of TCM, Shanghai, China;_ 2 _Cedars-Sinai Medical Center, Los Angeles, CA;_ 3 _Cedars-Sinai Medical Center & UCLA, Los Angeles, CA_.

Background: Cigarette smoking is an established risk factor for pancreatic ductal adenocarcinoma (PDAC). Among patients with metastatic PDAC, 90% have liver and 50% have lung metastasis. In this project we investigated the effect of smoking on pancreatic cancer invasion and metastasis in vitro and in vivo.

Methods: Pancreatic cancer cells were treated with different doses of 4-(methylnitro-s-amino)-1-(3-pyridyl)-1-butanone (NNK) and cigarette smoke extract (CSE) and cancer cell survival, markers of epithelial to mesenchymal transition (EMT) as well as invasion were measured by MTT assay, Western, RT-PCR and Matrigel Invasion Assay. In a syngeneic mouse model of pancreatic cancer mice were pre-treated with NNK for 4 weeks and mouse cancer cells were injected in the spleen of mice. Progression of the disease including metastasis to liver and lung was monitored by imaging and tissues were collected and analyzed after 6 weeks.

Results: Smoking compounds slightly but significantly increased cancer cell survival. They significantly promoted expression of EMT markers and promoted invasion of the cancer cells. Inhibition of histone deacetylase (HDAC) (class I and II), and especially HDAC4, prevented the pro-EMT and invasion effect of smoking compounds. NNK increased the protein level of HDAC4 and the level of Yes-associated protein 1 (Yap) translocated to the nucleus. Inhibition of HDAC4 reversed this effect. Analysis of Yap binding proteins showed presence of HDAC4 in the Yap protein complex. HDAC inhibition decreased the level of Yap-HDAC4 complex formation. In vivo, NNK promoted metastasis of pancreatic cancer cells to mice lung and liver; whereas, inhibition of HDAC decreased the number of metastases to a level lower than control treated mice. Smoking promoted liver and lung fibrosis and inflammation and measured by the level of collagen staining, stellate cells activation and immune cells staining. HDAC inhibition reversed these effects of smoking.

Conclusion: We showed for the first time in an animal model that smoking promotes metastasis of PDAC to the lung. We found that HDAC4 is key mediator of this effect. Inhibiting HDAC4 is a promising strategy for preventing PDAC metastasis.

#2736

A molecular and histopathological map of cancer metastasis.

Francisco Sanchez-Vega, Christopher Fong, Walid Chatila, Philip Jonsson, Alexander V. Penson, Renzo G. DiNatale, Michael F. Berger, Ahmet Zehir, Ed Reznik, Jianjiong Gao, Karuna Ganesh, Barry S. Taylor, Nikolaus Schultz. _Memorial Sloan Kettering, New York, NY_.

Metastatic disease is responsible for the vast majority of cancer-related deaths. Patterns of metastatic spread and colonization are, however, not random: routes of metastatic dissemination are cancer-type specific, and different cancer types exhibit varying levels of affinity for specific organs as metastatic target sites.

We analyzed a cohort of more than 30,000 patients that were treated at Memorial Sloan Kettering Cancer Center (MSK) and had their tumors sequenced using the clinical MSK-IMPACT test, a targeted next-generation sequencing assay that identifies mutations, copy number changes and structural rearrangements in 468 genes. By querying the electronic medical record, we catalogued all metastatic events in the clinical history of these patients and mapped them to a set of 32 tissue sites. The primary diagnoses spanned 30 different cancer types, which were further stratified into >50 relevant molecular subtypes, based on a combination of molecular and histopathological features. Observed patterns of metastatic dissemination were cancer type specific and, in some cases, exhibited strong differences across molecular subtypes of the same cancer type. For example, HER2+ subtypes of breast cancer exhibited increased rates of brain (34% vs. 18%, p<0.0001) and lung (31% vs. 19%, p=0.006) metastasis when compared to HER2- subtypes.

The median number of organs affected by metastatic disease per patient was 4 (range 1-14). The number of affected organs exhibited a strong positive correlation with the overall level of aneuploidy, as measured by the total fraction of genome altered by copy number changes (P<0.0001, Spearman correlation), but not with overall mutational burden. We identified genomic alterations that were significantly enriched in tumors from patients with metastasis to specific organs in a cancer-type dependent manner. For example, 244/556 (44%) of lung adenocarcinoma patients with EGFR amplification or activating mutations presented bone metastasis at some point of their clinical history, as opposed to only 400/1,124 (36%) of EGFR wild type patients (p=0.001, Fisher's exact test). In the case of HR+/ER- breast adenocarcinoma patients, bone metastases were reported in 22/30 (73%) of patients with NF1 inactivated tumors, as opposed to only 330/745 (44%) of patients with NF1 wild-type (p=0.002, Fisher's exact test).

We present the first pan-cancer study that combines molecular and histopathological features to draw a comprehensive atlas of tumor metastasis. Our results provide novel insights into the biology of metastatic tumors and can have a direct impact on patient management and care by optimizing the use and frequency of specific imaging studies for metastasis surveillance.

#2737

PRAME misexpression alters protein ubiquitination and leads to increased invasion and metastasis in uveal melanoma.

Margaret I. Sanchez,1 Stefan Kurtenbach,1 Jeffim N. Kuznetsov,1 Daniel A. Rodriguez,1 Dien G. Pham,1 Henry Barreras,2 J. Williams Harbour1. 1 _Bascom Palmer Eye Institute, Sylvester Comprehensive Cancer Center and Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, Miami, FL;_ 2 _University of Miami, Miami, FL_.

Uveal melanoma (UM) is the most common primary intraocular malignancy in human, and is associated with fatal metastases in up to 50% of patients despite effective primary tumor treatment. We previously showed that UMs can be divided into two prognostic subgroups based on gene expression profile (GEP), where class 1 GEP tumors have a low risk of metastasis, and those with the class 2 GEP have a high risk. Recently, we identified PRAME (Preferentially Expressed Antigen in Melanoma) as an independent predictor of metastasis in both class 1 and class 2 UM. In order to gain insight into the mechanisms by which PRAME drives tumor progression, we developed Tet-inducible cell lines allowing for the inducible expression (MEL290 Tet-On PRAME) and knockdown (FP41 Tet-shPRAME) of PRAME. PRAME expression correlates strongly with invasion in cell culture experiments. Further, we developed a Nod-Scid Gamma (NSG) mouse xenograft model using a specialized suprachoroidal injection technique that results in a pattern of metastasis that mimics human UM. In this model, UM cells that express high levels of PRAME developed significantly more and larger liver metastasis and had decreased survival rates compared to the same cell line in which PRAME was depleted using shRNA (p<0.028). These results indicate that PRAME is not only a biomarker of poor outcome, but a functional mediator of metastasis in UM. Furthermore, we performed mass spectrometry after immunoprecipitation and found that components of the Cul2 ubiquitin ligase complex were the most abundant PRAME binding proteins, which is similar to previous findings in leukemia, where PRAME was found to function as a substrate specificity motif for the Cul2 complex. To identify novel PRAME interacting partners and potential substrates of the PRAME-Cul2 complex, we then used a biotin ligase proximity labelling approach (BioID2), followed by biotin pulldown and mass spectrometry. This approach revealed multiple interacting partners from pathways known to modulate proliferation, cell cycle, and invasion. To quantify ubiquitination changes on these newly discovered interacting partners, we performed mass spectrometry after enriching for ubiquitinated peptides (PTMScan®). We observed that PRAME expression significantly changes the ubiquitome of the cells, including several of the interacting partners found by the BioID2 approach. In addition, global mass spectrometry was used to quantify changes in protein levels, allowing to correlate altered ubiquitination of proteins with their degradation. Taken together, these findings show that PRAME misexpression in UM promotes invasion and metastasis by effecting ubiquitomic and proteomic alterations. These findings could serve as a basis for novel drug discovery in the treatment of advanced UM.

#2738

Wnt11 regulates migration and invasion in pancreatic ductal adenocarcinoma.

Tara Hughes, Xinqun Li, Bingbing Dai, Jenying Deng, Christian Siangco, Kaberi Das, Shanshan Bai, Min Hu, Emi Sei, Tapsi Seth, Nicholas Navin, Michael Kim. _MD Anderson Cancer Center, Houston, TX_.

Canonical Wnt/β-catenin signaling is strongly associated with cancer development and metastasis, but non-canonical Wnt signaling and its role in pancreatic ductal adenocarcinoma (PDAC) metastasis is not well understood. Wnt11 is implicated in cancer cell motility through non-canonical Wnt signaling pathways, however, previously described mechanisms are diverse and tissue specific. We investigated Wnt11 expression and its effects on PDAC cell motility/invasion and correlated these with gene expression signatures generated through single cell RNA-sequencing of PDAC tumors. Expression of Wnt11 was confirmed by RT-qPCR in PDAC cell lines derived from genetically engineered mouse models (KPC) and PDAC patient derived xenografts. Wnt11 was overexpressed in three PDAC cell lines following transfection with Wnt11 cDNA cloned into a pcDNA3.1 backbone and confirmed by RT-qPCR and western blot. siRNAs targeting Wnt11 were used to silence Wnt11 in PDAC cell lines relative to non-targeting controls and confirmed by RT-qPCR. Transwell migration/invasion assays were performed in triplicate with matched MTT proliferation assays in 3 PDAC cell lines with stable Wnt11 overexpression and 3 PDAC cell lines following Wnt11 siRNA knockdown. For single cell transcriptomic analysis, single cell suspensions were generated from 14 unique patient PDAC tumors. 10x GemCode microfluidics technology was used to isolate single cells into gel-bead in emulsion (GEM) units and were individually barcoded, lysed and reverse-transcribed. Barcoded cDNA libraries were then amplified and underwent next-generation sequencing. PDAC cell subpopulations with >4-fold levels of Wnt11 expression relative to all sequenced cells were identified and compared to the remaining tumor cell population to develop differentially expressed gene profiles. Pathways analysis of this gene set was performed using ENRICHR. Single cell transcriptomic analysis of PDAC cells derived from 14 different PDAC patients confirmed strong Wnt11 expression in subpopulations of PDAC cells. Wnt11 overexpression resulted in a 3.2-fold increase in migration and invasion (p<0.001) and RNAi-mediated depletion of Wnt11 resulted in a 6.6-fold reduction in migration and invasion (p<0.001). In PDAC cell populations with increased (>4-fold) Wnt11 expression, ERICHR analysis using the NCI pathway interaction database revealed significant enrichment in β-integrin signaling (p<0.001, z = -1.16). Wnt11 is strongly expressed in subpopulations of human PDAC cells and significantly enhances PDAC cell motility and invasion. Ongoing tail vein injection experiments with Wnt11-null PDAC cell lines and Wnt11 wildtype controls will test the in vivo role of Wnt11 in PDAC metastasis. β-integrin signaling is associated with Wnt11 expression in human PDAC tumors and may be involved in PDAC cell motility and invasion.

#2739

EZH2 promotes breast cancer bone metastasis.

Jingkun Qu, Lin Zhang, Zhifen Zhou, Dihua Yu. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Metastasis causes about 90% of cancer deaths and approximately 55% of breast cancer patients develop bone metastases. Although significant advances in the cure of breast cancer, effective therapies targeted bone metastases are still not available. To develop novel therapies for patients with bone metastasis, better understanding of underlying mechanisms of bone metastasis is critical. EZH2 is a classic histone methyl transferase that generally suppresses downstream target genes through tri-methylation of H3K27. EZH2 is considered as a breast cancer oncogene and is frequently over expressed in breast cancer, but the effect of EZH2 on bone metastasis is unknown. To explore the function of EZH2 in bone metastasis, we intracardially injected into nude mice control MDA.MB.231 breast cancer cells expressing readily detectable EZH2 or a MDA.MB.231 subline having EZH2 knocked out (231.EZH2.ko). We found that the mice injected with 231.EZH2.ko had prolonged bone metastasis-free survival compared to the control group of mice. To gain initial insight on the underlying mechanism of EZH2-mediated bone metastasis, we co-cultured MDA.MB.231 cells or 231.EZH2.ko with pre-osteoclasts (RAW264.7) and osteoblasts (MC3T3) under TGF-β treatment (triple co-culture) to mimic bone metastasis tumor microenvironment. We examined osteoclast maturation and breast cancer cell proliferation because osteolysis induced by mature osteoclasts is critical for breast cancer cell growth in the bone metastasis. In triple co-culture of MDA.MB.231 cells, more RAW264.7 pre-osteoclasts were differentiated to mature osteoclasts, as measured by TRAP+ staining, a marker of mature osteoclasts, and MDA.MB.231 cells grew faster than the triple co-culture of 231.EZH2.ko cells. Furthermore, we found that the mRNA level of PTHrP, a master regulator of bone metastasis and a TGF-β downstream cytokine, was expressed at significantly higher level in MDA.MB.231 cells than that in 231.EZH2.ko cells under TGF-β stimulation. PTHrP was known to promote the "vicious cycle" of bone destruction in bone metastasis. The data suggests EZH2 promotes breast cancer bone metastasis through PTHrP. Currently, we are further exploring how EZH2 regulates PTHrP in breast cancer cells to promote bone metastasis. Our studies may reveal novel biomarkers and/or therapeutic targets for bone metastasis.

#2740

MET exon 14 skipping mutation promotes metastasis via activation of the PI3K/Akt-Rac1 pathway in non-small cell lung cancer.

Feng Wang, Elaine Shum, Roman Perez-Soler, Haiying Cheng, Balazs Halmos. _Albert Einstein College of Medicine /Montefiore Medical Center, Bronx, NY_.

The MET proto-oncogene plays crucial roles in cell growth and proliferation, epithelial-mesenchymal (EMT) transition, invasion/metastasis and drug resistance in a broad range of malignancies. In non–small cell lung cancer (NSCLC), aberrant MET signaling can occur through a number of mechanisms that include MET or HGF protein overexpression, MET gene amplification, MET gene mutation or aberrations in downstream signaling or regulatory components. Among the various MET alterations, recurrent MET exon 14 skipping (MET∆14) mutations occur in approximately 3%-5% of NSCLC, and recently emerged as an actionable oncogenic driver. Moreover, MET∆14 was found to be mutually exclusive with other recognized drivers, which further supports its potential oncogenic role.

In the current study, we sought to perform an array of experiments to determine the intrinsic tumorigenic role of MET∆14 in NSCLC. We firstly established several MET∆14 NSCLC cell models using a CRISPR gene editing system. Using these models, we dissected the biochemical events underlying MET∆14 oncogenic activity. Through combining a biotin-labeled receptor internalization assay and fluorescence imaging analysis of receptor endocytic trafficking we identified that MET∆14 decreases ligand-dependent receptor internalization and lysosomal degradation but does not affect a recycling process. Our results indicate that MET∆14 does not affect receptor dimerization but impairs receptor endocytic degradation, which eventually leads to an extended activation of HGF/MET signaling. In subsequent functional analyses, we identified that MET∆14 significantly increases HGF-dependent cell scatter, migration and invasion capacity in vitro as well as metastasis in vivo compared to wild-type MET. In order to define the underlying mechanisms we pursued further experiments and demonstrate that MET∆14 increases cell movement at least partially via regulation of the PI3K/Akt-GTPase Rac1 pathway. Accordingly, MET∆14 significantly increased the HGF-dependent activation of the Rac1 and cell ruffling formation whereas Rac1 knockdown contrastively repressed HGF/MET∆14-mediated Rac1 activation and cell invasion. Separately, the PI3K inhibitor, LY294002 repressed HGF/MET∆14-mediated Rac1 activity and cell movement. Therefore, these data demonstrate that MET∆14 increases cell movement, at least partially via regulation of the PI3K/Akt-Rac1 pathway.

We finally tested the effect of a recently developed potent MET inhibitor, MGCD516, and found that MGCD516 significantly inhibited MET∆14-mediated PI3K/Akt and MAPK activation, repressed Rac1 activation and cell invasion. These results suggested that MET inhibition with agents such as MGCD516 might be potent therapeutic options for MET∆14-mutated NSCLC patients.

#2741

Comprehensive molecular profiling of primary tumors and paired distant metastases in non-small cell lung cancer.

Won-Chul Lee,1 Daniel Gomez,1 Jianhua Zhang,1 Alexandre Reuben,1 Ali Jalali,2 Whijae Roh,3 Chia Chin Wu,1 Wei Lu,1 Chi-Wan Chow,1 Junya Fujimoto,1 Mara Antonoff,1 Cesar Moran,1 Erik Sulman,1 Ganesh Rao,1 Stephen Swisher,1 John Heymach,1 Ignacio I. Wistuba,1 Andrew Futreal,1 Jianjun Zhang1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Baylor College of Medicine, Houston, TX;_ 3 _The Broad Institute, Houston, MA_.

Despite complete resection, many non-small cell lung cancer (NSCLC) patients still develop and succumb to distant metastases, which is a major cause of cancer-related death worldwide. However, our understanding of the molecular mechanisms driving metastasis is rudimentary. We performed whole exome sequencing, RNA sequencing (RNA-seq), methylation microarray, and immunohistochemistry using multiple immune markers on 8 pairs of NSCLC primary tumors and matched distant metastases including 7 metachronous brain and 1 synchronous liver metastases. On average, 60% of all somatic mutations (22% to 90%) and 86% of canonical cancer gene mutations were shared between primary tumors and paired distant metastases. Metastases also resembled paired primary tumors closely in regard to their mutational spectrum, copy number aberrations, allelic imbalance, and methylation profiles. Genomic aberrations unique to metastases were rather patient-specific than commonly observed across multiple patients. Of particular interest, subclonal architecture analysis suggested that monoclonal metastatic seeding is a prevalent mode of metastasis in NSCLC. We validated these findings in a large published dataset consisting of 38 pairs of primary lung tumors and matched distant metastases. RNA-seq showed gene expression profiles in metastases were similar to those of paired primary tumors. Metastases commonly up-regulated metabolism-associated pathways and down-regulated immune-related pathways, consistent with the immunohistochemical staining data for CD3, CD4, CD8, CD20, CD68, PD1, and PD-L1. Our data suggest that distant metastasis is a late event during carcinogenesis and that a majority of genomic and epigenetic aberrations occur before metastatic outgrowth. While molecular mechanisms underlying postsurgical distant metastasis seem to be variable among NSCLC patients, immune suppression may be a common characteristic of cancer cells with metastatic plasticity.

### Signaling in the Tumor Microenvironment

#2742

Bone marrow-derived stromal cells in the tumor microenvironment might be associated with the progression of gastric cancer.

Masakazu Yashiro, Hiroaki Kasashima, Syuhei Kushiyama, Sadaaki Nishimura, Kenji Kuroda, Shingo Togano, Tomohisa Okuno, Yuichiro Miki, Tatsunari Fukuoka, Hirohisa Nakamae, Masayuki Hino, Masaichi Ohira. _Osaka City Univ. Grad. School of Medicine, Osaka, Japan_.

Purpose: It has been reported that the interaction between cancer cells and cancer-associated fibroblasts (CAFs) is closely associated with the progression of various types of cancer. The aim of this study is to clarify the role of bone marrow-derived stromal cells (BM-SCs), which are supposed to be the origin of CAFs in the tumor microenvironment, on the development of gastric cancer.

Methods: The effect of human BM-SCs which frequently express CD271 on the proliferation and motility of six gastric cancer cell lines, OCUM-2M, OCUM-2MD3, OCUM-12, KATO-III, NUGC-3, and MKN-74, was examined. CD271 expression levels in BM-SCs were analyzed by flow cytometry. We also generated a gastric tumor model by orthotopic inoculation of OCUM-2MD3 cells in mice that had received transplantation of bone marrow from CAG-EGFP mice. The correlation between the clinicopathological features of 279 primary gastric carcinomas and CD271 expression in tumor stroma was examined by immunohistochemistry.

Results: Numerous BM-SCs infiltrated the gastric tumor microenvironment; CD271 expression was found approximately 25% of BM-SCs. Conditioned medium from BM-SCs significantly increased the proliferation of gastric cancer cell lines. Furthermore, conditioned medium from gastric cancer cells significantly increased the number of BM-SCs, while migration of OCUM-12 and NUGC-3 cells was significantly increased by conditioned medium from BM-SCs. CD271 expression in stromal cells was significantly associated with macroscopic type-4 cancers, diffuse-type tumors, and tumor invasion depth. The overall survival of patients (n=279) with CD271-positive stromal cells was significantly worse than that of patients with CD271-negative stromal cells. This is the first report of the significance of BM-SCs in gastric cancer progression.

Conclusions: BM-SCs were abundant in the tumor microenvironment of gastric cancer in vivo. In vitro study also indicated that the crosstalk between BM-SCs and gastric cancer cells might play an important role for the development of gastric cancer. CD271-positive tumor stromal cells might be a useful predictive prognostic factor for patients with gastric cancer. BM-SCs, therefore, might be one of therapeutic targets for cancer treatment.

#2743

Mesothelioma exosomes as potential new cancer therapy.

Phillip B. Munson,1 Elizabeth M. Hall,2 Arti Shukla1. 1 _Univ. of Vermont, Burlington, VT;_ 2 _William and Mary, VA_.

Malignant mesothelioma (MM) is a highly invasive cancer arising on the mesothelial surface of organ cavities, predominantly the pleura, and is a direct result of asbestos exposure. Once diagnosed, the disease is virtually incurable with a median lifespan of 6-12 months. Additionally, there is no means of early diagnosis prior to disease symptoms, and the standard of care chemotherapeutics do not extend the life of patients. Taken together, these facts indicate a deep chasm of knowledge that needs to be filled. Our group recently delved into MM tumor biology from the perspective of exosome contained microRNAs (miRNAs). Exosomes are vesicles secreted from every known mammalian cell type and present in essentially all body fluids. Most notably, exosomes play an outsize role in cancer biology. We initially sought to uncover the exosomal miRNAs secreted from MM tumor cells and found a unique signature associated with MM cancer exosomes compared to their mesothelial cell counterparts. The surprising discovery, however, was that the most abundant miRNAs in MM cancer exosomes were tumor suppressors, particularly the pro-apoptotic miR-16-5p. This led us to hypothesize that MM preferentially secreted tumor-suppressor miRNAs via exosomes in order to avoid their effect of killing the tumor cells. Through three novel avenues of potential therapeutic advance, coupled with the possibility of using these exosomal miRNAs as future biomarkers, we have embarked on an innovative strategy to kill MM tumor cells. We have employed small molecule inhibitors to block exosome secretion, thereby reducing miR-16 exosome loss and rebuilding the cellular miR-16 leading to cell death, decreased migration/invasion, and loss of miR-16 target oncogenic proteins CCND1 and BCL2. In addition, we have force-fed concentrated MM tumor exosomes back to MM tumor cells, which led to remarkable levels of cell death, and a reduction in the same oncogenes, CCND1 and BCL2 as seen in our exosome inhibition trials. We have recapitulated these results with direct transfection of miR-16, leading to very comprehensive and convincing evidence that MM tumor cells do in fact secrete high levels of miR-16, and that when re-delivered back to the producer tumor cells through exosome inhibition, exosome force-feeding, or direct transfection, leads to cancer cell death and loss of oncogenic proteins CCND1 and BCL2. Our data, thus far, provide striking new information on a tumorigenic mechanism of MM tumor cells by preferential miR-16 secretion. Moreover we indicate possible miRNA biomarkers through exosomes, and show a never before proposed approach to targeting the death of MM cancer cells by blocking exosome secretion or force-feeding back their own exosomes. This study is supported majorly by Department of Defense, UVM REACH, the UVM Department of Pathology Fellowship, and in small part by NIH RO1.

#2744

Blocking CXCR4 alleviates desmoplasia, increases T-lymphocyte infiltration, and improves immunotherapy in metastatic breast cancer.

Ivy X. Chen,1 Vikash P. Chauhan,1 Jessica Posada,1 Mei R. Ng,1 Michelle Wu,1 Pichet Adstamongkonkul,2 Peigen Huang,1 Neal Lindeman,3 Robert Langer,4 Rakesh K. Jain1. 1 _Massachusetts General Hospital, Boston, MA;_ 2 _Harvard University, Cambridge, MA;_ 3 _Brigham and Women's Hospital, Boston, MA;_ 4 _Massachusetts Institute of Technology, Cambridge, MA_.

Metastatic breast cancers (mBCs) are largely resistant to immune checkpoint blockade, but the mechanisms remain unclear. Primary breast cancers are characterized by a dense fibrotic stroma, which is considered immunosuppressive in multiple malignancies, but the stromal composition of breast cancer metastases and its role in immunosuppression are largely unknown. Here we show that liver and lung metastases of human breast cancers tend to be highly fibrotic, and unlike primary breast tumors, they exclude cytotoxic T-lymphocytes (CTLs). Unbiased analysis of the TCGA database of human breast tumors revealed a set of genes that are associated with stromal T-lymphocyte exclusion. Among these we focused on CXCL12 as a relevant target based on its known roles in immunosuppression in other cancer types. We found that the CXCL12 receptor CXCR4 is highly expressed in both human primary tumors and metastases. To gain insight into the role of CXCL12/CXCR4 axis, we inhibited CXCR4 and found that CXCR4 blockade decreases fibrosis, alleviates solid stress, decompresses blood vessels, increases CTL infiltration, and decreases immunosuppression in murine mBC models. We confirmed that these immunosuppressive effects are dependent on cancer-associated fibroblast (CAF) signaling through genetic deletion of Cxcr4 in aSMA+ cells. Accordingly, CXCR4 inhibition more than doubles the response to immune checkpoint blockers in mice bearing mBCs. These findings demonstrate that CXCL12/CXCR4-mediated desmoplasia in mBC promotes immunosuppression and is a potential target for overcoming therapeutic resistance to immune checkpoint blockade in mBC patients.

#2745

Tumor microenvironment and host genetics impact glioma progression in a Collaborative Cross-based orthotopic allograft model.

Kasey Skinner,1 Martin Ferris,1 Ryan Bash,1 Abigail Shelton,1 Erin Smithberger,1 Steve Angus,1 Brian Golitz,1 Noah Sciaky,1 Jeremy Simon,1 Jason Stein,1 Glenn Matsushima,1 Quinn Ostrom,2 Lindsay Stetson,3 Jill Barnholtz-Sloan,3 Harshil Dhruv,4 Michael Berens,4 Fernando Pardo Manuel de Villena,1 C. Ryan Miller1. 1 _UNC Chapel Hill, Chapel Hill, NC;_ 2 _Baylor College of Medicine, Houston, TX;_ 3 _Case Western Reserve University, Cleveland, OH;_ 4 _Translational Genomics Research Institute, Phoenix, AZ_.

Gliomas are diffusely invasive brain tumors with fatal outcomes and few effective treatments. Precision medicine focuses on targeting the genetics of individual tumors, but not host genetics, despite studies that have linked germline polymorphisms with glioma risk. Accordingly, glioma survival studies in mice utilize genetically variable tumors on identical host genetic backgrounds, which fails to differentiate between cancer cell-autonomous (CCA) and tumor microenvironment (TME) effects on glioma progression and host survival. The Collaborative Cross (CC) is a panel of genetically diverse mouse strains derived from both wild- and traditional inbred laboratory strains that facilitates high-resolution genetic mapping in models of complex disease. Here, we implement a novel platform to discover genetic modifiers of both CCA and TME phenotypes using genetically defined orthotopic murine allograft gliomas and CC hosts. We stereotactically injected Nf1;Trp53-/- oligodendrocyte progenitor-derived mouse tumor cells into syngeneic C57BL/6 control mice and 14 different CC strains. Seven strains survived significantly longer than controls (P<0.05), suggesting slower tumor growth (Gs, growth slow). The remaining 7 strains survived similarly to controls, suggesting fast growth (Gf, growth fast). Variable tumor growth in CC mice suggests that genetic background influences molecular processes in the TME that inhibit or potentiate tumor growth, respectively. To identify candidate genes, we performed RNA sequencing on 36 tumors from 3 Gf strains, 4 Gs strains, and controls. 134 genes were differentially expressed among Gf, Gs, and control tumors (P<0.05). Hierarchical clustering on these genes revealed that Gs strains clustered separately from Gf and controls. Gene ontology analysis using GOrilla showed 30 enriched processes, (FDR q<0.001), all of which were involved in immune responses or extracellular matrix biology. These results suggest that Gs strains activate immune and TME processes that slow tumor growth. Quantitative trait locus (QTL) analyses of host genetics and tumor data are pending and will facilitate identification of genetic variants that influence TME effects on tumor progression.

#2746

Stat3 labels a subpopulation of reactive astrocytes required for brain metastasis.

Neibla Priego,1 Lucía Zhu,1 Catia Monteiro,1 Manon Mulders,1 David Wasilewski,2 Wendy Bindeman,1 Laura Doglio,3 Elena Martínez-Saez,4 Santiago Ramón y Cajal,4 Coral Fustero-Torre,1 Elena Piñeiro-Yáñez,1 Aurelio Hernández-Laín,5 Valeria Poli,6 Javier A. Menéndez,7 Ricardo Soffietti,6 Joaquim Bosch-Barrera,7 Manuel Valiente1. 1 _CNIO, Spain;_ 2 _University Medical Center Hamburg-Eppendorf, Germany;_ 3 _King's College, United Kingdom;_ 4 _Vall d'Hebron Hospital, Spain;_ 5 _Hospital Universitario 12 de Octubre Research Institute, Spain;_ 6 _University and City of Health and Science University Hospital of Turin, Italy;_ 7 _Catalan Institute of Oncology, Spain_.

The diagnosis of brain metastasis involves high morbidity and mortality and remains as an unmet clinical need in spite of being the most common tumor in the brain. Brain metastasis affects between 10-30% of cancer patients with 200.000 new cases yearly only in the US. Increasing evidences point out the relevance of the microenvironment to understand the biology of brain colonization by metastatic cells, however translation strategies targeting it are lacking. We have identified altered signaling pathways in pro-metastatic reactive astrocytes (RA), and translated that into a novel therapeutic application for brain metastasis. Specifically we have identified a subpopulation of RA characterized by activated STAT3 pathway (pSTAT3+). This subpopulation is located in the vicinity of metastatic lesions intermingled with pSTAT3- RA in several experimental models and in 89% of human brain metastases independently of the primary tumor source. The pro-metastatic behaviour of RA is regulated by the activation of the STAT3 pathway, which modulates the immune system locally promoting the survival of cancer cells. Specifically the secretome of pSTAT3+ RA decreases the anti-tumor activity of CD8+ T cells as well as promotes the expansion of the pro-tumor population of CD74+ microglia/macrophage, which infiltrates metastasis cores. Interestingly, the immunosuppressive nature of pSTAT3+ RA is linked to the acquisition of stem cell-like properties, which might reflect the misuse of brain responses to injury instigated by the presence of cancer cells. Genetic and pharmacologic approaches targeting STAT3 in RA impair the progression of brain metastasis, even at advanced stages of the disease. Moreover, a safe and orally bioavailable STAT3 inhibitor reduced brain metastasis in 75% of 18 stage IV lung adenocarcinoma patients with established brain metastases, improving the outcome of the disease by increasing patient survival from 4 to 15 months. Besides increasing therapeutic opportunities for patients with brain metastasis, we have described for the first time the role of reactive astrocytes as regulators of local immunosuppression in brain metastasis as well as the importance of uncovering the heterogeneity within the metastasis-associated microenvironment.

#2747

Loss of S100A10 gene suppresses mammary tumor progression in PyMT mouse tumor model.

Alamelu Bharadwaj,1 Ryan Holloway,1 Patricia Colp,1 Rong-Zong Liu,2 Rosaline Godbout,2 Penny Barnes,1 Paola A. Marignani,1 David Waisman1. 1 _Dalhousie University, Halifax, Nova Scotia, Canada;_ 2 _University of Alberta, Edmonton, Alberta, Canada_.

Introduction: S100A10 (p11) is a plasminogen receptor that we have shown is a master regulator of cellular plasmin generation by cancer cells and is also is essential and sufficient for macrophage migration to tumor sites. In the current study we have investigated the role of p11 in breast cancer tumor progression.

Methods: We have employed gene profiling analysis of breast cancer patient tumors and an in depth study of the Polyoma Middle T (PyMT) transgenic mouse breast cancer model to interrogate the possible role of p11 in breast cancer.

Results: Gene expression profiling from 176 primary breast cancer samples obtained through the CBCF tumor bank showed that p11 mRNA levels were significantly higher in tumors with high Ki67 immunoreactivity, was upregulated in high grade breast tumors, and was also significantly associated with poor patient prognosis (hazard ratio of 3.34). To further investigate the function of p11 in breast cancer, we established the MMTV-PyMT transgenic mouse mammary breast cancer model and generated two groups of animals – PyMT/p11-WT and PyMT/p11-null mice. We observed a significant delay in tumor onset and appearance of palpable tumours ias well as a dramatic reduction (5-fold at 20 weeks) in tumor growth in mice lacking p11 compared to WT controls. Importantly, the total tumor burden at the time of sacrifice was 3.5-fold lower for the p11-null mice concomitant with decreased tumour cell proliferation (Ki67), vascularity (CD31) and macrophage infiltration (F4/80). The histopathological progression to late carcinoma was delayed in PyMT/p11-null mice, with only 6% of PyMT/p11-null mice showing late carcinoma as compared to 69% PyMT/p11-WT at 20 weeks. Consistently, there was a 5-fold decrease in total metastatic foci in the lungs of the PyMT/p11-null mice. Experimental metastasis assay of p11-WT cells injected in p11-WT and p11-null mice showed a dramatic decrease in metastatic burden in p11-null mice suggesting a stromal involvement. Interestingly loss of p11 did not decrease plasmin generation in the PyMT tumors and cells obtained from tumors. Surprisingly, we also observed decreased plasmin independent migration and invasion with loss of p11.

Conclusions: These studies suggest that p11 plays a critical role in breast tumor growth and metastasis, independent of its role as a plasminogen receptor. Future studies are aimed at elucidating the tumor cell or stroma specific role of p11 in breast cancer progression and identifying the intracellular function of p11 in this process.

#2748

The role of TMPRSS2_ERG fusions in modulating tumor microenvironment in prostate cancer.

Hubert Pakula,1 Caroline F. Ribeiro,1 Giuseppe N. Fanelli,2 Rory Kirchner,3 Sudeepa Syamala,1 Basudev Chowdhury,1 Giorgia Zadra,1 Paolo Chetta,4 Zhe Li,5 Massimo Loda1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _University of Padua, Padua, Italy;_ 3 _Harvard T.H. Chan School of Public Health, Boston, MA;_ 4 _University of Milan, Milan, Italy;_ 5 _Brigham and Women's Hospital, Boston, Boston, MA_.

The control region of TMPRSS2 fuses with ETS transcription factor family members, particularly ERG. This occurs in ~20-50% of human prostate cancers depending on the ethnicity. TMPRSS2-ERG (T2E) fusions, however, do not display a notable phenotype in genetically engineered mouse models (GEMM) recapitulating the fusion expression and T2E also has no prognostic significance in humans, despite its high incidence. Wnt signaling has been implicated in prostate cancer. Here, we report a discovery of a role for T2E fusion that impacts the prostate stroma in pre-clinical models. The composition of stromal cells within the microenvironment in T2E mice was studied by single cell RNAseq analysis. We identified stromal cell clusters in T2E mice that differed from wild type mice in the expression of Pdgfrβ, Pdgfrα and Col1a1. Interestingly, these clusters showed an increase of expression of Wnt receptors such as Lgr5 and Fzd7 as well as Wnt ligands such as Sfrp2, Wnt2 and Wnt6. T2E GEMMs also exhibited upregulation of the Wnt-secretion regulator porcupine (PORCN-Protein-serine O-palmitoleoyltransferase) in the stroma adjacent to the T2E-expression prostate epithelial cells. Furthermore, lineage-tracing with the Wnt reporter and epithelial stem cell marker Lgr5 showed that Wnt-active cells were increased in prostatic intraepithelial neoplasia (PIN) lesions in T2E;Pten+/- mice. Strikingly, Lgr5+ cells were also found in the prostate stroma surrounding these PINs. Since, TMPRSS2/ERG fusions represent an early event in prostate tumorigenesis, here we provide a mechanism whereby induction of Wnt signaling in the stroma by T2E-expressing prostate epithelial cells increases the stem cell compartments in both epithelial and stromal cells. These data suggest that aberrant ERG-expressing prostate epithelial cells activate upregulation of Wnt signaling in stromal cells, which provides a possible route for enhancing prostate carcinogenesis. 
